AU745110B2 - Novel bacillus thuringiensis strains active against lepidopteran and coleopteran pests - Google Patents

Description

S F Ref: 357200D1

AUSTRALIA

PATENT ACT 1990 COMPLETE SPECFICATION FOR A STANDARD PATENT

ORIGINAL

*w S.* Name and Address of Applicant: Abbott Laboratories.

100 Abbott Park Road Abbett Park' llnis 60064 I INITT~fl CTAT~E nE* AKAOTf(A U1 -z>~c Actual Inventor(s): Address for Service: Chi-LI Liu, Lee Fremont Adams, Patricia A. Lufburrow and Michael David Thomas.

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Novel Bacillus Thuringiensis Strains Active Against Lepidopteran and Coleopteran Pests Invention Title: 'The following statement is a best method of performing it full description of known to me/us:this invention, including the 5845 NOVEL BACILLUS THURINGIENSIS STRAINS ACTIVE AGAINST

LEPIDOPTERAN

SAND COLEOPTERAN

PESTS

1. FIELD OF THE INVENTION The invention is related to novel delta-endotoxins obtained from a biologically pure Bacillus thuringiensis strain(s) active against lepidopteran and coleopteran pests which produces a bipyramidal crystal consisting essentially of at least two delta-endotoxins 1 0 having a molecular weight of about 130,000 daltons and a rhomboidal crystal consisting essentially of two delta-endotoxins, each having a molecular weight of about 33,000 daltons.

The invention also relates to nucleic acid sequences encoding these delta-endotoxins, as well as .DNA constructs, vectors and/or hosts cells comprising these nucleic acid sequences.

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 broad spectrum pesticides, chemical pesticides with a broad range of activity. However, there are a number of disadvantages to using such chemical .pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests.

Additionally, these chemical pesticides are frequently toxic to animals and humans, and 2 5 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 are naturally occurring organisms that produce a toxin(s), a substance toxic to the infesting agent which is generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.

The most widely used biopesticide is Bacillus thuringiensis B.t. is a widely distributed, rod shaped, aerobic and spore forming microorganism. During its sporulation cycle, B.t. produces a protein(s) known as a delta-endotoxin(s), that forms crystalline inclusion bodies within the cell The delta-endotoxins have molecular weights 3 5 ranging from 27-140 kD and kill insect larvae upon ingestion.

Delta-endotoxins have been produced by recombinant DNA methods (see, for example, Tailor etal., 1992, Molecular Microbiology 6:1211-1217; toxin is active against lepidopteran and coleopteran pests; Payne et al., U.S. Patent No. 5,045,469; toxin is active against lepidopteran pests). The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form.

A number ofB.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 bipyramidal and cuboidal crystal proteins in each cell during sporulation (Liithy 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 three crylA genes (Aronson et al., 1986, Microbiol. Rev. 50:1-50). B.t. subsp. kurstaki HD-73 crystal delta-endotoxin contains the CryIA(c) protein (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 1 0 protein that differs from the holotype CryIA(a) protein by 24 amino acids; B.t. subsp.

subtoxicus HD-10 contains CrylA and CryIB proteins; B.t. subsp. tolworthi HD-121 contains *CrylA and CryII proteins; and B.t. subsp. aizawai HD-68 contains CrylA proteins (Hfte and Whiteley, 1989, Microbiol. Reviews 53:242-255). Payne, U.S. Patent No. 4,990,332, issued February 5, 1993, discloses an isolate of PS85AI, and a mutant of the isolate, 1 5 which both have activity against Plutella xylostella, a lepidopteran pest, and produce alkaline 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 alkaline soluble proteins having a molecular weight of 130,000 and 60,000 daltons and has activity against Spodoptera exigua and T. ni; the toxin gene from PS81F appears to have little homology 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 alkaline-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. Bernier et al., U.S.

25 Patent No. 5,061,489 and WO 90/03434 discloses strain A20 producing a delta-endotoxin encoded by at least three genes: and 4.5-type genes (crylA(a), crylA(b), and crylA(c)). Chestukhina et al., 1988, FEBS Lett. 232:249-51, disclose that B.t. subsp.

galleriae produces two delta-endotoxins, both of whichl are active against lepidopteran pests.

Other strains, e.g. Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Patent No. 4,766,203), have been found to be specific for Coleoptera. The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Hernnstadt et al.

Bio/Technology vol. 4, 305-308, 1986, US patent 4,764,372, 1988). This strain, designated "Bacillus thuringiensis subsp. san diego", M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publicly acknowledged that Bacillus thuringiensis subsp. san diego is Bacillus thuringiensis subsp. tenebrionis.

Other isolated strains have been found to be active against two orders of pests.

2

L.

j "i -~i rc ii 3 l'adua. 1990. Microbiol. Lett. 66:257-262. discloses the isolation of two mutants containing two delta-endotoxins, a 144 kD protein having activity against a lepidopteran pest and a 66 kD protein having activity against mosquitoes. Bradfish el al., U.S. Patent No. 5.208.017. discloses B.I. isolates PS86AI and PS86Q3 which produce alkaline soluble proteins having a molecular weight of 58,000 and 45,000 daltons and 155,000, 135,000, 98.00. 62.000. and 58,000 daltons, respectively and which have activity against lepidopteran and coleopteran pests. PCT Application No. WO 90/13651 and Tailor el al., 1992. Molecular Microbiology 6:1211-1217, disclose a B.I. strain which is toxic against Lcpidoptera and Coleoptera and which produces a toxin having a molecular weight of i0 ~S k)D.

It is advantageous to produce new delta-endotoxins so that there exists a wider spectrum of biopesticides for any given insect pest.

Summary of the Invention According to one embodiment of this invention there is provided a purified and i: isolated nucleic acid having a nucleic acid sequence encoding a delta-endotoxin having a molecular weight of about 33,000 daltons and an amino acid sequence essentially as depicted in SEQ ID NO:37 or a portion of said defta-endotoxin having insecticidal activity against an insect pest of the order Lepidoptera.

According to another embodiment of this invention there is provided the nucleic 2 acid of the invention wherein the nucleic acid sequence is essentially depicted in SEQ ID NO:39 or SEQ ID NO:44.

According to a further embodiment of this invention there is provided a purified and isolated nucleic acid having a nucleic acid sequence encoding a delta-endotoxin having a molecular weight of about 33,000 daltons and an amino acid sequence essentially as 2 depicted in SEQ ID NO:38 or a portion of said delta-endotoxin having insecticidal activity against an insect pest of the order Lepidoptera.

CAccording to yet a further embodiment of this invention there is provided a nucleic acid of the invention wherein the nucleic acid is as essentially depicted in SEQ ID or SEQ ID According to yet another embodiment of this invention there is provided a purified and isolated nucleic acid having a nucleic acid sequence essentially as depicted in SEQ ID NO:41.

According to another embodiment of this invention there is provided a DNA construct comprising the nucleic acid of the invention.

[R:\LIBXX]02756.doc:aak 3;a According to a further embodiment of this invention there is provided a ircolminant DNA vector comprising the DNA construct of the invention; a promoter operably linked to the DNA sequence of and a selectable marker.

According to another embodiment of this invention there is provided a host cell comprising the DNA construct of the invention.

According to a further embodiment of this invention there is provided a deltacndlotoin product of the expression in a procaryotic or eucaryotic host cell of a nucleic icid of the invention.

According to a further embodiment of this invention there is provided an ;it insecticidal composition comprising the delta-endotoxin product of the invention in association with an insecticidal carrier.

According to another embodiment of this invention there is provided the insecticidal composition of the invention in which the insecticidal composition further comnprises spores of biologically pure Bacillus thuringiensis strain.

i. According to a further embodiment of this invention there is provided the insecticidal composition of the invention which further comprises at least two deltaendotoxins having a molecular weight of about 130,000 and activity against an insect pest the order Lepidoptera.

According to another embodiment of this invention there is provided a method for 20 controlling an insect pest of the order Lepidoptera comprising exposing the pest to an insect-controlling effective amount of an insecticidal composition of the invention.

According to a further embodiment of this invention there is provided an insecticidal composition of the invention when used for controlling an insect pest of the order Lepidoptera.

According to another embodiment of this invention there is provided use of a deltacndotoxin product according to the invention for the preparation of an insecticidal composition for controlling an insect pest of the order Lepidoptera.

According to another embodiment of this invention there is provided an insecticidal composition of the invention when used for controlling an insect pest of the order .13 Coleoptera.

According to another embodiment of this invention there is provided use of a deltacndotoxin product according to the invention for the preparation of an insecticidal composition for controlling an insect pest of the order Coleoptera.

The invention is related to two novel delta-endotoxins, both having molecular w \veights of about 33,000 daltons. One of the 33,000 dalton delta-endotoxins has an amino IR:\LIBXX]02756.doc:aak 3b acid sequence essentially as depicted in SEQ ID NO:37 (hereinafter referred to as the .M IV\DL protein6). The other 33,000 dalton delta-endotoxin has an amino acid sequence csscnially as depicted in SEQ ID NO:38 (hereinafter referred to as the "MK-HHK irmcin The delta-endotoxins of the present invention are obtainable from a hiOIlogically pure Bacillus Ihuringiensis strain(s) or a crystal(s) or mutant(s) thereof which :;rain or mutant has activity against an insect pest of the order Lepicloptera and an insect pest of the order Coleoptera and produces at east two delta-endotoxins having a 1mlecular \veight of about 130,000 daltons and two delta-endotoxins both having molecular \weights of about 33,000 daltons. The 130,000 delta-endotoxins have insecticidal activity against insect pests of the order Lepidoptera. In a specific cmbodiment of the invention, the B. Ihuringiensis strain is EMCCOO75 and EMCC0076 having the identifying characteristics of NRRL B-21019 and NRRL B-21020 respectively nmd is disclosed in W094/13785.

hlie invention also relates to an isolated nucleic acid fragment containing a nucleic acid sequence encoding each of the delta-endotoxins of the present invention or a portion of the delta-endotoxin having insecticidal activity against a pest. In one embodiment, the nucleic acid fri-agment contains a nucleic acid sequence encoding the MIVDL protein and may have the nucleic acid sequence essentially as depicted in SEQ ID NO:39. In another embodiment, the nucleic acid fragment contains a nucleic acid sequence encoding the N MIKHH-1K protein and may have the nucleic acid sequence essentially as depicted in SEQ 11ID NO:40. The invention is also directed to a genomic sequence comprising nucleic acid sequence encoding the MKHHK and/or MIVDL and may have the nucleic acid sequence essentially as depicted in SEQ ID NOS:41 (MKHHK and MIVDL), 44 (MKHHK), and 45 (MIVDL).

[R:\LIBXX]02756.doc:aak I; i S* (I The invention also provides vectors, DNA constructs and recombinant host cells comprising the claimed nucleic acid fragment(s), which vectors, DNA constructs and recombinant host cells are useful in the recombinant production of the delta-endotoxins of the present invention. The nucleic acid fragment may be operably linked to transcription and translation signals capable of directing expression of the delta-endotoxin in the host cell of choice. Recombinant production of the delta-endotoxin(s) of the invention is achieved by culturing a host cell transformed or transfected with the nucleic acid fragment of the invention, or progeny thereof, under conditions suitable for expression of the delta-endotoxin, and recovering the delta-endotoxin from the culture.

1 0 The invention is further related to an oligonucleotide probe having a nucleotide sequence essentially as depicted in SEQ ID NO:20 which can be used to detected the MIVDL protein and oligonucleotide probe essentially as depicted in SEQ ID NO:21 which can be used to detect the MKHHK protein.

The novel Bacillus thuringiensis delta-endotoxins may within the scope of this 1 5 invention each be formulated into insecticidal compositions. In one embodiment, the deltaendotoxins may be combined with an insecticidal carrier. The compositions of the present invention may comprise one or both delta-endotoxins of the present invention. The compositions may further comprise 130,000 molecular weight delta-endotoxins having activity against an insect pest of the order Lepidoptera. These insecticidal compositions may be used to 2 0 control insect pests of the order Lepidoptera in a method comprising exposing the pest to an insect-controlling effective amount of such an insecticidal composition. The compositions of the present invention may also comprise a spore of a biologically pure strain or mutant thereof which strain or mutant has activity against an insect pest of the order Lepidoptera and an insect 2. .pest of the order Coleoptera, produces at least two delta-endotoxins having a molecular weight 25 of about 130,000 daltons and two delta-endotoxins both having molecular weights of about 33,000 daltons. These compositions may be effective against an insect pest of the order Lepidoptera or Coleoptera.

Furthermore, the compositions or delta-endotoxins of the present invention may be used to enhance the insecticidal activity of another Bacillus-related insecticide. As defined herein, "a Bacillus related insecticide" is a Bacillus Bacillus thuringiensis, specifically, Bacillus thuringiensis subsp. kurstaki or Bacillus thuringiensis subsp. tenebrionis or Bacillus subtilis) strain, spore, or substance, protein or fragment thereof having activity against or which kill insects; a substance that provides plant protection, e.g. antifeeding substance; or a microorganism capable of expressing a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kills insects Bacillus thuringiensis deltaendotoxin) and an acceptable carrier (see Section infra, for examples of such carriers). A microorganism capable of expressing a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill insects inhabits the phylloplane (the surface of the plant leaves), and/or the rhizosphere (the soil surrounding plant roots), and/or aquatic environments, and is capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms and provide for the stable maintenance and expression of a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill insects. Examples of such microorganisms include but are not limited to bacteria, genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, 1 0 Alcaligenes, and Clostridium; algae, e.g. families Cyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae, Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

In a specific embodiment, the delta-endotoxins or compositions of the present S invention may act together with Bacillus-related insecticides in a synergistic fashion. In another embodiment, Bacillus strains active against insect pests of the order Coleoptera may act together in a synergistic fashion with delta-endotoxins, Bacillus strains or spores thereof active 2 0 against insect pests of the order Lepidoptera to kill insect pests of the order Coleoptera. In yet another embodiment, the delta-endotoxins of the present invention may act together in a synergistic fashion.

4. BRIEF DESCRIPTION OF THE FIGURES S* 2 5 Figure 1 shows the results of PCR analysis of Bacillus thuringiensis strains for cryl genes by agarose gel electrophoresis. Lane 1 shows molecular weight markers (1 kb ladder, BRL-GIBCO). Lanes 2 and 3 show analysis of strains EMCC0075 and EMCC0076 with crylD oligonucleotide primers described in Figure 1. Lanes 4-6 show the analysis of Bacillus thuringiensis subsp. tenebrionis, an unknown Bacillus thuringiensis strain, and Bacillus thuringiensis subsp. aizawai with crylD oligonucleotide primers. Bacillus thuringiensis subsp. renebrionis contains only the crylllA gene; the unknown Bacillus thuringiensis strain does not contain the crylD gene; and Bacillus thuringiensis subsp. aizawai contains several cryl genes including crylD.

Figure 2 shows the cloned DNA fragments which encode the MKHHK and MIVDL proteins.

i (I I Figures 3A and 3B shows the homology of the "MIVDL" protein to the 34 kDa protein of Bacillus thuringiensis subsp. thompsoni and the CryIA(a) protein of Bacillus thuringiensis subsp. kurstaki.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. OBTAINING DELTA-ENDOTOXINS The delta-endotoxins of the present invention may be obtainable from a biologically pure Bacillus thuringiensis strain(s) or a crystal(s) or mutant(s) thereof which 1 0 strain or mutant has activity against an insect pest of the order Lepidoptera and an insect pest of the order Coleoptera and produces at least two delta-endotoxins having a molecular weight of about 130,000 daltons and two delta-endotoxins both having molecular weights of about 33,000 daltons. The 130,000 delta-endotoxins have insecticidal activity against insect pests of the order Lepidoptera. In a specific embodiment of the invention, the B.thuringiensis strain is .1 5 EMCC0075 and EMCC0076 having the identifying characteristics of NRRL B-21019 and NRRL B-21020 respectively and is disclosed in W094/13785. The strains 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., .2 0 Academic Press, 1980). Upon completion of the fermentation cycle, the crystals and spores can be harvested by separating B.t. spores and crystals from the fermentation broth by means well known in the art, e.g. centrifugation. The spores and crystals are contained in the pellet As noted in Section 2, supra, crystals consist essentially of a delta- 2 5 endotoxin(s). The strains of the present invention produce two types of crystals. One is a bipyramidal crystal consisting essentially of at least two 130,000 dalton delta-endotoxins. The other is a rhomboidal crystal consisting essentially of the two 33,000 dalton delta-endotoxins.

Purification of the crystals or delta-endotoxins can be carried out by various procedures known in the art, including, but not limited to, density gradient centrifugation, 3 0 chromatography ion exchange, affinity, hydrophobic and size exclusion), electrophoretic procedures, differential solubility, or any other standard technique for the purification of proteins.

The delta-endotoxins may also be obtained from a recombinant DNA expression system. Specifically, DNA encoding each toxin as, for example, essentially depicted in SEQ ID NOS:39, 40, 44, and 45 is cloned into a suitable DNA expression vector. Alternatively, one genomic DNA fragment comprising nucleic acid sequences encoding each delta endotoxin as, for example, essentially depicted in SEQ ID NO:41 may be cloned.

T--

Identification of the specific DNA fragment encoding the delta-endotoxin may be accomplished in a number of ways, including, but not limited to, electrophoretic separation of the fragments (Southern, 1975, J. Mol. Biol. 98:503) in agarose, transfer of the separated DNA fragments to nitrocellulose, nylon, or other suitable support medium, and probing of the transferred fragments with a degenerate oligonucleotide probe(s) based on the amino acid sequence of the protein as determined by sequential Edman degradation. Alternatively, one may probe with a labeled gene fragment corresponding to the open reading frame of a protein with suspected high homology to the protein of interest High homology to the gene of interest may be determined by alignment of a family of related proteins and identification of highly 1 0 conserved regions in the encoding DNA segments (see, for example, Gribskov, and J.

Devereux, eds., in Sequence Analysis Primer, Stockton Press, 1991). An elegant and reliable method is to determine the amino acid sequences of at least two peptide fragments, generated by enzymatic or chemical means from the protein of interest, design degenerate oligonucleotides that will recognize the DNA encoding those regions, and then to apply 1 5 polymerase chain reaction (PCR) techniques to amplify perfect or near-perfect copies of the i: intervening region of DNA. This PCR-generated segment of DNA can then be labeled and used as a highly specific probe for cloning the delta-endotoxin-encoding gene.

Once identified, the DNA fragment harboring the gene encoding the deltaendotoxin or a pesticidally active fragment thereof may be cloned by ligation of a size-selected 0 library of fragments expected to harbor the gene of interest into a suitable vector, including, but not limited to, pBR322, pUC118, pACYC194, and pBCSK plasmids and their variants for transformation into Escherichia coli; or pUB110, pBD64, pBC16, pHP13, pE194, pC194, and their variants, for transformation into Bacillus spp. Bacteriophage vectors, such as lambda and its derivatives, may also be used for cloning of the gene(s) into E. coli.

5 Production of the delta-endotoxin or a pesticidally active fragment thereof at commercially useful levels can be achieved by subcloning the encoding gene into plasmid vectors that permit stable expression and maintenance in a suitable host. Frequently, acceptable expression can be achieved using the native regulatory elements present on the DNA fragment encoding the delta-endotoxin. However, one might wish to add or alter transcriptional regulatory signals (promoters, initiation start sites, operators, activator regions, terminators) and translational regulatory signals (ribosomal binding sites, initiation codons) for enhanced or more regulated expression of the delta-endotoxin gene within the chosen host cell In addition to plasmids, delta-endotoxin genes and the appropriate regulatory elements may be introduced into one of the native plasmids of Bacillus thuringiensis and/or other chosen host, or into the chromosomal DNA, via "gene conversion" Iglesias and Trautner, 1983, Mol Gen. Genet. 189:73-76; Duncan et al., 1978, Proc. Natl. Acad. Sci.

U.S.A. 75:3664-3665) or homologous recombination Ferrari et al., 1983, J. Bacteriol.

154:1513-1515) at sites of shared DNA homology between the vector and the host strain. An efficient "two-plasmid" system may be used for introduction of genes into Bacilli via homologous recombination (see, for example, PCT Patent W091/09129). Transposons may also be used to introduce cry genes into the selected host strain. For example, in the Bacilli, transposons such as Tn917 and its derivatives may be used (Youngman et al., 1989, In Regulation of Prokaryotic Development, I. Smith, R. Slepecky, and P. Setlow, eds. American Society for Microbiology, Washington, Transfer of cloned delta-endotoxin genes into Bacillus thuringiensis, as well as into other organisms, may be achieved by a variety of techniques, including, but not limited to, 1 0 protoplasting of cells (Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115; Crawford et al., 1987, J. Bacteriol. 169: 5423-5428); electroporation Schurter et al., 1989, Mol.

Gen. Genet. 218: 177-181 and Macaluso et al., 1991, J. Bacteriol. 173: 1353-1356); particle bombardment Shark et al., 1991, Appl. Environ. Microbiol. 57:480-485); silicon S carbide fiber-mediated transformation of cells (Kaeppler et al., 1992, Theor. AppL Genet.

1 5 84:560-566); conjugation (Gonzalez et al., 1982, Proc. Natl. Acad. Sci. U.S.A.

.79:6951-6955); or transduction by bacteriophage Lecadet et al., 1992, Appl. Environ.

*.Microbiol. 58: 840-849). Transformed colonies may be detected by their ability to produce crystal delta-endotoxin, to bind antibody directed against that specific delta-endotoxin, or to kill susceptible pests, arthropods or nematodes, in bioassay.

2 0 Criteria for selection of a particular host for production include, but are not limited to, ease of introducing the gene into the host, availability of expression systems, and stable maintenance and expression of the gene encoding the delta-endotoxin. The host may be a microorganism, such as Bacillus thuringiensis itself, or an inhabitant of the phytosphere, the phylloplane (the surface of plants), and/or the rhizosphere (the soil surrounding plant 25 roots), and/or aquatic environments, and should be capable of competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms. Examples of such microorganisms include but are not limited to bacteria, e.g. genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobiumn, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, 3 0 Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes, and Clostridium; algae, e.g. families Cyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae, Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, e.g. genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

The gene(s) encoding the delta-endotoxin(s) of the present invention or a portion thereof can also be inserted into an appropriate cloning vector for subsequent 3t__ introduction into the genomes of suitable plants that are known to be infested with insects susceptible to the delta-endotoxin(s), or into specific baculoviruses which can in turn be directly used as insecticides.

Those skilled in the art will recognize that the invention is not limited to use of the nucleic acid fragments specifically disclosed herein, for example, in SEQ ID NO:39 OR It will be apparent that the invention also encompasses those nucleotide sequences that encode the same amino acid sequences as depicted in SEQ ID NO:39 OR 40, but which differ from those specifically depicted nucleotide sequences by virtue of the degeneracy of the genetic code. The invention specifically encompasses any variant nucleotide sequence, and the protein 1 0 encoded thereby, which protein retains at least about an 80%, preferably 90%, and most preferably 95% homology or identity with one or the other of the amino acid sequences depicted in Figure 2 and retains the activity of the sequences described herein. In particular, variants which retain a high level 80%) of homology at highly conserved regions of said delta-endotoxin are contemplated. Furthermore, the invention encompasses any variant 1 5 that hybridizes to the nucleotide sequence of the delta-endotoxin under the following S conditions:presoaking in 5X SSC and prehydbridizing for 1 hr. at about 40°C in a solution of 20% formamide, 5X Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 ug denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 uM ATP for 18 hrs. at about 40'C, followed by a wash in 0.4X SSC 0 at a temperature of about Useful variants within the categories defined above include, for example, ones in which conservative amino acid substitutions have been made, which substitutions do not significantly affect the activity of the protein. By conservative substitution is meant that amino acids of the same class may be substituted by any other of that class. For example, the nonpolar aliphatic 5 residues Ala, Val, Leu, and Ile may be interchanged, as may be the basic residues Lys and Arg, or the acidic residues Asp and Glu. Similarly, Ser and Thr are conservative substitutions for each other, as are Asn and Gin. It will be apparent to the skilled artisan that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active delta-endotoxin. Retention of the desired activity can readily be determined by using the assay procedures described below.

5.2. MUTANTS A mutant B.t. strain may produce a larger amount of and/or larger crystals than the parental strain. A "parental strain" as defined herein is the original Bacillus thuringiensis strain before mutagenesis.

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 .lilL~- Iljit- methanesulfonate, or by irradiation with gamma rays, X-rays or UV. Specifically, in one method of mutating Bacillus thuringiensis strains and selecting such mutants the following procedure is used: i) the parental strain is treated with a mutagen; ii) the thus presumptive mutants are grown in a medium suitable for the selection of a mutant strain; and iii) the mutant strain is selected for increased production of delta-endotoxin.

According to a preferred embodiment of this method, the selected colonies are grown in a production medium, and a final selection for strains capable of increased delta- 1 0 endotoxin production is performed.

Alternatively, the mutant(s) may be obtained using recombinant DNA methods known in the art. For example, a DNA sequence containing a gene coding for a deltaendotoxin may be inserted into an appropriate expression vector and subsequently introduced into the parental strain using procedures known in the art. Alternatively, a DNA sequence 1 5 containing a gene coding for a delta-endotoxin may be inserted into an appropriate vector for recombination into the genome and subsequent amplification.

5.3. BIOASSAY The activity of the B.t. strains or spores, mutants, crystals, or delta-endotoxins 2 0 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.

5.4. COMPOSITIONS 2 5 The delta-endotoxins of the present invention described supra can be formulated with an acceptable carrier into an insecticidal composition(s) 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. The compositions of the present invention may further comprise the 130,000 MW delta-endotoxins obtainable from the strains disclosed in W094/13785. Alternatively, the compositions of the invention may further comprise the spore of the strains disclosed in W094/13785.

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 Nacylsarcosinate; 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 alkylbenzene 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 1 0 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 S *5 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 oxygencontaining amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked 0 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, phyllosilicates, carbonates, sulfates, phosphates; organic materials such as sugars, starch or cyclodextrin or botanical materials such as wood products, cork, powdered i *5 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 powder 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 delta-endotoxins 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

-L

-i- -irr 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, 1 0 sorghum and related crops), beets (sugar beet and fodder beet), drupes, pomes and soft fruit (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 5 fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other brassicae, carrots, onions, tomatoes, potatoes, paprika), lauraceae S (avocados, cinnamon, camphor), deciduous trees and conifers 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, 0 as well as ornamentals. In most cases, the preferred mode of application is by foliar spraying.

The preferred mode of application for soil pests is by furrow application or by "lay-by" application. 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 compositions 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.gAchroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, 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., Cochylis hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmiafuneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella, Erannis tiliaria, Estigmene acrea, Eulia salubricola, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellumn, Hyphantria cunea, Keiferia lycopersicella, Lam dinafiscellariafiscellaria, Lambdinafisceilaria lugubrosa, Leucorna salicis, Lobesia botrana, Loxostege sticticoiis, LYmtantria dispar, Macalla thyrsisalis, Malacosomna sp., Mainestra brassicae, Mamnestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testidalis, Melanchra picta, Operophtera brumnata, Orgyic sp., Ostrinia nubilalis, Paleacrita vernauz, Papilic cresphontes, Pectinophora gossypiella, Ph'yganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota sultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylosteila, Pontia protodice, Pseudaletia unipuncta, Pseudoplusia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga 1 0 cerealella, Spilonota ocellana, Spodoptera sp., Thaurnstopoea pityocaMpa, Tineola bisseiliella.

Trichoplusia ni, Udea rubigalis, Xylomnyges curialis, Yponomeuta padella; Coleoptera, e.g., Leptinotarsa sp., Acanthoscelides obtectus, Callosobruchus chinensis, Epilachna varivestis, Pyrrhalta luteola, Cylasformicarius elegantulus, Listronotus oregonensis, Sitophilus sp., C-yclocephala borealis, C'yclocephala immaculata, Macro dactylus subspinosus, Pop illia *7 aponica, Rhizotrogus majalis, Aiphitobius diaperinus, Palorus ratzeburgi, Tenebric mzolitor, Tenebric obscurus, Triboliurn castaneum, Tibolium confusum, Tibolius destructor.

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

0 6. EXAMPLES 6.1. EXAMPLE 1: CULTIVATING B.t. STRAINS EMCCO075

AND

EMCCO076 Subcultures of EMCCO075 and EMCCOO76, maintained on Nutrient Broth 2 5 Agar slants, are used to inoculate 250 ml baffled shake flasks containing 50 mil of medium with the following composition: Corn Steep liquor 15 g/l Maltrin-100 40 g1 Potato Starch 30 g/1

KH

2

PO

4 1.77 g/l

K

2

HPO

4 4.53 g/l The pH of the medium is adjusted to 7.0 using 10 N NaQH.

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 centrifuge.

6.2. EXAMPLE 2: TESTING OF B.t. STRAINS EMCC0075 AND EMCC0076 SPORES AND CRYSTALS EMCC0075 and EMCC0076 are cultivated in shake flasks as described in Example 1, supra. To determine if EMCC0075 and EMCC0076 are active against lepidopteran pests, a 1:50 dilution of culture broth is made. 5 ml of such diluted culture broth is transferred into a 50 ml polypropylene centrifuge tube. 20 ml of artificial insect diet containing antibiotics 1 0 is added into the centrifuge tube. The mixture is subsequently dispensed into bioassay trays.

Three to six eggs either of beet armyworm (Spodoptera exigua) or tobacco budworm (Heliothis virescens) are applied on the surface of the "diet". Mylar is ironed onto the bioassay trays and the trays are incubated at 28°C. Scoring is carried out at 7 and 11 days.

.To determine if EMCC0075 and EMCC0076 are active against insect pests of the order Coleoptera, 5 ml of the culture broths are removed from the shake flasks and Stransferred directly into the 50 ml polypropylene centrifuge tubes. 20 ml of artificial insect diet (containing known antibiotics) are then added into the tubes (final testing concentration=20 w/w) and mixed vigorously. The mixtures are then dispensed into bioassay trays. Three to six eggs of corn rootworm (Diabrotica undecimpunctata) are applied to the surface of the "diet".

g Mylar is ironed onto the bioassay trays and the trays are incubated at 28'C. Scoring is carried out at 7 and 11 days.

The bioactivity of EMCC0075 and EMCC0076 towards Spodoptera exigua and Diabrotica undecimpunctata is expressed in terms of stunt score The stunt score is determined after incubating the trays for 7 days. In this system, 4=full size larvae (control 2 5 larvae); 3=3/4 size of control larvae; 2=1/2 size of control larvae; 1= 1/4 size of control larvae; and 0=mortality. The smaller the number, the higher the B.t. activity. The results are shown in Table I. It is evident that EMCC0075 and EMCC0076 possess activity against both lepidopteran and coleopteran pests.

TABLE I Spodoptera Diabrotica Heliothis exigua undecimpunctata virescens EMCC0075 1.7 0.9 EMCC0076 1.8 1.8 1.8 Control 4.0 4.0 6.3. EXAMPLE 3: cry GENE PROFILE FOR EMCC0075 AND EMCC0076 The cry gene profile for EMCC0075 and EMCC0076 is determined by using the PCR method which is described in the Perkin Elmer Cetus Gene Amp® PCR Reagent Kit 5 literature. Double-stranded DNA is heat-denatured and the two oligonucleotides corresponding S to the crylA(a) gene (listed in the Sequence Listing as SEQ ID NO:3 and SEQ ID NO:4 S respectively), crylA(b) gene (listed in the Sequence Listing as SEQ ID NO:5 and SEQ ID NO:6 respectively), crylA(c) gene (listed in the Sequence Listing as SEQ ID NO:7 and SEQ ID NO:8 respectively), crylD gene (listed in the Sequence Listing as SEQ ID NO:9 and SEQ ID 2 0 respectively), cryIIA gene (listed in the Sequence Listing as SEQ ID NO:11 and SEQ ID S NO:12 respectively), crylllB gene (listed in the Sequence Listing as SEQ ID NO: 13 and SEQ ID NO:14 respectively), cryIIC gene (listed in the Sequence Listing as SEQ ID NO:15 and SEQ ID NO:16 respectively), and cryllID gene (listed in the Sequence Listing as SEQ ID NO:17 and SEQ ID NO:18 respectively), are annealed at low temperature and then extended at an intermediate temperature.

PCR analysis indicated that both strains contain a crylD-like gene (see Figure A probe specific to crylD also detected a crylD-like gene in Southern analysis of restricted genomic DNA from both strains. No PCR amplifications are observed with primers to crylA(a), crylA(b), crylA(c), crylB (SEQ ID NOS:22 and 23), crylC (SEQ ID NOS:24 and 3 0 25), crylD, crylE (SEQ ID NOS:26 and 27), crylF (SEQ ID NOS:28 and 29), or crylG (SEQ ID NOS:30 and 31), nor to cryllA (SEQ ID NOS:32 and 33), cryllB (SEQ ID NOS:34 and 33), or crylIC (SEQ ID NOS: 35 and 36), nor to crylllA, crylllB, crylllC, or cryIID.

However, Southern analysis of a restriction fragment from genomic DNA from EMCC0075 and EMCC0076 with a probe that can detect crylA(a), crylA(b), and crylA(c) confirmed the presence of a crylA-like gene.

L-

6.4. EXAMPLE 4: PURIFICATION OF EMCC0075 BIPYRAMIDAL AND RHOMBOIDAL CRYSTALS A subculture of EMCC0075, maintained on a Nutrient Broth agar plate, is used to inoculate a 2.0 liter baffled shake flask containing 500 ml of medium with the same composition as described in Example 5, infra. After inoculation, the shake flask is incubated at on a rotary shaker for 72 hours at 250 rpm. The crystals and spores are recovered by centrifugation at 10,000 rpm (Sorvall GSA rotor) for 30 minutes. The pellets are washed with deionized water, centrifuged at 15,000 rpm (Sorvall SS34 rotor), and resuspended in deionized water by sonication to a concentration of 0.1 g wet weight per ml. 1 g wet weight crude 1 0 crystals are diluted to 33.2 ml with deionized water and placed in a 250 ml separatory funneL The bottom phase solution comprised of 10 ml 3M sodium chloride, 23.4 ml 20% polyethylene glycol 8000, and 33.4 ml 20% sedium dextran sulfate is added to the 250 ml separatory funnel and mixed, followed by 100 ml of a polyethylene glycol upper phase solution comprised of 0.3 g sodium dextran sulfate, 70.3 g polyethylene glycol 8000, and 17.5 g sodium chloride per liter deionized water. The suspension is shaken vigorously, and the two phases are allowed to separate at room temperature for 30 minutes.

The upper phase which contains large quantities of spores is removed with a pipet The lower phase contains crystals and residual spores. The extraction is repeated several times until the upper phase contains essentially no spores. The lower phase is then S2 0 diluted with 100 ml deionized water, and centrifuged at 10,000 rpm (Sorvall GSA rotor) for minutes at 5*C to recover the crystals. The recovered crystals are washed with 200 ml deionized water, and recentrifuged as before. The spores from the upper phase are also recovered using the above washing procedure.

The bipyramidal and rhomboidal crystals are then further purified by density .2*5 gradient centrifugation using a discontinuous LudoxTM HS-40 (DuPont) gradient comprised of 3.8 ml each of 75%, 50%, and 38% Ludox T v/v adjusted to pH 2.5 with 0.2M Tris-HCL mg of crystals in 100 p1 deionized water are layered on the top of the gradient, and centrifuged in a Beckman Ultracentrifuge at 10,000 rpm (Beckman 41 Ti rotor) for 15 minutes at 20 0

C.

Four separate bands are obtained. One contains pure rhomboidal crystals and another contains pure bipyramidal crystals. The two other bands contains mixtures of the two crystal types.

The pure crystal bands are recovered, washed with deionized water, and used for bioassay.

EXAMPLE 5: SDS-PAGE ANALYSIS OF THE DELTA- ENDOTOXINS FROM EMCC0075 and EMCC0076 Subcultures of EMCC0075 and EMCC0076, maintained on Nutrient Broth agar plates, are used to inoculate 250 ml baffled shake flasks containing 50 ml of medium with the following composition: iYllil i;-r Glucose 2.0 g/1

KH

2

PO

4 0.86 g/1

K

2

HPO

4 0.55 g/1 Sodium Citrate 2.0 g/1 CaCI 2 0.1 g/1 MnCl2-4H 2 0 0.16 g/1 MgCl 2 .6H 2 0 0.43 g/1 ZnCl2 0.007 g/1 FeC1 3 0.003 g/1 Casamino Acids 5 g/1 After inoculation, the shake flasks are incubated at 30 0 C on a rotary shaker for 5 72 hours at 250 rpm. The B.t crystals obtained in the above fermentations of EMCC0075 and 5 EMCC0076 are recovered by centrifugation at 10,000 rpm (Sorvall GSA rotor) for 30 minutes.

The B.t. crystals are then purified by biphasic extraction using sodium dextran sulfate and polyethylene glycol as outlined in Example 4, supra.

B.t. crystal preparations from EMCC0075 and EMCC0076 are analyzed by SDS-PAGE. Specifically, the SDS-PAGE is carried out on 10-15% gradient gels using Pharmacia's Phast System

T

The protein bands are analyzed on a Pharmacia densitometer using Pharmacia Gelscan T Software. The results indicated that the crystals produced by both strains contain at least two proteins with molecular weights of approximately 130,000 daltons and 33,000 daltons.

25 6.6. EXAMPLE 6: BIOASSAY USING SPODOPTERA EXIGUA TO DETERMINE ACTIVITY OF NOVEL LEPIDOPTERAN ACTIVE Bacillus thuringiensis STRAINS To determine if purified bipyramidal and rhomboidal crystals are active against lepidopteran pests, the crystals are bioassayed against Spodoptera exigua using a surface overlay assay. Samples of crystal preparations are applied to individual wells of a jelly tray containing 500 pll of solidified artificial insect diet per well. The trays containing the various samples are air dried. Two to four 2nd or early 3rd instar Spodoptera exigua are added to each well containing the dried test sample. The trays are then sealed with Mylar punched with holes for air exchange and are incubated for 3 days at 30 0 C. The degree of stunting, as described in Example 2, supra, is then recorded.

The results are shown in Table II. It is evident that, surprisingly, both the bipyramidal crystal and the rhomboidal crystal possess activity against Spodoptera exigua. The spores also show activity against Spodoptera exigua.

TABLE II No crystals or spores

C

C

*30 Rhomboidal bipyramidal crystals and spores Both crystals, no spores Bipyramidal crystals Rhomboidal crystals Spores Wet Weight 2.5 mg/well 5.0 mg/well 2.5 mg/well 10 mg/well 0.092 mg/well 0.48 mg/well 0.05 mg/well 0.1 mg/well mg/well 10 mg/well 20 mg/well Stunt score 1 0-1 1 0-1 1 0-1 1 0-1 0 0-1 0 *.35 6.7. EXAMPLE 7: BIOASSAY AGAINST DIABROTICA

UNDECIMPUNCTATA

The coleopteran activity of the whole culture broth of EMCC0075, prepared as described in EXAMPLE 1, is bioassayed against Diabrotica undecimpunctata using a microdiet incorporation bioassay. Specifically, artificial diet is prepared comprised of water, agar, 4 0 sugar, casein, wheat germ, methyl paraben, sorbic acid, linseed oil, cellulose, salts, propionic acid, phosphoric acid, streptomycin, chlortetracycline, and vitamins. The artificial diet is developed to allow samples consisting of rehydrated dry powders and liquids to be incorporated at a rate of 20% v/v. The test sample is prepared in microcentrifuge tubes to yield eight serial dilutions. The whole broth sample is tested neat at 200 pl/ml, and then diluted in 1 0.1% Tween 20T m to contain 132 pl/ml, 87 p./ml, 66 pl/mi, 44 pl/ml, 30 V/ml, 20 pI/ml, and 13 gV/ml. The molten mixture is vortexed and pipetted in 0.1 ml aliquots into 10 wells of a 96 well microtiter plate. Control samples containing 0.1% Tween 20 T M are dispensed into 16 wells. Once the diet has cooled and solidified, two neonateDiabrotica undecimpunctata larvae are added to each well, and the trays are covered with a perforated sheet of clear mylar. The trays are then incubated for five days at 28 2 0 C and 65% relative humidity.

After five days, insect mortality is rated. The mylar sheet is removed and each well of the microtiter plate is inspected using a dissecting microscope. Larvae that do not move when prodded with a dissecting needle are counted as dead. Percent mortality is calculated, and the data is analyzed via parallel probit analysis. The LCso, LC 90 slope of regression lines, coefficient of variation and potencies are determined.

i The results as shown in Table III indicate the whole culture broth from EMCCi: 0075 has a LC 50 and a LC90 of 51 p1/ml diet and 170 pl/ml diet, respectively, against Diabrotica undecimpunctata.

TABLE m *4

LC

0 o LCo Slope CV

N

.Ipml 51 170 2 7 8 6.8. EXAMPLE 8: PROTEIN SEQUENCING OF THE DELTA- ENDOTOXINS FROM THE RHOMBOIDAL

CRYSTAL

PROTEINS OF EMCC0075 60 .1 of 50% trifluoroacetic acid (TFA) are added to 25 pg of rhomboidal crystals. Four 15 pl aliquots of the mixture are spot dried onto a Biobrene-coated and TFApretreated microcartridge glass fiber filter. N-terminal sequencing is performed on a Applied Biosystems Inc. Protein Sequencer Model 476A with on-line HPLC and liquid phase TFA delivery. HPLC determination of phenylthiohydantoin-amino acids is achieved by using the Premix buffer system (ABI Inc.). Data is collected on a Macintosh IIsi using ABI's 610 data analysis software.

A double sequence is observed at approximately a 60/40 ratio. Data are analyzed and the sequences are sorted as follows: "MIVDL": MIVDLYRYLGGLAAVNAVLHFYEPRP (SEQ ID NO:1) "MKHHK": MKHHKNFDHI (SEQ ID NO:2) 6.9. EXAMPLE 9: CLONING OF THE GENES ENCODING THE "MIVDL" AND "MKHHK" PROTEINS" The amino acid sequence initially determined for the "MIVDL" protein, MIVDLYRYLGGLAAVNAVLHFYEPRP, is encoded by the sequence ATG ATH GTN GAY YTN TAY MGN TAY YTN GGN GGN YTN GCN GCN GTN AAY GCN GTN YTN CAY TTY TAY GAR CCN MGN CCN (SEQ ID NO:19). Based on this sequence, a 71 nt .5 oligomer is designed, where mixed deoxynucleotides are used at the 2-fold redundant positions and deoxyinosine at the 4-fold redundant positions to decrease both base discrimination at mismatches and selectivity at incorrect bases (Martin, F. and M. M. Castro, 1985, Nucleic Acids Res. 13: 892-8938): ATG ATI GTI GAY YTI TAY MGI TAY YTI GGI GGI YTI GCI GCI GTI AAY GCI GTI YTI CAY TTY TAY GAR CC (SEQ ID :2.0 The amino acid sequence determined for the "MKHHK" protein, namely, MKHHKNFDHI, permitted design of a more discriminating probe because of the absence of amino acids specified by more than two codons. Further discrimination is permitted by the assumption that As or Ts would be used in the coding sequence in preference to Gs or Cs, due to the overall low G C content of B. t. strains (approx 34 moles Claus, and R. C.

.25 W. Berkeley. 1986. Genus Bacillus, p. 1112. In P. H. A. Sneath Bergey's manual of systematic bacteriology, v. 2. The Williams and Wilkins Co., Baltimore). The following probe is synthesized: ATG AAA CAT AAA AAT TIT GAT CAT AT (SEQ ID NO:21). Both the MIVDL and the MKHHK probes are tailed with digoxygenin-dUTP according to the manufacturer's instructions (Boerhinger-Mannheim Genius System TM Users Guide, Version EMCC0075 genomic DNA is digested with EcoRI, EcoRV, HindII, PstI, or combinations of those enzymes overnight in buffers supplied by the manufacturers, electrophoresed through 0.8% agarose in 0.5X TBE (TRIS-borate-EDTA buffer; Sambrook et al., 1989, in Molecular Cloning, a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, transferred in 10X SSC to Boehringer Mannheim nylon membrane with a Stratagene Posiblotter in 10X SSC, and then probed as described below. The MIVDL probe, after hybridization and stringent washing at 48 0 C with 0.5X SSC, detected EcoRV and PstI fragments 12 kb or more in size, an EcoRI fragment of approx 10 kb, and a HindII fragment of approx 3.5 kb. The MKHHK probe, after hybridization and stringent washing at 48 0 C with 5 X SSC, detected the same size EcoRI, EcoRV, and PstI fragments as did the MIVDL probe. This result indicates that the two genes lie in close proximity to each other.

Additionally, the MKHHK probe detected a HindmII fragment of approx 6 kb.

To clone the HindI fragments encoding at least part of the "MIVDL" and "MKHHK" proteins, pUC 18 is digested with Hind and then treated with calf intestinal phosphatase to dephosphorylate the 5' ends and thus prevent vector religation. Restricted and phosphatased pUC118 is then mixed with EMCC0075 genomic DNA that had been previously 1 0 digested to completion with HindmI. After ligation, the reaction mix is used to transform

E.

coli strain XL1-Blue MRF' (Stratagene, Inc., La Jolla, CA). Colonies harboring the desired DNA fragment are detected by "colony hybridization" with the aforementioned "MIVDL" and "MKHHK" probes by the procedure described by Sambrook et al., 1989, Molecular cloning, A Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. Three .1 5 fragments are cloned with the "MIVDL" and "MKHHK" probes (see Figure E. coli containing the "13D" MIVDL gene fragment are referred to as EMCC0117 cells; E. coli containing the "8D-1" MKHHK gene fragment are referred to as EMCC0118 cells; E. coli containing the "2B" fragment of the MIVDL and MKHHK genes are referred to as EMCC0118 cells.

6.10. EXAMPLE 10: SEQUENCING OF THE GENES ENCODING

THE

"MIDVDL" AND "MKHHK"

PROTEINS

Nested deletions of three cloned fragments described in EXAMPLE 9 are performed according to the method of Henikoff (Gene 28:351-359, 1984) with a Promega 25 "Erase-a-Base" kit. Nested deletion sets encompassing the region of interest are sequenced by the dideoxy method (Sanger et al., 1977, PNAS USA 74:5463-5467) with an ABI 373A sequencer. Sequence correction is performed with SeqEd v 1.0.3; sequence is assembled with MacVector 4.1.1 and AssemblyLIGN v 1.0.7; and additional alignments and searches are performed with the IntelliGenetics Suite Programs, v 5.4.

The determined nucleotide (nt) sequence encoding the MKHHK and MIVDL proteins are shown in SEQ ID NO:39 and 40. The deduced amino acid sequence of the MKHHK and MIVDL proteins is shown underneath their corresponding DNA sequence. The amino acid sequence determined by N-terminal Edman degradation as described in EXAMPLE 8 is in complete agreement with the sequences deduced from the nucleotide sequence. The genomic DNA sequence is shown in SEQ ID NOS:41 (MKHHK and MIVDL), 44 (MKHHK), and 45 (MIVDL).

~r~-22 The MKHHK and MIVDL genes encode proteins with calculated molecular masses of 32,719 and 32,866 daltons. The MKHHK protein aligns poorly with any deduced protein from the EMBL, GeneSeq, or GenBank sequence databases. The MIVDL protein has weak regional homology with the 34 kdal gene of B. thuringiensis subsp. thompsoni as shown in Figure 3 (SEQ ID NO:42) (Brown and Whiteley, 1990, J. Bacteriology 174:549-557). In addition, the MIVDL protein has weak regional homologies with CryIA(a) (SEQ ID NO:43) (see Figure These weak homologies do not correspond to the any of the 5 conserved blocks of Cry toxins described by Hdfte and Whiteley (Microbiol. Rev. 53:242-255, 1989).

A nucleotide analysis of the region encoding the MKHHK and MIVDL genes 1 0 shows ribosome binding sites (AAGGAGT and AAGGTGG, respectively) that differ by one nucleotide with the canonical ribosome binding site of B. subtilis (AAGGAGG, which is presumably similar to the B. thuringiensis RBS). There is a reasonable transcriptional terminator downstream of the MIVDL gene.

T5 7. DEPOSIT OF MICROORGANISMS The following strains of Bacillus thuringiensis have been deposited in the Agricultural Research Service Patent Culture Collection Laboratory (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604, USA.

Strain Accession Number Deposit Date EMCC0075 NRRL B-21019 December 3, 1992 EMCC0076 NRRL B-21020 December 3, 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 2 5 Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 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 I I r-" 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.

o SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Novo Nordisk Entotech, Inc.

STREET: 1497 Drew Avenue CITY: Davis, California COUNTRY: United States of America POSTAL CODE (ZIP): 95616-4880 TELEPHONE: (916) 757-4700 TELEFAX: (916) 757-4789 (ii) TITLE OF INVENTION: NOVEL BACILLUS THURINGIENSIS STRAINS ACTIVE AGAINST LEPIDOPTERAN AND COLEOPTERAN PESTS (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Novo Nordisk of North America, Inc.

STREET: 405 Lexington Avenue, 64th Floor CITY: New York STATE: New York COUNTRY: USA ZIP: 10174-6401 COMPUTER READABLE FORM: MEDIUM TYPE: Tape COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE: 15-JUN-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/991,073 FILING DATE: 15-DEC-1992 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/264,100 FILING DATE: 22-JUN-1994 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/337,358 FILING DATE: 10-NOV-1994 (viii) ATTORNEY/AGENT INFORMATION: NAME: Agris Dr., Cheryl H.

REGISTRATION NUMBER: 34,086 REFERENCE/DOCKET NUMBER: 3778.404-WO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 212-867-0123 TELEFAX: 212-878-9655 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Met Ile Val Asp Leu Tyr Arg Tyr Leu Gly Gly Leu Ala Ala Val Asn 1 5 10 Ala Val Leu His Phe Tyr Glu Pro Arg Pro INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys His His Lys Asn Phe Asp His Ile 1 5 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTGCTCCAGC TGCTTGGCTC INFORMATION FOR SEQ ID NO:4: o SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTATACT TGGTTCAGGC CC 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GCACACCTTA CATTTTAAAG CA 22 I-r* INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGATTACAAG CGGATACCAA CATCGCG 27 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGGCACTTTC AAAATAACCA A 21 S INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCATCGGATA GTATTACTCA AATCCC 26 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGCTCTAACA TAGACCTTAT AA 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID t

-I

GACATTTCAT TAGGGCTTAT TAATTT 26 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CAGCGGACGG CCAGACCGCA AG 22 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GTCGGAGTCA ACAACCTTAG GGGC 24 24 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATCCGGAAAA GCCGCTATGT

C

INFORMATION FOR SEQ ID NO:14: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATCCGGAAAA GCCGCTATGT C 21 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGCCAGAAAA TGGAAAAATT TGGG 24 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GTGGGTACAG GAGGTACCAA A 21 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTGGGTACAG GAGGTACCAA A 21 S INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGAAATACTA TGAGTGTAAC TGC 23 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 54 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: YTNGGNGGNY TNGCNGCNGT NAAYGCNGTN YTNCAYTTYT AYGARCCNMG NCCN 54 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID ATGATGTGAY YTTAYMGTAY YTGGGGYTGC GCGTAAYGCG TYTCAYTTYT AYGARCC 57 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: ATGAAACATC ATAAAAATTT TGATCATAT 29 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: S: TGAATTCAT ATCTACTAAT GAGCAATCGA A 31 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CCACACGCCT AGATTCTCAT GC 22 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 46 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CGGGATCCAC AGTTACAGTC TGTAGCTCAA TTACCTACTT TTAACG 46 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid I STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGCCAAGGTT GCTGTAATAA TCG 23 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: S CTCAATATTC TCGAAGCTGG GGCC 24 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GCAGTCTGTA CGGAATTTAT ACA 23 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CGAGGGTTAG CAGATAGCTA TG 22 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AAGATGGGGC GGTCTAACTC C 21 INFORMATION FOR SEQ ID S S

C

*55* *5 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GACCGTTATC GGGTGAATCT

TTAG

INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TCGGCTGCAC TCTAAATTGT

TGAG

INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: TATTGAGTGA ATTATGGGGG AT INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ATGTTCTAAA TTCTAACATA

TCG

INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: TTATACCTAG ATCCTATTGT

TG

24 24 22 23 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID TAACATTTCC ACAC

T

TTTCA ATC 23 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: AAGGCTAGCG ACTGCTGTC 19 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 287 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Met Lys His His Lys Asn Phe Asp His Ile Val Trp Asp Phe Ala Glu 1 5 10 Lys Trp Thr Glu Gln Lys Gly Val Asp Leu Lys Arg Val Ser Tyr Val 20 25 Asp Pro Ile Thr Gly Glu Asp Thr Leu Glu Phe Ile Thr Lys Phe Asn 40 Tyr Val Gly Lys Leu Glu Glu Lys Ala Tyr Cys Pro Glu Val Ile Glu 55 Thr Gln Ser Phe Ser Asn Ser Asn Cys Asp Val Ser Arg Glu Phe Leu 70 75 Lys Lys Lys Val Asp Arg Lys Glu Cys Tyr Leu Trp Asp Ile Asp Tyr 90 Gly Phe Ile Ile Pro Thr Ser Val Leu Thr Asn Pro Leu Leu Pro Pro 100 105 110 Thr Leu Asn Glu Lys Ile Asn Pro Ala Met Glu Val Asp Leu Phe Lys 115 120 125 Ser Ala Asn Leu Phe Glu Ser Lys Leu Asn Asn Tyr Arg Met Ile Glu 130 135 140 a.

a a *aa.

a a Ala Gly Val Tyr Ile Glu Pro Asn Gin Al 145 150 Val Th-r Pro Lys Gin Val Gin Gin Asp Ty: 165 174 Ser Gly Ser Ile Ile Ile Giu Leu Lys Asj 180 185 Asp Lys Giu Thr Ile Giu Thr Ile Phe Ty3 195 200 Ile Tyr Arg Ser Giu Leu Aia His Asn His 210 215 Glu Thr Val Ile Phe Thr Gly Lys Gly Thi 225 230 Ser Asn Ile Phe Vai Glu Gly Glu Arg Phe 245 250 Cys Leu Gly Lys Tyr Val Ile Pro Leu Ser 260 265 Val Asp Cys Ile Ser Ile Phe Leu Asn Ser 275 280 INFORMATION FOR SEQ ID NO:38: SEQUENCE

CHARACTERISTICS:

LENGTH: 294 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Ile Val Asp Leu Tyr Arg Tyr Leu Gly 1 5 10 Ala Val Leu His Phe Tyr Glu Pro Arg Pro 25 Ser Glu Glu Tyr Asn Leu Ile Val Phe Gly 40 Ser Ile Asp Pro Ser Gin Ile Asn Ile Asn 55 Pro Val Asp Giu Ile Thr Ile Asn Asn Val 70 Ser Ser Arg Phe Glu Asn Thr Gly Phe Val 90 Thr Pro Giu Leu Ser Arg Thr Val Val Asn 100 105 Thr Thr Gly Tyr Lys Tyr Thr Gin Ser Leu 115 120 Ser Phe Asn Phe Pro Val Ala Giy Ala Giu 130 135 Val Gly Phe Giu Gin Asn Leu Ser Thr Thr aVal 155 r Cys 0 Ala *Thr Ser Phe 235 *Asp Sle Glu Thr Ile Tyr Val Phe 220 Lys Ser Glu Lys *Ala *Ser Asn Pro 205 His Giy Gin Lys Gly 285 Ser Leu Ala 190 Ilie Leu Leu Thr Lys 270 Gly Ilie Met 160 Giu Ile 175 Cys Thr Ala Asp Asp Gly Ilie Cys 240 Gly Gu 255 Asn Asn Ile Gly Asp Asp Asn Arg 75 Asp Ser Thr Asn Glu Leu Ile Arg Leu 60 Ser Thr Ile Val Asn 140 Thr Ala Cys Ile Ser Ile Glu Ser Ser 125 Ile Lys Ala Arg Pro Val Gin Asn Thr 110 Ser Ser Thr Val1 Asn Thr Asp Leu Tyr Ser Lys Phe Glu Asn Ile Phe Thr Ile Phe Thr Phe Ser Ser 145 Thr Ser Thr Leu Met 165 Arg Thr Ala Lys Arg 180 150 155 Arg Ile Pro Pro Gin Pro Val Ser Val Arg Pro 175 Ile Pro Val Giu Ile Ser 185 Phe Giu Leu Arg Ile Gin 195 Ile Ser Asn 210 Asn Giu Ile Ser His Ile Ser Gly 200 Ser Asp 215, Ser Tyr Gly Leu Phe Val Thr Gly Leu Pro Thr Asp Ser 225 Ile Asp Thr Gly Leu Cys Pro His Glu Asn 245 Asn Leu Thr 260 Asn 230 Arg Leu Tyr Ala Ile Asn Arg 235 Gly Leu Gin Asp Asp Phe Leu Arg 240 Leu Thr Arg Thr Gly Phe Gly Phe 265 250 Ala Ser Leu 255 Ser Leu Asp Ile Arg Thr 270 Ile Lys Arg Asp Leu Pro 275 Ser Asn Thr Ile Ile 280 Asn Ile Giu Asn Giu 285 Ala His 290 Ile Leu Thr Gin INFORMATION FOR SEQ ID NO:39: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 864 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoxnic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

ATGAAACATC

CAAAAGGGGG

TTIAGAGTTTA

GAAGTAATAG

AAGAAAAAAG

CCAACTTCGG

GCAATGGAAG

AGAATGATAG

G ITACACCAA ATCA TTGAGC

?I'CTATACCG

CATTAGATG

TCTAATATAT

ATAAAAATTr

TAGAT'ITAAA

TAACCAAATT

AAACACAATC

TAGACAGGAA

TACTTACAAA

TGGACTTAITr

AAGCAGGTGT

AACAAGTACA

TGAAAGATGC

TGCCAATTGC

GAGAAACTGT

TTGTAAGG

TGATCACATA G ITGGGACT TCGCTGAAAA

AAGGGTCAGT

TAATTATG'r P1'TTTCAAAC GGAATGTT1AT

TCCAITATTA

TAAAAGTGCA

TTATATTGAA

GCAAGATTAT

TTATAATIGCT

AGATATATAC

AATA'TrACA GGAAAGA'PrC

TATGTAGATC

GGGAAA ITAG

TCAAATTGTG

'ITATGGGATA

CCCCCCACTC

AACCTGTI'TG

CCAAATCAAG

TGTA ITAGCC

TGTACAGATA

AGATCCGAGC

GGGAAAGGTA

GATT~CTCAAA

CCATTACTGG

AAGAAAAAGC

ACGTrCGAG

TAGACTATOG

TCAATGAAAA

AATCCAAACT

CAGTAACCGC

TTGAGATTC

AAGAAACTAT

TTGCCCATAA

CGTTTAAAGG

CGGGGGAATG

GTGGACTGAA

TGAAGATACA

TTATTCCA

GGAA'TrCTA

GTTATATA

AATTAATCCA

AAATAA'PrAT

CAGCATAATG

AGGTAGTAT

TGAAACAATA

CCA'ITCCTTr

CTTAATATGT

?TTGGGGAAA

120 180 240 300 360 420 480 540 600 660 720 780 TATGTGATCC CAT'rAAGTAT AGAAAAGAAA AATAATGTAG ATI'GTATCTIC TATATITpTTA -AA'rrCAGAAA AAGGTGGGAT

TTAA

INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 885 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID ATGATAGTAG

A!PPTATATAG

TTITATGAGC

TT'TGGAGACC

TCTIGTGGACA

TCTAGTCGTr

TCTAGAACAG

TCCCTTACTG

ATTCATTTT

AC'rrCAACGC

AGGGTGAAA~

TITGTAACAG

TrAACACGGA

ATAGATCATG

ACATCATTAG

CACGCCCTGA

GTATAccA~c CTCCAGT3GA

TTGAAAATAC

TTIGTAAATAG

TITCATCCAA

CAGTAGGTTT

TTATGCGTAT

TATCGCTCTr

GTACTCTTCC

CTGATAGCCT.

AAAATAGGGG

ATT'GcAAT ATATTrrAGGT

TATATGTAGG

TTTAGCATA

TGAAATAACT

AGGATPI'GTC

CATATCTACA

ATTCTCC'PrT

TGAACAAAAC

ACCTCCACAA

TGAATT1GGCA

AACAAT'ITCA

ATGCCCTAAT

TrGGGA'PrA

IAGAACTACT

AAGAGCCCAT

GGATTGGCAG

AAT.ATA.AGCG

GATCCTTCGC

ATTAATAACG

GATACTGAAA

TOGACTACTA

AATTTCCCAG

C71wrCAACTA.

CCAGT'I'CCG

ATCCCTAGAA

AATTCGCATA

TCATATATTA

CAAGGCTrCG GAATATGA'pr

ATACTCACAC

CAGTAAA~c

AAGAATATAA

AAATAAATAT

T13AGAAGTAT ATTATTTAc

CAGGATATAA

TTGCGGGTGC

CAGAAACTAA

TAAGACCCAG

TACAAAACGA

TTT~CCGATCT

ACCGAGATGA

GTrCTCTCAC

TACCTTCAAA

AGTAA

k.GGAGTAATA2 k.AAGTGGACT C

E'GTGAAAT)

=TCT'ATrGT C I AGGGAATTTr c

CGTACTTCAC

CC11'ATAGTA TAACAATrrrA ACAArIWIAATA

TCCTGAAITA

GTACACTCAA

AGAAAATAAT

AACAGAAAGT

AACAGCAAAA

AArNTrCCGGA

TTATGCTGTA

CI'ITrAAGA

TGGAAATTT'A

TACAATTATA

kTTATGAAAC

AACAAAAGG

=CTTAGAG3T

CAGAAGTAA

TAAAGAAAA

120 180 240 300 360 420 480 540 600 660 720 780 840 885 120 180 240 300 AATATAGAGA

ACGAAATA

INFORM'ATION FOR SEQ, ID NO:41: SEQUENCE

CHARACTERISTICS:

LENGTH: 2101 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOL.ECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: ATTAAACACT AATACATTC ACATATT~CT

AACAAAGAAA.

ATCATAAAA TTITGATCAC ATAGTT1GGG

ACTTCGCTGA

GGGTAGATTr AAAAGGTC AGTTATGTAG ATCCCArrrAC TrATAACCAA ATTTAATTAT G'N'GGGAAAT

TAGAAGAAAA

TAGAAACACA ATCITIrCXA AACTCAAATr

GTGACGTTTC

AAGTAGACAG GAAGGAM'GT TAfIWTATCGG ATATAGACTA TGGGTTrA1'I ATACCAACTrr eGGTACTprA( AAGTGGAC7

TAGAAGCAG(

CAAAACAAGJ

AGCTGAAAG)

CCGTGCCAA9

ATGGAGAAAC

TATTG'o1-; TCCCATrTAAC

AAAAAGGTGC.

GTAAATGCCG

GATrGGCAGC

ATATAAGCGA

ATCC ITCGCA

TTIAATAACGT

ATACTGAAAA

CGACTACTAC

ATTTCCCAGT

TTCAACTAC

CAGT'rC CGT

TCCCTAGAAT

ATTCGCATAT

CATATkArA3A AAGGCTrCGG

AATATGAT

TACTCACACA

CTAGTGACAG

GTACCGTCAG

T'ITTAACAAC

:AAATCCATp r' ATITrAAAAJ ;TGTITAT7 P ACAGCAAGAT

TGCTTATAAT

TOCAGATATA

TGTAATATT

LAGGGGAAAGA

ITATAGAAAAG

GA'TrAACAT TACTTCACITr

AGTAAATGCC

AGAATATAAC

AATAAATATT

GAGAAGTATA

TTATITrACT

AGGATATAAG

TGCGGGTGCA

AGAAACTAAA

AAGACCCAGA

ACAAAAcGAA rTCCGATCT'r

CCGAGATGAC

PTCTCTCACT

ACC'ITCAAAT

GTAAITAATA

GGTTCTTG'CT

GAAAATGCG

GTTAAGAAAG

'ITACCCCCCA

GCAAACCTGT

GAACCAAATC

TATTGTATTA

GCTTGTACAG

TACAGATCCG

ACAGGGAAAG

ITCGATTCTC

AAAAATAATC

GATAGTAGAT

GATITAAACA

GTACI'CACT

CTTATAGTAT

AACAATTTAT

CAA'ITAATAT

CCTGAATTAT

TACACTCAAT

GAAAATAATA

ACAGAAAGTA

ACAGCAAAAA

ATTTCCGGAT

TATGCTGTAT

TTTAAGAA

GAAATTTAA

ACAATTATAA

GAAATAGACC

GTCGGACCGCA

ATT'TACAACG

TTTCAATGGT

CTCTCAATGA

TTGAATCCAA

AAGCAGTAAC

GCCTI'GAGAT

ATAAAGAAAC

ACCTTGCCCA

GTACGTTTAA

AAACGGGGGA

TAGMWITGTAT

TITATATAGAT

TGATAGTAGA

TTTATGAGCC

TTGGAGACCG

CTGTGGACAC

CTAGTCGT'T

CTAGAACAGT

CCCTTACTGT

TITCXITTTC

CTTCAACGCT

GGGTI'GAAAT

ITGTAACAGG

TAACACGGAC

TAGATCATCA,

CATCA ITAGA

ATATAGAGAA

GATAATCGGT

AGGTAGCAAA

CTAAGCCCAT

CTTAAAGAAT

AAAAATTAAT

ACTAAATAAT

CGCCAGCATA

TCAGGTAGT

TATGAAACA

TAACCAiITCC

AGCP'AATA

ATGITI'GGGG

CTCTATATT

A'rTAGGTGG TrTATATAGA

ACGCCCTGAT

TATACCAACT

TCCAGTGGAT

TGAAAATACA

TGTAAATAGc

TTCATCCAAA

AGTAGGT'r

TATGCGTATA

ATCGCTCTT 4

TACTCTTCCA.

T'GATAGCCTA

A.AATAGGGGT

7rI*IrPGCAATr

CGAAATAAAA

'ITrCCCCCTG

TITCTGAAGA

TITCCTGACG2

CTAATGAGAT

CCAGCAATlGG TATAGAATG3A

ATGGTTACAC

ATrATCA7'IrG

ATATTCTATA

TTTCATrTAG

TGTTCTAATA

AAATATGTGA

TTAAATTCAG

ATTGGCAGCA

TATTTrAGGTG

ATATGTAGGA

TTTAGCATAG

GAAATAACTA

GGATTTGTCG

ATATCTACAT

7TTCTC'TA GAACAAAACc

CCTCCACAAC

GAATTGGCAA

ACAATTTCAA

I'GCCCTAATT

TTGGGATTAC

AGAACTACTG

k.GAGCCCATA rCAAGTAGGC

:CCATATGGG

kTTCCCCCAT

.ATITCTCC

360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2101 INFORMATION FOR SEQ ID NO:42: Wi SEQUENCE CHARACTERISTICS: LENGTH: 310 amino acids TYPE: amino acid 36' (ii) (xi) STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID NO:42: Met Ala Ile Met Asn Pro Arg Pro Asp Ala Thr Thr Ser 65 Thr Thr Ala Thr Thr 145 Val1 Asp Gly Leu Giu 225 Ser Ser Gly Ser Pro 305 LTri Asr Prc 50 Val Ala Lys Thr 130 Asn Pro Phe Gin Tyr 210 His Asn Gin Tyr Asn 290 Asp 1Arg Pro *Ser *Ala *Val Phe 115 Thr Thr Pro Thr Thr 195 Ala Ile Giy Gly SerC 275 Ser Ile Gin Gly Gin Ser Thr 100 Asn Val1 Val His Val1 180 Gly Val1 kla 74 1 eu 6 0 er Ile Leu Asn Asp Phe 85 His Ala Ser Thr Ser 165 Pro Thr Aia Pro Gin 245 Tyr Glu Ala Phe Leu Leu 70 As n Gly Lys Thr Arg 150 Arg Val Leu Thr Pro 230 Ala Ser Thr Gly Asn Asn 55 Phe Giu Val1 Ile Giu 135 Gly Val1 Leu Pro Tyr 215 Ala Ile Val1 Arg *Pro *Tyr 40 Phe Asn .Ser Lys Leu 120 Tyr Trp Thr Gin Ala 200 Giu Leu Trp, Val1 Thr 280 Phe 25 Gin Ser Asn Arg Ser 105 Val Asn Ser Ala Asn 185 Gly Asn Phe Arg Arg 265 Giu Ile Ile Ile Val Thr Thr 90 Gly Lys Phe Ile Thr 170 Glu Asn Thr Arg Gly 250 Ile Tyr Ala Gin Asp Ala Arg Gly Val Ser 75 Val Val Ser Ser Pro 155 Leu Leu Pro Leu Ala 235 Thr Asp Tyr *Pro Asn Pro Val Val Thr le Ser 140 Ala Gin Ser Ser Leu 220 Ser Ala Giu Leu Leu 300 Gly Ile Glu Gin Glu Val Giu 125 Thr Gin Ile Leu Phe 205 Gly Asn rhr Arg Pro 285 Thr Giu Leu Gin Thr Ser 110 Gin Thr Pro Tyr Arg 190 Pro Arg Ala rhr Pro 270 /ai Ala Thr Vai Asp Ser Thr Ala Thr Thr Val1 Lys 175 Val Ser Ile Tyr Arg 255 Leu Thr Arg Pro Giu Phe Gin Ser Ser le Arg Leu 160 Gly Tyr Asp Arg Ile 240 ~ia Gin Ile Leu 295 Thr Pro Gly Ser 31y Ser Giu Ile Ile Ile Asn Pro INFOMTION FOR SEQ ID N4O:43: SEQUENCE CHARACTERISTICS: LENGTH: 358 amino acids TYPE: amino acid STRAN~DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Trp Val Arg Tyr Asn Gin Phe Arg Arg Giu Leu Thr Leu Thr Val Leu 1 5 10 Asp Ile Val Ala Leu Phe Ser Asn Tyr Asp Ser Arg Arg Tyr Pro Gly 25 Gly Ile Arg Thiz Val Ser Gin Leu Thr Arg Giu Ile Tyr Thr Asn Pro 40 Val Leu Cys Glu Asn Phe Ser Glu Asp Gly Ser Phe Arg Giy Met Ala 5560 Gin Arg Ile Glu Gin Asn Ile Arg Gin Pro His Leu Met Asp Ile Leu 70 75 Asn Ser Ile Thr Ile Tyr Thr Asp Val His Arg Giy Phe Asn Tyr Trp *85 90 Ser Gly His Gin Ile Thr Ala Ser Pro Vai Gly Phe Ser Giy Pro Giu 100 105 110 Phe Ala Phe Pro Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro Pro Val 115 120 125 Leu Val Ser Leu Thr Gly Leu Gly Ile Phe Arg Thr Leu Ser Ser Pro 130 135 140- Leu Tyr Arg Tyr Thr Gin Arg Ile Ile Leu Giy Ser Gly Pro Asn Asn .:145 150 155 160 Gin Giu Leu Phe Val Leu Asp Gly Thr Giu Asn Asn Phe Ser Phe Ala 165 170 175 Ser Leu Thr Thr Asn Leu Pro Ser Thr Ile Tyr Arg Gin Arg Gly Thr 180 185 190 Val Asp Ser Leu Asp Val Ile Pro Pro Gin Asp Asn Ser Val Pro Pro 195 200 205 Arg Ala Gly Lys Arg Val Glu Phe Ser Leu His Arg Leu Ser His Val 210 215 220 Thr Met Leu Ser Gin Ala Ala Gly Ala Val Tyr Thr Leu Arg Ala Pro 225 230 235 240 Thr Phe Ser Trp Gin His Arg Ser Ala Giu Phe Asn Asn Ile Ile Pro 245 250 255 Ser Ser Gin Ser Leu Ile Thr Gin Ile Pro Leu Thr Lys Ser Thr Asn 260 265 270 Leu Giy Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly 275 280 285 Asp Ile Leu Arg Arg Thr Ser Pro Gly Gin Ile Ser Thr Leu Arg Val 290 295 Asn Ile Thr Ala Pro Leu Ser Gin Arg Tyr Arg 305 310 315 Ala Ser Thr Thr Asn Leu Gin Phe His Thr Ser 325 330 Ile Asn Gin Gly Asn Phe Ser Ala Thr Met Ser 340 345 Gin Ser Gly Ser Phe Arg INFORMATION FOR SEQ ID NO:44: SEQUENCE

CHARACTERISTICS:

LENGTH: 980 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 300 Val Arg Ile Arg Tyr 320 Ile Asp Gly Arg Pro 335 Ser Gly Ser Asn Leu 350 9 9*

A'ITAAACACT

ATCATAAAAA

GGGTAGATTT

'rrATAAccAA

TAGAAACACA

AAGTAGACAG

CGGTACTTAC

AAGTGGAC'pr

TAGAAGCAGG

CAAAACA3AGT

AGCTGAAAGA

CCGTGCCAAT

ATGGAGAAAC

TATTTrGTTGA

TCCCATTAAG

AAAAAGGTGG

AAATACAITC

TTITIGATCAC

AAAAAGGGTC

ATTTAATTAT

ATC'rm'prCA

GAAGGAATGT

AAATCCArTrA A ITrAAAAGT TbTTTATATT

ACAGCAAGAT

TGCTTATAAT

TGCAGATATA

TGTAATAT'TT

AGGGGAAAGA

TATAGAAAAG

GATITrAACAT

ACAITA'ITCT

ATAGI'TGGG

AGTTATGTAG

GTTGGGAAAT

AACTCAAA'pr TATrTATGGG 'rrACCCCCCA

GCAAACCTGT

GAACCAAATC

TATTGTA7ITA

GCTTGTACAG

TACAGATCCG

ACAGGGAAAG,

ITCGATTCTC

AAAAATAATG

GATAGTAGAT

AACAAAGAAA

ACTTCGCTGA

ATCCCATrAC

TAGAAGAAAA

GTGACGTrC

ATATAGACTA

CTCTCAATGA

TTGAATCCAA

AAGCAGTAAC

GCCII'GAGAT

ATAAAGAAAC

AGCETrGCCCA

GTACGTTTAA

AAACGGGGGA

TAGATITGTAT

TTATATAGAT

AGGAGTAATA

AAAGTGGACT

TGGTGAAGAT

AGCTTATTGT

GAGGGAATT

TGGGTTTATT

AAAAAT'rAAT

ACTAAATAAT

CGCCAGCATA

TCAGGTAGT

TATTGAAACA

TAACCA'ITCC

AGGCTTAATA

ATG TTjGGGG

CTCTATATT

ATT'TAGGTGG

A'ITATGAAAC

GAACAAAAGG

ACATTAGAGT

CCAGAAGTAA

CTAAAGAAAA

ATACCAACTr

CCAGCAATOG

TATAGAATGA

ATGGTTACAC

ATTATCA'pIG ATA'PrCTATA ITTCAITrAG

TGTTCTAATA

AAATATGTGA

TTAAATTCAG

ATGGCAGCA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 980 GTAAATGCCG

TACTTCACTTI

INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 1121 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

A

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ 9* C

C

C C

C.

GAI'TAAACA

GTACTITCACT

C'ITATAGTAT

AACAATTITAT

CAATTAATAT

CCTGAXITTAT

TACACTCAAT

GAAAATAATA

ACAGAAAGTA

ACAGCAAAAA

ATTTCCGGAT

TATCCTCTAT

?TTI'AAGAA

GGAAXIVTrAA

ACAATTATAA

GAAATAGACC

GTGGACCGCA

ATTTACAACG

PITCAATGGT

TGATAGTAGA

TI'ATGAGCC

'IGGAGACCG

CTIGTGGACAC

CTAGTCGTTT

CTAGAACAGT

CCCTTACTGT

TTTCA ETITC

CTTCAACGCT

GGGTTGAAAT

TDGTAACAGG

TAACACGGAC

TAGATCATGA

CATCATITAGA

ATATAGAGAA

GATAATCGGT

AGGTAGCAAA

CTAAGCCCAT

CTTIAAAGAAT

TTT~ATATAGA

ACGCCCTGAT

TATACCAACT

TCCAGTGGAT

TCAAAATACA

TGTAAATAGC

TT1CATCCAAA

AGTAGGTTTIT

TATGCGTATA

ATCGCTC?1'T TACTC ITCCA

TGATAGCCTA

AAATAGGCGT

T'ICAITT

CGAAATAAAA

CTTCCCCCTG

TTCTGAAGA

TTTCCTGACG

CTAATGAGAT

TA TTAGGTG

ATATGTAGGA

TTTAGCATAG

GAAATAACTA

GGA7?IwrTCG

ATATCTACAT

TTCTCCTTTA

GAACAAAACC

CCTCCACAAC

GAAT'rGGCAA

ACAATTTCAA

TGCCCTAAT

TTGrGGA'ITAC

AGAACTACTC

AGAGCCCATA

TCAAGTAGGC

CCCATATGCG

ATT'CCCCCAT

GATTGGCAGC

ATATAAGCGA

ATCCTTCCCA

TAATAACGT

ATACTGAAAA

CGACTACTAC

A'TI'CCCAGT

TTTICAACTAC

CAGTTTCCGT

TCCCTAGAAT

A ITCGCATAT

CATATATTAA

AAGGCTTCGG

AATATGATTT

TACTCACACA

CTAGTGACAG

GTACCGTCAG

lliAACAAC

AGTAAATGCC

AGAATATAAc

AATAAATATT

GAGAAGTATA

TTAN~TrACT

AGCATATAAG

TGCGGGTGCA

AGAAACTAAA

AAGACCCAGA

ACAAAACGAA

TrCCGATCTr

CCGAGATGAC

TTCTCTCACT

ACC TTCAAAT

GTAATT~AATA

GGTI'CTTGCT

GAAAATGCGG

GTTAAGAAAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1121 CATTTTCTCC G

Claims (12)

1. 2. The nucleic acid of claim 1 wherein the nucleic acid sequence is essentially Jepicted in SEQ ID NO:39 or SEQ ID NO:44.
2. 3. A purified and isolated nucleic acid having a nucleic acid sequenceencodi.ng a; dclta-ndotoxin having a molecular weight of about 33.000 daltons and an amino acid seqluence essentially as depicted in SEQ ID NO:38 or a portion of said delta-endotoxin having insecticidal activity against an insect pest of the order Lepidoptera.
3. 4. A- nucleic acid of claim 3 wherein the nucleic acid is as essentially depicted in S1:Q ID NO:40 or SEQ ID A purified and isolated nucleic acid having a nucleic acid sequence essentially ais depicted in SEQ ID NO:41.
4. 6. A DNA construct comprising the nucleic acid of any one of claims
5. 7. A recombinant DNA vector comprising the DNA construct of claim 6; (b) S a promoter operably linked to the DNA sequence of(a); and a selectable marker.
6. 8. A host cell comprising the DNA construct of claim 6.
7. 9. A delta-endotoxin product of the expression in a procaryotic or eucaryotic host cell of a nucleic acid of any one of claims
8. 10. An insecticidal composition comprising the delta-endotoxin product of claim in association with an insecticidal carrier. II. The insecticidal composition of claim 10 in which the insecticidal composition further comprises spores of biologically pure Bacillus thuringiensis strain.
9. 12. The insecticidal composition of claim 10 or 11 which further comprises at ;o least two delta-endotoxins having a molecular weight of about 130,000 and activity a lagainst an insect pest of the order Lepidoptera.
10. 13. A method for controlling an insect pest of the order Lepidoptera comprising exposing the pest to an insect-controlling effective amount of an insecticidal composition of any one of claims 10-12.
11. 14. A method for controlling an insect pest of the order Coleoptera comprising exposing the pest to an insect-controlling effective amount of an insecticidal composition Ool claim 12. [R:\LIBXX]02756.doc:aak 42 An insecticidal composition of any one -of claims 10- 12 When used for controlling an insect pest of the order Lepidoptera. 1 6. Use of a delta-endotoxin product according(- to claim 9 for the preparation of din insecticidal composition for controlling an insect pest of the order Lepidoptera.
12. 17. An insecticidal composition of any one of claims 10-1 2 when used for controlling an insect pest of the order Coleoptera. 1 8. Use of a delta-endotoxin product according to claim 9 for the preparation of ain insecticidal composition for controlling an insect pest of the order Coleoptera. Dated 18 June, 2001 I Abbott Laboratories Patent Attorneys for the Applicant/Nominated Pecrson SPRUSON FERGUSON 41* R:LI BXX]02 756.doc: aak