NOVEL PESTICIDAL PROTEINS AND STRAINS

The present invention is drawn to methods and compositions for controlling plant and non-plant pests. Particularly, new pesticidal proteins are disclosed which are isolatable from the vegetative growth stage of Bacillus. Bacillus strains, proteins, and genes encoding the proteins are provided. The methods and compositions of the invention may be used in a variety of systems for controlling plant and non-plant pests.

Insect pests are a major factor in the loss of the world's commercially important agricultural crops. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. There is, however, substantial interest in developing effective alternative pesticides.

Microbial pesticides have played an important role as alternatives to chemical pest control. The most extensively used microbial product is based on the bacterium Bacillus thuringiensis (Bt). Bt is a gram-positive spore forming Bacillus which produces an insecticidal crystal protein (ICP) during sporulation.

Numerous varieties of Bt are known that produce more than 25 different but related ICP's. The majority of ICP's made by Bt are toxic to larvae of certain insects in the orders Lepidoptera, Diptera and Coleoptera. In general, when an ICP is ingested by a susceptible insect the crystal is solubilized and transformed into a toxic moiety by the insect gut proteases. None of the ICP's active against coleopteran larvae such as Colorado potato beetle (Leptiπotarsa decemlineata) or Yellow mealworm (Tenebrio molitor) have demonstrated significant effects on members of the genus Diabrotica particularly Diabrotica virgifera the western corn rootworm (WCRW) or Diabrotica longicornis barbed, the northern corn rootworm.

Bacillus cereus (Be) is closely related to Bt. A major distinguishing characteristic is the absence of a parasporal crystal in Be. Be is a widely distributed bacterium that is commonly found in soil and has been isolated from a variety of foods and drugs. The organism has been implicated in the spoilage of food.

Although Bt has been very useful in controlling insect pests, there is a need to expand the number of potential biological control agents.

Within the present invention compositions and methods for controlling plant pests are provided. In particular, novel pesticidal proteins are provided which are produced during vegetative growth of Bacillus strains. The proteins are useful as pesticidal agents.

More specifically, the present invention relates to a substantially purified Bacillus strain which produces a pesticidal protein during vegetative growth wherein said Bacillus is not B. sphaericus SSI 1-1. Preferred are a Bacillus cereus strain having Accession No. NRRL B-21058 and Bacillus thυriπgiensis strain having Accession No. NRRL B-21060. Also preferred is a Bacillus strain selected from Accession Numbers NRRL B-21224, NRRL B-21225, NRRL B-21226, NRRL B-21227, NRRL B-21228, NRRL B-21229, NRRL B-21230, and NRRL B-21439.

The invention further relates to an insect-specific protein isolatable during the vegetative growth phase of Bacillus spp, but preferably of a Bacillus thυringiensis and B. cereus strain, and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1. The insect-specific protein of the invention is preferably toxic to Coleoptera or Lepidoptera insects and has a molecular weight of about 30 kDa or greater, preferably of about 60 to about 100 kDa, and more preferably of about 80 kDa.

More particularly, the insect-specific protein of the invention has a spectrum of insecticidal activity that includes an activity against Agrotis and/or Spodoptera species, but preferably a black cutworm [Agrotis ipsilon ; BCW] and/or fall armyworm [Spodoptera frugiperda] and/or beet armyworm [Spodoptera exigυa ] and/or tobacco budworm and/or corn earworm [Helicoverpa zea] activity.

The insect-specific protein of the invention can preferably be isolated, for example, from Bacillus cereus having Accession No. NRRL B-21058, or from Bacillus thuringiensis having Accession No. NRRL B-21060.

The insect-specific protein of the invention can also preferably be isolated from a Bacillus spp strain selected from Accession Numbers NRRL B-21224, NRRL B- 21225, NRRL B-21226, NRRL B-21227, NRRL B-21228, NRRL B-21229, NRRL B- 21230, and NRRL B-21439.

The present invention especially encompasses an insect-specific protein that has the amino acid sequence selected from the group consisting of SEQ ID NO:5 and

SEQ ID NO:7, including any proteins that are structurally and/or functionally homologous thereto.

Further preferred is an insect-specific protein, wherein said protein has the sequence selected from the group consisting of SEQ ID NO.20, SEQ ID NO:21 , SEQ ID NO:29 SEQ ID NO.32 and SEQ ID NO:2, including any proteins that are structurally and/or functionally homologous thereto.

Especially preferred is an insect-specific protein, wherein said protein has the sequence selected from the group consisting of SEQ ID NO.29 and SEQ ID NO:32, including any proteins that are structurally and/or functionally homologous thereto.

A further preferred embodiment of the invention comprises an insect-specific protein of the invention, wherein the sequences representing the secretion signal have been removed or inactivated.

The present invention further encompasses auxiliary proteins which enhance the insect-specific activity of an insect-specific protein. The said auxiliary proteins preferably have a molecular weight of about 50 kDa and can be isolated, for example, from the vegetative growth phase of a Bacillus cereus strain, but especially of Bacillus cereus strain AB78.

A preferred embodiment of the invention relates to an auxiliary protein, wherein the sequences representing the secretion signal have been removed or inactivated.

The present invention further relates to multimeric pesticidal proteins, which comprise more than one polypeptide chain and wherein at least one of the said polypeptide chains represents an insect-specific protein of the invention and at least one of the said polypeptide chains represents an auxiliary protein of the invention, which activates or enhances the pesticidal activity of the said insect-specific protein.

The multimeric pesticidal proteins according to the invention preferably have a molecular weight of about 50 kDa to about 200 kDa.

The invention especially encompasses a multimeric pesticidal protein, which comprises an insect-specific protein of the invention and an auxiliary protein according to the invention, which activates or enhances the pesticidal activity of the said insect- specific protein.

The present invention further relates to fusion proteins comprising several protein domains including at least an insect-specific protein of the invention and/or an auxiliary protein according to the invention produced by in frame genetic fusions,

which, when translated by ribosomes, produce a fusion protein with at least the combined attributes of the insect-specific protein of the invention and/or an auxiliary protein according to the invention and, optionally, of the other components used in the fusion.

A specific embodiment of the invention relates to a fusion protein comprising a ribo¬ nuclease S-protein, an insect-specific protein of the invention and an auxiliary protein according to the invention.

A further specific embodiment of the invention relates to a fusion protein comprising an insect-specific protein according to the invention and an auxiliary protein according to the invention having either the insect-specific protein or the auxiliary protein at the N-terminal end of the said fusion protein.

Preferred is a fusion protein, which comprises an insect-specific protein as given in SEQ ID NO:5 and an auxiliary protein as given in SEQ ID NO: 2 resulting in the protein given in SEQ ID NO: 23, including any proteins that are structurally and/or functionally homologous thereto.

Also preferred is a fusion protein, which comprises an insect-specific protein as given in SEQ ID NO.35 and an auxiliary protein as given in SEQ ID NO: 27 resulting in the protein given in SEQ ID NO: 50, including any proteins that are structurally and/or functionally homologous thereto.

The invention further relates to a fusion protein comprising an insect-specific protein of the invention and/or an auxiliary protein according to the invention fused to a signal sequence, preferably a secretion signal sequence or a targeting sequence that directs the transgene product to a specific organelle or cell compartment, which signal sequence is of herterologous origin with respect to the recipient protein.

Especially preferred within this invention is a fusion protein wherein the said protein has a sequence as given in SEQ ID NO: 43, or in SEQ ID NO: 46, including any proteins that are structurally and/or functionally homologous thereto.

As used in the present application, substantial sequence homology means close structural relationship between sequences of amino acids. For example, substantially homologous proteins may be 40% homologous, preferably 50% and most preferably 60% or 80% homologous, or more. Homology also includes a relationship wherein one or several subsequences of amino acids are missing, or subsequences with additional amino acids are interdispersed.

A further aspect of the invention relates to a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein isolatable during the vegetative growth phase of Bacillus spp. and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1. In particular, the present invention relates to a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein wherein the spectrum of insecticidal activity includes an activity against Agrotis and/or Spodoptera species, but preferably a black cutworm [Agrotis ipsilon ; BCW] and/or fall armyworm [Spodoptera frυgiperda] and/or beet armyworm [Spodoptera exigua ] and/or tobacco budworm and or corn earworm [Helicoveφa zea] activity.

Preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ, ID NO: 4, or SEQ ID NO: 6, including any DNA molecules that are structurally and/or functionally homologous thereto.

Also preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given SEQ ID NO:19, SEQ ID NO.28, SEQ ID NO.31 , or SEQ ID NO:1 , including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to a DNA molecule comprising a nucleotide sequence which encodes an auxiliary protein according to the invention which enhances the insect-specific activity of an insect-specific protein.

Preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given SEQ ID NO:19, including any DNA molecules that are structurally and/or functionally homologous thereto.

A further embodiment of the invention relates to a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein isolatable during the vegetative growth phase of Bacillus spp. and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1 , which nucleotide sequence has been optimized for expression in a microorganism or a plant.

Preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO:17 or SEQ ID NO:18, including any DNA molecules that are structurally and/or functionally homologous thereto.

Also preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO:27, or

SEQ ID NO:30, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to a DNA molecule which comprises a nucleotide sequence encoding a multimeric pesticidal protein, which comprises more than one polypeptide chains and wherein at least one of the said polypeptide chains represents an insect-specific protein of the invention and at least one of the said polypeptide chains represents an auxiliary protein according to the invention, which activates or enhances the pesticidal activity of the said insect-specific protein.

Preferred is a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein of the invention and an auxiliary protein according to the invention, which activates or enhances the pesticidal activity of the said insect-specific protein.

Especially preferred is a DNA molecule, wherein said molecule comprises a nucleotide sequence as given in SEQ ID NO:1 or SEQ ID NO:19, including any nucleotide sequences that are structurally and/or functionally homologous thereto. A further embodiment of the invention relates to a DNA molecule which comprises a nucleotide sequence encoding a fusion protein comprising several protein domains including at least an insect-specific protein of the invention and/or an auxiliary protein according to the invention produced by in frame genetic fusions, which, when translated by ribosomes, produce a fusion protein with at least the combined attributes of the insect-specific protein of the invention and/or an auxiliary protein according to the invention and, optionally, of the other components used in the fusion.

Preferred within the invention is a DNA molecule which comprises a nucleotide sequence encoding a fusion protein comprising an insect-specific protein according to the invention and an auxiliary protein according to the invention having either the insect-specific protein or the auxiliary protein at the N-terminal end of the said fusion protein. Especially preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO:22, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to a DNA molecule which comprises a nucleotide sequence encoding a fusion protein comprising an insect-specific protein of the invention and/or an auxiliary protein of the invention fused to a signal sequence, preferably a secretion signal sequence or a targeting sequence that directs the

transgene product to a specific organelle or cell compartment, which signal sequence is of herterologous origin with respect to the recipient DNA.

The present invention further encompasses a DNA molecule comprising a nucleotide sequence encoding a fusion protein or a mulitmeric protein according to the invention that has been optimized for expression in a microorganism or plant.

Preferred is an optimized DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO.42, SEQ ID NO.45, or SEQ ID NO.49, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to an optimized DNA molecule, wherein the sequences encoding the secretion signal have been removed from its 5' end, but especially to an optimized DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO: 35 or SEQ ID NO.39, including any DNA molecules that are structurally and/or functionally homologous thereto. As used in the present application, substantial sequence homology means close structural relationship between sequences of nucleotides. For example, substantially homologous DNA molecules may be 60% homologous, preferably 80% and most preferably 90% or 95% homologous, or more. Homology also includes a relationship wherein one or several subsequences of nucleotides or amino acids are missing, or subsequences with additional nucleotides or amino acids are interdispersed.

Also comprised by the present invention are DNA molecules which hybridizes to a DNA molecule according to the invention as defined hereinbefore, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said insect-specific protein at least 10 nucleotides in length, under moderately stringent conditions and which molecules have insect-specific activity and also the insect-specific proteins being encoded by the said DNA molecules.

Preferred are DNA molecules, wherein hybridization occurs at 65°C in a buffer comprising 7% SDS and 0.5 M sodium phosphate.

Especially preferred is a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein according to the invention obtainable by a process comprising

(a) obtaining a DNA molecule comprising a nucleotide sequence encoding an insect- specific protein; and

(b) hybridizing said DNA molecule with an oligonucleotide probe acording to claim 107 obtained from a DNA molecule comprising a nucleotide sequence as given in SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 31 ; and

(c) isolating said hybridized DNA.

The invention further relates to an insect-specific protein, wherein the said protein is encoded by a DNA molecule according to the invention.

Also encompassed by the invention is an expression cassette comprising a DNA molecule according to the invention operably linked to expression sequences including the transcriptional and translational regulatory signals necessary for expression of the associated DNA constructs in a host organism, preferably a microorganism or a plant, and optionally further regulatory sequences.

The invention further relates to a vector molecule comprising an expression cassette according to the invention.

The expression cassette and/or the vector molecule according to the invention are preferably part of the plant genome.

A further embodiment of the invention relates to a host organism, preferably a host organism selected from the group consisting of plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes and algae, comprising a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette, preferably stably incorporated into the genome of the host organism.

The invention further relates to a transgenic plant, but preferably a maize plant, including parts as well as progeny and seed thereof comprising a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette, preferably stably incorporated into the plant genome.

Preferred is a transgenic plant including parts as well as progeny and seed thereof which has been stably transformed with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette.

Also preferred is a transgenic plant including parts as well as progeny and seed thereof which expresses an insect-specific protein according to the invention.

The invention further relates to a transgenic plant, preferably a maize plant, according to the invention as defined hereinbefore, which further expresses a second distinct insect control principle, but preferably a Bt δ-endotoxin. The said plant is preferably a hybrid plant.

Parts of transgenic plants are to be understood within the scope of the invention to comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed with a DNA molecule according to the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.

The invention further relates to plant propagating material of a plant according to the invention, which is treated with a seed protectant coating.

The invention further encompasses a microorganism transformed with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette, wherein the said microorganism is preferably a microorganism that multiply on plants and more preferably a root colonizing bacterium.

A further embodiment of the invention relates to an encapsulated insect-specific protein which comprises a microorganism comprising an insect specific protein according to the invention.

The invention also relates to an entomocidal composition comprising a host organism of the invention, but preferably a purified Bacillus strain, in an insecticidally- effective amount together with a suitable carrier.

Further comprised by the invention is an entomocidal composition comprising an isolated protein molecule according to the invention, alone or in combination with a host organism of the invention and/or an encapsulated insect-specific protein according to the invention, in an insecticidally-effective amount, together with a suitable carrier.

A further embodiment of the invention relates to a method of obtaining a purified insect-specific protein according to the invention, said method comprising applying a

solution comprising said insect-specific protein to a NAD column and eluting bound protein.

Also comprised is a method for identifying insect activity of an insect-specific protein according to the invention, said method comprising: growing a Bacillus strain in a culture; obtaining supernatant from said culture; allowing insect larvae to feed on diet with said supernatant; and, determining mortality. Another aspect of the invention relates to a method for isolating an insect-specific protein according to the invention, said method comprising: growing a Bacillus strain in a culture; obtaining supernatant from said culture; and, isolating said insect-specific protein from said supernatant. The invention also encompasses a method for isolating a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein exhibiting the insecticidal activity of the proteins according to the invention, said method comprising: obtaining a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein; and hybridizing said DNA molecule with DNA obtained from a Bacillus species; and isolating said hybridized DNA. The invention further relates to a method of increasing insect target range by using an insect specific protein according to the invention in combination with at least one second insecticidal protein that is different from the insect specific protein according to the invention, but preferably with an insecticidal protein selected from the group consisting of Bt δ-endotoxins, protease inhibitors, lectins, α-amylases and peroxidases.

Preferred is a method for increasing insect target range within a plant by expressing within the said plant a insect specific protein according to the invention in combination with at least one second insecticidal protein that is different from the insect specific protein according to the invention, but preferably with an insecticidal protein selected from the group consisting of Bt δ-endotoxins, protease inhibitors, lectins, α-amylases and peroxidases.

Also comprised is a method of protecting plants against damage caused by an insect pest, but preferably by Spodoptera and/or Agrotis species, and more preferably by an insect pest selected from the group consisting of black cutworm [Agrotis ipsilon ; BCW], fall armyworm [Spodoptera frugiperda], beet armyworm [Spodoptera exigua ], tobacco budworm and corn earworm [Helicoverpa zea] comprising applying to the plant or the growing area of the said plant an entomocidal composition or a toxin protein according to the invention.

The invention further relates to method of protecting plants against damage caused by an insect pest, but preferably by Spodoptera and/or Agrotis species, and more preferably by an insect pest selected from the group consisting of black cutworm [Agrotis ipsilon ; BCW], fall armyworm [Spodoptera frugiperda], beet armyworm [Spodoptera exigua ], tobacco budworm and corn earworm [Helicoveφa zea] comprising planting a transgenic plant expressing a insect-specific protein according to the invention within an area where the said insect pest may occur.

The invention also encompasses a method of producing a host organism which comprises stably integrated into its genome a DNA molecule according to the invention and preferably expresses an insect-specific protein according to the invention comprising transforming the said host organism with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette.

A further embodiment of the invention relates to a method of producing a transgenic plant or plant cell which comprises stably integrated into the plant genome a DNA molecule according to the invention and preferably expresses an insect- specific protein according to the invention comprising transforming the said plant and plant cell, respectively, with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette.

The invention also relates to a method of producing an entomocidal composition comprising mixing an isolated Bacillus strain and/or a host organism and/or an isolated protein molecule, and/or an encapsulated protein according to the invention in an insecticidally-effective amount with a suitable carrier.

The invention also encompasses a method of producing transgenic progeny of a transgenic parent plant comprising stably incorporated into the plant genome a DNA

molecule comprising a nucleotide sequence encoding an insect-specific protein according to the invention comprising transforming the said parent plant with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette and transferring the pesticidal trait to the progeny of the said transgenic parent plant involving known plant breeding techniques.

Also encompassed by the invention is oligonucleotide probe capable of specifically hybridizing to a nucleotide sequence encoding a insect-specific protein isolatable during the vegetative growth phase of Bacillus spp. and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1 , wherein said probe comprises a contiguous portion of the coding sequence for the said insect- specific protein at least 10 nucleotides in length and the use of the said oligonucleotide probe for screening of any Bacillus strain or other organisms to determine whether the insect-specific protein is naturally present or whether a particular transformed organism includes the said gene

The present invention recognizes that pesticidal proteins are produced during vegetative growth of Bacillus strains. Having recognized that such a class exists, the present invention embraces all vegetative insecticidal proteins, hereinafter referred to as VIPs, except for the mosquitocidal toxin from B. sphaericus.

The present VIPs are not abundant after sporulation and are particularly expressed during log phase growth before stationary phase. For the purpose of the present invention vegetative growth is defined as that period of time before the onset of sporulation. Genes encoding such VIPs can be isolated, cloned and transformed into various delivery vehicles for use in pest management programs.

For purposes of the present invention, pests include but are not limited to insects, fungi, bacteria, nematodes, mites, ticks, protozoan pathogens, animal-parasitic liver flukes, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.

Tables 1 - 10 gives a list of pests associated with major crop plants and pests of human and veterinary importance. Such pests are included within the scope of the present invention.

TABLE 1

Lepidoptera (Butterflies and Moth)

Maize

Ostrinia nubilalis, European corn borer Agrotis ipsilon, black cutworm Helicoverpa zea, corn earworm Spodoptera frugiperda, fall armyworm Diatraea grandiosella, southwestern corn borer Elasmopalpus lignosellus, lesser cornstalk borer Diatraea saccharalis, sugarcane borer

Sorghum

Chilo partellus, sorghum borer Spodoptera frugiperda, fall armyworm Helicoverpa zea, corn earworm Elasmopalpus lignosellus, lesser cornstalk borer Feltia subterranea, granulate cutworm

Wheat

Pseudaletia υnipunctata, army worm Spodoptera frugiperda, fall armyworm Elasmopalpus lignosellus, lesser cornstalk borer Agrotis orthogonia, pale western cutworm Elasmopalpus lignosellus, lesser cornstalk borer

Sunflower

Suleima helianthana, sunflower bud moth Homoeosoma electellum, sunflower moth

Cotton

Heliothis virescens, cotton boll worm Helicoverpa zea, cotton bollworm Spodoptera exigua, beet armyworm Pectinophora gossypiella, pink bollworm

Rice

Diatraea saccharalis, sugarcane borer Spodoptera frugiperda, fall armyworm Helicoverpa zea, corn earworm

Soybean

Pseudoplusia includens, soybean looper Anticarsia gemmatalis, velvetbean caterpillar Plathypena scabra, green cloverworm Ostrinia nubilalis, European corn borer Agrotis ipsilon, black cutworm Spodoptera exigua, beet armyworm Heliotnis virescens, cotton boll worm Helicoverpa zea, cotton bollworm

Barley

Ostrinia nubilalis, European corn borer Agrotis ipsilon, black cutworm

TABLE 2 Coleoptera (Beetles)

Maize

Diabrotica virgifera virgifera, western corn rootworm Diabrotica longicornis barberi, northern corn rootworm Diabrotica undecimpunctata howardi, southern corn rootworm Melanotus spp., wireworms

Cyclocephala borealis, northern masked chafer (white grub) Cyclocephala immaculata, southern masked chafer (white grub) Popillia japonica, Japanese beetle Chaetocnema pulicaria, corn flea beetle Sphenophorus maidis, maize billbug

Sorghum

Phyllophaga crinita, white grub Eleodes, Conoderυs, and Aeolus spp., wireworms Oulema melanopus, cereal leaf beetle Chaetocnema pulicaria, corn flea beetle Sphenophorus maidis, maize billbug

Wheat

Oulema melanopus, cereal leaf beetle

Hypera punctata, clover leaf weevil

Diabrotica undecimpunctata howardi, southern corn rootworm

Sunflower

Zygogramma exclamationis, sunflower beetle Bothyrus gibbosus, carrot beetle

Cotton

Anthonomus grandis, boll weevil

Rice

Colaspis brunnea, grape colaspis Lissorhoptrus oryzophilus, rice water weevil Sitophilus oryzae, rice weevil

Soybean

Epilachna varivestis, Mexican bean beetle

TABLE 3

Homoptera (Whiteflies. Aphids etc..)

Maize

Rhopalosiphum maidis, corn leaf aphid Anuraphis maidiradicis, corn root aphid

Sorghum

Rhopalosiphum maidis, corn leaf aphid Sipha flava, yellow sugarcane aphid

Wheat

Russian wheat aphid Schizaphis graminυm, greenbug Macrosiphum avenae, English grain aphid

Cotton

Aphis gossypii, cotton aphid Pseudatomoscelis seriatus, cotton fleahopper Trialeurodes abutilonea, bandedwinged whitefly

Rice

Nephotettix nigropictus, rice leafhopper

Soybean

Myzus persicae, green peach aphid Empoasca fabae, potato leafhopper

Barley

Schizaphis graminum, greenbug

Oil Seed Rape

Brevicoryne brassicae, cabbage aphid

TABLE 4 Hemiptera (Bugs)

Maize

Blissus leucopterus leucopterus, chinch bug

Sorghum

Blissus leucopterus leucopterus, chinch bug

Cotton

Lygus lineolaris, tarnished plant bug

Rice

Blissus leucopterus leucopterus, chinch bug Acrosternum hilare, green stink bug

Soybean

Acrosternum hilare, green stink bug

Barley

Blissus leucopterus leucopterus, chinch bug Acrosternum hilare, green stink bug Euschistus servus, brown stink bug

TABLE 5

Orthoptera (Grasshoppers, Crickets, and Cockroaches)

Maize

Melanoplus femurrubrum, redlegged grasshopper Melanoplus sangυinipes, migratory grasshopper

Wheat

Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper Melanoplus sanguinipes, migratory grasshopper

Cotton

Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper

Soybean

Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper

Structural/Household

Periplaneta americana, American cockroach Blattella germanica, German cockroach Blatta orientalis, oriental cockroach

TABLE 6

Diptera (Flies and Mosquitoes)

Maize

Hylemya platura, seedcorn maggot Agromyza parvicornis, corn blotch leafminer

Sorghum

Contarinia sorghicola, sorghum midge

Wheat

Mayetiola destructor, Hessian fly Sitodiplosis mosellana, wheat midge Meromyza americana, wheat stem maggot Hylemya coarctata, wheat bulb fly

Sunflower

Neolasioptera murtfeldtiana, sunflower seed midge

Soybean

Hylemya platura, seedcorn maggot

Barley

Hylemya platura, seedcorn maggot Mayetiola destructor, Hessian fly

Insects attacking humans and animals and disease carriers

Aedes aegypti, yellowfever mosquito Aedes albopictus, forest day mosquito Phlebotomus papatasii, sand fly Musca domestica, house fly Tabanus atratus, black horse fly Cochliomyia hominivorax, screwworm fly

TABLE 7 Thvsanoptera (Thrips)

Maize

Anaphothrips obscurus, grass thrips

Wheat

Frankliniella fusca, tobacco thrips

Cotton

Thrips tabaci, onion thrips Frankliniella fusca, tobacco thrips

Soybean

Sericothrips variabilis, soybean thrips Thrips tabaci, onion thrips

TABLE 8

Hymenoptera (Sawflies, Ants. Wasps, etc.)

Maize

Solenopsis milesta, thief ant

Wheat

Cephus cinctus, wheat stem sawfly

TABLE 9

Other Orders and Representative Species

Dermaptera (Earwigs)

Forficula auricυlaria, European earwig

Isoptera (Termites)

Reticulitermes flavipes, eastern subterranean termite

Mallophaga (Chewing Lice)

Cuclotogaster heterographa, chicken head louse Bovicola bovis, cattle biting louse

Anoplura (Sucking Lice)

Pediculus humanυs, head and body louse

Siphonaptera (Fleas)

Ctenocephalides felis, cat flea

TABLE 10

Acari (Mites and Ticks)

Maize

Tetranychus urticae, twospotted spider mite

Sorghum

Tetranychus cinnabarinus, carmine spider mite Tetranychus urticae, twospotted spider mite

Wheat

Aceria tulipae, wheat curl mite

Cotton

Tetranychus cinnabarinus, carmine spider mite Tetranychus urticae, twospotted spider mite

Soybean

Tetranychus turkestani, strawberry spider mite Tetranychus urticae, twospotted spider mite

Barley

Petrobia latens, brown wheat mite

Important human and animal Acari

Demacentor variabilis, American dog tick

Argas persicus, fowl tick

Dermatophagoides farinae, American house dust mite

Dermatophagoides pteronyssinus, European house dust mite

Now that it has been recognized that pesticidal proteins can be isolated from the vegetative growth phase of Bacillus, other strains can be isolated by standard techniques and tested for activity against particular plant and non-plant pests. Generally Bacillus strains can be isolated from any environmental sample, including soil, plant, insect, grain elevator dust, and other sample material, etc., by methods

known in the art. See, for example, Travers et al. (1987) Appl. Environ. Microbiol. 53:1263-1266; Saleh et al. (1969) Can J. Microbiol. 15:1101-1 104; DeLucca et al. (1981 ) Can. J. Microbiol. 27:865-870; and Norris, et al. (1981) "The genera Bacillus and Sporolactobacillus," In Starr et al. (eds.), The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria, Vol. II, Springer-Verlog Berlin Heidelberg. After isolation, strains can be tested for pesticidal activity during vegetative growth. In this manner, new pesticidal proteins and strains can be identified.

Such Bacillus microorganisms which find use in ^' the invention include Bacillus cereus and Bacillus thuringiensis, as well as those Bacillus species listed in Table 1 1.

TABLE 1 1 List of Bacillus species

Morphological Group 1

B. megaterium

B. cereus ^*

B. cereus var. mycoides

B. thuringiensis ^*

B. licheniformis

B. subtilis ^*

B. pumilus

B. firmus ^*

B. coagulans

Morphological Group 2

B. polymyxa

B. macerans

B. circulans

B. stearothermophilus

B. alvei ^*

B. laterosporus ^*

B. brevis

B. pulvifaciens

B. popilliae ^*

B. lentimorbus ^*

B. larvae ^*

Morphological Group 3

B. sphaericus ^* B. pasteurii

Unassigned Strains

Subgroup A

B. apiarυs ^* B. filicolonicus B. thiaminolyticus B. alcalophilus

Subgroup B

B. cirroflagellosus B. chitinosporus B. lentus

Subgroup C

B. badius B. aneυrinolyticus B. macroides B. freundenreichii

Subgroup D

B. pantothenticus B. epiphytus

Subgroup E1

B. aminovorans B. globisporus B. insolitυs B. psychrophilus

Subgroup E2

B. psychrosaccharolyticus B. macquariensis

^* =Those Bacillus strains that have been previously found associated with insects Grouping according to Parry, J.M. et al. (1983) Color Atlas of Bacillus species, Wolfe Medical Publications, London.

In accordance with the present invention, the pesticidal proteins produced during vegetative growth can be isolated from Bacillus. In one embodiment, insecticidal proteins produced during vegetative growth, can be isolated. Methods for protein isolation are known in the art. Generally, proteins can be purified by conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. Such methods are known in the art. See for example Current Protocols in Molecular Biology. Vols. 1 and 2, Ausubel et al. (eds.), John Wiley & Sons, NY (1988). Additionally, antibodies can be prepared against substantially pure preparations of the protein. See, for example, Radka et al. (1983) J_ Immunol. 128:2804; and Radka et al. (1984) Immunooenetics 19:63. Any combination of methods may be utilized to purify protein having pesticidal properties. As the protocol is being formulated, pesticidal activity is determined after each purification step.

Such purification steps will result in a substantially purified protein fraction. By "substantially purified" or "substantially pure" is intended protein which is substantially free of any compound normally associated with the protein in its natural state. "Substantially pure" preparations of protein can be assessed by the absence of other detectable protein bands following SDS-PAGE as determined visually or by densitometry scanning. Alternatively, the absence of other amino-terminal sequences or N-terminal residues in a purified preparation can indicate the level of purity. Purity can be verified by rechromatography of "pure" preparations showing the absence of other peaks by ion exchange, reverse phase or capillary electrophoresis. The terms "substantially pure" or "substantially purified" are not meant to exclude artificial or synthetic mixtures of the proteins with other compounds. The terms are also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the protein, and which may be present, for example, due to incomplete purification.

Once purified protein is isolated, the protein, or the polypeptides of which it is comprised, can be characterized and sequenced by standard methods known in the art. For example, the purified protein, or the polypeptides of which it is comprised, may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem. 257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res. 21 :209-215). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and electroblotting onto PVDF membranes, and subjected to amino acid sequencing. To accomplish this task, the peptides are preferably analyzed by automated sequenators. It is recognized that N-terminal, C-terminal, or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the gene encoding the pesticidal protein.

It is recognized that the pesticidal proteins may be oligomeric and will vary in molecular weight, number of protomers, component peptides, activity against particular pests, and in other characteristics. However, by the methods set forth herein, proteins active against a variety of pests may be isolated and characterized.

Once the purified protein has been isolated and characterized it is recognized that it may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the pesticidal proteins can be prepared by mutations in the DNA. Such variants will possess the desired pesticidal activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

In this manner, the present invention encompasses the pesticidal proteins as well as components and fragments thereof. That is, it is recognized that component protomers, polypeptides or fragments of the proteins may be produced which retain pesticidal activity. These fragments include truncated sequences, as well as N-terminal, C-terminal, internal and internally deleted amino acid sequences of the proteins.

Most deletions, insertions, and substitutions of the protein sequence are not expected to produce radical changes in the characteristics of the pesticidal protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.

The proteins or other component polypeptides described herein may be used alone or in combination. That is, several proteins may be used to control different insect pests.

Some proteins are single polypeptide chains while many proteins consist of more than one polypeptide chain, i.e., they are oligomeric. Additionally, some VIPs are pesticidally active as oligomers. In these instances, additional protomers are utilized to enhance the pesticidal activity or to activate pesticidal proteins. Those protomers which enhance or activate are referred to as auxiliary proteins. Auxiliary proteins activate or enhance a pesticidal protein by interacting with the pesticidal protein to form an oligomeric protein having increased pesticidal activity compared to that observed in the absence of the auxiliary protein.

Auxiliary proteins activate or increase the activity of pesticidal proteins such as the VIP1 protein from AB78. Such auxiliary proteins are exemplified by, but not limited to, the VIP2 protein from AB78. As demonstrated in the Experimental section of the application, auxiliary proteins can activate a number of pesticidal proteins. Thus, in one embodiment of the invention, a plant, Parent 1 , can be transformed with an auxiliary protein. This Parent 1 can be crossed with a number of Parent 2 plants transformed with one or more pesticidal proteins whose pesticidal activities are activated by the auxiliary protein.

Amongst the pesticidal proteins of the invention a new class of insect-specific proteins could be surprisingly identified within the scope of the present invention. The said proteins, which are designated throughout this application as VIP3, can be obtained from Bacillus spp strains, but preferably from Bacillus thuringiensis strains and most preferably from Bacillus thuringiensis strains AB88 and AB424. The said VIPs are present mostly in the supernatants of Bacillus cultures amounting to at least 75% of the total in strain AB88. The VIP3 proteins are further characterized by their unique spectrum of insectical acitivity, which includes an activity against Agrotis and/or Spodoptera species, but especially a black cutworm [BCW] and/or fall

armyworm and/or beet armyworm and/or tobacco budworm and/or corn earworm activity.

Black cutworm is an agronomically important insect quite resistant to δ-endotoxins. Macintosh et al (1990) J Invertebr Pathol 56, 258-266 report that the δ-endotoxins CrylA(b) and CrylA(c) possesses insecticidal properties against BCW with LC∞ of more than 80 μg and 18 μg ml of diet respectively. The vip3A insecticidal proteins according to the invenition provide >50% mortality when added in an amount of protein at least 10 to 500, preferably 50 to 350, and more preferably 200 to 300 fold lower than the amount of CrylA proteins needed to achieve just 50% mortality. Especially preferred within the invention are vip3A insecticidal proteins which provide 100% mortality when added in an amount of protein at least 260 fold lower than the amount of CrylA proteins needed to achieve just 50% mortality.

The vip3 insecticidal proteins according to the invention are present mostly in the supernatants of the cultures and are therefore are to be classified as secreted proteins. They preferably contain in the N-terminal sequence a number of positively charged residues followed by a hydrophobic core region and are not N-terminally processed during export.

As the other pesticidal proteins reported hereto within the scope of the invention, the VIP3 proteins can be detected in growth stages prior to sporulation establishing a further clear distinction from other proteins that belong to the δ-endotoxin family. Preferably, expression of the insect-specific protein starts during mid-log phase and continues during sporulation. Owing to the specific expression pattern in combination with the high stability of the VIP3 proteins, large amounts of the VIP3 proteins can be found in supernatants of sporulating cultures. Especially preferred are the VIP3 proteins identified in SEQ ID NO.29 and SEQ ID NO.32 and the corresponding DNA molecules comprising nucleotide sequences encoding the said proteins, but especially those DNA molecules comprising the nucleotide sequences given in SEQ ID NO:28, SEQ ID NO.30 and SEQ ID NO:31.

The pesticidal proteins of the invention can be used in combination with Bt endotoxins or other insecticidal proteins to increase insect target range. Furthermore, the use of the VIPs of the present invention in combination with Bt δ-endotoxins or other insecticidal principles of a distinct nature has particular utility for the prevention and/or management of insect resistance. Other insecticidal principles include

protease inhibitors (both serine and cysteine types), lectins, α-amylase and peroxidase. In one preferred embodiment, expression of VIPs in a transgenic plant is accompanied by the expression of one or more Bt δ-endotoxins. 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 VIPs. A second plant, Parent 2, can be genetically engineered for the expression of Bt δ-endotoxin. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2. Particularly preferred Bt δ-endotoxins are those disclosed in EP-A 0618976, herein incorporated by reference.

A substantial number of cytotoxic proteins, though not all, are binary in action. Binary toxins typically consist of two protein domains, one called the A domain and the other called the B domain (see Sourcebook of Bacterial Protein Toxins. J. E. Alouf and J. H. Freer eds.(1991 ) Academic Press). The A domain possesses a potent cytotoxic activity. The B domain binds an external cell surface receptor before being internalized. Typically, the cytotoxic A domain must be escorted to the cytoplasm by a translocation domain. Often the A and B domains are separate polypeptides or protomers, which are associated by a protein-protein interaction or a di-sulfide bond. However, the toxin can be a single polypeptide which is proteolytically processed within the cell into two domains as in the case for Pseudomonas exotoxin A. In summary binary toxins typically have three important domains, a cytotoxic A domain, a receptor binding B domain and a translocation domain. The A and B domain are often associated by protein-protein interacting domains.

The receptor binding domains of the present invention are useful for delivering any protein, toxin, enzyme, transcription factor, nucleic acid, chemical or any other factor into target insects having a receptor recognized by the receptor binding domain of the binary toxins described in this patent. Similarly, since binary toxins have translocation domains which penetrate phosopholipid bilayer membranes and escort cytotoxins across those membranes, such translocation domains may be useful in escorting any protein, toxin, enzyme, transcription factor, nucleic acid, chemical or any other factor across a phospholipid bilayer such as the plasma membrane or a vesicle membrane. The translocation domain may itself perforate membranes, thus having toxic or insecticidal properties. Further, all binary toxins have cytotoxic domains; such a

cytotoxic domain may be useful as a lethal protein, either alone or when delivered into any target cell(s) by any means.

Finally, since binary toxins comprised of two polypeptides often form a complex, it is likely that there are protein-protein interacting regions within the components of the binary toxins of the invention. These protein-protein interacting domains may be useful in forming associations between any combination of toxins, enzymes, transcription factors, nucleic acids, antibodies, cell binding moieties, or any other chemicals, factors, proteins or protein domains.

Toxins, enzymes, transcription factors, antibodies, cell binding moieties or other protein domains can be fused to pesticidal or auxiliary proteins by producing in frame genetic fusions which, when translated by ribosomes, would produce a fusion protein with the combined attributes of the VIP and the other component used in the fusion. Furthermore, if the protein domain fused to the VIP has an affinity for another protein, nucleic acid, carbohydrate, lipid, or other chemical or factor, then a three-component complex can be formed. This complex will have the attributes of all of its components. A similar rationale can be used for producing four or more component complexes. These complexes are useful as insecticidal toxins, pharmaceuticals, laboratory reagents, and diagnostic reagents, etc. Examples where such complexes are currently used are fusion toxins for potential cancer therapies, reagents in ELISA assays and immunoblot analysis.

One strategy of altering pesticidal or auxiliary proteins is to fuse a 15-amino-acid "S-tag" to the protein without destroying the insect cell binding domain(s), translocation domains or protein-protein interacting domains of the proteins. The S- tag has a high affinity (K _d = 10 ^'9 M) for a ribonuclease S-protein, which, when bound to the S-tag, forms an active ribonuclease (See F. M. Richards and H. W. Wyckoff (1971) in 'The Enzymes", Vol. IV (Boyer, P.D. ed.). pp. 647-806. Academic Press, New York). The fusion can be made in such a way as to destroy or remove the cytotoxic activity of the pesticidal or auxiliary protein, thereby replacing the VIP cytotoxic activity with a new cytotoxic ribonuclease activity. The final toxin would be comprised of the S-protein, a pesticidal protein and an auxiliary protein, where either the pesticidal protein or the auxiliary protein is produced as translational fusions with the S-tag. Similar strategies can be used to fuse other potential cytotoxins to pesticidal or auxiliary proteins including (but not limited to) ribosome inactivating

proteins, insect hormones, hormone receptors, transcription factors, proteases, phosphatases, Pseudomonas exotoxin A, or any other protein or chemical factor that is lethal when delivered into cells. Similarly, proteins can be delivered into cells which are not lethal, but might alter cellular biochemistry or physiology.

The spectrum of toxicity toward different species can be altered by fusing domains to pesticidal or auxiliary proteins which recognize cell surface receptors from other species. Such domains might include (but are not limited to) antibodies, transferrin, hormones, or peptide sequences isolated from phage displayed affinity selectable libraries. Also, peptide sequences which are bound to nutrients, vitamins, hormones, or other chemicals that are transported into cells could be used to alter the spectrum of toxicity. Similarly, any other protein or chemical which binds a cell surface receptor or the membrane and could be internalized might be used to alter the spectrum of activity of VIP1 and VIP2.

The pesticidal proteins of the present invention are those proteins which confer a specific pesticidal property. Such proteins may vary in molecular weight, having component polypeptides at least a molecular weight of 30 kDa or greater, preferably about 50 kDa or greater.

The auxiliary proteins of the invention may vary in molecular weight, having at least a molecular weight of about 15 kDa or greater, preferably about 20 kDa or greater; more preferably, about 30 kDa or greater. The auxiliary proteins themselves may have component polypeptides.

It is possible that the pesticidal protein and the auxiliary protein may be components of a multimeric, pesticidal protein. Such a pesticidal protein which includes the auxiliary proteins as one or more of its component polypeptides may vary in molecular weight, having at least a molecular weight of 50 kDa up to at least 200 kDa, preferably about 100 kDa to 150 kDa.

An auxiliary protein may be used in combination with the pesticidal proteins of the invention to enhance activity or to activate the pesticidal protein. To determine whether the auxiliary protein will affect activity, the pesticidal protein can be expressed alone and in combination with the auxiliary protein and the respective activities compared in feeding assays for pesticidal activity.

It may be beneficial to screen strains for potential pesticidal activity by testing activity of the strain alone and in combination with the auxiliary protein. In some

instances an auxiliary protein in combination with the native proteins of the strains yields pesticidal activity where none is seen in the absence of an auxiliary protein.

The auxiliary protein can be modified, as described above, by various methods known in the art. Therefore, for purposes of the invention, the term "Vegetative Insecticidal Protein" (VIP) encompasses those proteins produced during vegetative growth which alone or in combination can be used for pesticidal activity. This includes pesticidal proteins, auxiliary proteins and those proteins which demonstrate activity only in the presence of the auxiliary protein or the polypeptide components of these proteins.

It is recognized that there are alternative methods available to obtain the nucleotide and amino acid sequences of the present proteins. For example, to obtain the nucleotide sequence encoding the pesticidal protein, cosmid clones, which express the pesticidal protein, can be isolated from a genomic library. From larger active cosmid clones, smaller subclones can be made and tested for activity. In this manner, clones which express an active pesticidal protein can be sequenced to determine the nucleotide sequence of the gene. Then, an amino acid sequence can be deduced for the protein. For general molecular methods, see, for example, Molecular Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and the references cited therein.

The present invention also encompasses nucleotide sequences from organisms other than Bacillus, where the nucleotide sequences are isolatable by hybridization with the Bacillus nucleotide sequences of the invention. Proteins encoded by such nucleotide sequences can be tested for pesticidal activity. The invention also encompasses the proteins encoded by the nucleotide sequences. Furthermore, the invention encompasses proteins obtained from organisms other than Bacillus wherein the protein cross-reacts with antibodies raised against the proteins of the invention. Again the isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or others well-known in the art.

Once the nucleotide sequences encoding the pesticidal proteins of the invention have been isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants.

The pesticidal genes of the invention can be optimized for enhanced expression in plants. See, for example EP-A 0618976; EP-A 0359472; EP-A 0385962; WO 91/16432; Perlak etal. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17: 477-498. In this manner, the genes can be synthesized utilizing plant preferred codons. That is the preferred codon for a particular host is the single codon which most frequently encodes that amino acid in that host. The maize preferred codon, for example, for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is found in Murray et al. (1989), Nucleic Acids Research 17:477- 498, the disclosure of which is incorporated herein by reference. Synthetic genes can also be made based on the distribution of codons a particular host uses for a particular amino acid.

In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

In like manner, the nucleotide sequences can be optimized for expression in any microorganism. For Bacillus preferred codon usage, see, for example US Patent No. 5,024,837 and Johansen et al. (1988) Gene 65:293-304.

Methodologies for the construction of plant expression cassettes as well as the introduction of foreign DNA into plants are described in the art. Such expression cassettes may include promoters, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the pesticidal protein coding sequence. It is further recognized that promoters or terminators of the VIP genes can be used in expression cassettes.

Generally, for the introduction of foreign DNA into plants Ti plasmid vectors have been utilized for the delivery of foreign DNA as well as direct DNA uptake, liposomes, electroporation, micro-injection, and the use of microprojectiles. Such methods had been published in the art. See, for example, Guerche et al., (1987) Plant Science 52:1 1 1 -1 16; Neuhause et al., (1987) Theor. APPI. Genet. 75:30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265: Horsch et al., (1985) Science 227: 1229-1231 ; DeBlock et al., (1989) Plant Physiology 91 :694-701 ; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski,

eds.) Academic Press, Inc. (1989). See also US patent application serial no. 08/008,374 herein incorporated by reference. See also, EP-A 0193259 and EP-A 0451878. It is understood that the method of transformation will depend upon the plant cell to be transformed.

It is further recognized that the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. See, for example Periak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; Murray et al., (1989) Nucleic Acids Research 17:477-498; and WO 91/16432.

The construct may also include any other necessary regulators such as terminators, (Guerineau et al., (1991). Moi. Gen. Genet.. 226:141-144; Proudfoot, (1991), CeH, 64:671 -674; Sanfacon et al., (1991). Genes Dev.. 5:141 -149; Mogen et al., (1990). Plant Cell. 2:1261 -1272; Munroe et al., (1990), Gene, 91 :151 -158; Ballas et al et al., (1989). Nucleic Acids Res.. 17:7891 -7903; Joshi et al., (1987). Nucleic Acid Res.. 15:9627-9639); plant translational consensus sequences (Joshi, C.P., (1987), Nucleic Acids Research. 15:6643-6653), introns (Luehrsen and Walbot, (1991), Moi. Gen. Genet- 225:81 -93) and the like, operably linked to the nucleotide sequence. It may be beneficial to include 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translational leaders are known in the art and include:

Picornavirus leaders, for example, EMCV leader (encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. (1989) PNAS USA 86:6126-6130);

Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology. 154:9-20), and

Human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G., and Sarnow, P., (1991 ), Nature. 353:90-94;

Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., and Gehrke, L, (1987), Nature. 325:622-625;

Tobacco mosaic virus leader (TMV), (Gallie, D.R. et al., (1989), Molecular Biology of RNA. pages 237-256; and

Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S.A. et al., (1991 ), Virology. 81.382-385. See also, Della-Cioppa et al., (1987), Plant Physiology. 84:965-968.

A plant terminator may be utilized in the expression cassette. See, Rosenberg et al., (1987), Gene, 56:125; Guerineau et al., (1991 ). Moi. Gen. Genet.. 226:141-144; Proudfoot, (1991), CeJ], 64:671-674; Sanfacon et al., (1991 ), Genes Dev.. 5:141-149; Mogen et al., (1990). Plant Cell. 2.J 261 -1272; Munroe et al., (1990), Gene. 91 :151-158; Ballas et al., (1989), Nucleic Acids Res.. 17:7891 -7903; Joshi et al., (1987). Nucleic Acid Res.. 15:9627-9639.

For tissue specific expression, the nucleotide sequences of the invention can be operably linked to tissue specific promoters. See, for example, EP-A 0618976, herein incorporated by reference.

Further comprised within the scope of the present invention are transgenic plants, in particular transgenic fertile plants transformed by means of the aforedescribed processes and their asexual and/or sexual progeny, which comprise and preferably also express the pesticidal protein according to the invention. Especially preferred are hybrid plants.

The transgenic plant according to the invention may be a dicotyledonous or a monocotyledonous plant. Preferred are monocotyledonous plants of the Graminaceae family involving Loliυm. Zea. Triticυm. Triticale. Sorghum. Saccharum. Bromus. Orvzae, Avena. Hordeum. Secale and Setaria plants.

Especially preferred are transgenic maize, wheat, barley, sorghum, rye, oats, turf grasses and rice.

Among the dicotyledonous plants soybean, cotton, tobacco, sugar beet, oilseed rape, and sunflower are especially preferred herein.

The expression 'progeny' is understood to embrace both, "asexually" and "sexually" generated progeny of transgenic plants. This definition is also meant to include all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initially transformed parent plant, together with all crossing and fusion products of the transformed plant material.

Another object of the invention concerns the proliferation material of transgenic plants.

The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells.

calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.

Parts of plants, such as for example flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed by means of the process of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.

Before the plant propagation material [fruit, tuber, grains, seed], but expecially seed is sold as a commerical product, it is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.

In order to treat the seed, the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, eg treatment directed at the buds or the fruit.

The plant seed according to the invention comprising a DNA molecule comprising a nucleotide sequence encoding a pesticidal protein according to the invention may be treated with a seed protectant coating comprising a seed treatment compound, such as, for example, captan, carboxin, thiram (TMTD ^® ), methalaxyl (Apron ^® ) and pirimiphos-methyl (Actellic ^® ) and others that are commonly used in seed treatment. Preferred within the scope of the invention are seed protectant coatings comprising an entomocidal composition according to the invention alone or in combination with one of the a seed protectant coating customarily used in seed treatment.

It is thus a further object of the present invention to provide plant propagation material for cultivated plants, but especially plant seed that is treated with a seed protectant coating as defined hereinbefore.

It is recognized that the genes encoding the pesticidal proteins can be used to transform insect pathogenic organisms. Such organisms include Baculoviruses, fungi, protozoa, bacteria and nematodes.

The Bacillus strains of the invention may be used for protecting agricultural crops and products from pests. Alternatively, a gene encoding the pesticide may be

introduced via a suitable vector into a microbial host, and said host applied to the environment or plants or animals. Microorganism hosts may be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild- type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacilius, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Klυyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandir, and phytosphere yeast species such as Rhodotorula rubra, R. glυtinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Klυyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A number of ways are available for introducing a gene expressing the pesticidal protein into the microorganism host under conditions which allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed which include the DNA constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the DNA constructs, and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include but are not limited to promoter, transcriptional initiation start site, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals,

and the like. See, for example, US Patent 5,039,523; US Patent No. 4,853,331 ; EPO 0480762A2; Sambrook et al. supra; Molecular Cloning, a Laboratory Manual, Maniatis et al. (eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Advanced Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980); and the references cited therein.

Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of the target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such a Saccharomyces and Schizosaccharromyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane

organisms such as Pseudomonas sp., Erwinia sp_ and Flavobacterium sp.; or such other organisms as Escherichia, LactoBacillus sp.. Bacillus sp., and the like. Specific organisms include Pseudomonas aeurginosa, Pseudomonas flυorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.

VIP genes can be introduced into micro-organisms that multiply on plants (epiphytes) to deliver VIP proteins to potential target pests. Epiphytes can be gram- positive or gram-negative bacteria for example.

Root colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain which colonizes roots could be isolated from roots of a plant ( for example see J. Handelsman, S. Raffel, E. Mester, L Wunderlich and C. Grau, AppI. Environ. Microbiol. 56:713-718, (1990)). VIP1 and/or VIP2 and/or VIP3 could be introduced into a root colonizing Bacillus cereus by standard methods known in the art.

Specifically, VIP1 and/or VIP2 derived from Bacillus cereus strain AB78 can be introduced into a root colonizing Bacillus cereus by means of conjugation using standard methods (J. Gonzalez, B. Brown and B. Carlton, Proc. Natl. Acad. Sci. 79:6951 -6955, (1982)).

Also, VIP1 and/or VIP2 and/or VIP3 or other VIPs of the invention can be introduced into the root colonizing Bacillus by means of electro-transformation. Specifically, VIPs can be cloned into a shuttle vector, for example, pHT3101 (D. Lereclus et al., FEMS Microbiol. Letts.. 60:211 -218 (1989)) as described in Example 10. The shuttle vector pHT3101 containing the coding sequence for the particular VIP can then be transformed into the root colonizing Bacillus by means of electroporation (D. Lereclus et al. 1989, FEMS Microbiol. Letts. 60:21 1-218).

Expression systems can be designed so that VIP proteins are secreted outside the cytoplasm of gram negative bacteria, E. coli, for example. Advantages of having VIP proteins secreted are (1 ) it avoids potential toxic effects of VIP proteins expressed within the cytoplasm and (2) it can increase the level of VIP protein expressed and (3) can aid in efficient purification of VIP protein.

VIP proteins can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the VIP signal peptide or replacing the VIP signal peptide with the E. coli signal peptide. Signal peptides

recognized by E. coli can be found in proteins already known to be secreted in E. coli, for example the OmpA protein (J. Ghrayeb, H. Kimura, M. Takahara, Y. Masui and M. Inouye, EMBO J.. 3:2437-2442 (1984)). OmpA is a major protein of the E. coli outer membrane and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide ( G. Duffaud, P. March and M. Inouye, Methods in Enzvmoloov. 153:492 (1987)).

Specifically, unique BamHI restriction sites can be introduced at the amino- terminal and carboxy-terminal ends of the VIP coding sequences using standard methods known in the art. These BamHI fragments can be cloned, in frame, into the vector plN-lll-ompA1 , A2 or A3 (J. Ghrayeb, H. Kimura, M. Takahara, H. Hsiung, Y. Masui and M. Inouye, EMBO J.. 3:2437-2442 (1984)) thereby creating ompA:VIP fusion gene which is secreted into the periplasmic space. The other restriction sites in the polylinker of pIN-lll-ompA can be eliminated by standard methods known in the art so that the VIP amino-terminal amino acid coding sequence is directly after the ompA signal peptide cleavage site. Thus, the secreted VIP sequence in E. coli would then be identical to the native VIP sequence.

When the VIP native signal peptide is not needed for proper folding of the mature protein, such signal sequences can be removed and replaced with the ompA signal sequence. Unique BamHI restriction sites can be introduced at the amino-termini of the proprotein coding sequences directly after the signal peptide coding sequences of VIP and at the carboxy-termini of VIP coding sequence. These BamHI fragments can then be cloned into the pIN-lll-ompA vectors as described above.

General methods for employing the strains of the invention in pesticide control or in engineering other organisms as pesticidal agents are known in the art. See, for example US Patent No. 5,039,523 and EP 0480762A2.

VIPs can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of a VIP(s) which is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the VIP protein(s) into the growth medium during the fermentation process. The VIPs are retained within the cell

and the cells are then processed to yield the encapsulated VIPs. Any suitable microorganism can be used for this purpose. Psuedomonas has been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide. (H. Gaertner et al. 1993, In Advanced Engineered Pesticides, L. Kim ed.)

Various strains of Bacillus thuringiensis are used in this manner. Such Bt strains produce endotoxin protein(s) as well as VIPs. Alternatively, such strains can produce only VIPs. A sporulation deficient strain of Bacillus sυbtilis has been shown to produce high levels of the CrylllA endotoxin from Bacillus thuringiensis (Agaisse, H. and Lereclus, D., "Expression in Bacillus subtilis of the Bacillus thuringiensis CrylllA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spoOA mutant", J. Bacteriol.. 176:4734-4741 (1994)). A similar spoOA mutant can be prepared in Bacillus thuringiensis and used to produce encapsulated VIPs which are not secreted into the medium but are retained within the cell.

To have VIPs maintained within the Bacillus cell the signal peptide can be disarmed so that it no longer functions as a secretion signal. Specifically, the putative signal peptide for VIP1 encompasses the first 31 amino acids of the protein with the putative consensus cleavage site, Ala-X-Ala, at the C-terminal portion of this sequence (G. von Heijne , J. Moi. Biol. 184:99-105 (1989)) and the putative signal peptide for VIP2 encompasses the first 40 amino acids of the protein with the putative cleavage site after Ala40. The cleavage sites in either VIP1 or VIP2 can be mutated with methods known in the art to replace the cleavage site consensus sequence with alternative amino acids that are not recognized by the signal peptidases.

Alternatively, the signal peptides of VIP1 , VIP2 and/or other VIPs of the invention can be eliminated from the sequence thereby making them unrecognizable as secretion proteins in Bacillus. Specifically, a methionine start site can be engineered in front of the proprotein sequence in VIP1 , starting at Asp32, or the proprotein sequence in VIP2, starting at Glu41 using methods known in the art.

VIP genes can be introduced into micro-organisms that mutiply on plants (epiphytes) to deliver VIP proteins to potential target pests. Epiphytes can be gram- positive or gram-negative bacteria for example.

The Bacillus strains of the invention or the microorganisms which have been genetically altered to contain the pesticidal gene and protein may be used for

protecting agricultural crops and products from pests. In one aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).

Alternatively, the pesticides are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.

The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, mollusicides 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 employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

Preferred methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention which contains at least one of the insect-specific proteins produced by the bacterial strains of the present invention are leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.

The present invention thus further provides an entomocidal composition comprising as an active ingrdient at least one of the novel insect-specific proteins

according to the invention and/or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect- specific proteins in recombinant form, but especially a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis, containing at least one one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or a derivative or mutant thereof, together with an agricultural adjuvant such as a carrier, diluent, surfactant or application-promoting adjuvant. The composition may also contain a further biologically active compound. The 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 employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers

The composition may comprise from 0J 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 acitve ingredient comprising at least one of the novel insect-specific proteins according to the invention or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect- specific proteins in recombinant form, but especially a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or a derivative or mutant thereof, or the composition containing the said acitve 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. It may be administered as a dust, a suspension, a wettable powder or in any other material form suitable for agricultural application.

The invention further provides methods for for controlling or inhibiting of insect pests by applying an active ingredient comprising at least one of the novel insect- specific proteins according to the invention or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form or a composition comprising the said active ingredient to (a) an environment in which the insect pest may occur, (b) a plant or plant part in order to protect said plant or plant part from damage caused by an insect pest, or (c) seed in order to protect a plant which develops from said seed from damage caused by an insect 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. However, the active ingredient may also penetrate the plants through the roots (systemic action) if the locus of the plants is impregnated with a liquid formulation or if the active ingredient is incorporated in solid form into the locus of the plants, for example into the soil, e.g. in granular form (soil application). In paddy rice crops, such granules may be applied in metered amounts to the flooded rice field.

The compositions 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 acitve 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.

The compositions according to the invention comprising as an active ingredient a recombinant microorganism containing at least one of the novel toxin genes in recombinant form, but especially a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or a derivative or mutant thereof 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 comprise, e.g., the following species of plants:

cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beet (sugar beet and fodder beet), forage grasses (orchardgrass, fescue, and the like), drupes, pomes and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries), leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons) fiber plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other Brassicae, onions, tomatoes, potatoes, paprika), lauraceae (avocados, carrots, cinnamon, camphor), deciduous trees and conifers (e.g. linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs, larches, pines), or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, as well as ornamentals (including composites).

A recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain, containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form is normally applied in the form of entomocidal compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with further biologically active compounds. These compounds may be both fertilizers or micronutrient donors or other preparations that influence plant growth. They may also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.

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 are normally from about 50 g to about 5 kg of active ingredient (a.i.) per hectare ("ha", approximately 2.471 acres), preferably from about 100 g to about 2kg a.i./ha. Important rates of application are about 200 g to about 1 kg a.i./ha and 200g to 500g a.i./ha.

For seed dressing advantageous application rates are 0.5 g to 1000 g a.i.per 100 kg seed, preferably 3 g to 100 g a.i. per 100 kg seed or 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 containing the recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form as an active ingredient or combinations thereof with other active ingredients, and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, e.g., by homogeneously mixing and/or grinding the active ingredients with extenders, e.g., solvents, solid carriers, and in some cases surface-active compounds (surfactants).

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 giycols 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, e.g., 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 surface-active 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 ₁₀ -C22), 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 ₈ -C 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 benzimidazole 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 N-substituent, at least one C ₈ -C 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, methyisulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride or benzyldi-(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, e.g., 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, Munich/Vienna.

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, δ-endotoxin crystals are solubilized and thus become more accessible to proteolytic inactivation. 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 of 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 δ-endotoxin genes in a microbe. For biological encapsulation, the intact microbe containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form 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 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 -0 097 571. 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)] various 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 1th Volume, ASTM STP 1 112 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.

The entomocidal compositions of the invention usually contain from about OJ to about 99%, preferably about OJ to about 95%, and most preferably from about 3 to about 90% of the active ingredient, from aboutJ 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 OJ to about 25%, and most preferably from about OJ to about 20% of a surfactant.

In a preferred embodiment of the invention the entomocidal compositions usually contain 0J to 99%, preferably 0J to 95%, of a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or combination thereof with other active ingredients, 1 to 99.9% of a solid or liquid adjuvant, and 0 to 25%, preferably 0J to 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, antifoams, viscosity regulators, binders, tackifiers as well as fertilizers or other active ingredients in order to obtain special effects.

In one embodiment of the invention a Bacillus cereus microorganism has been isolated which is capable of killing Diabrotica virgifera virgifera, and Diabrotica longicornis barberi. The novel B. cereus strain AB78 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, IL 61604, USA and given Accession No. NRRL B-21058.

A fraction protein has been substantially purified from the B. cereus strain. This purification of the protein has been verified by SDS-PAGE and biological activity. The

protein has a molecular weight of about 60 to about 100 kDa, particularly about 70 to about 90 kDa, more particularly about 80 kDa, hereinafter VIP.

Amino-terminal sequencing has revealed the N-terminal amino-acid sequence to be:

N^-Lys-Arg-Glu-lle-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp-Se r-lle-Pro- (SEQ ID NO:8) where Asx represents either Asp or Asn. The entire amino acid sequence is given in SEQ ID NO:7. The DNA sequence which encodes the amino acid sequence of SEQ ID NO:7 is disclosed in SEQ ID NO:6.

An oligonuleotide probe for the region of the gene encoding amino acids 3-9 of the NH ₂ -terminus has been generated. The probe was synthesized based on the codon usage of a Bacillus thuringiensis (Bt) δ-endotoxin gene. The nucleotide sequence of the oligonucleotide probe used for Southern hybridizations was as follows:

5'- GAA ATT GAT CAA GAT ACN GAT -3' (SEQ ID NO:9) where N represents any base.

In addition, the DNA probe for the Be AB78 VIP1 gene described herein, permits the screening of any Bacillus strain or other organisms to determine whether the VIP1 gene (or related gene) is naturally present or whether a particular transformed organism includes the VIP1 gene.

The invention now being generally described, the same will be better understood by reference to the following detailed examples that are provided for the purpose of illustration and are not to be considered limiting of the invention unless so specified.

A standard nomenclature has been developed based on the sequence identity of the proteins encompassed by the present invention. The gene and protein names for the detailed examples which follow and their relationship to the names used in the parent application [US application serial no 314594/08] are shown below.

VIP2A(a) VIP2 VIP2 from strain AB78 as disclosed in SEQ ID NO:2.

VIP1A(b) VIP1 VIP1 from Bacillus thuringiensis var. homolog tenebrionis as disclosed in SEQ ID NO:21.

VIP2A(b) VIP2 VIP2 from Bacillus thuringiensis var. homolog tenebrionis as disclosed in SEQ ID NO.20.

VIP3A(a) VIP from strain AB88 as disclosed in SEQ ID NO:28 of the present application

VIP3A(b) VIP from strain AB424 as disclosed in SEQ ID NO:31 of the present application

EXPERIMENTAL

Formulation Examples

The active ingredient used in the following formulation examples are Bacillus cereus strain AB78 having Accession No. NRRL B-21058; Bacillus thuringiensis strains having Accession Nos. NRRL B-21060, NRRL B-21224, NRRL B-21225, NRRL B- 21226, NRRL B-21227, and NRRL B-21439; and Bacillus spp strains having Accession Nos NRRL B-21228, NRRL B-21229, and NRRL B-21230. All the mentioned strains are natural isolates comprising the insect-specific proteins according to the invention.

Alternatively, the isolated insect-specific proteins are used as the active ingredient alone or in combination with the above-mentioned Bacillus strains.

A1. Wettable powders

a) b) c)

The spores are thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations.

A2. Emulsifiable concentrate

Bacillus thuringiensis spores 10% Ό octylphenol polyethylene glycol ether (4-5 moles ethylene oxide) 3% clacium dodecylbenzensulfonate 3% o

castor oil polyglycol ether (36 moles of ethylene oxide) 4% cyclohexanone 30% o xylene mixture 50% Ό

Emulsions of any required concentration can be obtained from this concentrate by dilution with water.

A3. Dusts

Bacillus thuringiensis spores talcum kaolin - 92%

Ready for use dusts are obtained by mixing the active ingredient with the carriers and grinding the mixture in a suitable mill.

A4. Extruder Granulate

Bacillus thuringiensis spores 10% sodium lignosulfonate 2% carboxymethylcellulose 1% kaolin 87%

The active ingredient or combination is mixed and ground with the adjuvants and the mixture is subsequently moistened with water. The mixture is extruded, granulated and the dried in a stream of air.

A5. Coated Granule

Bacillus thuringiensis spores 3% o polyethylene glycol (moi wt 200) 3% kaolin 94%

The active ingredient or combination is uniformly applied in a mixer to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner.

A6. Suspension Concentrate

Bacillus thuringiensis spores 40% ethylene glycol 10% nonylphenol polyethylene glycol ether (15 moles of ethylene oxide) 6% sodium lignosulfonate 10% carboxymethylcellulose 1%

37% aqueous formaldehyde solution 0.2%> silicone oil in the form of a 75% aqueous solution 0.8% water 32%

The active ingredient or combination is intimately mixed with the adjuvants giving a suspension concentrate from which suspensions of any desired concentration can be obtained by dilution with water.

EXAMPLE 1. AB78 ISOLATION AND CHARACTERIZATION

Bacillus cereus strain AB78 was isolated as a plate contaminant in the laboratory on T3 media (per liter: 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate (pH 6.8), and 0.005 g MnCI ₂ ; Travers, R.S. 1983). During log phase growth, AB78 gave significant activity against western corn rootworm. Antibiotic activity against gram-positive Bacillus spp. was also demonstrated (Table 12).

TABLE 12

Antibiotic activity of AB78 culture supernatant

Zone of inhibition(cm)

Morphological characteristics of AB78 are as follows: Vegetative rods straight, 3J-5.0 mm long and 0.5-2.0 mm wide. Cells with rounded ends, single in short chains. Single subterminal, cylindrical-oval, endospore formed per cell. No parasporal crystal formed. Colonies opaque, erose, lobate and flat. No pigments produced. Cells motile. Flagella present.

Growth characteristics of AB78 are as follows:

Facultative anaerobe with optimum growth temperature of 21 -30°C. Will grow at 15, 20, 25, 30 and 37°C. Will not grow above 40°C. Grows in 5-7% NaCl.

Table 13 provides the biochemical profile of AB78.

TABLE 13 Biochemical characteristics of B. cereus strain AB78.

A subculture of Be strain AB78 was used to inoculate the following medium, known as TB broth:

The potassium phosphate was added to the autoclaved broth after cooling. Flasks were incubated at 30°C on a rotary shaker at 250 rpm for 24 h-36 h, which represents an early to mid-log growth phase.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

During vegetative growth, usually 24-36 h. after starting the culture, which represents an early to mid-log growth phase, AB78 bacteria were centrifuged from the culture supernatant. The culture supernatant containing the active protein was used in bioassays.

EXAMPLE 3. INSECT BIOASSAYS

B. cereus strain AB78 was tested against various insects as described below.

Western, Northern and Southern corn rootworm, Diabrotica virgifera virgifera, D. longcornis barberi and D. undecempunctata howardi, respectively: dilutions were made of AB78 culture supernatant grown 24-36 h., mixed with molten artificial diet (Marrone et al. (1985) J of Economic Entomology 78:290-293) and allowed to solidify. Solidified diet was cut and placed in dishes. Neonate larvae were placed on the diet and held at 30 C. Mortality was recorded after 6 days.

E coli clone bioassav: E. coli cells were grown overnight in broth containing 100 μg/ml ampicillin at 37°C. Ten ml culture was sonicated 3X for 20 sec each. 500 μl of sonicated culture was added to molten western corn rootworm diet.

Colorado potato beetle, Leptinotarsa decemlineata: dilutions in Triton X-100 (to give final concentration of 0.1% TX-100) were made of AB78 culture supernatant grown 24-36 h. Five cm ² potato leaf pieces were dipped into these dilutions, air dried, and placed on moistened filter paper in plastic dishes. Neonate larvae were placed on the leaf pieces and held at 30°C. Mortality was recorded after 3-5 days.

Yellow mealworm, Tenebrio molitor. dilutions were made of AB78 culture supernatant grown 24-36 h., mixed with molten artificial diet (Bioserv #F9240) and allowed to solidify. Solidified diet was cut and placed in plastic dishes. Neonate larvae were placed on the diet and held at 30°C. Mortality was recorded after 6-8 days.

European corn borer, black cutworm, tobacco budworm, tobacco hornworm and beet armyworm; Ostrinia nubilalis, Agrotis ipsilon, Heliothis virescens, Manduca sexta and Spodoptera exigua, respectively: dilutions, in TX-100 (to give final concentration of 0.1% TX-100), were made of AB78 culture supernatant grown 24-36 hrs. 100 μl was pipetted onto the surface of 18 cm of solidified artificial diet (Bioserv #F9240) and allowed to air dry. Neonate larvae were then placed onto the surface of the diet and held at 30°C. Mortality was recorded after 3-6 days. Northern house mosquito, Ct /e / / ^' eπs:-dilutions were made of AB78 culture supernatant grown 24-36 h. 100 μl was pipetted into 10 ml water in a 30 ml plastic cup. Third instar larvae were added to the water and held at room temperature. Mortality was recorded after 24-48 hours. The spectrum of entomocidal activity of AB78 is given in Table 14.

TABLE 14 Activity of AB78 culture supernatant against various insect species

Insect species tested to date Order Activity

Western corn rootworm {Diabrotica virgifera virgifera) Col +++

Northern corn rootworm

{Diabrotica longicornis barberi) Col +++

Southern corn rootworm {Diabrotica undecimpunctata howardi) Col

Colorado potato beetle {Leptinotarsa decemlineata) Col Yellow mealworm ( Tenebrio molitoή Col

European corn borer

{Ostrinia nubilalis) Lep

Tobacco budworm

{Heliothis virescens) Lep

Tobacco hornworm

{Manduca sexta) Lep

Beet armyworm

{Spodoptera exigua) Lep

Black cutworm

{Agrotis ipsilon) Lep

Northern house mosquito

{Cυlex pipiens) Dip

The newly discovered B. cereus strain AB78 showed a significantly different spectrum of insecticidal activity as compared to known coleopteran active δ-endotoxins from Bt. In particular, AB78 showed more selective activity against beetles than known coleopteran-active Bt strains in that it was specifically active against Diabrotica sββ. More specifically, it was most active against D. virgifera virgifera and D. longicornis barberi but not D. undecimpunctata howardi.

A number of Bacillus strains were bioassayed for activity during vegetative growth (Table 15) against western corn rootworm. The results demonstrate that AB78 is unique in that activity against western corn rootworm is not a general phenomenon.

TABLE 15 Activity of culture supernatants from various Bacillus spp. against western corn rootworm

Specific activity of AB78 against western corn rootworm is provided in Table 16.

TABLE 16

Activity of AB78 culture supernatant against neonate western corn rootworm

The LC50 was calculated to be 6.2 μl of culture supernatant per ml of western corn rootworm diet.

The cell pellet was also bioassayed and had no activity against WCRW. Thus, the presence of activity only in the supernatant indicates that this VIP is an exotoxin.

EXAMPLE 4. ISOLATION AND PURIFICATION OF CORN ROOTWORM ACTIVE PROTEINS FROM AB78.

Culture media free of cells and debris was made to 70% saturation by the addition of solid ammonium sulfate (472 g/L). Dissolution was at room temperature followed by cooling in an ice bath and centrifugation at 10,000 X g for thirty minutes to pellet the precipitated proteins. The supernatant was discarded and the pellet was dissolved in 1/10 the original volume of 20 mM TRIS-HCI at pH 7.5. The dissolved pellet was desalted either by dialysis in 20 mM TRIS-HCI pH 7.5, or passing through a desalting column.

The desalted material was titrated to pH 3.5 using 20 mM sodium citrate pH 2.5. Following a thirty minute room temperature incubation the solution was centrifuged at

3000 X g for ten minutes. The supernatant at this stage contained the greatest amount of active protein.

Following neutralization of the pH to 7.0 the supernatant was applied to a Mono-Q, anion exchange, column equilibrated with 20 mM TRIS pH 7.5 at a flow rate of 300 mL/min. The column was developed with a stepwise and linear gradient employing 400 mM NaCl in 20 mM TRIS pH 7.5.

Bioassay of the column fractions and SDS-PAGE analysis were used to confirm the active fractions. SDS-PAGE analysis identified the biologically active protein as having components of a molecular weight in the range of about 80 kDa and 50 kDa.

EXAMPLE 5. SEQUENCE ANALYSIS OF THE CORN ROOTWORM ACTIVE PROTEIN

The 80 kDa component isolated by SDS-PAGE was transferred to PVDF membrane and was subjected to amino-terminal sequencing as performed by repetitive Edman cycles on an ABI 470 pulsed-liquid sequencer. Transfer was carried out in 10 mM CAPS buffer with 10% methanol pH 1 1.0 as follows:

Incubation of the gel following electrophoresis was done in transfer buffer for five minutes. ProBlott PVDF membrane was wetted with 100% MeOH briefly then equilibrated in transfer buffer. The sandwich was arranged between foam sponges and filter paper squares with the configuration of cathode-gel-membrane-anode.

Transfer was performed at 70 V constant voltage for 1 hour.

Following transfer, the membrane was rinsed with water and stained for two minutes with 0.25% Coomassie Blue R-250 in 50% MeOH.

Destaining was done with several rinses with 50% MeOH 40% water 10% acetic acid.

Following destaining the membrane was air dried prior to excision of the bands for sequence analysis. A BlottCartridge and appropriate cycles were utilized to achieve maximum efficiency and yield. Data analysis was performed using model 610 Sequence Analysis software for identifying and quantifying the PTH-amino acid derivatives for each sequential cycle.

The N-terminal sequence was determined to be: NH2-Lys-Arg-Glu-lle-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp-Ser- lle-Pro-

(SEQ ID NO:8) where Asx represents Asp or Asn. The complete amino acid sequence for the 80 kDa component is disclosed in SEQ ID NO:7. The DNA sequence which encodes SEQ ID NO:7 is disclosed in SEQ ID NO:6.

EXAMPLE6. CONSTRUCTIONOFDNAPROBE

An oligonucleotide probe for the region of the gene encoding amino acids 3-9 of the N-terminal sequence (Example 5) was generated. The probe was synthesized based on the codon usage of a Bacillus thuringiensis (Bt) δ-endotoxin gene. The nucleotide sequence

5'- GAA ATT GAT CAA GAT ACN GAT -3' (SEQ ID NO:9) was used as a probe in Southern hybridizations. The oligonucleotide was synthesized using standard procedures and equipment.

EXAMPLE 7. ISOELECTRIC POINT DETERMINATION OF THE CORN ROOTWORM ACTIVE PROTEIN

Purified protein from step 5 of the purification process was analyzed on a 3-9 pi isoelectric focusing gel using the Phastgel electrophoresis system (Pharmacia). Standard operating procedures for the unit were followed for both the separation and silver staining development procedures. The pi was approximated at about 4.9.

EXAMPLE 8. PCR DATA ON AB78

PCR analysis (See, for example US patent application serial no. 08/008,006; and, Carozzi et al. (1991 ) AppI. Environ. Microbiol. 57(11 ):3057-3061 , herein incorporated by reference.) was used to verify that the B. cereus strain AB78 did not contain any insecticidal crystal protein genes of B. thuringiensis or B. sphaericus (Table 17).

TABLE 17 Bacillus insecticidal crystal protein gene primers tested by PCR against AB78 DNA.

EXAMPLE 9. COSMID CLONING OF TOTAL DNA FROM B. CEREUS STRAIN AB78

The VIP1A(a) gene was cloned from total DNA prepared from strain AB78 as follows:

Isolation of AB78 DNA was as follows:

1. Grow bacteria in 10 ml L-broth overnight. (Use 50 ml sterile centrifuge tube)

2. Add 25 ml of fresh L-broth and ampicillin (30 μg/ml).

3. Grow cells 2-6 h. at 30°C with shaking.

4. Spin cells in a 50 ml polypropylene orange cap tube in IEC benchtop clinical centrifuge at 3/4 speed.

5. Resuspend cell pellet in 10 ml TES (TES = 50 mM TRIS pH 8.0, 100 mM EDTA, 15 mM NaCl).

6. Add 30 mg lysozyme and incubate 2 hrs at 37°C.

7. Add 200 μl 20% SDS and 400 μl Proteinase K stock (20 mg/ml). Incubate at 37°C.

8. Add 200 μl fresh Proteinase K. Incubate 1 hr. at 55°C. Add 5 ml TES to make 15 ml final volume.

9. Phenol extract twice (10 ml phenol, spin at room temperature at 3/4 speed in an IEC benchtop clinical centrifuge). Transfer supernatant (upper phase) to a clean tube using a wide bore pipette.

10. Extract once with 1 :1 vol. phenol hloroform/isoamyl alcohol (24:1 ratio).

1 1. Precipitate DNA with an equal volume of cold isopropanol; Centrifuge to pellet DNA.

12. Resuspend pellet in 5 ml TE.

13. Precipitate DNA with 0.5 ml 3M NaOAc pH 5.2 and 1 1 ml 95% ethanol. Place at -20°C for 2 h.

14. "Hook" DNA from tube with a plastic loop, transfer to a microfuge tube, spin, pipette off excess ethanol, dry in vacuo.

15. Resuspend in 0.5 ml TE. Incubate 90 min. at 65 ^C C to help get DNA back into solution.

16. Determine concentration using standard procedures.

Cosmid Cloning of AB78

All procedures, unless indicated otherwise, were performed according to Stratagene Protocol, Supercos 1 Instruction Manual, Cat. No. 251301.

Generally, the steps were as follows:

A. Sau 3A partial digestion of the AB78 DNA.

B. Preparation of vector DNA

C. Ligation and packaging of DNA

D. Tittering the cosmid library

1. Start a culture of HB101 cells by placing 50 ml of an overnight culture in 5 mis of TB with 0.2% maltose. Incubate 3.5 hrs. at 37°C.

2. Spin out cells and resuspend in 0.5 ml 10 mM MgSO4.

3. Add together: 100 I cells

100 I diluted packaging mixture 100 I 10 mM MgSO4

30 I TB

4. Adsorb at room temperature for 30 minutes with no shaking.

5. Add 1 ml TB and mix gently. Incubate 30 minutes at 37°C.

6. Plate 200 I onto L-amp plates. Incubate at 37°C overnight.

At least 400 cosmid clones were selected at random and screened for activity against western corn rootworm as described in Example 3. DNA from 5 active clones and 5 non-active clones were used in Southern hybridizations. Results demonstrated that hybridization using the above described oligonucleotide probe correlated with western corn rootworm activity (Table 18).

Cosmid clones P3-12 and P5-4 have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21061 and NRRL B-21059 respectively.

TABLE 18 Activity of AB78 cosmid clones against western corn rootworm.

Mean Clone percent mortality (N=4)

Clones which hybridize with probe

P1 -73 47

P1 -83 64

P2-2 69

P3-12 85

P5-4 97

Clones which do not hybridize with probe

P1 -2 5

P3-8 4

P3-9 12

P3- 8 0

P4-6 9

EXAMPLE 10. IDENTIFICATION OF A 6 KB REGION ACTIVE AGAINST WESTERN CORN ROOTWORM.

DNA from P3-12 was partially digested with restriction enzyme Sau 3A, and ligated into the E coli vector pUC19 and transformed into E. coli. A DNA probe specific for the 80 kDa VIP1 A(a) protein was synthesized by PCR amplification of a portion of P3-12 DNA. Oligonucleotides MK113 and MK117, which hybridize to portions of VIP1 A(a), were synthesized using the partial amino acid sequence of the 80 kDa protein. Plasmid subclones were identified by colony hybridization to the PCR-generated probe, and tested for activity against western corn rootworm. One such clone, PL2, hybridized to the PCR-generated fragment, and was active against western corn rootworm in the assay previously described.

A 6 kb Cla I restriction fragment from pl_2 was cloned into the Sma I site of the E coli-βaciflus shuttle vector pHT 3101 (Lereclus, D. etal., FEMS Microbiology Letters 60:211 -218 (1989)) to yield pCIB6201. This construct confers anti-western corn rootworm activity upon both Bacillus and E.co// strains, in either orientation. pCIB6022 contains this same 6 kb Cla I fragment in pBluescript SK(+) (Stratagene), produces equivalent VIP1 A(a) protein (by western blot), and is also active against western corn rootworm.

The nucleotide sequence of pCIB6022 was determined by the dideoxy termination method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase® Terminator Double-Stranded DNA Sequencing Kit and analyzed on an ABI 373 automatic sequencer. The sequence is given in SEQ ID NO:1. The 6 kb fragment encodes both VIP1 A(a) and VIP2A(a), as indicated by the open reading frames described in SEQ ID NO:1. The sequence encoding VIP2A(a) is further disclosed in SEQ ID NO:4. The relationship between VIP1 A(a) and VIP2A(a) within the 6 kb fragment found in pCIB6022 is depicted in Table 19. pCIB6022 was

deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given the Accession No. NRRL B-21222.

EXAMPLE 11. FUNCTIONAL DISSECTION OF THE VIP1 A(a) DNA REGION.

To confirm that the VIP1 A(a) open reading frame (ORF) is necessary for insecticidal activity a translational frameshift mutation was created in the gene. The restriction enzyme Bgl II recognizes a unique site located 857 bp into the coding region of VIP1A(a). pCIB6201 was digested with Bgl II, and the single-stranded ends filled-in with DNA polymerase (Klenow fragment) and dNTPS. The plasmid was re- ligated and transformed into E coli. The resulting plasmid, pCIB6203, contains a four nucleotide insertion in the coding region of VIP1 A(a). pCIB6203 does not confer WCRW insecticidal activity, confirming that VIP1A(a) is an essential component of western corn rootworm activity.

To further define the region necessary to encode VIP1 A(a), subclones of the VIP1 A(a) and VIP2A(a) (auxiliary protein) region were constructed and tested for their ability to complement the mutation in pCIB6203. pCIB6023 contains the 3.7kb Xba I- EcoRV fragment in pBluescript SK(+) (Stratagene). Western blot analysis indicates that pCIB6023 produces VIP1A(a) protein of equal size and quantity as clones PL2 and pCIB6022. pCIB6023 contains the entire gene encoding the 80 kD protein. pCIB6023 was deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given the Accession No. NRRL B-21223N. pCIB6206 contains the 4.3 kb Xba l-CIa I fragment from pCIB6022 in pBluescript SK(+) (Stratagene). pCIB6206 was also deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given the Accession No. NRRL B- 21321 . pCIB6023, pCIB6206, and pCIB6203 do not produce detectable western corn rootworm activity when tested individually. However, a mixture of cells containing pCIB6203 (VIP1 A(a)-mutated, plus VIP2A(a)) and cells containing pCIB6023 (only

VIP1 A(a)) shows high activity against western corn rootworm. Similarly, a mixture of cells containing pCIB6206 and cells containing pCIB6203 shows high activity against western corn rootworm.

To further define the limits of VIP2A(a), we constructed pCIB6024, which contains the entirety of VIP2A(a), but lacks most of the VIP1 A(a) coding region. pCIB6024 was constructed by gel purifying the 2.2 kb Cla l-Sca I restriction fragment from pCIB6022, filling in the single-stranded ends with DNA polymerase (Klenow fragment) and dNTPs, and ligating this fragment into pBluescript SK(+) vector (Stratagene) digested with the enzyme Eco RV. Cells containing pCIB6024 exhibit no activity against western corn rootworm. However, a mixture of cells containing pCIB6024 and cells containing pCIB6023 shows high activity against western corn rootworm .(See Table 19).

Thus, pCIB6023 and pCIB6206 must produce a functional VIP1 A(a) gene product, while pCIB6203 and pCIB6024 must produce a functional VIP2A(a) gene product. These results suggest a requirement for a gene product(s) from the VIP2A(a) region, in combination with VIP1A(a), to confer maximal western com rootworm activity. (See Table 19.)

Table 19 Characterization of pCIB6022

Activity vs. WCRW

Ri B RV C

I viP (a) J pCIB6022 +++

3 1 pCIB6203 —

_- pCIB6023 —

Z I pCIB6206 —

pCIB6024

Functional Complementation of VIP

pCIB6203

+++

►βs ^ϋ»«ββBC pCIB6023

pCIB6203

+++ PCIB6206

pCIB6023

+++

L pCIB6024

Boxed regions represent the extent of VIP1 A(a) and VIP2A(a). White box represents the portion of VIP1 encoding the 80 kDa peptide observed in Bacillus. Dark box represents the N- terminal 'propeptide' of VIP1 A(a) predicted by DNA sequence analysis. Stippled box represents the VIP2A(a) coding region. Large 'X' represents the location of the frameshift mutation introduced into VIP1 A(a). Arrows represent constructs transcribed by the beta-galactosidase

EXAMPLE 12. AB78 ANTIBODY PRODUCTION

Antibody production was initiated in 2 Lewis rats to allow for both the possibility of moving to production of hybridoma cell lines and also to produce enough serum for limited screening of genomic DNA library. Another factor was the very limited amount of antigen available and the fact that it could only be produced to purity by PAGE and subsequent electrotransfer to nitrocellulose.

Due to the limited availability of antigen on nitrocellulose, the nitrocellulose was emulsified in DMSO and injected into the hind footpads of the animals to elicit B-cell production in the popliteal lymph nodes just upstream. A strong reacting serum was produced as judged by western blot analysis with the first production bleed. Several subsequent injections and bleeds produced enough serum to accomplish all of the screening required.

Hybridoma production with one of the rats was then initiated. The popliteal lymph node was excised, macerated, and the resulting cells fused with mouse myeloma P3x63Ag8.653. Subsequent cell screening was accomplished as described below. Four initial wells were selected which gave the highest emulsified antigen reaction to be moved to limited dilution cloning. An additional 10 wells were chosen for expansion and cryoperservation.

Procedure to Emulsify AB78 on nitrocellulose in DMSO for ELISA screening:

After electrotransfer of AB78 samples run on PAGE to nitrocellulose, the reversible strain Ponceau S is used to visualize all protein transferred. The band corresponding to AB78 toxin, previously identified and N-terminal sequenced, was identified and excised from nitrocellulose. Each band is approximately 1 mm x 5 mm in size to minimize the amount of nitrocellulose emulsified. A single band is placed in a microfuge tube with 250 μl of DMSO and macerated using a plastic pestle (Kontes, Vineland, NJ). To aid in emulsification, the DMSO mixture is heated for 2-3 minutes at 37 C-45 C. Some further maceration might be necessary following heating; however, all of the nitrocellulose should be emulsified. Once the AB78 sample is emulsified, it is placed on ice. In preparation for microtiter plate coating with the emulsified antigen, the sample must be diluted in borate buffered saline as follows: 1 :5, 1 :10, 1 :15, 1 :20, 1 :30, 1 :50, 1 :100, and 0. The coating antigen must be prepared fresh immediately prior to use.

ELISA protocol:

1. Coat with AB78/DMSO in BBS. Incubate overnight at 4°C.

2. Wash plate 3X with 1 X ELISA wash buffer.

3. Block (1% BSA & 0.05% Tween 20 in PBS) for 30 minutes at Room Temperature.

4. Wash plate 3X with 1 X ELISA wash buffer.

5. Add rat serum. Incubate 1.5 hours at 37°C.

6. Wash plate 3X with 1 X ELISA wash buffer.

7. Add goat anti-rat at a concentration of 2 μg/ml in ELISA diluent. Incubate 1 hr. at 37°C.

8. Wash plate 3X with 1 X ELISA wash buffer.

9. Add rabbit anti-goat alkaline phosphatase at 2 μg/ml in ELISA diluent. Incubate 1 hr. at 37°C.

10. Wash 3X with 1X ELISA wash buffer.

1 1. Add Substrate. Incubate 30 minutes at room temperature.

12. Stop with 3N NaOH after 30 minutes.

Preparation of VIP2A(a) Antisera

A partially purified AB78 culture supernatant was separated by discontinuous SDS PAGE (Novex) following manufacturer's instructions. Separated proteins were electrophoresed to nitrocellulose (S&S #21640) as described by Towbin et al., (1979). The nitrocellulose was stained with Ponceau S and the VIP2A(a) band identified. The VIP2A(a) band was excised and emulsified in DMSO immediately prior to injection. A rabbit was initially immunized with emulsified VIP2A(a) mixed approximately 1 :1 with Freund's Complete adjuvant by intramuscular injection at four different sites. Subsequent immunizations occurred at four week intervals and were identical to the first, except for the use of Freund' Incomplete adjuvant. The first serum harvested following immunization reacted with VIP2A(a) protein. Western blot analysis of AB78 culture supernatant using this antisera identifies predominately full length VIP2A(a) protein.

EXAMPLE 13. ACTIVATION OF INSECTICIDAL ACTIVITY OF NON-ACTIVE BT

STRAINS WITH AB78 VIP CLONES.

Adding pCIB6203 together with a 24 h culture (early to mid-log phase) supernatant from Bt strain GC91 produces 100% mortality in Diabrotica virgifera virgifera. Neither pCIB6203 nor GC91 is active on Diabrotica virgifera virgifera by itself. Data are shown below:

Test material Percent Diabrotica mortality pCIB6203 ^'■ 0

GC91 16 pCIB6203 + GC91 100

Control 0

EXAMPLE 14. ISOLATION AND BIOLOGICAL ACTIVITY OF B. CEREUS AB81.

A second a cereus strain, designated AB81 , was isolated from grain bin dust samples by standard methodologies. A subculture of AB81 was grown and prepared for bioassay as described in Example 2. Biological activity was evaluated as described in Example 3. The results are as follows:

Insect species Percent tested Mortality

Ostrinia nubilalis 0

Agrotis ipsilon 0

Diabrotica virgifera virgifera 55

EXAMPLE 5. ISOLATION AND BIOLOGICAL ACTIVITY OF g. THURINGIENSIS AB6.

A a thuringiensis strain, designated AB6, was isolated from grain bin dust samples by standard methods known in the art. A subculture of AB6 was grown and prepared for bioassay as described in Example 2. Half of the sample was autoclaved 15 minutes to test for the presence of β-exotoxin.

Biological activity was evaluated as described in Example 3. The results are as follows:

The reduction of insecticidal acitivity of the culture supernatant to insignificant levels by autoclaving indicates that the active principle is not β-exotoxin.

Strain AB6 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given Accession No. NRRL B-21060.

EXAMPLE 16. ISOLATION AND BIOLOGICAL CHARACTERIZATION OF B. THURINGIENSIS AB88.

A Bt strain, designated AB88, was isolated from grain bin dust samples by standard methodologies. A subculture of AB88 was grown and prepared for bioassay as described in Example 2. Half of the sample was autoclaved 15 minutes to test for the presence of β-exotoxin. Biological activity was evaluated against a number of insect species as described in Example 3. The results are as follows:

The reduction of insecticidal acitivity of the culture supernatant to insignificant levels by autoclaving indicates that the active principle is not β-exotoxin.

Delta-endotoxin crystals were purified from strain AB88 by standard methodologies. No activity from pure crystals was observed when bioassayed against Agrotis ipsilon.

EXAMPLE 17. PURIFICATION OF VIPS FROM STRAIN AB88:

Bacterial liquid culture was grown overnight [for 12h] at 30°C in TB media. Cells were centrifuged at 5000 x g for 20 minutes and the supernatant retained. Proteins present in the supernatant were precipitated with ammonium sulfate (70% saturation),

centrifuged [at 5000 x g for 15 minutes] and the pellet retained. The pellet was resuspended in the original volume of 20 mM Tris pH 7.5 and dialyzed overnight against the same buffer at 4°C. AB88 dialysate was more turbid than comparable material from AB78. The dialysate was titrated to pH 4.5 using 20 mM sodium citrate (pH 2.5) and, after 30 min incubation at room temperature, the solution was centrifuged at 3000 x g for 10 min. The protein pellet was redissolved in 20 mM Bis- Tris-Propane pH 9.0.

AB88 proteins have been separated by several different methods following clarification including isoeiectric focusing (Rotofor, BioRad, Hercules, CA), precipitation at pH 4.5, ion-exchange chromotography, size exclusion chromatography and ultrafiltration.

Proteins were separated on a Poros HQ/N anion exchange column (PerSeptive Biosystems, Cambridge, MA) using a linear gradient from 0 to 500 mM NaCl in 20 mM Bis-Tris-Propane pH 9.0 at a flow rate of 4 ml/min. The insecticidal protein eluted at 250 mM NaCl.

European corn borer (ECB)-active protein remained in the pellet obtained by pH 4.5 precipitation of dialysate. When preparative IEF was done on the dialysate using pH 3-10 ampholytes, ECB insecticidal activity was found in all fractions with pH of 7 or greater. SDS-PAGE analysis of these fractions showed protein bands of MW -60 kDa and -80 kDa. The 60 kDa and 80 kDa bands were separated by anion exchange HPLC on a Poros-Q column (PerSeptive Biosystems, Cambridge, MA). N-terminal sequence was obtained from two fractions containing proteins of slightly differing MW, but both of approximately 60 kDa in size. The sequences obtained were similar to each other and to some δ-endotoxins. anion exchange fraction 23 (smaller): xEPFVSAxxxQxxx (SEQ ID NOJ0) anion exchange fraction 28 (larger): xEYENVEPFVSAx (SEQ ID NO:1 1 )

When the ECB-active pH 4.5 pellet was further separated by anion exchange on a Poros-Q column, activity was found only in fractions containing a major band of -60 kDa.

Black cutworm-active protein also remained in the pellet when AB88 dialysate was brought down to pH 4.5. In preparative IEF using pH 3-10 ampholytes, activity was not found in the ECB-active IEF fractions; instead, it was highest in a fraction of pH 4.5-5.0. Its major components have molecular weights of -35 and -80 kDa.

The pH 4.5 pellet was separated by anion exchange HPLC to yield fractions containing only the 35 kDa material and fractions containing both 35 kDa and 80 kDa bands.

EXAMPLE 18. CHARACTERIZATION OF AB88 VIP.

Fractions containing the various iepidopteran active vegetative proteins were generated as described in Example 17. Fractions with insecticidal acitivity were separated in 8 to 16% SDS-polyacrylamide gels and transferred to PVDF membranes [LeGendre et al, (1989) in: A Practical Guide to Protein and Peptide Purification for Microsequencing, ed Matsudaria PT (Academic Press Inc, New York.]. Biological analysis of fractions demonstrated that different VIPs were responsible for the different Iepidopteran species activity.

The Agrotis ipsilon activity is due to an 80 kDa and/or a 35 kDa protein, either delivered singly or in combination. These proteins are not related to any δ-endotoxins from Bt as evidenced by the lack of sequence homology of known Bt δ-endotoxin sequences. The vip3A(a) insecticidal protein from strain AB88 is present mostly (at least 75% of the total) in supernatants of AB88 cultures.

Also, these proteins are not found in the AB88 δ-endotoxin crystal. N-terminal sequences of the major δ-endotoxin proteins were compared with the N-terminal sequences of the 80 kDa and 35 kDa VIP and revealed no sequence homology. The N-terminal sequence of the vip3A(a) insecticidal protein posses a number of positively charged residues (from Asn2 to Asn7) followed by a hydrophobic core region (from Thr8 to Ile34). Unlike most of the known secretion proteins, the vip3A(a) insecticidal protein from strain AB88 is not N-terminally processed during export.

A summary of the results follows:

Agrotis VIP N-terminal sequences N-terminal sequence of major δ-endotoxin proteins

130 kDa

MDNNPNINE (SEQ ID NO:14)

80 kDa 80 kDa

MNKNNTKLPTRALP (SEQ ID MDNNPNINE (SEQ ID

NO:12) NO:15)

60 kDa

MNVLNSGRTTI (SEQ ID

NO:16)

35 kDa

ALSENTGKDGGYIVP (SEQ ID

NO:13)

The Ostrinia nubilalis activity is due to a 60 kDa VIP and the Spodoptera frugiperda activity is due to a VIP of unknown size.

Bacillus thuringiensis strain AB88 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA and given the Accession No. NRRL B-21225.

EXAMPLE 18A. ISOLATION AND BIOLOGICAL ACTIVITY OF ft THURINGIENSIS AB424

A a thuringiensis strain, designated AB424, was isolated from a moss covered pine cone sample by standard methods known in the art. A subculture of AB424 was grown and prepared for bioassay as described in Example 2.

Biological activity was evaluated as described in Example 3. The results are as follows:

Insect species tested Percent mortality

Ostrinia nubilalis 100 Agrotis ipsilon 100 Diabrotica virgifera 0 virgifera

Strain AB424 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given Accession No. NRRL B-21439.

EXAMPLE 18B. CLONING OF THE VIP3A(a) and VIP3A(b) GENES WHICH ENCODE PROTEINS ACTIVE AGAINST BLACK CUTWORM.

Total DNA from isolates AB88 and AB424 was isolated [Ausubel et al (1988), in: Current Protocols in Molecular Biology (John Wiley & Sons, NY)] and digested with the restriction enzymes Xbal [library of 4.0 to 5.0 Kb size-fractionated Xbal fragments of B thuringiensis AB88 DNA] and EcoRI [library of 4.5 to 6.0 Kb size-fractionated EcoRI fragments B thuringiensis AB424 DNA] respectively, ligated into pBluescript vector previously linearized with the same enzymes and dephosphorylated, and transformed into E co//DH5α strain. Recombinant clones were blotted onto nitrocellulose filters which were subsequently probed with a ^M P labeled 33-bases long oligonucleotide corresponding to the 11-N terminal amino acids of the 80 kDa protein active against Agrotis ipsilon (black cutworm). Hybridization was carried out at 42°C in 2 x SSC/0.1% SDS (1 x SSC = 0.15 m NaCI/0.015 M sodium citrate, pH 7.4) for 5 min and twice at 50°C in 1 x SSC/0.1 SDS for 10 min. Four out of 400 recombinant clones were positive. Insect bioassays of the positive recombinants exhibited toxicity to black cutworm larvae comparable to that of AB88 or AB424 supernantants.

Plasmid pCIB7104 contains a 4.5 Kb Xbal fragment of AB88 DNA. Subclones were constructed to define the coding region of the insecticidal protein. co// ^' pCIB7105 was constructed by cloning the 3.5 Kb Xbal-Accl fragment of pCIB7104 into pBluescript.

Plasmid pCIB7106 contained a 5.0 Kb EcoRI fragment of AB424 DNA. This fragment was further digested with Hindi to render a 2.8 kb EcoRI-Hincll nsert (pCIB7107), which still encoded a functional insecticidal protein.

The nucleotide sequence of pCIB7104, a positive recombinant clone from AB88, and of pCIB7107, a positive recombinant clone from AB424, was determined by the dideoxy termination method of Sanger etal., Proc. Natl. Acad. Sci. USA, 74: 5463- 5467 (1977), using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase® Terminator Double-Stranded DNA Sequencing Kit and analysed on an ABI 373 automatic sequencer.

The clone pCIB7104 contains the VIP3A(a) gene whose coding region is disclosed in SEQ ID NO.28 and the encoded protein sequence is disclosed in SEQ ID NO.29. A synthetic version of the coding region designed to be highly expressed in maize is given in SEQ ID NO.30. Any number of synthetic genes can be designed based on the amino acid sequence given in SEQ ID NO:29.

The clone pCIB7107 contains the VIP3A(b) gene whose coding region is disclosed in SEQ ID NO:31 and the encoded protein is disclosed in SEQ ID NO.32. Both pCIB7104 and pCIB7107 have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21422 and B- 21423, respectively.

The VIP3A(a) gene contains an open reading frame (ORF) that extends form nucleotide 732 to 3105. This ORF encodes a peptide of 791 amino acids corresponding to a molecular mass of 88,500 daltons. A Shine-Dalgarno (SD) sequence is located 6 bases before the first methionine and its sequence identifies a strong SD for Bacillus.

The VIP3A(b) gene is 98% identical to VIP3A(a).

When blost of total DNA isolated from AB88 B thuringiensis cells were probed with a 33.base fragment that spans the N-terminal region of the VIP3A-insecticidal protein, single bands could be observed in different restriction digests. This result was

confirmed by using larger probes spanning the coding region of the gene. A search of the GenBank data base revealed no homology to known proteins.

EXAMPLE 18C. EXPRESSION OF THE VIP3A INSECTICIDAL PROTEINS

The time course for expression of the VIP3A(a) insecticidal protein was analyzed by western blot. Samples from Bacillus thuringiensis Ab88 clutures were taken throughout ist growth curve and sporulation. The VIP3A(a) insecticidal protein can be detected in the supernatants of AB88 cultures during logarithmic phase, as early as 15 h after initiating the culture. It reached its maximum level during early stages of stationary phase and remained at high levels during and after sporulation. Similar results were obtained when supernatants of AB424 Bacillus cereus cultures were used. The levels of VIP3A(a) insecticidal protein reflected the expression of the VIP3A(a) gene as determined by Northern blot. The initiation of the sporulation was determined by direct microscopic observations and by analyzing the presence of δ- endotoxins in cell pellets. Cry-I type prtoeins could be detected late in the stationary phase , during and after sporulation.

EXAMPLE 18D. IDENTIFICATION OF NOVEL VIP3-LIKE GENES BY HYBRIDIZATION

To identify Bacillus containing genes related to the VIP3A(a) from isolate AB88, a collection of Bacillus isolates was screened by hybridization. Cultures of 463 Bacillus strains were grown in microtiter wells until sporulation. A 96-pin colony stampel was used to transfer the cultures to 150 mm plates containing L-agar. Inoculated plates were kept at 30°C for 10 hours, then at 4°C overnight. Colonies were blotted onto nylon filters and probed with a 1.2Kb Hindlll VIP3A(a) derived fragment. Hybridization was performed overnight at 62°C using hybridization conditions of Maniatis et al. Molecular Cloning: A Laboratory Manual (1982). Filters were washed with 2xSSC/0J% SDS at 62°C and exposed to X-ray film.

Of the 463 Bacillus strains screened, 60 contain VIP3-like genes that could detected by hybridization. Further characterization of some of them (AB6 and AB426)

showed that their supernatants contain a BCW insecticidal protein similar to the Vip3 protein that are active against black cutworm.

EXAMPLE 18E. CHARACTERIZATION OF A ft thuringiensis STRAIN M2194 CONTAINING A CRYPTIC VIP3-LIKE GENE

A a thuringiensis strain, designated M2194, was shown to contain VIP3-like gene(s) by colony hybridization as described in Example 18C. The M2194 VIP3 like gene is considered cryptic since no expression can be detected throughout the bacterial growth phases either by immunoblot analysis using polyclonal antibodies raised against the VIP3A(a) protein isolated from AB88 or by bioassay as described in Example 3.

Antiserum against purified VIP3A(a) insecticidal protein was produced in rabbits. Nictrocellulose-bound protein (50 μg) was dissolved in DMSO and emulsified with Freund's complete adjuvant (Difco). Two rabbits were given subcutaneous injections each month for three month. They were bled 10 days after the second and third injection and the serum was recovered from the blood sample [Harlow et al (1988) in : Antibodies: A Laboratory Manual (Cold Spring Harbor Lab Press, Plainview, NY)].

The M2194 VIP3-like gene was cloned into pKS by following the protocol described in Example 9, which created pCIB7108. E coli containing pCIB7108 which comprises the M2194 VIP3 gene were active against black cutworm demonstrating that the gene encodes a functional protein with insecticidal activity. The plasmid pCIB7108 has been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession No. NRRL B-21438.

EXAMPLE 18F. INSECTICIDAL ACITIVITY OF VIP3A PROTEINS

The activity spectrum of VIP3A insecticidal proteins was qualitatively determined in insect bioassays in which recombinant E coli carrying the VIP ^* A genes were fed to larvae. In these assays, cells carrying the VIP3A(a) and VIP3A(b) genes were insecticidal to Agrotis ipsilon, Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens and Helicoverpa zea. Under the same expermimental conditions, bacterial extracts containing VIP3A proteins did not show any activity against Ostrinia nubilalis.

Effect of VIP'A insecticidal proteins on Agrotis ipsilon larvae

BCW = Black Cut Worm; FAW = Fall Army Worm; BAW = Beet Army Worm; TBW = Tobacco Bud Worm; CEW = Corn Ear Worm; ECB = European Corn Borer

EXAMPLE 19. ISOLATION AND BIOLOGICAL ACTIVITY OF OTHER BACILLUS SP.

Other Bacillus species have been isolated which produce proteins with insecticidal activity during vegetative growth. These strains were isolated from environmental samples by standard methodologies. Isolates were prepared for bioassay and assayed as described in Examples 2 and 3 respectively. Isolates which produced insecticidal proteins during vegetative growth with activity against Agrotis ipsilon in the bioassay are tabulated below. No correlation was observed between the presence of a δ-endotoxin crystal and vegetative insecticidal protein production.

Isolates AB289, AB294 and AB359 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria II 61604, USA and given the Accession Numbers NRRL B-21227, NRRL B-21229, and NRRL B-21226 respectively.

Bacillus isolates which produce insecticidal proteins during vegetative growth with activity against Diabrotica virgifera virgifera are tabulated below.

Isolates AB59 and AB256 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Illinois 61604, USA, and given the Accession Numbers NRRL B-21228 and NRRL B-21230, respectively.

EXAMPLE 20. IDENTIFICATION OF NOVEL VIP1/VIP2 LIKE GENES BY

HYBRIDIZATION

To identify strains containing genes related to those found in the VIP1 A(a)/VIP2A(a) region of AB78, a collection of Bacillus strains was screened by hybridization. Independent cultures of 463 Bacillus strains were grown in wells of 96 well microtiter dishes (five plates total) until the cultures sporulated. Of the strains tested, 288 were categorized as Bacillus thuringiensis, and 175 were categorized as other Bacillus species based on the presence or absence of δ-endotoxin crystals. For each microtiter dish, a 96-pin colony stamper was used to transfer approximately 10 μl of spore culture to two 150 mm plates containing L-agar. Inoculated plates were grown 4-8 hours at 30 °C, then chilled to 4 °C. Colonies were transferred to nylon filters, and the cells lysed by standard methods known in the art. The filters were hybridized to a DNA probe generated from DNA fragments containing both VIP1 A(a) and VIP2A(a) DNA sequences. Hybridization was performed overnight at 65 °C using the hybridization conditions of Church and Gilbert (Church, G.M., and W. Gilbert,

PNAS, 81 J 991-1995 (1984)). Filters were washed with 2x SSC containing 0.1% SDS at 65 °C and exposed to X-Ray film.

Of the 463 Bacillus strains screened, 55 strains were identified that hybridized to the VIP1 A(a)/VIP2A(a) probe. DNA was isolated from 22 of these strains, and analyzed using a Southern blot with VIP1 A(a)/VIP2A(a) DNA as probes. These strains were grouped into 8 classes based on their Southern blot pattern. Each class differed in Southern blot pattern from AB78. One class had a pattern identical to that of the VIP1 A(a)/VIP2A(a) homologs from Bacillus thuringiensis var tenebrionis (see below). Each of the 22 strains was tested for activity against western corn rootworm (WCRW). Three strains, AB433, AB434, and AB435 were found to be active on WCRW. Western blot analysis using VIP2A(a) antisera revealed that strains AB6, AB433, AB434, AB435, AB444, and AB445 produce a protein(s) of equivalent size to VIP2A(a).

Notable among the strains identified was Bacillus thuringiensis strain AB6, (NRRL B-21060) which produced a VIP active against black cutworm {Agrotis ipsilon) as described in Example 15. Western blot analysis with polyclonal antisera to VIP2A(a) and polyclonal antisera to VIP1 A(a) suggests that AB6 produces proteins similar to VIP2A(a) and VIP1 A(a). Thus, AB6 may contain VIPs similar to VIP1 A(a) and VIP2A(a), but with a different spectrum of insecticidal activity.

EXAMPLE 21. CLONING OF A VIP1 A(a IP2A(a) HOMOLOG FROM BACILLUS THURINGIENSIS VAR. TENEBRIONIS.

Several previously characterized Bacillus strains were tested for presence of DNA similar to VIP1 A(a)/VIP2A(a) by Southern blot analysis. DNA from Bacillus strains AB78, AB88, GC91 , HD-1 and ATCC 10876 was analyzed for presence of VIP1A(a)/VIP2A(a) like sequences. DNA from Bt strains GC91 and HD-1 , and the Be strain ATCC 10876 did not hybridize to VIP2A(a)/VIP1A(a) DNA, indicating they lack DNA sequences similar to VIP1 A(a)/VIP2A(a) genes. Similarly, DNA from the insecticidal strain AB88 (Example 16) did not hybridize to VIP1A(a)/VIP2A(a) DNA region, suggesting that the VIP activity produced by this strain does not result from VIP1A(a)/VIP2A(a) homologs. In contrast, Bacillus thuringiensis var. tenebrionis (Btt)

contained sequences that hybridized to the VIP1A(a)/VIP2A(a) region. Further analysis confirmed that Btt contains VIP1 A(a)/VIP2A(a) like sequences.

To characterize the Btt homologs of VIP2A(a) and VIP1 A(a), the genes encoding these proteins were cloned. Southern blot analysis identified a 9.5 kb Eco Rl restriction fragment likely to contain the coding regions for the homologs. Genomic DNA was digested with Eco Rl, and DNA fragments of approximately 9.5 kb in length were gel-purified. This DNA was ligated into pBluescript SK(+) digested with Eco Rf, and transformed into E coli to generate a plasmid library. Approximately 10,000 colonies were screened by colony hybridization for the presence of VIP2A(a) homologous sequences. Twenty eight positive colonies were identified. All twenty eight clones are identical, and contain VIP1A(a)/VIP2A(a) homologs. Clone pCIB7100 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Illinois 61604, USA, and given the Accession Number B-21322. Several subclones were constructed from pCIB7100. A 3.8 kb Xba I fragment from pCIB7100 was cloned into pBluescript SK(+) to yield pCIB7101. A 1.8 kb Hind III fragment and a 1.4 kb Hind III fragment from pCIB7100 were cloned into pBluescript SK(+) to yield pCIB7102 and pCIB7103, respectively. Subclones pCIB7101, pCIB7102 and pCIB7103 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Illinois 61604, USA, and given the Accession Numbers B-21323, B-21324 and B-21325 respectively.

The DNA sequence of the region of pCIB7100 containing the VIP2A(a)/VIP1A(a) homologs was determined by the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). Reactions were performed using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase® Terminator Double-Stranded DNA Sequencing Kits, and analyzed on an ABI model 373 automated sequencer. Custom oligonucleotides were used as primers to determine the DNA sequence in certain regions. The DNA sequence of this region is shown in SEQ ID NO:19.

The 4 kb region shown in SEQ ID NO:19 contains two open readings frames (ORFs), which encode proteins with a high degree of similarity to VIP1 A(a) and VIP2A(a) proteins from strain AB78. The amino acid sequence of the VIP2A(a)

homolog, designated as VIP2A(b) using the standardized nomenclature, is found at SEQ ID NO:20 and the amino acid sequence of the VIP1 A(a) homolog, designated as VIP1A(b) using the standardized nomenclature, is disclosed at SEQ ID NO.21. The VIP2A(b) protein exhibits 91% amino acid identity to VIP2A(a) from AB78. An alignment of the amino acid sequences of the two VIP2 proteins is provided in Table 20. The VIP1A(b) protein exhibits 77 % amino acid identity to VIP1 A(a) from AB78. An alignment of these two VIP1 proteins is provided in Table 21. The alignment shown in Table 21 discloses the similarity between VIP1A(b) and VIP1 A(a) from AB78. This alignment reveals that the amino terminal regions of the two VIP1 proteins share higher amino acid identity in the amino-terminal region than in the carboxy terminal region. In fact, the amino terminal two thirds (up to aa 618 of the VIP1 A(b) sequence shown in Table 21 ) of the two proteins exhibit 91% identity, while the carboxy-terminal third (from aa 619-833 of VIP1 A(b)) exhibit only 35% identity.

Western blot analysis indicated that Bacillus thuringiensis var. tenebrionis (Btt) produces both VIP1 A(a) like and VIP2A(a) like proteins. However, these proteins do not appear to have activity against western corn rootworm. Bioassay for activity against western corn rootworm was performed using either a 24 h culture supernatant from Btt or E coli clone pCIB7100 (which contains the entire region of the VIP1 A(a)/VIP2A(a) homologs). No activity against western corn rootworm was detected in either case.

Given the similarity between the VIP2 proteins from Btt and AB78, the ability of VIP2A(b) from Btt to substitute for VIP2A(a) from AB78 was tested. Cells containing pCIB6206 (which produces AB78 VIP1 A(a) but not VIP2A(a) protein) were mixed with Btt culture supernatant, and tested for activity against western corn rootworm. While neither Btt culture supernatant nor cells containing pCIB6206 had activity on WCRW, the mixture of Btt and pCIB6206 gave high activity against WCRW. Furthermore, additional bioassay showed that the Btt clone pCIB7100, which contains the Btt VIP1 A(b)/VIP2A(b) genes in E coli, also confers activity against WCRW when mixed with pCIB6206. Thus, the VIP2A(b) protein produced by Btt is functionally equivalent to the VIP2A(a) protein produced by AB78.

Thus, the ability to identify new strains with insecticidal activity by using VIP DNA as hybridization probes has been demonstrated. Furthermore, Bacillus strains that contain VIP1A(a)/VIP2A(a) like sequences, produce VIPlA(a)/VIP2A(a) like protein.

yet demonstrate toxicity toward different insect pests. Similar methods can identify many more members of the VIP1/VIP2 family. Furthermore, use of similar methods can identify homologs of other varieties of VIPs (for example, the VIPs from AB88).

TABLE 20

Alignment of VIP2 Amino Acid Sequences from Bacillus thuringiensis var. tenebrionis (VIP2A(b)) vs. AB78 (VIP2A(a))

Btt 1 MQRMEGK___FWSKTLQWTRTVI_LSTW^ 50 SEQ ID NO: 20

I . I I I I I I I : I I I • I I I I I : I I I I I I I : I I . I I I I I I I I : I I I I I I I I I AB78 1 MKRi_EGKLFWSKKI_QVVTKTVl^ 50 SEQ ID NO: 2

51 Ϊ ra_QNLKIPDNAEDFKEDK3KAKEWGKE_KGE_E ro 100

I I I I I I M I • I .. M I I I I I: I I I I II i I I :• M: . I I I I I . I I I II I I 51 YTNLQNLKITDKVEDFKEDKEKAK^^ 100

101 K_SroiKTNYKEITFSJ^GSCTϊ)EIKD.^ 150

M M I I I I I I I I M M I I I I I I I . I I I I :| I I . I I I . I I I I I I I I I I 101 KiroimrϊKEITFS^GSFEDEIKDIi-EIDK^ 150

151 ATIGFNKSLTEGNTINSDA^QFKEQFKSΦMKFDSYLDTHLTAQQVSS 200 . II II I II II II I I II I II II II I II II :: I Ml I I II II II II II II I

151 TTIGFNKSLTEGNTINSDA AQFKEQFK)PDIKFDSYLDTHLTAQQVSSK 200

201 KRVILKVTVPSC^GSTTPTKAGVT:...^^ 250

. II II I I II II II I I I I I I I M II II I . I I M II I I I I :M I I M II II I 201 ERVIIitt /PSGKGSTTPTKAGVIIJ-JNSEYKMLIDNGY 250

251 KG ECLQVEGTI_KKSI_DFK^roINAE^^SWGMKIYEDWA NLTASQRE_AI__D 300

MMMIMMMMMM I I I I I MUM I I I I : I MMIM I Mill 251 KGVECIiQIEGTIiKSI_DFK DINAEAHS GMKNYEE AKDLTDSQREA_l__D 300

301 GYArøDYKEINNYIiWQfJGSGNEKLDAQIi^NISDALGKKPIPENITVyP^ 350

I I II II I II II II I I II I I I I I I I I II 1:11 I I I II II II I II I I II III 301 GYARQDYKEIl^IYLrøQGGSGNEK^AQIKNISDALGKKPIPENITvYRW 350

351 CG_FEFGYQISDPI_PSI__KDFEEQFLNTIKEXlKGYMSTSLSSERLAAFGSR 400

I MM III I I M M II II II II I II I II II III III II I I I II II MM I

351 CG PEFGYQISD I_PSI_KDFEEQF ^r^I EDKGYMSTSLSSERLAAFGSR 400

401 KIILRLQWKGSTGAYLSMGGFASEKEILLDKDSKYHIDKATEV IKGV 450

II I I I II III I I II I I I I II I I I II I I I I I I I I II M I I I l I I II I II

401 KIILRIiQVPKGSTGAYLSMGGFASEKEILLDKDSKYHIDKVTEVTIKGV 450

451 KRYWDATLLTN 462

I I I f I I I I I I I I

451 KRYWDATLLTN 462

TABLE 21

Alignment of VIP1 Amino Acid Sequences from Bacillus thuringiensis var. tenebrionis (VIP1A(b)) vs. AB78(VIP1A(a))

Btt 1 MKNMKKKLASVVTCMLLAP]^^ 50 SEQ ID NO: 21

I I I I I I I I I I I I I I Mill II MM I II I I I I . I I I I I I I . I I I I I I I

Ab78 1 MKNMKKKIJ^VVTCTLI_APMFLNGNVNAVYAD 50 SEQ ID NO: 5

51 RKGIiLGYYFKGKDFN^TMFAPTRDNTI^^ 100

II II II I I II I II l M I II II I Ml Ml II I II I I I I I I I I I I I I I I

51 RKGIJ£YYFKGKDFSNLT^APTRDSTLIYDQQTANKIJ__DKKQQEYQSIR 100

101 WIGLIQRKETGDFTFNLSKDEQAIIEIDGKIISNKGKEKQWHLEKEKLV 150

I I I I I I. I I I I I I I I I I l I I I I I I I :| I I I I I I I I I I I I I I I I |:| I I 101 WIGLIQSKETGDFTFNLSEDEQAIIEINGKIISNKGKEKQWHLEKGKLV 150

151 PIKIEYQSDTKFNIDSKTFKELKLFKIDSQNQSQQVQ...LRNPEFNKKE 197 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I.I I I I I I I I I I II I I

151 PIKIEYQSDTKFNIDSKTFKEL LFKIDSQNQPQQVQQDEI_R_N!PEFNKKE 200

198 SQE_FI_AKASK ]_ ₁ FKQK^^KRDIDEDTDTDGDSIPDL EENG TIQ K AV 247

I I I I I I : I I . I I I M I I I I : I I I I I I I I I M I I I I I I I I I I I I I M M I 201 SQEFLAKPSKINLFTQK KREIDEDTDTOGDSIPDLWEENGYTIQNRIAV 250

248 KWDDSLASKGYTKFVSl llDSHTVGDPYTDYEKAAR^ 297

I M I I I M i l l M I N I I M I I I M i l l I I I M I N I M i l l M i l l I I I 251 KWDDSLASKGYTKFVSNPLESHTVGDPYTDYEKAARDLDLSNAKETFNPL 300

298 VAAFPSV ^SME-KVII^PNENLSNSVESHSSTNWSYTNTEGASIEAGGGP 347

I II I I II II II II II II II I II I II II II II II I I M I II II M II I II

301 VAAFPSVNVS1EKVII^PNENLSNSVESHSSTNWSYTNTEGASVEAGIGP 350

348 LG]_5FGVSV YQHSETVAQEM^STG TSQF^^rASAG LNA RY^πIV^ 397

1: 1 I I I I I. MI N I M I N I M l lll lll l l I I I I I I I M i l l Mi l l 351 KGISFGVSVϊTϊ'QHSETVAQEWGTSTGNTSQFNTASAGYLNANVRYNNVGT 400

398 GAIYDVlΦTTSFVIJJNNTIATITAKSNSTALRISPGDSYPEIGENAIAIT 447

M I I I I I I I I I I I I M I I I I I I I I I I I I I Ml I I M I I I . M I M I I 401 GMYDV1ΦTTSFVI_NNDT_IATITAKSNSTALNISPGESYPKKGQNGIAIT 450

448 S^DFNSHPITIJ^QQVNQLINNKPIM___ETDQTDGVYKIRDTHGNIVT 497

II I II II I M I I IM IM. Ml II M I I I Mill II II Ml II I II III

451 SrøDF^SHPITI_NKKQVDNLLNNKPMM]__ETNQTDGVYKIKDTHGNI^ 500

498 EWNGVTQQIKAKTASIIVDDGKQVAEKRVAAKDYGHPEDKTPPLTLKDTL 547

II I I I . I I I I I I I I I I I I I I I .. I I I I I I I I I I I : : I I I I I I . I I I I I . I 501 EWNGVIQQIKAKTASIIVDDGERVAEKRV7 ^KDYENPEDKTPSLTLKDAL 550

548 KLSYPDEIKETNGLLYYDDKPIYESSV1OTYLDENTAKEVKKQINDTTGKF 597

I I I I I I I I I I . : l I I I I . : l I I I I I I I I I I I I I I I I I I I . I 1 : 1 I I I I I I 551 KLSYPDEIKEIEGIiYYKNKPIYESSVMTYI-DENTAKEVTKQLNDTTGKF 600

598 KDVNHLYDVKLTPKMNFTIKMASLYDGAENNHNSK?IWYLTYNVAGGNTG 647

M I I I I I I II I I I I. I I I:. I I M I I -M MM. I I l I Ml 601 KDVSHL DVKLTPKlyπSIVTIKI^ILYD ^[ AESNDNSIGK TN NIVSGGNNG 650

648 KRQYRSMSCAHVALSSEAKKK QNANYYLSMYMKADSTTEPTIEV^ 697

I : I I . I . : . I : : . I .. : I .. I I I . I : I I : I : I I I . : .. I : .. I . : . I I

698 KS TSKKVKLNNQNYQRvDILVKNSERNPrøKIYIRGNGTTNVYGDDVT 747

M l. I. I.: I.: I I. I. Ml:.. I .. I I : .. : . I ^* : . I : ... : : ||:. 701 IYPITTKT\^VNKDNYKRLDIIAHNIKSNPISSLHIKTNDEITIJWDDIS 750

748 IPEVSAINPASLSDEEIQEIFKDSTIEYGNPSFVADAVTFK 788

|.:|..|.|..|.|.| |.:|:. .|..::. ::.. ..:. 751 ITDVASIKPENLTDSEIKQIYSRYGIKLEDGILIDKKGGIHYGEFINEAS 800

789 .NIKPLQNYVKEYEIYHK SHRYEKKTVFDIMGVHYEYSIAREQ 830

II. I I I I I |.. I.: .. I. . I...::. .:.:::. ...

801 FNIEPLQISTYVTKYKVTYSSEI.GQNVSDTLESDKIY 850

831 KKA 833

851 EQG 853

EXAMPLE 22. FUSION OF VIP PROTEINS TO MAKE A SINGLE

POLYPEPTIDE

VIP proteins may occur in nature as single polypeptides, or as two or more interacting polypeptides. When an active VIP is comprised of two or more interacting protein chains, these protein chains can be produced as a single polypeptide chain from a gene resulting from the fusion of the two (or more) VIP coding regions. The genes encoding the two chains are fused by merging the coding regions of the genes to produce a single open reading frame encoding both VIP polypeptides. The composite polypeptides can be fused to produce the smaller polypeptide as the NH ₂ terminus of the fusion protein, or they can be fused to produce the larger of the

polypeptides as the NH ₂ terminus of the fusion protein. A linker region can optionally be used between the two polypeptide domains. Such linkers are known in the art. This linker can optionally be designed to contain protease cleavage sites such that once the single fused polypeptide is ingested by the target insect it is cleaved in the linker region to liberate the two polypeptide components of the active VIP molecule.

VIP1A(a) and VIP2A(a) from B. cereus strain AB78 are fused to make a single polypeptide by fusing their coding regions. The resulting DNA comprises a sequence given in SEQ ID NO:22 with the encoded protein given in SEQ ID NO:23. In like manner, other fusion proteins may be produced. .

The fusion of the genes encoding VIP1 A(a) and VIP2A(a) is accomplished using standard techniques of molecular biology. The nucleotides deleted between the VIP1A(a) and VIP2A(a) coding regions are deleted using known mutagenesis techniques or, alternatively, the coding regions are fused using PCR techniques.

The fused VIP polypeptides can be expressed in other organisms using a synthetic gene, or partially synthetic gene, optimized for expression in the alternative host. For instance, to express the fused VIP polypeptide from above in maize, one makes a synthetic gene using the maize preferred codons for each amino acid, see for example EP-A 0618976, herein incorporated by reference. Synthetic DNA sequences created according to these methods are disclosed in SEQ ID NO:17 (maize optimized version of the 100 kDa VIP1 A(a) coding sequence), SEQ ID NO:18 (maize optimized version of the 80 kDa VIP1 A(a) coding sequence) and SEQ ID NO.24 (maize optimized version of the VIP2A(a) coding sequence).

Synthetic VIP1 and VIP2 genes optimized for expression in maize can be fused using PCR techniques, or the synthetic genes can be designed to be fused at a common restriction site. Alternatively, the synthetic fusion gene can be designed to encode a single polypeptide comprised of both VIP1 and VIP2 domains.

Addition of a peptide linker between the VIP1 and VIP2 domains of the fusion protein can be accomplished by PCR mutagenesis, use of a synthetic DNA linker encoding the linker peptide, or other methods known in the art.

The fused VIP polypeptides can be comprised of one or more binding domains. If more than one binding domain is used in the fusion, multiple target pests are controlled using such a fusion. The other binding domains can be obtained by using all or part of other VIPs; Bacillus thuringiensis endotoxins, or parts thereof; or other

proteins capable of binding to the target pest or appropriate biding domains derived from such binding proteins.

One example of a fusion construction comprising a maize optimized DNA sequence encoding a single polypeptide chain fusion having VIP2A(a) at the N- terminal end and VIP1A(a) at the C-terminal end is provided by pCIB5531. A DNA sequence encoding a linker with the peptide sequence PSTPPTPSPSTPPTPS (SEQ ID NO:47) has been inserted between the two coding regions. The sequence encoding this linker and relevant cloning sites is 5'- CCC GGG CCT TCT ACT CCC CCA ACT CCC TCT CCT AGC ACG CCT CCG ACA.CCT AGC GAT ATC GGA TC C -3' (SEQ ID NO:48). Oligonucleotides were synthesized to represent both the upper and lower strands and cloned into a pUC vector following hybridization and phosphorylation using standard procedures. The stop codon in VIP2A(a) was removed using PCR and replaced by the Bglll restriction site with a Smal site. A translation fusion was made by ligating the Bam HI / Pstl fragment of the VIP2A(a) gene from pCIB5522 (see Example 24), a PCR fragment containing the Pstl-end fragment of the VIP2A(a) gene (identical to that used to construct pCIB5522), a synthetic linker having ends that would ligate with a blunt site at the 5' end and with BamHI at the 3' end and the modified synthetic VIP1 A(a) gene from pCIB5526 described below {See SEQ ID NO:35). The fusion was obtained by a four way ligation that resulted in a plasmid containing the VIP2A(a) gene without a translation stop codon, with a linker and the VIP1 A(a) coding region without the Bacillus secretion signal. The DNA sequence for this construction is disclosed in SEQ ID NO:49, which encodes the fusion protein disclosed in SEQ ID NO:50. A single polypeptide fusion where VIP1 A(a) is at the N-terminal end and VIP2A(a) is at the C-terminal end can be made in a similar fashion. Furthermore, either one or both genes can be linked in a translation fusion with or without a linker at either the 5' or the 3' end to other molecules like toxin encoding genes or reporter genes.

EXAMPLE 23. TARGETING OF VIP2 TO PLANT ORGANELLES

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the

chloroplast is controlled by a signal sequence found at the amino-terminal end of various proteins. This signal is cleaved during chloroplast import, yielding the mature protein {e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products such as VIP2 to effect the import of those products into the chloroplast (van den Broeck et al. Nature 313: 358- 363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5 ^* end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized.

Other gene products are localized to other organelles such as the mitochondrion and the peroxisome {e.g. Unger et al. Plant Molec. Biol. 13: 41 1 -418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products such as VIP2 to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Similarly, targeting to cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

By the fusion of the appropriate targeting sequences described above to coding sequences of interest such as VI P2 it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino-terminal ATG of the transgene. The signal sequence selected should include the known cleavage site and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the start codon ATG, or alternatively replacement of some amino acids within the coding sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by (Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp 1081 - 1091 (1982); Wasmann et al. Moi. Gen. Genet. 205: 446-453 (1986)). These

construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The above described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.

A DNA sequence encoding a secretion signal is present in the native Bacillus VIP2 gene. This signal is not present in the mature protein which has the N-terminal sequence of LKITDKVEDF (amino acid residues 57 to 66 of SEQ ID NO:2). It is possible to engineer VIP2 to be secreted out of the plant cell or to be targeted to subcellular organelles such as the endoplasmic reticulum, vacuole, mitochondria or plastids including chloroplasts. Hybrid proteins made by fusion of a secretion signal peptide to a marker gene have been successfully targeted into the secretion pathway. (Itirriaga G. et al., The Plant Cell. J.: 381-390 (1989) , Denecke et al., The Plant Cell. 2:51-59 (1990). Amino-terminal sequences have been identified that are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)).

The presence of additional signals are required for the protein to be retained in the endoplasmic reticulum or the vacuole. The peptide sequence KDEL/HDEL at the carboxy-terminal of a protein is required for its retention in the endoplasmic reticulum (reviewed by Pelham, Annual Review Cell Biol.. 5:1-23 (1989). The signals for retention of proteins in the vacuole have also been characterized. Vacuolar targeting signals may be present either at the amino-terminal portion, (Holwerda et al., The Plant Cell. 4:307-318 (1992), Nakamura et al.. Plant Phvsiol- 101 :1 -5 (1993)), carboxy- terminal portion, or in the internal sequence of the targeted protein. (Tague et al., The Plant Cell. 4:307-318 (1992), Saalbach et al.. The Plant Cell. 3:695-708 (1991)). Additionally, amino-terminal sequences in conjunction with carboxy-terminal sequences are responsible for vacuolar targeting of gene products (Shinshi etal. Plant Molec. Biol. 14: 357-368 (1990)). Similarly, proteins may be targeted to the mitochondria or plastids using specific carboxy terminal signal peptide fusions (Heijne et al., Eur. J. Biochem.. 180:535-545 (1989), Archer and Keegstra, Plant Molecular Biology. 23:1105-1 1 15 (1993)).

In order to target VIP2, either for secretion or to the various subcellular organelles, a maize optimized DNA sequence encoding a known signal peptide(s) may be designed to be at the 5' or the 3' end of the gene as required. To secrete VIP2 out of the cell, a DNA sequence encoding the eukaryotic secretion signal peptide MGWSWIFLFLLSGAAGVHCL (SEQ ID NO:25) from PCT application No. IB95/00497 or any other described in the literature (Itirriaga et al., The Plant Cell. 1 :381-390 (1989) , Denecke, et al., The Plant Cell. 2:51-59 (1990)) may be added to the 5' end of either the complete VIP2 gene sequence or to the sequence truncated to encode the mature protein or the gene truncated to nucleotide 286 or encoding a protein to start at amino acid residue 94 (methionine). To target VIP2 to be retained in the endoplasmic reticulum, a DNA sequence encoding the ER signal peptide KDEL /HDEL, in addition to the secretion signal, can be added to the 3' end of the gene. For vacuolar targeting a DNA sequence encoding the signal peptide SSSSFADSNPIRVTDRAAST (SEQ ID NO:3; Holwerda etal., The Plant Cell.4:307- 318 (1992)) can be designed to be adjacent to the secretion signal or a sequence encoding a carboxyl signal peptide as described by Dombrowski et al., The Plant Cell. 5:587-596 (1993) or a functional variation may be inserted at the 3' end of the gene. Similarly, VIP2 can be designed to be targeted to either the mitochondria or the plastids, including the chloroplasts, by inserting sequences in the VIP2 sequence described that would encode the required targeting signals. The bacterial secretion signal present in VIP2 may be retained or removed from the final construction.

One example of a construction which incorporates a eukaryotic secretion signal fused to a coding sequence for a VIP is provided by pCIB5528. Oligonucleotides corresponding to both the upper and lower strand of sequences encoding the secretion signal peptide of SEQ ID NO.25 was synthesized and has the sequence 5'- GGATCCACC ATG GGC TGG AGC TGG ATC TTC CTG TTC CTG CTG AGC GGC GCC GCG GGC GTG CAC TGC CTGCAG-3' (SEQ ID NO.41 ). When hybridized, the 5' end of the secretion signal resembled "sticky-ends" corresponding to restriction sites BamHI and Pstl. The oligonucleotide was hybridized and phosphorylated and ligated into pCIB5527 (construction described in Example 23A) which had been digested with BamHI/ Pstl using standard procedures. The resulting maize optimized coding sequence is disclosed in SEQ ID NO:42 which encodes the protein disclosed

in SEQ ID NO:43. This encoded protein comprises the eukaryotic secretion signal in place of the Bacillus secretion signal.

One example of a construction which incorporates a vacuolar targe tting signal fused to a coding sequence for a VIP is provided by pCIB5533. Oligonucleotides corresponding to both the upper and lower strand of sequences encoding the vacuolar targetting peptide of SEQ ID NO:3 was synthesized and has the sequence 5'-CCG CGG GCG TGC ACT GCC TCA GCA GCA GCA GCT TCG CCG ACA GCA ACC CCA TCC GCG TGA CCG ACC GCG CCG CCA GCA CCC TGC AG-3' (SEQ ID NO:44). When hybridized, the 5" end of the vacuolar targetting signal resembled "sticky-ends" corresponding to restriction sites Sacll and Pstl. The oligonucleotide was hybridized and phosphorylated and ligated into pCIB5528 (construction described above) which had been digested with Sacll / Pstl using standard procedures. The resulting maize optimized coding sequence is disclosed in SEQ ID NO:45 which encodes the protein disclosed in SEQ ID NO:46. This encoded protein comprises the vacuolar targetting peptide in addition to the eukaryotic secretion signal.

The VIP1 gene can also be designed to be secreted or targeted to subcellular organelles by similar procedures.

EXAMPLE 23A. REMOVAL OF BACILLUS SECRETION SIGNAL FROM VIP1A(a) AND VIP2A(a)

VIP1A(a) and VIP2A(a) are secreted during the growth of strain AB78. The nature of peptide sequences that act as secretion signals has been described in the literature (Simonen and Palva, Microbiological reviews, pg. 109-137 (1993)). Following the information in the above publication, the putative secretion signal was identified in both genes. In VIP1 A(a) this signal is composed of amino acids 1-33 {See SEQ ID NO:5). Processing of the secretion signal probably occurs after the serine at amino acid 33. The secretion signal in VIP2A(a) was identified as amino acids 1-49 {See SEQ ID NO:2). N-terminal peptide analysis of the secreted mature VIP2A(a) protein revealed the N-terminal sequence LKITDKVEDFKEDK. This sequence is found beginning at amino acid 57 in SEQ ID NO:2. The genes encoding these proteins have been modified by removal of the Bacillus secretion signals.

A maize optimized VIP1 A(a) coding region was constructed which had the sequences encoding the first 33 amino acids, i.e., the secretion signal, removed from its 5' end. This modification was obtained by PCR using an forward primer that

contained the sequence 5'-GGA TCC ACC ATG AAG ACC AAC CAG ATC AGC-3' (SEQ ID NO.33), which hybridizes with the maize optimized gene (SEQ ID NO:26) at nucleotide position 100, and added a BamHI restriction site and a eukaryotic translation start site consensus including a start codon. The reverse primer that contained the sequence 5"-AAG CTT CAG CTC CTT G-3' (SEQ ID NO:34) hybridizes on the complementary strand at nucelotide position 507. A 527 bp amplification product was obtained containing the restriction sites BamHI at the 5' end and Hindlll site at the 3' end. The amplification product was cloned into a T- vector (described in Example 24, below) and sequenced to ensure the correct DNA sequence. The BamHI / Hindlll fragment was then obtained by restriction digest and used to replace the BamHI/Hindlll fragment of the maize optimized VIP1A(a) gene cloned in the root- preferred promoter cassette. The construct obtained was designated pCIB5526. The maize optimized coding region for VIP1A(a) with the Bacillus secretion signal removed is disclosed as SEQ ID NO.35 and the encoded protein is disclosed as SEQ ID NO.36.

The gene encoding the processed form of VIP2A(a), i.e., a coding region with the secretion signal removed, was constructed by a procedure similar to that described for that used to construct the processed form of VIP1 A(a), above. The modification was obtained by PCR using the forward primer 5'-GGA TCC ACC ATG CTG CAG AAC CTG AAG ATC AC -3' (SEQ ID NO:37). This primer hybridizes at nucleotide position 150 of the maize optimized VIP2A(a) gene (SEQ ID NO:27). A silent mutation has been inserted at nucleotide position 15 of this primer to obtain a Pstl restriction site. The reverse primer has the sequence 5'-AAG CTT CCA CTC CTT CTC-3' (SEQ ID NO:38). A 259 bp product was obtained with Hindlll restriction site at the 3' end. The amplification product was cloned into a T- vector, sequenced and ligated to a BamHI /Hindlll digested root-preferred promoter cassette containing the maize optimized VIP2A(a). The construct obtained was designated pCIB5527. The maize optimized coding region for VIP2A(a) with the Bacillus secretion signal removed is disclosed as SEQ ID NO:39 and the encoded protein is disclosed as SEQ ID NO:40.

EXAMPLE 24. CONSTRUCTION AND CLONING OF THE VIP1 Ate) AND VIP2A(a) MAIZE OPTIMIZED GENES

Design: The maize optimized genes were designed by reverse translation of the native VIP1A(a) and VIP2A(a) protein sequences using codons that are used most often in maize (Murray et al., Nucleic Acid Research. 17:477-498 (1989)). To facilitate cloning, the DNA sequence was further modified to incorporate unique restriction sites at intervals of every 200-360 nucleotides. VIP1 A(a) was designed to be cloned in 11 such fragments and VIP2A(a) was cloned in 5 fragments. Following cloning of the individual fragments, adjacent fragments were joined using the restriction sites common to both fragments, to obtain the complete gene. To clone each fragment, oligonucleotides (50-85 nucleotides) were designed to represent both the upper and the lower strand of the DNA. The upper oligo of the first oligo pair was designed to have a 15 bp single stranded region at the 3' end which was homologous to a similar single stranded region of the lower strand of the next oligo pair to direct the orientation and sequence of the various oligo pairs within a given fragment. The oligos are also designed such that when the all the oligos representing a fragment are hybridized, the ends have single stranded regions corresponding to the particular restriction site to be formed. The structure of each oligomer was examined for stable secondary structures such as hairpin loops using the OLIGO program from NBI Inc. Whenever neccesary, nucleotides were changed to decrease the stability of the secondary structure without changing the amino acid sequence of the protein. A plant ribosomal binding site consensus sequence, TAAACAATG (Joshi et al., Nucleic Acid Res.. 15:6643-6653 (1987)) or eukaryotic ribosomal binding site concensus sequence CCACCATG (Kozak, Nucleic Acid Research. 12:857-872 (1984)) was inserted at the translational start codon of the gene.

Cloning: Oligos were synthesized by IDT Inc., and were supplied as lyophilized powders. They were resuspended at a concentration of 200 μM. To 30 μl of each oligo formamide was added a final concentration of 25-50% and the sample was boiled for two minutes before separation on a premade 10% polyacryamide / urea gel obtained from Novex. After electrophoresis, the oligo was detected by UV shadowing by placing the gel on a TLC plate containing a fluorescent indicator and exposing it to UV light. The region containing DNA of the correct size was excised and extracted

from the polyacryamide by an overnight incubation of the minced gel fragment in a buffer containing 0.4 M LiCI, OJ mM EDTA. The DNA was separated from the gel residue by centrifugation through a Millipore UFMC filter. The extracted DNA was ethanol precipitated by the addition of 2 volumes of absolute alcohol. After centrifugation, the precipitate was resuspended in dH ₂ 0 at a concentration of 2.5 μM. Fragments were cloned either by hybridization of the oligos and ligation with the appropriate vector or by amplification of the hybridized fragment using a equimolar mixture of all the oligos for a particular fragment as a template and end-specific PCR primers.

Cloning by hybridization and ligation: Homologous double stranded oligo pairs were obtained by mixing 5 μl of the upper and of the lower oligo for each oligo pair with buffer containing 1X polynucleotide kinase (PNK) buffer (70 mM Tris-HCl (pH 7.6), 10 mM MgC. ₂ .5 mM dithiothreitol (DTT)), 50 mM KCl, and 5 % formamide in a final volume of 50 μl. The oligos were boiled for 10 minutes and slow cooled to 37° C or room temperature. 10 μl was removed for analysis on a 4% agarose in a TAE buffer system (Metaphore®; FMC). Each hybridized oligo pair was kinased by the addition of ATP at a final concentration of 1 mM, BSA at a final concentration of 100 μg per ml and 200 units of polynucleotide kinase and 1 μl of 10X PNK buffer in a volume of 10 μl. Following hybridization and phosphorylation, the reaction was incubated at 37° C for 2 hours to overnight. 10 μl of each of the oligo pairs for a particular fragment, were mixed in a final volume of 50 μl. The oligo pairs were hybridized by heating at 80° C for 10 minutes and slow cooling to 37° C. 2 μl of oligos was mixed with about 100 ng of an appropriate vector and ligated using a buffer containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCI ₂ , 10 mM DTT, 1 mM ATP. The reaction was incubated at room temp, for 2 hours to overnight and transformed into DH5α strain of E.coli , plated on L- plates containing ampicillin at a concentration of 100 μg/ml using standard procedures. Positive clones were further characterized and confirmed by PCR miniscreen described in detail in EP-A 0618976 using the universal primers "Reverse" and M13 "-20 " as primers. Positive clones were identified by digestion of DNA with appropriate enzymes followed by sequencing. Recombinants that had the expected DNA sequence were then selected for further work.

PCR Amplification and cloning into T- vector:

PCR amplification was carried out by using a mixture of all the oligomers that represented the upper and the lower strand of a particular fragment ( final concentration 5 mM each) as template, specific end primers for the particular fragment ( final concentration 2 μM) 200 μM of each dATP, dTTP, dCTP and dGTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCI ₂ ,0.01% gelatin and 5 units of Taq polymerase in a final reaction volume of 50 μl. The amplification reaction was carried out in a Perkin Elmer thermocycler 9600 by incubation at 95° C for 1 min (1 cycle ), followed by 20 cycles of 95 °C for 45 sec, 50 °C for 45 sec, 72 °C for 30 sec. Finally the reaction was incubated for 5 min at 72°C before analyzing the product. 10 μl of the reaction was analyzed on a 2.5% Nusieve (FMC) agarose gel in a TAE buffer system. The correct size fragment was gel purified and used for cloning into a PCR cloning vector or T-vector. T-vector construction was as described by Marchuk et al., Nucleic Acid Research. 19:1154 (1991). pBluescriptsk- (Stratagene®, Ca.) was used as the parent vector. Transformation and identification of the correct clone was carried out as described above.

Fragments 1 , 3, 4, 5, 6, 8, and 9 of VIP1 A(a) and fragments 2 and 4 of VIP2A(a) were obtained by cloning of PCR amplification products; whereas, fragments 2, 7, 10 and 11 of VIP1 A(a) and fragments 1 , 3, and 5 of VIP2A(a) were obtained by hybridization/ ligation.

Once fragments with the desired sequence were obtained, the complete gene was assembled by cloning together adjacent fragments. The complete gene was resequenced and tested for activity against WCRW before moving it into plant expression vectors containing the root preferred promoter (disclosed in U.S. patent application serial no. 08/017,209, herein incorporated by reference) and the rice actin promoter.

One such plant expression vector is pCIB5521. The maize optimized VIP1 A(a) coding region (SEQ ID NO:26) was cloned in a plant expression vector containing the root preferred promoter at the 5" of the gene with the PEP Carboxylase intron #9 followed by the 35S terminator at the 3' end. The plasmid also contains sequences for ampicillin resistance from the plasmid pUC19. Another plant expression vector is pCIB5522, which contains the maize optimized VIP2A(a) coding region (SEQ ID

NO:27) fused to the root preferred promoter at the 5' of the gene with the PEP Carboxylase intron #9 followed by the 35S terminator at the 3' end.

EXAMPLE 25. NAD AFFINITY CHROMATOGRAPHY

A purification strategy was used based on the affinity of VIP2 for the substrate NAD. The supernatant from the pH 3.5 sodium citrate buffer treatment described in Example 4 was dialyzed in 20 mM TRIS pH 7.5 overnight. The neutralized supernatant was added to an equal volume of washed NAD agarose and incubated with gentle rocking at 4° C overnight. The resin and protein solution were added to a 10 ml disposable polypropylene column and the protein solution allowed to flow out. The column was washed with 5 column volumes of 20 mM TRIS pH 7.5 then washed with 2-5 column volumes of 20 mM TRIS pH 7.5, 100 mM NaCl, followed by 2-5 column volumes of 20 mM TRIS 7.5. The VIP proteins were eluted in 20 mM TRIS pH 7.5 supplemented with 5 mM NAD. Approximately 3 column volumes of the effluent were collected and concentrated in a Centricon -10. Yield is typically about 7-15 μg of protein per ml of resin.

When the purified proteins were analyzed by SDS-PAGE followed by silver staining, two polypeptides were visible, one with Mr of approximately 80,000 and one with Mr of approximately 45,000. N-terminal sequencing revealed that the Mr 80,000 protein corresponded to a proteolytically processed form of VIP1 A(A) and the Mr 45,000 form corresponded to a proteolytically processed form of VIP2A(a). The co- purification of VIP1 A(a) with VIP2A(a) indicates that the two proteins probably form a complex and have protein-protein interacting regions. VIP1A(a) and VIP2A(a) proteins purified in this manner were biologically active against western com rootworm.

EXAMPLE 26. EXPRESSION OF MAIZE OPTIMIZED VIP1 Ate) AND VIP2A(a)

E coli strains containing different plasmids comprising VIP genes were assayed for expression of VIPs. E coli strains harboring the individual plasmids were grown overnight in L-broth and expressed protein was extracted from the culture as described in Example 3, above. Protein expression was assayed by Western Blot analysis using antibodies developed using standard methods known in the art, similar

to those described in Example 12, above. Also, insecticidal activity of the expressed proteins were tested against Western corn rootworm according to the method in Example 3, above. The results of the E coli expression assays are described below.

Expression of VIPs in E. coli

Extract of E. coli Strain Assay Assay Protein Harboring Indicated Plasmid No. 1 No. 2 Detected

% Mortality

The DNA from these plasmids was used to transiently express the VIPs in a maize protoplast expression system. Protoplasts were isolated from maize 2717 Line 6 suspension cultures by digestion of the cell walls using Cellulase RS and Macerase R10 in appropriate buffer. Protoplasts were recovered by sieving and centrifugation. Protoplasts were transformed by a standard direct gene transfer method using approximately 75 g plasmid DNA and PEG-40. Treated protoplasts were incubated overnight in the dark at room temperature. Analysis of VIP expression was

accomplished on protoplast explants by Western blot analysis and insecticidal activity against Western corn rootworm as described above for the expression in E coli. The results of the maize protoplast expression assays are described below.

Expression of VIPs in Plant Protoplasts

(p) = extract of protoplast culture transformed with indicated plasmid

(e) = extract of E coli strain harboring indicated plasmid

The expression data obtained with both E coli and maize protoplasts show that the maize optimized VIP1A(a) and VIP2A(a) genes make the same protein as the native VIP1 A(a) and VIP2A(a) genes, respectively, and that the proteins encoded by the maize optimized genes are functionally equivalent to the proteins encoded by the native genes.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The following deposits have been made at Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA:

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(A) NAME: CIBA-GEIGY AG

(B) STREET: Klybeckstr. 141

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : 4002

(G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991

(ii) TITLE OF INVENTION: Novel Pesticidal Proteins and Strains (iii) NUMBER OF SEQUENCES: 52

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS .

(D) SOFTWARE: Patentln Release #1.0, Version #1.30B

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6049 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus cereus

(B) STRAIN: AB78

(C) INDIVIDUAL ISOLATE: NRRL B-21058

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1082..2467

(D) OTHER INFORMATION: /product= "VIP2A(a)"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 2475..5126

(D) OTHER INFORMATION: /note= "Coding sequence for the 100 kd VIPlA(a) protein. This coding sequence is repeated in SEQ ID NO:4 and translated separately."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATCGATACAA TGTTGTTTTA CTTAGACCGG TAGTCTCTGT AATTTGTTTA ATGCTATATT 60

CTTTACTTTG ATACATTTTA ATAGCCATTT CAACCTTATC AGTATGTTTT TGTGGTCTTC 120

CTCCTTTTTT TCCACGAGCT CTAGCTGCGT TTAATCCTGT TTTGGTACGT TCGCTAATAA 180

TATCTCTTTC TAATTCTGCA ATACTTGCCA TCATTCGAAA GAAGAATTTC CCCATAGCAT 240

TAGAGGTATC AATGTTGTCA TGAATAGAAA TAAAATCTAC ACCTAGCTCT TTGAATTTTT 300

CACTTAACTC AATTAGGTGT TTTGTAGAGC GAGAAATTCG ATCAAGTTTG TAAACAACTA 360

TCTTATCGCC TTTACGTAAT ACTTTTAGCA ACTCTTCGAG ^* TTGAGGGCGC TCTTTTTTTA 420

TTCCTGTTAT TTTCTCCTGA TATAGCCTTT CTACACCATA TTGTTGCAAA GCATCTATTT 480

GCATATCGAG ATTTTGTTCT TCTGTGCTGA CACGAGCATA ACCAAAAATC AAATTGGTTT 540

CACTTCCTAT CTAAATATAT CTATTAAAAT AGCACCAAAA ACCTTATTAA ATTAAAATAA 600

GGAACTTTGT TTTTGGATAT GGATTTTGGT ACTCAATATG GATGAGTTTT TAACGCTTTT 660

GTTAAAAAAC AAACAAGTGC CATAAACGGT CGTTTTTGGG ATGACATAAT AAATAATCTG 720

TTTGATTAAC CTAACCTTGT ATCCTTACAG CCCAGTTTTA TTTGTACTTC AACTGACTGA 780

ATATGAAAAC AACATGAAGG TTTCATAAAA TTTATATATT TTCCATAACG GATGCTCTAT 840

CTTTAGGTTA TAGTTAAATT ATAAGAAAAA AACAAACGGA GGGAGTGAAA AAAAGCATCT 900

TCTCTATAAT TTTACAGGCT CTTTAATAAG AAGGGGGGAG ATTAGATAAT AAATATGAAT 960

ATCTATCTAT AATTGTTTGC TTCTACAATA ACTTATCTAA CTTTCATATA CAACAACAAA 1020

ACAGACTAAA TCCAGATTGT ATATTCATTT TCAGTTGTTC CTTTATAAAA TAATTTCATA 1080

A ATG AAA AGA ATG GAG GGA AAG TTG TTT ATG GTG TCA AAA AAA TTA 1126 Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu 1 5 10 15

CAA GTA GTT ACT AAA ACT GTA TTG CTT AGT ACA GTT TTC TCT ATA TCT 1174 Gin Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser He Ser 20 25 30

TTA TTA AAT AAT GAA GTG ATA AAA GCT GAA CAA TTA AAT ATA AAT TCT 1222 Leu Leu Asn Asn Glu Val He Lys Ala Glu Gin Leu Asn He Asn Ser 35 40 45

CAA AGT AAA TAT ACT AAC TTG CAA AAT CTA AAA ATC ACT GAC AAG GTA 1270 Gin Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys Val 50 55 60

GAG GAT TTT AAA GAA GAT AAG GAA AAA GCG AAA GAA TGG GGG AAA GAA 1318

Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu 65 70 75

AAA GAA AAA GAG TGG AAA CTA ACT GCT ACT GAA AAA GGA AAA ATG AAT 1366 Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn 80 85 90 95

AAT TTT TTA GAT AAT AAA AAT GAT ATA AAG ACA AAT TAT AAA GAA ATT 1414 Asn Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He 100 105 110

ACT TTT TCT ATG GCA GGC TCA TTT GAA GAT GAA ATA AAA GAT TTA AAA 1462 Thr Phe Ser Met Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys 115 120 125

GAA ATT GAT AAG ATG TTT GAT AAA ACC AAT CTA TCA AAT TCT ATT ATC 1510 Glu He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He 130 135 140

ACC TAT AAA AAT GTG GAA CCG ACA ACA ATT GGA TTT AAT AAA TCT TTA 1558 Thr Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu 145 150 155

ACA GAA GGT AAT ACG ATT AAT TCT GAT GCA ATG GCA CAG TTT AAA GAA 1606 Thr Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu 160 165 170 175

CAA TTT TTA GAT AGG GAT ATT AAG TTT GAT AGT TAT CTA GAT ACG CAT 1654 Gin Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His 180 185 190

TTA ACT GCT CAA CAA GTT TCC AGT AAA GAA AGA GTT ATT TTG AAG GTT 1702 Leu Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val 195 200 205

ACG GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA ACA AAA GCA GGT GTC 1750 Thr Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val 210 215 220

ATT TTA AAT AAT AGT GAA TAC AAA ATG CTC ATT GAT AAT GGG TAT ATG 1798 He Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met 225 230 235

GTC CAT GTA GAT AAG GTA TCA AAA GTG GTG AAA AAA GGG GTG GAG TGC 1846 Val His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys 240 245 250 255

TTA CAA ATT GAA GGG ACT TTA AAA AAG AGT CTT GAC TTT AAA AAT GAT 1894 Leu Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp 260 265 270

ATA AAT GCT GAA GCG CAT AGC TGG GGT ATG AAG AAT TAT GAA GAG TGG 1942 He Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp 275 280 285

GCT AAA GAT TTA ACC GAT TCG CAA AGG GAA GCT TTA GAT GGG TAT GCT 1990 Ala Lys Asp Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala 290 295 300

AGG CAA GAT TAT AAA GAA ATC AAT AAT TAT TTA AGA AAT CAA GGC GGA 2038 Arg Gin Asp Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly Gly 305 310 315

AGT GGA AAT GAA AAA CTA GAT GCT CAA ATA AAA AAT ATT TCT GAT GCT 2086 Ser Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala 320 325 330 335

TTA GGG AAG AAA CCA ATA CCG GAA AAT ATT ACT GTG TAT AGA TGG TGT 2134 Leu Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys 340 345 350

GGC ATG CCG GAA TTT GGT TAT CAA ATT AGT GAT CCG TTA CCT TCT TTA 2182 Gly Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu 355 360 365

AAA GAT TTT GAA GAA CAA TTT TTA AAT ACA ATC AAA GAA GAC AAA GGA 2230 Lys Asp Phe Glu Glu Gin Phe Leu Asn Thr He Lys Glu Asp Lys Gly 370 375 380

TAT ATG AGT ACA AGC TTA TCG AGT GAA CGT CTT GCA GCT TTT GGA TCT 2278 Tyr Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser 385 390 395

AGA AAA ATT ATA TTA CGA TTA CAA GTT CCG AAA GGA AGT ACG GGT GCG 2326 Arg Lys He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala 400 405 410 415

TAT TTA AGT GCC ATT GGT GGA TTT GCA AGT GAA AAA GAG ATC CTA CTT 2374 Tyr Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu 420 425 430

GAT AAA GAT AGT AAA TAT CAT ATT GAT AAA GTA ACA GAG GTA ATT ATT 2422 Asp Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He 435 440 445

AAA GGT GTT AAG CGA TAT GTA GTG GAT GCA ACA TTA TTA ACA AAT 2467 Lys Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 450 455 460

TAAGGAGATG AAAAATATGA AGAAAAAGTT AGCAAGTGTT GTAACGTGTA CGTTATTAGC 2527

TCCTATGTTT TTGAATGGAA ATGTGAATGC TGTTTACGCA GACAGCAAAA CAAATCAAAT 2587

TTCTACAACA CAGAAAAATC AACAGAAAGA GATGGACCGA AAAGGATTAC TTGGGTATTA 2647

TTTCAAAGGA AAAGATTTTA GTAATCTTAC TATGTTTGCA CCGACACGTG ATAGTACTCT 2707

TATTTATGAT CAACAAACAG CAAATAAACT ATTAGATAAA AAACAACAAG AATATCAGTC 2767

TATTCGTTGG ATTGGTTTGA TTCAGAGTAA AGAAACGGGA GATTTCACAT TTAACTTATC 2827

TGAGGATGAA CAGGCAATTA TAGAAATCAA TGGGAAAATT ATTTCTAATA AAGGGAAAGA 2887

AAAGCAAGTT GTCCATTTAG AAAAAGGAAA ATTAGTTCCA ATCAAAATAG AGTATCAATC 2947

AGATACAAAA TTTAATATTG ACAGTAAAAC ATTTAAAGAA CTTAAATTAT TTAAAATAGA 3007

TAGTCAAAAC CAACCCCAGC AAGTCCAGCA AGATGAACTG AGAAATCCTG AATTTAACAA 3067

GAAAGAATCA CAGGAATTCT TAGCGAAACC ATCGAAAATA AATCTTTTCA CTCAAAAAAT 3127

GAAAAGGGAA ATTGATGAAG ACACGGATAC GGATGGGGAC TCTATTCCTG ACCTTTGGGA 3187

AGAAAATGGG TATACGATTC ACAATAGAAT CGCTGTAAAG TGGGACGATT CTCTAGCAAG 3247

TAAAGGGTAT ACGAAATTTG TTTCAAATCC ACTAGAAAGT CACACAGTTG GTGATCCTTA 3307

TACAGATTAT GAAAAGGCAG CAAGAGATCT AGATTTGTCA AATGCAAAGG AAACGTTTAA 3367

CCCATTGGTA GCTGCTTTTC CAAGTGTGAA TGTTAGTATG GAAAAGGTGA TATTATCACC 3427

AAATGAAAAT TTATCCAATA GTGTAGAGTC TCATTCATCC ACGAATTGGT CTTATACAAA 3487

TACAGAAGGT GCTTCTGTTG AAGCGGGGAT TGGACCAAAA GGTATTTCGT TCGGAGTTAG 3547

CGTAAACTAT CAACACTCTG AAACAGTTGC ACAAGAATGG GGAACATCTA CAGGAAATAC 3607

TTCGCAATTC AATACGGCTT CAGCGGGATA TTTAAATGCA AATGTTCGAT ATAACAATGT 3667

AGGAACTGGT GCCATCTACG ATGTAAAACC TACAACAAGT TTTGTATTAA ATAACGATAC 3727

TATCGCAACT ATTACGGCGA AATCTAATTC TACAGCCTTA AATATATCTC CTGGAGAAAG 3787

TTACCCGAAA AAAGGACAAA ATGGAATCGC AATAACATCA ATGGATGATT TTAATTCCCA 3847

TCCGATTACA TTAAATAAAA AACAAGTAGA TAATCTGCTA AATAATAAAC CTATGATGTT 3907

GGAAACAAAC CAAACAGATG GTGTTTATAA GATAAAAGAT ACACATGGAA ATATAGTAAC 3967

TGGCGGAGAA TGGAATGGTG TCATACAACA AATCAAGGCT AAAACAGCGT CTATTATTGT 4027

GGATGATGGG GAACGTGTAG CAGAAAAACG TGTAGCGGCA AAAGATTATG AAAATCCAGA 4087

AGATAAAACA CCGTCTTTAA CTTTAAAAGA TGCCCTGAAG CTTTCATATC CAGATGAAAT 41 7

AAAAGAAATA GAGGGATTAT TATATTATAA AAACAAACCG ATATACGAAT CGAGCGTTAT 4207

GACTTACTTA GATGAAAATA CAGCAAAAGA AGTGACCAAA CAATTAAATG ATACCACTGG 4267

GAAATTTAAA GATGTAAGTC ATTTATATGA TGTAAAACTG ACTCCAAAAA TGAATGTTAC 4327

AATCAAATTG TCTATACTTT ATGATAATGC TGAGTCTAAT GATAACTCAA TTGGTAAATG 4387

GACAAACACA AATATTGTTT CAGGTGGAAA TAACGGAAAA AAACAATATT CTTCTAATAA 4447

TCCGGATGCT AATTTGACAT TAAATACAGA TGCTCAAGAA AAATTAAATA AAAATCGTGA 4507

CTATTATATA AGTTTATATA TGAAGTCAGA AAAAAACACA CAATGTGAGA TTACTATAGA 4567

TGGGGAGATT TATCCGATCA CTACAAAAAC AGTGAATGTG AATAAAGACA ATTACAAAAG 4627

ATTAGATATT ATAGCTCATA ATATAAAAAG TAATCCAATT TCTTCACTTC ATATTAAAAC 4687

GAATGATGAA ATAACTTTAT TTTGGGATGA TATTTCTATA ACAGATGTAG CATCAATAAA 4747

ACCGGAAAAT TTAACAGATT CAGAAATTAA ACAGATTTAT AGTAGGTATG GTATTAAGTT 4807

AGAAGATGGA ATCCTTATTG ATAAAAAAGG TGGGATTCAT TATGGTGAAT TTATTAATGA 4867

AGCTAGTTTT AATATTGAAC CATTGCAAAA TTATGTGACC'AAATATGAAG TTACTTATAG 4927

TAGTGAGTTA GGACCAAACG TGAGTGACAC ACTTGAAAGT GATAAAATTT ACAAGGATGG 4987

GACAATTAAA TTTGATTTTA CCAAATATAG TAAAAATGAA CAAGGATTAT TTTATGACAG 5047

TGGATTAAAT TGGGACTTTA AAATTAATGC TATTACTTAT GATGGTAAAG AGATGAATGT 5107

TTTTCATAGA TATAATAAAT AGTTATTATA TCTATGAAGC TGGTGCTAAA GATAGTGTAA 5167

AAGTTAATAT ACTGTAGGAT TGTAATAAAA GTAATGGAAT TGATATCGTA CTTTGGAGTG 5227

GGGGATACTT TGTAAATAGT TCTATCAGAA ACATTAGACT AAGAAAAGTT ACTACCCCCA 5287

CTTGAAAATG AAGATTCAAC TGATTACAAA CAACCTGTTA AATATTATAA GGTTTTAACA 5347

AAATATTAAA CTCTTTATGT TAATACTGTA ATATAAAGAG TTTAATTGTA TTCAAATGAA 5407

GCTTTCCCAC AAAATTAGAC TGATTATCTA ATGAAATAAT CAGTCTAATT TTGTAGAACA 5467

GGTCTGGTAT TATTGTACGT GGTCACTAAA AGATATCTAA TATTATTGGG CAAGGCGTTC 5527

CATGATTGAA TCCTCGAATG TCTTGCCCTT TTCATTTATT TAAGAAGGAT TGTGGAGAAA 5587

TTATGGTTTA GATAATGAAG AAAGACTTCA CTTCTAATTT TTGATGTTAA ATAAATCAAA 5647

ATTTGGCGAT TCACATTGTT TAATCCACTG ATAAAACATA CTGGAGTGTT CTTAAAAAAT 5707

«GCTTTTTT CTTTATAAAA TTTTGCTTAG CGTACGAAAT TCGTGTTTTG TTGGTGGGAC 5767

CCCATGCCCA TCAACTTAAG AGTAAATTAG TAATGAACTT TCGTTCATCT GGATTAAAAT 5827

AACCTCAAAT TAGGACATGT TTTTAAAAAT AAGCAGACCA AATAAGCCTA GAATAGGTAT 5887

CATTTTTAAA AATTATGCTG CTTTCTTTTG TTTTCCAAAT CCATTATACT CATAAGCAAC 5947

ACCCATAATG TCAAAGACTG TTTTTGTCTC ATATCGATAA GCTTGATATC GAATTCCTGC 6007

AGCCCGGGGG ATCCACTAGT TCTAGAGCGG CCGCCACCGC GG 6049

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (D) 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 15

Val Val Thr Lys Thr Val Leu Leu Ser Thr Val -Phe Ser He Ser Leu 20 25 30

Leu Asn Asn Glu Val He Lys Ala Glu Gin Leu Asn He Asn Ser Gin 35 40 45

Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu 50 55 60

Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys 65 70 75 80

Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn 85 90 95

Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr 100 105 110

Phe Ser Met Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu 115 120 125

He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr 130 135 140

Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr 145 150 155 160

Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 165 170 175

Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 180 185 190

Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr 195 200 205

Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He 210 215 220

Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val 225 230 235 240

His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu 245 250 255

Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He 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 He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 305 310 315- 320

Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu 325 330 335

Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly 340 345 350

Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys 355 360 365

Asp Phe Glu Glu Gin Phe Leu Asn Thr He 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 He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 405 410 415

Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp 420 425 430

Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He Lys 435 440 445

Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 450 455 460

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..20

(D) OTHER INFORMATION: /note= "Signal peptide for vacuolar arge ting"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro He Arg Val Thr Asp Arg 1 5 10 15

Ala Ala Ser Thr 20

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2655 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus cereus

(B) STRAIN: AB78

(C) INDIVIDUAL ISOLATE: NRRL B-21058

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..2652

(D) OTHER INFORMATION: /product= "100 kDa protein VIPlA(a)" /note= "This sequence is identical to the portion of SEQ ID NO:l between and including nucleotide 2475 to 5126."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

ATG AAA AAT ATG AAG AAA AAG TTA GCA AGT GTT GTA ACG TGT ACG TTA 48

Met Lys Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu 465 470 475

TTA GCT CCT ATG TTT TTG AAT GGA AAT GTG AAT GCT GTT TAC GCA GAC 96

Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp

480 485 490

AGC AAA ACA AAT CAA ATT TCT ACA ACA CAG AAA AAT CAA CAG AAA GAG 144

Ser Lys Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin Lys Glu 495 500 505 510

ATG GAC CGA AAA GGA TTA CTT GGG TAT TAT TTC AAA GGA AAA GAT TTT 192 Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe 515 520 525

AGT AAT CTT ACT ATG TTT GCA CCG ACA CGT GAT AGT ACT CTT ATT TAT 240 Ser Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr 530 535 540

GAT CAA CAA ACA GCA AAT AAA CTA TTA GAT AAA AAA CAA CAA GAA TAT 288 Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin Glu Tyr 545 550 555

CAG TCT ATT CGT TGG ATT GGT TTG ATT CAG AGT AAA GAA ACG GGA GAT 336 Gin Ser He Arg Trp He Gly Leu He Gin Ser Lys Glu Thr Gly Asp 560 565 ^• 570

TTC ACA TTT AAC TTA TCT GAG GAT GAA CAG GCA ATT ATA GAA ATC AAT 384 Phe Thr Phe Asn Leu Ser Glu Asp Glu Gin Ala He He Glu He Asn 575 580 585 590

GGG AAA ATT ATT TCT AAT AAA GGG AAA GAA AAG CAA GTT GTC CAT TTA 432 Gly Lys He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu 595 600 605

GAA AAA GGA AAA TTA GTT CCA ATC AAA ATA GAG TAT CAA TCA GAT ACA 480 Glu Lys Gly Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr 610 615 620

AAA TTT AAT ATT GAC AGT AAA ACA TTT AAA GAA CTT AAA TTA TTT AAA 528 Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys 625 630 635

ATA GAT AGT CAA AAC CAA CCC CAG CAA GTC CAG CAA GAT GAA CTG AGA 576 He Asp Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu Leu Arg 640 645 650

AAT CCT GAA TTT AAC AAG AAA GAA TCA CAG GAA TTC TTA GCG AAA CCA 624 Asn Pro Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Pro 655 660 665 670

TCG AAA ATA AAT CTT TTC ACT CAA AAA ATG AAA AGG GAA ATT GAT GAA 672 Ser Lys He Asn Leu Phe Thr Gin Lys Met Lys Arg Glu He Asp Glu 675 680 685

GAC ACG GAT ACG GAT GGG GAC TCT ATT CCT GAC CTT TGG GAA GAA AAT 720 Asp Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn 690 695 700

GGG TAT ACG ATT CAA AAT AGA ATC GCT GTA AAG TGG GAC GAT TCT CTA 768 Gly Tyr Thr He Gin Asn Arg He Ala Val Lys Trp Asp Asp Ser Leu 705 710 715

GCA AGT AAA GGG TAT ACG AAA TTT GTT TCA AAT CCA CTA GAA AGT CAC 816 Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His 720 725 730

ACA GTT GGT GAT CCT TAT ACA GAT TAT GAA AAG GCA GCA AGA GAT CTA 864 Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu 735 740 745 750

GAT TTG TCA AAT GCA AAG GAA ACG TTT AAC CCA TTG GTA GCT GCT TTT 912 Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe 755 760 765

CCA AGT GTG AAT GTT AGT ATG GAA AAG GTG ATA TTA TCA CCA AAT GAA 960 Pro Ser Val Asn Val Ser Met Glu Lys Val He Leu Ser Pro Asn Glu 770 775 780

AAT TTA TCC AAT AGT GTA GAG TCT CAT TCA TCC ACG AAT TGG TCT TAT 1008 Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr 785 790 795

ACA AAT ACA GAA GGT GCT TCT GTT GAA GCG GGG ATT GGA CCA AAA GGT 1056 Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gly He Gly Pro Lys Gly 800 805 810

ATT TCG TTC GGA GTT AGC GTA AAC TAT CAA CAC TCT GAA ACA GTT GCA 1104 He Ser Phe Gly Val Ser Val Asn Tyr Gin His Ser Glu Thr Val Ala 815 820 825 830

CAA GAA TGG GGA ACA TCT ACA GGA AAT ACT TCG CAA TTC AAT ACG GCT 1152 Gin Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala 835 840 845

TCA GCG GGA TAT TTA AAT GCA AAT GTT CGA TAT AAC AAT GTA GGA ACT 1200 Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr 850 855 860

GGT GCC ATC TAC GAT GTA AAA CCT ACA ACA AGT TTT GTA TTA AAT AAC 1248 Gly Ala He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn 865 870 875

GAT ACT ATC GCA ACT ATT ACG GCG AAA TCT AAT TCT ACA GCC TTA AAT 1296 Asp Thr He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn 880 885 890

ATA TCT CCT GGA GAA AGT TAC CCG AAA AAA GGA CAA AAT GGA ATC GCA 1344 He Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly He Ala 895 900 905 910

ATA ACA TCA ATG GAT GAT TTT AAT TCC CAT CCG ATT ACA TTA AAT AAA 1392 He Thr Ser Met Asp Asp Phe Asn Ser His Pro He Thr Leu Asn Lys 915 920 925

AAA CAA GTA GAT AAT CTG CTA AAT AAT AAA CCT ATG ATG TTG GAA ACA 1440 Lys Gin Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr 930 935 940

AAC CAA ACA GAT GGT GTT TAT AAG ATA AAA GAT ACA CAT GGA AAT ATA 1488 Asn Gin Thr Asp Gly Val Tyr Lys He Lys Asp Thr His Gly Asn He

945 950 955

GTA ACT GGC GGA GAA TGG AAT GGT GTC ATA CAA CAA ATC AAG GCT AAA 1536 Val Thr Gly Gly Glu Trp Asn Gly Val He Gin Gin He Lys Ala Lys 960 965 970

ACA GCG TCT ATT ATT GTG GAT GAT GGG GAA CGT GTA GCA GAA AAA CGT 1584 Thr Ala Ser He He Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg 975 980 985 990

GTA GCG GCA AAA GAT TAT GAA AAT CCA GAA GAT AAA ACA CCG TCT TTA 1632 Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu 995 1000 1005

ACT TTA AAA GAT GCC CTG AAG CTT TCA TAT CCA GAT GAA ATA AAA GAA 1680 Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Glu 1010 1015 1020

ATA GAG GGA TTA TTA TAT TAT AAA AAC AAA CCG ATA TAC GAA TCG AGC 1728 He Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pro He Tyr Glu Ser Ser 1025 1030 1035

GTT ATG ACT TAC TTA GAT GAA AAT ACA GCA AAA GAA GTG ACC AAA CAA 1776 Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin 1040 1045 1050

TTA AAT GAT ACC ACT GGG AAA TTT AAA GAT GTA AGT CAT TTA TAT GAT 1824 Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp 1055 1060 1065 1070

GTA AAA CTG ACT CCA AAA ATG AAT GTT ACA ATC AAA TTG TCT ATA CTT 1872 Val Lys Leu Thr Pro Lys Met Asn Val Thr He Lys Leu Ser He Leu 1075 1080 1085

TAT GAT AAT GCT GAG TCT AAT GAT AAC TCA ATT GGT AAA TGG ACA AAC 1920 Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser He Gly Lys Trp Thr Asn 1090 1095 1100

ACA AAT ATT GTT TCA GGT GGA AAT AAC GGA AAA AAA CAA TAT TCT TCT 1968 Thr Asn He Val Ser Gly Gly Asn Asn Gly Lys Lys Gin Tyr Ser Ser 1105 1110 1115

AAT AAT CCG GAT GCT AAT TTG ACA TTA AAT ACA GAT GCT CAA GAA AAA 2016 Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin Glu Lys 1120 1125 1130

TTA AAT AAA AAT CGT GAC TAT TAT ATA AGT TTA TAT ATG AAG TCA GAA 2064 Leu Asn Lys Asn Arg Asp Tyr Tyr He Ser Leu Tyr Met Lys Ser Glu 1135 1140 1145 1150

AAA AAC ACA CAA TGT GAG ATT ACT ATA GAT GGG GAG ATT TAT CCG ATC 2112 Lys Asn Thr Gin Cys Glu He Thr He Asp Gly Glu He Tyr Pro He 1155 1160 1165

ACT ACA AAA ACA GTG 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

ATT ATA GCT CAT AAT ATA AAA AGT AAT CCA ATT TCT TCA CTT CAT ATT 2208 He He Ala His Asn He Lys Ser Asn Pro He Ser Ser Leu His He 1185 1190 1195

AAA ACG AAT GAT GAA ATA ACT TTA TTT TGG GAT GAT ATT TCT ATA ACA 2256 Lys Thr Asn Asp Glu He Thr Leu Phe Trp Asp Asp He Ser He Thr 1200 1205 1210

GAT GTA GCA TCA ATA AAA CCG GAA AAT TTA ACA GAT TCA GAA ATT AAA 2304 Asp Val Ala Ser He Lys Pro Glu Asn Leu Thr Asp Ser Glu He Lys 1215 1220 1225 1230

CAG ATT TAT AGT AGG TAT GGT ATT AAG TTA GAA GAT GGA ATC CTT ATT 2352 Gin He Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly He Leu He 1235 1240 1245

GAT AAA AAA GGT GGG ATT CAT TAT GGT GAA TTT ATT AAT GAA GCT AGT 2400 Asp Lys Lys Gly Gly He His Tyr Gly Glu Phe He Asn Glu Ala Ser 1250 1255 1260

TTT AAT ATT GAA CCA TTG CAA AAT TAT GTG ACC AAA TAT GAA GTT ACT 2448 Phe Asn He Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr 1265 1270 1275

TAT AGT AGT GAG TTA 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 ATT AAA TTT GAT TTT ACC AAA TAT AGT 2544 Lys He Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys Tyr Ser 1295 1300 1305 1310

AAA AAT GAA CAA GGA TTA TTT TAT GAC AGT GGA TTA AAT TGG GAC TTT 2592 Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe 1315 1320 1325

AAA ATT AAT GCT ATT ACT TAT GAT GGT AAA GAG ATG AAT GTT TTT CAT 2640 Lys He Asn Ala He 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

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 884 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Lys Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu 1 5 10 15

Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp 20 25 30

Ser Lys Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin Lys Glu 35 40 45

Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe 50 55 60

Ser Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr 65 70 75 80

Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin Glu Tyr 85 90 95

Gin Ser He Arg Trp He Gly Leu He Gin Ser Lys Glu Thr Gly Asp 100 105 110

Phe Thr Phe Asn Leu Ser Glu Asp Glu Gin Ala He He Glu He Asn 115 120 125

Gly Lys He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu 130 135 140

Glu Lys Gly Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr 145 150 155 160

Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys 165 170 175

He Asp Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu Leu Arg 180 185 190

Asn Pro Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Pro 195 200 205

Ser Lys He Asn Leu Phe Thr Gin Lys Met Lys Arg Glu He Asp Glu 210 215 220

Asp Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn 225 230 235 240

Gly Tyr Thr He Gin Asn Arg He Ala Val Lys Trp Asp Asp Ser Leu 245 250 255

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 He 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 He Gly Pro Lys Gly 340 345 350

He 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 He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn 405 410 415

Asp Thr He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn 420 425 430

He Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly He Ala 435 440 445

He Thr Ser Met Asp Asp Phe Asn Ser His Pro He 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 He Lys Asp Thr His Gly Asn He 485 490 495

Val Thr Gly Gly Glu Trp Asn Gly Val He Gin Gin He Lys Ala Lys 500 505 510

Thr Ala Ser He He 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

Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Glu 545 550 555 560

He Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pro He Tyr Glu Ser Ser

565 570 575

Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin 580 585 590

Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp 595 600 605

Val Lys Leu Thr Pro Lys Met Asn Val Thr He Lys Leu Ser He Leu 610 615 620

Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser He Gly Lys Trp Thr Asn 625 630 635 640

Thr Asn He Val Ser Gly Gly Asn Asn Gly Lys Lys Gin Tyr Ser Ser 645 650 655

Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin Glu Lys 660 665 670

Leu Asn Lys Asn Arg Asp Tyr Tyr He Ser Leu Tyr Met Lys Ser Glu 675 680 685

Lys Asn Thr Gin Cys Glu He Thr He Asp Gly Glu He Tyr Pro He 690 695 700

Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp 705 710 715 720

He He Ala His Asn He Lys Ser Asn Pro He Ser Ser Leu His He 725 730 735

Lys Thr Asn Asp Glu He Thr Leu Phe Trp Asp Asp He Ser He Thr 740 745 750

Asp Val Ala Ser He Lys Pro.Glu Asn Leu Thr Asp Ser Glu He Lys 755 760 765

Gin He Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly He Leu He 770 775 780

Asp Lys Lys Gly Gly He His Tyr Gly Glu Phe He Asn Glu Ala Ser 785 790 795 800

Phe Asn He Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr 805 810 815

Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu Ser Asp 820 825 830

Lys He Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys Tyr Ser 835 840 845

Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe 850 855 860

Lys He Asn Ala He Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His 865 870 875 880

Arg Tyr Asn Lys

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2004 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus cereus

(B) STRAIN: AB78

(C) INDIVIDUAL ISOLATE: NRRL B-21058

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..2001

(D) OTHER INFORMATION: /product= "80 kDa protein VIPlA(a)" /note= "This sequence is identical to that found in SEQ ID NO:l between and including nucleotide positions 3126 and 5126"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

ATG AAA AGG GAA ATT GAT GAA GAC ACG GAT ACG GAT GGG GAC TCT ATT 48 Met Lys Arg Glu He Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser He 885 890 895 900

CCT GAC CTT TGG GAA GAA AAT GGG TAT ACG ATT CAA AAT AGA ATC GCT 96 Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr He Gin Asn Arg He Ala 905 910 915

GTA AAG TGG GAC GAT TCT CTA GCA AGT AAA GGG TAT ACG AAA TTT GTT 144 Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Val 920 925 930

TCA AAT CCA CTA GAA AGT CAC ACA GTT GGT GAT CCT TAT ACA GAT TAT 192 Ser Asn Pro Leu Glu Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr 935 940 945

GAA AAG GCA GCA AGA GAT CTA GAT TTG TCA AAT GCA AAG GAA ACG TTT 240 Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe 950 955 960

AAC CCA TTG GTA GCT GCT TTT CCA AGT GTG AAT GTT AGT ATG GAA AAG 288 Asn Pro Leu Val Ala Ala Phe Pro Ser Val Asn Val Ser Met Glu Lys 965 970 975 980

GTG ATA TTA TCA CCA AAT GAA AAT TTA TCC AAT AGT GTA GAG TCT CAT 336 Val He Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu Ser His 985 990 995

TCA TCC ACG AAT TGG TCT TAT ACA AAT ACA GAA GGT GCT TCT GTT GAA 384 Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val Glu 1000 1005 1010

GCG GGG ATT GGA CCA AAA GGT ATT TCG TTC GGA GTT AGC GTA AAC TAT 432 Ala Gly He Gly Pro Lys Gly He Ser Phe Gly Val Ser Val Asn Tyr 1015 1020 1025

CAA CAC TCT GAA ACA GTT GCA CAA GAA TGG GGA ACA TCT ACA GGA AAT 480 Gin His Ser Glu Thr Val Ala Gin Glu Trp Gly Thr Ser Thr Gly Asn 1030 1035 1040

ACT TCG CAA TTC AAT ACG GCT TCA GCG GGA TAT TTA AAT GCA AAT GTT 528 Thr Ser Gin Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val 1045 1050 1055 1060

CGA TAT AAC AAT GTA GGA ACT GGT GCC ATC TAC GAT GTA AAA CCT ACA 576 Arg Tyr Asn Asn Val Gly Thr Gly Ala He Tyr Asp Val Lys Pro Thr 1065 1070 1075

ACA AGT TTT GTA TTA AAT AAC GAT ACT ATC GCA ACT ATT ACG GCG AAA 624 Thr Ser Phe Val Leu Asn Asn Asp Thr He Ala Thr He Thr Ala Lys 1080 1085 1090

TCT AAT TCT ACA GCC TTA AAT ATA TCT CCT GGA GAA AGT TAC CCG AAA 672 Ser Asn Ser Thr Ala Leu Asn He Ser Pro Gly Glu Ser Tyr Pro Lys 1095 1100 1105

AAA GGA CAA AAT GGA ATC GCA ATA ACA TCA ATG GAT GAT TTT AAT TCC 720 Lys Gly Gin Asn Gly He Ala He Thr Ser Met Asp Asp Phe Asn Ser 1110 1115 1120

CAT CCG ATT ACA TTA AAT AAA AAA CAA GTA GAT AAT CTG CTA AAT AAT 768 His Pro He Thr Leu Asn Lys Lys Gin Val Asp Asn Leu Leu Asn Asn 1125 1130 1135 1140

AAA CCT ATG ATG TTG GAA ACA AAC CAA ACA GAT GGT GTT TAT AAG ATA 816 Lys Pro Met Met Leu Glu Thr Asn Gin Thr Asp Gly Val Tyr Lys He 1145 1150 1155

AAA GAT ACA CAT GGA AAT ATA GTA ACT GGC GGA GAA TGG AAT GGT GTC 864 Lys Asp Thr His Gly Asn He Val Thr Gly Gly Glu Trp Asn Gly Val 1160 1165 1170

ATA CAA CAA ATC AAG GCT AAA ACA GCG TCT ATT ATT GTG GAT GAT GGG 912 He Gin Gin He Lys Ala Lys Thr Ala Ser He He Val Asp Asp Gly

1175 1180 1185

GAA CGT GTA GCA GAA AAA CGT GTA GCG GCA AAA GAT TAT GAA AAT CCA 960 Glu Arg Val Ala Glu Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn Pro 1190 1195 1200

GAA GAT AAA ACA CCG TCT TTA ACT TTA AAA GAT GCC CTG AAG CTT TCA 1008 Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Ser 1205 1210 1215 1220

TAT CCA GAT GAA ATA AAA GAA ATA GAG GGA TTA TTA TAT TAT AAA AAC 1056 Tyr Pro Asp Glu He Lys Glu He Glu Gly Leu Leu Tyr Tyr Lys Asn 1225 1230 1235

AAA CCG ATA TAC GAA TCG AGC GTT ATG ACT TAC TTA GAT GAA AAT ACA 1104 Lys Pro He Tyr Glu Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr 1240 1245 1250

GCA AAA GAA GTG ACC AAA CAA TTA AAT GAT ACC ACT GGG AAA TTT AAA 1152 Ala Lys Glu Val Thr Lys Gin Leu Asn Asp Thr Thr Gly Lys Phe Lys 1255 1260 1265

GAT GTA AGT CAT TTA TAT GAT GTA AAA CTG ACT CCA AAA ATG AAT GTT 1200 Asp Val Ser His Leu Tyr Asp Val Lys Leu Thr Pro Lys Met Asn Val 1270 1275 1280

ACA ATC AAA TTG TCT ATA CTT TAT GAT AAT GCT GAG TCT AAT GAT AAC 1248 Thr He Lys Leu Ser He Leu Tyr Asp Asn Ala Glu Ser Asn Asp Asn 1285 1290 1295 1300

TCA ATT GGT AAA TGG ACA AAC ACA AAT ATT GTT TCA GGT GGA AAT AAC 1296 Ser He Gly Lys Trp Thr Asn Thr Asn He Val Ser Gly Gly Asn Asn 1305 1310 1315

GGA AAA AAA CAA TAT TCT TCT AAT AAT CCG GAT GCT AAT TTG ACA TTA 1344 Gly Lys Lys Gin Tyr Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr Leu 1320 1325 1330

AAT ACA GAT GCT CAA GAA AAA TTA AAT AAA AAT CGT GAC TAT TAT ATA 1392 Asn Thr Asp Ala Gin Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr He 1335 1340 1345

AGT TTA TAT ATG AAG TCA GAA AAA AAC ACA CAA TGT GAG ATT ACT ATA 1440 Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr Gin Cys Glu He Thr He 1350 1355 1360

GAT GGG GAG ATT TAT CCG ATC ACT ACA AAA ACA GTG AAT GTG AAT AAA 1488 Asp Gly Glu He Tyr Pro He Thr Thr Lys Thr Val Asn Val Asn Lys 1365 1370 1375 1380

GAC AAT TAC AAA AGA TTA GAT ATT ATA GCT CAT AAT ATA AAA AGT AAT 1536 Asp Asn Tyr Lys Arg Leu Asp He He Ala His Asn He Lys Ser Asn 1385 1390 1395

CCA ATT TCT TCA CTT CAT ATT AAA ACG AAT GAT GAA ATA ACT TTA TTT 1584

Pro He Ser Ser Leu His He Lys Thr Asn Asp Glu He Thr Leu Phe 1400 1405 1410

TGG GAT GAT ATT TCT ATA ACA GAT GTA GCA TCA ATA AAA CCG GAA AAT 1632 Trp Asp Asp He Ser He Thr Asp Val Ala Ser He Lys Pro Glu Asn 1415 1420 1425

TTA ACA GAT TCA GAA ATT AAA CAG ATT TAT AGT AGG TAT GGT ATT AAG 1680 Leu Thr Asp Ser Glu He Lys Gin He Tyr Ser Arg Tyr Gly He Lys 1430 1435 1440

TTA GAA GAT GGA ATC CTT ATT GAT AAA AAA GGT GGG ATT CAT TAT GGT 1728 Leu Glu Asp Gly He Leu He Asp Lys Lys Gly Gly He His Tyr Gly 1445 1450 1455 1460

GAA TTT ATT AAT GAA GCT AGT TTT AAT ATT GAA CCA TTG CCA AAT TAT 1776 Glu Phe He Asn Glu Ala Ser Phe Asn He Glu Pro Leu Pro Asn Tyr 1465 1470 1475

GTG ACC AAA TAT GAA GTT ACT TAT AGT AGT GAG TTA GGA CCA AAC GTG 1824 Val Thr Lys Tyr Glu Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn Val 1480 1485 1490

AGT GAC ACA CTT GAA AGT GAT AAA ATT TAC AAG GAT GGG ACA ATT AAA 1872 Ser Asp Thr Leu Glu Ser Asp Lys He Tyr Lys Asp Gly Thr He Lys 1495 1500 1505

TTT GAT TTT ACC AAA TAT AGT AAA AAT GAA CAA GGA TTA TTT TAT GAC 1920 Phe Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gin Gly Leu Phe Tyr Asp 1510 1515 1520

AGT GGA TTA AAT TGG GAC TTT AAA ATT AAT GCT ATT ACT TAT GAT GGT 1968 Ser Gly Leu Asn Trp Asp Phe Lys He Asn Ala He Thr Tyr Asp Gly 1525 1530 1535 1540

AAA GAG ATG AAT GTT TTT CAT AGA TAT AAT AAA TAG 2004

Lys Glu Met Asn Val Phe His Arg Tyr Asn Lys 1545 1550

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 667 amino acids ^■

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Lys Arg Glu He Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser He 1 5 10 15

Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr He Gin Asn Arg He Ala

20 25 30

Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Val 35 40 45

Ser Asn Pro Leu Glu Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr 50 55 60

Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe 65 70 75 80

Asn Pro Leu Val Ala Ala Phe Pro Ser Val Asn Val Ser Met Glu Lys 85 90 95

Val He 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 He Gly Pro Lys Gly He 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 He Tyr Asp Val Lys Pro Thr 180 185 190

Thr Ser Phe Val Leu Asn Asn Asp Thr He Ala Thr He Thr Ala Lys 195 200 205

Ser Asn Ser Thr Ala Leu Asn He Ser Pro Gly Glu Ser Tyr Pro Lys 210 215 220

Lys Gly Gin Asn Gly He Ala He Thr Ser Met Asp Asp Phe Asn Ser 225 230 235 240

His Pro He 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 He 260 265 270

Lys Asp Thr His Gly Asn He Val Thr Gly Gly Glu Trp Asn Gly Val 275 280 285

He Gin Gin He Lys Ala Lys Thr Ala Ser He He Val Asp Asp Gly 290 295 300

Glu Arg Val Ala Glu Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn Pro 305 310 315 320

Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Ser 325 330 335

Tyr Pro Asp Glu He Lys Glu He Glu Gly Leu Leu Tyr Tyr Lys Asn 340 345 350

Lys Pro He Tyr Glu Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr 355 360 365

Ala Lys Glu Val Thr Lys Gin Leu Asn Asp Thr Thr Gly Lys Phe Lys 370 375 380

Asp Val Ser His Leu Tyr Asp Val Lys Leu Thr Pro Lys Met Asn Val 385 390 395 400

Thr He Lys Leu Ser He Leu Tyr Asp Asn Ala Glu Ser Asn Asp Asn 405 410 415

Ser He Gly Lys Trp Thr Asn Thr Asn He Val Ser Gly Gly Asn Asn 420 425 430

Gly Lys Lys Gin Tyr Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr Leu 435 440 445

Asn Thr Asp Ala Gin Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr He 450 455 460

Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr Gin Cys Glu He Thr He 465 470 475 480

Asp Gly Glu He Tyr Pro He Thr Thr Lys Thr Val Asn Val Asn Lys 485 490 495

Asp Asn Tyr Lys Arg Leu Asp He He Ala His Asn He Lys Ser Asn 500 505 510

Pro He Ser Ser Leu His He Lys Thr Asn Asp Glu He Thr Leu Phe 515 520 525

Trp Asp Asp He Ser He Thr Asp Val Ala Ser He Lys Pro Glu Asn 530 535 540

Leu Thr Asp Ser Glu He Lys Gin He Tyr Ser Arg Tyr Gly He Lys 545 550 555 560

Leu Glu Asp Gly He Leu He Asp Lys Lys Gly Gly He His Tyr Gly 565 570 575

Glu Phe He Asn Glu Ala Ser Phe Asn He Glu Pro Leu Pro Asn Tyr 580 585 590

Val Thr Lys Tyr Glu Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn Val 595 600 605

Ser Asp Thr Leu Glu Ser Asp Lys He Tyr Lys Asp Gly Thr He Lys 610 615 620

Phe Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gin Gly Leu Phe Tyr Asp 625 630 635 640

Ser Gly Leu Asn Trp Asp Phe Lys He Asn Ala He Thr Tyr Asp Gly 645 650 655

Lys Glu Met Asn Val Phe His Arg Tyr Asn Lys 660 665

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus cereus

(B) STRAIN: AB78

(C) INDIVIDUAL ISOLATE: NRRL B-21058

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..16

(D) OTHER INFORMATION: /note= "N-terminal sequence of protein purified from strain AB78"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Lys Arg Glu He Asp Glu Asp Thr Asp Thr Asx Gly Asp Ser He Pro 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..21

(D) 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: GAAATTGATC AAGATACNGA T 21

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis

(B) STRAIN: AB88

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..14

(D) OTHER INFORMATION: /note---* "N-terminal amino acid sequence of protein known as anion exchange fraction 23 (smaller) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Xaa Glu Pro Phe Val Ser Ala Xaa Xaa Xaa Gin Xaa Xaa Xaa

1 5 10

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: N-terminal

(vi) ORIGINAL SOURCE:

(A) 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 10

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: N-terminal

(vi) ORIGINAL SOURCE:

(A) 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 10

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis

(B) STRAIN: AB88

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..15

(D) 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 He Val Pro 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Met Asp Asn Asn Pro Asn He Asn Glu 1 5

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..9

(D) OTHER INFORMATION: /note= "N-terminal sequence of 80 kDa delta-endotoxin"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Met Asp Asn Asn Pro Asn He Asn Glu 1 5

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..11

(D) OTHER INFORMATION: /note= "N-terminal sequence from 60 kDa delta-endotoxin"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Met Asn Val Leu Asn Ser Gly Arg Thr Thr He 1 5 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2655 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..2652

(D) OTHER INFORMATION: /note= "Maize optimized DNA sequence for 100 kd VIPlA(a) protein from AB78"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

ATGAAGAACA TGAAGAAGAA GCTGGCCAGC GTGGTGACCT GCACCCTGCT GGCCCCCATG 60

TTCCTGAACG GCAACGTGAA CGCCGTGTAC GCCGACAGCA AGACCAACCA GATCAGCACC 120

ACCCAGAAGA ACCAGCAGAA GGAGATGGAC CGCAAGGGCC TGCTGGGCTA CTACTTCAAG 180

GGCAAGGACT TCAGCAACCT GACCATGTTC GCCCCCACGC GTGACAGCAC CCTGATCTAC 2 0

GACCAGCAGA CCGCCAACAA GCTGCTGGAC AAGAAGCAGC AGGAGTACCA GAGCATCCGC 300

TGGATCGGCC TGATCCAGAG CAAGGAGACC GGCGACTTCA CCTTCAACCT GAGCGAGGAC 360

GAGCAGGCCA TCATCGAGAT CAACGGCAAG ATCATCAGCA ACAAGGGCAA GGAGAAGCAG 420

GTGGTGCACC TGGAGAAGGG CAAGCTGGTG CCCATCAAGA TCGAGTACCA GAGCGACACC 480

AAGTTCAACA TCGACAGCAA GACCTTCAAG GAGCTGAAGC TTTTCAAGAT CGACAGCCAG 540

AACCAGCCCC AGCAGGTGCA GCAGGACGAG CTGCGCAACC CCGAGTTCAA CAAGAAGGAG 600

AGCCAGGAGT TCCTGGCCAA GCCCAGCAAG ATCAACCTGT TCACCCAGCA GATGAAGCGC 660

GAGATCGACG AGGACACCGA CACCGACGGC GACAGCATCC CCGACCTGTG GGAGGAGAAC 720

GGCTACACCA TCCAGAACCG CATCGCCGTG AAGTGGGACG ACAGCCTGGC TAGCAAGGGC 780

TACACCAAGT TCGTGAGCAA CCCCCTGGAG AGCCACACCG TGGGCGACCC CTACACCGAC 840

TACGAGAAGG CCGCCCGCGA CCTGGACCTG AGCAACGCCA AGGAGACCTT CAACCCCCTG 900

GTGGCCGCCT TCCCCAGCGT GAACGTGAGC ATGGAGAAGG TGATCCTGAG CCCCAACGAG 960

AACCTGAGCA ACAGCGTGGA GAGCCACTCG AGCACCAACT GGAGCTACAC CAACACCGAG 1020

GGCGCCAGCG TGGAGGCCGG CATCGGTCCC AAGGGCATCA GCTTCGGCGT GAGCGTGAAC 1080

TACCAGCACA GCGAGACCGT GGCCCAGGAG TGGGGCACCA GCACCGGCAA CACCAGCCAG 1140

TTCAACACCG CCAGCGCCGG CTACCTGAAC GCCAACGTGC GCTACAACAA CGTGGGCACC 1200

GGCGCCATCT ACGACGTGAA GCCCACCACC AGCTTCGTGC TGAACAACGA CACCATCGCC 1260

ACCATCACCG CCAAGTCGAA TTCCACCGCC CTGAACATCA GCCCCGGCGA GAGCTACCCC 1320

AAGAAGGGCC AGAACGGCAT CGCCATCACC AGCATGGACG ACTTCAACAG CCACCCCATC 1380

ACCCTGAACA AGAAGCAGGT GGACAACCTG CTGAACAACA AGCCCATGAT GCTGGAGACC 1440

AACCAGACCG ACGGCGTCTA CAAGATCAAG GACACCCACG GCAACATCGT GACCGGCGGC 1500

GAGTGGAACG GCGTGATCCA GCAGATCAAG GCCAAGACCG CCAGCATCAT CGTCGACGAC 1560

GGCGAGCGCG TGGCCGAGAA GCGCGTGGCC GCCAAGGACT ACGAGAACCC CGAGGACAAG 1620

ACCCCCAGCC TGACCCTGAA GGACGCCCTG AAGCTGAGCT ACCCCGACGA GATCAAGGAG 1680

ATCGAGGGCC TGCTGTACTA CAAGAACAAG CCCATCTACG AGAGCAGCGT GATGACCTAT 1740

CTAGACGAGA ACACCGCCAA GGAGGTGACC AAGCAGCTGA ACGACACCAC CGGCAAGTTC 1800

AAGGACGTGA GCCACCTGTA CGACGTGAAG CTGACCCCCA AGATGAACGT GACCATCAAG 1860

CTGAGCATCC TGTACGACAA CGCCGAGAGC AACGACAACA GCATCGGCAA GTGGACCAAC 1920

ACCAACATCG TGAGCGGCGG CAACAACGGC AAGAAGCAGT ACAGCAGCAA CAACCCCGAC 1980

GCCAACCTGA CCCTGAACAC CGACGCCCAG GAGAAGCTGA ACAAGAACCG CGACTACTAC 2040

ATCAGCCTGT ACATGAAGAG CGAGAAGAAC ACCCAGTGCG AGATCACCAT CGACGGCGAG 2100

ATATACCCCA TCACCACCAA GACCGTGAAC GTGAACAAGG ACAACTACAA GCGCCTGGAC 2160

ATCATCGCCC ACAACATCAA GAGCAACCCC ATCAGCAGCC TGCACATCAA GACCAACGAC 2220

GAGATCACCC TGTTCTGGGA CGACATATCG ATTACCGACG TCGCCAGCAT CAAGCCCGAG 2280

AACCTGACCG ACAGCGAGAT CAAGCAGATA TACAGTCGCT ACGGCATCAA GCTGGAGGAC 2340

GGCATCCTGA TCGACAAGAA GGGCGGCATC CACTACGGCG AGTTCATCAA CGAGGCCAGC 2400

TTCAACATCG AGCCCCTGCA GAACTACGTG ACCAAGTACG AGGTGACCTA CAGCAGCGAG 2460

CTGGGCCCCA ACGTGAGCGA CACCCTGGAG AGCGACAAGA TTTACAAGGA CGGCACCATC 2520

AAGTTCGACT TCACCAAGTA CAGCAAGAAC GAGCAGGGCC TGTTCTACGA CAGCGGCCTG 2580

AACTGGGACT TCAAGATCAA CGCCATCACC TACGACGGCA AGGAGATGAA CGTGTTCCAC 2640

CGCTACAACA AGTAG 2655 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2004 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..2004

(D) OTHER INFORMATION: /note= "Maize optimized DNA sequence for VIPlA(a) 80 kd protein from AB78"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATGAAGCGCG AGATCGACGA GGACACCGAC ACCGACGGCG ACAGCATCCC CGACCTGTGG 60

GAGGAGAACG GCTACACCAT CCAGAACCGC ATCGCCGTGA AGTGGGACGA CAGCCTGGCT 120

AGCAAGGGCT ACACCAAGTT CGTGAGCAAC CCCCTGGAGA GCCACACCGT GGGCGACCCC 180

TACACCGACT ACGAGAAGGC CGCCCGCGAC CTGGACCTGA GCAACGCCAA GGAGACCTTC 240

AACCCCCTGG TGGCCGCCTT CCCCAGCGTG AACGTGAGCA TGGAGAAGGT GATCCTGAGC 300

CCCAACGAGA ACCTGAGCAA CAGCGTGGAG AGCCACTCGA GCACCAACTG GAGCTACACC 360

AACACCGAGG GCGCCAGCGT GGAGGCCGGC ATCGGTCCCA AGGGCATCAG CTTCGGCGTG 420

AGCGTGAACT ACCAGCACAG CGAGACCGTG GCCCAGGAGT GGGGCACCAG CACCGGCAAC 480

ACCAGCCAGT TCAACACCGC CAGCGCCGGC TACCTGAACG CCAACGTGCG CTACAACAAC 540

GTGGGCACCG GCGCCATCTA CGACGTGAAG CCCACCACCA GCTTCGTGCT GAACAACGAC 600

ACCATCGCCA CCATCACCGC CAAGTCGAAT TCCACCGCCC TGAACATCAG CCCCGGCGAG 660

AGCTACCCCA AGAAGGGCCA GAACGGCATC GCCATCACCA GCATGGACGA CTTCAACAGC 720

CACCCCATCA CCCTGAACAA GAAGCAGGTG GACAACCTGC TGAACAACAA GCCCATGATG 780

CTGGAGACCA ACCAGACCGA CGGCGTCTAC AAGATCAAGG ACACCCACGG CAACATCGTG 840

ACCGGCGGCG AGTGGAACGG CGTGATCCAG CAGATCAAGG CCAAGACCGC CAGCATCATC 900

GTCGACGACG GCGAGCGCGT GGCCGAGAAG CGCGTGGCCG CCAAGGACTA CGAGAACCCC 960

GAGGACAAGA CCCCCAGCCT GACCCTGAAG GACGCCCTGA AGCTGAGCTA CCCCGACGAG 1020

ATCAAGGAGA TCGAGGGCCT GCTGTACTAC AAGAACAAGC CCATCTACGA GAGCAGCGTG 1080

ATGACCTATC TAGACGAGAA CACCGCCAAG GAGGTGACCA AGCAGCTGAA CGACACCACC 1140

GGCAAGTTCA AGGACGTGAG CCACCTGTAC GACGTGAAGC TGACCCCCAA GATGAACGTG 1200

ACCATCAAGC TGAGCATCCT GTACGACAAC GCCGAGAGCA ACGACAACAG CATCGGCAAG 1260

TGGACCAACA CCAACATCGT GAGCGGCGGC AACAACGGCA AGAAGCAGTA CAGCAGCAAC 1320

AACCCCGACG CCAACCTGAC CCTGAACACC GACGCCCAGG AGAAGCTGAA CAAGAACCGC 1380

GACTACTACA TCAGCCTGTA CATGAAGAGC GAGAAGAACA CCCAGTGCGA GATCACCATC 1440

GACGGCGAGA TATACCCCAT CACCACCAAG ACCGTGAACG TGAACAAGGA CAACTACAAG 1500

CGCCTGGACA TCATCGCCCA CAACATCAAG AGCAACCCCA TCAGCAGCCT GCACATCAAG 1560

ACCAACGACG AGATCACCCT GTTCTGGGAC GACATATCGA TTACCGACGT CGCCAGCATC 1620

AAGCCCGAGA ACCTGACCGA CAGCGAGATC AAGCAGATAT ACAGTCGCTA CGGCATCAAG 1680

CTGGAGGACG GCATCCTGAT CGACAAGAAG GGCGGCATCC ACTACGGCGA GTTCATCAAC 1740

GAGGCCAGCT TCAACATCGA GCCCCTGCAG AACTACGTGA CCAAGTACGA GGTGACCTAC 1800

AGCAGCGAGC TGGGCCCCAA CGTGAGCGAC ACCCTGGAGA GCGACAAGAT TTACAAGGAC 1860

GGCACCATCA AGTTCGACTT CACCAAGTAC AGCAAGAACG AGCAGGGCCT GTTCTACGAC 1920

AGCGGCCTGA ACTGGGACTT CAAGATCAAC GCCATCACCT ACGACGGCAA GGAGATGAAC 1980

GTGTTCCACC GCTACAACAA GTAG 2004 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4074 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1386

(D) OTHER INFORMATION: /product= "VIP2A(b) from Btt"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1394..3895

(D) OTHER INFORMATION: /product---- "VIPlA(b) from Btt"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..4074

(D) OTHER INFORMATION: /note= "Cloned DNA 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 TTG TTT GTG GTG TCA AAA ACA TTA CAA 48 Met Gin Arg Met Glu Gly Lys Leu Phe Val Val Ser Lys Thr Leu Gin 670 675 680

GTA GTT ACT AGA ACT GTA TTG CTT AGT ACA GTT TAC TCT ATA ACT TTA 96 Val Val Thr Arg Thr Val Leu Leu Ser Thr Val Tyr Ser He Thr Leu 685 690 695

TTA AAT AAT GTA GTG ATA AAA GCT GAC CAA TTA AAT ATA AAT TCT CAA 144 Leu Asn Asn Val Val He Lys Ala Asp Gin Leu Asn He Asn Ser Gin 700 705 710 715

AGT AAA TAT ACT AAC TTG CAA AAT CTA AAA ATC CCT GAT AAT GCA GAG 192 Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Pro Asp Asn Ala Glu

720 725 730

GAT TTT AAA GAA GAT AAG GGG AAA GCG AAA GAA TGG GGG AAA GAG AAA 240 Asp Phe Lys Glu Asp Lys Gly Lys Ala Lys Glu Trp Gly Lys Glu Lys 735 740 745

GGG GAA GAG TGG AGG CCT CCT GCT ACT GAG AAA GGA GAA ATG AAT AAT 288 Gly Glu Glu Trp Arg Pro Pro Ala Thr Glu Lys Gly Glu Met Asn Asn 750 755 760

TTT TTA GAT AAT AAA AAT GAT ATA AAG ACC AAT TAT AAA GAA ATT ACT 336 Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr 765 770 775

TTT TCT ATG GCA GGT TCA TGT GAA GAT GAA ATA AAA GAT TTA GAA GAA 384 Phe Ser Met Ala Gly Ser Cys Glu Asp Glu He Lys Asp Leu Glu Glu 780 785 790 795

ATT GAT AAG ATC TTT GAT AAA GCC AAT CTC TCG AGT TCT ATT ATC ACC 432 He Asp Lys He Phe Asp Lys Ala Asn Leu Ser Ser Ser He He Thr 800 805 810

TAT AAA AAT GTG GAA CCA GCA ACA ATT GGA TTT AAT AAA TCT TTA ACA 480 Tyr Lys Asn Val Glu Pro Ala Thr He Gly Phe Asn Lys Ser Leu Thr 815 820 825

GAA GGT AAT ACG ATT AAT TCT GAT GCA ATG GCA CAG TTT AAA GAA CAA 528 Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 830 835 840

TTT TTA GGT AAG GAT ATG AAG TTT GAT AGT TAT CTA GAT ACT CAT TTA 576 Phe Leu Gly Lys Asp Met Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 845 850 855

ACT GCT CAA CAA GTT TCC AGT AAA AAA AGA GTT ATT TTG AAG GTT ACG 624 Thr Ala Gin Gin Val Ser Ser Lys Lys Arg Val He Leu Lys Val Thr 860 865 870 875

GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA ACA AAA GCA GGT GTC ATT 672 Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He 880 885 890

TTA AAC AAT AAT GAA TAC AAA ATG CTC ATT GAT AAT GGG TAT GTG CTC 720 Leu Asn Asn Asn Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Val Leu 895 900 905

CAT GTA GAT AAG GTA TCA AAA GTA GTA AAA AAA GGG ATG GAG TGC TTA 768 His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Met Glu Cys Leu 910 915 920

CAA GTT GAA GGG ACT TTA AAA AAG AGT CTC GAC TTT AAA AAT GAT ATA 816 Gin Val Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He 925 930 935

AAT GCT GAA GCG CAT AGC TGG GGG ATG AAA ATT TAT GAA GAC TGG GCT 864

Asn Ala Glu Ala His Ser Trp Gly Met Lys He Tyr Glu Asp Trp Ala 940 945 950 955

AAA AAT TTA ACC GCT TCG CAA AGG GAA GCT TTA GAT GGG TAT GCT AGG 912 Lys Asn Leu Thr Ala Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg 960 965 970

CAA GAT TAT AAA GAA ATC AAT AAT TAT TTG CGC AAT CAA GGC GGG AGT 960 Gin Asp Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 975 980 985

GGA AAT GAA AAG CTG GAT GCC CAA TTA AAA AAT ATT TCT GAT GCT TTA 1008 Gly Asn Glu Lys Leu Asp Ala Gin Leu Lys Asn He Ser Asp Ala Leu 990 995 1000

GGG AAG AAA CCC ATA CCA GAA AAT ATT ACC GTG TAT AGA TGG TGT GGC 1056 Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly 1005 1010 1015

ATG CCG GAA TTT GGT TAT CAA ATT AGT GAT CCG TTA CCT TCT TTA AAA 1104 Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys 1020 1025 1030 1035

GAT TTT GAA GAA CAA TTT TTA AAT ACA ATT AAA GAA GAC AAA GGG TAT 1152 Asp Phe Glu Glu Gin Phe Leu Asn Thr He Lys Glu Asp Lys Gly Tyr 1040 1045 1050

ATG AGT ACA AGC TTA TCG AGT GAA CGT CTT GCA GCT TTT GGA TCT AGA 1200 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg 1055 1060 1065

AAA ATT ATA TTA CGC TTA CAA GTT CCG AAA GGA AGT ACG GGG GCG TAT 1248 Lys He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 1070 1075 1080

TTA AGT GCC ATT GGT GGA TTT GCA AGT GAA AAA GAG ATC CTA CTT GAT 1296 Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp 1085 1090 1095

AAA GAT AGT AAA TAT CAT ATT GAT AAA GCA ACA GAG GTA ATC ATT AAA 1344 Lys Asp Ser Lys Tyr His He Asp Lys Ala Thr Glu Val He He Lys 1100 1105 1110 1115

GGT GTT AAG CGA TAT GTA GTG GAT GCA ACA TTA TTA ACA AAT 1386

Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 1120 1125

TAAGGAG ATG AAA AAT ATG AAG AAA AAG TTA GCA AGT GTT GTA ACC TGT 1 35 Met Lys Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys 1 5 10

ATG TTA TTA GCT CCT ATG TTT TTG AAT GGA AAT GTG AAT GCT GTT AAC 1483 Met Leu Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Asn 15 20 25 30

GCG GAT AGT AAA ATA AAT CAG ATT TCT ACA ACG CAG GAA AAC CAA CAG 1531 Ala Asp Ser Lys He Asn Gin He Ser Thr Thr Gin Glu Asn Gin Gin 35 40 45

AAA GAG ATG GAC CGA AAG GGA TTA TTG GGA TAT TAT TTC AAA GGA AAA 1579 Lys Glu Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys 50 55 60

GAT TTT AAT AAT CTT ACT ATG TTT GCA CCG ACA CGT GAT AAT ACC CTT 1627 Asp Phe Asn Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Asn Thr Leu 65 70 75

ATG TAT GAC CAA CAA ACA GCG AAT GCA TTA TTA GAT AAA AAA CAA CAA 1675 Met Tyr Asp Gin Gin Thr Ala Asn Ala Leu Leu Asp Lys Lys Gin Gin 80 85 90

GAA TAT CAG TCC ATT CGT TGG ATT GGT TTG ATT CAG CGT AAA GAA ACG 1723 Glu Tyr Gin Ser He Arg Trp He Gly Leu He Gin Arg Lys Glu Thr 95 100 105 110

GGC GAT TTC ACA TTT AAC TTA TCA AAG GAT GAA CAG GCA ATT ATA GAA 1771 Gly Asp Phe Thr Phe Asn Leu Ser Lys Asp Glu Gin Ala He He Glu 115 120 125

ATC GAT GGG AAA ATC ATT TCT AAT AAA GGG AAA GAA AAG CAA GTT GTC 1819 He Asp Gly Lys He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val 130 135 140

CAT TTA GAA AAA GAA AAA TTA GTT CCA ATC AAA ATA GAG TAT CAA TCA 1867 His Leu Glu Lys Glu Lys Leu Val Pro He Lys He Glu Tyr Gin Ser 145 150 155

GAT ACG AAA TTT AAT ATT GAT AGT AAA ACA TTT AAA GAA CTT AAA TTA 1915 Asp Thr Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu 160 165 170

TTT AAA ATA GAT AGT CAA AAC CAA TCT CAA CAA GTT CAA CTG AGA AAC 1963 Phe Lys He Asp Ser Gin Asn Gin Ser Gin Gin Val Gin Leu Arg Asn 175 180 185 190

CCT GAA TTT AAC AAA AAA GAA TCA CAG GAA TTT TTA GCA AAA GCA TCA 2011 Pro Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Ala Ser 195 200 205

AAA ACA AAC CTT TTT AAG CAA AAA ATG AAA AGA GAT ATT GAT GAA GAT 2059 Lys Thr Asn Leu Phe Lys Gin Lys Met Lys Arg Asp He Asp Glu Asp 210 215 220

ACG GAT ACA GAT GGA GAC TCC ATT CCT GAT CTT TGG GAA GAA AAT GGG 2107 Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn Gly 225 230 235

TAC ACG ATT CAA AAT AAA GTT GCT GTC AAA TGG GAT GAT TCG CTA GCA 2155 Tyr Thr He Gin Asn Lys Val Ala Val Lys Trp Asp Asp Ser Leu Ala 240 245 250

AGT AAG GGA TAT ACA AAA TTT GTT TCG AAT CCA TTA GAC AGC CAC ACA 2203 Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Asp Ser His Thr 255 260 265 270

GTT GGC GAT CCC TAT ACT GAT TAT GAA AAG GCC GCA AGG GAT TTA GAT 2251 Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp 275 280 285

TTA TCA AAT GCA AAG GAA ACG TTC AAC CCA TTG GTA GCT GCT TTT CCA 2299 Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro 290 295 300

AGT GTG AAT GTT AGT ATG GAA AAG GTG ATA TTA TCA CCA AAT GAA AAT 2347 Ser Val Asn Val Ser Met Glu Lys Val He Leu Ser Pro Asn Glu Asn 305 310 315

TTA TCC AAT AGT GTA GAG TCT CAT TCA TCC ACG AAT TGG TCT TAT ACG 2395 Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr 320 325 330

AAT ACA GAA GGA GCT TCC ATT GAA GCT GGT GGC GGT CCA TTA GGC CTT 2443 Asn Thr Glu Gly Ala Ser He Glu Ala Gly Gly Gly Pro Leu Gly Leu 335 340 345 350

TCT TTT GGC GTG AGT GTT ACT TAT CAA CAC TCT GAA ACA GTT GCA CAA 2491 Ser Phe Gly Val Ser Val Thr Tyr Gin His Ser Glu Thr Val Ala Gin 355 360 365

GAA TGG GGA ACA TCT ACA GGA AAT ACT TCA CAA TTC AAT ACG GCT TCA 2539 Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala Ser 370 375 380

GCG GGA TAT TTA AAT GCA AAT GTT CGG TAT AAC AAT GTA GGG ACT GGT 2587 Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly 385 390 395

GCC ATC TAT GAT GTA AAA CCT ACA ACA AGT TTT GTA TTA AAT AAC AAT 2635 Ala He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asn 400 405 410

ACC ATC GCA ACG ATT ACA GCA AAA TCA AAT TCA ACA GCT TTA CGT ATA 2683 Thr He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Arg He 415 420 425 430

TCT CCG GGG GAT AGT TAT CCA GAA ATA GGA GAA AAC GCT ATT GCG ATT 2731 Ser Pro Gly Asp Ser Tyr Pro Glu He Gly Glu Asn Ala He Ala He 435 440 445

ACA TCT ATG GAT GAT TTT AAT TCT CAT CCA ATT ACA TTA AAT AAA CAA 2779 Thr Ser Met Asp Asp Phe Asn Ser His Pro He Thr Leu Asn Lys Gin 450 455 460

CAG GTA AAT CAA TTG ATA AAT AAT AAG CCA ATT ATG CTA GAG ACA GAC 2827 Gin Val Asn Gin Leu He Asn Asn Lys Pro He Met Leu Glu Thr Asp

465 470 475

CAA ACA GAT GGT GTT TAT AAA ATA AGA GAT ACA CAT GGA AAT ATT GTA 2875 Gin Thr Asp Gly Val Tyr Lys He Arg Asp Thr His Gly Asn He Val 480 485 490

ACT GGT GGA GAA TGG AAT GGT GTA ACA CAA CAA ATT AAA GCA AAA ACA 2923 Thr Gly Gly Glu Trp Asn Gly Val Thr Gin Gin He Lys Ala Lys Thr 495 500 505 510

GCG TCT ATT ATT GTG GAT GAC GGG AAA CAG GTA GCA GAA AAA CGT GTG 2971 Ala Ser He He Val Asp Asp Gly Lys Gin Val Ala Glu Lys Arg Val 515 520 525

GCG GCA AAA GAT TAT GGT CAT CCA GAA GAT AAA ACA CCA CCT TTA ACT 3019 Ala Ala Lys Asp Tyr Gly His Pro Glu Asp Lys Thr Pro Pro Leu Thr 530 535 540

TTA AAA GAT ACC CTG AAG CTT TCA TAC CCA GAT GAA ATA AAA GAA ACT 3067 Leu Lys Asp Thr Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Glu Thr 545 550 555

AAT GGA TTG TTG TAC TAT GAT GAC AAA CCA ATC TAT GAA TCG AGT GTC 3115 Asn Gly Leu Leu Tyr Tyr Asp Asp Lys Pro He Tyr Glu Ser Ser Val 560 565 570

ATG ACT TAT CTG GAT GAA AAT ACG GCA AAA GAA GTC AAA AAA CAA ATA 3163 Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Lys Lys Gin He 575 580 585 590

AAT GAT ACA ACC GGA AAA TTT AAG GAT GTA AAT CAC TTA TAT GAT GTA 3211 Asn Asp Thr Thr Gly Lys Phe Lys Asp Val Asn His Leu Tyr Asp Val 595 600 605

AAA CTG ACT CCA AAA ATG AAT TTT ACG ATT AAA ATG GCT TCC TTG TAT 3259 Lys Leu Thr Pro Lys Met Asn Phe Thr He Lys Met Ala Ser Leu Tyr 610 615 620

GAT GGG GCT GAA AAT AAT CAT AAC TCT TTA GGA ACC TGG TAT TTA ACA 3307 Asp Gly Ala Glu Asn Asn His Asn Ser Leu Gly Thr Trp Tyr Leu Thr 625 630 635

TAT AAT GTT GCT GGT GGA AAT ACT GGG AAG AGA CAA TAT CGT TCA GCT 3355 Tyr Asn Val Ala Gly Gly Asn Thr Gly Lys Arg Gin Tyr Arg Ser Ala 640 645 650

CAT TCT TGT GCA CAT GTA GCT CTA TCT TCA GAA GCG AAA AAG AAA CTA 3403 His Ser Cys Ala His Val Ala Leu Ser Ser Glu Ala Lys Lys Lys Leu 655 660 665 670

AAT CAA AAT GCG AAT TAC TAT CTT AGC ATG TAT ATG AAG GCT GAT TCT 3451 Asn Gin Asn Ala Asn Tyr Tyr Leu Ser Met Tyr Met Lys Ala Asp Ser 675 680 685

ACT ACG GAA CCT ACA ATA GAA GTA GCT GGG GAA AAA TCT GCA ATA ACA 3499

Thr Thr Glu Pro Thr He Glu Val Ala Gly Glu Lys Ser Ala He Thr 690 695 700

AGT AAA AAA GTA AAA TTA AAT AAT CAA AAT TAT CAA AGA GTT GAT ATT 3547 Ser Lys Lys Val Lys Leu Asn Asn Gin Asn Tyr Gin Arg Val Asp He 705 710 715

TTA GTG AAA AAT TCT GAA AGA AAT CCA ATG GAT AAA ATA TAT ATA AGA 3595 Leu Val Lys Asn Ser Glu Arg Asn Pro Met Asp Lys He Tyr He Arg 720 725 730

GGA AAT GGC ACG ACA AAT GTT TAT GGG GAT GAT GTT ACT ATC CCA GAG 3643 Gly Asn Gly Thr Thr Asn Val Tyr Gly Asp Asp Val Thr He Pro Glu 735 740 745 750

GTA TCA GCT ATA AAT CCG GCT AGT CTA TCA GAT GAA GAA ATT CAA GAA 3691 Val Ser Ala He Asn Pro Ala Ser Leu Ser Asp Glu Glu He Gin Glu 755 760 765

ATA TTT AAA GAC TCA ACT ATT GAA TAT GGA AAT CCT AGT TTC GTT GCT 3739 He Phe Lys Asp Ser Thr He Glu Tyr Gly Asn Pro Ser Phe Val Ala 770 775 780

GAT GCC GTA ACA TTT AAA AAT ATA AAA CCT TTA CAA AAT TAT GTA AAG 3787 Asp Ala Val Thr Phe Lys Asn He Lys Pro Leu Gin Asn Tyr Val Lys 785 790 795

GAA TAT GAA ATA TAT CAT AAA TCT CAT CGA TAT GAA AAG AAA ACG GTC 3835 Glu Tyr Glu He Tyr His Lys Ser His Arg Tyr Glu Lys Lys Thr Val 800 805 810

TTT GAT ATC ATG GGT GTT CAT TAT GAG TAT AGT ATA GCT AGG GAA CAA 3883 Phe Asp He Met Gly Val His Tyr Glu Tyr Ser He Ala Arg Glu Gin 815 820 825 830

AAG AAA GCC GCA TAATTTTAAA AATAAAACTC GTTAGAGTTT ATTTAGCATG 3935 Lys Lys Ala Ala

GTATTTTTAA GAATAATCAA TATGTTGAAC CGTTTGTAGC TGTTTTGGAA GGGAATTTCA 3995 TTTTATTTGG TCTCTTAAGT TGATGGGCAT GGGATATGTT CAGCATCCAA GCGTTTNGGG 4055 GGTTANAAAA TCCAATTTT 4074

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Met Gin Arg Met Glu Gly Lys Leu Phe Val Val Ser Lys Thr Leu Gin 1 5 10 15

Val Val Thr Arg Thr Val Leu Leu Ser Thr Val Tyr Ser He Thr Leu 20 25 30

Leu Asn Asn Val Val He Lys Ala Asp Gin Leu Asn He Asn Ser Gin 35 40 45

Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Pro Asp Asn Ala Glu 50 55 60

Asp Phe Lys Glu Asp Lys Gly Lys Ala Lys Glu Trp Gly Lys Glu Lys 65 70 75 80

Gly Glu Glu Trp Arg Pro Pro Ala Thr Glu Lys Gly Glu Met Asn Asn 85 90 95

Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr 100 105 110

Phe Ser Met Ala Gly Ser Cys Glu Asp Glu He Lys Asp Leu Glu Glu 115 120 125

He Asp Lys He Phe Asp Lys Ala Asn Leu Ser Ser Ser He He Thr 130 135 140

Tyr Lys Asn Val Glu Pro Ala Thr He Gly Phe Asn Lys Ser Leu Thr 145 150 155 160

Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 165 170 175

Phe Leu Gly Lys Asp Met Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 180 185 190

Thr Ala Gin Gin Val Ser Ser Lys Lys Arg Val He Leu Lys Val Thr 195 200 205

Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He 210 215 220

Leu Asn Asn Asn Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Val Leu 225 230 235 240

His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Met Glu Cys Leu 245 250 255

Gin Val Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He 260 265 270

Asn Ala Glu Ala His Ser Trp Gly Met Lys He Tyr Glu Asp Trp Ala 275 280 285

Lys Asn Leu Thr Ala Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg 290 295 300

Gin Asp Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 305 310 315 320

Gly Asn Glu Lys Leu Asp Ala Gin Leu Lys Asn He Ser Asp Ala Leu 325 330 335

Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly 340 345 350

Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys 355 360 365

Asp Phe Glu Glu Gin Phe Leu Asn Thr He 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 He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 405 410 415

Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp 420 425 430

Lys Asp Ser Lys Tyr His He Asp Lys Ala Thr Glu Val He He Lys 435 440 445

Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 450 455 460

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 834 amino acids

(B) TYPE: amino acid (D) 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 15

Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Asn Ala Asp 20 25 30

Ser Lys He Asn Gin He Ser Thr Thr Gin Glu Asn Gin Gin Lys Glu 35 40 45

Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe 50 55 60

Asn Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Asn Thr Leu Met Tyr 65 70 75 80

Asp Gin Gin Thr Ala Asn Ala Leu Leu Asp Lys Lys Gin Gin Glu Tyr 85 90 95

Gin Ser He Arg Trp He Gly Leu He Gin Arg Lys Glu Thr Gly Asp 100 105 110

Phe Thr Phe Asn Leu Ser Lys Asp Glu Gin Ala He He Glu He Asp 115 120 125

Gly Lys He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu 130 135 140

Glu Lys Glu Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr 145 150 155 160

Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys 165 170 175

He Asp Ser Gin Asn Gin Ser Gin Gin Val Gin Leu Arg Asn Pro Glu 180 185 190

Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Ala Ser Lys Thr 195 200 205

Asn Leu Phe Lys Gin Lys Met Lys Arg Asp He Asp Glu Asp Thr Asp 210 215 220

Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr 225 230 235 240

He Gin Asn Lys Val Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys 245 250 255

Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Asp Ser His Thr Val Gly 260 265 270

Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser 275 280 285

Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val 290 295 300

Asn Val Ser Met Glu Lys Val He Leu Ser Pro Asn Glu Asn Leu Ser 305 310 315 320

Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr 325 330 335

Glu Gly Ala Ser He Glu Ala Gly Gly Gly Pro Leu Gly Leu Ser Phe

340 345 350

Gly Val Ser Val Thr Tyr Gin His Ser Glu Thr Val Ala Gin Glu Trp 355 360 365

Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala Ser Ala Gly 370 375 380

Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala He 385 390 395 400

Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asn Thr He 405 410 415

Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Arg He Ser Pro 420 425 430

Gly Asp Ser Tyr Pro Glu He Gly Glu Asn Ala He Ala He Thr Ser 435 440 445

Met Asp Asp Phe Asn Ser His Pro He Thr Leu Asn Lys Gin Gin Val 450 455 460

Asn Gin Leu He Asn Asn Lys Pro He Met Leu Glu Thr Asp Gin Thr 465 470 475 480

Asp Gly Val Tyr Lys He Arg Asp Thr His Gly Asn He Val Thr Gly 485 490 495

Gly Glu Trp Asn Gly Val Thr Gin Gin He Lys Ala Lys Thr Ala Ser 500 505 510

He He Val Asp Asp Gly Lys Gin Val Ala Glu Lys Arg Val Ala Ala 515 520 525

Lys Asp Tyr Gly His Pro Glu Asp Lys Thr Pro Pro Leu Thr Leu Lys 530 535 540

Asp Thr Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Glu Thr Asn Gly 545 550 555 560

Leu Leu Tyr Tyr Asp Asp Lys Pro He Tyr Glu Ser Ser Val Met Thr 565 570 575

Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Lys Lys Gin He Asn Asp 580 585 590

Thr Thr Gly Lys Phe Lys Asp Val Asn His Leu Tyr Asp Val Lys Leu 595 600 605

Thr Pro Lys Met Asn Phe Thr He Lys Met Ala Ser Leu Tyr Asp Gly 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 He Glu Val Ala Gly Glu Lys Ser Ala He Thr Ser Lys 690 695 700

Lys Val Lys Leu Asn Asn Gin Asn Tyr Gin Arg Val Asp He Leu Val 705 710 715 720

Lys Asn Ser Glu Arg Asn Pro Met Asp Lys He Tyr He Arg Gly Asn 725 730 735

Gly Thr Thr Asn Val Tyr Gly Asp Asp Val Thr He Pro Glu Val Ser 740 745 750

Ala He Asn Pro Ala Ser Leu Ser Asp Glu Glu He Gin Glu He Phe 755 760 765

Lys Asp Ser Thr He Glu Tyr Gly Asn Pro Ser Phe Val Ala Asp Ala 770 775 780

Val Thr Phe Lys Asn He Lys Pro Leu Gin Asn Tyr Val Lys Glu Tyr 785 790 795 800

Glu He Tyr His Lys Ser His Arg Tyr Glu Lys Lys Thr Val Phe Asp 805 810 815

He Met Gly Val His Tyr Glu Tyr Ser He Ala Arg Glu Gin Lys Lys 820 825 830

Ala Ala

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4041 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..4038

(D) OTHER INFORMATION: /product= "VIPlA(a) /VIP2A(a) fusion

product "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

ATG AAA AGA ATG GAG GGA AAG TTG TTT ATG GTG TCA AAA AAA TTA CAA 48 Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gin 835 840 845 850

GTA GTT ACT AAA ACT GTA TTG CTT AGT ACA GTT TTC TCT ATA TCT TTA 96 Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser He Ser Leu 855 860 865

TTA AAT AAT GAA GTG ATA AAA GCT GAA CAA TTA AAT ATA AAT TCT CAA 144 Leu Asn Asn Glu Val He Lys Ala Glu Gin Leu Asn He Asn Ser Gin 870 875 880

AGT AAA TAT ACT AAC TTG CAA AAT CTA AAA ATC ACT GAC AAG GTA GAG 192 Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu 885 890 895

GAT TTT AAA GAA GAT AAG GAA AAA GCG AAA GAA TGG GGG AAA GAA AAA 240 Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys 900 905 910

GAA AAA GAG TGG AAA CTA ACT GCT ACT GAA AAA GGA AAA ATG AAT AAT 288 Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn 915 920 925 930

TTT TTA GAT AAT AAA AAT GAT ATA AAG ACA AAT TAT AAA GAA ATT ACT 336 Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr 935 940 945

TTT TCT ATG GCA GGC TCA TTT GAA GAT GAA ATA AAA GAT TTA AAA GAA 384 Phe Ser Met Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu 950 955 960

ATT GAT AAG ATG TTT GAT AAA ACC AAT CTA TCA AAT TCT ATT ATC ACC 432 He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr 965 970 975

TAT AAA AAT GTG GAA CCG ACA ACA ATT GGA TTT AAT AAA TCT TTA ACA 480 Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr 980 985 990

GAA GGT AAT ACG ATT AAT TCT GAT GCA ATG GCA CAG TTT AAA GAA CAA 528 Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 995 1000 1005 1010

TTT TTA GAT AGG GAT ATT AAG TTT GAT AGT TAT CTA GAT ACG CAT TTA 576 Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 1015 1020 1025

ACT GCT CAA CAA GTT TCC AGT AAA GAA AGA GTT ATT TTG AAG GTT ACG 624 Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr

1030 1035 1040

GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA ACA AAA GCA GGT GTC ATT 672 Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He 1045 1050 1055

TTA AAT AAT AGT GAA TAC AAA ATG CTC ATT GAT AAT GGG TAT ATG GTC 720 Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val 1060 1065 1070

CAT GTA GAT AAG GTA TCA AAA GTG GTG AAA AAA GGG GTG GAG TGC TTA 768 His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu 1075 1080 1085 1090

CAA ATT GAA GGG ACT TTA AAA AAG AGT CTT GAC TTT AAA AAT GAT ATA 816 Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He 1095 1100 1105

AAT GCT GAA GCG CAT AGC TGG GGT ATG AAG AAT TAT GAA GAG TGG GCT 864 Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp Ala 1110 1115 1120

AAA GAT TTA ACC GAT TCG CAA AGG GAA GCT TTA GAT GGG TAT GCT AGG 912 Lys Asp Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg 1125 1130 1135

CAA GAT TAT AAA GAA ATC AAT AAT TAT TTA AGA AAT CAA GGC GGA AGT 960 Gin Asp Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 1140 1145 1150

GGA AAT GAA AAA CTA GAT GCT CAA ATA AAA AAT ATT TCT GAT GCT TTA 1008 Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu 1155 1160 1165 1170

GGG AAG AAA CCA ATA CCG GAA AAT ATT ACT GTG TAT AGA TGG TGT GGC 1056 Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly 1175 1180 1185

ATG CCG GAA TTT GGT TAT CAA ATT AGT GAT CCG TTA CCT TCT TTA AAA 1104 Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys 1190 1195 1200

GAT TTT GAA GAA CAA TTT TTA AAT ACA ATC AAA GAA GAC AAA GGA TAT 1152 Asp Phe Glu Glu Gin Phe Leu Asn Thr He Lys Glu Asp Lys Gly Tyr 1205 1210 1215

ATG AGT ACA AGC TTA TCG AGT GAA CGT CTT GCA GCT TTT GGA TCT AGA 1200 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg 1220 1225 1230

AAA ATT ATA TTA CGA TTA CAA GTT CCG AAA GGA AGT ACG GGT GCG TAT 1248 Lys He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 1235 1240 1245 1250

TTA AGT GCC ATT GGT GGA TTT GCA AGT GAA AAA GAG ATC CTA CTT GAT 1296

Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp 1255 1260 1265

AAA GAT AGT AAA TAT CAT ATT GAT AAA GTA ACA GAG GTA ATT ATT AAA 1344 Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He Lys 1270 1275 1280

GGT GTT AAG CGA TAT GTA GTG GAT GCA ACA TTA TTA ACA AAT ATG AAA 1392 Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn Met Lys 1285 1290 1295

AAT ATG AAG AAA AAG TTA GCA AGT GTT GTA ACG TGT ACG TTA TTA GCT 1440 Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu Leu Ala 1300 1305 1310

CCT ATG TTT TTG AAT GGA AAT GTG AAT GCT GTT TAC GCA GAC AGC AAA 1488 Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser Lys 1315 1320 1325 1330

ACA AAT CAA ATT TCT ACA ACA CAG AAA AAT CAA CAG AAA GAG ATG GAC 1536 Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin Lys Glu Met Asp 1335 1340 1345

CGA AAA GGA TTA CTT GGG TAT TAT TTC AAA GGA AAA GAT TTT AGT AAT 1584 Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn 1350 1355 1360

CTT ACT ATG TTT GCA CCG ACA CGT GAT AGT ACT CTT ATT TAT GAT CAA 1632 Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr Asp Gin 1365 1370 1375

CAA ACA GCA AAT AAA CTA TTA GAT AAA AAA CAA CAA GAA TAT CAG TCT 1680 Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin Glu Tyr Gin Ser 1380 1385 1390

ATT CGT TGG ATT GGT TTG ATT CAG AGT AAA GAA ACG GGA GAT TTC ACA 1728 He Arg Trp He Gly Leu He Gin Ser Lys Glu Thr Gly Asp Phe Thr 1395 1400 1405 1410

TTT AAC TTA TCT GAG GAT GAA CAG GCA ATT ATA GAA ATC AAT GGG AAA 1776 Phe Asn Leu Ser Glu Asp Glu Gin Ala He He Glu He Asn Gly Lys 1415 1420 1425

ATT ATT TCT AAT AAA GGG AAA GAA AAG CAA GTT GTC CAT TTA GAA AAA 1824 He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu Glu Lys 1430 1435 1440

GGA AAA TTA GTT CCA ATC AAA ATA GAG TAT CAA TCA GAT ACA AAA TTT 1872 Gly Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr Lys Phe 1445 1450 1455

AAT ATT GAC AGT AAA ACA TTT AAA GAA CTT AAA TTA TTT AAA ATA GAT 1920 Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys He Asp 1460 1465 1470

AGT CAA AAC CAA CCC CAG CAA GTC CAG CAA GAT GAA CTG AGA AAT CCT 1968 Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu Leu Arg Asn Pro 1475 1480 1485 1490

GAA TTT AAC AAG AAA GAA TCA CAG GAA TTC TTA GCG AAA CCA TCG AAA 2016 Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Pro Ser Lys 1495 1500 1505

ATA AAT CTT TTC ACT CAA AAA ATG AAA AGG GAA ATT GAT GAA GAC ACG 2064 He Asn Leu Phe Thr Gin Lys Met Lys Arg Glu He Asp Glu Asp Thr 1510 1515 1520

GAT ACG GAT GGG GAC TCT ATT CCT GAC CTT TGG GAA GAA AAT GGG TAT 2112 Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn Gly Tyr 1525 1530 1535

ACG ATT CAA AAT AGA ATC GCT GTA AAG TGG GAC GAT TCT CTA GCA AGT 2160 Thr He Gin Asn Arg He Ala Val Lys Trp Asp Asp Ser Leu Ala Ser 1540 1545 1550

AAA GGG TAT ACG AAA TTT GTT TCA AAT CCA CTA GAA AGT CAC ACA GTT 2208 Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His Thr Val 1555 1560 1565 1570

GGT GAT CCT TAT ACA GAT TAT GAA AAG GCA GCA AGA GAT CTA GAT TTG 2256 Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu 1575 1580 1585

TCA AAT GCA AAG GAA ACG TTT AAC CCA TTG GTA GCT GCT TTT CCA AGT 2304 Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser 1590 1595 1600

GTG AAT GTT AGT ATG GAA AAG GTG ATA TTA TCA CCA AAT GAA AAT TTA 2352 Val Asn Val Ser Met Glu Lys Val He Leu Ser Pro Asn Glu Asn Leu 1605 1610 1615

TCC AAT AGT GTA GAG TCT CAT TCA TCC ACG AAT TGG TCT TAT ACA AAT 2400 Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn 1620 1625 1630

ACA GAA GGT GCT TCT GTT GAA GCG GGG ATT GGA CCA AAA GGT ATT TCG 2448 Thr Glu Gly Ala Ser Val Glu Ala Gly He Gly Pro Lys Gly He Ser 1635 1640 1645 1650

TTC GGA GTT AGC GTA AAC TAT CAA CAC TCT GAA ACA GTT GCA CAA GAA 2496 Phe Gly Val Ser Val Asn Tyr Gin His Ser Glu Thr Val Ala Gin Glu 1655 1660 1665

TGG GGA ACA TCT ACA GGA AAT ACT TCG CAA TTC AAT ACG GCT TCA GCG 2544 Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala Ser Ala 1670 1675 1680

GGA TAT TTA AAT GCA AAT GTT CGA TAT AAC AAT GTA GGA ACT GGT GCC 2592 Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala 1685 1690 1695

ATC TAC GAT GTA AAA CCT ACA ACA AGT TTT GTA TTA AAT AAC GAT ACT 260 He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asp Thr 1700 1705 1710

ATC GCA ACT ATT ACG GCG AAA TCT AAT TCT ACA GCC TTA AAT ATA TCT 2688 He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn He Ser 1715 1720 1725 1730

CCT GGA GAA AGT TAC CCG AAA AAA GGA CAA AAT GGA ATC GCA ATA ACA 2736 Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly He Ala He Thr 1735 1740 1745

TCA ATG GAT GAT TTT AAT TCC CAT CCG ATT ACA TTA AAT AAA AAA CAA 2784 Ser Met Asp Asp Phe Asn Ser His Pro He Thr Leu Asn Lys Lys Gin 1750 1755 1760

GTA GAT AAT CTG CTA AAT AAT AAA CCT ATG ATG TTG GAA ACA AAC CAA 2832 Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr Asn Gin 1765 1770 1775

ACA GAT GGT GTT TAT AAG ATA AAA GAT ACA CAT GGA AAT ATA GTA ACT 2880 Thr Asp Gly Val Tyr Lys He Lys Asp Thr His Gly Asn He Val Thr 1780 1785 1790

GGC GGA GAA TGG AAT GGT GTC ATA CAA CAA ATC AAG GCT AAA ACA GCG 2928 Gly Gly Glu Trp Asn Gly Val He Gin Gin He Lys Ala Lys Thr Ala 1795 1800 1805 1810

TCT ATT ATT GTG GAT GAT GGG GAA CGT GTA GCA GAA AAA CGT GTA GCG 2976 Ser He He Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg Val Ala 1815 1820 1825

GCA AAA GAT TAT GAA AAT CCA GAA GAT AAA ACA CCG TCT TTA ACT TTA 3024 Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu 1830 1835 1840

AAA GAT GCC CTG AAG CTT TCA TAT CCA GAT GAA ATA AAA GAA ATA GAG 3072 Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Glu He Glu 1845 1850 1855

GGA TTA TTA TAT TAT AAA AAC AAA CCG ATA TAC GAA TCG AGC GTT ATG 3120 Gly Leu Leu Tyr Tyr Lys Asn Lys Pro He Tyr Glu Ser Ser Val Met 1860 1865 1870

ACT TAC TTA GAT GAA AAT ACA GCA AAA GAA GTG ACC AAA CAA TTA AAT 3168 Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin Leu Asn 1875 1880 1885 1890

GAT ACC ACT GGG AAA TTT AAA GAT GTA AGT CAT TTA TAT GAT GTA AAA 3216 Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val Lys 1895 1900 1905

CTG ACT CCA AAA ATG AAT GTT ACA ATC AAA TTG TCT ATA CTT TAT GAT 3264 Leu Thr Pro Lys Met Asn Val Thr He Lys Leu Ser He Leu Tyr Asp

1910 1915 1920

AAT GCT GAG TCT AAT GAT AAC TCA ATT GGT AAA TGG ACA AAC ACA AAT 3312 Asn Ala Glu Ser Asn Asp Asn Ser He Gly Lys Trp Thr Asn Thr Asn 1925 1930 1935

ATT GTT TCA GGT GGA AAT AAC GGA AAA AAA CAA TAT TCT TCT AAT AAT 3360 He Val Ser Gly Gly Asn Asn Gly Lys Lys Gin Tyr Ser Ser Asn Asn 1940 1945 1950

CCG GAT GCT AAT TTG ACA TTA AAT ACA GAT GCT CAA GAA AAA TTA AAT 3408 Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin Glu Lys Leu Asn 1955 1960 1965 1970

AAA AAT CGT GAC TAT TAT ATA AGT TTA TAT ATG AAG TCA GAA AAA AAC 3456 Lys Asn Arg Asp Tyr Tyr He Ser Leu Tyr Met Lys Ser Glu Lys Asn 1975 1980 1985

ACA CAA TGT GAG ATT ACT ATA GAT GGG GAG ATT TAT CCG ATC ACT ACA 3504 Thr Gin Cys Glu He Thr He Asp Gly Glu He Tyr Pro He Thr Thr 1990 1995 2000

AAA ACA GTG AAT GTG AAT AAA GAC AAT TAC AAA AGA TTA GAT ATT ATA 3552 Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp He He 2005 2010 2015

GCT CAT AAT ATA AAA AGT AAT CCA ATT TCT TCA CTT CAT ATT AAA ACG 3600 Ala His Asn He Lys Ser Asn Pro He Ser Ser Leu His He Lys Thr 2020 2025 2030

AAT GAT GAA ATA ACT TTA TTT TGG GAT GAT ATT TCT ATA ACA GAT GTA 3648 Asn Asp Glu He Thr Leu Phe Trp Asp Asp He Ser He Thr Asp Val 2035 2040 2045 2050

GCA TCA ATA AAA CCG GAA AAT TTA ACA GAT TCA GAA ATT AAA CAG ATT 3696 Ala Ser He Lys Pro Glu Asn Leu Thr Asp Ser Glu He Lys Gin He 2055 2060 2065

TAT AGT AGG TAT GGT ATT AAG TTA GAA GAT GGA ATC CTT ATT GAT AAA 3744 Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly He Leu He Asp Lys 2070 2075 2080

AAA GGT GGG ATT CAT TAT GGT GAA TTT ATT AAT GAA GCT AGT TTT AAT 3792 Lys Gly Gly He His Tyr Gly Glu Phe He Asn Glu Ala Ser Phe Asn 2085 2090 2095

ATT GAA CCA TTG CAA AAT TAT GTG ACC AAA TAT GAA GTT ACT TAT AGT 3840 He Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr Tyr Ser 2100 2105 2110

AGT GAG TTA GGA CCA AAC GTG AGT GAC ACA CTT GAA AGT GAT AAA ATT 3888 Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu Ser Asp Lys He 2115 2120 2125 2130

TAC AAG GAT GGG ACA ATT AAA TTT GAT TTT ACC AAA TAT AGT AAA AAT 3936

Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys Tyr Ser Lys Asn 2135 2140 2145

GAA CAA GGA TTA TTT TAT GAC AGT GGA TTA AAT TGG GAC TTT AAA ATT 3984 Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe Lys He 2150 2155 2160

AAT GCT ATT ACT TAT GAT GGT AAA GAG ATG AAT GTT TTT CAT AGA TAT 4032 Asn Ala He Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His Arg Tyr 2165 2170 2175

AAT AAA TAG 4041

Asn Lys 2180

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1346 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gin 1 5 10 15

Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser He Ser Leu 20 25 30

Leu Asn Asn Glu Val He Lys Ala Glu Gin Leu Asn He Asn Ser Gin 35 40 45

Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu 50 55 60

Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys 65 70 75 80

Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn 85 90 95

Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr 100 105 110

Phe Ser Met Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu 115 120 125

He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr 130 135 140

Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr

145 150 155 160

Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 165 170 175

Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 180 185 190

Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr 195 200 205

Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He 210 215 220

Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val 225 230 235 240

His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu 245 250 255

Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He 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 He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 305 310 315 320

Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu 325 330 335

Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly 340 345 350

Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys 355 360 365

Asp Phe Glu Glu Gin Phe Leu Asn Thr He 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 He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 405 410 415

Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp 420 425 430

Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He Lys 435 440 445

Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn Met Lys 450 455 460

Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu Leu Ala 465 470 475 480

Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser Lys 485 490 495

Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin Lys Glu Met Asp 500 505 510

Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn 515 520 525

Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr Asp Gin 530 535 540

Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin Glu Tyr Gin Ser 545 550 555 560

He Arg Trp He Gly Leu He Gin Ser Lys Glu Thr Gly Asp Phe Thr 565 570 575

Phe Asn Leu Ser Glu Asp Glu Gin Ala He He Glu He Asn Gly Lys 580 585 590

He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu Glu Lys 595 600 605

Gly Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr Lys Phe 610 615 620

Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys He Asp 625 630 635 640

Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu Leu Arg Asn Pro 645 650 655

Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Pro Ser Lys 660 665 670

He Asn Leu Phe Thr Gin Lys Met Lys Arg Glu He Asp Glu Asp Thr 675 680 685

Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn Gly Tyr 690 695 700

Thr He Gin Asn Arg He Ala Val Lys Trp Asp Asp Ser Leu Ala Ser 705 710 715 720

Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His Thr Val 725 730 735

Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu 740 745 750

Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser 755 760 765

Val Asn Val Ser Met Glu Lys Val He Leu Ser Pro Asn Glu Asn Leu 770 775 780

Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn 785 790 795 800

Thr Glu Gly Ala Ser Val Glu Ala Gly He Gly Pro Lys Gly He Ser 805 810 815

Phe Gly Val Ser Val Asn Tyr Gin His Ser Glu Thr Val Ala Gin Glu 820 825 830

Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala Ser Ala 835 840 845

Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala 850 855 860

He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asp Thr 865 870 875 880

He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn He Ser 885 890 895

Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly He Ala He Thr 900 905 910

Ser Met Asp Asp Phe Asn Ser His Pro He Thr Leu Asn Lys Lys Gin 915 920 925

Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr Asn Gin 930 935 940

Thr Asp Gly Val Tyr Lys He Lys Asp Thr His Gly Asn He Val Thr 945 950 955 960

Gly Gly Glu Trp Asn Gly Val He Gin Gin He Lys Ala Lys Thr Ala 965 970 975

Ser He He Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg Val Ala 980 985 990

Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu 995 1000 1005

Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Glu He Glu 1010 1015 1020

Gly Leu Leu Tyr Tyr Lys Asn Lys Pro He 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 He Lys Leu Ser He Leu Tyr Asp 1075 1080 1085

Asn Ala Glu Ser Asn Asp Asn Ser He Gly Lys Trp Thr Asn Thr Asn 1090 1095 1100

He 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 He Ser Leu Tyr Met Lys Ser Glu Lys Asn 1140 1145 1150

Thr Gin Cys Glu He Thr He Asp Gly Glu He Tyr Pro He Thr Thr 1155 1160 1165

Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp He He 1170 1175 1180

Ala His Asn He Lys Ser Asn Pro He Ser Ser Leu His He Lys Thr 1185 1190 1195 1200

Asn Asp Glu He Thr Leu Phe Trp Asp Asp He Ser He Thr Asp Val 1205 1210 1215

Ala Ser He Lys Pro Glu Asn Leu Thr Asp Ser Glu He Lys Gin He 1220 1225 1230

Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly He Leu He Asp Lys 1235 1240 1245

Lys Gly Gly He His Tyr Gly Glu Phe He Asn Glu Ala Ser Phe Asn 1250 1255 1260

He 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 He 1285 1290 1295

Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys Tyr Ser Lys Asn 1300 1305 1310

Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe Lys He 1315 1320 1325

Asn Ala He Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His Arg Tyr 1330 1335 1340

Asn Lys 1345

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1399 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..1386

(D) OTHER INFORMATION: /note= "Maize optimized DNA sequence for VIP2A(a) protein from AB78"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ATGAAGCGCA TGGAGGGCAA GCTGTTCATG GTGAGCAAGA AGCTCCAGGT GGTGACCAAG 60

ACCGTGCTGC TGAGCACCGT GTTCAGCATC AGCCTGCTGA ACAACGAGGT GATCAAGGCC 120

GAGCAGCTGA ACATCAACAG CCAGAGCAAG TACACCAACC TCCAGAACCT GAAGATCACC 180

GACAAGGTGG AGGACTTCAA GGAGGACAAG GAGAAGGCCA AGGAGTGGGG CAAGGAGAAG 240

GAGAAGGAGT GGAAGCTTAC CGCCACCGAG AAGGGCAAGA TGAACAACTT CCTGGACAAC 300

AAGAACGACA TCAAGACCAA CTACAAGGAG ATCACCTTCA GCATGGCCGG CAGCTTCGAG 360

GACGAGATCA AGGACCTGAA GGAGATCGAC AAGATGTTCG ACAAGACCAA CCTGAGCAAC 420

AGCATCATCA CCTACAAGAA CGTGGAGCCC ACCACCATCG GCTTCAACAA GAGCCTGACC 480

GAGGGCAACA CCATCAACAG CGACGCCATG GCCCAGTTCA AGGAGCAGTT CCTGGACCGC 540

GACATCAAGT TCGACAGCTA CCTGGACACC CACCTGACCG CCCAGCAGGT GAGCAGCAAG 600

GAGCGCGTGA TCCTGAAGGT GACCGTCCCC AGCGGCAAGG GCAGCACCAC CCCCACCAAG 660

GCCGGCGTGA TCCTGAACAA CAGCGAGTAC AAGATGCTGA TCGACAACGG CTACATGGTG 720

CACGTGGACA AGGTGAGCAA GGTGGTGAAG AAGGGCGTGG AGTGCCTCCA GATCGAGGGC 780

ACCCTGAAGA AGAGTCTAGA CTTCAAGAAC GACATCAACG CCGAGGCCCA CAGCTGGGGC 840

ATGAAGAACT ACGAGGAGTG GGCCAAGGAC CTGACCGACA GCCAGCGCGA GGCCCTGGAC 900

GGCTACGCCC GCCAGGACTA CAAGGAGATC AACAACTACC TGCGCAACCA GGGCGGCAGC 960

GGCAACGAGA AGCTGGACGC CCAGATCAAG AACATCAGCG ACGCCCTGGG CAAGAAGCCC 1020

ATCCCCGAGA ACATCACCGT GTACCGCTGG TGCGGCATGC CCGAGTTCGG CTACCAGATC 1080

AGCGACCCCC TGCCCAGCCT GAAGGACTTC GAGGAGCAGT TCCTGAACAC CATCAAGGAG 1140

GACAAGGGCT ACATGAGCAC CAGCCTGAGC AGCGAGCGCC TGGCCGCCTT CGGCAGCCGC 1200

AAGATCATCC TGCGCCTGCA GGTGCCCAAG GGCAGCACCG GCGCCTACCT GAGCGCCATC 1260

GGCGGCTTCG CCAGCGAGAA GGAGATCCTG CTGGACAAGG ACAGCAAGTA CCACATCGAC 1320

AAGGTGACCG AGGTGATCAT CAAGGGCGTG AAGCGCTACG TGGTGGACGC CACCCTGCTG 1380

ACCAACTAGA TCTGAGCTC 1399 (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..19

(D) OTHER INFORMATION: /note= "Secretion signal peptide to secrete VIP2 out of a cell"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Gly Trp Ser Trp He Phe Leu Phe Leu Leu Ser Gly Ala Ala Gly Val 1 5 10 15

His Cys Leu

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2655 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA" ( ii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..2655

(D) OTHER INFORMATION: /note= "maize optimized DNA sequence encoding VEPlA(a)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

ATGAAGAACA TGAAGAAGAA GCTGGCCAGC GTGGTGACCT GCACCCTGCT GGCCCCCATG 60

TTCCTGAACG GCAACGTGAA CGCCGTGTAC GCCGACAGCA AGACCAACCA GATCAGCACC 120

ACCCAGAAGA ACCAGCAGAA GGAGATGGAC CGCAAGGGCC TGCTGGGCTA CTACTTCAAG 180

GGCAAGGACT TCAGCAACCT GACCATGTTC GCCCCCACGC GTGACAGCAC CCTGATCTAC 240

GACCAGCAGA CCGCCAACAA GCTGCTGGAC AAGAAGCAGC AGGAGTACCA GAGCATCCGC 300

TGGATCGGCC TGATCCAGAG CAAGGAGACC GGCGACTTCA CCTTCAACCT GAGCGAGGAC 360

GAGCAGGCCA TCATCGAGAT CAACGGCAAG ATCATCAGCA ACAAGGGCAA GGAGAAGCAG 420

GTGGTGCACC TGGAGAAGGG CAAGCTGGTG CCCATCAAGA TCGAGTACCA GAGCGACACC 480

AAGTTCAACA TCGACAGCAA GACCTTCAAG GAGCTGAAGC TTTTCAAGAT CGACAGCCAG 540

AACCAGCCCC AGCAGGTGCA GCAGGACGAG CTGCGCAACC CCGAGTTCAA CAAGAAGGAG 600

AGCCAGGAGT TCCTGGCCAA GCCCAGCAAG ATCAACCTGT TCACCCAGCA GATGAAGCGC 660

GAGATCGACG AGGACACCGA CACCGACGGC GACAGCATCC CCGACCTGTG GGAGGAGAAC 720

GGCTACACCA TCCAGAACCG CATCGCCGTG AAGTGGGACG ACAGCCTGGC TAGCAAGGGC 780

TACACCAAGT TCGTGAGCAA CCCCCTGGAG AGCCACACCG TGGGCGACCC CTACACCGAC 840

TACGAGAAGG CCGCCCGCGA CCTGGACCTG AGCAACGCCA AGGAGACCTT CAACCCCCTG 900

GTGGCCGCCT TCCCCAGCGT GAACGTGAGC ATGGAGAAGG TGATCCTGAG CCCCAACGAG 960

AACCTGAGCA ACAGCGTGGA GAGCCACTCG AGCACCAACT GGAGCTACAC CAACACCGAG 1020

GGCGCCAGCG TGGAGGCCGG CATCGGTCCC AAGGGCATCA GCTTCGGCGT GAGCGTGAAC 1080

TACCAGCACA GCGAGACCGT GGCCCAGGAG TGGGGCACCA GCACCGGCAA CACCAGCCAG 11 0

TTCAACACCG CCAGCGCCGG CTACCTGAAC GCCAACGTGC GCTACAACAA CGTGGGCACC 1200

GGCGCCATCT ACGACGTGAA GCCCACCACC AGCTTCGTGC TGAACAACGA CACCATCGCC 1260

ACCATCACCG CCAAGTCGAA TTCCACCGCC CTGAACATCA GCCCCGGCGA GAGCTACCCC 1320

AAGAAGGGCC AGAACGGCAT CGCCATCACC AGCATGGACG ACTTCAACAG CCACCCCATC 1380

ACCCTGAACA AGAAGCAGGT GGACAACCTG CTGAACAACA AGCCCATGAT GCTGGAGACC 1440

AACCAGACCG ACGGCGTCTA CAAGATCAAG GACACCCACG GCAACATCGT GACGGGCGGC 1500

GAGTGGAACG GCGTGATCCA GCAGATCAAG GCCAAGACCG CCAGCATCAT CGTCGACGAC 1560

GGCGAGCGCG TGGCCGAGAA GCGCGTGGCC GCCAAGGACT ACGAGAACCC CGAGGACAAG 1620

ACCCCCAGCC TGACCCTGAA GGACGCCCTG AAGCTGAGCT ACCCCGACGA GATCAAGGAG 1680

ATCGAGGGCT TGCTGTACTA CAAGAACAAG CCCATCTACG AGAGCAGCGT GATGACCTAT 1740

CTAGACGAGA ACACCGCCAA GGAGGTGACC AAGCAGCTGA ACGACACCAC CGGCAAGTTC 1800

AAGGACGTGA GCCACCTGTA CGACGTGAAG CTGACCCCCA AGATGAACGT GACCATCAAG 1860

CTGAGCATCC TGTACGACAA CGCCGAGAGC AACGACAACA GCATCGGCAA GTGGACCAAC 1920

ACCAACATCG TGAGCGGCGG CAACAACGGC AAGAAGCAGT ACAGCAGCAA CAACCCCGAC 1980

GCCAACCTGA CCCTGAACAC CGACGCCCAG GAGAAGCTGA ACAAGAACCG CGACTACTAC 2040

ATCAGCCTGT ACATGAAGAG CGAGAAGAAC ACCCAGTGCG AGATCACCAT CGACGGCGAG 2100

ATATACCCCA TCACCACCAA GACCGTGAAC GTGAACAAGG ACAACTACAA GCGCCTGGAC 2160

ATCATCGCCC ACAACATCAA GAGCAACCCC ATCAGCAGCC TGCACATCAA GACCAACGAC 2220

GAGATCACCC TGTTCTGGGA CGACATATCG ATTACCGACG TCGCCAGCAT CAAGCCCGAG 2280

AACCTGACCG ACAGCGAGAT CAAGCAGATA TACAGTCGCT ACGGCATCAA GCTGGAGGAC 2340

GGCATCCTGA TCGACAAGAA AGGCGGCATC CACTACGGCG AGTTCATCAA CGAGGCCAGC 2400

TTCAACATCG AGCCCCTGCA GAACTACGTG ACCAAGTACG AGGTGACCTA CAGCAGCGAG 2460

CTGGGCCCCA ACGTGAGCGA CACCCTGGAG AGCGACAAGA TTTACAAGGA CGGCACCATC 2520

AAGTTCGACT TCACCAAGTA CAGCAAGAAC GAGCAGGGCC TGTTCTACGA CAGCGGCCTG 2580

AACTGGGACT TCAAGATCAA CGCCATCACC TACGACGGCA AGGAGATGAA CGTGTTCCAC 2640

CGCTACAACA AGTAG 2655 (2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1389 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..1389

(D) OTHER INFORMATION: /note= "maize optimized DNA sequence encoding VIP2A(a)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

ATGAAGCGCA TGGAGGGCAA GCTGTTCATG GTGAGCAAGA AGCTCCAGGT GGTGACCAAG 60

ACCGTGCTGC TGAGCACCGT GTTCAGCATC AGCCTGCTGA ACAACGAGGT GATCAAGGCC 120

GAGCAGCTGA ACATCAACAG CCAGAGCAAG TACACCAACC TCCAGAACCT GAAGATCACC 180

GACAAGGTGG AGGACTTCAA GGAGGACAAG GAGAAGGCCA AGGAGTGGGG CAAGGAGAAG 240

GAGAAGGAGT GGAAGCTTAC CGCCACCGAG AAGGGCAAGA TGAACAACTT CCTGGACAAC 300

AAGAACGACA TCAAGACCAA CTACAAGGAG ATCACCTTCA GCATAGCCGG CAGCTTCGAG 360

GACGAGATCA AGGACCTGAA GGAGATCGAC AAGATGTTCG ACAAGACCAA CCTGAGCAAC 420

AGCATCATCA CCTACAAGAA CGTGGAGCCC ACCACCATCG GCTTCAACAA GAGCCTGACC 480

GAGGGCAACA CCATCAACAG CGACGCCATG GCCCAGTTCA AGGAGCAGTT CCTGGACCGC 540

GACATCAAGT TCGACAGCTA CCTGGACACC CACCTGACCG CCCAGCAGGT GAGCAGCAAG 600

GAGCGCGTGA TCCTGAAGGT GACCGTCCCC AGCGGCAAGG GCAGCACCAC CCCCACCAAG 660

GCCGGCGTGA TCCTGAACAA CAGCGAGTAC AAGATGCTGA TCGACAACGG CTACATGGTG 720

CACGTGGACA AGGTGAGCAA GGTGGTGAAG AAGGGCGTGG AGTGCCTCCA GATCGAGGGC 780

ACCCTGAAGA AGAGTCTAGA CTTCAAGAAC GACATCAACG CCGAGGCCCA CAGCTGGGGC 840

ATGAAGAACT ACGAGGAGTG GGCCAAGGAC CTGACCGACA GCCAGCGCGA GGCCCTGGAC 900

GGCTACGCCC GCCAGGACTA CAAGGAGATC AACAACTACC TGCGCAACCA GGGCGGCAGC 960

GGCAACGAGA AGCTGGACGC CCAGATCAAG AACATCAGCG ACGCCCTGGG CAAGAAGCCC 1020

ATCCCCGAGA ACATCACCGT GTACCGCTGG TGCGGCATGC CCGAGTTCGG CTACCAGATC 1080

AGCGACCCCC TGCCCAGCCT GAAGGACTTC GAGGAGCAGT TCCTGAACAC CATCAAGGAG 1140

GACAAGGGCT ACATGAGCAC CAGCCTGAGC AGCGAGCGCC TGGCCGCCTT CGGCAGCCGC 1200

AAGATCATCC TGCGCCTGCA GGTGCCCAAG GGCAGCACTG GTGCCTACCT GAGCGCCATC 1260

GGCGGCTTCG CCAGCGAGAA GGAGATCCTG CTGGATAAGG ACAGCAAGTA CCACATCGAC 1320

AAGGTGACCG AGGTGATCAT CAAGGGCGTG AAGCGCTACG TGGTGGACGC CACCCTGCTG 1380

ACCAACTAG 1389 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2378 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..2375

(D) OTHER INFORMATION: /note= "Native DNA sequence encoding VIP3A(a) protein frcm AB88 as contained in pCIB7104"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

AGATGAAC ATG AAC AAG AAT AAT ACT AAA TTA AGC ACA AGA GCC TTA CCA 50 Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro 1 5 10

AGT TTT ATT GAT TAT TTT AAT GGC ATT TAT GGA TTT GCC ACT GGT ATC 98 Ser Phe He Asp Tyr Phe Asn Gly He Tyr Gly Phe Ala Thr Gly He 15 20 25 30

AAA GAC ATT ATG AAC ATG ATT TTT AAA ACG GAT ACA GGT GGT GAT CTA 146 Lys Asp He Met Asn Met He Phe Lys Thr Asp Thr Gly Gly Asp Leu 35 40 45

ACC CTA GAC GAA ATT TTA AAG AAT CAG CAG TTA CTA AAT GAT ATT TCT 194 Thr Leu Asp Glu He Leu Lys Asn Gin Gin Leu Leu Asn Asp He Ser 50 55 60

GGT AAA TTG GAT GGG GTG AAT GGA AGC TTA AAT GAT CTT ATC GCA CAG 242 Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu He Ala Gin 65 70 75

GGA AAC TTA AAT ACA GAA TTA TCT AAG GAA ATA TTA AAA ATT GCA AAT 290 Gly Asn Leu Asn Thr Glu Leu Ser Lys Glu He Leu Lys He Ala Asn 80 85 90

GAA CAA AAT CAA GTT TTA AAT GAT GTT AAT AAC AAA CTC GAT GCG ATA 338

Glu Gin Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala He

95 100 105 110

AAT ACG ATG CTT CGG GTA TAT CTA CCT AAA ATT ACC TCT ATG TTG AGT 386 Asn Thr Met Leu Arg Val Tyr Leu Pro Lys He Thr Ser Met Leu Ser 115 120 125

GAT GTA ATG AAA CAA AAT TAT GCG CTA AGT CTG CAA ATA GAA TAC TTA 434 Asp Val Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin He Glu Tyr Leu 130 135 140

AGT AAA CAA TTG CAA GAG ATT TCT GAT AAG TTG GAT ATT ATT AAT GTA 482 Ser Lys Gin Leu Gin Glu He Ser Asp Lys Leu Asp He He Asn Val 145 150 155

AAT GTA CTT ATT AAC TCT ACA CTT ACT GAA ATT ACA CCT GCG TAT CAA 530 Asn Val Leu He Asn Ser Thr Leu Thr Glu He Thr Pro Ala Tyr Gin 160 165 170

AGG ATT AAA TAT GTG AAC GAA AAA TTT GAG GAA TTA ACT TTT GCT ACA 578 Arg He Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr 175 180 185 190

GAA ACT AGT TCA AAA GTA AAA AAG GAT GGC TCT CCT GCA GAT ATT CTT 626 Glu Thr Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp He Leu 195 200 205

GAT GAG TTA ACT GAG TTA ACT GAA CTA GCG AAA AGT GTA ACA AAA AAT 674 Asp Glu Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn 210 215 220

GAT GTG GAT GGT TTT GAA TTT TAC CTT AAT ACA TTC CAC GAT GTA ATG 722 Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met 225 230 235

GTA GGA AAT AAT TTA TTC GGG CGT TCA GCT TTA AAA ACT GCA TCG GAA 770 Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ma Ser Glu 240 245 250

TTA ATT ACT AAA GAA AAT GTG AAA ACA AGT GGC AGT GAG GTC GGA AAT 818 Leu He Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn 255 260 265 270

GTT TAT AAC TTC TTA ATT GTA TTA ACA GCT CTG CAA GCC CAA GCT TTT 866 Val Tyr Asn Phe Leu He Val Leu Thr Ala Leu Gin Ala Gin Ala Phe 275 280 285

CTT ACT TTA ACA ACA TGC CGA AAA TTA TTA GGC TTA GCA GAT ATT GAT 914 Leu Thr Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp He Asp 290 295 300

TAT ACT TCT ATT ATG AAT GAA CAT TTA AAT AAG GAA AAA GAG GAA TTT 962 Tyr Thr Ser He Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe

305 310 315

AGA GTA AAC ATC CTC CCT ACA CTT TCT AAT ACT TTT TCT AAT CCT AAT 1010 Arg Val Asn He Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn 320 325 330

TAT GCA AAA GTT AAA GGA AGT GAT GAA GAT GCA AAG ATG ATT GTG GAA 1058 Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met He Val Glu 335 340 345 350

GCT AAA CCA GGA CAT GCA TTG ATT GGG TTT GAA ATT AGT AAT GAT TCA 1106 Ala Lys Pro Gly His Ala Leu He Gly Phe Glu He Ser Asn Asp Ser 355 360 365

ATT ACA GTA TTA AAA GTA TAT GAG GCT AAG CTA AAA CAA AAT TAT CAA 1154 He Thr Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gin Asn Tyr Gin 370 375 380

GTC GAT AAG GAT TCC TTA TCG GAA GTT ATT TAT GGT GAT ATG GAT AAA 1202 Val Asp Lys Asp Ser Leu Ser Glu Val He Tyr Gly Asp Met Asp Lys 385 390 395

TTA TTG TGC CCA GAT CAA TCT GAA CAA ATC TAT TAT ACA AAT AAC ATA 1250 Leu Leu Cys Pro Asp Gin Ser Glu Gin He Tyr Tyr Thr Asn Asn He 400 405 410

GTA TTT CCA AAT GAA TAT GTA ATT ACT AAA ATT GAT TTC ACT AAA AAA 1298 Val Phe Pro Asn Glu Tyr Val He Thr Lys He Asp Phe Thr Lys Lys 415 420 425 430

ATG AAA ACT TTA AGA TAT GAG GTA ACA GCG AAT TTT TAT GAT TCT TCT 1346 Met Lys Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser 435 440 445

ACA GGA GAA ATT GAC TTA AAT AAG AAA AAA GTA GAA TCA AGT GAA GCG 1394 Thr Gly Glu He Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala 450 455 460

GAG TAT AGA ACG TTA AGT GCT AAT GAT GAT GGG GTG TAT ATG CCG TTA 1442 Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu 465 470 475

GGT GTC ATC AGT GAA ACA TTT TTG ACT CCG ATT AAT GGG TTT GGC CTC 1490 Gly Val He Ser Glu Thr Phe Leu Thr Pro He Asn Gly Phe Gly Leu 480 485 490

CAA GCT GAT GAA AAT TCA AGA TTA ATT ACT TTA ACA TGT AAA TCA TAT 1538 Gin Ala Asp Glu Asn Ser Arg Leu He Thr Leu Thr Cys Lys Ser Tyr 495 500 505 510

TTA AGA GAA CTA CTG CTA GCA ACA GAC TTA AGC AAT AAA GAA ACT AAA 1586 Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys 515 520 525

TTG ATC GTC CCG CCA AGT GGT TTT ATT AGC AAT ATT GTA GAG AAC GGG 1634

Leu He Val Pro Pro Ser Gly Phe He Ser Asn He Val Glu Asn Gly 530 535 540

TCC ATA GAA GAG GAC AAT TTA GAG CCG TGG AAA GCA AAT AAT AAG AAT 1682 Ser He Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn 545 550 555

GCG TAT GTA GAT CAT ACA GGC GGA GTG AAT GGA ACT AAA GCT TTA TAT 1730 Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr 560 565 570

GTT CAT AAG GAC GGA GGA ATT TCA CAA TTT ATT GGA GAT AAG TTA AAA 1778 Val His Lys Asp Gly Gly He Ser Gin Phe He Gly Asp Lys Leu Lys 575 580 585 590

CCG AAA ACT GAG TAT GTA ATC CAA TAT ACT GTT AAA GGA AAA CCT TCT 1826 Pro Lys Thr Glu Tyr Val He Gin Tyr Thr Val Lys Gly Lys Pro Ser 595 600 605

ATT CAT TTA AAA GAT GAA AAT ACT GGA TAT ATT CAT TAT GAA GAT ACA 1874 He His Leu Lys Asp Glu Asn Thr Gly Tyr He His Tyr Glu Asp Thr 610 615 620

AAT AAT AAT TTA GAA GAT TAT CAA ACT ATT AAT AAA CGT TTT ACT ACA 1922 Asn Asn Asn Leu Glu Asp Tyr Gin Thr He Asn Lys Arg Phe Thr Thr 625 630 635

GGA ACT GAT TTA AAG GGA GTG TAT TTA ATT TTA AAA AGT CAA AAT GGA 1970 Gly Thr Asp Leu Lys Gly Val Tyr Leu He Leu Lys Ser Gin Asn Gly 640 645 650

GAT GAA GCT TGG GGA GAT AAC TTT ATT ATT TTG GAA ATT AGT CCT TCT 2018 Asp Glu Ala Trp Gly Asp Asn Phe He He Leu Glu He Ser Pro Ser 655 660 665 670

GAA AAG TTA TTA AGT CCA GAA TTA ATT AAT ACA AAT AAT TGG ACG AGT 2066 Glu Lys Leu Leu Ser Pro Glu Leu He Asn Thr Asn Asn Trp Thr Ser 675 680 685

ACG GGA TCA ACT AAT ATT AGC GGT AAT ACA CTC ACT CTT TAT CAG GGA 2114 Thr Gly Ser Thr Asn He Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly 690 695 700

GGA CGA GGG ATT CTA AAA CAA AAC CTT CAA TTA GAT AGT TTT TCA ACT 2162 Gly Arg Gly He Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr 705 710 715

TAT AGA GTG TAT TTT TCT GTG TCC GGA GAT GCT AAT GTA AGG ATT AGA 2210 Tyr Arg Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg He Arg 720 725 730

AAT TCT AGG GAA GTG TTA TTT GAA AAA AGA TAT ATG AGC GGT GCT AAA 2258 Asn Ser Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys 735 740 745 750

GAT GTT TCT GAA ATG TTC ACT ACA AAA TTT GAG AAA GAT AAC TTT TAT 2306 Asp Val Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr 755 760 765

ATA GAG CTT TCT CAA GGG AAT AAT TTA TAT GGT GGT CCT ATT GTA CAT 2354 He Glu Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro He Val His 770 775 780

TTT TAC GAT GTC TCT ATT AAG TAA 2378 Phe Tyr Asp Val Ser He Lys 785

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 789 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe 1 5 10 15

He Asp Tyr Phe Asn Gly He Tyr Gly Phe Ala Thr Gly lie Lys Asp 20 25 30

He Met Asn Met He Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 35 40 45

Asp Glu He Leu Lys Asn Gin Gin Leu Leu Asn Asp He Ser Gly Lys 50 55 60

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu He Ala Gin Gly Asn 65 70 75 80

Leu Asn Thr Glu Leu Ser Lys Glu He Leu Lys He Ala Asn Glu Gin 85 90 95

Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala He Asn Thr 100 105 110

Met Leu Arg Veil Tyr Leu Pro Lys He Thr Ser Met Leu Ser Asp Val 115 120 125

Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin He Glu Tyr Leu Ser Lys 130 135 140

Gin Leu Gin Glu He Ser Asp Lys Leu Asp He He Asn Val Asn Val 145 150 155 160

Leu He Asn Ser Thr Leu Thr Glu He Thr Pro Ala Tyr Gin Arg He

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 He Leu 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 He 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 He Val Leu Thr Ala Leu Gin Ala Gin Ala Phe Leu Thr 275 280 285

Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp He Asp Tyr Thr 290 295 300

Ser He Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 305 310 315 320

Asn He 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 He Val Glu Ala Lys 340 345 350

Pro Gly His Ala Leu He Gly Phe Glu He Ser Asn Asp Ser He 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 He Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400

Cys Pro Asp Gin Ser Glu Gin He Tyr Tyr Thr Asn Asn He Val Phe 405 410 415

Pro Asn Glu Tyr Val He Thr Lys He 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 He 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

He Ser Glu Thr Phe Leu Thr Pro He Asn Gly Phe Gly Leu Gin Ala 485 490 495

Asp Glu Asn Ser Arg Leu He 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 He 515 520 525

Val Pro Pro Ser Gly Phe He Ser Asn He Val Glu Asn Gly Ser He 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

Lys Asp Gly Gly He Ser Gin Phe He Gly Asp Lys Leu Lys Pro Lys 580 585 590

Thr Glu Tyr Val He Gin Tyr Thr Val Lys Gly Lys Pro Ser He His 595 600 605

Leu Lys Asp Glu Asn Thr Gly Tyr He His Tyr Glu Asp Thr Asn Asn 610 615 620

Asn Leu Glu Asp Tyr Gin Thr He Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640

Asp Leu Lys Gly Val Tyr Leu He Leu Lys Ser Gin Asn Gly Asp Glu 645 650 655

Ala Trp Gly Asp Asn Phe He He Leu Glu He Ser Pro Ser Glu Lys 660 665 670

Leu Leu Ser Pro Glu Leu He Asn Thr Asn Asn Trp Thr Ser Thr Gly 675 680 685

Ser Thr Asn He Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg 690 695 700

Gly He Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg 705 710 715 720

Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg He Arg Asn Ser 725 730 735

Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val 740 745 750

Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr He Glu 755 760 765

Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro He Val His Phe Tyr 770 775 780

Asp Val Ser He Lys 785

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2403 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 11..2389

(D) OTHER INFORMATION: /note= "maize optimized DNA sequence encoding VIP3A(a)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

GGATCCACCA ATGAACATGA ACAAGAACAA CACCAAGCTG AGCACCCGCG CCCTGCCGAG 60

CTTCATCGAC TACTTCAACG GCATCTACGG CTTCGCCACC GGCATCAAGG ACATCATGAA 120

CATGATCTTC AAGACCGACA CCGGCGGCGA CCTGACCCTG GACGAGATCC TGAAGAACCA 180

GCAGCTGCTG AACGACATCA GCGGCAAGCT GGACGGCGTG AACGGCAGCC TGAACGACCT 240

GATCGCCCAG GGCAACCTGA ACACCGAGCT GAGCAAGGAG ATCCTTAAGA TCGCCAACGA 300

GCAGAACCAG GTGCTGAACG ACGTGAACAA CAAGCTGGAC GCCATCAACA CCATGCTGCG 360

CGTGTACCTG CCGAAGATCA CCAGCATGCT GAGCGACGTG ATGAAGCAGA ACTACGCCCT 420

GAGCCTGCAG ATCGAGTACC TGAGCAAGCA GCTGCAGGAG ATCAGCGACA AGCTGGACAT 480

CATCAACGTG AACGTCCTGA TCAACAGCAC CCTGACCGAG ATCACCCCGG CCTACCAGCG 540

CATCAAGTAC GTGAACGAGA AGTTCGAAGA GCTGACCTTC GCCACCGAGA CCAGCAGCAA 600

GGTGAAGAAG GACGGCAGCC CGGCCGACAT CCTGGACGAG CTGACCGAGC TGACCGAGCT 660

GGCCAAGAGC GTGACCAAGA ACGACGTGGA CGGCTTCGAG TTCTACCTGA ACACCTTCCA 720

CGACGTGATG GTGGGCAACA ACCTGTTCGG CCGCAGCGCC CTGAAGACCG CCAGCGAGCT 780

GATCACCAAG GAGAACGTGA AGACCAGCGG CAGCGAGGTG GGCAACGTGT ACAACTTCCT 840

GATCGTGCTG ACCGCCCTGC AGGCCCAGGC CTTCCTGACC CTGACCACCT GTCGCAAGCT 900

GCTGGGCCTG GCCGACATCG ACTACACCAG CATCATGAAC GAGCACTTGA ACAAGGAGAA 960

GGAGGAGTTC CGCGTGAACA TCCTGCCGAC CCTGAGCAAC ACCTTCAGCA ACCCGAACTA 1020

CGCCAAGGTG AAGGGCAGCG ACGAGGACGC CAAGATGATC GTGGAGGCTA AGCCGGGCCA 1080

CGCGTTGATC GGCTTCGAGA TCAGCAACGA CAGCATCACC GTGCTGAAGG TGTACGAGGC 1140

CAAGCTGAAG CAGAACTACC AGGTGGACAA GGACAGCTTG AGCGAGGTGA TCTACGGCGA 1200

CATGGACAAG CTGCTGTGTC CGGACCAGAG CGAGCAAATC TACTACACCA ACAACATCGT 1260

GTTCCCGAAC GAGTACGTGA TCACCAAGAT CGACTTCACC AAGAAGATGA AGACCCTGCG 1320

CTACGAGGTG ACCGCCAACT TCTACGACAG CAGCACCGGC GAGATCGACC TGAACAAGAA 1380

GAAGGTGGAG AGCAGCGAGG CCGAGTACCG CACCCTGAGC GCGAACGACG ACGGCGTCTA 1440

CATGCCACTG GGCGTGATCA GCGAGACCTT CCTGACCCCG ATCAACGGCT TTGGCCTGCA 1500

GGCCGACGAG AACAGCCGCC TGATCACCCT GACCTGTAAG AGCTACCTGC GCGAGCTGCT 1560

GCTAGCCACC GACCTGAGCA ACAAGGAGAC CAAGCTGATC GTGCCACCGA GCGGCTTCAT 1620

CAGCAACATC GTGGAGAACG GCAGCATCGA GGAGGACAAC CTGGAGCCGT GGAAGGCCAA 1680

CAACAAGAAC GCCTACGTGG ACCACACCGG CGGCGTGAAC GGCACCAAGG CCCTGTACGT 1740

GCACAAGGAC GGCGGCATCA GCCAGTTCAT CGGCGACAAG CTGAAGCCGA AGACCGAGTA 1800

CGTGATCCAG TACACCGTGA AGGGCAAGCC ATCGATTCAC CTGAAGGACG AGAACACCGG 1860

CTACATCCAC TACGAGGACA CCAACAACAA CCTGGAGGAC TACCAGACCA TCAACAAGCG 1920

CTTCACCACC GGCACCGACC TGAAGGGCGT GTACCTGATC CTGAAGAGCC AGAACGGCGA 1980

CGAGGCCTGG GGCGACAACT TCATCATCCT GGAGATCAGC CCGAGCGAGA AGCTGCTGAG 2040

CCCGGAGCTG ATCAACACCA ACAACTGGAC CAGCACCGGC AGCACCAACA TCAGCGGCAA 2100

CACCCTGACC CTGTACCAGG GCGGCCGCGG CATCCTGAAG CAGAACCTGC AGCTGGACAG 2160

CTTCAGCACC TACCGCGTGT ACTTCAGCGT GAGCGGCGAC GCCAACGTGC GCATCCGCAA 2220

CAGCCGCGAG GTGCTGTTCG AGAAGAGGTA CATGAGCGGC GCCAAGGACG TGAGCGAGAT 2280

GTTCACCACC AAGTTCGAGA AGGACAACTT CTACATCGAG CTGAGCCAGG GCAACAACCT 2340

GTACGGCGGC CCGATCGTGC ACTTCTACGA CGTGAGCATC AAGTTAACGT AGAGCTCAGA 2400 TCT 2403

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2612 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (gencπάc) (iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 118..2484

(D) OTHER INFORMATION: /note= "Native DNA sequence encoding VD?3A(b) from AB424"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

ATTGAAATTG ATAAAAAGTT ATGAGTGTTT AATAATCAGT AATTACCAAT AAAGAATTAA 60

GAATACAAGT TTACAAGAAA TAAGTGTTAC AAAAAATAGC TGAAAAGGAA GATGAAC 117

ATG AAC AAG AAT AAT ACT AAA TTA AGC ACA AGA GCC TTA CCA AGT TTT 165 Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe 790 795 800 805

ATT GAT TAT TTC AAT GGC ATT TAT GGA TTT GCC ACT GGT ATC AAA GAC 213 He Asp Tyr Phe Asn Gly He Tyr Gly Phe Ala Thr Gly He Lys Asp 810 815 820

ATT ATG AAC ATG ATT TTT AAA ACG GAT ACA GGT GGT GAT CTA ACC CTA 261 He Met Asn Met He Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 825 830 835

GAC GAA ATT TTA AAG AAT CAG CAG CTA CTA AAT GAT ATT TCT GGT AAA 309 Asp Glu He Leu Lys Asn Gin Gin Leu Leu Asn Asp He Ser Gly Lys 840 845 850

TTG GAT GGG GTG AAT GGA AGC TTA AAT GAT CTT ATC GCA CAG GGA AAC 357 Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu He Ala Gin Gly Asn 855 860 865

TTA AAT ACA GAA TTA TCT AAG GAA ATA TTA AAA ATT GCA AAT GAA CAA 405 Leu Asn Thr Glu Leu Ser Lys Glu He Leu Lys He Ala Asn Glu Gin 870 875 880 885

AAT CAA GTT TTA AAT GAT GTT AAT AAC AAA CTC GAT GCG ATA AAT ACG 453

Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala He Asn Thr 890 895 900

ATG CTT CGG GTA TAT CTA CCT AAA ATT ACC TCT ATG TTG AGT GAT GTA 501 Met Leu Arg Val Tyr Leu Pro Lys He Thr Ser Met Leu Ser Asp Val 905 910 915

ATG AAA CAA AAT TAT GCG CTA AGT CTG CAA ATA GAA TAC TTA AGT AAA 549 Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin He Glu Tyr Leu Ser Lys 920 925 930

CAA TTG CAA GAG ATT TCT GAT AAG TTG GAT ATT ATT AAT GTA AAT GTA 597 Gin Leu Gin Glu He Ser Asp Lys Leu Asp He He Asn Val Asn Val 935 940 945

CTT ATT AAC TCT ACA CTT ACT GAA ATT ACA CCT GCG TAT CAA AGG ATT 645 Leu He Asn Ser Thr Leu Thr Glu He Thr Pro Ala Tyr Gin Arg He 950 955 960 965

AAA TAT GTG AAC GAA AAA TTT GAG GAA TTA ACT TTT GCT ACA GAA ACT 693 Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr 970 975 980

AGT TCA AAA GTA AAA AAG GAT GGC TCT CCT GCA GAT ATT CGT GAT GAG 741 Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp He Arg Asp Glu 985 990 995

TTA ACT GAG TTA ACT GAA CTA GCG AAA AGT GTA ACA AAA AAT GAT GTG 789 Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val 1000 1005 1010

GAT GGT TTT GAA TTT TAC CTT AAT ACA TTC CAC GAT GTA ATG GTA GGA 837 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly 1015 1020 1025

AAT AAT TTA TTC GGG CGT TCA GCT TTA AAA ACT GCA TCG GAA TTA ATT 885 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu He 1030 1035 1040 1045

ACT AAA GAA AAT GTG AAA ACA AGT GGC AGT GAG GTC GGA AAT GTT TAT 933 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr 1050 1055 1060

AAC TTC CTA ATT GTA TTA ACA GCT CTG CAA GCA AAA GCT TTT CTT ACT 981 Asn Phe Leu He Val Leu Thr Ala Leu Gin Ala Lys Ala Phe Leu Thr 1065 1070 1075

TTA ACA CCA TGC CGA AAA TTA TTA GGC TTA GCA GAT ATT GAT TAT ACT 1029 Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp He Asp Tyr Thr 1080 1085 1090

TCT ATT ATG AAT GAA CAT TTA AAT AAG GAA AAA GAG GAA TTT AGA GTA 1077 Ser He Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 1095 1100 1105

AAC ATC CTC CCT ACA CTT TCT AAT ACT TTT TCT AAT CCT AAT TAT GCA 1125 Asn He Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala 1110 1115 1120 1125

AAA GTT AAA GGA AGT GAT GAA GAT GCA AAG ATG ATT GTG GAA GCT AAA 1173 Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met He Val Glu Ala Lys 1130 1135 1140

CCA GGA CAT GCA TTG ATT GGG TTT GAA ATT AGT AAT GAT TCA ATT ACA 1221 Pro Gly His Ala Leu He Gly Phe Glu He Ser Asn Asp Ser He Thr 1145 1150 1155

GTA TTA AAA GTA TAT GAG GCT AAG CTA AAA CAA AAT TAT CAA GTC GAT 1269 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gin Asn Tyr Gin Val Asp 1160 1165 1170

AAG GAT TCC TTA TCG GAA GTT ATT TAT GGC GAT ATG GAT AAA TTA TTG 1317 Lys Asp Ser Leu Ser Glu Val He Tyr Gly Asp Met Asp Lys Leu Leu 1175 1180 1185

TGC CCA GAT CAA TCT GGA CAA ATC TAT TAT ACA AAT AAC ATA GTA TTT 1365 Cys Pro Asp Gin Ser Gly Gin He Tyr Tyr Thr Asn Asn He Val Phe 1190 1195 1200 1205

CCA AAT GAA TAT GTA ATT ACT AAA ATT GAT TTC ACT AAA AAA ATG AAA 1413 Pro Asn Glu Tyr Val He Thr Lys He Asp Phe Thr Lys Lys Met Lys 1210 1215 1220

ACT TTA AGA TAT GAG GTA ACA GCG AAT TTT TAT GAT TCT TCT ACA GGA 1461 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 1225 1230 1235

GAA ATT GAC TTA AAT AAG AAA AAA GTA GAA TCA AGT GAA GCG GAG TAT 1509 Glu He Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr 1240 1245 1250

AGA ACG TTA AGT GCT AAT GAT GAT GGG GTG TAT ATG CCG TTA GGT GTC 1557 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val 1255 1260 1265

ATC AGT GAA ACA TTT TTG ACT CCG ATT AAT GGG TTT GGC CTC CAA GCT 1605 He Ser Glu Thr Phe Leu Thr Pro He Asn Gly Phe Gly Leu Gin Ala 1270 1275 1280 1285

GAT GAA AAT TCA AGA TTA ATT ACT TTA ACA TGT AAA TCA TAT TTA AGA 1653 Asp Glu Asn Ser Arg Leu He Thr Leu Thr Cys Lys Ser Tyr Leu Arg 1290 1295 1300

GAA CTA CTG CTA GCA ACA GAC TTA AGC AAT AAA GAA ACT AAA TTG ATC 1701 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu He 1305 1310 1315

GTC CCG CCA AGT GGT TTT ATT AGC AAT ATT GTA GAG AAC GGG TCC ATA 1749 Val Pro Pro Ser Gly Phe He Ser Asn He Val Glu Asn Gly Ser He 1320 1325 1330

GAA GAG GAC AAT TTA GAG CCG TGG AAA GCA AAT AAT AAG AAT GCG TAT 1797 Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr 1335 1340 1345

GTA GAT CAT ACA GGC GGA GTG AAT GGA ACT AAA GCT TTA TAT GTT CAT 1845 Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His 1350 1355 1360 1365

AAG GAC GGA GGA ATT TCA CAA TTT ATT GGA GAT AAG TTA AAA CCG AAA 1893 Lys Asp Gly Gly He Ser Gin Phe He Gly Asp Lys Leu Lys Pro Lys 1370 1375 1380

ACT GAG TAT GTA ATC CAA TAT ACT GTT AAA GGA AAA CCT TCT ATT CAT 1941 Thr Glu Tyr Val He Gin Tyr Thr Val Lys Gly Lys Pro Ser He His 1385 1390 1395

TTA AAA GAT GAA AAT ACT GGA TAT ATT CAT TAT GAA GAT ACA AAT AAT 1989 Leu Lys Asp Glu Asn Thr Gly Tyr He His Tyr Glu Asp Thr Asn Asn 1400 1405 1410

AAT TTA GAA GAT TAT CAA ACT ATT AAT AAA CGT TTT ACT ACA GGA ACT 2037 Asn Leu Glu Asp Tyr Gin Thr He Asn Lys Arg Phe Thr Thr Gly Thr 1415 1420 1425

GAT TTA AAG GGA GTG TAT TTA ATT TTA AAA AGT CAA AAT GGA GAT GAA 2085 Asp Leu Lys Gly Val Tyr Leu He Leu Lys Ser Gin Asn Gly Asp Glu 1430 1435 1440 1445

GCT TGG GGA GAT AAC TTT ATT ATT TTG GAA ATT AGT CCT TCT GAA AAG 2133 Ala Trp Gly Asp Asn Phe He He Leu Glu He Ser Pro Ser Glu Lys 1450 1455 1460

TTA TTA AGT CCA GAA TTA ATT AAT ACA AAT AAT TGG ACG AGT ACG GGA 2181 Leu Leu Ser Pro Glu Leu He Asn Thr Asn Asn Trp Thr Ser Thr Gly 1465 1470 1475

TCA ACT AAT ATT AGC GGT AAT ACA CTC ACT CTT TAT CAG GGA GGA CGA 2229 Ser Thr Asn He Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg 1480 1485 1490

GGG ATT CTA AAA CAA AAC CTT CAA TTA GAT AGT TTT TCA ACT TAT AGA 2277 Gly He Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg 1495 1500 1505

GTG TAT TTC TCT GTG TCC GGA GAT GCT AAT GTA AGG ATT AGA AAT TCT 2325 Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg He Arg Asn Ser 1510 1515 1520 1525

AGG GAA GTG TTA TTT GAA AAA AGA TAT ATG AGC GGT GCT AAA GAT GTT 2373 Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val 1530 1535 1540

TCT GAA ATG TTC ACT ACA AAA TTT GAG AAA GAT AAC TTC TAT ATA GAG 2421 Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr He Glu

1545 1550 1555

CTT TCT CAA GGG AAT AAT TTA TAT GGT GGT CCT ATT GTA CAT TTT TAC 2469 Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro He Val His Phe Tyr 1560 1565 1570

GAT GTC TCT ATT AAG TAAGATCGGG ATCTAATATT AACAGTTTTT AGAAGCTAAT 2524 Asp Val Ser He Lys 1575

TCTTGTATAA TGTCCTTGAT TATGGAAAAA CACAATTTTG TTTGCTAAGA TGTATATATA 2584

GCTCACTCAT TAAAAGGCAA TCAAGCTT 2612

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 789 amino acids

(B) TYPE: amino acid (D) 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 15

He Asp Tyr Phe Asn Gly He Tyr Gly Phe Ala Thr Gly He Lys Asp 20 25 30

He Met Asn Met He Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 35 40 45

Asp Glu He Leu Lys Asn Gin Gin Leu Leu Asn Asp He Ser Gly Lys 50 55 60

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu He Ala Gin Gly Asn 65 70 75 80

Leu Asn Thr Glu Leu Ser Lys Glu He Leu Lys He Ala Asn Glu Gin 85 90 95

Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala He Asn Thr 100 105 110

Met Leu Arg Val Tyr Leu Pro Lys He Thr Ser Met Leu Ser Asp Val 115 120 125

Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin He Glu Tyr Leu Ser Lys 130 135 140

Gin Leu Gin Glu He Ser Asp Lys Leu Asp He He Asn Val Asn Val 145 150 155 160

Leu He Asn Ser Thr Leu Thr Glu He Thr Pro Ala Tyr Gin Arg He 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 He 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 He 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 He 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 He Asp Tyr Thr 290 295 300

Ser He Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 305 310 315 320

Asn He 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 He Val Glu Ala Lys 340 345 350

Pro Gly His Ala Leu He Gly Phe Glu He Ser Asn Asp Ser He 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 He Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400

Cys Pro Asp Gin Ser Gly Gin He Tyr Tyr Thr Asn Asn He Val Phe 405 410 415

Pro Asn Glu Tyr Val He Thr Lys He 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 He 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

He Ser Glu Thr Phe Leu Thr Pro He Asn Gly Phe Gly Leu Gin Ala 485 490 495

Asp Glu Asn Ser Arg Leu He 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 He 515 520 525

Val Pro Pro Ser Gly Phe He Ser Asn He Val Glu Asn Gly Ser He 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

Lys Asp Gly Gly He Ser Gin Phe He Gly Asp Lys Leu Lys Pro Lys 580 585 590

Thr Glu Tyr Val He Gin Tyr Thr Val Lys Gly Lys Pro Ser He His 595 600 605

Leu Lys Asp Glu Asn Thr Gly Tyr He His Tyr Glu Asp Thr Asn Asn 610 615 620

Asn Leu Glu Asp Tyr Gin Thr He Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640

Asp Leu Lys Gly Val Tyr Leu He Leu Lys Ser Gin Asn Gly Asp Glu 645 650 655

Ala Trp Gly Asp Asn Phe He He Leu Glu He Ser Pro Ser Glu Lys 660 665 670

Leu Leu Ser Pro Glu Leu He Asn Thr Asn Asn Trp Thr Ser Thr Gly 675 680 685

Ser Thr Asn He Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg 690 695 700

Gly He Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg 705 710 715 720

Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg He Arg Asn Ser 725 730 735

Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val

740 745 750

Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr He Glu 755 760 765

Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro He Val His Phe Tyr 770 775 780

Asp Val Ser He Lys 785

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "forward primer used to make pCIB5526"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GGATCCACCA TGAAGACCAA CCAGATCAGC 30

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "reverse primer used to make pCIB5526"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: AAGCTTCAGC TCCTT 15

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2576 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..2564

(D) OTHER INFORMATION: /note= "Maize optimized sequence encoding VIPlA(a) with the Bacillus secretion signal removed as contained in pCIB5526"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

GATCCACC ATG AAG ACC AAC CAG ATC AGC ACC ACC CAG AAG AAC CAG CAG 50 Met Lys Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin 825 830 835

AAG GAG ATG GAC CGC AAG GGC CTG CTG GGC TAC TAC TTC AAG GGC AAG 98 Lys Glu Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys 840 845 850

GAC TTC AGC AAC CTG ACC ATG TTC GCC CCC ACG CGT GAC AGC ACC CTG 146 Asp Phe Ser Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu 855 860 865

ATC TAC GAC CAG CAG ACC GCC AAC AAG CTG CTG GAC AAG AAG CAG CAG 194 He Tyr Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin 870 875 880

GAG TAC CAG AGC ATC CGC TGG ATC GGC CTG ATC CAG AGC AAG GAG ACC 242 Glu Tyr Gin Ser He Arg Trp He Gly Leu He Gin Ser Lys Glu Thr 885 890 895

GGC GAC TTC ACC TTC AAC CTG AGC GAG GAC GAG CAG GCC ATC ATC GAG 290 Gly Asp Phe Thr Phe Asn Leu Ser Glu Asp Glu Gin Ala He He Glu 900 905 910 915

ATC AAC GGC AAG ATC ATC AGC AAC AAG GGC AAG GAG AAG CAG GTG GTG 338 He Asn Gly Lys He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val 920 925 930

CAC CTG GAG AAG GGC AAG CTG GTG CCC ATC AAG ATC GAG TAC CAG AGC 386 His Leu Glu Lys Gly Lys Leu Val Pro He Lys He Glu Tyr Gin Ser 935 940 945

GAC ACC AAG TTC AAC ATC GAC AGC AAG ACC TTC AAG GAG CTG AAG CTT 434

Asp Thr Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu 950 955 960

TTC AAG ATC GAC AGC CAG AAC CAG CCC CAG CAG GTG CAG CAG GAC GAG 482 Phe Lys He Asp Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu 965 970 975

CTG CGC AAC CCC GAG TTC AAC AAG AAG GAG AGC CAG GAG TTC CTG GCC 530 Leu Arg Asn Pro Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala 980 985 990 995

AAG CCC AGC AAG ATC AAC CTG TTC ACC CAG CAG ATG AAG CGC GAG ATC 578 Lys Pro Ser Lys He Asn Leu Phe Thr Gin Gin Met Lys Arg Glu He 1000 1005 1010

GAC GAG GAC ACC GAC ACC GAC GGC GAC AGC ATC CCC GAC CTG TGG GAG 626 Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu 1015 1020 1025

GAG AAC GGC TAC ACC ATC CAG AAC CGC ATC GCC GTG AAG TGG GAC GAC 674 Glu Asn Gly Tyr Thr He Gin Asn Arg He Ala Val Lys Trp Asp Asp 1030 1035 1040

AGC CTG GCT AGC AAG GGC TAC ACC AAG TTC GTG AGC AAC CCC CTG GAG 722 Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu 1045 1050 1055

AGC CAC ACC GTG GGC GAC CCC TAC ACC GAC TAC GAG AAG GCC GCC CGC 770 Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg 1060 1065 1070 1075

GAC CTG GAC CTG AGC AAC GCC AAG GAG ACC TTC AAC CCC CTG GTG GCC 818 Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala 1080 1085 1090

GCC TTC CCC AGC GTG AAC GTG AGC ATG GAG AAG GTG ATC CTG AGC CCC 866 Ala Phe Pro Ser Val Asn Val Ser Met Glu Lys Val He Leu Ser Pro 1095 1100 1105

AAC GAG AAC CTG AGC AAC AGC GTG GAG AGC CAC TCG AGC ACC AAC TGG 914 Asn Glu Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp 1110 1115 1120

AGC TAC ACC AAC ACC GAG GGC GCC AGC GTG GAG GCC GGC ATC GGT CCC 962 Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gly He Gly Pro 1125 1130 1135

AAG GGC ATC AGC TTC GGC GTG AGC GTG AAC TAC CAG CAC AGC GAG ACC 1010 Lys Gly He Ser Phe Gly Val Ser Val Asn Tyr Gin His Ser Glu Thr 1140 1145 1150 1155

GTG GCC CAG GAG TGG GGC ACC AGC ACC GGC AAC ACC AGC CAG TTC AAC 1058 Val Ala Gin Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn 1160 1165 1170

ACC GCC AGC GCC GGC TAC CTG AAC GCC AAC GTG CGC TAC AAC AAC GTG 1106 Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val 1175 1180 1185

GGC ACC GGC GCC ATC TAC GAC GTG AAG CCC ACC ACC AGC TTC GTG CTG 1154 Gly Thr Gly Ala He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu 1190 1195 1200

AAC AAC GAC ACC ATC GCC ACC ATC ACC GCC AAG TCG AAT TCC ACC GCC 1202 Asn Asn Asp Thr He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala 1205 1210 1215

CTG AAC ATC AGC CCC GGC GAG AGC TAC CCC AAG AAG GGC CAG AAC GGC 1250 Leu Asn He Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly 1220 1225 1230 1235

ATC GCC ATC ACC AGC ATG GAC GAC TTC AAC AGC CAC CCC ATC ACC CTG 1298 He Ala He Thr Ser Met Asp Asp Phe Asn Ser His Pro He Thr Leu 1240 1245 1250

AAC AAG AAG CAG GTG GAC AAC CTG CTG AAC AAC AAG CCC ATG ATG CTG 1346 Asn Lys Lys Gin Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu 1255 1260 1265

GAG ACC AAC CAG ACC GAC GGC GTC TAC AAG ATC AAG GAC ACC CAC GGC 1394 Glu Thr Asn Gin Thr Asp Gly Val Tyr Lys He Lys Asp Thr His Gly 1270 1275 1280

AAC ATC GTG ACG GGC GGC GAG TGG AAC GGC GTG ATC CAG CAG ATC AAG 1442 Asn He Val Thr Gly Gly Glu Trp Asn Gly Val He Gin Gin He Lys 1285 1290 1295

GCC AAG ACC GCC AGC ATC ATC GTC GAC GAC GGC GAG CGC GTG GCC GAG 1490 Ala Lys Thr Ala Ser He He Val Asp Asp Gly Glu Arg Val Ala Glu 1300 1305 1310 1315

AAG CGC GTG GCC GCC AAG GAC TAC GAG AAC CCC GAG GAC AAG ACC CCC 1538 Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro 1320 1325 1330

AGC CTG ACC CTG AAG GAC GCC CTG AAG CTG AGC TAC CCC GAC GAG ATC 1586 Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu He 1335 1340 1345

AAG GAG ATC GAG GGC TTG CTG TAC TAC AAG AAC AAG CCC ATC TAC GAG 1634 Lys Glu He Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pro He Tyr Glu 1350 1355 1360

AGC AGC GTG ATG ACC TAT CTA GAC GAG AAC ACC GCC AAG GAG GTG ACC 1682 Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr 1365 1370 1375

AAG CAG CTG AAC GAC ACC ACC GGC AAG TTC AAG GAC GTG AGC CAC CTG 1730 Lys Gin Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu 1380 1385 1390 1395

TAC GAC GTG AAG CTG ACC CCC AAG ATG AAC GTG ACC ATC AAG CTG AGC 1778 Tyr Asp Val Lys Leu Thr Pro Lys Met Asn Val Thr He Lys Leu Ser 1400 1405 1410

ATC CTG TAC GAC AAC GCC GAG AGC AAC GAC AAC AGC ATC GGC AAG TGG 1826 He Leu Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser He Gly Lys Trp 1415 1420 1425

ACC AAC ACC AAC ATC GTG AGC GGC GGC AAC AAC GGC AAG AAG CAG TAC 1874 Thr Asn Thr Asn He Val Ser Gly Gly Asn Asn Gly Lys Lys Gin Tyr 1430 1435 1440

AGC AGC AAC AAC CCC GAC GCC AAC CTG ACC CTG AAC ACC GAC GCC CAG 1922 Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin 1445 1450 1455

GAG AAG CTG AAC AAG AAC CGC GAC TAC TAC ATC AGC CTG TAC ATG AAG 1970 Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr He Ser Leu Tyr Met Lys 1460 1465 1470 1475

AGC GAG AAG AAC ACC CAG TGC GAG ATC ACC ATC GAC GGC GAG ATA TAC 2018 Ser Glu Lys Asn Thr Gin Cys Glu He Thr He Asp Gly Glu He Tyr 1480 1485 1490

CCC ATC ACC ACC AAG ACC GTG AAC GTG AAC AAG GAC AAC TAC AAG CGC 2066 Pro He Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg 1495 1500 1505

CTG GAC ATC ATC GCC CAC AAC ATC AAG AGC AAC CCC ATC AGC AGC CTG 2114 Leu Asp He He Ala His Asn He Lys Ser Asn Pro He Ser Ser Leu 1510 1515 1520

CAC ATC AAG ACC AAC GAC GAG ATC ACC CTG TTC TGG GAC GAC ATA TCG 2162 His He Lys Thr Asn Asp Glu He Thr Leu Phe Trp Asp Asp He Ser 1525 1530 1535

ATT ACC GAC GTC GCC AGC ATC AAG CCC GAG AAC CTG ACC GAC AGC GAG 2210 He Thr Asp Val Ala Ser He Lys Pro Glu Asn Leu Thr Asp Ser Glu 1540 1545 1550 1555

ATC AAG CAG ATA TAC AGT CGC TAC GGC ATC AAG CTG GAG GAC GGC ATC 2258 He Lys Gin He Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly He 1560 1565 1570

CTG ATC GAC AAG AAA GGC GGC ATC CAC TAC GGC GAG TTC ATC AAC GAG 2306 Leu He Asp Lys Lys Gly Gly He His Tyr Gly Glu Phe He Asn Glu 1575 1580 1585

GCC AGC TTC AAC ATC GAG CCC CTG CAG AAC TAC GTG ACC AAG TAC GAG 2354 Ala Ser Phe Asn He Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu 1590 1595 1600

GTG ACC TAC AGC AGC GAG CTG GGC CCC AAC GTG AGC GAC ACC CTG GAG 2402 Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu

1605 1610 1615

AGC GAC AAG ATT TAC AAG GAC GGC ACC ATC AAG TTC GAC TTC ACC AAG 2450 Ser Asp Lys He Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys 1620 1625 1630 1635

TAC AGC AAG AAC GAG CAG GGC CTG TTC TAC GAC AGC GGC CTG AAC TGG 2498 Tyr Ser Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp 1640 1645 1650

GAC TTC AAG ATC AAC GCC ATC ACC TAC GAC GGC AAG GAG ATG AAC GTG 2546 Asp Phe Lys He Asn Ala He Thr Tyr Asp Gly Lys Glu Met Asn Val 1655 1660 1665

TTC CAC CGC TAC AAC AAG TAGATCTGAG CT 2576

Phe His Arg Tyr Asn Lys 1670

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 852 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Met Lys Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin Lys Glu 1 5 10 15

Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe 20 25 30

Ser Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr 35 40 45

Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin Glu Tyr 50 55 60

Gin Ser He Arg Trp He Gly Leu He Gin Ser Lys Glu Thr Gly Asp 65 70 75 80

Phe Thr Phe Asn Leu Ser Glu Asp Glu Gin Ala He He Glu He Asn 85 90 95

Gly Lys He He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu 100 105 110

Glu Lys Gly Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr 115 120 125

Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys

130 135 140

He 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 He Asn Leu Phe Thr Gin Gin Met Lys Arg Glu He Asp Glu 180 185 190

Asp Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn 195 200 205

Gly Tyr Thr He Gin Asn Arg He 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 He 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 He Gly Pro Lys Gly 305 310 315 320

He 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 He Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn 370 375 380

Asp Thr He Ala Thr He Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn 385 390 395 400

He Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly He Ala 405 410 415

He Thr Ser Met Asp Asp Phe Asn Ser His Pro He 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 He Lys Asp Thr His Gly Asn He 450 455 460

Val Thr Gly Gly Glu Trp Asn Gly Val He Gin Gin He Lys Ala Lys 465 470 475 480

Thr Ala Ser He He 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 He Lys Glu 515 520 525

He Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pro He 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 He Lys Leu Ser He Leu 580 585 590

Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser He Gly Lys Trp Thr Asn 595 600 605

Thr Asn He 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 He Ser Leu Tyr Met Lys Ser Glu 645 650 655

Lys Asn Thr Gin Cys Glu He Thr He Asp Gly Glu He Tyr Pro He 660 665 670

Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp 675 680 685

He He Ala His Asn He Lys Ser Asn Pro He Ser Ser Leu His He 690 695 700

Lys Thr Asn Asp Glu He Thr Leu Phe Trp Asp Asp He Ser He Thr 705 710 715 720

Asp Val Ala Ser He Lys Pro Glu Asn Leu Thr Asp Ser Glu He Lys 725 730 735

Gin He Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly He Leu He 740 745 750

Asp Lys Lys Gly Gly He His Tyr Gly Glu Phe He Asn Glu Ala Ser 755 760 765

Phe Asn He 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 He Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys Tyr Ser 805 810 815

Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe 820 825 830

Lys He Asn Ala He Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His 835 840 845

Arg Tyr Asn Lys 850

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "forward primer used to make pCIB5527"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGATCCACCA TGCTGCAGAA CCTGAAGATC AC 32

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "reverse primer used to make pCIB5527"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: AAGCTTCCAC TCCTTCTC 18

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1241 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..1238

(D) OTHER INFORMATION: /note= "Maize optimized DNA sequence encoding VTP2A(a) with the Bacillus secretion signal removed as contained in pCIB5527"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

GATCCACC ATG CTG CAG AAC CTG AAG ATC ACC GAC AAG GTG GAG GAC TTC 50 Met Leu Gin Asn Leu Lys He 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 CTT ACC GCC ACC GAG AAG GGC AAG ATG AAC AAC TTC 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 TTC AGC 194 Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr Phe Ser 900 905 910

ATA GCC GGC AGC TTC GAG GAC GAG ATC AAG GAC CTG AAG GAG ATC GAC 242

He Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu He Asp 915 920 925 930

AAG ATG TTC GAC AAG ACC AAC CTG AGC AAC AGC ATC ATC ACC TAC AAG 290 Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr Tyr Lys 935 940 945

AAC GTG GAG CCC ACC ACC ATC GGC TTC AAC AAG AGC CTG ACC GAG GGC 338 Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr Glu Gly 950 955 960

AAC ACC ATC AAC AGC GAC GCC ATG GCC CAG TTC AAG GAG CAG TTC CTG 386 Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin Phe Leu 965 970 975

GAC CGC GAC ATC AAG TTC GAC AGC TAC CTG GAC ACC CAC CTG ACC GCC 434 Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala 980 985 990

CAG CAG GTG AGC AGC AAG GAG CGC GTG ATC CTG AAG GTG ACC GTC CCC 482 Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr Val Pro 995 1000 1005 1010

AGC GGC AAG GGC AGC ACC ACC CCC ACC AAG GCC GGC GTG ATC CTG AAC 530 Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He Leu Asn 1015 1020 1025

AAC AGC GAG TAC AAG ATG CTG ATC GAC AAC GGC TAC ATG GTG CAC GTG 578 Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val His Val 1030 1035 1040

GAC AAG GTG AGC AAG GTG GTG AAG AAG GGC GTG GAG TGC CTC CAG ATC 626 Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu Gin He 1045 1050 1055

GAG GGC ACC CTG AAG AAG AGT CTA GAC TTC AAG AAC GAC ATC AAC GCC 674 Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He Asn Ala 1060 1065 1070

GAG GCC CAC AGC TGG GGC ATG AAG AAC TAC GAG GAG TGG GCC AAG GAC 722 Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp 1075 1080 1085 1090

CTG ACC GAC AGC CAG CGC GAG GCC CTG GAC GGC TAC GCC CGC CAG GAC 770 Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gin Asp 1095 1100 1105

TAC AAG GAG ATC AAC AAC TAC CTG CGC AAC CAG GGC GGC AGC GGC AAC 818 Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser Gly Asn 1110 1115 1120

GAG AAG CTG GAC GCC CAG ATC AAG AAC ATC AGC GAC GCC CTG GGC AAG 866 Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu Gly Lys 1125 1130 1135

AAG CCC ATC CCC GAG AAC ATC ACC GTG TAC CGC TGG TGC GGC ATG CCC 914 Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly Met Pro 1140 1145 1150

GAG TTC GGC TAC CAG ATC AGC GAC CCC CTG CCC AGC CTG AAG GAC TTC 962 Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe 1155 1160 1165 1170

GAG GAG CAG TTC CTG AAC ACC ATC AAG GAG GAC AAG GGC TAC ATG AGC 1010 Glu Glu Gin Phe Leu Asn Thr He Lys Glu Asp Lys Gly Tyr Met Ser 1175 1180 1185

ACC AGC CTG AGC AGC GAG CGC CTG GCC GCC TTC GGC AGC CGC AAG ATC 1058 Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg Lys He 1190 1195 1200

ATC CTG CGC CTG CAG GTG CCC AAG GGC AGC ACT GGT GCC TAC CTG AGC 1106 He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser 1205 1210 1215

GCC ATC GGC GGC TTC GCC AGC GAG AAG GAG ATC CTG CTG GAT AAG GAC 1154 Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp Lys Asp 1220 1225 1230

AGC AAG TAC CAC ATC GAC AAG GTG ACC GAG GTG ATC ATC AAG GGC GTG 1202 Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He Lys Gly Val 1235 1240 1245 1250

AAG CGC TAC GTG GTG GAC GCC ACC CTG CTG ACC AAC TAG 1241

Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 1255 1260

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 410 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Met Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu Asp Phe Lys Glu 1 5 10 15

Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp 20 25 30

Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu Asp Asn 35 40 45

Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr Phe Ser He Ala 50 55 60

Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu He Asp Lys Met 65 70 75 80

Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr Tyr Lys Asn Val 85 90 95

Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr Glu Gly Asn Thr 100 105 110

He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin Phe Leu Asp Arg 115 120 125

Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala Gin Gin 130 135 140

Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr Val Pro Ser Gly 145 150 155 160

Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He Leu Asn Asn Ser 165 170 175

Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val His Val Asp Lys 180 185 190

Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu Gin He Glu Gly 195 200 205

Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He Asn Ala Glu Ala 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 He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser Gly Asn Glu Lys 260 265 270

Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu Gly Lys Lys Pro 275 280 285

He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly Met Pro Glu Phe 290 295 300

Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe Glu Glu 305 310 315 320

Gin Phe Leu Asn Thr He 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 He He Leu 340 345 350

Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser Ala He 355 360 365

Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp Lys Asp Ser Lys 370 375 380

Tyr His He Asp Lys Val Thr Glu Val He He Lys Gly Val Lys Arg 385 390 395 400

Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 405 410

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide encoding eukaryotic secretion signal used to construct pCIB5527"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GGATCCACCA TGGGCTGGAG CTGGATCTTC CTGTTCCTGC TGAGCGGCGC CGCGGGCGTG 60 CACTGCCTGC AG 72

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1241 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..1238

(D) OTHER INFORMATION: /note= "Maize optimized DNA sequence encoding VIP2A(a) with the Bacillus secretion signal removed and the eukaryotic secretion signal inserted as

contained in pCIB5528"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

GATCCACC ATG CTG CAG AAC CTG AAG ATC ACC GAC AAG GTG GAG GAC TTC 50

Met Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu Asp Phe

415 420

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 425 430 435 440

GAG TGG AAG CTT ACC GCC ACC GAG AAG GGC AAG ATG AAC AAC TTC CTG 146 Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu 445 450 455

GAC AAC AAG AAC GAC ATC AAG ACC AAC TAC AAG GAG ATC ACC TTC AGC 194 Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr Phe Ser 460 465 470

ATA GCC GGC AGC TTC GAG GAC GAG ATC AAG GAC CTG AAG GAG ATC GAC 242 He Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu He Asp 475 480 485

AAG ATG TTC GAC AAG ACC AAC CTG AGC AAC AGC ATC ATC ACC TAC AAG 290 Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr Tyr Lys 490 495 500

AAC GTG GAG CCC ACC ACC ATC GGC TTC AAC AAG AGC CTG ACC GAG GGC 338 Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr Glu Gly 505 510 515 520

AAC ACC ATC AAC AGC GAC GCC ATG GCC CAG TTC AAG GAG CAG TTC CTG 386 Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin Phe Leu 525 530 535

GAC CGC GAC ATC AAG TTC GAC AGC TAC CTG GAC ACC CAC CTG ACC GCC 434 Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala 540 545 550

CAG CAG GTG AGC AGC AAG GAG CGC GTG ATC CTG AAG GTG ACC GTC CCC 482 Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr Val Pro 555 560 565

AGC GGC AAG GGC AGC ACC ACC CCC ACC AAG GCC GGC GTG ATC CTG AAC 530 Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He Leu Asn 570 575 580

AAC AGC GAG TAC AAG ATG CTG ATC GAC AAC GGC TAC ATG GTG CAC GTG 578 Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val His Val 585 590 595 600

GAC AAG GTG AGC AAG GTG GTG AAG AAG GGC GTG GAG TGC CTC CAG ATC 626 Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu Gin He

605 610 615

GAG GGC ACC CTG AAG AAG AGT CTA GAC TTC AAG AAC GAC ATC AAC GCC 674 Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He Asn Ala 620 625 630

GAG GCC CAC AGC TGG GGC ATG AAG AAC TAC GAG GAG TGG GCC AAG GAC 722 Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp 635 640 645

CTG ACC GAC AGC CAG CGC GAG GCC CTG GAC GGC TAC GCC CGC CAG GAC 770 Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gin Asp 650 655 660

TAC AAG GAG ATC AAC AAC TAC CTG CGC AAC CAG GGC GGC AGC GGC AAC 818 Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser Gly Asn 665 670 675 680

GAG AAG CTG GAC GCC CAG ATC AAG AAC ATC AGC GAC GCC CTG GGC AAG 866 Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu Gly Lys 685 690 695

AAG CCC ATC CCC GAG AAC ATC ACC GTG TAC CGC TGG TGC GGC ATG CCC 914 Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly Met Pro 700 705 710

GAG TTC GGC TAC CAG ATC AGC GAC CCC CTG CCC AGC CTG AAG GAC TTC 962 Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe 715 720 725

GAG GAG CAG TTC CTG AAC ACC ATC AAG GAG GAC AAG GGC TAC ATG AGC 1010 Glu Glu Gin Phe Leu Asn Thr He Lys Glu Asp Lys Gly Tyr Met Ser 730 735 740

ACC AGC CTG AGC AGC GAG CGC CTG GCC GCC TTC GGC AGC CGC AAG ATC 1058 Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg Lys He 745 750 755 760

ATC CTG CGC CTG CAG GTG CCC AAG GGC AGC ACT GGT GCC TAC CTG AGC 1106 He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser 765 770 775

GCC ATC GGC GGC TTC GCC AGC GAG AAG GAG ATC CTG CTG GAT AAG GAC 1154 Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp Lys Asp 780 785 790

AGC AAG TAC CAC ATC GAC AAG GTG ACC GAG GTG ATC ATC AAG GGC GTG 1202 Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He Lys Gly Val 795 800 805

AAG CGC TAC GTG GTG GAC GCC ACC CTG CTG ACC AAC TAG 1241 Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 810 815 820

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 410 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Met Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu Asp Phe Lys Glu

1 5 10 15

Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp 20 25 30

Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu Asp Asn 35 40 45

Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr Phe Ser He Ala 50 55 60

Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu He Asp Lys Met 65 70 75 80

Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr Tyr Lys Asn Val 85 90 95

Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr Glu Gly Asn Thr 100 105 110

He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin Phe Leu Asp Arg 115 120 125

Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala Gin Gin 130 135 140

Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr Val Pro Ser Gly 145 150 155 160

Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He Leu Asn Asn Ser 165 170 175

Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val His Val Asp Lys 180 185 190

Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu Gin He Glu Gly 195 200 205

Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He Asn Ala Glu Ala 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 He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser Gly Asn Glu Lys 260 265 270

Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu Gly Lys Lys Pro 275 280 285

He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly Met Pro Glu Phe 290 295 300

Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe Glu Glu 305 310 315 320

Gin Phe Leu Asn Thr He 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 He He Leu 340 345 350

Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser Ala He 355 360 365

Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp Lys Asp Ser Lys 370 375 380

Tyr His He Asp Lys Val Thr Glu Val He He Lys Gly Val Lys Arg 385 390 395 400

Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 405 410

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 86 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide encoding vacuolar targetting peptide used to construct pCIB5533"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CCGCGGGCGT GCACTGCCTC AGCAGCAGCA GCTTCGCCGA CAGCAACCCC ATCCGCGTGA 60

CCGACCGCGC CGCCAGCACC CTGCAG 86

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1358 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 9..1355

(D) 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 NO:45:

GATCCACC ATG GGC TGG AGC TGG ATC TTC CTG TTC CTG CTG AGC GGC GCC 50

Met Gly Trp Ser Trp He Phe Leu Phe Leu Leu Ser Gly Ala

415 420

GCG GGC GTG CAC TGC CTC AGC AGC AGC AGC TTC GCC GAC AGC AAC CCC 98 Ala Gly Val His Cys Leu Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 425 430 435 440

ATC CGC GTG ACC GAC CGC GCC GCC AGC ACC CTG CAG AAC CTG AAG ATC 146 He Arg Val Thr Asp Arg Ala Ala Ser Thr Leu Gin Asn Leu Lys He 445 450 455

ACC GAC AAG GTG GAG GAC TTC AAG GAG GAC AAG GAG AAG GCC AAG GAG 194 Thr Asp Lys Val Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu 460 465 470

TGG GGC AAG GAG AAG GAG AAG GAG TGG AAG CTT ACC GCC ACC GAG AAG 242 Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys 475 480 485

GGC AAG ATG AAC AAC TTC CTG GAC AAC AAG AAC GAC ATC AAG ACC AAC 290 Gly Lys Met Asn Asn Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn 490 495 500

TAC AAG GAG ATC ACC TTC AGC ATA GCC GGC AGC TTC GAG GAC GAG ATC 338 Tyr Lys Glu He Thr Phe Ser He Ala Gly Ser Phe Glu Asp Glu He 505 510 515 520

AAG GAC CTG AAG GAG ATC GAC AAG ATG TTC GAC AAG ACC AAC CTG AGC 386

Lys Asp Leu Lys Glu He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser 525 530 535

AAC AGC ATC ATC ACC TAC AAG AAC GTG GAG CCC ACC ACC ATC GGC TTC 434 Asn Ser He He Thr Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe 540 545 550

AAC AAG AGC CTG ACC GAG GGC AAC ACC ATC AAC AGC GAC GCC ATG GCC 482 Asn Lys Ser Leu Thr Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala 555 560 565

CAG TTC AAG GAG CAG TTC CTG GAC CGC GAC ATC AAG TTC GAC AGC TAC 530 Gin Phe Lys Glu Gin Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr 570 575 580

CTG GAC ACC CAC CTG ACC GCC CAG CAG GTG AGC AGC AAG GAG CGC GTG 578 Leu Asp Thr His Leu Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val 585 590 595 600

ATC CTG AAG GTG ACC GTC CCC AGC GGC AAG GGC AGC ACC ACC CCC ACC 626 He Leu Lys Val Thr Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr 605 610 615

AAG GCC GGC GTG ATC CTG AAC AAC AGC GAG TAC AAG ATG CTG ATC GAC 674 Lys Ala Gly Val He Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp 620 625 630

AAC GGC TAC ATG GTG CAC GTG GAC AAG GTG AGC AAG GTG GTG AAG AAG 722 Asn Gly Tyr Met Val His Val Asp Lys Val Ser Lys Val Val Lys Lys 635 640 645

GGC GTG GAG TGC CTC CAG ATC GAG GGC ACC CTG AAG AAG AGT CTA GAC 770 Gly Val Glu Cys Leu Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp 650 655 660

TTC AAG AAC GAC ATC AAC GCC GAG GCC CAC AGC TGG GGC ATG AAG AAC 818 Phe Lys Asn Asp He Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn 665 670 675 680

TAC GAG GAG TGG GCC AAG GAC CTG ACC GAC AGC CAG CGC GAG GCC CTG 866 Tyr Glu Glu Trp Ala Lys Asp Leu Thr Asp Ser Gin Arg Glu Ala Leu 685 690 695

GAC GGC TAC GCC CGC CAG GAC TAC AAG GAG ATC AAC AAC TAC CTG CGC 914 Asp Gly Tyr Ala Arg Gin Asp Tyr Lys Glu He Asn Asn Tyr Leu Arg 700 705 710

AAC CAG GGC GGC AGC GGC AAC GAG AAG CTG GAC GCC CAG ATC AAG AAC 962 Asn Gin Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn 715 720 725

ATC AGC GAC GCC CTG GGC AAG AAG CCC ATC CCC GAG AAC ATC ACC GTG 1010 He Ser Asp Ala Leu Gly Lys Lys Pro He Pro Glu Asn He Thr Val 730 735 740

TAC CGC TGG TGC GGC ATG CCC GAG TTC GGC TAC CAG ATC AGC GAC CCC 1058 Tyr Arg Trp Cys Gly Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro 745 750 755 760

CTG CCC AGC CTG AAG GAC TTC GAG GAG CAG TTC CTG AAC ACC ATC AAG 1106 Leu Pro Ser Leu Lys Asp Phe Glu Glu Gin Phe Leu Asn Thr He Lys 765 770 775

GAG GAC AAG GGC TAC ATG AGC ACC AGC CTG AGC AGC GAG CGC CTG GCC 1154 Glu Asp Lys Gly Tyr Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala 780 785 790

GCC TTC GGC AGC CGC AAG ATC ATC CTG CGC CTG CAG GTG CCC AAG GGC 1202 Ala Phe Gly Ser Arg Lys He He Leu Arg Leu Gin Val Pro Lys Gly 795 800 805

AGC ACT GGT GCC TAC CTG AGC GCC ATC GGC GGC TTC GCC AGC GAG AAG 1250 Ser Thr Gly Ala Tyr Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys 810 815 820

GAG ATC CTG CTG GAT AAG GAC AGC AAG TAC CAC ATC GAC AAG GTG ACC 1298 Glu He Leu Leu Asp Lys Asp Ser Lys Tyr His He Asp Lys Val Thr 825 830 835 840

GAG GTG ATC ATC AAG GGC GTG AAG CGC TAC GTG GTG GAC GCC ACC CTG 1346 Glu Val He He Lys Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu 845 850 855

CTG ACC AAC TAG 1358

Leu Thr Asn

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 449 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Met Gly Trp Ser Trp He Phe Leu Phe Leu Leu Ser Gly Ala Ala Gly

1 5 10 15

Val His Cys Leu Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro He Arg 20 25 30

Val Thr Asp Arg Ala Ala Ser Thr Leu Gin Asn Leu Lys He Thr Asp 35 40 45

Lys Val Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly 50 55 60

Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys 65 70 75 80

Met Asn Asn Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys 85 90 95

Glu He Thr Phe Ser He Ala Gly Ser Phe Glu Asp Glu He Lys Asp 100 105 110

Leu Lys Glu He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser 115 120 125

He He Thr Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys 130 135 140

Ser Leu Thr Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe 145 150 155 160

Lys Glu Gin Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp 165 170 175

Thr His Leu Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He 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 He Leu Asn Asn Ser Glu Tyr Lys Met Leu He 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 He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys 245 250 255

Asn Asp He 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 He Asn Asn Tyr Leu Arg Asn Gin 290 295 300

Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser 305 310 315 320

Asp Ala Leu Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg 325 330 335

Trp Cys Gly Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro 340 345 350

Ser Leu Lys Asp Phe Glu Glu Gin Phe Leu Asn Thr He 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 He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr 385 390 395 400

Gly Ala Tyr Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He 405 410 415

Leu Leu Asp Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val 420 425 430

He He Lys Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr 435 440 445

Asn

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..16

(D) OTHER INFORMATION: /note- "linker peptide for fusion of VIPlA(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 15

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA encoding linker peptide used to construct pCIB5533"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CCCGGGCCTT CTACTCCCCC AACTCCCTCT CCTAGCACGC CTCCGACACC TAGCGATATC 60 GGATCC 66

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4031 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic DNA"

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 6..4019

(D) OTHER INFORMATION: /note= "Maize optimized DNA sequence encoding a VTP2A(a) - VIPlA(a) fusion protein as contained in pCIB5531"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GATCC ATG AAG CGC ATG GAG GGC AAG CTG TTC ATG GTG AGC AAG AAG 47 Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys 450 455 460

CTC CAG GTG GTG ACC AAG ACC GTG CTG CTG AGC ACC GTG TTC AGC ATC 95 Leu Gin Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser He 465 470 475

AGC CTG CTG AAC AAC GAG GTG ATC AAG GCC GAG CAG CTG AAC ATC AAC 143 Ser Leu Leu Asn Asn Glu Val He Lys Ala Glu Gin Leu Asn He Asn 480 485 490 495

AGC CAG AGC AAG TAC ACC AAC CTC CAG AAC CTG AAG ATC ACC GAC AAG 191 Ser Gin Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys 500 505 510

GTG GAG GAC TTC AAG GAG GAC AAG GAG AAG GCC AAG GAG TGG GGC AAG 239

Val Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys 515 520 525

GAG AAG GAG AAG GAG TGG AAG CTT ACC GCC ACC GAG AAG GGC AAG ATG 287 Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met 530 535 540

AAC AAC TTC CTG GAC AAC AAG AAC GAC ATC AAG ACC AAC TAC AAG GAG 335 Asn Asn Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu 545 550 555

ATC ACC TTC AGC ATA GCC GGC AGC TTC GAG GAC GAG ATC AAG GAC CTG 383 He Thr Phe Ser He Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu 560 565 570 575

AAG GAG ATC GAC AAG ATG TTC GAC AAG ACC AAC CTG AGC AAC AGC ATC 431 Lys Glu He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He 580 585 590

ATC ACC TAC AAG AAC GTG GAG CCC ACC ACC ATC GGC TTC AAC AAG AGC 479 He Thr Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser 595 600 605

CTG ACC GAG GGC AAC ACC ATC AAC AGC GAC GCC ATG GCC CAG TTC AAG 527 Leu Thr Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys 610 615 620

GAG CAG TTC CTG GAC CGC GAC ATC AAG TTC GAC AGC TAC CTG GAC ACC 575 Glu Gin Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr 625 630 635

CAC CTG ACC GCC CAG CAG GTG AGC AGC AAG GAG CGC GTG ATC CTG AAG 623 His Leu Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys 640 645 650 655

GTG ACC GTC CCC AGC GGC AAG GGC AGC ACC ACC CCC ACC AAG GCC GGC 671 Val Thr Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly 660 665 670

GTG ATC CTG AAC AAC AGC GAG TAC AAG ATG CTG ATC GAC AAC GGC TAC 719 Val He Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr 675 680 685

ATG GTG CAC GTG GAC AAG GTG AGC AAG GTG GTG AAG AAG GGC GTG GAG 767 Met Val His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu 690 695 700

TGC CTC CAG ATC GAG GGC ACC CTG AAG AAG AGT CTA GAC TTC AAG AAC 815 Cys Leu Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn 705 710 715

GAC ATC AAC GCC GAG GCC CAC AGC TGG GGC ATG AAG AAC TAC GAG GAG 863 Asp He Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu 720 725 730 735

TGG GCC AAG GAC CTG ACC GAC AGC CAG CGC GAG GCC CTG GAC GGC TAC 911 Trp Ala Lys Asp Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr 740 745 750

GCC CGC CAG GAC TAC AAG GAG ATC AAC AAC TAC CTG CGC AAC CAG GGC 959 Ala Arg Gin Asp Tyr Lys Glu He Asn Asn Tyr Leu Arg Asn Gin Gly 755 760 765

GGC AGC GGC AAC GAG AAG CTG GAC GCC CAG ATC AAG AAC ATC AGC GAC 1007 Gly Ser Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp 770 775 780

GCC CTG GGC AAG AAG CCC ATC CCC GAG AAC ATC ACC GTG TAC CGC TGG 1055 Ala Leu Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp 785 790 795

TGC GGC ATG CCC GAG TTC GGC TAC CAG ATC AGC GAC CCC CTG CCC AGC 1103 Cys Gly Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser 800 805 810 815

CTG AAG GAC TTC GAG GAG CAG TTC CTG AAC ACC ATC AAG GAG GAC AAG 1151 Leu Lys Asp Phe Glu Glu Gin Phe Leu Asn Thr He Lys Glu Asp Lys 820 825 830

GGC TAC ATG AGC ACC AGC CTG AGC AGC GAG CGC CTG GCC GCC TTC GGC 1199 Gly Tyr Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly 835 840 845

AGC CGC AAG ATC ATC CTG CGC CTG CAG GTG CCC AAG GGC AGC ACT GGT 1247 Ser Arg Lys He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly 850 855 860

GCC TAC CTG AGC GCC ATC GGC GGC TTC GCC AGC GAG AAG GAG ATC CTG 1295 Ala Tyr Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu 865 870 875

CTG GAT AAG GAC AGC AAG TAC CAC ATC GAC AAG GTG ACC GAG GTG ATC 1343 Leu Asp Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val He 880 885 890 895

ATC AAG GGC GTG AAG CGC TAC GTG GTG GAC GCC ACC CTG CTG ACC AAC 1391 He Lys Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 900 905 910

TCC CGG GGG CCT TCT ACT CCC CCA ACT CCC TCT CCT AGC ACG CCT CCG 1439 Ser Arg Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro 915 920 925

ACA CCT AGC GAT ATC GGA TCC ACC ATG AAG ACC AAC CAG ATC AGC ACC 1 87 Thr Pro Ser Asp He Gly Ser Thr Met Lys Thr Asn Gin He Ser Thr 930 935 940

ACC CAG AAG AAC CAG CAG AAG GAG ATG GAC CGC AAG GGC CTG CTG GGC 1535 Thr Gin Lys Asn Gin Gin Lys Glu Met Asp Arg Lys Gly Leu Leu Gly 945 950 955

TAC TAC TTC AAG GGC AAG GAC TTC AGC AAC CTG ACC ATG TTC GCC CCC 1583 Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn Leu Thr Met Phe Ala Pro 960 965 970 975

ACG CGT GAC AGC ACC CTG ATC TAC GAC CAG CAG ACC GCC AAC AAG CTG 1631 Thr Arg Asp Ser Thr Leu He Tyr Asp Gin Gin Thr Ala Asn Lys Leu 980 985 990

CTG GAC AAG AAG CAG CAG GAG TAC CAG AGC ATC CGC TGG ATC GGC CTG 1679 Leu Asp Lys Lys Gin Gin Glu Tyr Gin Ser He Arg Trp He Gly Leu 995 1000 1005

ATC CAG AGC AAG GAG ACC GGC GAC TTC ACC TTC AAC CTG AGC GAG GAC 1727 He Gin Ser Lys Glu Thr Gly Asp Phe Thr Phe Asn Leu Ser Glu Asp 1010 1015 1020

GAG CAG GCC ATC ATC GAG ATC AAC GGC AAG ATC ATC AGC AAC AAG GGC 1775 Glu Gin Ala He He Glu He Asn Gly Lys He He Ser Asn Lys Gly 1025 1030 1035

AAG GAG AAG CAG GTG GTG CAC CTG GAG AAG GGC AAG CTG GTG CCC ATC 1823 Lys Glu Lys Gin Val Val His Leu Glu Lys Gly Lys Leu Val Pro He 1040 1045 1050 1055

AAG ATC GAG TAC CAG AGC GAC ACC AAG TTC AAC ATC GAC AGC AAG ACC 1871 Lys He Glu Tyr Gin Ser Asp Thr Lys Phe Asn He Asp Ser Lys Thr 1060 1065 1070

TTC AAG GAG CTG AAG CTT TTC AAG ATC GAC AGC CAG AAC CAG CCC CAG 1919 Phe Lys Glu Leu Lys Leu Phe Lys He Asp Ser Gin Asn Gin Pro Gin 1075 1080 1085

CAG GTG CAG CAG GAC GAG CTG CGC AAC CCC GAG TTC AAC AAG AAG GAG 1967 Gin Val Gin Gin Asp Glu Leu Arg Asn Pro Glu Phe Asn Lys Lys Glu 1090 1095 1100

AGC CAG GAG TTC CTG GCC AAG CCC AGC AAG ATC AAC CTG TTC ACC CAG 2015 Ser Gin Glu Phe Leu Ala Lys Pro Ser Lys He Asn Leu Phe Thr Gin 1105 1110 1115

CAG ATG AAG CGC GAG ATC GAC GAG GAC ACC GAC ACC GAC GGC GAC AGC 2063 Gin Met Lys Arg Glu He Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser 1120 1125 ^' 1130 1135

ATC CCC GAC CTG TGG GAG GAG AAC GGC TAC ACC ATC CAG AAC CGC ATC 2111 He Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr He Gin Asn Arg He 1140 1145 1150

GCC GTG AAG TGG GAC GAC AGC CTG GCT AGC AAG GGC TAC ACC AAG TTC 2159 Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe 1155 1160 1165

GTG AGC AAC CCC CTG GAG AGC CAC ACC GTG GGC GAC CCC TAC ACC GAC 2207 Val Ser Asn Pro Leu Glu Ser His Thr Val Gly Asp Pro Tyr Thr Asp

1170 1175 1180

TAC GAG AAG GCC GCC CGC GAC CTG GAC CTG AGC AAC GCC AAG GAG ACC 2255 Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr 1185 1190 1195

TTC AAC CCC CTG GTG GCC GCC TTC CCC AGC GTG AAC GTG AGC ATG GAG 2303 Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val Asn Val Ser Met Glu 1200 1205 1210 1215

AAG GTG ATC CTG AGC CCC AAC GAG AAC CTG AGC AAC AGC GTG GAG AGC 2351 Lys Val He Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu Ser 1220 1225 1230

CAC TCG AGC ACC AAC TGG AGC TAC ACC AAC ACC GAG GGC GCC AGC GTG 2399 His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val 1235 1240 1245

GAG GCC GGC ATC GGT CCC AAG GGC ATC AGC TTC GGC GTG AGC GTG AAC 2447 Glu Ala Gly He Gly Pro Lys Gly He Ser Phe Gly Val Ser Val Asn 1250 1255 1260

TAC CAG CAC AGC GAG ACC GTG GCC CAG GAG TGG GGC ACC AGC ACC GGC 2495 Tyr Gin His Ser Glu Thr Val Ala Gin Glu Trp Gly Thr Ser Thr Gly 1265 1270 1275

AAC ACC AGC CAG TTC AAC ACC GCC AGC GCC GGC TAC CTG AAC GCC AAC 2543 Asn Thr Ser Gin Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn 1280 1285 1290 1295

GTG CGC TAC AAC AAC GTG GGC ACC GGC GCC ATC TAC GAC GTG AAG CCC 2591 Val Arg Tyr Asn Asn Val Gly Thr Gly Ala He Tyr Asp Val Lys Pro 1300 1305 1310

ACC ACC AGC TTC GTG CTG AAC AAC GAC ACC ATC GCC ACC ATC ACC GCC 2639 Thr Thr Ser Phe Val Leu Asn Asn Asp Thr He Ala Thr He Thr Ala 1315 1320 1325

AAG TCG AAT TCC ACC GCC CTG AAC ATC AGC CCC GGC GAG AGC TAC CCC 2687 Lys Ser Asn Ser Thr Ala Leu Asn He Ser Pro Gly Glu Ser Tyr Pro 1330 1335 1340

AAG AAG GGC CAG AAC GGC ATC GCC ATC ACC AGC ATG GAC GAC TTC AAC 2735 Lys Lys Gly Gin Asn Gly He Ala He Thr Ser Met Asp Asp Phe Asn 1345 1350 1355

AGC CAC CCC ATC ACC CTG AAC AAG AAG CAG GTG GAC AAC CTG CTG AAC 2783 Ser His Pro He Thr Leu Asn Lys Lys Gin Val Asp Asn Leu Leu Asn 1360 1365 1370 1375

AAC AAG CCC ATG ATG CTG GAG ACC AAC CAG ACC GAC GGC GTC TAC AAG 2831 Asn Lys Pro Met Met Leu Glu Thr Asn Gin Thr Asp Gly Val Tyr Lys 1380 1385 1390

ATC AAG GAC ACC CAC GGC AAC ATC GTG ACG GGC GGC GAG TGG AAC GGC 2879

Ile Lys Asp Thr His Gly Asn He Val Thr Gly Gly Glu Trp Asn Gly 1395 1400 1405

GTG ATC CAG CAG ATC AAG GCC AAG ACC GCC AGC ATC ATC GTC GAC GAC 2927 Val He Gin Gin He Lys Ala Lys Thr Ala Ser He He Val Asp Asp 1410 1415 1420

GGC GAG CGC GTG GCC GAG AAG CGC GTG GCC GCC AAG GAC TAC GAG AAC 2975 Gly Glu Arg Val Ala Glu Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn 1425 1430 1435

CCC GAG GAC AAG ACC CCC AGC CTG ACC CTG AAG GAC GCC CTG AAG CTG 3023 Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu 1440 1445 1450 1455

AGC TAC CCC GAC GAG ATC AAG GAG ATC GAG GGC TTG CTG TAC TAC AAG 3071 Ser Tyr Pro Asp Glu He Lys Glu He Glu Gly Leu Leu Tyr Tyr Lys 1460 1465 1470

AAC AAG CCC ATC TAC GAG AGC AGC GTG ATG ACC TAT CTA GAC GAG AAC 3119 Asn Lys Pro He Tyr Glu Ser Ser Val Met Thr Tyr Leu Asp Glu Asn 1475 1480 1485

ACC GCC AAG GAG GTG ACC AAG CAG CTG AAC GAC ACC ACC GGC AAG TTC 3167 Thr Ala Lys Glu Val Thr Lys Gin Leu Asn Asp Thr Thr Gly Lys Phe 1490 1495 1500

AAG GAC GTG AGC CAC CTG TAC GAC GTG AAG CTG ACC CCC AAG ATG AAC 3215 Lys Asp Val Ser His Leu Tyr Asp Val Lys Leu Thr Pro Lys Met Asn 1505 1510 1515

GTG ACC ATC AAG CTG AGC ATC CTG TAC GAC AAC GCC GAG AGC AAC GAC 3263 Val Thr He Lys Leu Ser He Leu Tyr Asp Asn Ala Glu Ser Asn Asp 1520 1525 1530 1535

AAC AGC ATC GGC AAG TGG ACC AAC ACC AAC ATC GTG AGC GGC GGC AAC 3311 Asn Ser He Gly Lys Trp Thr Asn Thr Asn He Val Ser Gly Gly Asn 1540 1545 1550

AAC GGC AAG AAG CAG TAC AGC AGC AAC AAC CCC GAC GCC AAC CTG ACC 3359 Asn Gly Lys Lys Gin Tyr Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr 1555 1560 1565

CTG AAC ACC GAC GCC CAG GAG AAG CTG AAC AAG AAC CGC GAC TAC TAC 3407 Leu Asn Thr Asp Ala Gin Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr 1570 1575 1580

ATC AGC CTG TAC ATG AAG AGC GAG AAG AAC ACC CAG TGC GAG ATC ACC 3455 He Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr Gin Cys Glu He Thr 1585 1590 1595

ATC GAC GGC GAG ATA TAC CCC ATC ACC ACC AAG ACC GTG AAC GTG AAC 3503 He Asp Gly Glu He Tyr Pro He Thr Thr Lys Thr Val Asn Val Asn 1600 1605 1610 1615

AAG GAC AAC TAC AAG CGC CTG GAC ATC ATC GCC CAC AAC ATC AAG AGC 3551 Lys Asp Asn Tyr Lys Arg Leu Asp He He Ala His Asn He Lys Ser 1620 1625 1630

AAC CCC ATC AGC AGC CTG CAC ATC AAG ACC AAC GAC GAG ATC ACC CTG 3599 Asn Pro He Ser Ser Leu His He Lys Thr Asn Asp Glu He Thr Leu 1635 1640 1645

TTC TGG GAC GAC ATA TCG ATT ACC GAC GTC GCC AGC ATC AAG CCC GAG 3647 Phe Trp Asp Asp He Ser He Thr Asp Val Ala Ser He Lys Pro Glu 1650 1655 1660

AAC CTG ACC GAC AGC GAG ATC AAG CAG ATA TAC AGT CGC TAC GGC ATC 3695 Asn Leu Thr Asp Ser Glu He Lys Gin He Tyr Ser Arg Tyr Gly He 1665 1670 1675

AAG CTG GAG GAC GGC ATC CTG ATC GAC AAG AAA GGC GGC ATC CAC TAC 3743 Lys Leu Glu Asp Gly He Leu He Asp Lys Lys Gly Gly He His Tyr 1680 1685 1690 1695

GGC GAG TTC ATC AAC GAG GCC AGC TTC AAC ATC GAG CCC CTG CAG AAC 3791 Gly Glu Phe He Asn Glu Ala Ser Phe Asn He Glu Pro Leu Gin Asn 1700 1705 1710

TAC GTG ACC AAG TAC GAG GTG ACC TAC AGC AGC GAG CTG GGC CCC AAC 3839 Tyr Val Thr Lys Tyr Glu Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn 1715 1720 1725

GTG AGC GAC ACC CTG GAG AGC GAC AAG ATT TAC AAG GAC GGC ACC ATC 3887 Val Ser Asp Thr Leu Glu Ser Asp Lys He Tyr Lys Asp Gly Thr He 1730 1735 1740

AAG TTC GAC TTC ACC AAG TAC AGC AAG AAC GAG CAG GGC CTG TTC TAC 3935 Lys Phe Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gin Gly Leu Phe Tyr 1745 1750 1755

GAC AGC GGC CTG AAC TGG GAC TTC AAG ATC AAC GCC ATC ACC TAC GAC 3983 Asp Ser Gly Leu Asn Trp Asp Phe Lys He Asn Ala He Thr Tyr Asp 1760 1765 1770 1775

GGC AAG GAG ATG AAC GTG TTC CAC CGC TAC AAC AAG TAGATCTGAG 4029

Gly Lys Glu Met Asn Val Phe His Arg Tyr Asn Lys 1780 1785

CT 4031

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1338 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gin 1 5 10 15

Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser He Ser Leu 20 25 30

Leu Asn Asn Glu Val He Lys Ala Glu Gin Leu Asn He Asn Ser Gin 35 40 45

Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu 50 55 60

Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys 65 70 75 80

Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn 85 90 95

Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu He Thr 100 105 110

Phe Ser He Ala Gly Ser Phe Glu Asp Glu He Lys Asp Leu Lys Glu 115 120 125

He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser He He Thr 130 135 140

Tyr Lys Asn Val Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu Thr 145 150 155 160

Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 165 170 175

Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 180 185 190

Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys Val Thr 195 200 205

Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He 210 215 220

Leu Asn Asn Ser Glu Tyr Lys Met Leu He Asp Asn Gly Tyr Met Val 225 230 235 240

His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu 245 250 255

Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp He 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 He Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 305 310 315 320

Gly Asn Glu Lys Leu Asp Ala Gin He Lys Asn He Ser Asp Ala Leu 325 330 335

Gly Lys Lys Pro He Pro Glu Asn He Thr Val Tyr Arg Trp Cys Gly 340 345 350

Met Pro Glu Phe Gly Tyr Gin He Ser Asp Pro Leu Pro Ser Leu Lys 355 360 365

Asp Phe Glu Glu Gin Phe Leu Asn Thr He 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 He He Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 405 410 415

Leu Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp 420 425 430

Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu Val He He 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 He Gly Ser Thr Met Lys Thr Asn Gin He 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 He Tyr Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp 530 535 540

Lys Lys Gin Gin Glu Tyr Gin Ser He Arg Trp He Gly Leu He Gin 545 550 555 560

Ser Lys Glu Thr Gly Asp Phe Thr Phe Asn Leu Ser Glu Asp Glu Gin 565 570 575

Ala He He Glu He Asn Gly Lys He He Ser Asn Lys Gly Lys Glu 580 585 590

Lys Gin Val Val His Leu Glu Lys Gly Lys Leu Val Pro He Lys He 595 600 605

Glu Tyr Gin Ser Asp Thr Lys Phe Asn He Asp Ser Lys Thr Phe Lys 610 615 620

Glu Leu Lys Leu Phe Lys He Asp Ser Gin Asn Gin Pro Gin Gin Val 625 630 635 640

Gin Gin Asp Glu Leu Arg Asn Pro Glu Phe Asn Lys Lys Glu Ser Gin 645 650 655

Glu Phe Leu Ala Lys Pro Ser Lys He Asn Leu Phe Thr Gin Gin Met 660 665 670

Lys Arg Glu He Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser He Pro 675 680 685

Asp Leu Trp Glu Glu Asn Gly Tyr Thr He Gin Asn Arg He Ala Val 690 695 700

Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser 705 710 715 720

Asn Pro Leu Glu Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu 725 730 735

Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn 740 745 750

Pro Leu Val Ala Ala Phe Pro Ser Val Asn Val Ser Met Glu Lys Val 755 760 765

He Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu Ser His Ser 770 775 780

Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val Glu Ala 785 790 795 800

Gly He Gly Pro Lys Gly He Ser Phe Gly Val Ser Val Asn Tyr Gin 805 810 815

His Ser Glu Thr Val Ala Gin Glu Trp Gly Thr Ser Thr Gly Asn Thr 820 825 830

Ser Gin Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg 835 840 845

Tyr Asn Asn Val Gly Thr Gly Ala He Tyr Asp Val Lys Pro Thr Thr 850 855 860

Ser Phe Val Leu Asn Asn Asp Thr He Ala Thr He Thr Ala Lys Ser 865 870 875 880

Asn Ser Thr Ala Leu Asn He Ser Pro Gly Glu Ser Tyr Pro Lys Lys 885 890 895

Gly Gin Asn Gly He Ala He Thr Ser Met Asp Asp Phe Asn Ser His 900 905 910

Pro He 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 He Lys 930 935 940

Asp Thr His Gly Asn He Val Thr Gly Gly Glu Trp Asn Gly Val He 945 950 955 960

Gin Gin He Lys Ala Lys Thr Ala Ser He He 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 He Lys Glu He Glu Gly Leu Leu Tyr Tyr Lys Asn Lys 1010 1015 1020

Pro He 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

He Lys Leu Ser He Leu Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser 1075 1080 1085

He Gly Lys Trp Thr Asn Thr Asn He 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 He Ser 1125 1130 1135

Leu Tyr Met Lys Ser Glu Lys Asn Thr Gin Cys Glu He Thr He Asp 1140 1145 1150

Gly Glu He Tyr Pro He Thr Thr Lys Thr Val Asn Val Asn Lys Asp

1155 1160 1165

Asn Tyr Lys Arg Leu Asp He He Ala His Asn He Lys Ser Asn Pro 1170 1175 1180

He Ser Ser Leu His He Lys Thr Asn Asp Glu He Thr Leu Phe Trp 1185 1190 1195 1200

Asp Asp He Ser He Thr Asp Val Ala Ser He Lys Pro Glu Asn Leu 1205 1210 1215

Thr Asp Ser Glu He Lys Gin He Tyr Ser Arg Tyr Gly He Lys Leu 1220 1225 1230

Glu Asp Gly He Leu He Asp Lys Lys Gly Gly He His Tyr Gly Glu 1235 1240 1245

Phe He Asn Glu Ala Ser Phe Asn He 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 He Tyr Lys Asp Gly Thr He 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 He Asn Ala He Thr Tyr Asp Gly Lys 1315 1320 1325

Glu Met Asn Val Phe His Arg Tyr Asn Lys 1330 1335

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2444 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 17..2444

(D) OTHER INFORMATION: /product- "3A(a) synthetic:native fusion"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

GGATCCACCA ATGAAC ATG AAC AAG AAC AAC ACC AAG CTG AGC ACC CGC 49 Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg 1 5 10

GCC CTG CCG AGC TTC ATC GAC TAC TTC AAC GGC ATC TAC GGC TTC GCC 97 Ala Leu Pro Ser Phe He Asp Tyr Phe Asn Gly He Tyr Gly Phe Ala 15 20 25

ACC GGC ATC AAG GAC ATC ATG AAC ATG ATC TTC AAG ACC GAC ACC GGC 145 Thr Gly He Lys Asp He Met Asn Met He Phe Lys Thr Asp Thr Gly 30 35 40

GGC GAC CTG ACC CTG GAC GAG ATC CTG AAG AAC CAG CAG CTG CTG AAC 193 Gly Asp Leu Thr Leu Asp Glu He Leu Lys Asn Gin Gin Leu Leu Asn 45 50 55

GAC ATC AGC GGC AAG CTG GAC GGC GTG AAC GGC AGC CTG AAC GAC CTG 241 Asp He Ser Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu 60 65 70 75

ATC GCC CAG GGC AAC CTG AAC ACC GAG CTG AGC AAG GAG ATC CTT AAG 289 He Ala Gin Gly Asn Leu Asn Thr Glu Leu Ser Lys Glu He Leu Lys 80 85 90

ATC GCC AAC GAG CAG AAC CAG GTG CTG AAC GAC GTG AAC AAC AAG CTG 337 He Ala Asn Glu Gin Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu 95 100 105

GAC GCC ATC AAC ACC ATG CTG CGC GTG TAC CTG CCG AAG ATC ACC AGC 385 Asp Ala He Asn Thr Met Leu Arg Val Tyr Leu Pro Lys He Thr Ser 110 115 120

ATG CTG AGC GAC GTG ATG AAG CAG AAC TAC GCC CTG AGC CTG CAG ATC 433 Met Leu Ser Asp Val Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin He 125 130 135

GAG TAC CTG AGC AAG CAG CTG CAG GAG ATC AGC GAC AAG CTG GAC ATC 481 Glu Tyr Leu Ser Lys Gin Leu Gin Glu He Ser Asp Lys Leu Asp He 140 145 150 155

ATC AAC GTG AAC GTC CTG ATC AAC AGC ACC CTG ACC GAG ATC ACC CCG 529 He Asn Val Asn Val Leu He Asn Ser Thr Leu Thr Glu He Thr Pro 160 165 170

GCC TAC CAG CGC ATC AAG TAC GTG AAC GAG AAG TTC GAA GAG CTG ACC 577 Ala Tyr Gin Arg He Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr 175 180 185

TTC GCC ACC GAG ACC AGC AGC AAG GTG AAG AAG GAC GGC AGC CCG GCC 625 Phe Ala Thr Glu Thr Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala 190 195 200

GAC ATC CTG GAC GAG CTG ACC GAG CTG ACC GAG CTG GCC AAG AGC GTG 673

Asp He Leu Asp Glu Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val 205 210 215

ACC AAG AAC GAC GTG GAC GGC TTC GAG TTC TAC CTG AAC ACC TTC CAC 721 Thr Lys Asn Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His 220 225 230 235

GAC GTG ATG GTG GGC AAC AAC CTG TTC GGC CGC AGC GCC CTG AAG ACC 769 Asp Val Met Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr 240 245 250

GCC AGC GAG CTG ATC ACC AAG GAG AAC GTG AAG ACC AGC GGC AGC GAG 817 Ala Ser Glu Leu He Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu 255 260 265

GTG GGC AAC GTG TAC AAC TTC CTG ATC GTG CTG ACC GCC CTG CAG GCC 865 Val Gly Asn Val Tyr Asn Phe Leu He Val Leu Thr Ala Leu Gin Ala 270 275 280

CAG GCC TTC CTG ACC CTG ACC ACC TGT CGC AAG CTG CTG GGC CTG GCC 913 Gin Ala Phe Leu Thr Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala 285 290 295

GAC ATC GAC TAC ACC AGC ATC ATG AAC GAG CAC TTG AAC AAG GAG AAG 961 Asp He Asp Tyr Thr Ser He Met Asn Glu His Leu Asn Lys Glu Lys 300 305 310 315

GAG GAG TTC CGC GTG AAC ATC CTG CCG ACC CTG AGC AAC ACC TTC AGC 1009 Glu Glu Phe Arg Val Asn He Leu Pro Thr Leu Ser Asn Thr Phe Ser 320 325 330

AAC CCG AAC TAC GCC AAG GTG AAG GGC AGC GAC GAG GAC GCC AAG ATG 1057 Asn Pro Asn Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met 335 340 345

ATC GTG GAG GCT G CCG GGC CAC GCG TTG ATC GGC TTC GAG ATC AGC 1105 He Val Glu Ala Lys Pro Gly His Ala Leu He Gly Phe Glu He Ser 350 355 360

AAC GAC AGC ATC ACC GTG CTG AAG GTG TAC GAG GCC AAG CTG AAG CAG 1153 Asn Asp Ser He Thr Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gin 365 370 375

AAC TAC CAG GTG GAC AAG GAC AGC TTG AGC GAG GTG ATC TAC GGC GAC 1201 Asn Tyr Gin Val Asp Lys Asp Ser Leu Ser Glu Val He Tyr Gly Asp 380 385 390 395

ATG GAC AAG CTG CTG TGT CCG GAC CAG AGC GAG CAA ATC TAC TAC ACC 1249 Met Asp Lys Leu Leu Cys Pro Asp Gin Ser Glu Gin He Tyr Tyr Thr 400 405 410

AAC AAC ATC GTG TTC CCG AAC GAG TAC GTG ATC ACC AAG ATC GAC TTC 1297 Asn Asn He Val Phe Pro Asn Glu Tyr Val He Thr Lys He Asp Phe 415 420 425

ACC AAG AAG ATG AAG ACC CTG CGC TAC GAG GTG ACC GCC AAC TTC TAC 1345 Thr Lys Lys Met Lys Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr 430 435 440

GAC AGC AGC ACC GGC GAG ATC GAC CTG AAC AAG AAG AAG GTG GAG AGC 1393 Asp Ser Ser Thr Gly Glu He Asp Leu Asn Lys Lys Lys Val Glu Ser 445 450 455

AGC GAG GCC GAG TAC CGC ACC CTG AGC GCG AAC GAC GAC GGC GTC TAC 1441 Ser Glu Ala Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr 460 465 470 475

ATG CCA CTG GGC GTG ATC AGC GAG ACC TTC CTG ACC CCG ATC AAC GGC 1489 Met Pro Leu Gly Val He Ser Glu Thr Phe Leu Thr Pro He Asn Gly 480 485 490

TTT GGC CTG CAG GCC GAC GAG AAC AGC CGC CTG ATC ACC CTG ACC TGT 1537 Phe Gly Leu Gin Ala Asp Glu Asn Ser Arg Leu He Thr Leu Thr Cys 495 500 505

AAG AGC TAC CTG CGC GAG CTG CTG CTA GCC ACC GAC CTG AGC AAC AAG 1585 Lys Ser Tyr Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys 510 515 520

GAG ACC AAG CTG ATC GTG CCA CCG AGC GGC TTC ATC AGC AAC ATC GTG 1633 Glu Thr Lys Leu He Val Pro Pro Ser Gly Phe He Ser Asn He Val 525 530 535

GAG AAC GGC AGC ATC GAG GAG GAC AAC CTG GAG CCG TGG AAG GCC AAC 1681 Glu Asn Gly Ser He Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn 540 545 550 555

AAC AAG AAC GCC TAC GTG GAC CAC ACC GGC GGC GTG AAC GGC ACC AAG 1729 Asn Lys Asn Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys 560 565 570

GCC CTG TAC GTG CAC AAG GAC GGC GGC ATC AGC CAG TTC ATC GGC GAC 1777 Ala Leu Tyr Val His Lys Asp Gly Gly He Ser Gin Phe He Gly Asp 575 580 585

AAG CTG AAG CCG AAG ACC GAG TAC GTG ATC CAG TAC ACC GTG AAG GGC 1825 Lys Leu Lys Pro Lys Thr Glu Tyr Val He Gin Tyr Thr Val Lys Gly 590 595 600

AAG CCA TCG ATT CAC CTG AAG GAC GAG AAC ACC GGC TAC ATC CAC TAC 1873 Lys Pro Ser He His Leu Lys Asp Glu Asn Thr Gly Tyr He His Tyr 605 610 615

GAG GAC ACC AAC AAC AAC CTG GAG GAC TAC CAG ACC ATC AAC AAG CGC 1921 Glu Asp Thr Asn Asn Asn Leu Glu Asp Tyr Gin Thr He Asn Lys Arg 620 625 630 635

TTC ACC ACC GGC ACC GAC CTG AAG GGC GTG TAC CTG ATC CTG AAG AGC 1969 Phe Thr Thr Gly Thr Asp Leu Lys Gly Val Tyr Leu He Leu Lys Ser 640 645 650

CAG AAC GGC GAC GAG GCC TGG GGC GAC AAC TTC ATC ATC CTG GAG ATC 2017 Gin Asn Gly Asp Glu Ala Trp Gly Asp Asn Phe He He Leu Glu He 655 660 665

AGC CCG AGC GAG AAG CTG CTG AGC CCG GAG CTG ATC AAC ACC AAC AAC 2065 Ser Pro Ser Glu Lys Leu Leu Ser Pro Glu Leu He Asn Thr Asn Asn 670 675 680

TGG ACC AGC ACC GGC AGC ACC AAC ATC AGC GGC AAC ACC CTG ACC CTG 2113 Trp Thr Ser Thr Gly Ser Thr Asn He Ser Gly Asn Thr Leu Thr Leu 685 690 695

TAC CAG GGC GGC CGG GGG ATT CTA AAA CAA AAC CTT CAA TTA GAT AGT 2161 Tyr Gin Gly Gly Arg Gly He Leu Lys Gin Asn Leu Gin Leu Asp Ser 700 705 710 715

TTT TCA ACT TAT AGA GTG TAT TTT TCT GTG TCC GGA GAT GCT AAT GTA 2209 Phe Ser Thr Tyr Arg Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val 720 725 730

AGG ATT AGA AAT TCT AGG GAA GTG TTA TTT GAA AAA AGA TAT ATG AGC 2257 Arg He Arg Asn Ser Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser 735 740 745

GGT GCT AAA GAT GTT TCT GAA ATG TTC ACT ACA AAA TTT GAG AAA GAT 2305 Gly Ala Lys Asp Val Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp 750 755 760

AAC TTT TAT ATA GAG CTT TCT CAA GGG AAT AAT TTA TAT GGT GGT CCT 2353 Asn Phe Tyr He Glu Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro 765 770 775

ATT GTA CAT TTT TAC GAT GTC TCT ATT AAG NAA GAT CGG GAT CTA ATA 2401 He Val His Phe Tyr Asp Val Ser He Lys Xaa Asp Arg Asp Leu He 780 785 790 795

TTA ACA GTT TTT AAA AGC NAA TTC TTG TAT AAT GTC CTT GAT T 2444

Leu Thr Val Phe Lys Ser Xaa Phe Leu Tyr Asn Val Leu Asp 800 805

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 809 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

He Asp Tyr Phe Asn Gly He Tyr Gly Phe Ala Thr Gly He Lys Asp 20 25 30

He Met Asn Met He Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 35 40 45

Asp Glu He Leu Lys Asn Gin Gin Leu Leu Asn Asp He Ser Gly Lys 50 55 60

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu He Ala Gin Gly Asn 65 70 75 80

Leu Asn Thr Glu Leu Ser Lys Glu He Leu Lys He Ala Asn Glu Gin 85 90 95

Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala He Asn Thr 100 105 110

Met Leu Arg Val Tyr Leu Pro Lys He Thr Ser Met Leu Ser Asp Val 115 120 125

Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin He Glu Tyr Leu Ser Lys 130 135 140

Gin Leu Gin Glu He Ser Asp Lys Leu Asp He He Asn Val Asn Val 145 150 155 160

Leu He Asn Ser Thr Leu Thr Glu He Thr Pro Ala Tyr Gin Arg He 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 He Leu 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 He 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 He Val Leu Thr Ala Leu Gin Ala Gin Ala Phe Leu Thr 275 280 285

Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp He Asp Tyr Thr 290 295 300

Ser He Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 305 310 315 320

Asn He 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 He Val Glu Ala Lys 340 345 350

Pro Gly His Ala Leu He Gly Phe Glu He Ser Asn Asp Ser He 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 He Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400

Cys Pro Asp Gin Ser Glu Gin He Tyr Tyr Thr Asn Asn He Val Phe 405 410 415

Pro Asn Glu Tyr Val He Thr Lys He 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 He 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

He Ser Glu Thr Phe Leu Thr Pro He Asn Gly Phe Gly Leu Gin Ala 485 490 495

Asp Glu Asn Ser Arg Leu He 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 He 515 520 525

Val Pro Pro Ser Gly Phe He Ser Asn He Val Glu Asn Gly Ser He 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

Lys Asp Gly Gly He Ser Gin Phe He Gly Asp Lys Leu Lys Pro Lys 580 585 590

Thr Glu Tyr Val He Gin Tyr Thr Val Lys Gly Lys Pro Ser He His

595 600 605

Leu Lys Asp Glu Asn Thr Gly Tyr He His Tyr Glu Asp Thr Asn Asn 610 615 620

Asn Leu Glu Asp Tyr Gin Thr He Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640

Asp Leu Lys Gly Val Tyr Leu He Leu Lys Ser Gin Asn Gly Asp Glu 645 650 655

Ala Trp Gly Asp Asn Phe He He Leu Glu He Ser Pro Ser Glu Lys 660 665 670

Leu Leu Ser Pro Glu Leu He Asn Thr Asn Asn Trp Thr Ser Thr Gly 675 680 685

Ser Thr Asn He Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg 690 695 700

Gly He Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg 705 710 715 720

Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg He Arg Asn Ser 725 730 735

Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val 740 745 750

Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr He Glu 755 760 765

Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro He Val His Phe Tyr 770 775 780

Asp Val Ser He Lys Xaa Asp Arg Asp Leu He Leu Thr Val Phe Lys 785 790 795 800

Ser Xaa Phe Leu Tyr Asn Val Leu Asp 805