Novel pesticidal proteins and strains

WO 96/10083

PCT/EP95/03826

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 (Leptinotarsa decemlineata) or Yellow mealworm (Tenebrio molitor) have demonstrated significant effects on members of the genus Diabrotica particularly Diabrotica virgifera virgifera^ the western corn rootworm (WCRW) or Diabrotica longicornis barberi, 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.

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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 thuringiensis 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 thuringiensis and B. cereus strain, and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SS11-1. 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 exigua ] 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 B21225, NRRL B-21226, NRRL B-21227, NRRL B-21228, NRRL B-21229, NRRL B21230, 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

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-3SEQ 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.

In a specific embodiment, the invention relates to a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp., wherein said vegetative insecticidal protein has at least 80% identity to SEQ ID NO: 29 or SEQ ID NO: 32.

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

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-3aprotein 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 10 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,

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-4which, 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 ribonuclease 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.

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-5A 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 SSI 1-1. 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 ipsilorr, BCW] and/or fall armyworm [Spodoptera frugiperda] and/or beet armyworm [Spodoptera exigua] and/or tobacco budworm and/or com earworm

[Helicoverpa 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 15 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.

In a specific embodiment, the invention relates to a DNA molecule comprising a nucleotide sequence which encodes a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp., wherein a complement of said nucleotide sequence hybridizes to SEQ ID NO: 28 or SEQ ID NO: 31 at 65°C in a buffer comprising 7% SDS and 0.5 M sodium phosphate.

In another specific embodiment, the invention relates to a DNA molecule comprising a nucleotide sequence which encodes a vegetative insecticidal protein secreted during the vegetative growth phase, wherein said nucleotide sequence has at least 80% identity to SEQ ID NO: 28 or SEQ ID NO: 31.

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-5aThe 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 5 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 SSI 1-1, 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

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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

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PCT/EP95/03826 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

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-8(a) obtaining a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein; and (b) hybridizing said DNA molecule with an oligonucleotide probe as described herein 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.

In a specific embodiment, the invention relates to a DNA molecule comprising a nucleotide sequence which encodes the vegetative insecticidal protein as described herein obtained by a process comprising: (a) obtaining a

DNA molecule comprising a nucleotide sequence encoding a vegetative insecticidal protein; and (b) hybridizing said DNA molecule to the complement of SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 31 at 65°O in a buffer comprising 7% SDS and 0.5 M sodium phosphate; 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 25 invention are preferably part of the plant genome.

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-8aA 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.

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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 insecticidallyeffective 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

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-10solution 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 10 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.

In a particular embodiment, the invention relates to a method for 15 isolating a vegetative insecticidal protein as described herein, said method comprising: (a) growing a Bacillus strain in a liquid culture; (b) obtaining supernatant from said culture; (c) precipitating secreted proteins from the supernatant of step (b); and (d) isolating said vegetative insecticidal protein from said supernatant.

The invention also encompasses a method for isolating a DNA 20 molecule comprising a nucleotide sequence encoding an insect-specific protein exhibiting the insecticidal activity of the proteins according to the invention, said method comprising:

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- 10aobtaining 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.

In a particular embodiment, the invention relates to a method for isolating a DNA molecule comprising a nucleotide sequence encoding a vegetative insecticidal protein as described herein, said method comprising: (a) obtaining a DNA molecule comprising a nucleotide sequence encoding a vegetative insecticidal protein; and (b) hybridizing said DNA molecule with SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 51; and (c) 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, a-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, a-amylases and peroxidases.

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- 11 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 [Helicoverpa 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 insectspecific 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

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-12molecule 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 an oligonucleotide probe capable of specifically hybridizing to a nucleotide sequence encoding 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 8. 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.

In a specific embodiment, the invention relates to an oligonucleotide probe for identifying a vegetative insecticidal protein coding sequence, wherein said probe is capable of specifically hybridizing at 65°C in a buffer comprising 7% SDS and 0.5 M sodium phosphate to a nucleotide sequence encoding a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp., wherein said probe comprises at least a 10 contiguous nucleotide portion of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 51.

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.

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-12aThe 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.

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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 unipunctata, 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

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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 Heliothis 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, Conoderus, 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

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Zygogramma exclamationis, sunflower beetle

Bothyrus gibbosus, carrot beetle

Cotton

Anthonomus grandis, boll weevil

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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 graminum, greenbug Macrosiphum avenae, English grain aphid

Cotton

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

Rice

Nephotettix nigropictus, rice leafhopper

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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

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TABLE 5

Orthoptera (Grasshoppers, Crickets, and Cockroaches)

Maize

Melanoplus femurrubrum, redlegged grasshopper Melanoplus sanguinipes, 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

Diotera (Flies and Mosquitoes)

Maize

Hylemya platura, seedcorn maggot Agromyza parvicornis, corn blotch leafminer

Sorghum

Contarinia sorghicola, sorghum midge

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- 18 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

Thysanoptera (Thrips)

Maize

Anaphothrips obscurus, grass thrips

Wheat

Frankliniella fusca, tobacco thrips

Cotton

Thrips tabaci, onion thrips Frankliniella fusca, tobacco thrips

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1 9904 9*

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- 19 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 auricularia, 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 humanus, head and body louse

Siphonaptera (Fleas)

Ctenocephalides felis, cat flea

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1 9 9 0 4 9

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-20 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

WO 96/10083

- 21 PCT/EP95/03826 known in the art. See, for example, Travers et al. (1987) Appl. Environ. Microbiol. 53:1263-1266; Saleh et al. (1969) Can J. Microbiol. 15:1101-1104; DeLucca etal. (1981) Can. J. Microbiol. 27:865-870; and Norris, etal. (1981) Thegenera 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 11

TABLE 11

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*

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- 22 Morphological Group 3

B. sphaericus*

B. pasteurii

Unassigned Strains

Subgroup A

B. apiarus*

B. filicolonicus B. thiaminolyticus B. alcalophilus

Subgroup B

B. cirroflagellosus B. chitinosporus B. lentus

Subgroup C

B. badius B. aneurinolyticus B. macroides B. freundenreichii

Subgroup D

B. pantothenticus B. epiphytus

Subgroup E1

B. aminovorans B. globisporus B. insolitus 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. etal. (1983) Color Atlas of Bacillus species, Wolfe

Medical Publications, London.

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-23 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) 1 Immunol. 128:2804; and Radka et al. (1984) Immunoqenetics 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.

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-24 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 etal. (1982) J. Biol. Chem. 257:9751-9758; Liu etal. (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.

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-25 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

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-26armyworm and/or beet armyworm and/or tobacco budworm and/or corn earworm activity.

Black cutworm is an agronomically important insect quite resistant to ô-endotoxins. Macintosh et al (1990) J Invertebr Pathol 56, 258-266 report that the ô-endotoxins CrylA(b) and CrylA(c) possesses insecticidal properties against BCW with LCso of more than 80 gg and 18 pg/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 Ô-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

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- 27 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.

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.

Alout 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

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-28cytotoxic 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 Stag 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 wày 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

-29 PCT/EP95/03826

WO 96/10083 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

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-30 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.

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-31 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 et al. (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 17:477498 . 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 52:111-116; Neuhause et al., (1987) Theor. Appl. Genet. 75:30-36; Klein etal., (1987) Nature 327: 70-73; Howell el al., (1980) Science 208:1265; Horsch etal., (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,

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-32 eds.) Academic Press, Inc. (1989). 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 Perlak 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), Mol. Gen. Genet., 226:141-144; Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., (1991). Genes Dey., 5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroe et al., (1990), Gene, 91:151-158; Balias et al etal., (1989), Nucleic Acids Res., 1 /:/891-/903: Joshi et al., (198/), 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), Mol. 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.

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-33 A plant terminator may be utilized in the expression cassette. See, Rosenberg et al., (1987), Gene, 56:125; Guerineau et al., (1991), Mol. Gen. Genet., 226:141-144; Proudfoot, (1991), Cell, 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; Balias 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.

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 Lolium, Zea, Triticum, Triticale, Sorghum, Saccharum, Bromus, Oryzae, 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,

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-34 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

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-35 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, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, 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. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces 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,

PCT/EP95/03826

WO 96/10083 •36and 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

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-37 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 fluorescens, 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 grampositive 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, Appl. 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 etal. 1989, FEMS Microbiol. Letts. 60:211-218).

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

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-38 2199049 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 Enzvmoloqy, 153:492 (1987)).

Specifically, unique BamHI restriction sites can be introduced at the aminoterminal 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. co//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

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-39 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 subtilis 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. Mol. 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 grampositive 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

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-40 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

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-41 according to the invention and/or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insectspecific 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 0.1 to 99% by weight of the active ingredient, from 1 to 99.9% by weight of a solid or liquid adjuvant, and from 0 to 25% by weight of a surfactant. The 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 insectspecific 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.

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-42 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 insectspecific 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

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-43 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,

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-44 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 glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethylsulfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils such as epoxidised coconut oil or soybean oil; or water.

The solid carriers used, 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

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-45 PCT/EP95/03826 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 (C₁₀ -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 C₈ -C22 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

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-46(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 C₈ -C22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or hydroxyl-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride or benzyldi-(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, 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

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-47PCT/EP95/03826 can be accomplished chemically (McGuire and Shasha, J Econ Entomol 85:14251433,1992) 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 ô-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.

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-48 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: 11th Volume, ASTM STP 1112 American Society for Testing and Materials, Philadelphia, 1992, pages 173 to 179 and can be used to formulate the recombinant microorganisms according to the invention.

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

In a preferred embodiment of the invention the entomocidal compositions usually contain 0.1 to 99%, preferably 0.1 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 0.1 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

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- 49 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;

NH2-Lys-Arg-Glu-lle-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp-Ser-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 NH2'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 [Canadian Patent Application

No. 2,157,297] are shown below.

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<td> Gene /Protein Name under Standard Nomenclature</td><td> Gene/ Protein Name in Parent</td><td> Description of Protein</td>

<td> VIP1A(a)</td><td> VIP1</td><td> VIP1 from strain AB78 as disclosed in SEQ ID NO:5.</td>

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

<td> VIP1 A(b)</td><td> VIP1 homolog</td><td> VIP1 from Bacillus thuringiensis var. tenebrionis as disclosed in SEQ ID NO:21.</td>

<td> VIP2A(b)</td><td> VIP2 homolog</td><td> VIP2 from Bacillus thuringiensis var. tenebrionis as disclosed in SEQ ID NO:20.</td>

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

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

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-51 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 B21226, 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

<td></td><td> a)</td><td> b)</td><td> c)</td>

<td> Bacillus thuringiensis spores</td><td> 25%</td><td> 50%</td><td> 75%</td>

<td> sodium lignosufonate</td><td> 5%</td><td> 5%</td><td> -</td>

<td> sodium laurylsulfate</td><td> 3%</td><td> -</td><td> 5%</td>

<td> sodium diisobutylnaphthalenesulfonate</td><td> -</td><td> 6%</td><td> 10%</td>

<td> octylphenol polyethylene glycol ether</td><td> -</td><td> 2%</td><td> -</td>

<td> (7-8 moles of ethylene oxid)</td><td></td><td></td><td></td>

<td> highly dispersed silicid acid</td><td> 5%</td><td> 10%</td><td> 10%</td>

<td> kaolin</td><td> 62%</td><td> 27%</td><td> —</td>

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 octylphenol polyethylene glycol ether (4-5 moles ethylene oxide) clacium dodecylbenzensulfonate

10%

3%

3%

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-52 castor oil polyglycol ether (36 moles of ethylene oxide) 4% cyclohexanone 30% xylene mixture 50%

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

A3. Dusts

a) b)

Bacillus thuringiensis spores 5% 8% talcum 95% 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% polyethylene glycol (mol wt 200) 3% kaolin 94%

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-53 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% ethyleneglycol 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 MnCI₂; 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).

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-54TABLE12

Antibiotic activity of AB78 culture supernatant

Zone of inhibition(cm)

Bacteria tested_AB78 Streptomycin

<td> E. coli</td><td> 0.0</td><td> 3.0</td>

<td> B. megaterium</td><td> 1.1</td><td> 2.2</td>

<td> B. mycoides</td><td> 1.3</td><td> 2.1</td>

<td> B. cereus CB</td><td> 1.0</td><td> 2.0</td>

<td> B. cereus 11950</td><td> 1.3</td><td> 2.1</td>

<td> B. cereus 14579</td><td> 1.0</td><td> 2.4</td>

<td> B. cereus AB78</td><td> 0.0</td><td> 2.2</td>

<td> Bt var. israelensis</td><td> 1.1</td><td> 2.2</td>

<td> Bt var. tenebrionis</td><td> 0.9</td><td> 2.3</td>

Morphological characteristics of AB78 are as follows:

Vegetative rods straight, 3.1-5.0 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.

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-55 TABLE 13

Biochemical characteristics of B. cereus strain AB78.

<td colspan="2"> Acid from L-arabinose</td><td> Methylene blue reoxidized</td><td> +</td>

<td> Gas from L-arabinose</td><td></td><td> Nitrate reduced</td><td> +</td>

<td> Acid from D-xylose</td><td> -</td><td> NOg reduced to NO₂</td><td> +</td>

<td> Gas from D-xylose -</td><td> -</td><td> VP</td><td> +</td>

<td> Acid from D-glucose</td><td> +</td><td> H₂O₂ decomposed</td><td> +</td>

<td> Gas from D-glucose</td><td> -</td><td> Indole</td><td> -</td>

<td> Acid from lactose</td><td> -</td><td> Tyrosine decomposed</td><td> +</td>

<td> Gas from lactose</td><td> -</td><td> Dihydroxiacetone</td><td> -</td>

<td> Acid from sucrose</td><td> -</td><td> Litmus milk acid</td><td> -</td>

<td> Gas from sucrose</td><td> -</td><td> Litmus milk coagulated</td><td> -</td>

<td> Acid from D-mannitol</td><td> -</td><td> Litmus milk alkaline</td><td> -</td>

<td> Gas from D-mannitol</td><td> -</td><td> Litmus milk peptonized</td><td> -</td>

<td> Proprionate utilization</td><td> +</td><td> Litmus milk reduced</td><td> -</td>

<td> Citrate utilization</td><td> +</td><td> Casein hydrolyzed</td><td> +</td>

<td> Hippurate hydrolysis</td><td> w</td><td> Starch hydrolyzed</td><td> +</td>

<td> Methylene blue reduced</td><td> +</td><td> Gelatin liquidified</td><td> +</td>

<td> Lecithinase produced</td><td> w</td><td></td><td></td>

w= weak reaction

EXAMPLE 2. BACTERIAL CULTURE

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

<td> Tryptone</td><td> 12</td><td> g/i</td>

<td> Yeast Extract</td><td> 24</td><td> g/i</td>

<td> Glycerol</td><td> 4</td><td> ml/l</td>

<td> kh₂po₄</td><td> 2.1</td><td> g/i</td>

<td> k₂hpo₄</td><td> 14.7</td><td> g/i</td>

<td> pH 7.4</td><td></td><td></td>

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- 56 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 BIQASSAYS

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

Western, Northern and Southern corn rootworm, Diabrotica virgifera virgifera, D. iongcomis 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. co//clone bioassay: E. co//cells were grown overnight in broth containing 100 μρ/πιΙ ampicillin at 37°C. Ten mi culture was sonicated 3X for 20 sec each. 500 μΙ 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 cm² 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.

*Trade-mark

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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 ul 2 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, Culexp/p/ens:-dilutions were made of AB78 culture supernatant grown 24-36 h. 100 μΙ 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

<td> tested to date</td><td> Order</td><td> Activity</td>

<td> Western corn rootworm (Diabrotica virgifera virgifera)</td><td> Col</td><td> +++</td>

<td> Northern corn rootworm (Diabrotica longicornis barberi)</td><td> Col</td><td> +++</td>

<td> Southern corn rootworm (Diabrotica undecimpunctata howardi)</td><td> Col</td><td></td>

<td> Colorado potato beetle (Leptinotarsa decemlineata)</td><td> Col</td><td> -</td>

<td> Yellow mealworm (Tenebrio ιποίϋοή</td><td> Col</td><td></td>

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<td colspan="2"> European corn borer</td>

<td> (Ostrinia nubilalis)</td><td> Lep</td>

<td> Tobacco budworm</td><td></td>

<td> (Heliothis virescens)</td><td> Lep</td>

<td> Tobacco hornworm</td><td></td>

<td> (Manduca sexta)</td><td> Lep</td>

<td> Beet armyworm (Spodoptera exigua) Black cutworm</td><td> Lep</td>

<td> (Agrotis ipsilon)</td><td> Lep</td>

<td> Northern house mosquito (Culex pipiens)</td><td> Dip</td>

The newly discovered β. 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 spp. 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.

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-59 TABLE 15

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

<td> Bacillus strain</td><td> Percent WCRW mortality</td>

<td> B. cereus AB78 (Bat.1 )</td><td> 100</td>

<td> B. cereus AB78 (Bat.2)</td><td> 100</td>

<td> B. cereus (Carolina Bio.)</td><td> 12</td>

<td> B. cereus ATCC 11950</td><td> 12</td>

<td> B. cereus ATCC 14579</td><td> 8</td>

<td> B. mycoides (Carolina Bio.)</td><td> 30</td>

<td> B. popilliae</td><td> 28</td>

<td> B. thuringiensis HD135</td><td> 41</td>

<td> B. thuringiensis HD191</td><td> 9</td>

<td> B. thuringiensis GC91</td><td> 4</td>

<td> B. thuringiensis isrealensis</td><td> 24</td>

<td> Water Control</td><td> 4</td>

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

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- 60 TABLE 16

Activity of AB78 culture supernatant against neonate western corn rootworm

Culture supernatant concentration (itl/ml)

100

2.5

Percent

WCRW mortality

100

The LC50 was calculated to be 6.2 μΙ 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

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- 61 PCT/EP95/03826

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 11.0 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-ProWO 96/10083 &gt;, 2 199049

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-62 (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.

EXAMPLE 6. CONSTRUCTION OF DNA PROBE

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 ) Appl. 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).

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TABLE 17

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

<td> Primers Tested Produi</td><td> :t Produced</td>

<td> 2 sets specific for Cry IIIA</td><td> Negative</td>

<td> CrylllB</td><td> Negative</td>

<td> 2 sets specific for CrylA</td><td> Negative</td>

<td> CrylA(a)</td><td> Negative</td>

<td> CrylA(b) specific</td><td> Negative</td>

<td> CrylB</td><td> Negative</td>

<td> CrylC specific</td><td> Negative</td>

<td> CrylE specific</td><td> Negative</td>

<td> 2 sets specific for B. sphaericus</td><td> Negative</td>

<td> 2 sets specific for CryIV</td><td> Negative</td>

<td> Bacillus control (PI-PLC)</td><td> Positive</td>

EXAMPLE 9. COSIVUD CLONING OF TOTAL DNA FROM B. CEREUS STRAIN

AB78

The VIP1 A(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 pg/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.

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-64 7. Add 200 μΙ 20% SDS and 400 μΙ Proteinase K stock (20 mg/ml). Incubate at 37°C.

8. Add 200 μΙ 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. phenohchloroform/isoamyl alcohol (24:1 ratio).

11. 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 11 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 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 100 110 mM MgSO4

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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

P3-8

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-66P3-9

P3-18

P4-6

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. col/Bacillus shuttle vector pHT 3101 (Lereclus, D. et al., FEMS Microbiology Letters 60:211-218 (1989)) to yield pCIB6201. This construct confers anti-western corn rootworm activity upon both Bacillus and Eco//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 VIP1A(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

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-67deposited 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 religated 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 VIP1 A(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 IEcoRV 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-Cla 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 B21321.

pCIB6023, pCIB6206, and pCIB6203 do not produce detectable western corn rootworm activity when tested individually. However, a mixture of cells containing pCIB6203 (VIPIA(a)-mutated, plus VIP2A(a)) and cells containing pCIB6023 (only

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-68 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 VIP1 A(a), to confer maximal western corn rootworm activity. (See Table 19.)

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-69 Table 19

Characterization of pCIB6022

Activity vs. WCRW

S Rl B

VIPlA(a)

RV C pCIB6022 ₊₊₊

<img file="CA2199049C_D0001.tif" />

<img file="CA2199049C_D0002.tif" />

pCIB6203 pCIB6023 pCIB6206 pCIB6024

Functional Complementation of VIP

<img file="CA2199049C_D0003.tif" />

PCIB6203 +++ pCIB6023

PCIB6203 +++ pCIB6206 pCIB6023 +++ 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 Nterminal ‘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

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- 70 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 μΙ 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:

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

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

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

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

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

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

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

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

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

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

11. 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&amp;S #21640) as described by Towbin etal., (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.

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- 72 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:

<td> Test material</td><td> Percent Diabrotica mortality</td>

<td> pCIB6203</td><td> 0</td>

<td> GC91</td><td> 16</td>

<td> pCIB6203 + GC91</td><td> 100</td>

<td> Control</td><td> 0</td>

EXAMPLE 14. ISOLATION AND BIOLOGICAL ACTIVITY OF B, CEREUS AB&amp;i.

A second B. 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

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1 9 9 0 4 9

- 73 EXAMPLE 15. ISOLATION AND BIOLOGICAL ACTIVITY OF

B. THURINGIENSIS ABG.

A B. 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:

<td> Insect species</td><td> Percent</td>

<td> tested</td><td> Mortality</td>

<td> Ostrinia nubilalis</td><td> 0</td>

<td> Agrotis ipsilon</td><td> 100</td>

<td> Agrotis ipsilon (autoclaved sample)</td><td> 0</td>

<td> Diabrotica virgifera virgifera</td><td> 0</td>

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 MMH.

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:

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<td rowspan="2"> Insect species tested</td><td rowspan="2"> Order</td><td colspan="2"> Percent mortality of culture supernatant Non-</td>

<td> autoclaved</td><td> Autoclav ed</td>

<td> Agrotis ipsilon</td><td> Lepidoptera</td><td> 100</td><td> 5</td>

<td> Ostrinia nubilalis Spodoptera</td><td> Lepidoptera</td><td> 100</td><td> 0</td>

<td> frugiperda</td><td> Lepidoptera</td><td> 100</td><td> 4</td>

<td> Helicoverpa zea</td><td> Lepidoptera</td><td> 100</td><td> 12</td>

<td> Heliothis virescens Leptinotarsa</td><td> Lepidoptera</td><td> 100</td><td> 12</td>

<td> decemlineata Diabrotica</td><td> Coleoptera</td><td> 0</td><td> 0</td>

<td> virgifera virgifera</td><td> Coleoptera</td><td> 0</td><td> 5</td>

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),

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- 75 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 BisTris-Propane pH 9.0.

AB88 proteins have been separated by several different methods following clarification including isoelectric 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 Ô-endotoxins.

anion exchange fraction 23 (smaller): xEPFVSAxxxQxxx (SEQIDNO:10) anion exchange fraction 28 (larger): xEYENVEPFVSAx (SEQ ID NO:11)

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.

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- 76 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 lepidopteran 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 Yorkl]. Biological analysis of fractions demonstrated that different VIPs were responsible for the different lepidopteran 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:

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<td> Agrotis VIP N-terminal sequences</td><td> N-terminal sequence of major δ-endotoxin proteins</td>

<td></td><td> 130 kDa MDNNPNINE (SEQ ID NO:14)</td>

<td> 80 kDa MNKNNTKLPTRALP (SEQ ID NO:12)</td><td> 80 kDa MDNNPNINE (SEQ ID NO:15)</td>

<td> 35 kDa ALSENTGKDGGYIVP (SEQ ID NO:13)</td><td> 60 kDa MNVLNSGRTTI (SEQ ID NO:16)</td>

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 B.

THURINGIENSIS AB424

A B. 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:

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<td> Insect species tested</td><td> Percent mortality</td>

<td> Ostrinia nubilalis</td><td> 100</td>

<td> Agrotis ipsilon</td><td> 100</td>

<td> Diabrotica virgifera</td><td> 0</td>

<td> virgifera</td><td></td>

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 VlP3A(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 &amp; Sons, NY)] and digested with the restriction enzymes Xba\ [library of 4.0 to 5.0 Kb size-fractionated Xbal fragments of B thuringiensis AB88 DNA] and EcoR\ [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. coli DH5a strain. Recombinant clones were blotted onto nitrocellulose filters which were subsequently probed with a³² 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.

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Plasmid pCIB7104 contains a 4.5 Kb Xbalfragment of AB88 DNA. Subclones were constructed to define the coding region of the insecticidal protein.

E coli pCIB7105 was constructed by cloning the 3.5 Kb Xbal-Acclfragment 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 insert (pCIB7107), which still encoded afunctional 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, 74:54635467 (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 B21423, 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

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-80confirmed 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 δendotoxins in cell pellets. Cry-I type prtoeins could be detected late in the stationary phase , during and after sporulation.

EXAMPLE 18D. IDENTIFICATION OF NOVEL V1P3-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 H/ndlll 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/0.1% 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)

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- 81 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 B. thuringiensis STRAW M2194

CONTAINING A CRYPTIC VIP3-LIKE GENE

A B. 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 pg) 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. co//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. Linder the same expermimental conditions, bacterial extracts containing VIP3A proteins did not show any activity against Ostrinia nubilalis.

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Treatment (%) Mortality

TB medium 5

AB88 Supernatant 100

Ab424 Supernatant 100

Buffer 7

E coli pKS 10

E coli pCIB7104 (AB88) 100

E coli pCIB7105 (AB88) 100

E coli pCIB7106 (AB424) 100

E coli pCIB7107 (AB424) 100

Effect of VIP3A insecticidal proteins on lepidopteran insect larvae

Treatment Insect (%) Mortality

E coli pKS BCW 10

FAW 5

BAW 10

TBW 8

CEW 10

ECB 5

Ecoli pCIB7105

Ecoli pCIB7107 BCW 100

FAW 100

BAW 100

TBW 100

CEW 50

ECB 10

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

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-83 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.

<td> Bacillus isolate</td><td> Presence of δ- endotoxin crystal</td><td> Percent mortality</td>

<td> AB6</td><td> +</td><td> 100</td>

<td> AB53</td><td> -</td><td> 80</td>

<td> AB88</td><td> +</td><td> 100</td>

<td> AB195</td><td> -</td><td> 60</td>

<td> AB211</td><td> -</td><td> 70</td>

<td> AB217</td><td> -</td><td> 83</td>

<td> AB272</td><td> -</td><td> 80</td>

<td> AB279</td><td> -</td><td> 70</td>

<td> AB289</td><td> +</td><td> 100</td>

<td> AB292</td><td> +</td><td> 80</td>

<td> AB294</td><td> -</td><td> 100</td>

<td> AB300</td><td> -</td><td> 80</td>

<td> AB359</td><td> -</td><td> 100</td>

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.

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<td> Bacillus isolate</td><td> Presence of δ- endotoxin crystal</td><td> Percent mortality</td>

<td> AB52</td><td> -</td><td> 50</td>

<td> AB59</td><td> -</td><td> 71</td>

<td> AB68</td><td> +</td><td> 60</td>

<td> AB78</td><td> -</td><td> 100</td>

<td> AB122</td><td> -</td><td> 57</td>

<td> AB218</td><td> -</td><td> 64</td>

<td> AB256</td><td> -</td><td> 64</td>

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 V1P1/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 μΙ 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,

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- 85 PNAS, 81:1991-1995 (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)A/IP2A(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 VIP1A(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 VIP 1 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)/VIP2A(a) HOMOLOG FROM

BACILLUS THURINGIENSIS MAR. TENEBRIONIS.

Several previously characterized Bacillus strains were tested for presence of DNA similar to VIP1 A(a)A/IP2A(a) by Southern blot analysis. DNA from Bacillus strains AB78, AB88, GC91, HD-1 and ATCC 10876 was analyzed for presence of VIP1A(a)A/IP2A(a) like sequences. DNA from Bt strains GC91 and HD-1, and the Be strain ATCC 10876 did not hybridize to VIP2A(a)A/IP1A(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)A/IP2A(a) DNA region, suggesting that the VIP activity produced by this strain does not result from VIP1 A(a)A/IP2A(a) homologs. In contrast, Bacillus thuringiensis var. tenebrionis (Btt)

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-86contained 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 Rl, 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)/VIP1 A(a) homologs was determined by the dideoxy chain termination method (Sanger etal., 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)

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- 87 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 VlP1A(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 VIP1 A(b) protein exhibits 77 % amino acid identity to VIP1A(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 VIP1A(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 VIP1 A(a)/VIP2A(a) like protein,

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-88yet 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 MQRMEGKLFWSKTLQVVTRTVLLSTVYSITLLNNWIKADQLNINSQSK 50 SEQ ID NO :20 I -1 I I I I I I : I I I -1 I I I I : I I I I 11 I : I I I I I I I I I I : I I I I I I I I I

AB78 1 MKRMEGKLFMVSKKLQVVTKTVLLSTVFSISLLNNEVIKAEQLNINSQSK 50 SEQ ID NO :2

YTN1QNLKIPDNAEDFKEDKGKAKEWGKEKGEEWRPPATEKGEMNNFLDN 100 lllllllll. I. .1111111:111111111:. ||: . I I I I I. I I I I I I I

YTND2NLKITDÎCVEDFKEDKEKAKEWGKEKEKEWKLTATEKGKMNNFLDN 100

101 KNDIKTOYKEITFSMAGSŒDEIKDLEEIDKIFDKANLSSSIITYKNVEP 150 Nil lllllllllllll 1111111 · I 111 : II I · I 11 11 111111II

101 Kt®IXTNYKEITFSMAGSFEDEIKDLKEIDKMFDKTNLSNSIITYKNVEP 150

151 ATIGFNKSLTEGNTINSDAMAQFKEQFLGKDMKFDSYLDTHLTAQQVSSK 200 I 11 I I 11 I I I I I I 111 I I I I 11 I 1111 :: I : I I I I I I I I I 11 I I I I I I I

151 TTIGFNKSLTEGNTINSDAMAQFKEQFLDRDIKFDSYLDTHLTAQQVSSK 200

201 KRVILKVTVPSGKGSTTPTKAGVILNNNEYKMLIDNGYVLHVDKVSKVVK 250 . 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 I I I I

201 ERVILKVTVPSGKGSTTPTKAGVILNNSEYKMLIDNGYMVHVDKVSKWK 250

251 KQ4ECLQVEGTLKKSLDFKNDINAEAHSWGMKIYEDWAKNLTASQREALD 300 ll:||||:|lllllllllllllllllllllll I I : I I I : I I -1 I I I I 11

251 KGVECDQIEGTLKKSLDFKNDINAEAHSWO4KNYEEWAKDLTDSQREALD 300

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-89 301 GYARQDYKEINNYLRNQGGSGNEKLDAQLKNISDALGKKPIPENITVYRW 350 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 I I I I I I I 301 GYARQDYKEINNYLRNQGGSGNEKLDAQIKNISDALGKKPIPENITVYRW 350

351 CGMPEFGYQISDPLPSLKDFEEQFLNTIKEDKGYMSTSLSSERLAAFGSR 400

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 I I I I I I I I

351 CGMPEFGYQISDPLPSLKDFEEQFLNTIKEDKGYMSTSLSSERLAAFGSR 400

401 KIILRLQVPKGSTGAYLSAIGGFASEKEILLDKDSKYHIDKATEVIIKGV 450

II 1111 I I 111 11 11 I I I I I 11 11 11 I I I 11 I I 11 I I I I I I I I I I I 111

401 KIILRLQVPKGSTGAYLSAIGGFASEKEILLDKDSKYHIDKVTEVIIKGV 450

451 KRYWDATLLTN 462 illlllllllll

451 KRYWDATLLTN 462

TABLE 21

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

Btt 1 MKNMKKKLASWTCMLLAPMFLNGNVNAVNADSKINQISTTQENQQKEMD 50 SEQ ID NO:21 I I 11 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 11 I I I I I I I

Ab78 1 MKNMKKKLASWTCTLLAPMFLNGNVNAVYADSKTNQISTTQKNQQKEMD 50 SEQ ID NO :5

RKGLLGYYFKGKDFNNLTMFAPTRDNTLMYDQQTANALLDKKQQEYQSIR 100 1111111 1111 I I I · I 11 I I I I I 11.1 I : I I I I I 11 I I I I I I I I 11111

RKGLLGYYFKGKDFSNLTMFAPTRDSTLIYDQQTANKLLDKKQQEYQSIR 100

101 WIGLIQRKETGDFTFNLSKDEQAIIEIDGKIISNKGKEKQWHLEKEKLV 150

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 I : I I I

101 WIGLIQSKETGDFTFNLSEDEQAIIEINGKIISNKGKEKQWHLEKGKLV 150

151 PIKIEYQSDTKFNIDSKTFKELKLFKIDSQNQSQQVQ...LRNPEFNKKE 197

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-90PIKIEYQSDTKFNIDSKTFKELKLFKIDSQNQPQQVQQDELRNPEFNKKE 200

SQEFLAKASKTNLFKQKMKRDIDEDTDTDGDSIPDLWEENGYTIQNKVAV 247 I I I I 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 SQEF1AKPSKINLFTQKMKREIDEDTDTDGDSIPDLWEENGYTIQNRIAV 250

KWDDSLASKGYTKFVSNPLDSHTVGDPYTDYEKAARDLDLSNAKETFNPL 297 ||||||HIIIIIIIIIII:|||IIIIIIIIIIII lllllllllllllll KWDDSLASKGYTKFVSNPLESHTVGDPYTDYEKAARDLDLSNAKETFNPL 300

VAAFPSVNVSMEKVILSPNENLSNSVESHSSTNWSYTNTEGASIEAGGGP 347 I111IIII11IIIII11IIIIII1111111111I I I I I I 11 I I : I 11 II VAAFPSVNVSMEKVILSPNENLSNSVESHSSTNWSYTNTEGASVEAGIGP 350

LGLSFGVSVTYQHSETVAQEWGTSTGNTSQFNTASAGYLNANVRYNNVGT 397 |:||llll.llllllllllllllllllllllllllllllllllllllll

KGISFGVSVNYQHSETVAQEWGTSTGNTSQFNTASAGYLNANVRYNNVGT 400

GAIYDVKPTTSFVLNNNTIATITAKSNSTALRISPGDSYPEIGENAIAIT 447 III II I 11 I I I 11 11 I : I I 111 I 11 I 11 11 Μ I I 1: 11 1. 1: 1: 11 I I GAIYDVKPTTSFVLNNDTIATITAKSNSTALNISPGESYPKKGQNGIAIT 450

SMDDFNSHPITLNKQQVNQLINNKPIMLETDQTDGVYKIRDTHGNIVTGG 497 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 I SMDDFNSHPITINKKQVDNIJHQNKPMMLETNQTDGVYKIKDTHGNIVTGG 500

EWGVTQQIKAKTASIIVDDGKQVAEKRVAAKDYGHPEDKrPPLTLKDTL 547 mil. lllllllllllllll. . 11111111 U t — 111111 · 11111 -1 EWNGVIQQIKAKTASIIVDDGERVAEKRVAAKDYENPEDKTPSLTLKDAL 550

PCLSYPDEIKETNGLLYYDDKPIYESSVMTYLDENTAKEVKKQINDTTGKF 597

I I I I I 11 I I I : I I I I I. : I I I I 11 I 11 I 111 I i I I I I I I m I I 11 I I

KLSYPDEIKEIEGLLYYKNKPIYESSVMTYLDENTAKEVTKQLNDTTGKF 600

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- 91 598 KDVNHLYDVKLTPKMNFTIKMASLYDGAENNHNSLGTWYLTYNVAGGNTG 647 . . . . 601 KDVSHLYDVKLTPKMNVTIKLSILYDNAESNDNSIGKWTNTNIVSGGNNG 650

648 WYRSAHSCAHVALSSEAKKKLNQNANYYLSlTiMKADSTrEPTIEVAGE 697

651 KKQYSSNNPDANLTLNTDAQEKLNKNRDYYISLYMKSEKNTQCEITIDΠ700

698 KSAITSKKVKLNNQNYQRVDILVKNSERNPMDKIYIRGNGTTNVYGDDVT 747

701 IYPITTKTVNVNKDNYKRLDIIAHNIKSNPISSLHIKTNDEITLFWDDIS 750

748 IPEVSAINPASLSDEEIQEIFKDSTIEYGNPSFVADAVTFK......... 788

751 ITDVASIKPENLTDSEIKQIYSRYGIKLEDGILIDKKGGIHYGEFINEAS 800

789 .NIKPLQNYVKEYEIYHK.......SHRYEKKTVFDIMGVHYEYSIAREQ 830

801 FNIEPLQNYVTKYKVTYSSELGQNVSDTLESDKIYKDGTIKFDFTKYSKN 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_Zterminus of the fusion protein, or they can be fused to produce the larger of the

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- 92 polypeptides as the NH₂ 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 VIP1A(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

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-93 PCT/EP95/03826 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 Nterminal 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

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-94 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. Cornai etal. 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 313:358363 (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 etal. Plant Molec. Biol. 13:411-418 (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 etal. (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 VIP2 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 etal. In: Edelmann etal. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp 1081 1091 (1982); Wasmann etal. Mol. Gen. Genet. 205:446-453 (1986)). These

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-95 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. 1.: 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 &amp; 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 Physiol., 101:1-5 (1993)), carboxy- terminal portion, or in the internal sequence of the targeted protein. (Tague etal., The Plant Cell. 4:307-318 (1992), Saalbach etal., 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 et al. 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. 23:1105-1115 (1993)).

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-96ln order to target VIP2, either for secretion or to the various subcellular organelles, a maize optimized DNA sequence encoding a known signai 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 Ceil. 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. 4:307318 (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 ÇTGÇAG-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

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- 97 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 targetting 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 GOG 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

VIP1 A(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

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-98contained the sequence 5'-GGA TGC 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 Hindi 11 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 / HindiII fragment was then obtained by restriction digest and used to replace the BamHI/Hindlll fragment of the maize optimized VIP1 A(a) gene cloned in the rootpreferred 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 VIP1A(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 HindiII restriction site at the 3' end. The amplification product was cloned into a T- vector, sequenced and ligated to a BamHI /HindiII 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 NQ:40.

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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 etal., 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 μΜ. To 30 μΙ 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

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- 100 from the polyacryamide by an overnight incubation of the minced gel fragment in a buffer containing 0.4 M LiCI, 0.1 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 dH₂0 at a concentration of 2.5 μΜ. 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 μΙ of the upper and of the lower oligo for each oligo pair with buffer containing 1X polynucleotide kinase (PNK) buffer (70 mM Tris-HCI (pH 7.6), 10 mM MgCI₂,5 mM dithiothreitol (DTT)), 50 mM KCI, and 5 % formamide in a final volume of 50 μΙ. The oligos were boiled for 10 minutes and slow cooled to 37° C or room temperature. 10 μΙ was removed for analysis on a 4% agarose in a TAE buffer system (Métaphore®; 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 μΙ of 10X PNK buffer in a volume of 10 μΙ. Following hybridization and phosphorylation, the reaction was incubated at 37° C for 2 hours to overnight. 10 μΙ of each of the oligo pairs for a particular fragment, were mixed in a final volume of 50 μΙ. The oligo pairs were hybridized by heating at 80° C for 10 minutes and slow cooling to 37° C. 2 μΙ of oligos was mixed with about 100 ng of an appropriate vector and ligated using a buffer containing 50 mM Tris-HCI (pH 7.8), 10 mM MgCI₂, 10 mM DTT, 1 mM ATP. The reaction was incubated at room temp, for 2 hours to overnight and transformed into DH5a 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.

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- 101 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 μΜ) 200 μΜ of each dATP, dTTP, dCTP and dGTP, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCI₂,0.01% gelatin and 5 units of Taq polymerase in a final reaction volume of 50 μΙ. 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 μΙ of the reaction was analyzed on a 2.5% Nusieve (FMG) 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 VIP1A(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

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- 102 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 gg 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 copurification 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 A(a) AND VIP2A(a)

E. coli strains containing different plasmids comprising VIP genes were assayed for expression of VIPs. E. co//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

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- 103 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

<td> Extract of E. coli Strain Harboring Indicated Plasmid</td><td colspan="2"> Assay Assay No. 1 No. 2 % Mortality</td><td> Protein Detected</td>

<td> Control</td><td> 0</td><td> 0</td><td> no</td>

<td> pCIB5521 (maize optimized VIP1 A(a))</td><td> 47</td><td> 27</td><td> yes</td>

<td> pCIB5522 (maize optimized VIP2A(a))</td><td> 7</td><td> 7</td><td> yes</td>

<td> pCIB6024 (native VIP2A(a))</td><td> 13</td><td> 13</td><td> yes</td>

<td> pCIB6206 (native VIP1A(a))</td><td> 27</td><td> 40</td><td> yes</td>

<td> Extracts pCIB5521 + pCIB5522 combined</td><td> 87</td><td> 47</td><td></td>

<td> Extracts pCIB5521 + pCIB6024 combined</td><td> 93</td><td> 100</td><td></td>

<td> Extracts pCIB5522 + pCIB6206 combined</td><td> 100</td><td> 100</td><td></td>

<td> Extracts pCIB6024 + pCIB6206 combined</td><td> 100</td><td> 100</td><td></td>

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

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- 104 accomplished on protoplast expiants 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

<td> Extract Tested</td><td> Assay No. 1 % Mo</td><td> Assay No. 2 rtality</td><td> Protein Detected</td>

<td> No DNA control</td><td> 27</td><td> 10</td><td> no</td>

<td> pCIB5521 (p) (maize</td><td> 20 (0)</td><td> 30</td><td> yes</td>

<td> optimized VIP1A(a)) pCIB5522 (p) (maize</td><td> 20 (0)</td><td> 20</td><td> yes</td>

<td> optmizied VIP2A(a)) Extracts pCIB5521 (p) +</td><td> 87 (82)</td><td> 90</td><td></td>

<td> pCIB5522 (p) combined Extracts pCIB5521 (p) +</td><td> 100</td><td></td><td></td>

<td> pCIB5522 (e) combined Extracts pCIB5522 (p) +</td><td> 53 (36)</td><td></td><td></td>

<td> pCIB5521 (e) combined Extracts pCIB5521 (p) +</td><td> 100</td><td></td><td></td>

<td> pCIB6024 (e) combined Extracts pCIB5522 (p) +</td><td> 100</td><td></td><td></td>

<td> pCIB6206 (e) combined pCIB6024(e) (native</td><td> 0</td><td></td><td> yes</td>

<td> VIP2A(a)) pCIB6206(e) (native</td><td> 20</td><td></td><td> yes</td>

<td> VIP1A(a)) PCIB5521 + pCIB 5522</td><td> 100</td><td> 100</td><td> yes</td>

(plasmids delivered by cotransformation) (p) = extract of protoplast culture transformed with indicated plasmid

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- 105 (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 VIP1 A(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.

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:

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<td colspan="2"> Strain designation</td><td> Deposition Number</td><td> Deposition Date</td>

<td> 1.</td><td> E. coli PL2</td><td> NRRL B-21221</td><td> March 09, 1994</td>

<td> 2.</td><td> E. coliPL2</td><td> NRRL B-21221 N</td><td> September 02,1994</td>

<td> 3.</td><td> E. coli pC IB6022</td><td> NRRL B-21222</td><td> March 09,1994</td>

<td> 4.</td><td> E. coli pCIB6023</td><td> NRRL B-21223</td><td> March 09,1994</td>

<td> 5.</td><td> E. coli pCIB6023</td><td> NRRL B-21223N</td><td> September 02,1994</td>

<td> 6.</td><td> Bacillus thuringiensis HD73-78VIP</td><td> NRRL B-21224</td><td> March 09, 1994</td>

<td> 7.</td><td> Bacillus thuringiensis AB88</td><td> NRRL B-21225</td><td> March 09,1994</td>

<td> 8.</td><td> Bacillus thuringiensis AB359</td><td> NRRL B-21226</td><td> March 09, 1994</td>

<td> 9.</td><td> Bacillus thuringiensis AB289</td><td> NRRL B-21227</td><td> March 09,1994</td>

<td> 10.</td><td> Bacillus sp. AB59</td><td> NRRL B-21228</td><td> March 09, 1994</td>

<td> 11.</td><td> Bacillus sp. AB294</td><td> NRRL B-21229</td><td> March 09,1994</td>

<td> 12.</td><td> Bacillus sp. AB256</td><td> NRRL B-21230</td><td> March 09,1994</td>

<td> 13.</td><td> E. coli P5-4</td><td> NRRL B-21059</td><td> March 18,1993</td>

<td> 14.</td><td> E. coli P3-12</td><td> NRRL B-21061</td><td> March 18, 1993</td>

<td> 15.</td><td> Bacillus cereus AB78</td><td> NRRL B-21058</td><td> March 18,1993</td>

<td> 16.</td><td> Bacillus thuringiensis AB6</td><td> NRRL B-21060</td><td> March 18, 1993</td>

<td> 17.</td><td> E. coli pCIB6202</td><td> NRRL B-21321</td><td> September 02,1994</td>

<td> 18.</td><td> E. coli pCIB7100</td><td> NRRL B-21322</td><td> September 02,1994</td>

<td> 19.</td><td> E. colipCIB7101</td><td> NRRL B-21323</td><td> September 02,1994</td>

<td> 20.</td><td> E. colipCIB7102</td><td> NRRL B-21324</td><td> September 02,1994</td>

<td> 21.</td><td> E. colipCIB7103</td><td> NRRL B-21325</td><td> September 02,1994</td>

<td> 22.</td><td> E. coli pC IB7104</td><td> NRRL B-21422</td><td> March 24, 1995</td>

<td> 23.</td><td> E. colipCIB7107</td><td> NRRL B-21423</td><td> March 24, 1995</td>

<td> 24.</td><td> E. coli pCIB7108</td><td> NRRL B-21438</td><td> May 05, 1995</td>

<td> 25.</td><td> Bacillus thuringiensis AB424</td><td> NRRL B-21439</td><td> May 05,1995</td>

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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.

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

(D) SOFTWARE: Patentln Release #1.0, Version #1.30B (2) INFORMATION FOR SEQ ID NO: 1:

(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.

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- 109 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

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

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

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

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

55 60

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

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<td> Glu</td><td> Asp 65</td><td> Phe</td><td> Lys</td><td> Glu</td><td> Asp</td><td> Lys 70</td><td> Glu</td><td> Lys</td><td> Ala</td><td> Lys</td><td> Glu 75</td><td> Trp</td><td> Gly</td><td> Lys</td><td> Glu</td><td></td>

<td> AAA</td><td> GAA</td><td> AAA</td><td> GAG</td><td> TGG</td><td> AAA</td><td> CTA</td><td> ACT</td><td> GCT</td><td> ACT</td><td> GAA</td><td> AAA</td><td> GGA</td><td> AAA</td><td> ATG</td><td> AAT</td><td> 1366</td>

<td> Lys 80</td><td> Glu</td><td> Lys</td><td> Glu</td><td> Trp</td><td> Lys 85</td><td> Leu</td><td> Thr</td><td> Ala</td><td> Thr</td><td> Glu 90</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Met</td><td> Asn 95</td><td></td>

<td> AAT</td><td> TTT</td><td> TTA</td><td> GAT</td><td> AAT</td><td> AAA</td><td> AAT</td><td> GAT</td><td> ATA</td><td> AAG</td><td> ACA</td><td> AAT</td><td> TAT</td><td> AAA</td><td> GAA</td><td> ATT</td><td> 1414</td>

<td> Asn</td><td> Phe</td><td> Leu</td><td> Asp</td><td> Asn 100</td><td> Lys</td><td> Asn</td><td> Asp</td><td> He</td><td> Lys 105</td><td> Thr</td><td> Asn</td><td> Tyr</td><td> Lys</td><td> Glu 110</td><td> He</td><td></td>

<td> ACT</td><td> TTT</td><td> TCT</td><td> ATG</td><td> GCA</td><td> GGC</td><td> TCA</td><td> TTT</td><td> GAA</td><td> GAT</td><td> GAA</td><td> ATA</td><td> AAA</td><td> GAT</td><td> TTA</td><td> AAA</td><td> 1462</td>

<td> Thr</td><td> Phe</td><td> Ser</td><td> Met 115</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Phe</td><td> Glu 120</td><td> Asp</td><td> Glu</td><td> He</td><td> Lys</td><td> Asp 125</td><td> Leu</td><td> Lys</td><td></td>

<td> GAA</td><td> ATT</td><td> GAT</td><td> AAG</td><td> ATG</td><td> TTT</td><td> GAT</td><td> AAA</td><td> ACC</td><td> AAT</td><td> CTA</td><td> TCA</td><td> AAT</td><td> TCT</td><td> ATT</td><td> ATC</td><td> 1510</td>

<td> Glu</td><td> He</td><td> Asp 130</td><td> Lys</td><td> Met</td><td> Phe</td><td> Asp</td><td> Lys 135</td><td> Thr</td><td> Asn</td><td> Leu</td><td> Ser</td><td> Asn 140</td><td> Ser</td><td> He</td><td> He</td><td></td>

<td> ACC</td><td> TAT</td><td> AAA</td><td> AAT</td><td> GTG</td><td> GAA</td><td> CCG</td><td> ACA</td><td> ACA</td><td> ATT</td><td> GGA</td><td> TTT</td><td> AAT</td><td> AAA</td><td> TCT</td><td> TTA</td><td> 1558</td>

<td> Thr</td><td> Tyr 145</td><td> Lys</td><td> Asn</td><td> Val</td><td> Glu</td><td> Pro 150</td><td> Thr</td><td> Thr</td><td> He</td><td> Gly</td><td> Phe 155</td><td> Asn</td><td> Lys</td><td> Ser</td><td> Leu</td><td></td>

<td> ACA</td><td> GAA</td><td> GGT</td><td> AAT</td><td> ACG</td><td> ATT</td><td> AAT</td><td> TCT</td><td> GAT</td><td> GCA</td><td> ATG</td><td> GCA</td><td> CAG</td><td> TTT</td><td> AAA</td><td> GAA</td><td> 1606</td>

<td> Thr 160</td><td> Glu</td><td> Gly</td><td> Asn</td><td> Thr</td><td> He 165</td><td> Asn</td><td> Ser</td><td> Asp</td><td> Ala</td><td> Met 170</td><td> Ala</td><td> Gin</td><td> Phe</td><td> Lys</td><td> Glu 175</td><td></td>

<td> CAA</td><td> TTT</td><td> TTA</td><td> GAT</td><td> AGG</td><td> GAT</td><td> ATT</td><td> AAG</td><td> TTT</td><td> GAT</td><td> AGT</td><td> TAT</td><td> CTA</td><td> GAT</td><td> ACG</td><td> CAT</td><td> 1654</td>

<td> Gin</td><td> Phe</td><td> Leu</td><td colspan="3"> Asp Arg Asp 180</td><td> He</td><td> Lys</td><td> Phe</td><td> Asp 185</td><td> Ser</td><td> Tyr</td><td> Leu</td><td> Asp</td><td> Thr 190</td><td> His</td><td></td>

<td> TTA</td><td> ACT</td><td> GCT</td><td> CAA</td><td> CAA</td><td> GTT</td><td> TCC</td><td> AGT</td><td> AAA</td><td> GAA</td><td> AGA</td><td> GTT</td><td> ATT</td><td> TTG</td><td> AAG</td><td> GTT</td><td> 1702</td>

<td> Leu</td><td> Thr</td><td> Ala</td><td> Gin 195</td><td> Gin</td><td> Val</td><td> Ser</td><td> Ser</td><td> Lys 200</td><td> Glu</td><td> Arg</td><td> Val</td><td> He</td><td> Leu 205</td><td> Lys</td><td> Val</td><td></td>

<td> ACG</td><td> GTT</td><td> CCG</td><td> AGT</td><td> GGG</td><td> AAA</td><td> GGT</td><td> TCT</td><td> ACT</td><td> ACT</td><td> CCA</td><td> ACA</td><td> AAA</td><td> GCA</td><td> GGT</td><td> GTC</td><td> 1750</td>

<td> Thr</td><td> Val</td><td> Pro 210</td><td> Ser</td><td> Gly</td><td> Lys</td><td> Gly</td><td> Ser 215</td><td> Thr</td><td> Thr</td><td> Pro</td><td> Thr</td><td> Lys 220</td><td> Ala</td><td> Gly</td><td> Val</td><td></td>

<td> ATT</td><td> TTA</td><td> AAT</td><td> AAT</td><td> AGT</td><td> GAA</td><td> TAC</td><td> AAA</td><td> ATG</td><td> CTC</td><td> ATT</td><td> GAT</td><td> AAT</td><td> GGG</td><td> TAT</td><td> ATG</td><td> 1798</td>

<td> He</td><td> Leu 225</td><td> Asn</td><td> Asn</td><td> Ser</td><td> Glu</td><td> Tyr 230</td><td> Lys</td><td> Met</td><td> Leu</td><td> He</td><td> Asp 235</td><td> Asn</td><td> Gly</td><td> Tyr</td><td> Met</td><td></td>

<td> GTC</td><td> CAT</td><td> GTA</td><td> GAT</td><td> AAG</td><td> GTA</td><td> TCA</td><td> AAA</td><td> GTG</td><td> GTG</td><td> AAA</td><td> AAA</td><td> GGG</td><td> GTG</td><td> GAG</td><td> TGC</td><td> 1846</td>

<td> Val 240</td><td> His</td><td> Val</td><td colspan="2"> Asp Lys</td><td> Val 245</td><td> Ser</td><td> Lys</td><td> Val</td><td> Val</td><td> Lys 250</td><td> Lys</td><td> Gly</td><td> Val</td><td> Glu</td><td> Cys 255</td><td></td>

<td> TTA</td><td> CAA</td><td> ATT</td><td> GAA</td><td> GGG</td><td> ACT</td><td> TTA</td><td> AAA</td><td> AAG</td><td> AGT</td><td> CTT</td><td> GAC</td><td> TTT</td><td> AAA</td><td> AAT</td><td> GAT</td><td> 1894</td>

<td> Leu</td><td> Gin</td><td> He</td><td> Glu</td><td> Gly 260</td><td> Thr</td><td> Leu</td><td> Lys</td><td> Lys</td><td> Ser 265</td><td> Leu</td><td> Asp</td><td> Phe</td><td> Lys</td><td> Asn 270</td><td> Asp</td><td></td>

<td> ATA</td><td> AAT</td><td> GCT</td><td> GAA</td><td> GCG</td><td> CAT</td><td> AGC</td><td> TGG</td><td> GGT</td><td> ATG</td><td> AAG</td><td> AAT</td><td> TAT</td><td> GAA</td><td> GAG</td><td> TGG</td><td> 1942</td>

<td> He</td><td> Asn</td><td> Ala</td><td> Glu</td><td> Ala</td><td> His</td><td> Ser</td><td> Trp</td><td> Gly</td><td> Met</td><td> Lys</td><td> Asn</td><td> Tyr</td><td> Glu</td><td> Glu</td><td> Trp</td><td></td>

275 280 285

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<td rowspan="2"> GCT Ala</td><td colspan="2"> AAA GAT</td><td rowspan="2"> TTA ACC GAT Leu Thr Asp</td><td rowspan="2"> TCG Ser</td><td colspan="5"> CAA AGG GAA GCT TTA GAT GGG TAT GCT</td><td rowspan="2"> 1990</td>

<td> Lys</td><td> Asp 290</td><td> Gin 295</td><td> Arg Glu</td><td> Ala</td><td> Leu Asp Gly 300</td><td> Tyr Ala</td>

<td> AGG</td><td> CAA</td><td> GAT</td><td> TAT AAA GAA</td><td> ATC</td><td> AAT</td><td> AAT TAT</td><td> TTA</td><td> AGA AAT CAA</td><td> GGC GGA</td><td> 2038</td>

<td> Arg</td><td> Gin 305</td><td> Asp</td><td> Tyr Lys Glu</td><td> He 310</td><td> Asn</td><td> Asn Tyr</td><td> Leu</td><td> Arg Asn Gin 315</td><td> Gly Gly</td><td></td>

<td> AGT</td><td> GGA</td><td> AAT</td><td> GAA AAA CTA</td><td> GAT</td><td> GCT</td><td> CAA ATA</td><td> AAA</td><td> AAT ATT TCT</td><td> GAT GCT</td><td> 2086</td>

<td> Ser 320</td><td> Gly</td><td> Asn</td><td> Glu Lys Leu 325</td><td> Asp</td><td> Ala</td><td> Gin He</td><td> Lys 330</td><td> Asn He Ser</td><td> Asp Ala 335</td><td></td>

<td> TTA</td><td> GGG</td><td> AAG</td><td> AAA CCA ATA</td><td> CCG</td><td> GAA</td><td> AAT ATT</td><td> ACT</td><td> GTG TAT AGA</td><td> TGG TGT</td><td> 2134</td>

<td> Leu</td><td> Gly</td><td> Lys</td><td> Lys Pro He 340</td><td> Pro</td><td> Glu</td><td> Asn He 345</td><td> Thr</td><td> Val Tyr Arg</td><td> Trp Cys 350</td><td></td>

<td> GGC</td><td> ATG</td><td> CCG</td><td> GAA TTT GGT</td><td> TAT</td><td> CAA</td><td> ATT AGT</td><td> GAT</td><td> CCG TTA CCT</td><td> TCT TTA</td><td> 2182</td>

<td> Gly</td><td> Met</td><td> Pro</td><td colspan="2"> Glu Phe Gly Tyr 355</td><td> Gin</td><td> He Ser 360</td><td> Asp</td><td> Pro Leu Pro 365</td><td> Ser Leu</td><td></td>

<td> AAA</td><td> GAT</td><td> TTT</td><td> GAA GAA CAA</td><td> TTT</td><td> TTA</td><td> AAT ACA</td><td> ATC</td><td> AAA GAA GAC</td><td> AAA GGA</td><td> 2230</td>

<td> Lys</td><td> Asp</td><td> Phe 370</td><td> Glu Glu Gin</td><td> Phe</td><td> Leu 375</td><td> Asn Thr</td><td> He</td><td colspan="2"> Lys Glu Asp Lys Gly 380</td><td></td>

<td> TAT</td><td> ATG</td><td> AGT</td><td> ACA AGC TTA</td><td> TCG</td><td> AGT</td><td> GAA CGT</td><td> CTT</td><td> GCA GCT TTT</td><td> GGA TCT</td><td> 2278</td>

<td> Tyr</td><td> Met 385</td><td> Ser</td><td> Thr Ser Leu</td><td> Ser 390</td><td> Ser</td><td> Glu Arg</td><td> Leu</td><td> Ala Ala Phe 395</td><td> Gly Ser</td><td></td>

<td> AGA</td><td> AAA</td><td> ATT</td><td> ATA TTA CGA</td><td> TTA</td><td> CAA</td><td> GTT CCG</td><td> AAA</td><td> GGA AGT ACG</td><td> GGT GCG</td><td> 2326</td>

<td> Arg 400</td><td> Lys</td><td> He</td><td> He Leu Arg 405</td><td> Leu</td><td> Gin</td><td> Val Pro</td><td> Lys 410</td><td> Gly Ser Thr</td><td> Gly Ala 415</td><td></td>

<td> TAT</td><td> TTA</td><td> AGT</td><td> GCC ATT GGT</td><td> GGA</td><td> TTT</td><td> GCA AGT</td><td> GAA</td><td> AAA GAG ATC</td><td> CTA CTT</td><td> 2374</td>

<td> Tyr</td><td> Leu</td><td> Ser</td><td> Ala He Gly 420</td><td> Gly</td><td> Phe</td><td> Ala Ser 425</td><td> Glu</td><td> Lys Glu He</td><td> Leu Leu 430</td><td></td>

<td> GAT</td><td> AAA</td><td> GAT</td><td> AGT AAA TAT</td><td> CAT</td><td> ATT</td><td> GAT AAA</td><td> GTA</td><td> ACA GAG GTA</td><td> ATT ATT</td><td> 2422</td>

<td> Asp</td><td> Lys</td><td> Asp</td><td> Ser Lys Tyr 435</td><td> His</td><td> He</td><td> Asp Lys 440</td><td> Val</td><td> Thr Glu Val 445</td><td> He He</td><td></td>

<td> AAA Lys</td><td> GGT Gly</td><td> GTT Val</td><td> AAG CGA TAT Lys Arg Tyr</td><td> GTA Val</td><td> GTG Val</td><td> GAT GCA Asp Ala</td><td> ACA Thr</td><td> TTA TTA ACA Leu Leu Thr</td><td> AAT Asn</td><td> 2467</td>

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

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<td> TGAGGATGAA</td><td> CAGGCAATTA</td><td> TAGAAATCAA</td><td> TGGGAAAATT</td><td> ATTTCTAATA</td><td> AAGGGAAAGA</td><td> 2887</td>

<td> AAAGCAAGTT</td><td> GTCCATTTAG</td><td> AAAAAGGAAA</td><td> ATTAGTTCCA</td><td> ATCAAAATAG</td><td> AGTATCAATC</td><td> 2947</td>

<td> AGATACAAAA</td><td> TTTAATATTG</td><td> ACAGTAAAAC</td><td> ATTTAAAGAA</td><td> CTTAAATTAT</td><td> TTAAAATAGA</td><td> 3007</td>

<td> TAGTCAAAAC</td><td> CAACCCCAGC</td><td> AAGTŒAGCA</td><td> AGATGAACTG</td><td> AGAAATCCTG</td><td> AATTTAACAA</td><td> 3067</td>

<td> GAAAGAATCA</td><td> CAGGAATTCT</td><td> TAGCGAAACC</td><td> ATCGAAAATA</td><td> AATCTTTTCA</td><td> CTCAAAAAAT</td><td> 3127</td>

<td> GAAAAGGGAA</td><td> ATTGATGAAG</td><td> ACACGGATAC</td><td> GGATGGGGAC</td><td> TCTATTCCTG</td><td> ACCTTTGGGA</td><td> 3187</td>

<td> AGAAAATGGG</td><td> TATACGATTC</td><td> ACAATAGAAT</td><td> CGCTGTAAAG</td><td> TGGGACGATT</td><td> CTCTAGCAAG</td><td> 3247</td>

<td> TAAAGGGTAT</td><td> ACGAAATTTG</td><td> TTTCAAATCC</td><td> ACTAGAAAGT</td><td> CACACAGTTG</td><td> GTGATCCTTA</td><td> 3307</td>

<td> TACAGATTAT</td><td> GAAAAGGCAG</td><td> CAAGAGATCT</td><td> AGATTTGTCA</td><td> AATGCAAAGG</td><td> AAACGTTTAA</td><td> 3367</td>

<td> CCCATTGGTA</td><td> GCTGCTTTTC</td><td> CAAGTGTGAA</td><td> TGTTAGTATG</td><td> GAAAAGGTGA</td><td> TATTATCACC</td><td> 3427</td>

<td> AAATGAAAAT</td><td> TTATCCAATA</td><td> GTGTAGAGTC</td><td> TCATTCATCC</td><td> ACGAATTGGT</td><td> CTTATACAAA</td><td> 3487</td>

<td> TACAGAAGGT</td><td> gcttctgttg</td><td> AAGCGGGGAT</td><td> TGGACCAAAA</td><td> GGTATTTCGT</td><td> TCGGAGTTAG</td><td> 3547</td>

<td> CGTAAACTAT</td><td> CAACACTCTG</td><td> AAACAGTTGC</td><td> ACAAGAATGG</td><td> GGAACATCTA</td><td> CAGGAAATAC</td><td> 3607</td>

<td> TTCGCAATTC</td><td> AATACGGCTT</td><td> CAGCGGGATA</td><td> TTTAAATGCA</td><td> AATGTTCGAT</td><td> ATAACAATGT</td><td> 3667</td>

<td> AGGAACTGGT</td><td> GCCATCTACG</td><td> ATGTAAAACC</td><td> TACAACAAGT</td><td> TTTGTATTAA</td><td> ATAACGATAC</td><td> 3727</td>

<td> TATCGCAACT</td><td> ATTACGGCGA</td><td> AATCTAATTC</td><td> TACAGCCTTA</td><td> AATATATCTC</td><td> CTGGAGAAAG</td><td> 3787</td>

<td> TTACCCGAAA</td><td> AAAGGACAAA</td><td> ATGGAATCGC</td><td> AATAACATCA</td><td> ATGGATGATT</td><td> TTAATTCCCA</td><td> 3847</td>

<td> TCCGATTACA</td><td> TTAAATAAAA</td><td> AACAAGTAGA</td><td> TAATCTGCTA</td><td> AATAATAAAC</td><td> CTATGATGTT</td><td> 3907</td>

<td> GGAAACAAAC</td><td> CAAACAGATG</td><td> GTGTTTATAA</td><td> GATAAAAGAT</td><td> ACACATGGAA</td><td> ATATAGTAAC</td><td> 3967</td>

<td> TGGCGGAGAA</td><td> TGGAATGGTG</td><td> TCATACAACA</td><td> AATCAAGGCT</td><td> AAAACAGCGT</td><td> CTATTATTGT</td><td> 4027</td>

<td> GGATGATGGG</td><td> GAACGTGTAG</td><td> CAGAAAAACG</td><td> TGTAGCGGCA</td><td> AAAGATTATG</td><td> AAAATCCAGA</td><td> 4087</td>

<td> AGATAAAACA</td><td> CCGTCTTTAA</td><td> CTTTAAAAGA</td><td> TGCCCTGAAG</td><td> CTTTCATATC</td><td> CAGATGAAAT</td><td> 4147</td>

<td> AAAAGAAATA</td><td> GAGGGATTAT</td><td> TATATTATAA</td><td> AAACAAACCG</td><td> ATATACGAAT</td><td> CGAGCGTTAT</td><td> 4207</td>

<td> GACTTACTTA</td><td> GATGAAAATA</td><td> CAGCAAAAGA</td><td> AGTGACCAAA</td><td> CAATTAAATG</td><td> ATACCACTGG</td><td> 4267</td>

<td> GAAATTTAAA</td><td> GATGTAAGTC</td><td> ATTTATATGA</td><td> TGTAAAACTG</td><td> ACTCCAAAAA</td><td> TGAATGTTAC</td><td> 4327</td>

<td> AATCAAATTG</td><td> TCTATACTTT</td><td> ATGATAATGC</td><td> TGAGTCTAAT</td><td> GATAACTCAA</td><td> TTGGTAAATG</td><td> 4387</td>

<td> GACAAACACA</td><td> AATATTGTTT</td><td> CAGGTGGAAA</td><td> TAACGGAAAA</td><td> AAACAATATT</td><td> CTTCTAATAA</td><td> 4447</td>

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<td> TCCGGATGCT</td><td> AATTTGACAT</td><td> TAAATACAGA</td><td> TGCTCAAGAA</td><td> AAATTAAATA</td><td> AAAATCGTGA</td><td> 4507</td>

<td> CTATTATATA</td><td> AGTTTATATA</td><td> TGAAGTCAGA</td><td> AAAAAACACA</td><td> CAATGTGAGA</td><td> TTACTATAGA</td><td> 4567</td>

<td> TGGGGAGATT</td><td> TATCCGATCA</td><td> CTACAAAAAC</td><td> AGTGAATGTG</td><td> AATAAAGACA</td><td> ATTACAAAAG</td><td> 4627</td>

<td> ATTAGATATT</td><td> ATAGCTCATA</td><td> ATATAAAAAG</td><td> TAATCCAATT</td><td> TCTTCACTTC</td><td> ATATTAAAAC</td><td> 4687</td>

<td> GAATGATGAA</td><td> ATAACTTTAT</td><td> TTTGGGATGA</td><td> TATTTCTATA</td><td> ACAGATGTAG</td><td> CATCAATAAA</td><td> 4747</td>

<td> ACCGGAAAAT</td><td> TTAACAGATT</td><td> CAGAAATTAA</td><td> ACAGATTTAT</td><td> AGTAGGTATG</td><td> GTATTAAGTT</td><td> 4807</td>

<td> AGAAGATGGA</td><td> ATCCTTATTG</td><td> ATAAAAAAGG</td><td> TGGGATTCAT</td><td> TATGGTGAAT</td><td> TTATTAATGA</td><td> 4867</td>

<td> AGCTAGTTTT</td><td> AATATTGAAC</td><td> CATTGCAAAA</td><td> TTATGTGACC</td><td> AAATATGAAG</td><td> TTACTTATAG</td><td> 4927</td>

<td> TAGTGAGTTA</td><td> GGACCAAACG</td><td> TGAGTGACAC</td><td> ACTTGAAAGT</td><td> GATAAAATTT</td><td> ACAAGGATGG</td><td> 4987</td>

<td> GACAATTAAA</td><td> TTTGATTTTA</td><td> CCAAATATAG</td><td> TAAAAATGAA</td><td> CAAGGATTAT</td><td> TTTATGACAG</td><td> 5047</td>

<td> TGGATTAAAT</td><td> TGGGACTTTA</td><td> AAATTAATGC</td><td> TATTACTTAT</td><td> GATGGTAAAG</td><td> AGATGAATGT</td><td> 5107</td>

<td> TTTTCATAGA</td><td> TATAATAAAT</td><td> AGTTATTATA</td><td> TCTATGAAGC</td><td> TGGTGCTAAA</td><td> GATAGTGTAA</td><td> 5167</td>

<td> AAGTTAATAT</td><td> ACTGTAGGAT</td><td> TGTAATAAAA</td><td> GTAATGGAAT</td><td> TGATATCGTA</td><td> CTTTGGAGTG</td><td> 5227</td>

<td> GGGGATACTT</td><td> TGTAAATAGT</td><td> TCTATCAGAA</td><td> ACATTAGACT</td><td> AAGAAAAGTT</td><td> ACTACCCCCA</td><td> 5287</td>

<td> CTTGAAAATG</td><td> AAGATTCAAC</td><td> TGATTACAAA</td><td> CAACCTGTTA</td><td> AATATTATAA</td><td> GGTTTTAACA</td><td> 5347</td>

<td> AAATATTAAA</td><td> CTCTTTATGT</td><td> TAATACTGTA</td><td> ATATAAAGAG</td><td> TTTAATTGTA</td><td> TTCAAATGAA</td><td> 5407</td>

<td> GCTTTCCCAC</td><td> AAAATTAGAC</td><td> TGATTATCTA</td><td> ATGAAATAAT</td><td> CAGTCTAATT</td><td> TTGTAGAACA</td><td> 5467</td>

<td> GGTCTGGTAT</td><td> TATTGTACGT</td><td> GGTCACTAAA</td><td> AGATATCTAA</td><td> TATTATTGGG</td><td> CAAGGCGTTC</td><td> 5527</td>

<td> CATGATTGAA</td><td> TCCTCGAATG</td><td> TCTTGCCCTT</td><td> TTCATTTATT</td><td> TAAGAAGGAT</td><td> TGTGGAGAAA</td><td> 5587</td>

<td> TTATGGTTTA</td><td> GATAATGAAG</td><td> AAAGACTTCA</td><td> CTTCTAATTT</td><td> TTGATGTTAA</td><td> ATAAATCAAA</td><td> 5647</td>

<td> ATTTGGCGAT</td><td> TCACATTGTT</td><td> TAATCCACTG</td><td> ATAAAACATA</td><td> CTGGAGTGTT</td><td> CTTAAAAAAT</td><td> 5707</td>

<td> CAGCTTTTTT</td><td> CTTTATAAAA</td><td> TTTTGCTTAG</td><td> CGTACGAAAT</td><td> TCGTGTTTTG</td><td> TTGGTGGGAC</td><td> 5767</td>

<td> CCCATGCCCA</td><td> TCAACTTAAG</td><td> AGTAAATTAG</td><td> TAATGAACTT</td><td> TCGTTCATCT</td><td> GGATTAAAAT</td><td> 5827</td>

<td> AACCTCAAAT</td><td> TAGGACATGT</td><td> TTTTAAAAAT</td><td> AAGCAGACCA</td><td> AATAAGCCTA</td><td> GAATAGGTAT</td><td> 5887</td>

<td> CATTTTTAAA</td><td> AATTATGCTG</td><td> CTTTCTTTTG</td><td> TTTTCCAAAT</td><td> CCATTATACT</td><td> CATAAGCAAC</td><td> 5947</td>

<td> ACCCATAATG</td><td> TCAAAGACTG</td><td> TTTTTGTCTC</td><td> ATATCGATAA</td><td> GCTTGATATC</td><td> GAATTCCTGC</td><td> 6007</td>

AGCCCGGGGG ATCCACTAGT TCTAGAGCGG CCGCCACCGC GG

6049

PCT/EP95/03826

WO 96/10083

- 114 (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

<td></td><td> (xi) SEQUENCE DESCRIPTION:</td><td> : SEQ ID NO:2:</td>

<td> Met 1</td><td> Lys Arg Met Glu Gly Lys Leu 5</td><td> Phe Met Val Ser Lys Lys Leu Gin 10 15</td>

<td> Val</td><td> Val Thr Lys Thr Val Leu Leu 20</td><td> Ser Thr Val Phe Ser He Ser Leu 25 30</td>

<td> Leu</td><td> Asn Asn Glu Val He Lys Ala 35 40</td><td> Glu Gin Leu Asn He Asn Ser Gin 45</td>

<td> Ser</td><td> Lys Tyr Thr Asn Leu Gin Asn 50 55</td><td> Leu Lys He Thr Asp Lys Val Glu 60</td>

<td> Asp 65</td><td> Phe Lys Glu Asp Lys Glu Lys 70</td><td> Ala Lys Glu Trp Gly Lys Glu Lys 75 80</td>

<td> Glu</td><td> Lys Glu Trp Lys Leu Thr Ala 85</td><td> Thr Glu Lys Gly Lys Met Asn Asn 90 95</td>

<td> Phe</td><td> Leu Asp Asn Lys Asn Asp He 100</td><td> Lys Thr Asn Tyr Lys Glu He Thr 105 110</td>

<td> Phe</td><td> Ser Met Ala Gly Ser Phe Glu 115 120</td><td> Asp Glu He Lys Asp Leu Lys Glu 125</td>

<td> He</td><td> Asp Lys Met Phe Asp Lys Thr 130 135</td><td> Asn Leu Ser Asn Ser He He Thr 140</td>

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

<td> Glu</td><td> Gly Asn Thr He Asn Ser Asp 165</td><td> Ala Met Ala Gin Phe Lys Glu Gin 170 175</td>

<td> Phe</td><td> Leu Asp Arg Asp He Lys Phe 180</td><td> Asp Ser Tyr Leu Asp Thr His Leu 185 190</td>

<td> Thr</td><td> Ala Gin Gin Val Ser Ser Lys 195 200</td><td> Glu Arg Val He Leu Lys Val Thr 205</td>

<td> Val</td><td> Pro Ser Gly Lys Gly Ser Thr 210 215</td><td> Thr Pro Thr Lys Ala Gly Val He 220</td>

<td> Leu 225</td><td> Asn Asn Ser Glu Tyr Lys Met 230</td><td> Leu He Asp Asn Gly Tyr Met Val 235 240</td>

&gt;2199049

WO 96/10083

PCT/EP95/03826

-115 -

<td colspan="2"> His Val Asp Lys Val Ser Lys Val</td><td colspan="2"> Val Lys Lys Gly Val Glu Cys Leu</td>

<td></td><td> 245</td><td> 250</td><td> 255</td>

<td> Gin He Glu</td><td> Gly Thr Leu Lys Lys 260</td><td> Ser Leu Asp Phe 265</td><td> Lys Asn Asp He 270</td>

<td> Asn Ala Glu 275</td><td> Ala His Ser Trp Gly 280</td><td> Met Lys Asn Tyr</td><td> Glu Glu Trp Ala 285</td>

<td> Lys Asp Leu 290</td><td> Thr Asp Ser Gin Arg 295</td><td> Glu Ala Leu Asp 300</td><td> Gly Tyr Ala Arg</td>

<td> Gin Asp Tyr 305</td><td> Lys Glu He Asn Asn 310</td><td> Tyr Leu Arg Asn 315</td><td> Gin Gly Gly Ser 320</td>

<td> Gly Asn Glu</td><td> Lys Leu Asp Ala Gin 325</td><td> He Lys Asn He 330</td><td> Ser Asp Ala Leu 335</td>

<td> Gly Lys Lys</td><td> Pro He Pro Glu Asn 340</td><td> He Thr Val Tyr 345</td><td> Arg Trp Cys Gly 350</td>

<td> Met Pro Glu 355</td><td> Phe Gly Tyr Gin He 360</td><td> Ser Asp Pro Leu</td><td> Pro Ser Leu Lys 365</td>

<td> Asp Phe Glu 370</td><td> Glu Gin Phe Leu Asn 375</td><td> Thr He Lys Glu 380</td><td> Asp Lys Gly Tyr</td>

<td> Met Ser Thr 385</td><td> Ser Leu Ser Ser Glu 390</td><td> Arg Leu Ala Ala 395</td><td> Phe Gly Ser Arg 400</td>

<td> Lys Ile He</td><td> Leu Arg Leu Gin Val 405</td><td> Pro Lys Gly Ser 410</td><td> Thr Gly Ala Tyr 415</td>

<td> Leu Ser Ala</td><td> He Gly Gly Phe Ala 420</td><td> Ser Glu Lys Glu 425</td><td> He Leu Leu Asp 430</td>

<td colspan="3"> Lys Asp Ser Lys Tyr His He Asp Lys Val Thr Glu 435 440 Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu 450 455 460 (2) INFORMATION FOR SEQ ID NO:3:</td><td> Val He He Lys 445 Thr Asn</td>

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:

PCT/EP95/03826

WO 96/10083

- 116 (A) NAME/KEY: Peptide (B) LOCATION: 1..20 (D) OTHER INFORMATION: /note® Signal peptide for vacuolar targetting (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

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

Ma 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: I..2652 (D) OTHER INFORMATION: /product® 100 kDa protein VIPlA(a) /note® This sequence is identical to the portion of SEQ ID NO:1 between and including nucleotide 2475 to 5126.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

<td colspan="3" rowspan="3"> ATG AAA AAT Met Lys Asn 465</td><td colspan="4"> ATG AAG AAA AAG</td><td colspan="9"> TTA GCA AGT GTT GTA ACG TGT ACG TTA</td><td rowspan="3"> 48</td>

<td colspan="2" rowspan="2"> Met Lys</td><td rowspan="2"> Lys</td><td rowspan="2"> Lys</td><td colspan="6"> Leu Ala Ser Val Val Thr</td><td colspan="3" rowspan="2"> Cys Thr Leu</td>

<td colspan="3"> 470</td><td colspan="3"> 475</td>

<td> TTA</td><td> GCT</td><td> CCT</td><td> ATG</td><td> TTT</td><td> TTG</td><td> AAT</td><td> GGA</td><td> AAT</td><td> GTG</td><td> AAT</td><td> GCT</td><td> GTT</td><td> TAC</td><td> GCA</td><td> GAC</td><td> 96</td>

<td> Leu</td><td> Ala</td><td> Pro</td><td> Met</td><td> Phe</td><td> Leu</td><td> Asn</td><td> Gly</td><td> Asn</td><td> Val</td><td> Asn</td><td> Ala</td><td> Val</td><td> Tyr</td><td> Ala</td><td> Asp</td><td></td>

<td></td><td> 480</td><td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td></td>

<td> AGC</td><td> AAA</td><td> ACA</td><td> AAT</td><td> CAA</td><td> ATT</td><td> TCT</td><td> ACA</td><td> ACA</td><td> CAG</td><td> AAA</td><td> AAT</td><td> CAA</td><td> CAG</td><td> AAA</td><td> GAG</td><td> 144</td>

<td> Ser</td><td> Lys</td><td> Thr</td><td> Asn</td><td> Gin</td><td> He</td><td> Ser</td><td> Thr</td><td> Thr</td><td> Gin</td><td> Lys</td><td> Asn</td><td> Gin</td><td> Gin</td><td> Lys</td><td> Glu</td><td></td>

<td> 495</td><td></td><td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td>

WO 96/10083 ' 2199049 • 117PCT/EP95/03826

<td colspan="2" rowspan="2"> ATG GAC Met Asp</td><td colspan="2" rowspan="2"> CGA AAA Arg Lys</td><td colspan="12"> GGA TTA CTT GGG TAT TAT TTC AAA GGA AAA GAT TTT</td><td rowspan="2"> 192</td>

<td> Gly 515</td><td> Leu</td><td> Leu</td><td> Gly</td><td> Tyr</td><td> Tyr 520</td><td> Phe</td><td> Lys</td><td> Gly</td><td colspan="3"> Lys Asp Phe 525</td>

<td> AGT</td><td> AAT</td><td> CTT</td><td> ACT</td><td> ATG</td><td> TTT</td><td> GCA</td><td> CCG</td><td> ACA</td><td> CGT</td><td> GAT</td><td> AGT</td><td> ACT</td><td> CTT</td><td> ATT</td><td> TAT</td><td> 240</td>

<td> Ser</td><td> Asn</td><td> Leu</td><td> Thr</td><td> Met</td><td> Phe</td><td> Ala</td><td> Pro</td><td> Thr</td><td colspan="2"> Arg Asp</td><td> Ser</td><td> Thr</td><td> Leu</td><td> He</td><td> Tyr</td><td></td>

<td></td><td></td><td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td>

<td> GAT</td><td> CAA</td><td> CAA</td><td> ACA</td><td> GCA</td><td> AAT</td><td> AAA</td><td> CTA</td><td> TTA</td><td> GAT</td><td> AAA</td><td> AAA</td><td> CAA</td><td> CAA</td><td> GAA</td><td> TAT</td><td> 288</td>

<td> Asp</td><td> Gin</td><td> Gin</td><td> Thr</td><td> Ala</td><td> Asn</td><td> Lys</td><td> Leu</td><td> Leu</td><td> Asp</td><td> Lys</td><td> Lys</td><td> Gin</td><td> Gin</td><td> Glu</td><td> Tyr</td><td></td>

<td></td><td></td><td> 545</td><td></td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td>

<td> CAG</td><td> TCT</td><td> ATT</td><td> CGT</td><td> TGG</td><td> ATT</td><td> GGT</td><td> TTG</td><td> ATT</td><td> CAG</td><td> AGT</td><td> AAA</td><td> GAA</td><td> ACG</td><td> GGA</td><td> GAT</td><td> 336</td>

<td> Gin</td><td> Ser</td><td> He</td><td colspan="2"> Arg Trp</td><td> He</td><td> Gly</td><td> Leu</td><td> He</td><td> Gin</td><td> Ser</td><td> Lys</td><td> Glu</td><td> Thr</td><td> Gly</td><td> Asp</td><td></td>

<td></td><td> 560</td><td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td></td>

<td> TTC</td><td> ACA</td><td> TTT</td><td> AAC</td><td> TTA</td><td> TCT</td><td> GAG</td><td> GAT</td><td> GAA</td><td> CAG</td><td> GCA</td><td> ATT</td><td> ATA</td><td> GAA</td><td> ATC</td><td> AAT</td><td> 384</td>

<td> Phe</td><td> Thr</td><td> Phe</td><td> Asn</td><td> Leu</td><td> Ser</td><td> Glu</td><td> Asp</td><td> Glu</td><td> Gin</td><td> Ala</td><td> He</td><td> He</td><td> Glu</td><td> He</td><td> Asn</td><td></td>

<td> 575</td><td></td><td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td>

<td> GGG</td><td> AAA</td><td> ATT</td><td> ATT</td><td> TCT</td><td> AAT</td><td> AAA</td><td> GGG</td><td> AAA</td><td> GAA</td><td> AAG</td><td> CAA</td><td> GTT</td><td> GTC</td><td> CAT</td><td> TTA</td><td> 432</td>

<td> Gly</td><td> Lys</td><td> He</td><td> He</td><td> Ser</td><td> Asn</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Glu</td><td> Lys</td><td> Gin</td><td> Val</td><td> Val</td><td> His</td><td> Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td>

<td> GAA</td><td> AAA</td><td> GGA</td><td> AAA</td><td> TTA</td><td> GTT</td><td> CCA</td><td> ATC</td><td> AAA</td><td> ATA</td><td> GAG</td><td> TAT</td><td> CAA</td><td> TCA</td><td> GAT</td><td> ACA</td><td> 480</td>

<td> Glu</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Leu</td><td> Val</td><td> Pro</td><td> He</td><td> Lys</td><td> He</td><td> Glu</td><td> Tyr</td><td> Gin</td><td> Ser</td><td> Asp</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td> 610</td><td></td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td>

<td> AAA</td><td> TTT</td><td> AAT</td><td> ATT</td><td> GAC</td><td> AGT</td><td> AAA</td><td> ACA</td><td> TTT</td><td> AAA</td><td> GAA</td><td> CTT</td><td> AAA</td><td> TTA</td><td> TTT</td><td> AAA</td><td> 528</td>

<td> Lys</td><td> Phe</td><td> Asn</td><td> He</td><td> Asp</td><td> Ser</td><td> Lys</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Glu</td><td> Leu</td><td> Lys</td><td> Leu</td><td> Phe</td><td> Lys</td><td></td>

<td></td><td></td><td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td>

<td> ATA</td><td> GAT</td><td> AGT</td><td> CAA</td><td> AAC</td><td> CAA</td><td> CCC</td><td> CAG</td><td> CAA</td><td> GTC</td><td> CAG</td><td> CAA</td><td> GAT</td><td> GAA</td><td> CTG</td><td> AGA</td><td> 576</td>

<td> He</td><td> Asp</td><td> Ser</td><td> Gin</td><td> Asn</td><td> Gin</td><td> Pro</td><td> Gin</td><td> Gin</td><td> Val</td><td> Gin</td><td> Gin</td><td> Asp</td><td> Glu</td><td> Leu</td><td> Arg</td><td></td>

<td></td><td> 640</td><td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td></td>

<td> AAT</td><td> CCT</td><td> GAA</td><td> TTT</td><td> AAC</td><td> AAG</td><td> AAA</td><td> GAA</td><td> TCA</td><td> CAG</td><td> GAA</td><td> TTC</td><td> TTA</td><td> GCG</td><td> AAA</td><td> CCA</td><td> 624</td>

<td> Asn</td><td> Pro</td><td> Glu</td><td> Phe</td><td> Asn</td><td> Lys</td><td> Lys</td><td> Glu</td><td> Ser</td><td> Gin</td><td> Glu</td><td> Phe</td><td> Leu</td><td> Ala</td><td> Lys</td><td> Pro</td><td></td>

<td> 655</td><td></td><td></td><td></td><td></td><td> 660</td><td></td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td>

<td> TCG</td><td> AAA</td><td> ATA</td><td> AAT</td><td> CTT</td><td> TTC</td><td> ACT</td><td> CAA</td><td> AAA</td><td> ATG</td><td> AAA</td><td> AGG</td><td> GAA</td><td> ATT</td><td> GAT</td><td> GAA</td><td> 672</td>

<td> Ser</td><td> Lys</td><td> He</td><td> Asn</td><td> Leu</td><td> Phe</td><td> Thr</td><td> Gin</td><td> Lys</td><td> Met</td><td> Lys</td><td> Arg</td><td> Glu</td><td> He</td><td> Asp</td><td> Glu</td><td></td>

<td></td><td></td><td></td><td></td><td> 675</td><td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td>

<td> GAC</td><td> ACG</td><td> GAT</td><td> ACG</td><td> GAT</td><td> GGG</td><td> GAC</td><td> TCT</td><td> ATT</td><td> CCT</td><td> GAC</td><td> CTT</td><td> TGG</td><td> GAA</td><td> GAA</td><td> AAT</td><td> 720</td>

<td> Asp</td><td> Thr</td><td> Asp</td><td> Thr</td><td> Asp</td><td colspan="2"> Gly Asp</td><td> Ser</td><td> He</td><td> Pro</td><td> Asp</td><td> Leu</td><td> Trp</td><td> Glu</td><td> Glu</td><td> Asn</td><td></td>

<td></td><td></td><td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td>

<td> GGG</td><td> TAT</td><td> ACG</td><td> ATT</td><td> CAA</td><td> AAT</td><td> AGA</td><td> ATC</td><td> GCT</td><td> GTA</td><td> AAG</td><td> TGG</td><td> GAC</td><td> GAT</td><td> TCT</td><td> CTA</td><td> 768</td>

<td> Gly</td><td> Tyr</td><td> Thr</td><td> He</td><td> Gin</td><td> Asn</td><td> Arg</td><td> He</td><td> Ala</td><td> Val</td><td> Lys</td><td colspan="3"> Trp Asp Asp</td><td> Ser</td><td> Leu</td><td></td>

<td></td><td></td><td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td>

<td> GCA</td><td> AGT</td><td> AAA</td><td> GGG</td><td> TAT</td><td> ACG</td><td> AAA</td><td> TTT</td><td> GTT</td><td> TCA</td><td> AAT</td><td> CCA</td><td> CTA</td><td> GAA</td><td> AGT</td><td> CAC</td><td> 816</td>

<td> Ala</td><td> Ser</td><td> Lys</td><td> Gly</td><td> Tyr</td><td> Thr</td><td> Lys</td><td> Phe</td><td> Val</td><td> Ser</td><td> Asn</td><td> Pro</td><td> Leu</td><td> Glu</td><td> Ser</td><td> His</td><td></td>

<td></td><td> 720</td><td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td></td>

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- 118 -

<td colspan="2" rowspan="2"> ACA GTT Thr Val 735</td><td colspan="2"> GGT GAT</td><td rowspan="2"> CCT Pro</td><td rowspan="2"> TAT Tyr 740</td><td colspan="3"> ACA GAT TAT</td><td colspan="7"> GAA AAG GCA GCA AGA GAT CTA</td><td rowspan="2"> 864</td>

<td> Gly</td><td> Asp</td><td> Thr</td><td> Asp</td><td> Tyr</td><td> Glu</td><td> Lys 745</td><td> Ala</td><td colspan="4"> Ala Arg Asp Leu 750</td>

<td> GAT</td><td> TTG</td><td> TCA</td><td> AAT</td><td> GCA</td><td> AAG</td><td> GAA</td><td> ACG</td><td> TTT</td><td> AAC</td><td> CCA</td><td> TTG</td><td> GTA</td><td> GCT</td><td> GCT</td><td> ITT</td><td> 912</td>

<td rowspan="2"> Asp</td><td> Leu</td><td> Ser</td><td> Asn</td><td> Ala</td><td> Lys</td><td> Glu</td><td> Thr</td><td> Phe</td><td> Asn</td><td> Pro</td><td> Leu</td><td> Val</td><td> Ala</td><td> Ala</td><td> Phe</td><td></td>

<td></td><td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td><td> 765</td><td></td><td></td>

<td> CCA</td><td> AGT</td><td> GTG</td><td> AAT</td><td> GTT</td><td> AGT</td><td> ATG</td><td> GAA</td><td> AAG</td><td> GTG</td><td> ATA</td><td> TTA</td><td> TCA</td><td> CCA</td><td> AAT</td><td> GAA</td><td> 960</td>

<td> Pro</td><td> Ser</td><td> Val</td><td> Asn</td><td> Val</td><td> Ser</td><td> Met</td><td> Glu</td><td> Lys</td><td> Val</td><td> He</td><td> Leu</td><td> Ser</td><td> Pro</td><td> Asn</td><td> Glu</td><td></td>

<td></td><td></td><td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td> 780</td><td></td><td></td><td></td>

<td> AAT</td><td> TTA</td><td> TCC</td><td> AAT</td><td> AGT</td><td> GTA</td><td> GAG</td><td> TCT</td><td> CAT</td><td> TCA</td><td> TCC</td><td> ACG</td><td> AAT</td><td> TGG</td><td> TCT</td><td> TAT</td><td> 1008</td>

<td> Asn</td><td> Leu</td><td> Ser</td><td> Asn</td><td> Ser</td><td> Val</td><td> Glu</td><td> Ser</td><td> His</td><td> Ser</td><td> Ser</td><td> Thr</td><td> Asn</td><td> Trp</td><td> Ser</td><td> Tyr</td><td></td>

<td></td><td></td><td> 785</td><td></td><td></td><td></td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td><td></td><td></td><td></td><td></td>

<td> ACA</td><td> AAT</td><td> ACA</td><td> GAA</td><td> GGT</td><td> GCT</td><td> TCT</td><td> GTT</td><td> GAA</td><td> GCG</td><td> GGG</td><td> ATT</td><td> GGA</td><td> CCA</td><td> AAA</td><td> GGT</td><td> 1056</td>

<td> Thr</td><td> Asn</td><td> Thr</td><td> Glu</td><td> Gly</td><td> Ala</td><td> Ser</td><td> Val</td><td> Glu</td><td> Ala</td><td> Gly</td><td> He</td><td> Gly</td><td> Pro</td><td> Lys</td><td> Gly</td><td></td>

<td></td><td> 800</td><td></td><td></td><td></td><td></td><td> 805</td><td></td><td></td><td></td><td></td><td> 810</td><td></td><td></td><td></td><td></td><td></td>

<td> ATT</td><td> TCG</td><td> TTC</td><td> GGA</td><td> GTT</td><td> AGC</td><td> GTA</td><td> AAC</td><td> TAT</td><td> CAA</td><td> CAC</td><td> TCT</td><td> GAA</td><td> ACA</td><td> GTT</td><td> GCA</td><td> 1104</td>

<td> He</td><td> Ser</td><td> Phe</td><td> Gly</td><td> Val</td><td> Ser</td><td> Val</td><td> Asn</td><td> Tyr</td><td> Gin</td><td> His</td><td> Ser</td><td> Glu</td><td> Thr</td><td> Val</td><td> Ala</td><td></td>

<td> 815</td><td></td><td></td><td></td><td></td><td> 820</td><td></td><td></td><td></td><td></td><td> 825</td><td></td><td></td><td></td><td></td><td> 830</td><td></td>

<td> CAA</td><td> GAA</td><td> TGG</td><td> GGA</td><td> ACA</td><td> TCT</td><td> ACA</td><td> GGA</td><td> AAT</td><td> ACT</td><td> TCG</td><td> CAA</td><td> TTC</td><td> AAT</td><td> ACG</td><td> GCT</td><td> 1152</td>

<td> Gin</td><td> Glu</td><td> Trp</td><td> Gly</td><td> Thr</td><td> Ser</td><td> Thr</td><td> Gly</td><td> Asn</td><td> Thr</td><td> Ser</td><td> Gin</td><td> Phe</td><td> Asn</td><td> Thr</td><td> Ala</td><td></td>

<td></td><td></td><td></td><td></td><td> 835</td><td></td><td></td><td></td><td></td><td> 840</td><td></td><td></td><td></td><td></td><td> 845</td><td></td><td></td>

<td> TCA</td><td> GCG</td><td> GGA</td><td> TAT</td><td> TTA</td><td> AAT</td><td> GCA</td><td> AAT</td><td> GTT</td><td> CGA</td><td> TAT</td><td> AAC</td><td> AAT</td><td> GTA</td><td> GGA</td><td> ACT</td><td> 1200</td>

<td> Ser</td><td> Ala</td><td> Gly</td><td> Tyr</td><td> Leu</td><td> Asn</td><td> Ala</td><td> Asn</td><td> Val</td><td> Arg</td><td> Tyr</td><td> Asn</td><td> Asn</td><td> Val</td><td> Gly</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td> 850</td><td></td><td></td><td></td><td></td><td> 855</td><td></td><td></td><td></td><td></td><td> 860</td><td></td><td></td><td></td>

<td> GGT</td><td> GCC</td><td> ATC</td><td> TAC</td><td> GAT</td><td> GTA</td><td> AAA</td><td> CCT</td><td> ACA</td><td> ACA</td><td> AGT</td><td> TTT</td><td> GTA</td><td> TTA</td><td> AAT</td><td> AAC</td><td> 1248</td>

<td> Gly</td><td> Ala</td><td> He</td><td colspan="2"> Tyr Asp</td><td> Val</td><td> Lys</td><td> Pro</td><td> Thr</td><td> Thr</td><td> Ser</td><td> Phe</td><td> Val</td><td> Leu</td><td> Asn</td><td> Asn</td><td></td>

<td></td><td></td><td> 865</td><td></td><td></td><td></td><td></td><td> 870</td><td></td><td></td><td></td><td></td><td> 875</td><td></td><td></td><td></td><td></td>

<td> GAT</td><td> ACT</td><td> ATC</td><td> GCA</td><td> ACT</td><td> ATT</td><td> ACG</td><td> GCG</td><td> AAA</td><td> TCT</td><td> AAT</td><td> TCT</td><td> ACA</td><td> GCC</td><td> TTA</td><td> AAT</td><td> 1296</td>

<td rowspan="2"> Asp</td><td> Thr</td><td> He</td><td> Ala</td><td> Thr</td><td> He</td><td> Thr</td><td> Ala</td><td> Lys</td><td> Ser</td><td> Asn</td><td> Ser</td><td> Thr</td><td> Ala</td><td> Leu</td><td> Asn</td><td></td>

<td> 880</td><td></td><td></td><td></td><td></td><td> 885</td><td></td><td></td><td></td><td></td><td> 890</td><td></td><td></td><td></td><td></td><td></td>

<td> ATA</td><td> TCT</td><td> CCT</td><td> GGA</td><td> GAA</td><td> AGT</td><td> TAC</td><td> CCG</td><td> AAA</td><td> AAA</td><td> GGA</td><td> CAA</td><td> AAT</td><td> GGA</td><td> ATC</td><td> GCA</td><td> 1344</td>

<td> He</td><td> Ser</td><td> Pro</td><td> Gly</td><td> Glu</td><td> Ser</td><td> Tyr</td><td> Pro</td><td> Lys</td><td> Lys</td><td> Gly</td><td> Gin</td><td> Asn</td><td> Gly</td><td> He</td><td> Ala</td><td></td>

<td> 895</td><td></td><td></td><td></td><td></td><td> 900</td><td></td><td></td><td></td><td></td><td> 905</td><td></td><td></td><td></td><td></td><td> 910</td><td></td>

<td> ATA</td><td> ACA</td><td> TCA</td><td> ATG</td><td> GAT</td><td> GAT</td><td> TTT</td><td> AAT</td><td> TCC</td><td> CAT</td><td> CCG</td><td> ATT</td><td> ACA</td><td> TTA</td><td> AAT</td><td> AAA</td><td> 1392</td>

<td> He</td><td> Thr</td><td> Ser</td><td> Met</td><td> Asp</td><td> Asp</td><td> Phe</td><td> Asn</td><td> Ser</td><td> His</td><td> Pro</td><td> He</td><td> Thr</td><td> Leu</td><td> Asn</td><td> Lys</td><td></td>

<td></td><td></td><td></td><td></td><td> 915</td><td></td><td></td><td></td><td></td><td> 920</td><td></td><td></td><td></td><td></td><td> 925</td><td></td><td></td>

<td> AAA</td><td> CAA</td><td> GTA</td><td> GAT</td><td> AAT</td><td> CTG</td><td> CTA</td><td> AAT</td><td> AAT</td><td> AAA</td><td> CCT</td><td> ATG</td><td> ATG</td><td> TTG</td><td> GAA</td><td> ACA</td><td> 1440</td>

<td> Lys</td><td> Gin</td><td> Val</td><td> Asp</td><td> Asn</td><td> Leu</td><td> Leu</td><td> Asn</td><td> Asn</td><td> Lys</td><td> Pro</td><td> Met</td><td> Met</td><td> Leu</td><td> Glu</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td> 930</td><td></td><td></td><td></td><td></td><td> 935</td><td></td><td></td><td></td><td></td><td> 940</td><td></td><td></td><td></td>

<td> AAC</td><td> CAA</td><td> ACA</td><td> GAT</td><td> GGT</td><td> GTT</td><td> TAT</td><td> AAG</td><td> ATA</td><td> AAA</td><td> GAT</td><td> ACA</td><td> CAT</td><td> GGA</td><td> AAT</td><td> ATA</td><td> 1488</td>

<td> Asn</td><td> Gin</td><td> Thr</td><td> Asp</td><td> Gly</td><td> Val</td><td> Tyr</td><td> Lys</td><td> He</td><td> Lys</td><td> Asp</td><td> Thr</td><td> His</td><td> Gly</td><td> Asn</td><td> He</td><td></td>

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945 950 955

<td colspan="5"> GTA ACT GGC GGA GAA</td><td colspan="4"> TGG AAT GGT GTC ATA CAA CAA ATC AAG GCT AAA</td><td rowspan="3"> 1536</td>

<td rowspan="2"> Val</td><td rowspan="2"> Thr 960</td><td rowspan="2"> Gly</td><td rowspan="2"> Gly</td><td rowspan="2"> Glu</td><td colspan="2"> Trp Asn Gly Val He Gin Gin</td><td rowspan="2"> He Lys</td><td rowspan="2"> Ala Lys</td>

<td> 965</td><td> 970</td>

<td> ACA</td><td> GCG</td><td> TCT</td><td> ATT</td><td> ATT</td><td> GTG GAT</td><td> GAT GGG GAA CGT GTA</td><td> GCA GAA</td><td> AAA CGT</td><td> 1584</td>

<td> Thr 975</td><td> Ala</td><td> Ser</td><td> He</td><td> He</td><td> Val Asp 980</td><td> Asp Gly Glu Arg Val 985</td><td> Ala Glu</td><td> Lys Arg 990</td><td></td>

<td> GTA</td><td> GCG</td><td> GCA</td><td> AAA</td><td> GAT</td><td> TAT GAA</td><td> AAT CCA GAA GAT AAA</td><td> ACA CCG</td><td> TCT TTA</td><td> 1632</td>

<td> Val</td><td> Ala</td><td> Ala</td><td> Lys</td><td> Asp 995</td><td> Tyr Glu</td><td> Asn Pro Glu Asp Lys 1000</td><td> Thr Pro</td><td> Ser Leu 1005</td><td></td>

<td> ACT</td><td> TTA</td><td> AAA</td><td> GAT</td><td> GCC</td><td> CTG AAG</td><td> CTT TCA TAT CCA GAT</td><td> GAA ATA</td><td> AAA GAA</td><td> 1680</td>

<td> Thr</td><td> Leu</td><td colspan="3"> Lys Asp Ala 1010</td><td> Leu Lys</td><td> Leu Ser Tyr Pro Asp 1015</td><td colspan="2"> Glu He Lys Glu 1020</td><td></td>

<td> ATA</td><td> GAG</td><td> GGA</td><td> TTA</td><td> TTA</td><td> TAT TAT</td><td> AAA AAC AAA CCG ATA</td><td> TAC GAA</td><td> TCG AGC</td><td> 1728</td>

<td> He</td><td> Glu</td><td colspan="2"> Gly Leu 1025</td><td> Leu</td><td> Tyr Tyr</td><td> Lys Asn Lys Pro He 1030</td><td> Tyr Glu 1035</td><td> Ser Ser</td><td></td>

<td> GTT</td><td> ATG</td><td> ACT</td><td> TAC</td><td> TTA</td><td> GAT GAA</td><td> AAT ACA GCA AAA GAA</td><td> GTG ACC</td><td> AAA CAA</td><td> 1776</td>

<td> Val</td><td colspan="2"> Met Thr 1040</td><td> Tyr</td><td> Leu</td><td colspan="3"> Asp Glu Asn Thr Ala Lys Glu Val Thr 1045 1050</td><td> Lys Gin</td><td></td>

<td> TTA</td><td> AAT</td><td> GAT</td><td> ACC</td><td> ACT</td><td> GGG AAA</td><td> TTT AAA GAT GTA AGT</td><td> CAT TTA</td><td> TAT GAT</td><td> 1824</td>

<td colspan="2"> Leu Asn 1055</td><td> Asp</td><td> Thr</td><td> Thr</td><td> Gly Lys 1060</td><td> Phe Lys Asp Val Ser 1065</td><td> His Leu</td><td> Tyr Asp 1070</td><td></td>

<td> GTA</td><td> AAA</td><td> CTG</td><td> ACT</td><td> CCA</td><td> AAA ATG</td><td> AAT GTT ACA ATC AAA</td><td> TTG TCT</td><td> ATA CTT</td><td> 1872</td>

<td> Val</td><td> Lys</td><td> Leu</td><td> Thr</td><td colspan="2"> Pro Lys Met 1075</td><td> Asn Val Thr He Lys 1080</td><td> Leu Ser</td><td> He Leu 1085</td><td></td>

<td> TAT</td><td> GAT</td><td> AAT</td><td> GCT</td><td> GAG</td><td> TCT AAT</td><td> GAT AAC TCA ATT GGT</td><td> AAA TGG</td><td> ACA AAC</td><td> 1920</td>

<td colspan="2"> Tyr Asp</td><td> Asn</td><td colspan="2"> Ala Glu 1090</td><td> Ser Asn</td><td colspan="3"> Asp Asn Ser He Gly Lys Trp Thr Asn 1095 1100</td><td></td>

<td> ACA</td><td> AAT</td><td> ATT</td><td> GTT</td><td> TCA</td><td> GGT GGA</td><td> AAT AAC GGA AAA AAA</td><td> CAA TAT</td><td> TCT TCT</td><td> 1968</td>

<td> Thr</td><td> Asn</td><td colspan="2"> He Val 1105</td><td> Ser</td><td> Gly Gly</td><td> Asn Asn Gly Lys Lys 1110</td><td> Gin Tyr 1115</td><td> Ser Ser</td><td></td>

<td> AAT</td><td> AAT</td><td> CCG</td><td> GAT</td><td> GCT</td><td> AAT TTG</td><td> ACA TTA AAT ACA GAT</td><td> GCT CAA</td><td> GAA AAA</td><td> 2016</td>

<td> Asn</td><td colspan="2"> Asn Pro 1120</td><td> Asp</td><td> Ala</td><td colspan="3"> Asn Leu Thr Leu Asn Thr Asp Ala Gin 1125 1130</td><td> Glu Lys</td><td></td>

<td> TTA</td><td> AAT</td><td> AAA</td><td> AAT</td><td> CGT</td><td> GAC TAT</td><td> TAT ATA AGT TTA TAT</td><td> ATG AAG</td><td> TCA GAA</td><td> 2064</td>

<td colspan="2"> Leu Asn 1135</td><td> Lys</td><td> Asn</td><td> Arg</td><td> Asp Tyr 1140</td><td> Tyr He Ser Leu Tyr 1145*</td><td> Met Lys</td><td> Ser Glu 1150</td><td></td>

<td> AAA</td><td> AAC</td><td> ACA</td><td> CAA</td><td> TGT</td><td> GAG ATT</td><td> ACT ATA GAT GGG GAG</td><td> ATT TAT</td><td> CCG ATC</td><td> 2112</td>

<td> Lys</td><td> Asn</td><td> Thr</td><td> Gin</td><td colspan="2"> Cys Glu He 1155</td><td> Thr He Asp Gly Glu 1160</td><td> He Tyr</td><td> Pro He 1165</td><td></td>

<td> ACT</td><td> ACA</td><td> AAA</td><td> ACA</td><td> GTG</td><td> AAT GTG</td><td> AAT AAA GAC AAT TAC</td><td> AAA AGA</td><td> TTA GAT</td><td> 2160</td>

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<td colspan="4"> Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp</td>

<td></td><td> 1170</td><td> 1175</td><td> 1180</td>

<td> ATT ATA</td><td> GCT CAT AAT</td><td> ATA AAA AGT AAT CCA ATT TCT TCA</td><td> CTT CAT ATT</td>

<td> He He</td><td> Ala His Asn 1185</td><td colspan="2"> He Lys Ser Asn Pro He Ser Ser Leu His He 1190 1195</td>

<td> AAA ACG</td><td> AAT GAT GAA</td><td> ATA ACT TTA TTT TGG GAT GAT ATT</td><td> TCT ATA ACA</td>

<td colspan="2"> Lys Thr Asn Asp Glu 1200</td><td> He Thr Leu Phe Trp Asp Asp He 1205 1210</td><td> Ser He Thr</td>

<td> GAT GTA</td><td> GCA TCA ATA</td><td> AAA CCG GAA AAT TTA ACA GAT TCA</td><td> GAA ATT AAA</td>

<td> Asp Val 1215</td><td> Ala Ser He</td><td> Lys Pro Glu Asn Leu Thr Asp Ser 1220 1225</td><td> Glu He Lys 1230</td>

<td> CAG ATT</td><td> TAT AGT AGG</td><td> TAT GGT ATT AAG TTA GAA GAT GGA</td><td> ATC CTT ATT</td>

<td> Gin He</td><td colspan="2"> Tyr Ser Arg Tyr Gly He Lys Leu Glu Asp Gly 1235 1240</td><td> He Leu He 1245</td>

<td> GAT AAA</td><td> AAA GGT GGG</td><td> ATT CAT TAT GGT GAA TTT ATT AAT</td><td> GAA GCT AGT</td>

<td> Asp Lys</td><td> Lys Gly Gly 1250</td><td> He His Tyr Gly Glu Phe He Asn 1255</td><td> Glu Ala Ser 1260</td>

<td> TTT AAT</td><td> ATT GAA CCA</td><td> TTG CAA AAT TAT GTG ACC AAA TAT</td><td> GAA GTT ACT</td>

<td> Phe Asn</td><td> He Glu Pro 1265</td><td colspan="2"> Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr 1270 1275</td>

<td> TAT AGT</td><td> AGT GAG TTA</td><td> GGA CCA AAC GTG AGT GAC ACA CTT</td><td> GAA AGT GAT</td>

<td colspan="2"> Tyr Ser Ser Glu Leu 1280</td><td> Gly Pro Asn Val Ser Asp Thr Leu 1285 1290</td><td> Glu Ser Asp</td>

<td> AAA ATT</td><td> TAC AAG GAT</td><td> GGG ACA ATT AAA TTT GAT TTT ACC</td><td> AAA TAT AGT</td>

<td> Lys He 1295</td><td colspan="2"> Tyr Lys Asp Gly Thr He Lys Phe Asp Phe Thr 1300 1305</td><td> Lys Tyr Ser 1310</td>

<td> AAA AAT</td><td> GAA CAA GGA</td><td> TTA TTT TAT GAC AGT GGA TTA AAT</td><td> TGG GAC TTT</td>

<td> Lys Asn</td><td colspan="2"> Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn 1315 1320</td><td> Trp Asp Phe 1325</td>

<td> AAA ATT</td><td> AAT GCT ATT</td><td> ACT TAT GAT GGT AAA GAG ATG AAT</td><td> GTT TTT CAT</td>

<td> Lys He AGA TAT Arg Tyr</td><td> Asn Ala He 1330 AAT AAA TAG Asn Lys 1345</td><td> Thr Tyr Asp Gly Lys Glu Met Asn 1335</td><td> Val Phe His 1340</td>

2208

2256

2304

2352

2400

2448

2496

2544

2592

2640

2655 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 884 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

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<td> (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:</td>

<td> Met Lys Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu</td>

<td> 15 10 15</td>

<td> Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp</td>

<td> 20 25 30</td>

<td> Ser Lys Thr Asn Gin He Ser Thr Thr Gin Lys Asn Gin Gin Lys Glu</td>

<td> 35 40 45</td>

<td> Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe</td>

<td> 50 55 - 60</td>

<td> Ser Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr</td>

<td> 65 70 75 80</td>

<td> Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys Gin Gin Glu Tyr</td>

<td> 85 90 95</td>

<td> Gin Ser Ile Arg Trp Ile Gly Leu He Gin Ser Lys Glu Thr Gly Asp</td>

<td> 100 105 110</td>

<td> Phe Thr Phe Asn Leu Ser Glu Asp Glu Gin Ala Ile Ile Glu He Asn</td>

<td> 115 120 125</td>

<td> Gly Lys Ile He Ser Asn Lys Gly Lys Glu Lys Gin Val Val His Leu</td>

<td> 130 135 140</td>

<td> Glu Lys Gly Lys Leu Val Pro Ile Lys He Glu Tyr Gin Ser Asp Thr</td>

<td> 145 150 155 160</td>

<td> Lys Phe Asn He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys</td>

<td> 165 170 175</td>

<td> He Asp Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu Leu Arg</td>

<td> 180 185 190</td>

<td> Asn Pro Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Pro</td>

<td> 195 200 205</td>

<td> Ser Lys He Asn Leu Phe Thr Gin Lys Met Lys Arg Glu He Asp Glu</td>

<td> 210 215 220</td>

<td> Asp Thr Asp Thr Asp Gly Asp Ser He Pro Asp Leu Trp Glu Glu Asn</td>

<td> 225 230 235 240</td>

<td> Gly Tyr Thr He Gin Asn Arg He Ala Val Lys Trp Asp Asp Ser Leu</td>

<td> 245 250 255</td>

<td> Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His</td>

<td> 260 265 270</td>

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<td colspan="4"> Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu</td>

<td></td><td> 275</td><td> 280</td><td> 285</td>

<td> Asp</td><td> Leu Ser Asn Ala Lys 290</td><td> Glu Thr 295</td><td> Phe Asn Pro Leu Val Ala Ala Phe 300</td>

<td> Pro 305</td><td> Ser Val Asn Val Ser 310</td><td> Met Glu</td><td> Lys Val Ile Leu Ser Pro Asn Glu 315 320</td>

<td> Asn</td><td> Leu Ser Asn Ser Val 325</td><td> Glu Ser</td><td> His Ser Ser Thr Asn Trp Ser Tyr 330 335</td>

<td> Thr</td><td> Asn Thr Glu Gly Ala 340</td><td> Ser Val</td><td> Glu Ala Gly Ile Gly Pro Lys Gly 345 350</td>

<td> Ile</td><td> Ser Phe Gly Val Ser 355</td><td> Val Asn 360</td><td> Tyr Gin His Ser Glu Thr Val Ala 365</td>

<td> Gin</td><td> Glu Trp Gly Thr Ser 370</td><td> Thr Gly 375</td><td> Asn Thr Ser Gin Phe Asn Thr Ala 380</td>

<td> Ser 385</td><td> Ala Gly Tyr Leu Asn 390</td><td> Ala Asn</td><td> Val Arg Tyr Asn Asn Val Gly Thr 395 400</td>

<td> Gly</td><td> Ala Ile Tyr Asp Val 405</td><td> Lys Pro</td><td> Thr Thr Ser Phe Val Leu Asn Asn 410 415</td>

<td> Asp</td><td> Thr Ile Ala Thr Ile 420</td><td> Thr Ala</td><td> Lys Ser Asn Ser Thr Ala Leu Asn 425 430</td>

<td> Ile</td><td> Ser Pro Gly Glu Ser 435</td><td> Tyr Pro 440</td><td> Lys Lys Gly Gin Asn Gly Ile Ala 445</td>

<td> Ile</td><td> Thr Ser Met Asp Asp 450</td><td> Phe Asn 455</td><td> Ser His Pro Ile Thr Leu Asn Lys 460</td>

<td> Lys 465</td><td> Gin Val Asp Asn Leu 470</td><td> Leu Asn</td><td> Asn Lys Pro Met Met Leu Glu Thr 475 480</td>

<td> Asn</td><td> Gin Thr Asp Gly Val 485</td><td> Tyr Lys</td><td> Ile Lys Asp Thr His Gly Asn Ile 490 495</td>

<td> Val</td><td> Thr Gly Gly Glu Trp 500</td><td> Asn Gly</td><td> Val Ile Gin Gin Ile Lys Ala Lys 505 510</td>

<td> Thr</td><td> Ala Ser Ile Ile Val 515</td><td> Asp Asp 520</td><td> Gly Glu Arg Val Ala Glu Lys Arg 525</td>

<td> Val</td><td> Ala Ala Lys Asp Tyr 530</td><td> Glu Asn 535</td><td> Pro Glu Asp Lys Thr Pro Ser Leu 540</td>

<td> Thr 545</td><td> Leu Lys Asp Ala Leu 550</td><td> Lys Leu</td><td> Ser Tyr Pro Asp Glu Ile Lys Glu 555 560</td>

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

199049

-123 PCT/EP95/03826

<img file="CA2199049C_D0004.tif" />

WO 96/10083

<td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td>

<td> Val</td><td> Met</td><td> Thr</td><td> Tyr 580</td><td> Leu</td><td> Asp</td><td> Glu</td><td> Asn</td>

<td> Leu</td><td> Asn</td><td> Asp 595</td><td> Thr</td><td> Thr</td><td> Gly</td><td> Lys</td><td> Phe 600</td>

<td> Val</td><td> Lys 610</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Lys</td><td> Met 615</td><td> Asn</td>

<td colspan="2"> Tyr Asp 625</td><td> Asn</td><td> Ala</td><td> Glu</td><td> Ser 630</td><td> Asn</td><td> Asp</td>

<td> Thr</td><td> Asn</td><td> He</td><td> Val</td><td> Ser 645</td><td colspan="2"> Gly Gly</td><td> Asn</td>

<td> Asn</td><td> Asn</td><td> Pro</td><td> Asp 660</td><td> Ala</td><td> Asn</td><td> Leu</td><td> Thr</td>

<td> Leu</td><td> Asn</td><td> Lys 675</td><td> Asn</td><td colspan="2"> Arg Asp</td><td colspan="2"> Tyr Tyr 680</td>

<td> Lys</td><td> Asn 690</td><td> Thr</td><td> Gin</td><td> Cys</td><td> Glu</td><td> He 695</td><td> Thr</td>

<td> Thr 705</td><td> Thr</td><td> Lys</td><td> Thr</td><td> Val</td><td> Asn 710</td><td> Val</td><td> Asn</td>

<td> He</td><td> He</td><td> Ala</td><td> His</td><td> Asn 725</td><td> He</td><td> Lys</td><td> Ser</td>

<td> Lys</td><td> Thr</td><td> Asn</td><td> Asp 740</td><td> Glu</td><td> He</td><td> Thr</td><td> Leu</td>

<td> Asp</td><td> Val</td><td> Ala 755</td><td> Ser</td><td> He</td><td> Lys</td><td> Pro</td><td> Glu 760</td>

<td> Gin</td><td> He 770</td><td> Tyr</td><td> Ser</td><td colspan="2"> Arg Tyr</td><td> Gly 775</td><td> He</td>

<td colspan="2"> Asp Lys 785</td><td> Lys</td><td colspan="2"> Gly Gly</td><td> He 790</td><td> His</td><td> Tyr</td>

<td> Phe</td><td> Asn</td><td> He</td><td> Glu</td><td> Pro 805</td><td> Leu</td><td> Gin</td><td> Asn</td>

<td> Tyr</td><td> Ser</td><td> Ser</td><td> Glu 820</td><td> Leu</td><td> Gly</td><td> Pro</td><td> Asn</td>

<td> Lys</td><td> He</td><td> Tyr 835</td><td> Lys</td><td> Asp</td><td> Gly</td><td> Thr</td><td> lie 840</td>

<td> 570</td><td> 575</td>

<td> Thr Ala Lys Glu 585</td><td> Val Thr Lys Gin 590</td>

<td> Lys Asp Val Ser</td><td> His Leu Tyr Asp 605</td>

<td> Val Thr He Lys 620</td><td> Leu Ser He Leu</td>

<td colspan="2"> Asn Ser He Gly Lys Trp Thr Asn 635 640</td>

<td> Asn Gly Lys' Lys 650</td><td> Gin Tyr Ser Ser 655</td>

<td> Leu Asn Thr Asp 665</td><td> Ala Gin Glu Lys 670</td>

<td> He Ser Leu Tyr</td><td> Met Lys Ser Glu 685</td>

<td> He Asp Gly Glu 700</td><td> He Tyr Pro He</td>

<td> Lys Asp Asn Tyr 715</td><td> Lys Arg Leu Asp 720</td>

<td> Asn Pro He Ser 730</td><td> Ser Leu His He 735</td>

<td> Phe Trp Asp Asp 745</td><td> He Ser He Thr 750</td>

<td> Asn Leu Thr Asp</td><td> Ser Glu He Lys 765</td>

<td colspan="2"> Lys Leu Glu Asp Gly He Leu He 780</td>

<td> Gly Glu Phe He 795</td><td> Asn Glu Ala Ser 800</td>

<td> Tyr Val Thr Lys 810</td><td> Tyr Glu Val Thr 815</td>

<td> Val Ser Asp Thr 825</td><td> Leu Glu Ser Asp 830</td>

<td> Lys Phe Asp Phe</td><td> Thr Lys Tyr Ser</td>

845

Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe

850 855 860

WO 96/10083

PCT/EP95/03826

- 124 Lys Ile Asn Ala Ile 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:1 between and including nucleotide positions 3126 and 5126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

<td rowspan="2"> ATG Met 885</td><td rowspan="2"> AAA AGG GAA ATT Lys Arg Glu He</td><td colspan="8"> GAT GAA GAC ACG GAT ACG GAT GGG GAC TCT ATT</td><td rowspan="2"> 48</td>

<td> Asp Glu 890</td><td> Asp</td><td> Thr Asp</td><td> Thr 895</td><td> Asp</td><td> Gly</td><td> Asp Ser</td><td> He 900</td>

<td> CCT</td><td> GAC CTT TGG GAA</td><td> GAA AAT</td><td> GGG</td><td> TAT ACG</td><td> ATT</td><td> CAA</td><td> AAT</td><td> AGA ATC</td><td> GCT</td><td> 96</td>

<td> Pro</td><td> Asp Leu Trp Glu 905</td><td> Glu Asn</td><td colspan="2"> Gly Tyr Thr 910</td><td> He</td><td> Gin</td><td> Asn</td><td> Arg He 915</td><td> Ala</td><td></td>

<td> GTA</td><td> AAG TGG GAC GAT</td><td> TCT CTA</td><td> GCA</td><td> AGT AAA</td><td> GGG</td><td> TAT</td><td> ACG</td><td> AAA TTT</td><td> GTT</td><td> 144</td>

<td> Val</td><td> Lys Trp Asp Asp 920</td><td> Ser Leu</td><td> Ala</td><td> Ser Lys 925</td><td> Gly</td><td> Tyr</td><td> Thr</td><td> Lys Phe 930</td><td> Val</td><td></td>

<td> TCA</td><td> AAT CCA CTA GAA</td><td> AGT CAC</td><td> ACA</td><td> GTT GGT</td><td> GAT</td><td> CCT</td><td> TAT</td><td> ACA GAT</td><td> TAT</td><td> 192</td>

<td> Ser</td><td> Asn Pro Leu Glu 935</td><td> Ser His</td><td> Thr 940</td><td colspan="2"> Val Gly Asp</td><td> Pro</td><td> Tyr 945</td><td colspan="2"> Thr Asp Tyr</td><td></td>

<td> GAA</td><td> AAG GCA GCA AGA</td><td> GAT CTA</td><td> GAT</td><td> TTG TCA</td><td> AAT</td><td> GCA</td><td> AAG</td><td> GAA ACG</td><td> TTT</td><td> 240</td>

<td> Glu</td><td> Lys Ala Ala Arg 950</td><td> Asp Leu 955</td><td> Asp</td><td> Leu Ser</td><td> Asn</td><td> Ala 960</td><td> Lys</td><td> Glu Thr</td><td> Phe</td><td></td>

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<td rowspan="2"> AAC CCA Asn Pro 965</td><td colspan="2"> TTG GTA</td><td rowspan="2"> GCT Ala</td><td rowspan="2"> GCT Ala 970</td><td colspan="3"> TTT CCA AGT</td><td colspan="4"> GTG AAT GTT AGT ATG GAA AAG</td><td rowspan="2"> 288</td>

<td> Leu</td><td> Val</td><td> Phe</td><td> Pro</td><td> Ser</td><td> Val</td><td> Asn 975</td><td> Val Ser Met</td><td> Glu Lys 980</td>

<td> GTG ATA</td><td> TTA</td><td> TCA</td><td> CCA</td><td> AAT</td><td> GAA</td><td> AAT</td><td> TTA</td><td> TCC</td><td> AAT</td><td> AGT GTA GAG</td><td> TCT CAT</td><td> 336</td>

<td> Val He</td><td> Leu</td><td> Ser</td><td> Pro 985</td><td> Asn</td><td> Glu</td><td> Asn</td><td> Leu</td><td> Ser 990</td><td> Asn</td><td> Ser Val Glu</td><td> Ser His 995</td><td></td>

<td> TCA TCC</td><td> ACG</td><td> AAT</td><td> TGG</td><td> TCT</td><td> TAT</td><td> ACA</td><td> AAT</td><td> ACA</td><td> GAA</td><td> GGT GCT TCT</td><td> GTT GAA</td><td> 384</td>

<td> Ser Ser</td><td> Thr</td><td colspan="2"> Asn Trp 1000</td><td> Ser</td><td> Tyr</td><td> Thr</td><td colspan="2"> Asn Thr 1005</td><td> Glu</td><td colspan="2"> Gly Ala Ser Val Glu 1010</td><td></td>

<td> GCG GGG</td><td> ATT</td><td> GGA</td><td> CCA</td><td> AAA</td><td> GGT</td><td> ATT</td><td> TCG</td><td> TTC</td><td> GGA</td><td> GTT AGC GTA</td><td> AAC TAT</td><td> 432</td>

<td> Ala Gly</td><td colspan="2"> He Gly 1015</td><td> Pro</td><td> Lys</td><td> Gly</td><td colspan="2"> He Ser 1020</td><td> Phe</td><td> Gly</td><td> Val Ser Val 1025</td><td> Asn Tyr</td><td></td>

<td> CAA CAC</td><td> TCT</td><td> GAA</td><td> ACA</td><td> GTT</td><td> GCA</td><td> CAA</td><td> GAA</td><td> TGG</td><td> GGA</td><td> ACA TCT ACA</td><td> GGA AAT</td><td> 480</td>

<td colspan="2"> Gin His Ser 1030</td><td> Glu</td><td> Thr</td><td> Val</td><td colspan="2"> Ala Gin 1035</td><td> Glu</td><td> Trp</td><td> Gly</td><td> Thr Ser Thr 1040</td><td> Gly Asn</td><td></td>

<td> ACT TCG</td><td> CAA</td><td> TTC</td><td> AAT</td><td> ACG</td><td> GCT</td><td> TCA</td><td> GCG</td><td> GGA</td><td> TAT</td><td> TTA AAT GCA</td><td> AAT GTT</td><td> 528</td>

<td> Thr Ser 1045</td><td> Gin</td><td> Phe</td><td> Asn</td><td colspan="2"> Thr Ala 1050</td><td> Ser</td><td> Ala</td><td colspan="3"> Gly Tyr Leu Asn Ala 1055</td><td> Asn Val 1060</td><td></td>

<td> CGA TAT</td><td> AAC</td><td> AAT</td><td> GTA</td><td> GGA</td><td> ACT</td><td> GGT</td><td> GCC</td><td> ATC</td><td> TAC</td><td> GAT GTA AAA</td><td> CCT ACA</td><td> 576</td>

<td> Arg Tyr</td><td> Asn</td><td> Asn</td><td colspan="2"> Val Gly 1065</td><td> Thr</td><td> Gly</td><td> Ala</td><td colspan="3"> He Tyr Asp Val Lys 1070</td><td> Pro Thr 1075</td><td></td>

<td> ACA AGT</td><td> TTT</td><td> GTA</td><td> TTA</td><td> AAT</td><td> AAC</td><td> GAT</td><td> ACT</td><td> ATC</td><td> GCA</td><td> ACT ATT ACG</td><td> GCG AAA</td><td> 624</td>

<td> Thr Ser</td><td> Phe</td><td colspan="2"> Val Leu 1080</td><td> Asn</td><td> Asn</td><td> Asp</td><td colspan="2"> Thr He 1085</td><td> Ala</td><td colspan="2"> Thr He Thr Ala Lys 1090</td><td></td>

<td> TCT AAT</td><td> TCT</td><td> ACA</td><td> GCC</td><td> TTA</td><td> AAT</td><td> ATA</td><td> TCT</td><td> CCT</td><td> GGA</td><td> GAA AGT TAC</td><td> CCG AAA</td><td> 672</td>

<td> Ser Asn</td><td colspan="2"> Ser Thr 1095</td><td> Ala</td><td> Leu</td><td> Asn</td><td colspan="2"> He Ser 1100</td><td> Pro</td><td> Gly</td><td> Glu Ser Tyr 1105</td><td> Pro Lys</td><td></td>

<td> AAA GGA</td><td> CAA</td><td> AAT</td><td> GGA</td><td> ATC</td><td> GCA</td><td> ATA</td><td> ACA</td><td> TCA</td><td> ATG</td><td> GAT GAT TTT</td><td> AAT TCC</td><td> 720</td>

<td colspan="2"> Lys Gly Gin 1110</td><td> Asn</td><td> Gly</td><td> He</td><td colspan="2"> Ala He 1115</td><td> Thr</td><td> Ser</td><td> Met</td><td> Asp Asp Phe 1120</td><td> Asn Ser</td><td></td>

<td> CAT CCG</td><td> ATT</td><td> ACA</td><td> TTA</td><td> AAT</td><td> AAA</td><td> AAA</td><td> CAA</td><td> GTA</td><td> GAT</td><td> AAT CTG CTA</td><td> AAT AAT</td><td> 768</td>

<td> His Pro 1125</td><td> He</td><td> Thr</td><td> Leu</td><td colspan="2"> Asn Lys 1130</td><td> Lys</td><td> Gin</td><td> Val</td><td colspan="2"> Asp Asn Leu Leu 1135</td><td> Asn Asn 1140</td><td></td>

<td> AAA CCT</td><td> ATG</td><td> ATG</td><td> TTG</td><td> GAA</td><td> ACA</td><td> AAC</td><td> CAA</td><td> ACA</td><td> GAT</td><td> GGT GTT TAT</td><td> AAG ATA</td><td> 816</td>

<td> Lys Pro</td><td> Met</td><td> Met</td><td colspan="2"> Leu Glu 1145</td><td> Thr</td><td> Asn</td><td> Gin</td><td colspan="3"> Thr Asp Gly Val Tyr 1150</td><td> Lys He 1155</td><td></td>

<td> AAA GAT</td><td> ACA</td><td> CAT</td><td> GGA</td><td> AAT</td><td> ATA</td><td> GTA</td><td> ACT</td><td> GGC</td><td> GGA</td><td> GAA TGG AAT</td><td> GGT GTC</td><td> 864</td>

<td> Lys Asp</td><td> Thr</td><td colspan="2"> His Gly 1160</td><td> Asn</td><td> He</td><td> Val</td><td colspan="2"> Thr Gly 1165</td><td> Gly</td><td colspan="2"> Glu Trp Asn Gly Val 1170</td><td></td>

<td> ATA CAA</td><td> CAA</td><td> ATC</td><td> AAG</td><td> GCT</td><td> AAA</td><td> ACA</td><td> GCG</td><td> TCT</td><td> ATT</td><td> ATT GTG GAT</td><td> GAT GGG</td><td> 912</td>

<td> He Gin</td><td> Gin</td><td> lie</td><td> Lys</td><td> Ala</td><td> Lys</td><td> Thr</td><td> Ala</td><td> Ser</td><td> He</td><td colspan="2"> He Val Asp Asp Gly</td><td></td>

WO 96/10083 2 1 9 9 0 4 9 PCT/EP95/03826

-1261175 1180 1185

<td colspan="6"> GAA CGT GTA GCA GAA AAA CGT GTA GCG GCA AAA GAT TAT GAA AAT CCA</td><td rowspan="3"> 960</td>

<td rowspan="2"> Glu</td><td colspan="2"> Arg Val Ala Glu Lys Arg Val</td><td rowspan="2"> Ala Ala</td><td rowspan="2"> Lys Asp Tyr 1200</td><td rowspan="2"> Glu Asn Pro</td>

<td> 1190</td><td> 1195</td>

<td> GAA</td><td> GAT AAA ACA</td><td> CCG TCT TTA ACT</td><td> TTA AAA</td><td> GAT GCC CTG</td><td> AAG CTT TCA</td><td> 1008</td>

<td> Glu</td><td> Asp Lys Thr</td><td> Pro Ser Leu Thr</td><td colspan="2"> Leu Lys Asp Ala Leu</td><td> Lys Leu Ser</td><td></td>

<td colspan="2"> 1205</td><td> 1210</td><td></td><td> 1215</td><td> 1220</td><td></td>

<td> TAT</td><td> CCA GAT GAA</td><td> ATA AAA GAA ATA</td><td colspan="2"> GAG GGA TTA TTA TAT</td><td> TAT AAA AAC</td><td> 1056</td>

<td> Tyr</td><td> Pro Asp Glu</td><td> Ile Lys Glu He</td><td colspan="3"> Glu Gly Leu Leu Tyr Tyr Lys Asn</td><td></td>

<td></td><td></td><td> 1225</td><td colspan="2"> 1230</td><td> 1235</td><td></td>

<td> AAA</td><td> CCG ATA TAC</td><td> GAA TCG AGC GTT</td><td> ATG ACT</td><td> TAC TTA GAT</td><td> GAA AAT ACA</td><td> 1104</td>

<td> Lys</td><td> Pro He Tyr</td><td> Glu Ser Ser Val</td><td> Met Thr</td><td> Tyr Leu Asp</td><td> Glu Asn Thr</td><td></td>

<td></td><td colspan="2"> 1240</td><td> 1245</td><td></td><td> 1250</td><td></td>

<td> GCA</td><td> AAA GAA GTG</td><td> ACC AAA CAA TTA</td><td> AAT GAT</td><td> ACC ACT GGG</td><td> AAA TTT AAA</td><td> 1152</td>

<td> Ala</td><td> Lys Glu Val</td><td> Thr Lys Gin Leu</td><td> Asn Asp</td><td colspan="2"> Thr Thr Gly Lys Phe Lys</td><td></td>

<td></td><td> 1255</td><td colspan="2"> 1260</td><td colspan="2"> 1265</td><td></td>

<td> GAT</td><td> GTA AGT CAT</td><td> TTA TAT GAT GTA</td><td> AAA CTG</td><td> ACT CCA AAA</td><td> ATG AAT GTT</td><td> 1200</td>

<td> Asp</td><td> Val Ser His</td><td> Leu Tyr Asp Val</td><td> Lys Leu</td><td> Thr Pro Lys</td><td> Met Asn Val</td><td></td>

<td></td><td> 1270</td><td> 1275</td><td></td><td> 1280</td><td></td><td></td>

<td> ACA</td><td> ATC AAA TTG</td><td> TCT ATA CTT TAT</td><td> GAT AAT</td><td> GCT GAG TCT</td><td> AAT GAT AAC</td><td> 1248</td>

<td> Thr</td><td> He Lys Leu</td><td colspan="2"> Ser He Leu Tyr Asp Asn</td><td> Ala Glu Ser</td><td> Asn Asp Asn</td><td></td>

<td colspan="2"> 1285</td><td> 1290</td><td></td><td> 1295</td><td> 1300</td><td></td>

<td> TCA</td><td> ATT GGT AAA</td><td> TGG ACA AAC ACA</td><td> AAT ATT</td><td> GTT TCA GGT</td><td> GGA AAT AAC</td><td> 1296</td>

<td> Ser</td><td> He Gly Lys</td><td> Trp Thr Asn Thr</td><td> Asn He</td><td colspan="2"> Val Ser Gly Gly Asn Asn</td><td></td>

<td></td><td></td><td> 1305</td><td colspan="2"> 1310</td><td> 1315</td><td></td>

<td> GGA</td><td> AAA AAA CAA</td><td> TAT TCT TCT AAT</td><td> AAT CCG</td><td> GAT GCT AAT</td><td> TTG ACA TTA</td><td> 1344</td>

<td> Gly</td><td> Lys Lys Gin</td><td> Tyr Ser Ser Asn</td><td> Asn Pro</td><td> Asp Ala Asn</td><td> Leu Thr Leu</td><td></td>

<td></td><td colspan="2"> 1320</td><td> 1325</td><td></td><td> 1330</td><td></td>

<td> AAT</td><td> ACA GAT GCT</td><td> CAA GAA AAA TTA</td><td> AAT AAA</td><td> AAT CGT GAC</td><td> TAT TAT ATA</td><td> 1392</td>

<td> Asn</td><td> Thr Asp Ala</td><td> Gin Glu Lys Leu</td><td> Asn Lys</td><td colspan="2"> Asn Arg Asp Tyr Tyr He</td><td></td>

<td></td><td> 1335</td><td colspan="2"> 1340</td><td colspan="2"> 1345</td><td></td>

<td> AGT</td><td> TTA TAT ATG</td><td> AAG TCA GAA AAA</td><td> AAC ACA</td><td> CAA TGT GAG</td><td> ATT ACT ATA</td><td> 1440</td>

<td> Ser</td><td> Leu Tyr Met</td><td> Lys Ser Glu Lys</td><td> Asn Thr</td><td> Gin Cys Glu</td><td> He Thr He</td><td></td>

<td></td><td> 1350</td><td> 1355</td><td></td><td> 1360</td><td></td><td></td>

<td> GAT</td><td> GGG GAG ATT</td><td> TAT CCG ATC ACT</td><td> ACA AAA</td><td> ACA GTG AAT</td><td> GTG AAT AAA</td><td> 1488</td>

<td colspan="2"> Asp Gly Glu He</td><td> Tyr Pro He Thr</td><td> Thr Lys</td><td colspan="2"> Thr Val Asn Val Asn Lys</td><td></td>

<td colspan="2"> 1365</td><td> 1370</td><td></td><td> 1375</td><td> 1380</td><td></td>

<td> GAC</td><td> AAT TAC AAA</td><td> AGA TTA GAT ATT</td><td> ATA GCT</td><td> CAT AAT ATA</td><td> AAA AGT AAT</td><td> 1536</td>

<td> Asp</td><td> Asn Tyr Lys</td><td> Arg Leu Asp He</td><td> He Ala</td><td> His Asn He</td><td> Lys Ser Asn</td><td></td>

<td></td><td></td><td> 1385</td><td colspan="2"> 1390</td><td> 1395</td><td></td>

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

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<td colspan="2"> Pro He Ser Ser Leu</td><td rowspan="2"> His He</td><td colspan="2"> Lys Thr Asn Asp Glu He Thr Leu Phe</td>

<td></td><td> 1400</td><td> 1405</td><td> 1410</td>

<td> TGG</td><td> GAT GAT ATT TCT</td><td> ATA ACA</td><td> GAT GTA GCA TCA</td><td> ATA AAA CCG GAA AAT 1632</td>

<td colspan="2"> Trp Asp Asp He Ser 1415</td><td> He Thr</td><td> Asp Val Ala Ser 1420</td><td> He Lys Pro Glu Asn 1425</td>

<td> TTA</td><td> ACA GAT TCA GAA</td><td> ATT AAA</td><td> CAG ATT TAT AGT</td><td> AGG TAT GGT ATT AAG 1680</td>

<td> Leu</td><td> Thr Asp Ser Glu 1430</td><td colspan="2"> He Lys Gin He Tyr Ser 1435</td><td> Arg Tyr Gly He Lys 1440</td>

<td> TTA</td><td> GAA GAT GGA ATC</td><td> CTT ATT</td><td> GAT AAA AAA GGT</td><td> GGG ATT CAT TAT GGT 1728</td>

<td colspan="2"> Leu Glu Asp Gly He 1445</td><td> Leu He 1450</td><td colspan="2"> Asp Lys Lys Gly Gly He His Tyr Gly 1455 1460</td>

<td> GAA</td><td> TTT ATT AAT GAA</td><td> GCT AGT</td><td> TTT AAT ATT GAA</td><td> CCA TTG CCA AAT TAT 1776</td>

<td> Glu</td><td colspan="2"> Phe He Asn Glu Ala Ser 1465</td><td> Phe Asn He Glu 1470</td><td> Pro Leu Pro Asn Tyr 1475</td>

<td> GTG</td><td> ACC AAA TAT GAA</td><td> GTT ACT</td><td> TAT AGT AGT GAG</td><td> TTA GGA CCA AAC GTG 1824</td>

<td> Val</td><td> Thr Lys Tyr Glu 1480</td><td> Val Thr</td><td> Tyr Ser Ser Glu 1485</td><td> Leu Gly Pro Asn Val 1490</td>

<td> AGT</td><td> GAC ACA CTT GAA</td><td> AGT GAT</td><td> AAA ATT TAC AAG</td><td> GAT GGG ACA ATT AAA 1872</td>

<td> Ser</td><td> Asp Thr Leu Glu 1495</td><td colspan="3"> Ser Asp Lys He Tyr Lys Asp Gly Thr He Lys 1500 1505</td>

<td> TTT</td><td> GAT TTT ACC AAA</td><td> TAT AGT</td><td> AAA AAT GAA CAA</td><td> GGA TTA TTT TAT GAC 1920</td>

<td> Phe</td><td> Asp Phe Thr Lys 1510</td><td colspan="2"> Tyr Ser Lys Asn Glu Gin 1515</td><td> Gly Leu Phe Tyr Asp 1520</td>

<td> AGT</td><td> GGA TTA AAT TGG</td><td> GAC TTT</td><td> AAA ATT AAT GCT</td><td> ATT ACT TAT GAT GGT 1968</td>

<td colspan="2"> Ser Gly Leu Asn Trp 1525 AAA GAG ATG AAT GTT Lys Glu Met Asn Val</td><td> Asp Phe 1530 TTT CAT Phe His</td><td colspan="2"> Lys He Asn Ala He Thr Tyr Asp Gly 1535 1540 AGA TAT AAT AAA TAG 2004 Arg Tyr Asn Lys</td>

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 Ile Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser He 15 10 15

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

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1 9 9 0 4 S

- 128 PCT/EP95/03826

<td></td><td> 20</td><td> 25</td><td> 30</td>

<td> Val Lys</td><td> Trp Asp Asp Ser 35</td><td> Leu Ala Ser Lys Gly Tyr Thr 40 45</td><td> Lys Phe Val</td>

<td> Ser Asn 50</td><td> Pro Leu Glu Ser</td><td> His Thr Val Gly Asp Pro Tyr 55 60</td><td> Thr Asp Tyr</td>

<td> Glu Lys 65</td><td> Ala Ala Arg Asp 70</td><td> Leu Asp Leu Ser Asn Ala Lys 75</td><td> Glu Thr Phe 80</td>

<td> Asn Pro</td><td> Leu Val Ala Ala 85</td><td> Phe Pro Ser Val Asn Val Ser 90</td><td> Met Glu Lys 95</td>

<td> Val Ile</td><td> Leu Ser Pro Asn 100</td><td> Glu Asn Leu Ser Asn Ser Val 105</td><td> Glu Ser His 110</td>

<td> Ser Ser</td><td> Thr Asn Trp Ser 115</td><td> Tyr Thr Asn Thr Glu Gly Ala 120 125</td><td> Ser Val Glu</td>

<td> Ala Gly 130</td><td> Ile Gly Pro Lys</td><td> Gly Ile Ser Phe Gly Val Ser 135 140</td><td> Val Asn Tyr</td>

<td> Gin His 145</td><td> Ser Glu Thr Val 150</td><td> Ala Gin Glu Trp Gly Thr Ser 155</td><td> Thr Gly Asn 160</td>

<td> Thr Ser</td><td> Gin Phe Asn Thr 165</td><td> Ala Ser Ala Gly Tyr Leu Asn 170</td><td> Ala Asn Val 175</td>

<td> Arg Tyr</td><td> Asn Asn Val Gly 180</td><td> Thr Gly Ala Ile Tyr Asp Val 185</td><td> Lys Pro Thr 190</td>

<td> Thr Ser</td><td> Phe Val Leu Asn 195</td><td> Asn Asp Thr Ile Ala Thr Ile 200 205</td><td> Thr Ala Lys</td>

<td> Ser Asn 210</td><td> Ser Thr Ala Leu</td><td> Asn Ile Ser Pro Gly Glu Ser 215 220</td><td> Tyr Pro Lys</td>

<td> Lys Gly 225</td><td> Gin Asn Gly Ile 230</td><td> Ala Ile Thr Ser Met Asp Asp 235</td><td> Phe Asn Ser 240</td>

<td> His Pro</td><td> Ile Thr Leu Asn 245</td><td> Lys Lys Gin Val Asp Asn Leu 250</td><td> Leu Asn Asn 255</td>

<td> Lys Pro</td><td> Met Met Leu Glu 260</td><td> Thr Asn Gin Thr Asp Gly Val 265</td><td> Tyr Lys Ile 270</td>

<td> Lys Asp</td><td> Thr His Gly Asn 275</td><td> Ile Val Thr Gly Gly Glu Trp 280 285</td><td> Asn Gly Val</td>

<td> Ile Gin 290</td><td> Gin Ile Lys Ala</td><td> Lys Thr Ala Ser Ile Ile Val 295 300</td><td> Asp Asp Gly</td>

<td> Glu Arg 305</td><td> Val Ala Glu Lys 310</td><td> Arg Val Ala Ala Lys Asp Tyr 315</td><td> Glu Asn Pro 320</td>

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<td rowspan="2"> Glu Asp Lys</td><td colspan="3"> Thr Pro Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Ser</td>

<td> 325</td><td> 330</td><td> 335</td>

<td> Tyr Pro Asp</td><td> Glu He 340</td><td> Lys Glu He Glu Gly Leu 345</td><td> Leu Tyr Tyr Lys Asn 350</td>

<td> Lys Pro He 355</td><td> Tyr Glu</td><td> Ser Ser Val Met Thr Tyr 360</td><td> Leu Asp Glu Asn Thr 365</td>

<td> Ala Lys Glu 370</td><td> Val Thr</td><td> Lys Gin Leu Asn Asp Thr 375</td><td> Thr Gly Lys Phe Lys 380</td>

<td> Asp Val Ser 385</td><td> His Leu</td><td> Tyr Asp Val Lys Leu Thr 390 395</td><td> Pro Lys Met Asn Val 400</td>

<td> Thr He Lys</td><td> Leu Ser 405</td><td> He Leu Tyr Asp Asn Ala 410</td><td> Glu Ser Asn Asp Asn 415</td>

<td> Ser He Gly</td><td> Lys Trp 420</td><td> Thr Asn Thr Asn He Val 425</td><td> Ser Gly Gly Asn Asn 430</td>

<td> Gly Lys Lys 435</td><td> Gin Tyr</td><td> Ser Ser Asn Asn Pro Asp 440</td><td> Ala Asn Leu Thr Leu 445</td>

<td> Asn Thr Asp 450</td><td> Ala Gin</td><td> Glu Lys Leu Asn Lys Asn 455</td><td> Arg Asp Tyr Tyr He 460</td>

<td> Ser Leu Tyr 465</td><td> Met Lys</td><td> Ser Glu Lys Asn Thr Gin 470 475</td><td> Cys Glu He Thr He 480</td>

<td> Asp Gly Glu</td><td> He Tyr 485</td><td> Pro He Thr Thr Lys Thr 490</td><td> Val Asn Val Asn Lys 495</td>

<td> Asp Asn Tyr</td><td> Lys Arg 500</td><td> Leu Asp He He Ala His 505</td><td> Asn He Lys Ser Asn 510</td>

<td> Pro He Ser 515</td><td> Ser Leu</td><td> His He Lys Thr Asn Asp 520</td><td> Glu He Thr Leu Phe 525</td>

<td> Trp Asp Asp 530</td><td> He Ser</td><td> He Thr Asp Val Ala Ser 535</td><td> He Lys Pro Glu Asn 540</td>

<td> Leu Thr Asp 545</td><td> Ser Glu</td><td> He Lys Gin He Tyr Ser 550 555</td><td> Arg Tyr Gly He Lys 560</td>

<td> Leu Glu Asp</td><td> Gly He 565</td><td> Leu He Asp Lys Lys Gly 570</td><td> Gly He His Tyr Gly 575</td>

<td> Glu Phe He</td><td> Asn Glu 580</td><td> Ala Ser Phe Asn He Glu 585</td><td> Pro Leu Pro Asn Tyr 590</td>

<td> Val Thr Lys 595</td><td> Tyr Glu</td><td> Val Thr Tyr Ser Ser Glu 600</td><td> Leu Gly Pro Asn Val 605</td>

WO 96/10083 &gt;2199049

PCT/EP95/03826 • 130 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 (ii) 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 15 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

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- 131 PCT/EP95/03826 (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 (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

W096/10083 2 1 9 9 Ο 4 9

PCT/EP95/03826

- 132 (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 15 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 15 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

WO 96/10083

PCT/EP95/03826 « 2199049

- 133 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ala Leu Ser Glu Asn Thr Gly Lys Asp Gly Gly Tyr He Val Pro 15 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

WO 96/10083 ^2199049

- 134 PCT/EP95/03826 (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:

kDa delta-endotoxin /note= N-terminal sequence from 60 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Met Asn Val Leu Asn Ser Gly Arg Thr Thr He 15 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

TTCCTGAACG GCAACGTGAA CGCCGTGTAC GCCGACAGCA

GCACCCTGCT

AGACCAACCA

GGCCCCCATG

GATCAGCACC

120

ACCCAGAAGA ACCAGCAGAA GGAGATGGAC CGCAAGGGCC TGCTGGGCTA CTACTTCAAG

180

PCT/EP95/03826

WO 96/10083 /2199049

-135-

<td> GGCAAGGACT</td><td> TCAGCAACCT</td><td> GACCATGTTC</td><td> GCCCCCACGC</td><td> GTGACAGCAC</td><td> CCTGATCTAC</td><td> 240</td>

<td> GACCAGCAGA</td><td> CCGCCAACAA</td><td> GCTGCTGGAC</td><td> AAGAAGCAGC</td><td> AGGAGTACCA</td><td> GAGCATCCGC</td><td> 300</td>

<td> TGGATCGGCC</td><td> TGATCCAGAG</td><td> CAAGGAGACC</td><td> GGCGACTTCA</td><td> CCTTCAACCT</td><td> GAGCGAGGAC</td><td> 360</td>

<td> GAGCAGGCCA</td><td> TCATCGAGAT</td><td> CAACGGCAAG</td><td> ATCATCAGCA</td><td> ACAAGGGCAA</td><td> GGAGAAGCAG</td><td> 420</td>

<td> GTGGTGCACC</td><td> TGGAGAAGGG</td><td> CAAGCTGGTG</td><td> CCCATCAAGA</td><td> TCGAGTACCA</td><td> GAGCGACACC</td><td> 480</td>

<td> AAGTTCAACA</td><td> TCGACAGCAA</td><td> GACCTTCAAG</td><td> GAGCTGAAGC</td><td> TTTTCAAGAT</td><td> CGACAGCCAG</td><td> 540</td>

<td> AACCAGCCCC</td><td> AGCAGGTGCA</td><td> GCAGGACGAG</td><td> CTGCGCAACC</td><td> CCGAGTTCAA</td><td> CAAGAAGGAG</td><td> 600</td>

<td> AGCCAGGAGT</td><td> TCCTGGCCAA</td><td> GCCCAGCAAG</td><td> ATCAACCTGT</td><td> TCACCCAGCA</td><td> GATGAAGCGC</td><td> 660</td>

<td> GAGATCGACG</td><td> AGGACACCGA</td><td> CACCGACGGC</td><td> GACAGCATCC</td><td> CCGACCTGTG</td><td> GGAGGAGAAC</td><td> 720</td>

<td> GGCTACACCA</td><td> TCCAGAACCG</td><td> CATCGCCGTG</td><td> AAGTGGGACG</td><td> ACAGCCTGGC</td><td> TAGCAAGGGC</td><td> 780</td>

<td> TACACCAAGT</td><td> TCGTGAGCAA</td><td> CCCCCTGGAG</td><td> AGCCACACCG</td><td> TGGGCGACCC</td><td> CTACACCGAC</td><td> 840</td>

<td> TACGAGAAGG</td><td> CCGCCCGCGA</td><td> CCTGGACCTG</td><td> AGCAACGCCA</td><td> AGGAGACCTT</td><td> CAACCCCCTG</td><td> 900</td>

<td> GTGGCCGCCT</td><td> TCCCCAGCGT</td><td> GAACGTGAGC</td><td> ATGGAGAAGG</td><td> TGATCCTGAG</td><td> CCCCAACGAG</td><td> 960</td>

<td> AACCTGAGCA</td><td> ACAGCGTGGA</td><td> GAGCCACTCG</td><td> AGCACCAACT</td><td> GGAGCTACAC</td><td> CAACACCGAG</td><td> 1020</td>

<td> GGCGCCAGCG</td><td> TGGAGGCCGG</td><td> CATCGGTCCC</td><td> AAGGGCATCA</td><td> GCTTCGGCGT</td><td> GAGCGTGAAC</td><td> 1080</td>

<td> TACCAGCACA</td><td> GCGAGACCGT</td><td> GGCCCAGGAG</td><td> TGGGGCACCA</td><td> GCACCGGCAA</td><td> CACCAGCCAG</td><td> 1140</td>

<td> TTCAACACCG</td><td> CCAGCGCCGG</td><td> CTACCTGAAC</td><td> GCCAACGTGC</td><td> GCTACAACAA</td><td> CGTGGGCACC</td><td> 1200</td>

<td> GGCGCCATCT</td><td> ACGACGTGAA</td><td> GCCCACCACC</td><td> AGCTTCGTGC</td><td> TGAACAACGA</td><td> CACCATCGCC</td><td> 1260</td>

<td> ACCATCACCG</td><td> CCAAGTCGAA</td><td> TTCCACCGCC</td><td> CTGAACATCA</td><td> GCCCCGGCGA</td><td> GAGCTACCCC</td><td> 1320</td>

<td> AAGAAGGGCC</td><td> AGAACGGCAT</td><td> CGCCATCACC</td><td> AGCATGGACG</td><td> ACTTCAACAG</td><td> CCACCCCATC</td><td> 1380</td>

<td> ACCCTGAACA</td><td> AGAAGCAGGT</td><td> GGACAACCTG</td><td> CTGAACAACA</td><td> AGCCCATGAT</td><td> GCTGGAGACC</td><td> 1440</td>

<td> AACCAGACCG</td><td> ACGGCGTCTA</td><td> CAAGATCAAG</td><td> GACACCCACG</td><td> GCAACATCGT</td><td> GACCGGCGGC</td><td> 1500</td>

<td> GAGTGGAACG</td><td> GCGTGATCCA</td><td> GCAGATCAAG</td><td> GCCAAGACCG</td><td> CCAGCATCAT</td><td> CGTCGACGAC</td><td> 1560</td>

<td> GGCGAGCGCG</td><td> TGGCCGAGAA</td><td> GCGCGTGGCC</td><td> GCCAAGGACT</td><td> ACGAGAACCC</td><td> CGAGGACAAG</td><td> 1620</td>

<td> ACCCCCAGCC</td><td> TGACCCTGAA</td><td> GGACGCCCTG</td><td> AAGCTGAGCT</td><td> ACCCCGACGA</td><td> GATCAAGGAG</td><td> 1680</td>

<td> ATCGAGGGCC</td><td> TGCTGTACTA</td><td> CAAGAACAAG</td><td> CCCATCTACG</td><td> AGAGCAGCGT</td><td> GATGACCTAT</td><td> 1740</td>

<td> CTAGACGAGA</td><td> ACACCGCCAA</td><td> GGAGGTGACC</td><td> AAGCAGCTGA</td><td> ACGACACCAC</td><td> CGGCAAGTTC</td><td> 1800</td>

<td> AAGGACGTGA</td><td> GCCACCTGTA</td><td> CGACGTGAAG</td><td> CTGACCCCCA</td><td> AGATGAACGT</td><td> GACCATCAAG</td><td> 1860</td>

PCT7EP95/03826

WO 96/10083 &gt;2199049 • 136CTGAGCATCC TGTACGACAA CGCCGAGAGC AACGACAACA GCATCGGCAA GTGGACCAAC

ACCAACATCG TGAGCGGCGG CAACAACGGC AAGAAGCAGT ACAGCAGCAA CAACCCCGAC

GCCAACCTGA CCCTGAACAC CGACGCCCAG GAGAAGCTGA ACAAGAACCG CGACTACTAC

ATCAGCCTGT ACATGAAGAG CGAGAAGAAC ACCCAGTGCG AGATCACCAT CGACGGCGAG

ATATACCCCA TCACCACCAA GACCGTGAAC GTGAACAAGG ACAACTACAA GCGCCTGGAC

ATCATCGCCC ACAACATCAA GAGCAACCCC ATCAGCAGCC TGCACATCAA GACCAACGAC

GAGATCACCC TGTTCTGGGA CGACATATCG ATTACCGACG TCGCCAGCAT CAAGCCCGAG

AACCTGACCG ACAGCGAGAT CAAGCAGATA TACAGTCGCT ACGGCATCAA GCTGGAGGAC

GGCATCCTGA TCGACAAGAA GGGCGGCATC CACTACGGCG AGTTCATCAA CGAGGCCAGC

TTCAACATCG AGCCCCTGCA GAACTACGTG ACCAAGTACG AGGTGACCTA CAGCAGCGAG

CTGGGCCCCA ACGTGAGCGA CACCCTGGAG AGCGACAAGA TTTACAAGGA CGGCACCATC

AAGTTCGACT TCACCAAGTA CAGCAAGAAC GAGCAGGGCC TGTTCTACGA CAGCGGCCTG

AACTGGGACT TCAAGATCAA CGCCATCACC TACGACGGCA AGGAGATGAA CGTGTTCCAC

CGCTACAACA AGTAG (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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

ATGAAGCGCG AGATCGACGA GGACACCGAC ACCGACGGCG ACAGCATCCC CGACCTGTGG

1920

1980

2040

2100

2160

2220

2280

2340

2400

2460

2520

2580

2640

2655 (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

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1990¾¾

PCT/EP95/03826

-137 -

<td> GAGGAGAACG</td><td> GCTACACCAT</td><td> CCAGAACCGC</td><td> ATCGCCGTGA</td><td> AGTGGGACGA</td><td> CAGCCTGGCT</td><td> 120</td>

<td> AGCAAGGGCT</td><td> ACACCAAGTT</td><td> CGTGAGCAAC</td><td> CCCCTGGAGA</td><td> GCCACACCGT</td><td> GGGCGACCCC</td><td> 180</td>

<td> TACACCGACT</td><td> ACGAGAAGGC</td><td> CGCCCGCGAC</td><td> CTGGACCTGA</td><td> GCAACGCCAA</td><td> GGAGACCTTC</td><td> 240</td>

<td> AACCCCCTGG</td><td> TGGCCGCCTT</td><td> CCCCAGCGTG</td><td> AACGTGAGCA</td><td> TGGAGAAGGT</td><td> GATCCTGAGC</td><td> 300</td>

<td> CCCAACGAGA</td><td> ACCTGAGCAA</td><td> CAGCGTGGAG</td><td> AGCCACTCGA</td><td> GCACCAACTG</td><td> GAGCTACACC</td><td> 360</td>

<td> AACACCGAGG</td><td> GCGCCAGCGT</td><td> GGAGGCCGGC</td><td> ATCGGTCCCA</td><td> AGGGCATCAG</td><td> CTTCGGCGTG</td><td> 420</td>

<td> AGCGTGAACT</td><td> ACCAGCACAG</td><td> CGAGACCGTG</td><td> GCCCAGGAGT</td><td> GGGGCACCAG</td><td> CACCGGCAAC</td><td> 480</td>

<td> ACCAGCCAGT</td><td> TCAACACCGC</td><td> CAGCGCCGGC</td><td> TACCTGAACG'</td><td> CCAACGTGCG</td><td> CTACAACAAC</td><td> 540</td>

<td> GTGGGCACCG</td><td> GCGCCATCTA</td><td> CGACGTGAAG</td><td> CCCACCACCA</td><td> GCTTCGTGCT</td><td> GAACAACGAC</td><td> 600</td>

<td> ACCATCGCCA</td><td> CCATCACCGC</td><td> CAAGTCGAAT</td><td> TCCACCGCCC</td><td> TGAACATCAG</td><td> CCCCGGCGAG</td><td> 660</td>

<td colspan="2"> AGCTACCCCA AGAAGGGCCA</td><td> GAACGGCATC</td><td> GCCATCACCA</td><td> GCATGGACGA</td><td> CTTCAACAGC</td><td> 720</td>

<td> CACCCCATCA</td><td> CCCTGAACAA</td><td> GAAGCAGGTG</td><td> GACAACCTGC</td><td> TGAACAACAA</td><td> GCCCATGATG</td><td> 780</td>

<td> CTGGAGACCA</td><td> ACCAGACCGA</td><td> CGGCGTCTAC</td><td> AAGATCAAGG</td><td> ACACCCACGG</td><td> CAACATCGTG</td><td> 840</td>

<td> ACCGGCGGCG</td><td> AGTGGAACGG</td><td> CGTGATCCAG</td><td> CAGATCAAGG</td><td> CCAAGACCGC</td><td> CAGCATCATC</td><td> 900</td>

<td> GTCGACGACG</td><td> GCGAGCGCGT</td><td> GGCCGAGAAG</td><td> CGCGTGGCCG</td><td> CCAAGGACTA</td><td> CGAGAACCCC</td><td> 960</td>

<td> GAGGACAAGA</td><td> CCCCCAGCCT</td><td> GACCCTGAAG</td><td> GACGCCCTGA</td><td> AGCTGAGCTA</td><td> CCCCGACGAG</td><td> 1020</td>

<td> ATCAAGGAGA</td><td> TCGAGGGCCT</td><td> GCTGTACTAC</td><td> AAGAACAAGC</td><td> CCATCTACGA</td><td> GAGCAGCGTG</td><td> 1080</td>

<td> ATGACCTATC</td><td> TAGACGAGAA</td><td> CACCGCCAAG</td><td> GAGGTGACCA</td><td> AGCAGCTGAA</td><td> CGACACCACC</td><td> 1140</td>

<td> GGCAAGTTCA</td><td> AGGACGTGAG</td><td> CCACCTGTAC</td><td> GACGTGAAGC</td><td> TGACCCCCAA</td><td> GATGAACGTG</td><td> 1200</td>

<td> ACCATCAAGC</td><td> TGAGCATCCT</td><td> GTACGACAAC</td><td> GCCGAGAGCA</td><td> ACGACAACAG</td><td> CATCGGCAAG</td><td> 1260</td>

<td> TGGACCAACA</td><td> CCAACATCGT</td><td> GAGCGGCGGC</td><td> AACAACGGCA</td><td> AGAAGCAGTA</td><td> CAGCAGCAAC</td><td> 1320</td>

<td> AACCCCGACG</td><td> CCAACCTGAC</td><td> CCTGAACACC</td><td> GACGCCCAGG</td><td> AGAAGCTGAA</td><td> CAAGAACCGC</td><td> 1380</td>

<td> GACTACTACA</td><td> TCAGCCTGTA</td><td> CATGAAGAGC</td><td> GAGAAGAACA</td><td> CCCAGTGCGA</td><td> GATCACCATC</td><td> 1440</td>

<td> GACGGCGAGA</td><td> TATACCCCAT</td><td> CACCACCAAG</td><td> ACCGTGAACG</td><td> TGAACAAGGA</td><td> CAACTACAAG</td><td> 1500</td>

<td> CGCCTGGACA</td><td> TCATCGCCCA</td><td> CAACATCAAG</td><td> AGCAACCCCA</td><td> TCAGCAGCCT</td><td> GCACATCAAG</td><td> 1560</td>

<td> ACCAACGACG</td><td> AGATCACCCT</td><td> GTTCTGGGAC</td><td> GACATATCGA</td><td> TTACCGACGT</td><td> CGCCAGCATC</td><td> 1620</td>

<td> AAGCCCGAGA</td><td> ACCTGACCGA</td><td> CAGCGAGATC</td><td> AAGCAGATAT</td><td> ACAGTCGCTA</td><td> CGGCATCAAG</td><td> 1680</td>

<td> CTGGAGGACG</td><td> GCATCCTGAT</td><td> CGACAAGAAG</td><td> GGCGGCATCC</td><td> ACTACGGCGA</td><td> GTTCATCAAC</td><td> 1740</td>

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- 138 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:

<td rowspan="2"> ATG Met</td><td colspan="5" rowspan="2"> CAA AGA ATG GAG GGA Gin Arg Met Glu Gly 670</td><td colspan="10"> AAG TTG TTT GTG GTG TCA AAA ACA TTA CAA</td><td rowspan="2"> 48</td>

<td> Lys</td><td> Leu 675</td><td> Phe</td><td> Val</td><td> Val</td><td colspan="3"> Ser Lys Thr 680</td><td colspan="2"> Leu Gin</td>

<td> GTA</td><td> GTT</td><td> ACT</td><td> AGA</td><td> ACT</td><td> GTA</td><td> TTG</td><td> CTT</td><td> AGT</td><td> ACA</td><td> GTT</td><td> TAC</td><td> TCT</td><td> ATA</td><td> ACT</td><td> TTA</td><td> 96</td>

<td> Val</td><td> Val</td><td> Thr</td><td> Arg</td><td> Thr</td><td> Val</td><td> Leu</td><td> Leu</td><td> Ser</td><td> Thr</td><td> Val</td><td> Tyr</td><td> Ser</td><td> He</td><td> Thr</td><td> Leu</td><td></td>

<td></td><td> 685</td><td></td><td></td><td></td><td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td></td>

<td> TTA</td><td> AAT</td><td> AAT</td><td> GTA</td><td> GTG</td><td> ATA</td><td> AAA</td><td> GCT</td><td> GAC</td><td> CAA</td><td> TTA</td><td> AAT</td><td> ATA</td><td> AAT</td><td> TCT</td><td> CAA</td><td> 144</td>

<td> Leu</td><td> Asn</td><td> Asn</td><td> Val</td><td> Val</td><td> He</td><td> Lys</td><td> Ala</td><td> Asp</td><td> Gin</td><td> Leu</td><td> Asn</td><td> He</td><td> Asn</td><td> Ser</td><td> Gin</td><td></td>

<td> 700</td><td></td><td></td><td></td><td></td><td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td>

<td> AGT</td><td> AAA</td><td> TAT</td><td> ACT</td><td> AAC</td><td> TTG</td><td> CAA</td><td> AAT</td><td> CTA</td><td> AAA</td><td> ATC</td><td> CCT</td><td> GAT</td><td> AAT</td><td> GCA</td><td> GAG</td><td> 192</td>

<td> Ser</td><td> Lys</td><td> Tyr</td><td> Thr</td><td> Asn</td><td> Leu</td><td> Gin</td><td> Asn</td><td> Leu</td><td> Lys</td><td> He</td><td> Pro</td><td> Asp</td><td> Asn</td><td> Ala</td><td> Glu</td><td></td>

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<td colspan="2"></td><td colspan="3"> 720</td><td colspan="9"> 725</td><td colspan="3"> 730</td>

<td> GAT</td><td> TTT</td><td> AAA</td><td> GAA</td><td> GAT</td><td> AAG</td><td> GGG</td><td> AAA</td><td> GCG</td><td> AAA</td><td> GAA</td><td> TGG</td><td> GGG</td><td> AAA</td><td> GAG</td><td> AAA</td><td> 240</td>

<td> Asp</td><td> Phe</td><td> Lys</td><td> Glu</td><td> Asp</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Ala</td><td> Lys</td><td> Glu</td><td> Trp</td><td> Gly</td><td> Lys</td><td> Glu</td><td> Lys</td><td></td>

<td></td><td></td><td></td><td> 735</td><td></td><td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td>

<td> GGG</td><td> GAA</td><td> GAG</td><td> TGG</td><td> AGG</td><td> CCT</td><td> CCT</td><td> GCT</td><td> ACT</td><td> GAG</td><td> AAA</td><td> GGA</td><td> GAA</td><td> ATG</td><td> AAT</td><td> AAT</td><td> 288</td>

<td> Gly</td><td> Glu</td><td> Glu</td><td> Trp</td><td> Arg</td><td> Pro</td><td> Pro</td><td> Ala</td><td> Thr</td><td> Glu</td><td> Lys</td><td> Gly</td><td> Glu</td><td> Met</td><td> Asn</td><td> Asn</td><td></td>

<td></td><td></td><td> 750</td><td></td><td></td><td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td>

<td> TTT</td><td> TTA</td><td> GAT</td><td> AAT</td><td> AAA</td><td> AAT</td><td> GAT</td><td> ATA</td><td> AAG</td><td> ACC</td><td> AAT</td><td> TAT</td><td> AAA</td><td> GAA</td><td> ATT</td><td> ACT</td><td> 336</td>

<td> Phe</td><td> Leu</td><td> Asp</td><td> Asn</td><td> Lys</td><td> Asn</td><td> Asp</td><td> He</td><td> Lys</td><td> Thr</td><td> Asn</td><td colspan="2"> Tyr Lys</td><td> Glu</td><td> He</td><td> Thr</td><td></td>

<td></td><td> 765</td><td></td><td></td><td></td><td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td></td>

<td> TTT</td><td> TCT</td><td> ATG</td><td> GCA</td><td> GGT</td><td> TCA</td><td> TGT</td><td> GAA</td><td> GAT</td><td> GAA</td><td colspan="2"> ATA AAA</td><td> GAT</td><td> TTA</td><td> GAA</td><td> GAA</td><td> 384</td>

<td> Phe</td><td> Ser</td><td> Met</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Cys</td><td> Glu</td><td> Asp</td><td> Glu</td><td> He</td><td> Lys</td><td> Asp</td><td> lieu</td><td> Glu</td><td> Glu</td><td></td>

<td> 780</td><td></td><td></td><td></td><td></td><td> 785</td><td></td><td></td><td></td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td><td></td>

<td> ATT</td><td> GAT</td><td> AAG</td><td> ATC</td><td> TTT</td><td> GAT</td><td> AAA</td><td> GCC</td><td> AAT</td><td> CTC</td><td> TCG</td><td> AGT</td><td> TCT</td><td> ATT</td><td> ATC</td><td> ACC</td><td> 432</td>

<td> He</td><td colspan="2"> Asp Lys</td><td> He</td><td> Phe</td><td colspan="2"> Asp Lys</td><td> Ala</td><td> Asn</td><td> Leu</td><td> Ser</td><td> Ser</td><td> Ser</td><td> He</td><td> He</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td></td><td> 800</td><td></td><td></td><td></td><td></td><td> 805</td><td></td><td></td><td></td><td></td><td> 810</td><td></td><td></td>

<td> TAT</td><td> AAA</td><td> AAT</td><td> GTG</td><td> GAA</td><td> CCA</td><td colspan="2"> GCA ACA</td><td> ATT</td><td> GGA</td><td> TTT</td><td> AAT</td><td> AAA</td><td> TCT</td><td> TTA</td><td> ACA</td><td> 480</td>

<td> Tyr</td><td> Lys</td><td> Asn</td><td> Val</td><td> Glu</td><td> Pro</td><td> Ala</td><td> Thr</td><td> He</td><td> Gly</td><td> Phe</td><td> Asn</td><td> Lys</td><td> Ser</td><td> Leu</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td> 815</td><td></td><td></td><td></td><td></td><td> 820</td><td></td><td></td><td></td><td></td><td> 825</td><td></td><td></td><td></td>

<td> GAA</td><td> GGT</td><td> AAT</td><td> ACG</td><td> ATT</td><td> AAT</td><td> TCT</td><td> GAT</td><td> GCA</td><td> ATG</td><td> GCA</td><td> CAG</td><td> TTT</td><td> AAA</td><td> GAA</td><td> CAA</td><td> 528</td>

<td> Glu</td><td> Gly</td><td> Asn</td><td> Thr</td><td> He</td><td> Asn</td><td> Ser</td><td> Asp</td><td> Ala</td><td> Met</td><td> Ala</td><td> Gin</td><td> Phe</td><td> Lys</td><td> Glu</td><td> Gin</td><td></td>

<td></td><td></td><td> 830</td><td></td><td></td><td></td><td></td><td> 835</td><td></td><td></td><td></td><td></td><td> 840</td><td></td><td></td><td></td><td></td>

<td> TTT</td><td> TTA</td><td> GGT</td><td> AAG</td><td> GAT</td><td> ATG</td><td> AAG</td><td> TTT</td><td> GAT</td><td> AGT</td><td> TAT</td><td> CTA</td><td> GAT</td><td> ACT</td><td> CAT</td><td> TTA</td><td> 576</td>

<td> Phe</td><td> Leu</td><td> Gly</td><td> Lys</td><td> Asp</td><td> Met</td><td> Lys</td><td> Phe</td><td> Asp</td><td> Ser</td><td> Tyr</td><td> Leu</td><td> Asp</td><td> Thr</td><td> His</td><td> Leu</td><td></td>

<td></td><td> 845</td><td></td><td></td><td></td><td></td><td> 850</td><td></td><td></td><td></td><td></td><td> 855</td><td></td><td></td><td></td><td></td><td></td>

<td> ACT</td><td> GCT</td><td> CAA</td><td> CAA</td><td> GTT</td><td> TCC</td><td> AGT</td><td> AAA</td><td> AAA</td><td> AGA</td><td> GTT</td><td> ATT</td><td> TTG</td><td> AAG</td><td> GTT</td><td> ACG</td><td> 624</td>

<td> Thr</td><td> Ala</td><td> Gin</td><td> Gin</td><td> Val</td><td> Ser</td><td> Ser</td><td> Lys</td><td> Lys</td><td> Arg</td><td> Val</td><td> He</td><td> Leu</td><td> Lys</td><td> Val</td><td> Thr</td><td></td>

<td> 860</td><td></td><td></td><td></td><td></td><td> 865</td><td></td><td></td><td></td><td></td><td> 870</td><td></td><td></td><td></td><td></td><td> 875</td><td></td>

<td> GTT</td><td> CCG</td><td> AGT</td><td> GGG</td><td> AAA</td><td> GGT</td><td> TCT</td><td> ACT</td><td> ACT</td><td> CCA</td><td> ACA</td><td> AAA</td><td> GCA</td><td> GGT</td><td> GTC</td><td> ATT</td><td> 672</td>

<td> Val</td><td> Pro</td><td> Ser</td><td> Gly</td><td> Lys</td><td> Gly</td><td> Ser</td><td> Thr</td><td> Thr</td><td> Pro</td><td> Thr</td><td> Lys</td><td> Ala</td><td> Gly</td><td> Val</td><td> He</td><td></td>

<td></td><td></td><td></td><td></td><td> 880</td><td></td><td></td><td></td><td></td><td> 885</td><td></td><td></td><td></td><td></td><td> 890</td><td></td><td></td>

<td> TTA</td><td> AAC</td><td> AAT</td><td> AAT</td><td> GAA</td><td> TAC</td><td> AAA</td><td> ATG</td><td> CTC</td><td> ATT</td><td> GAT</td><td> AAT</td><td> GGG</td><td> TAT</td><td> GTG</td><td> CTC</td><td> 720</td>

<td> Leu</td><td> Asn</td><td> Asn</td><td> Asn</td><td> Glu</td><td> Tyr</td><td> Lys</td><td> Met</td><td> Leu</td><td> He</td><td> Asp</td><td> Asn</td><td colspan="2"> Gly Tyr</td><td> Val</td><td> Leu</td><td></td>

<td></td><td></td><td></td><td> 895</td><td></td><td></td><td></td><td></td><td> 900</td><td></td><td></td><td></td><td></td><td> 905</td><td></td><td></td><td></td>

<td> CAT</td><td> GTA</td><td> GAT</td><td> AAG</td><td> GTA</td><td> TCA</td><td> AAA</td><td> GTA</td><td> GTA</td><td> AAA</td><td> AAA</td><td> GGG</td><td> ATG</td><td> GAG</td><td> TGC</td><td> TTA</td><td> 768</td>

<td> His</td><td> Val</td><td colspan="2"> Asp Lys</td><td> Val</td><td> Ser</td><td> Lys</td><td> Val</td><td> Val</td><td> Lys</td><td> Lys</td><td> Gly</td><td> Met</td><td> Glu</td><td> Cys</td><td> Leu</td><td></td>

<td></td><td></td><td> 910</td><td></td><td></td><td></td><td></td><td> 915</td><td></td><td></td><td></td><td></td><td> 920</td><td></td><td></td><td></td><td></td>

<td> CAA</td><td> GTT</td><td> GAA</td><td> GGG</td><td> ACT</td><td> TTA</td><td> AAA</td><td> AAG</td><td> AGT</td><td> CTC</td><td> GAC</td><td> TTT</td><td> AAA</td><td> AAT</td><td> GAT</td><td> ATA</td><td> 816</td>

<td> Gin</td><td> Val</td><td> Glu</td><td> Gly</td><td> Thr</td><td> Leu</td><td> Lys</td><td> Lys</td><td> Ser</td><td> Leu</td><td> Asp</td><td> Phe</td><td> Lys</td><td> Asn</td><td> Asp</td><td> He</td><td></td>

<td></td><td> 925</td><td></td><td></td><td></td><td></td><td> 930</td><td></td><td></td><td></td><td></td><td> 935</td><td></td><td></td><td></td><td></td><td></td>

<td> AAT</td><td> GCT</td><td> GAA</td><td> GCG</td><td> CAT</td><td> AGC</td><td> TGG</td><td> GGG</td><td> ATG</td><td> AAA</td><td> ATT</td><td> TAT</td><td> GAA</td><td> GAC</td><td> TGG</td><td> GCT</td><td> 864</td>

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<td rowspan="2"> Asn 940</td><td rowspan="2"> Ala</td><td rowspan="2"> Glu Ala His</td><td colspan="3"> Ser Trp Gly Met Lys He Tyr Glu Asp Trp Ala</td>

<td> 945</td><td> 950</td><td> 955</td>

<td> AAA</td><td> AAT</td><td> TTA ACC GCT</td><td> TCG CAA AGG</td><td> GAA GCT TTA GAT</td><td> GGG TAT GCT AGG 912</td>

<td> Lys</td><td> Asn</td><td> Leu Thr Ala</td><td> Ser Gin Arg</td><td> Glu Ala Leu Asp</td><td> Gly Tyr Ala Arg</td>

<td></td><td></td><td> 960</td><td></td><td> 965</td><td> 970</td>

<td> CAA</td><td> GAT</td><td> TAT AAA GAA</td><td> ATC AAT AAT</td><td> TAT TTG CGC AAT</td><td> CAA GGC GGG AGT 960</td>

<td> Gin</td><td colspan="2"> Asp Tyr Lys Glu</td><td> He Asn Asn</td><td> Tyr Leu Arg Asn</td><td> Gin Gly Gly Ser</td>

<td></td><td></td><td> 975</td><td></td><td> 980</td><td> 985</td>

<td> GGA</td><td> AAT</td><td> GAA AAG CTG</td><td> GAT GCC CAA</td><td> TTA AAA AAT ATT</td><td> TCT GAT GCT TTA 1008</td>

<td> Gly</td><td> Asn</td><td> Glu Lys Leu</td><td> Asp Ala Gin</td><td> Leu Lys Asn He</td><td> Ser Asp Ala Leu</td>

<td></td><td></td><td> 990</td><td> 995</td><td></td><td> 1000</td>

<td> GGG</td><td> AAG</td><td> AAA CCC ATA</td><td> CCA GAA AAT</td><td> ATT ACC GTG TAT</td><td> AGA TGG TGT GGC 1056</td>

<td> Gly</td><td> Lys</td><td> Lys Pro He</td><td> Pro Glu Asn</td><td colspan="2"> He Thr Val Tyr Arg Trp Cys Gly</td>

<td></td><td colspan="2"> 1005</td><td> 1010</td><td colspan="2"> 1015</td>

<td> ATG</td><td> CCG</td><td> GAA TTT GGT</td><td> TAT CAA ATT</td><td> AGT GAT CCG TTA</td><td> CCT TCT TTA AAA 1104</td>

<td> Met</td><td> Pro</td><td colspan="2"> Glu Phe Gly Tyr Gin He</td><td> Ser Asp Pro Leu</td><td> Pro Ser Leu Lys</td>

<td colspan="2"> 1020</td><td></td><td> 1025</td><td> 1030</td><td> 1035</td>

<td> GAT</td><td> TTT</td><td> GAA GAA CAA</td><td> TTT TTA AAT</td><td> ACA ATT AAA GAA</td><td> GAC AAA GGG TAT 1152</td>

<td> Asp</td><td> Phe</td><td> Glu Glu Gin</td><td> Phe Leu Asn</td><td> Thr He Lys Glu</td><td> Asp Lys Gly Tyr</td>

<td></td><td></td><td colspan="2"> 1040</td><td> 1045</td><td> 1050</td>

<td> ATG</td><td> AGT</td><td> ACA AGC TTA</td><td> TCG AGT GAA</td><td> CGT CTT GCA GCT</td><td> TTT GGA TCT AGA 1200</td>

<td> Met</td><td> Ser</td><td> Thr Ser Leu</td><td> Ser Ser Glu</td><td> Arg Leu Ala Ala</td><td> Phe Gly Ser Arg</td>

<td></td><td></td><td> 1055</td><td></td><td> 1060</td><td> 1065</td>

<td> AAA</td><td> ATT</td><td> ATA TTA CGC</td><td> TTA CAA GTT</td><td> CCG AAA GGA AGT</td><td> ACG GGG GCG TAT 1248</td>

<td> Lys</td><td> He</td><td> He Leu Arg</td><td> Leu Gin Val</td><td> Pro Lys Gly Ser</td><td> Thr Gly Ala Tyr</td>

<td></td><td></td><td> 1070</td><td colspan="2"> 1075</td><td> 1080</td>

<td> TTA</td><td> AGT</td><td> GCC ATT GGT</td><td> GGA TTT GCA</td><td> AGT GAA AAA GAG</td><td> ATC CTA CTT GAT 1296</td>

<td> Leu</td><td> Ser</td><td colspan="2"> Ala He Gly Gly Phe Ala</td><td> Ser Glu Lys Glu</td><td> He Leu Leu Asp</td>

<td></td><td colspan="2"> 1085</td><td> 1090</td><td colspan="2"> 1095</td>

<td> AAA</td><td> GAT</td><td> AGT AAA TAT</td><td> CAT ATT GAT</td><td> AAA GCA ACA GAG</td><td> GTA ATC ATT AAA 1344</td>

<td> Lys</td><td> Asp</td><td> Ser Lys Tyr</td><td> His He Asp</td><td> Lys Ala Thr Glu</td><td> Val He He Lys</td>

<td colspan="2"> 1100</td><td></td><td> 1105</td><td> 1110</td><td> 1115</td>

<td> GGT</td><td> GTT</td><td> AAG CGA TAT</td><td> GTA GTG GAT</td><td> GCA ACA TTA TTA</td><td> ACA AAT 1386</td>

<td colspan="2"> Gly Val</td><td> Lys Arg Tyr</td><td> Val Val Asp</td><td> Ala Thr Leu Leu</td><td> Thr Asn</td>

<td></td><td></td><td colspan="2"> 1120</td><td> 1125</td><td></td>

<td colspan="2"> TAAGGAG</td><td> ATG AAA AAT</td><td> ATG AAG AAA</td><td> AAG TTA GCA AGT</td><td> GTT GTA ACC TGT 1435</td>

<td></td><td></td><td> Met Lys Asn</td><td> Met Lys Lys</td><td> Lys Leu Ala Ser</td><td> Val Val Thr Cys</td>

<td></td><td></td><td> 1</td><td> 5</td><td> 10</td><td></td>

<td> ATG</td><td> TTA</td><td> TTA GCT CCT</td><td> ATG TTT TTG</td><td> AAT GGA AAT GTG</td><td> AAT GCT GTT AAC 1483</td>

<td> Met</td><td> Leu</td><td> Leu Ala Pro</td><td> Met Phe Leu</td><td> Asn Gly Asn Val</td><td> Asn Ala Val Asn</td>

<td> 15</td><td></td><td></td><td> 20</td><td> 25</td><td> 30</td>

WO 96/10083

- 141 PCT/EP95/03826

<td rowspan="2"> GCG GAT Ala Asp</td><td rowspan="2"> AGT AAA Ser Lys</td><td colspan="10"> ATA AAT CAG ATT TCT ACA ACG CAG GAA AAC CAA CAG</td><td rowspan="2"> 1531</td>

<td> He 35</td><td> Asn</td><td> Gin</td><td> He</td><td> Ser</td><td> Thr 40</td><td> Thr</td><td> Gin</td><td> Glu</td><td> Asn Gin Gin 45</td>

<td> AAA GAG</td><td> ATG GAC</td><td> CGA</td><td> AAG</td><td> GGA</td><td> TTA</td><td> TTG</td><td> GGA</td><td> TAT</td><td> TAT</td><td> TTC</td><td> AAA GGA AAA</td><td> 1579</td>

<td> Lys Glu</td><td colspan="2"> Met Asp Arg</td><td> Lys</td><td> Gly</td><td> Leu</td><td> Leu</td><td colspan="2"> Gly Tyr</td><td> Tyr</td><td> Phe</td><td> Lys Gly Lys</td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td>

<td> GAT TTT</td><td> AAT AAT</td><td> CTT</td><td> ACT</td><td> ATG</td><td> TTT</td><td> GCA</td><td> CCG</td><td> ACA</td><td> CGT</td><td> GAT</td><td> AAT ACC CTT</td><td> 1627</td>

<td> Asp Phe</td><td> Asn Asn</td><td> Leu</td><td> Thr</td><td> Met</td><td> Phe</td><td> Ala</td><td> Pro</td><td> Thr</td><td colspan="2"> Arg Asp</td><td> Asn Thr Leu</td><td></td>

<td></td><td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td>

<td> ATG TAT</td><td> GAC CAA</td><td> CAA</td><td> ACA</td><td> GCG</td><td> AAT</td><td> GCA</td><td> TTA</td><td> TTA</td><td colspan="2"> GAT AAA</td><td> AAA CAA CAA</td><td> 1675</td>

<td colspan="2"> Met Tyr Asp Gin</td><td> Gin</td><td> Thr</td><td> Ala</td><td> Asn</td><td> Ala</td><td> Leu</td><td> Leu</td><td colspan="2"> Asp Lys</td><td> Lys Gin Gin</td><td></td>

<td> 80</td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td> *</td><td> 90</td><td></td><td></td><td></td>

<td> GAA TAT</td><td> CAG TCC</td><td> ATT</td><td> CGT</td><td> TGG</td><td> ATT</td><td> GGT</td><td> TTG</td><td> ATT</td><td> CAG</td><td> CGT</td><td> AAA GAA ACG</td><td> 1723</td>

<td> Glu Tyr</td><td> Gin Ser</td><td> He</td><td> Arg</td><td> Trp</td><td> He</td><td> Gly</td><td> Leu</td><td> He</td><td> Gin</td><td colspan="2"> Arg Lys Glu Thr</td><td></td>

<td> 95</td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td> 110</td><td></td>

<td> GGC GAT</td><td> TTC ACA</td><td> TTT</td><td> AAC</td><td> TTA</td><td> TCA</td><td> AAG</td><td> GAT</td><td> GAA</td><td> CAG</td><td> GCA</td><td> ATT ATA GAA</td><td> 1771</td>

<td> Gly Asp</td><td> Phe Thr</td><td> Phe</td><td> Asn</td><td> Leu</td><td> Ser</td><td> Lys</td><td> Asp</td><td> Glu</td><td> Gin</td><td> Ala</td><td> He He Glu</td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td> 125</td><td></td>

<td> ATC GAT</td><td> GGG AAA</td><td> ATC</td><td> ATT</td><td> TCT</td><td> AAT</td><td> AAA</td><td> GGG</td><td> AAA</td><td> GAA</td><td> AAG</td><td> CAA GTT GTC</td><td> 1819</td>

<td colspan="2"> lie Asp Gly Lys</td><td> He</td><td> He</td><td> Ser</td><td> Asn</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Glu</td><td> Lys</td><td> Gin Val Val</td><td></td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td>

<td> CAT TTA</td><td> GAA AAA</td><td> GAA</td><td> AAA</td><td> TTA</td><td> GTT</td><td> CCA</td><td> ATC</td><td> AAA</td><td> ATA</td><td> GAG</td><td> TAT CAA TCA</td><td> 1867</td>

<td> His Leu</td><td> Glu Lys</td><td> Glu</td><td> Lys</td><td> Leu</td><td> Val</td><td> Pro</td><td> He</td><td> Lys</td><td> He</td><td> Glu</td><td> Tyr Gin Ser</td><td></td>

<td></td><td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td>

<td> GAT ACG</td><td> AAA TTT</td><td> AAT</td><td> ATT</td><td> GAT</td><td> AGT</td><td> AAA</td><td> ACA</td><td> TTT</td><td> AAA</td><td> GAA</td><td> CTT AAA TTA</td><td> 1915</td>

<td> Asp Thr</td><td> Lys Phe</td><td> Asn</td><td> He</td><td> Asp</td><td> Ser</td><td> Lys</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Glu</td><td> Leu Lys Leu</td><td></td>

<td> 160</td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td>

<td> TTT AAA</td><td> ATA GAT</td><td> AGT</td><td> CAA</td><td> AAC</td><td> CAA</td><td> TCT</td><td> CAA</td><td> CAA</td><td> GTT</td><td> CAA</td><td> CTG AGA AAC</td><td> 1963</td>

<td> Phe Lys</td><td> He Asp</td><td> Ser</td><td> Gin</td><td> Asn</td><td> Gin</td><td> Ser</td><td> Gin</td><td> Gin</td><td> Val</td><td> Gin</td><td> Leu Arg Asn</td><td></td>

<td> 175</td><td></td><td></td><td> 18.0</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td> 190</td><td></td>

<td> CCT GAA</td><td> TTT AAC</td><td> AAA</td><td> AAA</td><td> GAA</td><td> TCA</td><td> CAG</td><td> GAA</td><td> TTT</td><td> TTA</td><td> GCA</td><td> AAA GCA TCA</td><td> 2011</td>

<td> Pro Glu</td><td> Phe Asn</td><td> Lys</td><td> Lys</td><td> Glu</td><td> Ser</td><td> Gin</td><td> Glu</td><td> Phe</td><td> Leu</td><td> Ala</td><td> Lys Ala Ser</td><td></td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td>

<td> AAA ACA</td><td> AAC CTT</td><td> TTT</td><td> AAG</td><td> CAA</td><td> AAA</td><td> ATG</td><td> AAA</td><td> AGA</td><td> GAT</td><td> ATT</td><td> GAT GAA GAT</td><td> 2059</td>

<td> Lys Thr</td><td> Asn Leu</td><td> Phe</td><td> Lys</td><td> Gin</td><td> Lys</td><td> Met</td><td> Lys</td><td colspan="2"> Arg Asp</td><td> He</td><td> Asp Glu Asp</td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td>

<td> ACG GAT</td><td> ACA GAT</td><td> GGA</td><td> GAC</td><td> TCC</td><td> ATT</td><td> CCT</td><td> GAT</td><td> CTT</td><td> TGG</td><td> GAA</td><td> GAA AAT GGG</td><td> 2107</td>

<td> Thr Asp</td><td> Thr Asp</td><td> Gly</td><td> Asp</td><td> Ser</td><td> He</td><td> Pro</td><td> Asp</td><td> Leu</td><td> Trp</td><td> Glu</td><td> Glu Asn Gly</td><td></td>

<td></td><td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td>

<td> TAC ACG</td><td> ATT CAA</td><td> AAT</td><td> AAA</td><td> GTT</td><td> GCT</td><td> GTC</td><td> AAA</td><td> TGG</td><td> GAT</td><td> GAT</td><td> TCG CTA GCA</td><td> 2155</td>

<td> Tyr Thr</td><td> He Gin</td><td> Asn</td><td> Lys</td><td> Val</td><td> Ala</td><td> Val</td><td> Lys</td><td> Trp</td><td> Asp</td><td> Asp</td><td> Ser Leu Ala</td><td></td>

<td> 240</td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td>

PCT/EP95/03826

WO 96/10083

- 142 -

<img file="CA2199049C_D0005.tif" />

<td rowspan="2"> AGT AAG Ser Lys 255</td><td colspan="2"> GGA TAT</td><td rowspan="2"> ACA Thr</td><td rowspan="2"> AAA Lys 260</td><td colspan="3"> TTT GTT TCG</td><td colspan="5"> AAT CCA TTA GAC AGC CAC ACA</td><td rowspan="2"> 2203</td>

<td> Gly</td><td> Tyr</td><td> Phe</td><td> Val</td><td> Ser</td><td> Asn</td><td> Pro 265</td><td> Leu Asp Ser</td><td> His</td><td> Thr 270</td>

<td> GTT GGC</td><td> GAT</td><td> CCC</td><td> TAT</td><td> ACT</td><td> GAT</td><td> TAT</td><td> GAA</td><td> AAG</td><td> GCC</td><td> GCA AGG GAT</td><td> TTA</td><td> GAT</td><td> 2251</td>

<td> Val Gly</td><td> Asp</td><td> Pro</td><td> Tyr 275</td><td> Thr</td><td colspan="2"> Asp Tyr</td><td> Glu</td><td> Lys 280</td><td> Ala</td><td> Ala Arg Asp</td><td> Leu 285</td><td> Asp</td><td></td>

<td> TTA TCA</td><td> AAT</td><td> GCA</td><td> AAG</td><td> GAA</td><td> ACG</td><td> TTC</td><td> AAC</td><td> CCA</td><td> TTG</td><td> GTA GCT GCT</td><td> TTT</td><td> CCA</td><td> 2299</td>

<td> Leu Ser</td><td> Asn</td><td> Ala 290</td><td> Lys</td><td> Glu</td><td> Thr</td><td> Phe</td><td> Asn 295</td><td> Pro</td><td> Leu</td><td> Val Ala Ala 300</td><td> Phe</td><td> Pro</td><td></td>

<td> AGT GTG</td><td> AAT</td><td> GTT</td><td> AGT</td><td> ATG</td><td> GAA</td><td> AAG</td><td> GTG</td><td> ATA</td><td> TTA</td><td> TCA CCA AAT</td><td colspan="2"> GAA AAT</td><td> 2347</td>

<td> Ser Val</td><td> Asn 305</td><td> Val</td><td> Ser</td><td> Met</td><td> Glu</td><td> Lys 310</td><td> Val</td><td> He</td><td> Leu</td><td> 'Ser Pro Asn 315</td><td> Glu</td><td> Asn</td><td></td>

<td> TTA TCC</td><td> AAT</td><td> AGT</td><td> GTA</td><td> GAG</td><td> TCT</td><td> CAT</td><td> TCA</td><td> TCC</td><td> ACG</td><td> AAT TGG TCT</td><td> TAT</td><td> ACG</td><td> 2395</td>

<td> Leu Ser 320</td><td> Asn</td><td> Ser</td><td> Val</td><td> Glu</td><td> Ser 325</td><td> His</td><td> Ser</td><td> Ser</td><td> Thr</td><td> Asn Trp Ser 330</td><td> Tyr</td><td> Thr</td><td></td>

<td> AAT ACA</td><td> GAA</td><td> GGA</td><td> GCT</td><td> TCC</td><td> ATT</td><td> GAA</td><td> GCT</td><td> GGT</td><td> GGC</td><td> GGT CCA TTA</td><td> GGC</td><td> CTT</td><td> 2443</td>

<td> Asn Thr 335</td><td> Glu</td><td> Gly</td><td> Ala</td><td> Ser 340</td><td> He</td><td> Glu</td><td> Ala</td><td colspan="3"> Gly Gly Gly Pro Leu 345</td><td> Gly</td><td> Leu 350</td><td></td>

<td> TCT TTT</td><td> GGC</td><td> GTG</td><td> AGT</td><td> GTT</td><td> ACT</td><td> TAT</td><td> CAA</td><td> CAC</td><td> TCT</td><td> GAA ACA GTT</td><td> GCA</td><td> CAA</td><td> 2491</td>

<td> Ser Phe</td><td> Gly</td><td> Val</td><td> Ser 355</td><td> Val</td><td> Thr</td><td> Tyr</td><td> Gin</td><td> His 360</td><td> Ser</td><td> Glu Thr Val</td><td> Ala 365</td><td> Gin</td><td></td>

<td> GAA TGG</td><td> GGA</td><td> ACA</td><td> TCT</td><td> ACA</td><td> GGA</td><td> AAT</td><td> ACT</td><td> TCA</td><td> CAA</td><td> TTC AAT ACG</td><td> GCT</td><td> TCA</td><td> 2539</td>

<td> Glu Trp</td><td> Gly</td><td> Thr 370</td><td> Ser</td><td> Thr</td><td> Gly</td><td> Asn</td><td> Thr 375</td><td> Ser</td><td> Gin</td><td> Phe Asn Thr 380</td><td> Ala</td><td> Ser</td><td></td>

<td> GCG GGA</td><td> TAT</td><td> TTA</td><td> AAT</td><td> GCA</td><td> AAT</td><td> GTT</td><td> CGG</td><td> TAT</td><td> AAC</td><td> AAT GTA GGG</td><td> ACT</td><td> GGT</td><td> 2587</td>

<td> Ala Gly</td><td> Tyr 385</td><td> Leu</td><td> Asn</td><td> Ala</td><td> Asn</td><td> Val 390</td><td colspan="2"> Arg Tyr</td><td> Asn</td><td> Asn Val Gly 395</td><td> Thr</td><td> Gly</td><td></td>

<td> GCC ATC</td><td> TAT</td><td> GAT</td><td> GTA</td><td> AAA</td><td> CCT</td><td> ACA</td><td> ACA</td><td> AGT</td><td> TTT</td><td> GTA TTA AAT</td><td> AAC</td><td> AAT</td><td> 2635</td>

<td> Ala He 400</td><td colspan="2"> Tyr Asp</td><td> Val</td><td> Lys</td><td> Pro 405</td><td> Thr</td><td> Thr</td><td> Ser</td><td> Phe</td><td> Val Leu Asn 410</td><td> Asn</td><td> Asn</td><td></td>

<td> ACC ATC</td><td> GCA</td><td> ACG</td><td> ATT</td><td> ACA</td><td> GCA</td><td> AAA</td><td> TCA</td><td> AAT</td><td> TCA</td><td> ACA GCT TTA</td><td> CGT</td><td> ATA</td><td> 2683</td>

<td> Thr He 415</td><td> Ala</td><td> Thr</td><td> He</td><td> Thr 420</td><td> Ala</td><td> Lys</td><td> Ser</td><td> Asn</td><td> Ser 425</td><td> Thr Ala Leu</td><td> Arg</td><td> He 430</td><td></td>

<td> TCT CCG</td><td> GGG</td><td> GAT</td><td> AGT</td><td> TAT</td><td> CCA</td><td> GAA</td><td> ATA</td><td> GGA</td><td> GAA</td><td> AAC GCT ATT</td><td> GCG</td><td> ATT</td><td> 2731</td>

<td> Ser Pro</td><td> Gly</td><td> Asp</td><td> Ser 435</td><td> Tyr</td><td> Pro</td><td> Glu</td><td> He</td><td> Gly 440</td><td> Glu</td><td> Asn Ala He</td><td> Ala 445</td><td> He</td><td></td>

<td> ACA TCT</td><td> ATG</td><td> GAT</td><td> GAT</td><td> TTT</td><td> AAT</td><td> TCT</td><td> CAT</td><td> CCA</td><td> ATT</td><td> ACA TTA AAT</td><td> AAA</td><td> CAA</td><td> 2779</td>

<td> Thr Ser</td><td> Met</td><td colspan="2"> Asp Asp 450</td><td> Phe</td><td> Asn</td><td> Ser</td><td> His 455</td><td> Pro</td><td> He</td><td> Thr Leu Asn 460</td><td> Lys</td><td> Gin</td><td></td>

<td> CAG GTA</td><td> AAT</td><td> CAA</td><td> TTG</td><td> ATA</td><td> AAT</td><td> AAT</td><td> AAG</td><td> CCA</td><td> ATT</td><td> ATG CTA GAG</td><td> ACA</td><td> GAC</td><td> 2827</td>

<td> Gin Val</td><td> Asn</td><td> Gin</td><td> Leu</td><td> He</td><td> Asn</td><td> Asn</td><td> Lys</td><td> Pro</td><td> He</td><td> Met Leu Glu</td><td> Thr</td><td> Asp</td><td></td>

WO 96/10083

- 143 PCT/EP95/03826

465 470 475

<td colspan="5"> CAA ACA GAT GGT GTT</td><td colspan="3"> TAT AAA ATA AGA GAT ACA CAT GGA AAT ATT GTA</td><td rowspan="3"> 2875</td>

<td rowspan="2"> Gin</td><td rowspan="2"> Thr 480</td><td rowspan="2"> Asp</td><td rowspan="2"> Gly</td><td rowspan="2"> Val</td><td colspan="2"> Tyr Lys He Arg Asp Thr His</td><td rowspan="2"> Gly Asn He Val</td>

<td> 485</td><td> 490</td>

<td> ACT</td><td> GGT</td><td> GGA</td><td> GAA</td><td> TGG</td><td> AAT GGT</td><td> GTA ACA CAA CAA ATT</td><td> AAA GCA AAA ACA</td><td> 2923</td>

<td> Thr 495</td><td colspan="2"> Gly Gly</td><td> Glu</td><td> Trp</td><td> Asn Gly 500</td><td> Val Thr Gin Gin He 505</td><td> Lys Ala Lys Thr 510</td><td></td>

<td> GCG</td><td> TCT</td><td> ATT</td><td> ATT</td><td> GTG</td><td> GAT GAC</td><td> GGG AAA CAG GTA GCA</td><td> GAA AAA CGT GTG</td><td> 2971</td>

<td> Ala</td><td> Ser</td><td> He</td><td> He</td><td> Val 515</td><td colspan="2"> Asp Asp Gly Lys Gin Val Ala 520</td><td> Glu Lys Arg Val 525</td><td></td>

<td> GCG</td><td> GCA</td><td> AAA</td><td> GAT</td><td> TAT</td><td> GGT CAT</td><td> CCA GAA GAT AAA' ACA</td><td> CCA CCT TTA ACT</td><td> 3019</td>

<td> Ala</td><td> Ala</td><td colspan="3"> Lys Asp Tyr 530</td><td> Gly His</td><td> Pro Glu Asp Lys Thr 535</td><td> Pro Pro Leu Thr 540</td><td></td>

<td> TTA</td><td> AAA</td><td> GAT</td><td> ACC</td><td> CTG</td><td> AAG CTT</td><td> TCA TAC CCA GAT GAA</td><td> ATA AAA GAA ACT</td><td> 3067</td>

<td> Leu</td><td colspan="2"> Lys Asp 545</td><td> Thr</td><td> Leu</td><td> Lys Leu</td><td> Ser Tyr Pro Asp Glu 550</td><td> He Lys Glu Thr 555</td><td></td>

<td> AAT</td><td> GGA</td><td> TTG</td><td> TTG</td><td> TAC</td><td> TAT GAT</td><td> GAC AAA CCA ATC TAT</td><td> GAA TCG AGT GTC</td><td> 3115</td>

<td> Asn</td><td> Gly 560</td><td> Leu</td><td> Leu</td><td> Tyr</td><td colspan="2"> Tyr Asp Asp Lys Pro He Tyr 565 570</td><td> Glu Ser Ser Val</td><td></td>

<td> ATG</td><td> ACT</td><td> TAT</td><td> CTG</td><td> GAT</td><td> GAA AAT</td><td> ACG GCA AAA GAA GTC</td><td> AAA AAA CAA ATA</td><td> 3163</td>

<td> Met 575</td><td> Thr</td><td> Tyr</td><td> Leu</td><td> Asp</td><td> Glu Asn 580</td><td> Thr Ala Lys Glu Val 585</td><td> Lys Lys Gin He 590</td><td></td>

<td> AAT</td><td> GAT</td><td> ACA</td><td> ACC</td><td> GGA</td><td> AAA TTT</td><td> AAG GAT GTA AAT CAC</td><td> TTA TAT GAT GTA</td><td> 3211</td>

<td> Asn</td><td> Asp</td><td> Thr</td><td> Thr</td><td> Gly 595</td><td> Lys Phe</td><td> Lys Asp Val Asn His 600</td><td> Leu Tyr Asp Val 605</td><td></td>

<td> AAA</td><td> CTG</td><td> ACT</td><td> CCA</td><td> AAA</td><td> ATG AAT</td><td> TTT ACG ATT AAA ATG</td><td> GCT TCC TTG TAT</td><td> 3259</td>

<td> Lys</td><td> Leu</td><td> Thr</td><td> Pro 610</td><td> Lys</td><td> Met Asn</td><td> Phe Thr He Lys Met 615</td><td> Ala Ser Leu Tyr 620</td><td></td>

<td> GAT</td><td> GGG</td><td> GCT</td><td> GAA</td><td> AAT</td><td> AAT CAT</td><td> AAC TCT TTA GGA ACC</td><td> TGG TAT TTA ACA</td><td> 3307</td>

<td> Asp</td><td> Gly</td><td> Ala 625</td><td> Glu</td><td> Asn</td><td> Asn His</td><td> Asn Ser Leu Gly Thr 630</td><td> Trp Tyr Leu Thr 635</td><td></td>

<td> TAT</td><td> AAT</td><td> GTT</td><td> GCT</td><td> GGT</td><td> GGA AAT</td><td> ACT GGG AAG AGA CAA</td><td> TAT CGT TCA GCT</td><td> 3355</td>

<td> Tyr</td><td> Asn 640</td><td> Val</td><td> Ala</td><td> Gly</td><td> Gly Asn 645</td><td> Thr Gly Lys Arg Gin 650</td><td> Tyr Arg Ser Ala</td><td></td>

<td> CAT</td><td> TCT</td><td> TGT</td><td> GCA</td><td> CAT</td><td> GTA GCT</td><td> CTA TCT TCA GAA GCG</td><td> AAA AAG AAA CTA</td><td> 3403</td>

<td> His 655</td><td> Ser</td><td> Cys</td><td> Ala</td><td> His</td><td> Val Ala 660</td><td> Leu Ser Ser Glu Ala 665</td><td> Lys Lys Lys Leu 670</td><td></td>

<td> AAT</td><td> CAA</td><td> AAT</td><td> GCG</td><td> AAT</td><td> TAC TAT</td><td> CTT AGC ATG TAT ATG</td><td> AAG GCT GAT TCT</td><td> 3451</td>

<td> Asn</td><td> Gin</td><td> Asn</td><td> Ala</td><td> Asn 675</td><td> Tyr Tyr</td><td> Leu Ser Met Tyr Met 680</td><td> Lys Ala Asp Ser 685</td><td></td>

<td> ACT</td><td> ACG</td><td> GAA</td><td> CCT</td><td> ACA</td><td> ATA GAA</td><td> GTA GCT GGG GAA AAA</td><td> TCT GCA ATA ACA</td><td> 3499</td>

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<td> Thr</td><td> Thr</td><td> Glu</td><td> Pro 690</td><td> Thr</td><td> He</td><td> Glu</td><td> Val</td><td> Ala 695</td><td> Gly</td><td> Glu</td><td> Lys</td><td> Ser</td><td> Ala 700</td><td> He</td><td> Thr</td><td></td>

<td> AGT</td><td> AAA</td><td> AAA</td><td> GTA</td><td> AAA</td><td> TTA</td><td> AAT</td><td> AAT</td><td> CAA</td><td> AAT</td><td> TAT</td><td> CAA</td><td> AGA</td><td> GTT</td><td> GAT</td><td> ATT</td><td> 3547</td>

<td> Ser</td><td> Lys</td><td> Lys 705</td><td> Val</td><td> Lys</td><td> Leu</td><td> Asn</td><td> Asn 710</td><td> Gin</td><td> Asn</td><td> Tyr</td><td> Gin</td><td> Arg 715</td><td> Val</td><td> Asp</td><td> He</td><td></td>

<td> TTA</td><td> GTG</td><td> AAA</td><td> AAT</td><td> TCT</td><td> GAA</td><td> AGA</td><td> AAT</td><td> CCA</td><td> ATG</td><td> GAT</td><td> AAA</td><td> ATA</td><td> TAT</td><td> ATA</td><td> AGA</td><td> 3595</td>

<td> Leu</td><td> Val 720</td><td> Lys</td><td> Asn</td><td> Ser</td><td> Glu</td><td> Arg 725</td><td> Asn</td><td> Pro</td><td> Met</td><td> Asp</td><td> Lys 730</td><td> He</td><td> Tyr</td><td> He</td><td> Arg</td><td></td>

<td> GGA</td><td> AAT</td><td> GGC</td><td> ACG</td><td> ACA</td><td> AAT</td><td> GTT</td><td> TAT</td><td> GGG</td><td> GAT</td><td> GAT</td><td> GTT</td><td> ACT</td><td> ATC</td><td> CCA</td><td> GAG</td><td> 3643</td>

<td> Gly 735</td><td> Asn</td><td> Gly</td><td> Thr</td><td> Thr</td><td> Asn 740</td><td> Val</td><td> Tyr</td><td colspan="3"> Gly Asp Asp 745</td><td> Val</td><td> Thr</td><td> He</td><td> Pro</td><td> Glu 750</td><td></td>

<td> GTA</td><td> TCA</td><td> GCT</td><td> ATA</td><td> AAT</td><td> CCG</td><td> GCT</td><td> AGT</td><td> CTA</td><td> TCA</td><td> GAT</td><td> GAA</td><td> GAA</td><td> ATT</td><td> CAA</td><td> GAA</td><td> 3691</td>

<td> Val</td><td> Ser</td><td> Ala</td><td> He</td><td> Asn 755</td><td> Pro</td><td> Ala</td><td> Ser</td><td> Leu</td><td> Ser 760</td><td> Asp</td><td> Glu</td><td> Glu</td><td> He</td><td> Gin 765</td><td> Glu</td><td></td>

<td> ATA</td><td> TTT</td><td> AAA</td><td> GAC</td><td> TCA</td><td> ACT</td><td> ATT</td><td> GAA</td><td> TAT</td><td> GGA</td><td> AAT</td><td> CCT</td><td> AGT</td><td> TTC</td><td> GTT</td><td> GCT</td><td> 3739</td>

<td> He</td><td> Phe</td><td> Lys</td><td> Asp 770</td><td> Ser</td><td> Thr</td><td> He</td><td> Glu</td><td colspan="2"> Tyr Gly 775</td><td> Asn</td><td> Pro</td><td> Ser</td><td> Phe 780</td><td> Val</td><td> Ala</td><td></td>

<td> GAT</td><td> GCC</td><td> GTA</td><td> ACA</td><td> TTT</td><td> AAA</td><td> AAT</td><td> ATA</td><td> AAA</td><td> CCT</td><td> TTA</td><td> CAA</td><td> AAT</td><td> TAT</td><td> GTA</td><td> AAG</td><td> 3787</td>

<td> Asp</td><td> Ala</td><td> Val 785</td><td> Thr</td><td> Phe</td><td> Lys</td><td> Asn</td><td> He 790</td><td> Lys</td><td> Pro</td><td> Leu</td><td> Gin</td><td> Asn 795</td><td> Tyr</td><td> Val</td><td> Lys</td><td></td>

<td> GAA</td><td> TAT</td><td> GAA</td><td> ATA</td><td> TAT</td><td> CAT</td><td> AAA</td><td> TCT</td><td> CAT</td><td> CGA</td><td> TAT</td><td> GAA</td><td> AAG</td><td> AAA</td><td> ACG</td><td> GTC</td><td> 3835</td>

<td> Glu</td><td> Tyr 800</td><td> Glu</td><td> He</td><td> Tyr</td><td> His</td><td> Lys 805</td><td> Ser</td><td> His</td><td> Arg</td><td> Tyr</td><td> Glu 810</td><td> Lys</td><td> Lys</td><td> Thr</td><td> Val</td><td></td>

<td> TTT</td><td> GAT</td><td> ATC</td><td> ATG</td><td> GGT</td><td> GTT</td><td> CAT</td><td> TAT</td><td> GAG</td><td> TAT</td><td> AGT</td><td> ATA</td><td> GCT</td><td> AGG</td><td> GAA</td><td> CAA</td><td> 3883</td>

<td> Phe 815</td><td> Asp</td><td> He</td><td> Met</td><td> Gly</td><td> Val 820</td><td> His</td><td> Tyr</td><td> Glu</td><td> Tyr</td><td> Ser 825</td><td> He</td><td> Ala</td><td> Arg</td><td> Glu</td><td> Gin 830</td><td></td>

AAG AAA GCC GCA ΤΑΑΤΊΤΤΑΑΑ 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

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- 145 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

<td rowspan="2"> Met 1</td><td rowspan="2"> Gin Arg Met</td><td rowspan="2"> Glu 5</td><td colspan="2"> Gly Lys Leu Phe Val Val Ser Lys Thr Leu Gin</td>

<td> 10</td><td> 15</td>

<td> Val</td><td> Val Thr Arg 20</td><td> Thr</td><td> Val Leu Leu Ser Thr Val 25</td><td> Tyr Ser He Thr Leu 30</td>

<td> Leu</td><td> Asn Asn Val 35</td><td> Val</td><td> He Lys Ala Asp Gin Leu 40</td><td> Asn He Asn Ser Gin 45</td>

<td> Ser</td><td> Lys Tyr Thr 50</td><td> Asn</td><td> Leu Gin Asn Leu Lys He 55</td><td> Pro Asp Asn Ala Glu 60</td>

<td> Asp 65</td><td> Phe Lys Glu</td><td> Asp</td><td> Lys Gly Lys Ala Lys Glu 70 75</td><td> Trp Gly Lys Glu Lys 80</td>

<td> Gly</td><td colspan="2"> Glu Glu Trp Arg 85</td><td> Pro Pro Ala Thr Glu Lys 90</td><td> Gly Glu Met Asn Asn 95</td>

<td> Phe</td><td> Leu Asp Asn 100</td><td> Lys</td><td> Asn Asp He Lys Thr Asn 105</td><td> Tyr Lys Glu He Thr no</td>

<td> Phe</td><td> Ser Met Ala 115</td><td> Gly</td><td> Ser Cys Glu Asp Glu He 120</td><td> Lys Asp Leu Glu Glu 125</td>

<td> He</td><td> Asp Lys He 130</td><td> Phe</td><td> Asp Lys Ala Asn Leu Ser 135</td><td> Ser Ser He He Thr 140</td>

<td> Tyr 145</td><td> Lys Asn Val</td><td> Glu</td><td> Pro Ala Thr He Gly Phe 150 155</td><td> Asn Lys Ser Leu Thr 160</td>

<td> Glu</td><td> Gly Asn Thr</td><td> He 165</td><td> Asn Ser Asp Ala Met Ala 170</td><td> Gin Phe Lys Glu Gin 175</td>

<td> Phe</td><td> Leu Gly Lys 180</td><td> Asp</td><td> Met Lys Phe Asp Ser Tyr 185</td><td> Leu Asp Thr His Leu 190</td>

<td> Thr</td><td> Ala Gin Gin 195</td><td> Val</td><td> Ser Ser Lys Lys Arg Val 200</td><td> He Leu Lys Val Thr 205</td>

<td> Val</td><td> Pro Ser Gly 210</td><td> Lys</td><td> Gly Ser Thr Thr Pro Thr 215</td><td> Lys Ala Gly Val He 220</td>

<td> Leu 225</td><td> Asn Asn Asn</td><td> Glu</td><td> Tyr Lys Met Leu He Asp 230 235</td><td> Asn Gly Tyr Val Leu 240</td>

<td> His</td><td> Val Asp Lys</td><td> Val 245</td><td> Ser Lys Val Val Lys Lys 250</td><td> Gly Met Glu Cys Leu 255</td>

<td> Gin</td><td> Val Glu Gly 260</td><td> Thr</td><td> Leu Lys Lys Ser Leu Asp 265</td><td> Phe Lys Asn Asp He 270</td>

Asn Ala Glu Ala His Ser Trp Gly Met Lys He Tyr Glu Asp Trp Ala 275 280 285

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<td rowspan="2"> Lys</td><td rowspan="2"> Asn 290</td><td rowspan="2"> Leu Thr</td><td rowspan="2"> Ala</td><td rowspan="2"> Ser</td><td colspan="2"> Gin Arg Glu Ala Leu Asp</td><td rowspan="2"> Gly Tyr Ala Arg</td>

<td> 295</td><td> 300</td>

<td> Gin</td><td> Asp</td><td> Tyr Lys</td><td> Glu</td><td> He</td><td> Asn Asn Tyr</td><td> Leu Arg Asn</td><td> Gin Gly Gly Ser</td>

<td> 305</td><td></td><td></td><td></td><td> 310</td><td></td><td> 315</td><td> 320</td>

<td> Gly</td><td> Asn</td><td> Glu Lys</td><td> Leu</td><td> Asp</td><td> Ala Gin Leu</td><td> Lys Asn He</td><td> Ser Asp Ala Leu</td>

<td></td><td></td><td></td><td> 325</td><td></td><td></td><td> 330</td><td> 335</td>

<td colspan="2"> Gly Lys</td><td> Lys Pro</td><td> He</td><td> Pro</td><td> Glu Asn He</td><td colspan="2"> Thr Val Tyr Arg Trp Cys Gly</td>

<td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td> 350</td>

<td> Met</td><td> Pro</td><td> Glu Phe</td><td colspan="2"> Gly Tyr</td><td> Gin He Ser</td><td> Asp Pro Leu</td><td> Pro Ser Leu Lys</td>

<td></td><td></td><td> 355</td><td></td><td></td><td> 360</td><td></td><td> 365</td>

<td> Asp</td><td> Phe</td><td> Glu Glu</td><td> Gin</td><td> Phe</td><td> Leu Asn Thr</td><td> He Lys Glu</td><td> Asp Lys Gly Tyr</td>

<td></td><td> 370</td><td></td><td></td><td></td><td> 375</td><td> 380</td><td></td>

<td> Met</td><td> Ser</td><td> Thr Ser</td><td> Leu</td><td> Ser</td><td> Ser Glu Arg</td><td> Leu Ala Ala</td><td> Phe Gly Ser Arg</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td> 395</td><td> 400</td>

<td> Lys</td><td> He</td><td> He Leu</td><td> Arg</td><td> Leu</td><td> Gin Val Pro</td><td> Lys Gly Ser</td><td> Thr Gly Ala Tyr</td>

<td></td><td></td><td></td><td> 405</td><td></td><td></td><td> 410</td><td> 415</td>

<td> Leu</td><td> Ser</td><td> Ala He</td><td colspan="2"> Gly Gly</td><td> Phe Ala Ser</td><td> Glu Lys Glu</td><td> He Leu Leu Asp</td>

<td></td><td></td><td> 420</td><td></td><td></td><td> 425</td><td></td><td> 430</td>

<td> Lys</td><td> Asp</td><td colspan="2"> Ser Lys Tyr</td><td> His</td><td> He Asp Lys</td><td> Ala Thr Glu</td><td> Val He He Lys</td>

<td></td><td></td><td> 435</td><td></td><td></td><td> 440</td><td></td><td> 445</td>

<td> Gly</td><td> Val</td><td colspan="2"> Lys Arg Tyr</td><td> Val</td><td> Val Asp Ala</td><td> Thr Leu Leu</td><td> Thr Asn</td>

<td></td><td> 450</td><td></td><td></td><td></td><td> 455</td><td> 460</td><td></td>

<td> (2)</td><td colspan="2"> INFORMATION</td><td> FOR</td><td> SEQ</td><td> ID NO:21:</td><td></td><td></td>

(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:

<td> Met</td><td> Lys</td><td> Asn</td><td> Met</td><td> Lys</td><td> Lys</td><td> Lys</td><td> Leu</td><td> Ala</td><td> Ser</td><td> Val</td><td> Val</td><td> Thr</td><td> Cys</td><td> Met</td><td> Leu</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td> Leu</td><td> Ala</td><td> Pro</td><td> Met</td><td> Phe</td><td> Leu</td><td> Asn</td><td> Gly</td><td> Asn</td><td> Val</td><td> Asn</td><td> Ala</td><td> Val</td><td> Asn</td><td> Ala</td><td> Asp</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td> Ser</td><td> Lys</td><td> He</td><td> Asn</td><td> Gin</td><td> He</td><td> Ser</td><td> Thr</td><td> Thr</td><td> Gin</td><td> Glu</td><td> Asn</td><td> Gin</td><td> Gin</td><td> Lys</td><td> Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

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- 147-

<td colspan="2"> Met Asp Arg</td><td rowspan="2"> Lys</td><td colspan="2"> Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe</td>

<td></td><td> 50</td><td> 55</td><td> 60</td>

<td> Asn 65</td><td> Asn Leu</td><td> Thr</td><td> Met Phe Ala 70</td><td> Pro Thr Arg Asp Asn Thr Leu Met Tyr 75 80</td>

<td> Asp</td><td> Gin Gin</td><td> Thr</td><td> Ala Asn Ala 85</td><td> Leu Leu Asp Lys Lys Gin Gin Glu Tyr 90 95</td>

<td> Gin</td><td> Ser He</td><td colspan="2"> Arg Trp He Gly 100</td><td> Leu He Gin Arg Lys Glu Thr Gly Asp 105 110</td>

<td> Phe</td><td> Thr Phe 115</td><td> Asn</td><td> Leu Ser Lys</td><td> Asp Glu Gin Ala He He Glu He Asp 120 125</td>

<td colspan="2"> Gly Lys He 130</td><td> He</td><td> Ser Asn Lys 135</td><td> Gly Lys Glu Lys Gin Val Val His Leu 140</td>

<td> Glu 145</td><td> Lys Glu</td><td> Lys</td><td> Leu Val Pro 150</td><td> He Lys He Glu Tyr Gin Ser Asp Thr 155 160</td>

<td> Lys</td><td> Phe Asn</td><td> He</td><td> Asp Ser Lys 165</td><td> Thr Phe Lys Glu Leu Lys Leu Phe Lys 170 175</td>

<td> He</td><td> Asp Ser</td><td> Gin 180</td><td> Asn Gin Ser</td><td> Gin Gin Val Gin Leu Arg Asn Pro Glu 185 190</td>

<td> Phe</td><td> Asn Lys 195</td><td> Lys</td><td> Glu Ser Gin</td><td> Glu Phe Leu Ala Lys Ala Ser Lys Thr 200 205</td>

<td> Asn</td><td> Leu Phe 210</td><td> Lys</td><td> Gin Lys Met 215</td><td> Lys Arg Asp He Asp Glu Asp Thr Asp 220</td>

<td> Thr 225</td><td colspan="2"> Asp Gly Asp</td><td> Ser He Pro 230</td><td> Asp Leu Trp Glu Glu Asn Gly Tyr Thr 235 240</td>

<td> He</td><td> Gin Asn</td><td> Lys</td><td> Val Ala Val 245</td><td> Lys Trp Asp Asp Ser Leu Ala Ser Lys 250 255</td>

<td colspan="2"> Gly Tyr Thr</td><td> Lys 260</td><td> Phe Val Ser</td><td> Asn Pro Leu Asp Sêr His Thr Val Gly 265 270</td>

<td> Asp</td><td> Pro Tyr 275</td><td> Thr</td><td> Asp Tyr Glu</td><td> Lys Ala Ala Arg Asp Leu Asp Leu Ser 280 285</td>

<td> Asn</td><td> Ala Lys 290</td><td> Glu</td><td> Thr Phe Asn 295</td><td> Pro Leu Val Ala Ala Phe Pro Ser Val 300</td>

<td> Asn 305</td><td> Val Ser</td><td> Met</td><td> Glu Lys Val 310</td><td> He Leu Ser Pro Asn Glu Asn Leu Ser 315 320</td>

<td> Asn</td><td> Ser Val</td><td> Glu</td><td> Ser His Ser</td><td> Ser Thr Asn Trp Ser Tyr Thr Asn Thr</td>

325 330 335

Glu Gly Ala Ser lie Glu Ala Gly Gly Gly Pro Leu Gly Leu Ser Phe

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<td></td><td> 340</td><td> 345</td><td> 350</td>

<td> Gly</td><td> Val Ser Val Thr Tyr Gin</td><td> His Ser Glu Thr Val Ala</td><td> Gin Glu Trp</td>

<td></td><td> 355</td><td> 360 365</td><td></td>

<td> Gly</td><td> Thr Ser Thr Gly Asn Thr</td><td> Ser Gin Phe Asn Thr Ala</td><td> Ser Ala Gly</td>

<td></td><td> 370 375</td><td> 380</td><td></td>

<td> Tyr</td><td colspan="2"> Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr</td><td> Gly Ala He</td>

<td> 385</td><td> 390</td><td> 395</td><td> 400</td>

<td colspan="2"> Tyr Asp Val Lys Pro Thr Thr</td><td> Ser Phe Val Leu Asn Asn</td><td> Asn Thr He</td>

<td></td><td> 405</td><td> 410</td><td> 415</td>

<td> Ala</td><td> Thr He Thr Ala Lys Ser</td><td> Asn Ser Thr Ala Leu Arg</td><td> He Ser Pro</td>

<td></td><td> 420</td><td> 425</td><td> 430</td>

<td colspan="2"> Gly Asp Ser Tyr Pro Glu He</td><td> Gly Glu Asn Ala He Ala</td><td> He Thr Ser</td>

<td></td><td> 435</td><td> 440 445</td><td></td>

<td> Met</td><td> Asp Asp Phe Asn Ser His</td><td> Pro He Thr Leu Asn Lys</td><td> Gin Gin Val</td>

<td></td><td> 450 455</td><td> 460</td><td></td>

<td> Asn</td><td> Gin Leu He Asn Asn Lys</td><td> Pro He Met Leu Glu Thr</td><td> Asp Gin Thr</td>

<td> 465</td><td> 470</td><td> 475</td><td> 480</td>

<td colspan="3"> Asp Gly Val Tyr Lys He Arg Asp Thr His Gly Asn He</td><td> Val Thr Gly</td>

<td></td><td> 485</td><td> 490</td><td> 495</td>

<td colspan="2"> Gly Glu Trp Asn Gly Val Thr</td><td> Gin Gin He Lys Ala Lys</td><td> Thr Ala Ser</td>

<td></td><td> 500</td><td> 505</td><td> 510</td>

<td> He</td><td> He Val Asp Asp Gly Lys</td><td> Gin Val Ala Glu Lys Arg</td><td> Val Ala Ala</td>

<td></td><td> 515</td><td> 520 525</td><td></td>

<td colspan="2"> Lys Asp Tyr Gly His Pro Glu</td><td> Asp Lys Thr Pro Pro Leu</td><td> Thr Leu Lys</td>

<td></td><td> 530 535</td><td> 540</td><td></td>

<td> Asp</td><td> Thr Leu Lys Leu Ser Tyr</td><td> Pro Asp Glu He Lys Glu</td><td> Thr Asn Gly</td>

<td> 545</td><td> 550</td><td> 555</td><td> 560</td>

<td> Leu</td><td> Leu Tyr Tyr Asp Asp Lys</td><td> Pro He Tyr Glu Ser Ser</td><td> Val Met Thr</td>

<td></td><td> 565</td><td> 570</td><td> 575</td>

<td> Tyr</td><td> Leu Asp Glu Asn Thr Ala</td><td> Lys Glu Val Lys Lys Gin</td><td> He Asn Asp</td>

<td></td><td> 580</td><td> 585</td><td> 590</td>

<td> Thr</td><td> Thr Gly Lys Phe Lys Asp</td><td> Val Asn His Leu Tyr Asp</td><td> Val Lys Leu</td>

<td></td><td> 595</td><td> 600 605</td><td></td>

<td> Thr</td><td> Pro Lys Met Asn Phe Thr</td><td> He Lys Met Ala Ser Leu</td><td> Tyr Asp Gly</td>

<td></td><td> 610 615</td><td> 620</td><td></td>

<td> Ala</td><td> Glu Asn Asn His Asn Ser</td><td> Leu Gly Thr Trp Tyr Leu</td><td> Thr Tyr Asn</td>

<td> 625</td><td> 630</td><td> 635</td><td> 640</td>

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<td rowspan="2"> Val Ala</td><td rowspan="2"> Gly</td><td rowspan="2"> Gly Asn 645</td><td colspan="2"> Thr Gly Lys Arg Gin Tyr Arg Ser Ala His Ser</td>

<td> 650</td><td> 655</td>

<td> Cys Ala</td><td> His</td><td> Val Ala</td><td> Leu Ser Ser Glu Ala Lys Lys Lys Leu</td><td> Asn Gin</td>

<td></td><td></td><td> 660</td><td> 665 670</td><td></td>

<td> Asn Ala</td><td> Asn</td><td> Tyr Tyr</td><td> Leu Ser Met Tyr Met Lys Ala Asp Ser</td><td> Thr Thr</td>

<td></td><td> 675</td><td></td><td> 680 685</td><td></td>

<td> Glu Pro</td><td> Thr</td><td> He Glu</td><td> Val Ala Gly Glu Lys Ser Ala He Thr</td><td> Ser Lys</td>

<td> 690</td><td></td><td></td><td> 695 700</td><td></td>

<td> Lys Val</td><td> Lys</td><td> Leu Asn</td><td> Asn Gin Asn Tyr Gin Arg Val Asp He</td><td> Leu Val</td>

<td> 705</td><td></td><td></td><td> 710 715</td><td> 720</td>

<td> Lys Asn</td><td> Ser</td><td> Glu Arg</td><td> Asn Pro Met Asp Lys He Tyr He Arg</td><td> Gly Asn</td>

<td></td><td></td><td> 725</td><td> 730</td><td> 735</td>

<td> Gly Thr</td><td> Thr</td><td> Asn Val</td><td> Tyr Gly Asp Asp Val Thr He Pro Glu</td><td> Val Ser</td>

<td></td><td></td><td> 740</td><td> 745 750</td><td></td>

<td> Ala He</td><td> Asn</td><td> Pro Ala</td><td> Ser Leu Ser Asp Glu Glu He Gin Glu</td><td> He Phe</td>

<td></td><td> 755</td><td></td><td> 760 765</td><td></td>

<td> Lys Asp</td><td> Ser</td><td> Thr He</td><td> Glu Tyr Gly Asn Pro Ser Phe Val Ala</td><td> Asp Ala</td>

<td> 770</td><td></td><td></td><td> 775 780</td><td></td>

<td> Val Thr</td><td> Phe</td><td> Lys Asn</td><td> He Lys Pro Leu Gin Asn Tyr Val Lys</td><td> Glu Tyr</td>

<td> 785</td><td></td><td></td><td> 790 795</td><td> 800</td>

<td> Glu He</td><td> Tyr</td><td> His Lys</td><td> Ser His Arg Tyr Glu Lys Lys Thr Vàl</td><td> Phe Asp</td>

<td></td><td></td><td> 805</td><td> 810</td><td> 815</td>

<td> He Met</td><td> Gly</td><td> Val His</td><td> Tyr Glu Tyr Ser He Ala Arg Glu Gin</td><td> Lys Lys</td>

<td></td><td></td><td> 820</td><td> 825 830</td><td></td>

<td> Ala Ala</td><td></td><td></td><td></td><td></td>

<td colspan="3"> (2) INFORMATION FOR</td><td> SEQ ID NO:22:</td><td></td>

(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

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- 150 product (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

<td rowspan="2"> ATG AAA Met Lys 835</td><td colspan="9"> AGA ATG GAG GGA AAG TTG TTT ATG GTG TCA AAA AAA TTA CAA</td><td rowspan="2"> 48</td>

<td> Arg Met</td><td> Glu</td><td> Gly 840</td><td> Lys</td><td> Leu</td><td> Phe Met</td><td> Val 845</td><td> Ser Lys Lys</td><td> Leu Gin 850</td>

<td> GTA GTT</td><td> ACT AAA</td><td> ACT</td><td> GTA</td><td> TTG</td><td> CTT</td><td> AGT ACA</td><td> GTT</td><td> TTC TCT ATA</td><td> TCT TTA</td><td> 96</td>

<td> Val Val</td><td> Thr Lys</td><td> Thr</td><td> Val</td><td> Leu</td><td> Leu</td><td> Ser Thr</td><td> Val</td><td> Phe Ser He</td><td> Ser Leu</td><td></td>

<td></td><td></td><td> 855</td><td></td><td></td><td></td><td> 860</td><td></td><td></td><td> 865</td><td></td>

<td> TTA AAT</td><td> AAT GAA</td><td> GTG</td><td> ATA</td><td> AAA</td><td> GCT</td><td> GAA CAA</td><td> TTA</td><td> AAT ATA AAT</td><td> TCT CAA</td><td> 144</td>

<td> Leu Asn</td><td> Asn Glu</td><td> Val</td><td> He</td><td> Lys</td><td> Ala</td><td> Glu Gin</td><td> Leu</td><td> Asn He Asn</td><td> Ser Gin</td><td></td>

<td></td><td> 870</td><td></td><td></td><td></td><td></td><td> 875</td><td></td><td> 880</td><td></td><td></td>

<td> AGT AAA</td><td> TAT ACT</td><td> AAC</td><td> TTG</td><td> CAA</td><td> AAT</td><td> CTA AAA</td><td> ATC</td><td> ACT GAC AAG</td><td> GTA GAG</td><td> 192</td>

<td> Ser Lys</td><td> Tyr Thr</td><td> Asn</td><td> Leu</td><td> Gin</td><td> Asn</td><td> Leu Lys</td><td> He</td><td> Thr Asp Lys</td><td> Val Glu</td><td></td>

<td></td><td> 885</td><td></td><td></td><td></td><td> 890</td><td></td><td></td><td> 895</td><td></td><td></td>

<td> GAT TTT</td><td> AAA GAA</td><td> GAT</td><td> AAG</td><td> GAA</td><td> AAA</td><td> GCG AAA</td><td> GAA</td><td> TGG GGG AAA</td><td> GAA AAA</td><td> 240</td>

<td> Asp Phe</td><td> Lys Glu</td><td> Asp</td><td> Lys</td><td> Glu</td><td> Lys</td><td> Ala Lys</td><td> Glu</td><td> Trp Gly Lys</td><td> Glu Lys</td><td></td>

<td> 900</td><td></td><td></td><td></td><td> 905</td><td></td><td></td><td></td><td> 910</td><td></td><td></td>

<td> GAA AAA</td><td> GAG TGG</td><td> AAA</td><td> CTA</td><td> ACT</td><td> GCT</td><td> ACT GAA</td><td> AAA</td><td> GGA AAA ATG</td><td> AAT AAT</td><td> 288</td>

<td> Glu Lys</td><td> Glu Trp</td><td> Lys</td><td> Leu</td><td> Thr</td><td> Ala</td><td> Thr Glu</td><td> Lys</td><td> Gly Lys Met</td><td> Asn Asn</td><td></td>

<td> 915</td><td></td><td></td><td> 920</td><td></td><td></td><td></td><td> 925</td><td></td><td> 930</td><td></td>

<td> TTT TTA</td><td> GAT AAT</td><td> AAA</td><td> AAT</td><td> GAT</td><td> ATA</td><td> AAG ACA</td><td> AAT</td><td> TAT AAA GAA</td><td> ATT ACT</td><td> 336</td>

<td> Phe Leu</td><td> Asp Asn</td><td> Lys</td><td> Asn</td><td> Asp</td><td> He</td><td> Lys Thr</td><td> Asn</td><td> Tyr Lys Glu</td><td> He Thr</td><td></td>

<td></td><td></td><td> 935</td><td></td><td></td><td></td><td> 940</td><td></td><td></td><td> 945</td><td></td>

<td> TTT TCT</td><td> ATG GCA</td><td> GGC</td><td> TCA</td><td> TTT</td><td> GAA</td><td> GAT GAA</td><td> ATA</td><td> AAA GAT TTA</td><td> AAA GAA</td><td> 384</td>

<td> Phe Ser</td><td> Met Ala</td><td> Gly</td><td> Ser</td><td> Phe</td><td> Glu</td><td> Asp Glu</td><td> He</td><td> Lys Asp Leu</td><td> Lys Glu</td><td></td>

<td></td><td> 950</td><td></td><td></td><td></td><td></td><td> 955</td><td></td><td> 960</td><td></td><td></td>

<td> ATT GAT</td><td> AAG ATG</td><td> TTT</td><td> GAT</td><td> AAA</td><td> ACC</td><td> AAT CTA</td><td> TCA</td><td> AAT TCT ATT</td><td> ATC ACC</td><td> 432</td>

<td> He Asp</td><td> Lys Met</td><td> Phe</td><td> Asp</td><td> Lys</td><td> Thr</td><td> Asn Leu</td><td> Ser</td><td> Asn Ser He</td><td> He Thr</td><td></td>

<td></td><td> 965</td><td></td><td></td><td></td><td> 970</td><td></td><td></td><td> 975</td><td></td><td></td>

<td> TAT AAA</td><td> AAT GTG</td><td> GAA</td><td> CCG</td><td> ACA</td><td> ACA</td><td> ATT GGA</td><td> TTT</td><td> AAT AAA TCT</td><td> TTA ACA</td><td> 480</td>

<td> Tyr Lys</td><td> Asn Val</td><td> Glu</td><td> Pro</td><td> Thr</td><td> Thr</td><td> He Gly</td><td> Phe</td><td> Asn Lys Ser</td><td> Leu Thr</td><td></td>

<td> 980</td><td></td><td></td><td></td><td> 985</td><td></td><td></td><td></td><td> 990</td><td></td><td></td>

<td> GAA GGT</td><td> AAT ACG</td><td> ATT</td><td> AAT</td><td> TCT</td><td> GAT</td><td> GCA ATG</td><td> GCA</td><td> CAG TTT AAA</td><td> GAA CAA</td><td> 528</td>

<td> Glu Gly</td><td> Asn Thr</td><td> He</td><td> Asn</td><td> Ser</td><td> Asp</td><td> Ala Met</td><td> Ala</td><td> Gin Phe Lys</td><td> Glu Gin</td><td></td>

<td> 995</td><td></td><td></td><td colspan="2"> 1000</td><td></td><td></td><td colspan="2"> 1005</td><td> 1010</td><td></td>

<td> TTT TTA</td><td> GAT AGG</td><td> GAT</td><td> ATT</td><td> AAG</td><td> TTT</td><td> GAT AGT</td><td> TAT</td><td> CTA GAT ACG</td><td> CAT TTA</td><td> 576</td>

<td> Phe Leu</td><td colspan="2"> Asp Arg Asp</td><td> He</td><td> Lys</td><td> Phe</td><td> Asp Ser</td><td> Tyr</td><td> Leu Asp Thr</td><td> His Leu</td><td></td>

<td></td><td></td><td colspan="2"> 1015</td><td></td><td></td><td colspan="2"> 1020</td><td></td><td> 1025</td><td></td>

<td> ACT GCT</td><td> CAA CAA</td><td> GTT</td><td> TCC</td><td> AGT</td><td> AAA</td><td> GAA AGA</td><td> GTT</td><td> ATT TTG AAG</td><td> GTT ACG</td><td> 624</td>

<td> Thr Ala</td><td> Gin Gin</td><td> Val</td><td> Ser</td><td> Ser</td><td> Lys</td><td colspan="2"> Glu Arg Val</td><td> He Leu Lys</td><td> Val Thr</td><td></td>

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<td colspan="7"> GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA ACA AAA GCA GGT GTC ATT</td><td rowspan="3"> 672</td>

<td rowspan="2"> Val</td><td colspan="2"> Pro Ser Gly Lys Gly Ser Thr Thr</td><td rowspan="2"> Pro Thr</td><td> Lys Ala Gly</td><td rowspan="2"> Val</td><td rowspan="2"> He</td>

<td> 1045</td><td> 1050</td><td> 1055</td>

<td> TTA</td><td> AAT AAT AGT GAA TAC</td><td> AAA ATG CTC</td><td> ATT GAT</td><td> AAT GGG TAT</td><td> ATG</td><td> GTC</td><td> 720</td>

<td> Leu</td><td> Asn Asn Ser Glu Tyr 1060</td><td> Lys Met Leu 1065</td><td> He Asp</td><td> Asn Gly Tyr 1070</td><td> Met</td><td> Val</td><td></td>

<td> CAT</td><td> GTA GAT AAG GTA TCA</td><td> AAA GTG GTG</td><td> AAA AAA</td><td> GGG GTG GAG</td><td> TGC</td><td> TTA</td><td> 768</td>

<td colspan="3"> His Val Asp Lys Val Ser Lys Val Val 1075 1080</td><td colspan="2"> Lys Lys Gly Val Glu 1085</td><td> Cys</td><td> Leu 1090</td><td></td>

<td> CAA</td><td> ATT GAA GGG ACT TTA</td><td> AAA AAG AGT</td><td> CTT GAC</td><td> TTT AAA AAT</td><td> GAT</td><td> ATA</td><td> 816</td>

<td> Gin</td><td> He Glu Gly Thr Leu 1095</td><td> Lys Lys Ser</td><td> Leu Asp 1100</td><td> Phe Lys Asn</td><td colspan="2"> Asp lie 1105</td><td></td>

<td> AAT</td><td> GCT GAA GCG CAT AGC</td><td> TGG GGT ATG</td><td> AAG AAT</td><td> TAT GAA GAG</td><td> TGG</td><td> GCT</td><td> 864</td>

<td> Asn</td><td> Ala Glu Ala His Ser 1110</td><td colspan="2"> Trp Gly Met Lys Asn 1115</td><td colspan="2"> Tyr Glu Glu Trp 1120</td><td> Ala</td><td></td>

<td> AAA</td><td> GAT TTA ACC GAT TCG</td><td> CAA AGG GAA</td><td> GCT TTA</td><td> GAT GGG TAT</td><td> GCT</td><td> AGG</td><td> 912</td>

<td colspan="2"> Lys Asp Leu Thr Asp Ser 1125</td><td> Gin Arg Glu 1130</td><td> Ala Leu</td><td> Asp Gly Tyr 1135</td><td> Ala</td><td> Arg</td><td></td>

<td> CAA</td><td> GAT TAT AAA GAA ATC</td><td> AAT AAT TAT</td><td> TTA AGA</td><td> AAT CAA GGC</td><td colspan="2"> GGA AGT</td><td> 960</td>

<td> Gin</td><td> Asp Tyr Lys Glu He 1140</td><td> Asn Asn Tyr 1145</td><td> Leu Arg</td><td colspan="2"> Asn Gin Gly Gly 1150</td><td> Ser</td><td></td>

<td> GGA</td><td> AAT GAA AAA CTA GAT</td><td> GCT CAA ATA</td><td> AAA AAT</td><td> ATT TCT GAT</td><td> GCT</td><td> TTA</td><td> 1008</td>

<td colspan="3"> Gly Asn Glu Lys Leu Asp Ala Gin He 1155 1160</td><td colspan="2"> Lys Asn He Ser Asp 1165</td><td> Ala</td><td> Leu 1170</td><td></td>

<td> GGG</td><td> AAG AAA CCA ATA CCG</td><td> GAA AAT ATT</td><td> ACT GTG</td><td> TAT AGA TGG</td><td> TGT</td><td> GGC</td><td> 1056</td>

<td> Gly</td><td> Lys Lys Pro He Pro 1175</td><td> Glu Asn He</td><td> Thr Val 1180</td><td colspan="3"> Tyr Arg Trp Cys Gly 1185</td><td></td>

<td> ATG</td><td> CCG GAA TTT GGT TAT</td><td> CAA ATT AGT</td><td> GAT CCG</td><td> TTA CCT TCT</td><td> TTA</td><td> AAA</td><td> 1104</td>

<td> Met</td><td> Pro Glu Phe Gly Tyr 1190</td><td colspan="2"> Gin He Ser Asp Pro 1195</td><td colspan="2"> Leu Pro Ser Leu 1200</td><td> Lys</td><td></td>

<td> GAT</td><td> TTT GAA GAA CAA TTT</td><td> TTA AAT ACA</td><td> ATC AAA</td><td> GAA GAC AAA</td><td> GGA</td><td> TAT</td><td> 1152</td>

<td> Asp</td><td> Phe Glu Glu Gin Phe 1205</td><td> Leu Asn Thr 1210</td><td> He Lys</td><td> Glu Asp Lys 1215</td><td> Gly</td><td> Tyr</td><td></td>

<td> ATG</td><td> AGT ACA AGC TTA TCG</td><td> AGT GAA CGT</td><td> CTT GCA</td><td> GCT TTT GGA</td><td> TCT</td><td> AGA</td><td> 1200</td>

<td> Met</td><td> Ser Thr Ser Leu Ser 1220</td><td> Ser Glu Arg 1225</td><td> Leu Ala</td><td> Ala Phe Gly 1230</td><td> Ser</td><td> Arg</td><td></td>

<td> AAA</td><td> ATT ATA TTA CGA TTA</td><td> CAA GTT CCG</td><td> AAA GGA</td><td> AGT ACG GGT</td><td> GCG</td><td> TAT</td><td> 1248</td>

<td colspan="3"> Lys He He Leu Arg Leu Gin Val Pro 1235 1240</td><td colspan="2"> Lys Gly Ser Thr Gly 1245</td><td> Ala</td><td> Tyr 1250</td><td></td>

<td> TTA</td><td> AGT GCC ATT GGT GGA</td><td> TTT GCA AGT</td><td> GAA AAA</td><td> GAG ATC CTA</td><td> CTT</td><td> GAT</td><td> 1296</td>

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<td rowspan="2"> Leu</td><td colspan="3"> Ser Ala He Gly Gly Phe Ala Ser Glu Lys Glu He Leu Leu Asp</td>

<td> 1255</td><td> 1260</td><td> 1265</td>

<td> AAA</td><td> GAT AGT AAA TAT CAT ATT GAT</td><td> AAA GTA ACA GAG GTA ATT</td><td> ATT AAA 1344</td>

<td colspan="2"> Lys Asp Ser Lys Tyr His He Asp 1270</td><td colspan="2"> Lys Val Thr Glu Val He He Lys 1275 1280</td>

<td> GGT</td><td> GTT AAG CGA TAT GTA GTG GAT</td><td> GCA ACA TTA TTA ACA AAT</td><td> ATG AAA 1392</td>

<td> Gly</td><td colspan="2"> Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 1285 1290 1295</td><td> Met Lys</td>

<td> AAT</td><td> ATG AAG AAA AAG TTA GCA AGT</td><td> GTT GTA ACG TGT ACG TTA</td><td> TTA GCT 1440</td>

<td> Asn</td><td> Met Lys Lys Lys Leu Ala Ser 1300 1305</td><td> Val Val Thr Cys Thr Leu 1310</td><td> Leu Ala</td>

<td> CCT</td><td> ATG TTT TTG AAT GGA AAT GTG</td><td> AAT GCT GTT TAC GCA GAC</td><td> AGC AAA 1488</td>

<td colspan="2"> Pro Met Phe Leu Asn Gly Asn Val 1315 1320</td><td> Asn Ala Val Tyr Ala Asp 1325</td><td> Ser Lys 1330</td>

<td> ACA</td><td> AAT CAA ATT TCT ACA ACA CAG</td><td> AAA AAT CAA CAG AAA GAG</td><td> ATG GAC 1536</td>

<td> Thr</td><td> Asn Gin He Ser Thr Thr Gin 1335</td><td> Lys Asn Gin Gin Lys Glu 1340</td><td> Met Asp 1345</td>

<td> CGA</td><td> AAA GGA TTA CTT GGG TAT TAT</td><td> TTC AAA GGA AAA GAT TTT</td><td> AGT AAT 1584</td>

<td> Arg</td><td> Lys Gly Leu Leu Gly Tyr Tyr 1350</td><td colspan="2"> Phe Lys Gly Lys Asp Phe Ser Asn 1355 1360</td>

<td> CTT</td><td> ACT ATG TTT GCA CCG ACA CGT</td><td> GAT AGT ACT CTT ATT TAT</td><td> GAT CAA 1632</td>

<td> Leu</td><td colspan="3"> Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu He Tyr Asp Gin 1365 1370 1375</td>

<td> CAA</td><td> ACA GCA AAT AAA CTA TTA GAT</td><td> AAA AAA CAA CAA GAA TAT</td><td> CAG TCT 1680</td>

<td> Gin</td><td> Thr Ala Asn Lys Leu Leu Asp 1380 1385</td><td> Lys Lys Gin Gin Glu Tyr 1390</td><td> Gin Ser</td>

<td> ATT</td><td> CGT TGG ATT GGT TTG ATT CAG</td><td> AGT AAA GAA ACG GGA GAT</td><td> TTC ACA 1728</td>

<td colspan="2"> He Arg Trp He Gly Leu He Gin 1395 1400</td><td> Ser Lys Glu Thr Gly Asp 1405</td><td> Phe Thr 1410</td>

<td> TTT</td><td> AAC TTA TCT GAG GAT GAA CAG</td><td> GCA ATT ATA GAA ATC AAT</td><td> GGG AAA 1776</td>

<td> Phe</td><td> Asn Leu Ser Glu Asp Glu Gin 1415</td><td> Ala He He Glu He Asn 1420</td><td> Gly Lys 1425</td>

<td> ATT</td><td> ATT TCT AAT AAA GGG AAA GAA</td><td> AAG CAA GTT GTC CAT TTA</td><td> GAA AAA 1824</td>

<td> He</td><td> He Ser Asn Lys Gly Lys Glu 1430</td><td colspan="2"> Lys Gin Val Val His Leu Glu Lys 1435 1440</td>

<td> GGA</td><td> AAA TTA GTT CCA ATC AAA ATA</td><td> GAG TAT CAA TCA GAT ACA</td><td> AAA TTT 1872</td>

<td> Gly</td><td colspan="2"> Lys Leu Val Pro He Lys He Glu Tyr Gin Ser Asp Thr 1445 1450 1455</td><td> Lys Phe</td>

<td> AAT</td><td> ATT GAC AGT AAA ACA TTT AAA</td><td> GAA CTT AAA TTA TTT AAA</td><td> ATA GAT 1920</td>

<td> Asn</td><td colspan="2"> He Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys</td><td> He Asp</td>

1460 1465 1470

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<td colspan="2"> AGT CAA AAC CAA</td><td colspan="2"> CCC CAG CAA</td><td colspan="2"> GTC CAG CAA</td><td colspan="2" rowspan="2"> GAT GAA CTG AGA AAT CCT Asp Glu Leu Arg Asn Pro</td><td rowspan="3"> 1968</td>

<td rowspan="2"> Ser Gin 1475</td><td rowspan="2"> Asn Gin</td><td rowspan="2"> Pro</td><td rowspan="2"> Gin Gin 1480</td><td rowspan="2"> Val</td><td rowspan="2"> Gin Gin</td>

<td> 1485</td><td> 1490</td>

<td> GAA TTT</td><td> AAC AAG</td><td> AAA</td><td> GAA TCA</td><td> CAG</td><td> GAA TTC</td><td> TTA GCG AAA</td><td> CCA TCG AAA</td><td> 2016</td>

<td> Glu Phe</td><td> Asn Lys</td><td colspan="2"> Lys Glu Ser 1495</td><td> Gin</td><td colspan="2"> Glu Phe Leu Ala Lys 1500</td><td> Pro Ser Lys 1505</td><td></td>

<td> ATA AAT</td><td> CTT TTC</td><td> ACT</td><td> CAA AAA</td><td> ATG</td><td> AAA AGG</td><td> GAA ATT GAT</td><td> GAA GAC ACG</td><td> 2064</td>

<td> He Asn</td><td colspan="2"> Leu Phe Thr 1510</td><td> Gin Lys</td><td> Met</td><td> Lys Arg 1515</td><td> Glu He Asp</td><td> Glu Asp Thr 1520</td><td></td>

<td> GAT ACG</td><td> GAT GGG</td><td> GAC</td><td> TCT ATT</td><td> CCT</td><td> GAC CTT</td><td> TGG GAA GAA</td><td> AAT GGG TAT</td><td> 2112</td>

<td> Asp Thr</td><td colspan="2"> Asp Gly Asp 1525</td><td> Ser He</td><td colspan="2"> Pro Asp Leu 1530</td><td colspan="2"> Trp Glu Glu Asn Gly Tyr 1535</td><td></td>

<td> ACG ATT</td><td> CAA AAT</td><td> AGA</td><td> ATC GCT</td><td> GTA</td><td> AAG TGG</td><td> GAC GAT TCT</td><td> CTA GCA AGT</td><td> 2160</td>

<td colspan="2"> Thr He Gin Asn 1540</td><td> Arg</td><td colspan="2"> He Ala Val 1545</td><td colspan="2"> Lys Trp Asp Asp Ser 1550</td><td> Leu Ala Ser</td><td></td>

<td colspan="2"> AAA GGG TAT ACG</td><td> AAA</td><td> TTT GTT</td><td> TCA</td><td> AAT CCA</td><td> CTA GAA AGT</td><td> CAC ACA GTT</td><td> 2208</td>

<td colspan="2"> Lys Gly Tyr Thr 1555</td><td> Lys</td><td> Phe Val 1560</td><td> Ser</td><td> Asn Pro</td><td> Leu Glu Ser 1565</td><td> His Thr Val 1570</td><td></td>

<td> GGT GAT</td><td> CCT TAT</td><td> ACA</td><td> GAT TAT</td><td> GAA</td><td> AAG GCA</td><td> GCA AGA GAT</td><td> CTA GAT TTG</td><td> 2256</td>

<td> Gly Asp</td><td> Pro Tyr</td><td colspan="2"> Thr Asp Tyr 1575</td><td> Glu</td><td colspan="2"> Lys Ala Ala Arg Asp 1580</td><td> Leu Asp Leu 1585</td><td></td>

<td> TCA AAT</td><td> GCA AAG</td><td> GAA</td><td> ACG TTT</td><td> AAC</td><td> CCA TTG</td><td> GTA GCT GCT</td><td> TTT CCA AGT</td><td> 2304</td>

<td> Ser Asn</td><td colspan="2"> Ala Lys Glu 1590</td><td> Thr Phe</td><td> Asn</td><td> Pro Leu 1595</td><td> Val Ala Ala</td><td> Phe Pro Ser 1600</td><td></td>

<td> GTG AAT</td><td> GTT AGT</td><td> ATG</td><td> GAA AAG</td><td> GTG</td><td> ATA TTA</td><td> TCA CCA AAT</td><td> GAA AAT TTA</td><td> 2352</td>

<td> Val Asn</td><td> Val Ser 1605</td><td> Met</td><td> Glu Lys</td><td colspan="2"> Val He Leu 1610</td><td colspan="2"> Ser Pro Asn Glu Asn Leu 1615</td><td></td>

<td> TCC AAT</td><td> AGT GTA</td><td> GAG</td><td> TCT CAT</td><td> TCA</td><td> TCC ACG</td><td> AAT TGG TCT</td><td> TAT ACA AAT</td><td> 2400</td>

<td colspan="2"> Ser Asn Ser Val 1620</td><td> Glu</td><td colspan="2"> Ser His Ser 1625</td><td> Ser Thr</td><td> Asn Trp Ser 1630</td><td> Tyr Thr Asn</td><td></td>

<td> ACA GAA</td><td> GGT GCT</td><td> TCT</td><td> GTT GAA</td><td> GCG</td><td> GGG ATT</td><td> GGA CCA AAA</td><td> GGT ATT TCG</td><td> 2448</td>

<td> Thr Glu 1635</td><td> Gly Ala</td><td> Ser</td><td> Val Glu 1640</td><td> Ala</td><td> Gly He</td><td> Gly Pro Lys 1645</td><td> Gly He Ser 1650</td><td></td>

<td> TTC GGA</td><td> GTT AGC</td><td> GTA</td><td> AAC TAT</td><td> CAA</td><td> CAC TCT</td><td> GAA ACA GTT</td><td> GCA CAA GAA</td><td> 2496</td>

<td> Phe Gly</td><td> Val Ser</td><td colspan="2"> Val Asn Tyr 1655</td><td> Gin</td><td colspan="2"> His Ser Glu Thr Val 1660</td><td> Ala Gin Glu 1665</td><td></td>

<td> TGG GGA</td><td> ACA TCT</td><td> ACA</td><td> GGA AAT</td><td> ACT</td><td> TCG CAA</td><td> TTC AAT ACG</td><td> GCT TCA GCG</td><td> 2544</td>

<td> Trp Gly</td><td colspan="2"> Thr Ser Thr 1670</td><td> Gly Asn</td><td> Thr</td><td> Ser Gin 1675</td><td> Phe Asn Thr</td><td> Ala Ser Ala 1680</td><td></td>

<td> GGA TAT</td><td> TTA AAT</td><td> GCA</td><td> AAT GTT</td><td> CGA</td><td> TAT AAC</td><td> AAT GTA GGA</td><td> ACT GGT GCC</td><td> 2592</td>

<td> Gly Tyr</td><td> Leu Asn 1685</td><td> Ala</td><td> Asn Val</td><td colspan="2"> Arg Tyr Asn 1690</td><td colspan="2"> Asn Val Gly Thr Gly Ala 1695</td><td></td>

WO 96/10083

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- 154 -

<td rowspan="3"> ATC TAC GAT He Tyr Asp 1700</td><td rowspan="3"> GTA Val</td><td colspan="5"> AAA CCT ACA ACA AGT TTT GTA TTA AAT AAC GAT ACT</td><td rowspan="3"> 2640</td>

<td rowspan="2"> Lys</td><td colspan="2"> Pro Thr Thr Ser Phe Val Leu Asn</td><td rowspan="2"> Asn Asp</td><td rowspan="2"> Thr</td>

<td> 1705</td><td> 1710</td>

<td> ATC GCA ACT</td><td> ATT</td><td> ACG</td><td> GCG AAA TCT AAT</td><td> TCT ACA GCC TTA</td><td> AAT ATA</td><td> TCT</td><td> 2688</td>

<td> He Ala Thr 1715</td><td> He</td><td> Thr</td><td> Ala Lys Ser Asn 1720</td><td> Ser Thr Ala Leu 1725</td><td> Asn He</td><td> Ser 1730</td><td></td>

<td> CCT GGA GAA</td><td> AGT</td><td> TAC</td><td> CCG AAA AAA GGA</td><td> CAA AAT GGA ATC</td><td> GCA ÀTA</td><td> ACA</td><td> 2736</td>

<td> Pro Gly Glu</td><td> Ser</td><td colspan="2"> Tyr Pro Lys Lys Gly 1735</td><td> Gin Asn Gly He 1740</td><td colspan="2"> Ala He Thr 1745</td><td></td>

<td> TCA ATG GAT</td><td> GAT</td><td> TTT</td><td> AAT TCC CAT CCG</td><td> ATT ACA TTA AAT</td><td> AAA AAA</td><td> CAA</td><td> 2784</td>

<td colspan="3"> Ser Met Asp Asp Phe 1750</td><td colspan="2"> Asn Ser His Pro He Thr Leu Asn 1755</td><td> Lys Lys 1760</td><td> Gin</td><td></td>

<td> GTA GAT AAT</td><td> CTG</td><td> CTA</td><td> AAT AAT AAA CCT</td><td> ATG ATG TTG GAA</td><td> ACA AAC</td><td> CAA</td><td> 2832</td>

<td colspan="2"> Val Asp Asn Leu 1765</td><td> Leu</td><td> Asn Asn Lys Pro 1770</td><td colspan="2"> Met Met Leu Glu Thr Asn 1775</td><td> Gin</td><td></td>

<td> ACA GAT GGT</td><td> GTT</td><td> TAT</td><td> AAG ATA AAA GAT</td><td> ACA CAT GGA AAT</td><td> ATA GTA</td><td> ACT</td><td> 2880</td>

<td> Thr Asp Gly 1780</td><td> Val</td><td colspan="2"> Tyr Lys He Lys Asp 1785</td><td> Thr His Gly Asn 1790</td><td> He Val</td><td> Thr</td><td></td>

<td> GGC GGA GAA</td><td> TGG</td><td> AAT</td><td> GGT GTC ATA CAA</td><td> CAA ATC AAG GCT</td><td> AAA ACA</td><td> GCG</td><td> 2928</td>

<td> Gly Gly Glu 1795</td><td> Trp</td><td> Asn</td><td> Gly Val He Gin 1800</td><td> Gin He Lys Ala 1805</td><td> Lys Thr</td><td> Ala 1810</td><td></td>

<td> TCT ATT ATT</td><td> GTG</td><td> GAT</td><td> GAT GGG GAA CGT</td><td> GTA GCA GAA AAA</td><td> CGT GTA</td><td> GCG</td><td> 2976</td>

<td> Ser He He</td><td> Val</td><td colspan="2"> Asp Asp Gly Glu Arg 1815</td><td> Val Ala Glu Lys 1820</td><td colspan="2"> Arg Val Ala 1825</td><td></td>

<td> GCA AAA GAT</td><td> TAT</td><td> GAA</td><td> AAT CCA GAA GAT</td><td> AAA ACA CCG TCT</td><td> TTA ACT</td><td> TTA</td><td> 3024</td>

<td> Ala Lys Asp</td><td colspan="2"> Tyr Glu 1830</td><td colspan="2"> Asn Pro Glu Asp Lys Thr Pro Ser 1835</td><td> Leu Thr 1840</td><td> Leu</td><td></td>

<td> AAA GAT GCC</td><td> CTG</td><td> AAG</td><td> CTT TCA TAT CCA</td><td> GAT GAA ATA AAA</td><td> GAA ATA</td><td> GAG</td><td> 3072</td>

<td colspan="2"> Lys Asp Ala Leu 1845</td><td> Lys</td><td> Leu Ser Tyr Pro 1850</td><td colspan="2"> Asp Glu He Lys Glu He 1855</td><td> Glu</td><td></td>

<td> GGA TTA TTA</td><td> TAT</td><td> TAT</td><td> AAA AAC AAA CCG</td><td> ATA TAC GAA TCG</td><td> AGC GTT</td><td> ATG</td><td> 3120</td>

<td> Gly Leu Leu 1860</td><td colspan="2"> Tyr Tyr</td><td> Lys Asn Lys Pro 1865</td><td> He Tyr Glu Ser 1870</td><td> Ser Val</td><td> Met</td><td></td>

<td> ACT TAC TTA</td><td> GAT</td><td> GAA</td><td> AAT ACA GCA AAA</td><td> GAA GTG ACC AAA</td><td> CAA TTA</td><td> AAT</td><td> 3168</td>

<td> Thr Tyr Leu 1875</td><td> Asp</td><td> Glu</td><td> Asn Thr Ala Lys 1880</td><td> Glu Val Thr Lys 1885</td><td> Gin Leu</td><td> Asn 1890</td><td></td>

<td> GAT ACC ACT</td><td> GGG</td><td> AAA</td><td> TTT AAA GAT GTA</td><td> AGT CAT TTA TAT</td><td> GAT GTA</td><td> AAA</td><td> 3216</td>

<td> Asp Thr Thr</td><td colspan="3"> Gly Lys Phe Lys Asp Val 1895</td><td colspan="2"> Ser His Leu Tyr Asp Val 1900 1905</td><td> Lys</td><td></td>

<td> CTG ACT CCA</td><td> AAA</td><td> ATG</td><td> AAT GTT ACA ATC</td><td> AAA TTG TCT ATA</td><td> CTT TAT</td><td> GAT</td><td> 3264</td>

<td> Leu Thr Pro</td><td colspan="2"> Lys Met</td><td> Asn Val Thr He</td><td> Lys Leu Ser He</td><td colspan="2"> Leu Tyr Asp</td><td></td>

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PCT/EP95/03826

- 155 1910 1915 1920

<td colspan="8"> AAT GCT GAG TCT AAT GAT AAC TCA ATT GGT AAA TGG ACA AAC ACA AAT</td><td rowspan="3"> 3312</td>

<td rowspan="2"> Asn</td><td colspan="2"> Ala Glu Ser Asn Asp Asn Ser He</td><td rowspan="2"> Gly Lys</td><td rowspan="2"> Trp</td><td rowspan="2"> Thr Asn 1935</td><td rowspan="2"> Thr</td><td rowspan="2"> Asn</td>

<td> 1925</td><td> 1930</td>

<td> ATT</td><td> GTT TCA GGT GGA AAT</td><td> AAC GGA AAA</td><td> AAA CAA</td><td> TAT</td><td> TCT TCT</td><td> AAT</td><td> AAT</td><td> 3360</td>

<td> He</td><td> Val Ser Gly Gly Asn 1940</td><td> Asn Gly Lys 1945</td><td> Lys Gin</td><td colspan="2"> Tyr Ser Ser 1950</td><td> Asn</td><td> Asn</td><td></td>

<td> CCG</td><td> GAT GCT AAT TTG ACA</td><td> TTA AAT ACA</td><td> GAT GCT</td><td> CAA</td><td> GAA AAA</td><td> TTA</td><td> AAT</td><td> 3408</td>

<td colspan="3"> Pro Asp Ala Asn Leu Thr Leu Asn Thr 1955 1960</td><td colspan="2"> Asp Ala Gin 1965</td><td> Glu Lys</td><td> Leu</td><td> Asn 1970</td><td></td>

<td> AAA</td><td> AAT CGT GAC TAT TAT</td><td> ATA AGT TTA</td><td> TAT ATG·</td><td> AAG</td><td> TCA GAA</td><td> AAA</td><td> AAC</td><td> 3456</td>

<td> Lys</td><td> Asn Arg Asp Tyr Tyr 1975</td><td> He Ser Leu</td><td> Tyr Met 1980</td><td> Lys</td><td> Ser Glu</td><td colspan="2"> Lys Asn 1985</td><td></td>

<td> ACA</td><td> CAA TGT GAG ATT ACT</td><td> ATA GAT GGG</td><td> GAG ATT</td><td> TAT</td><td> CCG ATC</td><td> ACT</td><td> ACA</td><td> 3504</td>

<td> Thr</td><td> Gin Cys Glu He Thr 1990</td><td colspan="2"> He Asp Gly Glu He 1995</td><td> Tyr</td><td colspan="2"> Pro He Thr 2000</td><td> Thr</td><td></td>

<td> AAA</td><td> ACA GTG AAT GTG AAT</td><td> AAA GAC AAT</td><td> TAC AAA</td><td> AGA</td><td> TTA GAT</td><td> ATT</td><td> ATA</td><td> 3552</td>

<td> Lys</td><td> Thr Val Asn Val Asn 2005</td><td> Lys Asp Asn 2010</td><td> Tyr Lys</td><td> Arg</td><td> Leu Asp 2015</td><td> He</td><td> He</td><td></td>

<td> GCT</td><td> CAT AAT ATA AAA AGT</td><td> AAT CCA ATT</td><td> TCT TCA</td><td> CTT</td><td> CAT ATT</td><td> AAA</td><td> ACG</td><td> 3600</td>

<td> Ala</td><td> His Asn He Lys Ser 2020</td><td> Asn Pro He 2025</td><td> Ser Ser</td><td colspan="2"> Leu His He 2030</td><td> Lys</td><td> Thr</td><td></td>

<td> AAT</td><td> GAT GAA ATA ACT TTA</td><td> TTT TGG GAT</td><td> GAT ATT</td><td> TCT</td><td> ATA ACA</td><td> GAT</td><td> GTA</td><td> 3648</td>

<td colspan="5"> Asn Asp Glu He Thr Leu Phe Tip Asp Asp He Ser 2035 2040 2045</td><td> He Thr</td><td> Asp</td><td> Val 2050</td><td></td>

<td> GCA</td><td> TCA ATA AAA CCG GAA</td><td> AAT TTA ACA</td><td> GAT TCA</td><td> GAA</td><td> ATT AAA</td><td> CAG</td><td> ATT</td><td> 3696</td>

<td> Ala</td><td> Ser He Lys Pro Glu 2055</td><td> Asn Leu Thr</td><td> Asp Ser 2060</td><td> Glu</td><td> He Lys</td><td colspan="2"> Gin He 2065</td><td></td>

<td> TAT</td><td> AGT AGG TAT GGT ATT</td><td> AAG TTA GAA</td><td> GAT GGA</td><td> ATC</td><td> CTT ATT</td><td colspan="2"> GAT AAA</td><td> 3744</td>

<td> Tyr</td><td> Ser Arg Tyr Gly He 2070</td><td colspan="2"> Lys Leu Glu Asp Gly 2075</td><td> He</td><td colspan="3"> Leu He Asp Lys 2080</td><td></td>

<td> AAA</td><td> GGT GGG ATT CAT TAT</td><td> GGT GAA TTT</td><td> ATT AAT</td><td> GAA</td><td> GCT AGT</td><td> TTT</td><td> AAT</td><td> 3792</td>

<td> Lys</td><td> Gly Gly He His Tyr 2085</td><td> Gly Glu Phe 2090</td><td> He Asn</td><td> Glu</td><td> Ala Ser 2095</td><td> Phe</td><td> Asn</td><td></td>

<td> ATT</td><td> GAA CCA TTG CAA AAT</td><td> TAT GTG ACC</td><td> AAA TAT</td><td> GAA</td><td> GTT ACT</td><td> TAT</td><td> AGT</td><td> 3840</td>

<td> He</td><td> Glu Pro Leu Gin Asn 2100</td><td> Tyr Val Thr 2105</td><td> Lys Tyr</td><td colspan="2"> Glu Val Thr 2110</td><td> Tyr</td><td> Ser</td><td></td>

<td> AGT</td><td> GAG TTA GGA CCA AAC</td><td> GTG AGT GAC</td><td> ACA CTT</td><td> GAA</td><td> AGT GAT</td><td> AAA</td><td> ATT</td><td> 3888</td>

<td colspan="3"> Ser Glu Leu Gly Pro Asn Val Ser Asp 2115 2120</td><td colspan="2"> Thr Leu Glu 2125</td><td> Ser Asp</td><td> Lys</td><td> He 2130</td><td></td>

<td> TAC</td><td> AAG GAT GGG ACA ATT</td><td> AAA TTT GAT</td><td> TTT ACC</td><td> AAA</td><td> TAT AGT</td><td> AAA</td><td> AAT</td><td> 3936</td>

WO 96/10083 ^r 2 1 9 9 0 4 9

PCT/EP95/03826

-156Tyr 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

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

Asn Ala He Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His Arg Tyr

2165 2170 2175

AAT AAA TAG

Asn Lys 2180

3984

4032

4041 (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:

<td colspan="4"> Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gin</td>

<td> 1</td><td> 5</td><td> 10</td><td> 15</td>

<td> Val Val Thr</td><td> Lys Thr 20</td><td> Val Leu Leu Ser Thr Val 25</td><td> Phe Ser He Ser Leu 30</td>

<td> Leu Asn Asn 35</td><td> Glu Val</td><td> He Lys Ala Glu Gin Leu 40</td><td> Asn He Asn Ser Gin 45</td>

<td> Ser Lys Tyr 50</td><td> Thr Asn</td><td> Leu Gin Asn Leu Lys He 55</td><td> Thr Asp Lys Val Glu 60</td>

<td> Asp Phe Lys 65</td><td colspan="2"> Glu Asp Lys Glu Lys Ala Lys Glu 70 75</td><td> Trp Gly Lys Glu Lys 80</td>

<td> Glu Lys Glu</td><td> Trp Lys 85</td><td> Leu Thr Ala Thr Glu Lys 90</td><td> Gly Lys Met Asn Asn 95</td>

<td> Phe Leu Asp</td><td> Asn Lys 100</td><td> Asn Asp He Lys Thr Asn 105</td><td> Tyr Lys Glu He Thr 110</td>

<td> Phe Ser Met 115</td><td> Ala Gly</td><td> Ser Phe Glu Asp Glu He 120</td><td> Lys Asp Leu Lys Glu 125</td>

<td> He Asp Lys 130</td><td> Met Phe</td><td> Asp Lys Thr Asn Leu Ser 135</td><td> Asn Ser He He Thr 140</td>

<td> Tyr Lys Asn</td><td> Val Glu</td><td> Pro Thr Thr He Gly Phe</td><td> Asn Lys Ser Leu Thr</td>

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<td> 145</td><td> 150</td><td> 155</td><td> 160</td>

<td> Glu Gly Asn Thr</td><td> Ile Asn 165</td><td> Ser Asp Ala Met Ala Gin Phe Lys 170</td><td> Glu Gin 175</td>

<td colspan="2"> Phe Leu Asp Arg Asp Ile 180</td><td> Lys Phe Asp Ser Tyr Leu Asp Thr 185 190</td><td> His Leu</td>

<td colspan="2"> Thr Ala Gin Gin Val Ser 195</td><td> Ser Lys Glu Arg Val Ile Leu Lys 200 205</td><td> Val Thr</td>

<td colspan="2"> Val Pro Ser Gly Lys Gly 210</td><td> Ser Thr Thr Pro Thr Lys Ala Gly 215 220</td><td> Val Ile</td>

<td> Leu Asn Asn Ser 225</td><td> Glu Tyr 230</td><td> Lys Met Leu Ile Asp Asn Gly Tyr 235</td><td> Met Val 240</td>

<td> His Val Asp Lys</td><td> Val Ser 245</td><td> Lys Val Val Lys Lys Gly Val Glu 250</td><td> Cys Leu 255</td>

<td> Gin Ile Glu Gly 260</td><td> Thr Leu</td><td> Lys Lys Ser Leu Asp Phe Lys Asn 265 270</td><td> Asp Ile</td>

<td> Asn Ala Glu Ala 275</td><td> His Ser</td><td> Trp Gly Met Lys Asn Tyr Glu Glu 280 285</td><td> Trp Ala</td>

<td> Lys Asp Leu Thr 290</td><td> Asp Ser</td><td> Gin Arg Glu Ala Leu Asp Gly Tyr 295 300</td><td> Ala Arg</td>

<td> Gin Asp Tyr Lys 305</td><td> Glu Ile 310</td><td colspan="2"> Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 315 320</td>

<td> Gly Asn Glu Lys</td><td> Leu Asp 325</td><td colspan="2"> Ala Gin Ile Lys Asn Ile Ser Asp Ala Leu 330 335</td>

<td> Gly Lys Lys Pro 340</td><td> Ile Pro</td><td colspan="2"> Glu Asn Ile Thr Val Tyr Arg Trp Cys Gly 345 350</td>

<td> Met Pro Glu Phe 355</td><td> Gly Tyr</td><td> Gin Ile Ser Asp Pro Leu Pro Ser 360 365</td><td> Leu Lys</td>

<td> Asp Phe Glu Glu 370</td><td> Gin Phe</td><td> Leu Asn Thr Ile Lys Glu Asp Lys 375 380</td><td> Gly Tyr</td>

<td> Met Ser Thr Ser 385</td><td> Leu Ser 390</td><td> Ser Glu Arg Leu Ala Ala Phe Gly 395</td><td> Ser Arg 400</td>

<td> Lys Ile Ile Leu</td><td> Arg Leu 405</td><td> Gin Val Pro Lys Gly Ser Thr Gly 410</td><td> Ala Tyr 415</td>

<td> Leu Ser Ala Ile 420</td><td> Gly Gly</td><td> Phe Ala Ser Glu Lys Glu Ile Leu 425 430</td><td> Leu Asp</td>

<td> Lys Asp Ser Lys 435</td><td> Tyr His</td><td> Ile Asp Lys Val Thr Glu Val Ile 440 445</td><td> Ile Lys</td>

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<td rowspan="2"> Gly Val Lys Arg Tyr 450</td><td rowspan="2"> Val</td><td colspan="2"> Val Asp Ala Thr Leu Leu Thr Asn Met Lys</td>

<td> 455</td><td> 460</td>

<td> Asn Met Lys Lys Lys 465</td><td> Leu 470</td><td> Ala Ser Val Val</td><td> Thr Cys Thr Leu Leu Ala 475 480</td>

<td> Pro Met Phe Leu Asn 485</td><td> Gly</td><td> Asn Val Asn Ala 490</td><td> Val Tyr Ala Asp Ser Lys 495</td>

<td> Thr Asn Gin He Ser 500</td><td> Thr</td><td> Thr Gin Lys Asn 505</td><td> Gin Gin Lys Glu Met Asp 510</td>

<td> Arg Lys Gly Leu Leu 515</td><td colspan="2"> Gly Tyr Tyr Phe Lys 520</td><td> Gly Lys Asp Phe Ser Asn 525</td>

<td> Leu Thr Met Phe Ala 530</td><td> Pro</td><td> Thr Arg Asp Ser 535</td><td> Thr Leu He Tyr Asp Gin 540</td>

<td> Gin Thr Ala Asn Lys 545</td><td> Leu 550</td><td> Leu Asp Lys Lys</td><td> Gin Gin Glu Tyr Gin Ser 555 560</td>

<td> He Arg Trp He Gly 565</td><td> Leu</td><td> He Gin Ser Lys 570</td><td> Glu Thr Gly Asp Phe Thr 575</td>

<td> Phe Asn Leu Ser Glu 580</td><td> Asp</td><td> Glu Gin Ala He 585</td><td> He Glu He Asn Gly Lys 590</td>

<td> He He Ser Asn Lys 595</td><td colspan="2"> Gly Lys Glu Lys Gin 600</td><td> Val Val His Leu Glu Lys 605</td>

<td> Gly Lys Leu Val Pro 610</td><td> He</td><td> Lys He Glu Tyr 615</td><td> Gin Ser Asp Thr Lys Phe 620</td>

<td> Asn He Asp Ser Lys 625</td><td> Thr 630</td><td> Phe Lys Glu Leu</td><td> Lys Leu Phe Lys He Asp 635 640</td>

<td> Ser Gin Asn Gin Pro 645</td><td> Gin</td><td> Gin Val Gin Gin 650</td><td> Asp Glu Leu Arg Asn Pro 655</td>

<td> Glu Phe Asn Lys Lys 660</td><td> Glu</td><td> Ser Gin Glu Phe 665</td><td> Leu Ala Lys Pro Ser Lys 670</td>

<td> He Asn Leu Phe Thr 675</td><td> Gin</td><td> Lys Met Lys Arg 680</td><td> Glu He Asp Glu Asp Thr 685</td>

<td> Asp Thr Asp Gly Asp 690</td><td> Ser</td><td> He Pro Asp Leu 695</td><td> Trp Glu Glu Asn Gly Tyr 700</td>

<td> Thr He Gin Asn Arg 705</td><td> He 710</td><td colspan="2"> Ala Val Lys Trp Asp Asp Ser Leu Ala Ser 715 720</td>

<td> Lys Gly Tyr Thr Lys 725</td><td> Phe</td><td> Val Ser Asn Pro 730</td><td> Leu Glu Ser His Thr Val 735</td>

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<td colspan="2"> Gly Asp Pro Tyr</td><td rowspan="2"> Thr Asp</td><td colspan="2"> Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu</td>

<td></td><td> 740</td><td> 745</td><td> 750</td>

<td> Ser Asn Ala 755</td><td> Lys</td><td> Glu Thr</td><td> Phe Asn Pro 760</td><td> Leu Val Ala Ala Phe Pro Ser 765</td>

<td> Val Asn Val 770</td><td> Ser</td><td> Met Glu</td><td> Lys Val He 775</td><td> Leu Ser Pro Asn Glu Asn Leu 780</td>

<td> Ser Asn Ser 785</td><td> Val</td><td> Glu Ser 790</td><td> His Ser Ser</td><td> Thr Asn Trp Ser Tyr Thr Asn 795 800</td>

<td> Thr Glu Gly</td><td> Ala</td><td> Ser Val 805</td><td> Glu Ala Gly</td><td> He Gly Pro Lys Gly He Ser 810 815</td>

<td> Phe Gly Val</td><td> Ser 820</td><td> Val Asn</td><td> Tyr Gin His 825</td><td> Ser Glu Thr Val Ala Gin Glu 830</td>

<td> Trp Gly Thr 835</td><td> Ser</td><td> Thr Gly</td><td> Asn Thr Ser 840</td><td> Gin Phe Asn Thr Ala Ser Ala 845</td>

<td> Gly Tyr Leu 850</td><td> Asn</td><td> Ala Asn</td><td> Val Arg Tyr 855</td><td> Asn Asn Val Gly Thr Gly Ala 860</td>

<td> He Tyr Asp 865</td><td> Val</td><td> Lys Pro 870</td><td> Thr Thr Ser</td><td> Phe Val Leu Asn Asn Asp Thr 875 880</td>

<td> He Ala Thr</td><td> He</td><td> Thr Ala 885</td><td> Lys Ser Asn</td><td> Ser Thr Ala Leu Asn He Ser 890 895</td>

<td> Pro Gly Glu</td><td> Ser 900</td><td> Tyr Pro</td><td> Lys Lys Gly 905</td><td> Gin Asn Gly He Ala He Thr 910</td>

<td colspan="2"> Ser Met Asp Asp 915</td><td> Phe Asn</td><td> Ser His Pro 920</td><td> He Thr Leu Asn Lys Lys Gin 925</td>

<td> Val Asp Asn 930</td><td> Leu</td><td> Leu Asn</td><td> Asn Lys Pro 935</td><td> Met Met Leu Glu Thr Asn Gin 940</td>

<td> Thr Asp Gly 945</td><td> Val</td><td> Tyr Lys 950</td><td> He Lys Asp</td><td> Thr His Gly Asn He Val Thr 955 960</td>

<td> Gly Gly Glu</td><td> Trp</td><td> Asn Gly 965</td><td> Val He Gin</td><td> Gin He Lys Ala Lys Thr Ala 970 975</td>

<td> Ser He He</td><td> Val 980</td><td> Asp Asp</td><td> Gly Glu Arg 985</td><td> Val Ala Glu Lys Arg Val Ala 990</td>

<td colspan="2"> Ala Lys Asp Tyr 995</td><td> Glu Asn</td><td> Pro Glu Asp 1000</td><td> Lys Thr Pro Ser Leu Thr Leu 1005</td>

<td> Lys Asp Ala 1010</td><td> Leu</td><td> Lys Leu</td><td> Ser Tyr Pro 1015</td><td> Asp Glu He Lys Glu He Glu 1020</td>

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

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1025 1030 1035 1040

<td rowspan="2"> Thr</td><td rowspan="2"> Tyr Leu</td><td colspan="3"> Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin Leu Asn</td>

<td> 1045</td><td> 1050</td><td> 1055</td>

<td> Asp</td><td> Thr Thr</td><td> Gly Lys Phe</td><td> Lys Asp Val Ser His Leu</td><td> Tyr Asp Val Lys</td>

<td></td><td></td><td> 1060</td><td> 1065</td><td> 1070</td>

<td> Leu</td><td> Thr Pro</td><td> Lys Met Asn</td><td> Val Thr He Lys Leu Ser</td><td> He Leu Tyr Asp</td>

<td></td><td colspan="2"> 1075</td><td> 1080</td><td> 1085</td>

<td> Asn</td><td> Ala Glu</td><td> Ser Asn Asp</td><td> Asn Ser He Gly Lys Trp</td><td> Thr Asn Thr Asn</td>

<td></td><td> 1090</td><td></td><td colspan="2"> 1095 1100</td>

<td> He</td><td> Val Ser</td><td> Gly Gly Asn</td><td> Asn Gly Lys Lys Gin Tyr</td><td> Ser Ser Asn Asn</td>

<td colspan="2"> 1105</td><td colspan="2"> 1110 1115</td><td> 1120</td>

<td> Pro</td><td> Asp Ala</td><td> Asn Leu Thr</td><td> Leu Asn Thr Asp Ala Gin</td><td> Glu Lys Leu Asn</td>

<td></td><td></td><td> 1125</td><td> 1130</td><td> 1135</td>

<td> Lys</td><td colspan="2"> Asn Arg Asp Tyr Tyr</td><td> He Ser Leu Tyr Met Lys</td><td> Ser Glu Lys Asn</td>

<td></td><td></td><td> 1140</td><td> 1145</td><td> 1150</td>

<td> Thr</td><td colspan="2"> Gin Cys Glu He Thr</td><td> He Asp Gly Glu He Tyr</td><td> Pro He Thr Thr</td>

<td></td><td colspan="2"> 1155</td><td> 1160</td><td> 1165</td>

<td> Lys</td><td> Thr Val</td><td> Asn Val Asn</td><td> Lys Asp Asn Tyr Lys Arg</td><td> Leu Asp He He</td>

<td></td><td> 1170</td><td></td><td colspan="2"> 1175 1180</td>

<td> Ala</td><td> His Asn</td><td> He Lys Ser</td><td> Asn Pro He Ser Ser Leu</td><td> His He Lys Thr</td>

<td colspan="2"> 1185</td><td colspan="2"> 1190 1195</td><td> 1200</td>

<td> Asn</td><td> Asp Glu</td><td> He Thr Leu</td><td> Phe Trp Asp Asp He Ser</td><td> He Thr Asp Val</td>

<td></td><td></td><td> 1205</td><td> 1210</td><td> 1215</td>

<td> Ala</td><td> Ser He</td><td> Lys Pro Glu</td><td> Asn Leu Thr Asp Ser Glu</td><td> He Lys Gin He</td>

<td></td><td></td><td> 1220</td><td> 1225</td><td> 1230</td>

<td> Tyr</td><td colspan="2"> Ser Arg Tyr Gly He</td><td> Lys Leu Glu Asp Gly He</td><td> Leu He Asp Lys</td>

<td></td><td colspan="2"> 1235</td><td> 1240</td><td> 1245</td>

<td> Lys</td><td> Gly Gly</td><td> He His Tyr</td><td> Gly Glu Phe He Asn Glu</td><td> Ala Ser Phe Asn</td>

<td></td><td> 1250</td><td></td><td colspan="2"> 1255 1260</td>

<td> He</td><td> Glu Pro</td><td> Leu Gin Asn</td><td> Tyr Val Thr Lys Tyr Glu</td><td> Val Thr Tyr Ser</td>

<td colspan="2"> 1265</td><td colspan="2"> 1270 1275</td><td> 1280</td>

<td> Ser</td><td> Glu Leu</td><td> Gly Pro Asn</td><td> Val Ser Asp Thr Leu Glu</td><td> Ser Asp Lys He</td>

<td></td><td></td><td> 1285</td><td> 1290</td><td> 1295</td>

<td> Tyr</td><td colspan="3"> Lys Asp Gly Thr He Lys Phe Asp Phe Thr Lys</td><td> Tyr Ser Lys Asn</td>

<td></td><td></td><td> 1300</td><td> 1305</td><td> 1310</td>

Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe Lys He 1315 1320 1325

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- 161 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

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ATGAAGAACT ACGAGGAGTG GGCCAAGGAC CTGACCGACA GCCAGCGCGA GGCCCTGGAC

GGCTACGCCC GCCAGGACTA CAAGGAGATC AACAACTACC TGCGCAACCA GGGCGGCAGC

GGCAACGAGA AGCTGGACGC CCAGATCAAG AACATCAGCG ACGCCCTGGG CAAGAAGCCC

ATCCCCGAGA ACATCACCGT GTACCGCTGG TGCGGCATGC CCGAGTTCGG CTACCAGATC

AGCGACCCCC TGCCCAGCCT GAAGGACTTC GAGGAGCAGT TCCTGAACAC CATCAAGGAG

GACAAGGGCT ACATGAGCAC CAGCCTGAGC AGCGAGCGCC TGGCCGCCTT CGGCAGCCGC

AAGATCATCC TGCGCCTGCA GGTGCCCAAG GGCAGCACCG GCGCCTACCT GAGCGCCATC

GGCGGCTTCG CCAGCGAGAA GGAGATCCTG CTGGACAAGG ACAGCAAGTA CCACATCGAC

AAGGTGACCG AGGTGATCAT CAAGGGCGTG AAGCGCTACG TGGTGGACGC CACCCTGCTG

ACCAACTAGA TCTGAGCTC (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

900

960

1020

1080

1140

1200

1260

1320

1380

1399 (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 lie Phe Leu Phe Leu Leu Ser Gly Ala Ala Gly Val 15 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

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