BACILLUS THURINGIENSIS ISOLATES ACTIVE AGAINST LEPIDOPTERAN PESTS

1. FIELD OF THE INVENTION

The invention is related to a novel biologically pure Bacillus thuringiens±s (B. t . ) isolate which solely produces a CryIA(a)-like crystal delta- endotoxin having a molecular weight of about 130,000 daltoϊis and activity against lepidopteran pests as well as a spore, crystal delta-endotoxin and/or mutant thereof. The invention also relates to insecticidal compositions obtainable therefrom. The invention further relates to methods of using the insecticidal compositions to control an insect pest(s) from the order Lepidoptera.

2. BACKGROUND OF THE INVENTION

Every year, significant portions of the world's commercially important agricultural crops, including foods, textiles, and various domestic plants are lost to pest infestation, resulting in losses in the millions of dollars. Various strategies have been used in attempting to control such pests. One strategy is the use of chemical insecticides with a broad range of activity. However, there are a number of disadvantages to using such chemical insecticides. Specifically, because of their broad spectrum of activity, these insecticides may destroy non- target organisms such as beneficial insects and parasites of destructive pests. Additionally, these chemical insecticides are frequently toxic to animals and humans, and targeted pests frequently develop resistance when repeatedly exposed to such substances. Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations

of crops. Biopesticides comprise a bacterium which produces a toxin, a substance toxic to the pest. Biopesticides are generally less harmful to non-target organisms and the environment as a whole than chemical pesticides. The most widely used biopesticide is Bacillus thurlngiensis (B. t. ) . B. t. is a widely distributed, rod shaped, aerobic and spore forming microorganism.

During its sporulation cycle, B.t. produces an alkali soluble protein(s) in crystal form known as a crystal delta-endotoxin(s) having a molecular weight rangihg from 27-140 kd, which upon ingestion kills insect larvae. Toxic activity may reside in one or more of such crystal proteins in a given B. t. strain. Most delta- endotoxins are protoxins that are proteolytically converted into smaller toxic (truncated) polypeptides in the target insect midgut (Hδfte and Whiteley, 1989, Microbiol. Rev. 53:242-255). The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been.divided into six classes and several subclasses based on structural similarities and pesticidal specificity. The major classes are Lepidoptera-specific (cryj); Lepidoptera-and Diptera-specific ( cryJJ) ; Coleoptera-specific ( crylll) ; Diptera-specific (cryTF) (Hδfte and Whiteley, 1989, Microbiol. Rev. 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI by Feitelson et al., 1992, Bio/Technology 10:271-275).

Six cryJ genes have been identified: cryΙA(a) , cryΙA(b) , cryΙA(c) , crylB, crylC, and crylD (Hδfte and Whiteley, 1989, Microbiol. Rev. 53:242-255). Since cryΙA(a) , cryΙA(b) , and cryΙA(c) show more than 80% amino acid identity, they are considered to be part of the crylA group.

A number of B.t. strains have been isolated that have been found to be active against insect pests of the order Lepidoptera. B.t. subsp. kurstaki HD-1 produces a bipyramidal and cuboidal crystal proteins in each cell during sporulation (Lϋthy et al., in Microbial and Viral Pesticides, ed. E. Kurstak, Marcel Dekker, New York, 1982, pp.35-74); the bipyramidal crystal was found to be encoded by various crylA genes (Aronson et al., 1986, Microbiol. Rev. 50:1-50). B.t. subsp. kurstaki HD-73 contains the cryJA (c) gene for its crystal delta-endotoxin (Adang et al., 1985, Gene 36:289-300). B.t. subsp. dendrolimus HD-7 and HD-37 contain a CrylA and a Cryll protein; B.t. subsp. sotto contains an alkaline soluble protein that differs from the holotype CrylA(a) protein by 24 amino acids; B. t . subsp. subtoxicus HD-10 contains CrylA and CrylB proteins; B.t. subsp. tolworthi HD-121 contains CrylA and Cryll proteins; B.t. subsp. entomocidus HD-110, 4448 contains CrylA, CrylB, and CrylB proteins; and B.t. subsp. aizawai HD-68 contains CrylA proteins (Hδfte and Whiteley, 1989, Microbiol. Reviews 53:242-255). Bt. subsp. aizawai HD-11 contains a Cry IA protein as well as a P crystal. Padua,

1990, Microbiol. Lett. 66:257-262, discloses the isolation of two mutants containing two crystal delta-endotoxins, a 144 kD protein having activity against a lepidopteran pest and a 66 kD protein having activity against mosquitoes.

Payne, U.S. Patent No. 4,990,332, issued February 5, 1993, discloses an isolate of B.t. PS85A1 and a mutant of the isolate, PS85A1 which both have activity against Plutella xylostella, a Lepidopteran pest and produce alkali soluble proteins having a molecular weight of 130,000 and 60,000 daltons. Payne, U.S. Patent No. 5,045,469, issued September 3, 1991 discloses a B.t. isolate designated PS81F which also produces alkali soluble proteins having a molecular weight of 130,000 and 60,000 daltons and has activity against Spodoptera exigua and T. ni ; the toxin

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RECTIFIED SHEET (RULE 91) ISA/EP

Spodoptera exigua and T. ni; the toxin gene from PS81F appears to have little ho ology to the toxin gene from B. t. subsp. kurstaki HD-1. Payne, U.S. Patent No. 5,206,166, filed June 25, 1992, issued April 27, 1993, discloses B.t. isolates PS81A2 and PS81RR1 which produce 133,601 and 133,367 dalton alkali-soluble proteins; both have activity against Trichoplusia ni, Spodoptera exigua and Plutella xylostella and are different from B. t. subsp. kurstaki HD-1 and other B. t . isolates. Payne, U.S. Patent No. 5,169,629, filed November 1, 1988, issued December 2, 1992, 'discloses B.t. isolate PS81GG active against lepidopteran pests and which produces a bipyramidal (130,000 daltons) and a cuboidal (60,000 daltons) crystal delta-endotoxin. Payne, U.S. Patent No. 5,188,960, filed December 14, 1989, issued February 23, 1993, discloses

B. t . PS81I which produces a 130,000 dalton alkali soluble protein having a flagellar serotype of 7, aizawai which can be distinguished from HD-1 and is active against Spodoptera exigua, Plutella xylostella, and Choristoneura occidentalis. Bernier et al., U.S. Patent No. 5,061,489 and WO 90/03434 discloses strain A20 producing a delta- endotoxin encoded by at least three genes: 6.6-, 5.3-, and 4.5-type genes ( CryΙA(a)-like, cryΙA(b) , and cryΙA (c) ) . Bradfish et al., U.S. Patent No. 5,208,017, discloses B. t . isolates PS86A1 and PS86Q3 which respectively produce alkali-soluble proteins having a molecular weight of 58,000 and 45,000 daltons and 155,000, 135,000, 98,000, 62,000, and 58,000 daltons respectively and which have activity against lepidopteran and coleopteran pests. It is advantageous to isolate new strains of

Bacillus thuringiensis so that there exists a wider spectrum of biopesticides for any given insect pest.

3. SUMMARY OF THE INVENTION

The invention is related to a novel biologically pure Bacillus thuringiensis strain(s) or a spore(s) or mutant(s) thereof which strain or mutant in contrast to B.t. strains disclosed in the prior art, solely produces a CryΙA(a)-like crystal delta-endotoxin having activity against an insect pest of the order Lepidoptera and a molecular weight of about 130,000 daltons. As defined herein, a "biologically pure" B.t. strain is a strain essentiallly free of other microbial contaminants. In a specific embodiment of the invention, the thuringiensis strain of the present invention is EMCC-0073 and EMCC-0074 having the identifying characteristics of NRRL B-21014 and NRRL B-21015 respectively. As defined herein, a CryΙA(a)-like crystal delta-endotoxin is a protein in crystalline form substantially homologous to a CrylA(a) protein which is immunologically reactive with antibodies to the CryiA(a) protein and has essentially the same insecticidal activity as a CrylA(a) protein. Preferably, the CryΙA(a)-like protein has at least 90% homology to the CrylA(a) protein; more preferably at least 95% homology and most preferably at least 99% homology.

As detailed above, the prior art strains produce crystal delta-endotoxins encoded not only by the cryΙA (a) gene, but by other genes as well. The CryΙA(a)- like crystal delta-endotoxin is encoded by at least one copy of a cryΙA (a)-like gene. As defined herein, a " cryIA ( a )-like gene" is a DNA sequence encoding a CryΙA(a) -like protein defined above, in a specific embodiment, the crylA.a. ¹ -like gene has at least 90% homology to the crylA (a) gene, preferably at least 95% homolgy to the cryΙA (a) gene and most preferably at least 99% homolgy to the cryΙA (a) gene.

^■ 5-

RECTIFIED SHEET (RULE 91) ISA/EP

The invention is also related to a substantially pure crystal delta-endotoxin. As definded herein, a "substantially pure" crystal delta-endotoxin is substantially free (>95%) of other proteins and/or other contaminants. As will be detailed in Section 5, infra, the crystal delta-endotoxin of the present invention is obtainable from the strains of the present invention.

The novel Bacillus thuringiensis strains, spore, mutant or crystal delta-endotoxins may within the scope of this invention be formulated into an insecticidal composition. In one embodiment, the strain, spores, mutant or crystal delta-endotoxin may be combined with an insecticidal carrier. The insecticidal composition may be used to control an insect pest from the order Lepidoptera, particularly Spodoptera exigua, Heliothis zea, and

Heliothis virescens in a method comprising exposing the pest to an insect-controlling effective amount of such an insecticidal composition.

4. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results of PCR analysis of Bacillus thuringiensis strains for crylA genes by agarose gel electrophoresis. Lanes 1 and 14 show molecular weight markers (1 kb ladder, Bethesda Research Laboratories). Lanes 2-4 show analysis of strain EMCC-0073 with cryΙA(a) , cryΙA(b) , and cryΙA(c) oligonucleotide primers respectively; lanes 5-7 shows analysis of strain EMCC-0074 with cryΙA(a) , cryΙA(b) , and cryΙA(c) oligonucleotide primers respectively; lanes 8-10 shows analysis of strain EMCC-0086 with cryΙA(a) , cryΙA(b) , and cryΙA(c) oligonucleotide primers respectively; and lanes 11-13 shows analysis of a Bacillus thuringiensis subsp. tenebrionis strain containing only cryJJJA gene with cryΙA(a) , cryΙA(b) , and cryΙA(c) oligonucleotide primers respectively. EMCC-0086 is a Bacillus thuringiensis

subsp. kurstaki HD-1 strain containing all three crylA genes.

5. DETAILED DESCRIPTION OF THE INVENTION The spores and crystal delta-endotoxin of the present invention are obtainable from the strains of the present invention. The strains of the present invention may be cultured using media and fermentation techniques known in the art (see, for example, Rogoff et al., 1969, J. Invertebrate Path. 14:122-129; Dulmage et al., 1971, J. Invertebrate Path. 18:353-358; Dulmage et al., in Microbial Control of Pests and Plant Diseases, H.D. Burges, ed.. Academic Press, N.Y., 1980). Upon completion of the fermentation cycle, the bacteria can be harvested by separating B. t. spores and crystal delta- endotoxin from the fermentation broth by means well known in the art, e.g. centrifugation. The spores and crystal proteins are contained in the pellet.

Purification of the crystal delta-endotoxin can be carried out by various procedures known in the art, including but not limited to chromatography (e.g. ion exchange, affinity, hydrophobic and size exclusion), further centrifugation, electrophoretic procedures, differential solubility, or any other standard technique for the purification of proteins.

The invention is also directed to a mutant B. t. strain which produces a larger amount of and/or a larger crystal of CryIA(a)-like crystal delta-endotoxin than the parental strain. A "parental strain" as defined herein is the original Bacillus strain before mutagenesis. In a specific embodiment, the mutant contains more than one copy of the Cry IA(a)-like gene.

To obtain such mutants, the parental strain may, for example, be treated with a mutagen by chemical means such as N-methyl-N'-nitro-N-nitrosoguanidine or

ethyl methanesulfonate, gamma-irradiation, X-ray or UV- irradiation. Specifically, in one method of mutating Bacillus strains and selecting such mutants the parental strain is: i) treated with a mutagen; ii) the thus treated mutants are grown in a medium suitable for the selection of a mutant strain; iii) selection of a mutant strain.

According to a preferred embodiment of this method, the selected colonies are grown in a normal production medium, and a final selection for strains capable of increased CryIA(a)-like protein production is performed.

Alternatively, the mutant may be obtained used recombinant DNA methods known in the art. For example, a DNA sequence containing two or more copies of the CryΙA(a) - like gene may be inserted into an appropriate expression vector and subsequently introduced into the parental strain using procedures known in the art. The activity of the B. t. strains of the present invention or a spore(s) , mutant(s) or crystal delta- endotoxin thereof against various insect pests may be assayed using procedures known in the art, such as an artificial insect diet incorporation assay, artificial diet overlay, leaf painting, leaf dip, and foliar spray. Specific examples of such assays are given in Section 6, infra .

5.1. COMPOSITIONS The strain, spore(s), crystal delta-endotoxin, or mutant(s) of the present invention described supra can be formulated with an acceptable carrier into an insecticidal composition^) that is for example, a suspension, a solution, an emulsion, a dusting powder, a

dispersible granule, a wettable powder, an emulsifiable concentrate, an aerosol or impregnated granule.

Such compositions disclosed above may be obtained by the addition of a surface active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a U.V. protectant, a buffer, a flow agent, or other component to facilitate product handling and application for particular target pests. Suitable surface-active agents include but are not limited to anionic compounds such as a carboxylate, for example, a metal carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate or sodium cetyl sulphate; ethoxylated fatty alcohol sulphates ethoxylated alkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkyl aryl sulphonates such as alkyl-benzene sulphonates or lower alkylnaphthalene sulphonates, e.g. butyl-naphthalene sulphonate; salts of sulphonated naphthalene-formaldehyde condensates; salts of sulphonated phenol-formaldehyde condensates; or more complex sulphonates such as the amide sulphonates, e.g. the sulphonated condensation product of oleic acid and N- methyl taurine or the dialkyl sulphosuccinates, e.g. the sodium sulphonate or dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl- substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g. sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-

diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate; an oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide- linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllόsilicates, carbonates, sulfates, phosphates or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form for direct application or as a concentrate or primary composition which requires dilution with a suitable quantity of water or other diluent before application. The insecticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 1 to 98% of a solid or liquid inert carrier, and 0 to 50%, preferably 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product, preferably about 0.01 lb-5.0 lb per acre when in dry form and at about 0.01 pts-10 pts per acre when in liquid form.

In a further embodiment, the strain, spore, crystal delta-endotoxin or mutant of the present invention can be treated prior to formulation to prolong the pesticidal activity when applied to the environment of a target pest as long as the pretreatment is not deleterious to the crystal delta-endotoxin. Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties of the composition(s) . Examples of chemical reagents include but

are not limited to halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as isopropranol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason, Animal Tissue Techniques, W.H. Freeman and Co., 1967).

The compositions of the invention can be applied directly to the plant by, for example, spraying or dusting at the time when the pest has begun to appear on the plant or before the appearance of pests as a protective measure. Plants to be protected within the scope of the present invention include but are not limited to cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beets (sugar beet and fodder beet), drupes, pomes and soft ruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, and blackberries), leguminous plants (alfalfa, beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons), fibre plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other brassicae, carrots, onions, tomatoes, potatoes, paprika), lauraceae (avocados, 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, turf plants, tobacco, nuts, coffee, sugar cane, tea, vines hops, bananas and natural rubber plants, as well as ornamentals. In both cases, the preferred mode of application is by foliar spraying. It is generally important to obtain good control of pests in the early stages of plant growth as this is the time when the plant can be most severely damaged. The spray or dust can conveniently contain another pesticide if this is

thought necessary. In a preferred embodiment, the composition of the invention is applied directly to the plant.

The compositions of the present invention may be effective against pests including but not limited to pests of the order Lepidoptera, e.g. Achroia grisella, Acleris glover ana, Acleris variana, Adoxophyes or ana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp. , Argyrotaenia sp. , Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp. , Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia funeralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantria cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, ^' Macalla thyrsisalis, Malacosoma sp. , Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp. , Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana,

Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp. , Thaurnstopoea pityocampa, Tineola bisselliella, Trichoplusia ni, Udea rubigalis , Xylomyges curialis, Yponomeuta padella.

The following examples are presented by way of illustration, not by way of limitation.

6. EXAMPLES

6.1. CULTURING OF B.t. STRAINS EMCC-0073 AND EMCC-0074 A subculture of EMCC-0073 and EMCC-0074, maintained on a Nutrient Broth Agar slant is used to inoculate a 250 ml baffle shake flask containing 50 ml of medium with the following composition.

Corn Steep liquor 15 g/1 Maltrin-100 40 g/1

Potato Starch 30 g/1

KH ₂ P0 ₄ 1.77 g/1

K ₂ HP0 4.53 g/1

The pH of the medium is adjusted to 7.0 using 10 N NaOH. After inoculation, shake flasks are incubated at 30°C on a rotary shaker with 250 rpm shaking for 72 hours. The B.t. crystals and spores, obtained in the above fermentation are recovered by centrifugation at 15,000 rpm for 15 minutes using a Sorvall RC-5B centrifuge.

6.2. INSECTICIDAL ACTIVITY OF EMCC-0073 AND EMCC-0074

EMCC-0073 and EMCC-0074 are cultivated in shake flasks as described in Section 6.1., supra. A 1:50

dilution of culture broth was made. 5 ml of such diluted culture broth is transferred into a 50 ml propylene centrifuge tube. 20 ml of artificial insect diet containing antibiotics is added into the centrifuge tube. The mixture is subsequently dispensed into bioassay trays. Three to six eggs each of beet armyworm ( Spodoptera exigua) , corn earworm (Heliothis zea) and tobacco budworm (Heliothis virescens) are applied on the surface of the "diet". Mylar is ironed onto the bioassay trays and the trays were incubated at 28°C without photoperiod. Scoring is carried out at 7 and 11 days.

At the dosage tested, EMCC-0073 and EMCC-0074 stunted Spodoptera exigua and Heliothis zea. After seven days incubation, both Spodoptera exigua and Heliothis zea only grows to less than 25% of the size of the control larvae. At the same dosage, EMCC-0073 and EMCC-0074 kills 50% and 70% respectively, of the testing population of Heliothis virescens. In Table I, the bioactivity of EMCC- 0073 and EMCC-0074 towards Spodoptera exigua and Heliothis zea is expressed in terms of stunt score (SS). The stunt score is determined after incubating the trays for 7 days. In this system, 4=full size larvae (control larvae); 3=3/4 size of control larvae; 2=1/2 size of control larvae; and 1=1/4 size of control larvae. The smaller the number, the higher the B. t. activity. The bioactivity of EMCC-0073 and EMCC-0074 towards Heliothis virescens is determined in terms of % mortality and the live larvae (survivors) were scored by stunt score (SS) for their size.

6.3. CRY GENE PROFILE FOR EMCC-0073 AND EMCC-0074 The cry gene profile for EMCC-0073 and EMCC-

0074 is determined by using the PCR method which is described in the Perkin Elmer Cetus Gene Amp® PCR Reagent Kit literature with AmpliTaq® DNA Polymerase. The double- stranded DNA is heat-denatured and the two oligonucleotides of cryΙA(a) (SEQ ID NO:l and SEQ ID

NO:2), cryΙA(b) (SEQ ID NO:3 and SEQ ID NO:4), or cryΙA(c) (SEQ ID NO:5 and SEQ ID NO:6) are annealed at low temperature and then extended at an intermediate temperature. The results from the PCR analysis are shown in

Figure 1 and indicate that B.t. strains EMCC-0073 and EMCC- 0074 contain the cryΙA(a) , but not the cryΙA(b) nor the cryΙA(c) genes. Therefore, the crystal delta-endotoxin of B.t. strains EMCC-0073 and EMCC-0074 is encoded only by the cryΙA(a) gene.

Oligonucleotide primers for Polymerase Chain Reaction (PCR) amplification of the entire cryJAfa -like gene of EMCC-0073 were designed based on the sequence of the holotype cryΙA(a) gene cloned from Bacillus thuringiensis subsp. kurstaki HD-1 (Schnepf

et al., 1985, J. Biol. Chem. 260:6264-6272). The primers are shown in the Sequence Listing as SEQ ID NO:7 and SEQ ID NO:8. Fragments bearing the cryJA(a -like gene of EMCC-0073 (corresponding to nucleotides 380 to 4205 of the sequence reported by Schnepf et al., 1985, J. Biol. Chem. 260:6264-6272) were cloned from two separate PCRs were cloned in pCR™II (Invitrogen Corporation) or pBCSK+ (Stratagene Cloning Systems) from two separate PCRs. DNA sequencing was performed on the two clones using the Applied Biosystems 373A DNA Sequencer and PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit with synthetic oligonucleotides based on the sequence of the holotype crylA(a) gene. The clones shared two nucleotide differences relative to the holotype cryΙA(a) gene (C to T at nt 756 and C to G at nt 3551, according to the numbering of Schnepf et al . , 1985, J. Biol. Chem. 260:6264-6272), which corresponded to two amino acid changes relative to the holotype CrylAa protoxin (Pro to Leu at residue 77 and Leu to Val at residue 1009). The nucleotide sequence is shown in the Sequence Listing as SEQ ID NO:9 and the amino acid sequence is shown in the Sequence Listing as SEQ ID NO:10.

6.4. INSECTICIDAL ACTIVITY OF PURIFIED SPORES FROM EMCC-0073

The B. t. culture obtained from Section 6.1., supra is transferred into sterile 250 ml centrifuge bottles and centrifuged at 10,000 rpm in a Sorvall RC-5B centrifuge for 30 minutes at 5°C to collect crystals and spores. Pellets are then washed three times with sterile, de-ionized water. The pellets are resuspended into deionized water to 1 g. wet weight per 10 ml followed by sonicating the suspension on ice to disrupt any clumping. Each 10 ml suspension is further diluted to 33.2 ml with deionized water. 10 ml 3M NaCl, 23.4 ml 20% polyethylene

glycol, and 33.4 ml 20% sodium dextran sulfate are all added and mixed well in a separatory funnel with the previously diluted suspension (33.2 ml). An additional 100 ml 20% polyethylene glycol is then added to the separatory funnel and the mixture is shaken vigorously to mix the phases. The phase separation of the mixture is achieved by gravity at room temperature for 30 minutes. The upper phase consists of large quantities of spores which could be removed by pipetting. Purified spores are then bioassayed against

Spodoptera exigua, by using the diet incorporation bioassay described in Section 6.2., supra . The results are shown in Table II. 48 second instar larvae are used for each point. Mortality is recorded on the seventh day post-treatment.

TABLE II

% Mortality EMCC-0073 EMCC-0086 48 27

21 17

28 8

10 7

The spores from EMCC-0073 has significantly higher activity against Spodoptera exigua than spores from a B. t .k. type of reference strain.

7. DEPOSIT OF MICROORGANISMS

The following strains of Bacillus thuringiensis have been deposited in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional

Research Center, 1815 University Street, Peoria, Illinois, 61604, USA.

Strain Accession Number Deposit Date EMCC-0073 NRRL B-21014 November 16, 1992 EMCC-0074 NRRL B-21015 November 16, 1992

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122 and under conditions of the Budapest Treaty. The deposit represents a biologically pure culture of each deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Liu, Chi-Li

(ii) TITLE OF INVENTION: NOVEL BACILLUS THURINGIENSIS ISOLATES ACTIVE AGAINST LEPIDOPTERAN PESTS

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Novo Nordisk of North America, Inc.

(B) STREET: 405 Lexington Avenue

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: USA

(F) ZIP: 10174-6201

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/981,048

(B) FILING DATE: 24-NOV-1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Agris, Cheryl H.

(B) REGISTRATION NUMBER: 34,086

(C) REFERENCE/DOCKET NUMBER: 3674.204-WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-867-0123

(B) TELEFAX: 212-867-0298

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: CTGCTCCAGC TGCTTGGCTC 20

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAATTATACT TGGTTCAGGC CC 22

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCACACCTTA CATTTTAAAG CA 22

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGATTACAAG CGGATACCAA CATCGCG 27

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid (C). STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TGGCACTTTC AAAATAACCA A 21

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCATCGGATA GTATTACTCA ATCCC 25

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGGGATCCTG GGTCAAAAAT TGATATTTAG TAAAATTAG 39

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CCTGTCGACT AGAAAATAAC ATAGTAAAAC GGACATCACT CCG 43

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3826 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CCTGGGTCAA AAATTGATAT TTAGTAAAAT TAGTTGCACT TTGTGCATTT TTTCATAAGA 60

TGAGTCATAT GTTTTAAATT GTAGTAATGA AAAACAGTAT TATATCATAA TGAATTGGTA 120

TCTTAATAAA AGAGATGGAG GTAACTTATG GATAACAATC CGAACATCAA TGAATGCATT 180

CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT 240

GGTTACACCC CAATCGATAT TTCCTTGTCG CTAACGCAAT TTCTTTTGAG TGAATTTGTT 300

CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT TGGTCCCTCT 360

CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA ACCAAAGAAT AGAAGAATTC 420

GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA ATCTTTATCA AATTTACGCA 480

GAATCTTTTA GAGAGTGGGA AGCAGATCCT ACTAATCCAG CATTAAGAGA AGAGATGCGT 540

ATTCAATTCA ATGACATGAA CAGTGCCCTT ACAACCGCTA TTCCTCTTTT GGCAGTTCAA 600

AATTATCAAG TTCCTCTTTT ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 660

TTGAGAGATG TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT 720

CGTTATAATG ATTTAACTAG GCTTATTGGC AACTATACAG ATTATGCTGT GCGCTGGTAC 780

AATACGGGAT TAGAGCGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA 840

TTTAGAAGAG AGCTAACACT TACTGTATTA GATATCGTTG CTCTATTCTC AAATTATGAT 900

AGTCGAAGGT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA TACGAACCCA 960

GTATTAGAAA ATTTTGATGG TAGTTTTCGT GGAATGGCTC AGAGAATAGA ACAGAATATT 1020

AGGCAACCAC ATCTTATGGA TATCCTTAAT AGTATAACCA TTTATACTGA TGTGCATAGA 1080

GGCTTTAATT ATTGGTCAGG GCATCAAATA ACAGCTTCTC CTGTAGGGTT TTCAGGACCA 1140

GAATTCGCAT TCCCTTTATT TGGGAATGCG GGGAATGCAG CTCCACCCGT ACTTGTCTCA 1200

TTAACTGGTT TGGGGATTTT TAGAACATTA TCTTCACCTT TATATAGAAG AATTATACTT 1260

GGTTCAGGCC CAAATAATCA GGAACTGTTT GTCCTTGATG GAACGGAGTT TTCTTTTGCC 1320

TCCCTAACGA CCAACTTGCC TTCCACTATA TATAGACAAA GGGGTACAGT CGATTCACTA 1380

GATGTAATAC CGCCACAGGA TAATAGTGTA CCACCTCGTG CGGGATTTAG CCATCGATTG 1440

AGTCATGTTA CAATGCTGAG CCAAGCAGCT GGAGCAGTTT ACACCTTGAG AGCTCCAACG 1500

TTTTCTTGGC AGCATCGCAG TGCTGAATTT AATAATATAA TTCCTTCATC ACAAATTACA 1560

CAAATACCTT TAACAAAATC TACTAATCTT GGCTCTGGAA CTTCTGTCGT TAAAGGACCA 1620

GGATTTACAG GAGGAGATAT TCTTCGAAGA ACTTCACCTG GCCAGATTTC AACCTTAAGA 1680

GTAAATATTA CTGCACCATT ATCACAAAGA TATCGGGTAA GAATTCGCTA CGCTTCTACT 1740

ACAAATTTAC AATTCCATAC ATCAATTGAC GGAAGACCTA TTAATCAGGG TAATTTTTCA 1800

GCAACTATGA GTAGTGGGAG TAATTTACAG TCCGGAAGCT TTAGGACTGT AGGTTTTACT 1860

ACTCCGTTTA ACTTTTCAAA TGGATCAAGT GTATTTACGT TAAGTGCTCA TGTCTTCAAT 1920

TCAGGCAATG AAGTTTATAT AGATCGAATT GAATTTGTTC CGGCAGAAGT AACCTTTGAG 1980

GCAGAATATG ATTTAGAAAG AGCACAAAAG GCGGTGAATG AGCTGTTTAC TTCTTCCAAT 2040

CAAATCGGGT TAAAAACAGA TGTGACGGAT TATCATATTG ATCAAGTATC CAATTTAGTT 2100

GAGTGTTTAT CAGATGAATT TTGTCTGGAT GAAAAACAAG AATTGTCCGA GAAAGTCAAA 2160

CATGCGAAGC GACTTAGTGA TGAGCGGAAT TTACTTCAAG ATCCAAACTT CAGAGGGATC 2220

AATAGACAAC TAGACCGTGG CTGGAGAGGA AGTACGGATA TTACCATCCA AGGAGGCGAT 2280

GACGTATTCA AAGAGAATTA CGTTACGCTA TTGGGTACCT TTGATGAGTG CTATCCAACG 2340

TATTTATATC AAAAAATAGA TGAGTCGAAA TTAAAAGCCT ATACCCGTTA TCAATTAAGA 2400

GGGTATATCG AAGATAGTCA AGACTTAGAA ATCTATTTAA TTCGCTACAA TGCAAAACAT 2460

GAAACAGTAA ATGTGCCAGG TACGGGTTCC TTATGGCCGC TTTCAGCCCA AAGTCCAATC 2520

GGAAAGTGTG GAGAGCCGAA TCGATGCGCG CCACACCTTG AATGGAATCC TGACTTAGAT 2580

TGTTCGTGTA GGGATGGAGA AAAGTGTGCC CATCATTCGC ATCATTTCTC CTTAGACATT 2640

GATGTAGGAT GTACAGACTT AAATGAGGAC CTAGGTGTAT GGGTGATCTT TAAGATTAAG 2700

ACGCAAGATG GGCACGCAAG ACTAGGGAAT CTAGAGTTTC TCGAAGAGAA ACCATTAGTA 2760

GGAGAAGCGC TAGCTCGTGT GAAAAGAGCG GAGAAAAAAT GGAGAGACAA ACGTGAAAAA 2820

TTGGAATGGG AAACAAATAT CGTTTATAAA GAGGCAAAAG AATCTGTAGA TGCTTTATTT 2880

GTAAACTCTC AATATGATCA ATTACAAGCG GATACGAATA TTGCCATGAT TCATGCGGCA 2940

GATAAACGTG TTCATAGCAT TCGAGAAGCT TATCTGCCTG AGCTGTCTGT GATTCCGGGT 3000

GTCAATGCGG CTATTTTTGA AGAATTAGAA GGGCGTATTT TCACTGCATT CTCCCTATAT 3060

GATGCGAGAA ATGTCATTAA AAATGGTGAT TTTAATAATG GCTTATCCTG CTGGAACGTG 3120

AAAGGGCATG TAGATGTAGA AGAACAAAAC AACCAACGTT CGGTCCTTGT TGTTCCGGAA 3180

TGGGAAGCAG AAGTGTCACA AGAAGTTCGT GTCTGTCCGG GTCGTGGCTA TATCCTTCGT 3240

GTCACAGCGT ACAAGGAGGG ATATGGAGAA GGTTGCGTAA CCATTCATGA GATCGAGAAC 3300

AATACAGACG AACTGAAGTT TAGCAACTGC GTAGAAGAGG AAATCTATCC AAATAACACG 3360

GTAACGTGTA ATGATTATAC TGTAAATCAA GAAGAATACG GAGGTGCGTA CACTTCTCGT 3420

AATCGAGGAT ATAACGAAGC TCCTTCCGTA CCAGCTGATT ATGCGTCAGT CTATGAAGAA 3480

AAATCGTATA CAGATGGACG AAGAGAGAAT CCTTGTGAAT TTAACAGAGG GTATAGGGAT 3540

TACACGCCAC TACCAGTTGG TTATGTGACA AAAGAATTAG AATACTTCCC AGAAACCGAT 3600

AAGGTATGGA TTGAGATTGG AGAAACGGAA GGAACATTTA TCGTGGACAG CGTGGAATTA 3660

CTCCTTATGG AGGAATAGTC TCATGCAAAC TCAGGTTTAA ATATCGTTTT CAAATCAATT 3720

GTCCAAGAGC AGCATTACAA ATAGATAAGT AATTTGTTGT AATGAAAAAC GGACATCACC 3780

TCCATTGAAA CGGAGTGATG TCCGTTTTAC TATGTTATTT TCTAGT 3826 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1176 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D). TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Asp Asn Asn Pro Asn lie Asn Glu Cys lie Pro Tyr Asn Cys Leu 1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg lie Glu Thr Gly 20 25 30

Tyr Thr Pro lie Asp lie Ser Leu Ser Leu Thr Gin Phe Leu Leu Ser 35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp He He 50 55 60

Trp Gly He Phe Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80

Glu Gin Leu He Asn Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95

He Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gin He Tyr Ala Glu 100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125

Glu Met Arg He Gin Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140

He Pro Leu Leu Ala Val Gin Asn Tyr Gin Val Pro Leu Leu Ser Val 145 150 155 160

Tyr Val' Gin Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175

Val Phe Gly Gin Arg Trp Gly Phe Asp Ala Ala Thr He Asn Ser Arg 180 185 190

Tyr Asn Asp Leu Thr Arg Leu He Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220

Asp Trp Val Arg Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240

Leu Asp He Val Ala Leu Phe Ser Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255

He Arg Thr Val Ser Gin Leu Thr Arg Glu He Tyr Thr Asn Pro Val 260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Met Ala Gin Arg He Glu 275 280 285

Gin Asn He Arg Gin Pro His Leu Met Asp He Leu Asn Ser He Thr 290 295 300

He Tyr Thr Asp Val His Arg Gly Phe Asn Tyr Trp Ser Gly His Gin 305 310 315 320

He Thr Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Ala Phe Pro 325 330 335

Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro Pro Val Leu Val Ser Leu 340 345 350

Thr Gly Leu Gly He Phe Arg Thr Leu Ser Ser Pro Leu Tyr Arg Arg 355 360 365

He He Leu Gly Ser Gly Pro Asn Asn Gin Glu Leu Phe Val Leu Asp 370 375 380

Gly Thr Glu Phe Ser Phe Ala Ser Leu Thr Thr Asn Leu Pro Ser Thr 385 390 395 400

He Tyr Arg Gin Arg Gly Thr Val Asp Ser Leu Asp Val He Pro Pro 405 410 415

Gin Asp Asn Ser Val Pro Pro Arg Ala Gly Phe Ser His Arg Leu Ser 420 425 430

His Val Thr Met Leu Ser Gin Ala Ala Gly Ala Val Tyr Thr Leu Arg 435 440 445

Ala Pro Thr Phe Ser Trp Gin His Arg Ser Ala Glu Phe Asn Asn He 450 455 460

He Pro Ser Ser Gin He Thr Gin He Pro Leu Thr Lys Ser Thr Asn 465 - 470 475 480

Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly 485 490 495

Asp He Leu Arg Arg Thr Ser Pro Gly Gin He Ser Thr Leu Arg Val 500 505 510

Asn He> Thr Ala Pro Leu Ser Gin Arg Tyr Arg Val Arg He Arg Tyr 515 520 525

Ala Ser Thr Thr Asn Leu Gin Phe His Thr Ser He Asp Gly Arg Pro 530 535 540

He Asn Gin Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn Leu 545 550 555 560

Gin Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe 565 570 575

Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn Ser 580 585 590

Gly Asn Glu Val Tyr He Asp Arg He Glu Phe Val Pro Ala Glu Val 595 600 605

Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gin Lys Ala Val Asn 610 615 620

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

Asp Tyr His He Asp Gin Val Ser Asn Leu Val Glu Cys Leu Ser Asp 645 650 655

Glu Phe Cys Leu Asp Glu Lys Gin Glu Leu Ser Glu Lys Val Lys His 660 665 670

Ala Lys .Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe 675 680 685

Arg Gly He Asn Arg Gin Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp 690 695 700

He Thr He Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr 705 710 715 720

Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys 725 730 735

He Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Gly 740 745 750

Tyr He Glu Asp Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr Asn 755 760 765

Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro 770 775 780

Leu Ser Ala Gin Ser Pro He Gly Lys Cys Gly Glu Pro Asn Arg Cys 785 790 795 800

Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp 805 810 815

Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp He Asp 820 825 830

Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val He Phe 835 840 845

Lys He Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe 850 855 860

Leu Glu' Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg 865 870 875 880

Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr 885 890 895

Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val 900 905 910

Asn Ser Gin Tyr Asp Gin Leu Gin Ala Asp Thr Asn He Ala Met He 915 920 925

His Ala Ala Asp Lys Arg Val His Ser He Arg Glu Ala Tyr Leu Pro 930 935 940

Glu Leu Ser Val He Pro Gly Val Asn Ala Ala He Phe Glu Glu Leu 945 950 955 960

Glu Gly Arg He Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val 965 970 975

He Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys 980 985 990

Gly His Val Asp Val Glu Glu Gin Asn Asn Gin Arg Ser Val Leu Val 995 1000 1005

Val Pro Glu Trp Glu Ala Glu Val Ser Gin Glu Val Arg Val Cys Pro 1010 1015 1020

Gly Arg .Gly Tyr He Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly 1025 1030 1035 1040

Glu Gly Cys Val Thr He His Glu He Glu Asn Asn Thr Asp Glu Leu 1045 1050 1055

Lys Phe Ser Asn Cys Val Glu Glu Glu He Tyr Pro Asn Asn Thr Val 1060 1065 1070

Thr Cys Asn Asp Tyr Thr Val Asn Gin Glu Glu Tyr Gly Gly Ala Tyr 1075 1080 1085

Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp 1090 1095 1100

Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu 1105 1110 1115 1120

Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro 1125 1130 1135

Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys 1140 1145 1150

Val Trp He Glu He Gly Glu Thr Glu Gly Thr Phe He Val Asp Ser 1155 1160 1165

Val Glu Leu Leu Leu Met Glu Glu 1170 1175