AU9737601A - Bacillus thuringiensis toxins - Google Patents

Description

S&FRef: 476641D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Mycogen Corporation 5501 Oberlin Drive San Diego California 92121 United States of America H. Ernest Schnepf, Kenneth E. Narva, Judy Muller-Cohn Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Bacillus Thuringiensis Toxins The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c BACILLUS THURINGIENSIS TOXINS Background of the Invention The soil microbe Bacillus thuringiensis is a Gram-positive, spore-forming bacterium traditionally characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. toxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. toxin delivery vehicles (Gaertner, L. Kim [1988] TIBTECH 6:S4-S7; Beegle, T. Yamamoto, "History of Bacillus thuringiensis Berliner research and development," Can. Ent. 124:587-616). Thus, isolated B.t. toxin genes are 0 becoming commercially valuable. Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range oflepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline 6- endotoxin which is toxic to the larvae of a number of lepidopteran insects.

Investigators have now discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of namely israelensis and morrisoni (a.k.a.

tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F.H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R.M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, TL. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; and Beegle, C.C. (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, AM. Huger, GA. Langenbruch, W. Schnetter (1983) Z.

ang. Ent. 96:500-508 describe Bacillus thuringiensis var. tenebrionis, which is reportedly active 2 against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.

More recently, new subspecies of B.t. have been identified, and genes responsible for active 6-endotoxin proteins have been isolated (Hofte, H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were Cryl (Lepidoptera-specific), Cryl (Lepidoptera- and Diptera-specific), CryTIf (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. Payne, L. Kim [1992] Bio/Technology 10:271-275). CryV has been proposed to designate a class of toxin genes that are nematodespecific. Lambert et al. (Lambert, L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbiol 62(1):80-86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B. isolates active against lepidopteran pests. Gleave et al. ([1991] JGM 138:55-62), Shevelev et al. ([1993] FEBSLett. 336:79-82; and Smulevitch et al. ([1991] FEBS Lett. 293:25-26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.

The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described m the published literature (Schnepf, H.R. Whiteley [1981] Proc. Natl. Acad. Sci.

USA 78:2893-2897.). U.S. Patent 4,448,885 and U.S. Patent 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Patents 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose B.t. toxins having activity against lepidopterans. PCT application W096/05314 discloses PS86W1, PS86V1, and other B.t.

isolates active against lepidopteran pests. The PCT patent applications published as W094/24264 and W094/05771 describe B.t. isolates and toxins active against lepidopteran pests. B.t. proteins with activity against members of the family Noctuidae are described by Lambert et al., supra. U.S. Patents 4,797,276 and 4,853,331 disclose B. thuringiensis strain tenebrionis which can be used to control coleopteran pests in various environments. U.S. Patent No. 4,918,006 discloses B.t. toxins having activity against dipterans. U.S. Patent No. 5,151.363 and U.S. Patent No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Other U.S. patents which disclose activity against nematodes include 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; and 5,439,881.

A cry2Aa gene from HD263 kurstaki is disclosed by Donovan et al. in 264 JBC 4740 (1989). Another cry2Aa gene and a cry2Ab gene, from HD1 kurstaki, are disclosed by Widner Whiteley, 171 J. Bac. 965-974 (1989). Another cry2Ab gene from HDI kurstaki is disclosed by Dankocsik et al. in 4 Mol. Micro 2087-2094 (1990). A cry2Ac gene from B.t.S-1 (shanghai) is disclosed by Wu et al. in 81 FEMS 31-36 (1991).

An isolate known as PS192M4 is disclosed in U.S. Patent No. 5,273,746 as having activity against lice.

The PS8612 isolate is disclosed in U.S. Patent No. 5,686,069 as having activity against lepidopterans. PS91C2 is exemplified therein as producing a CryIF(b)-type of lepidopteranactive toxin, the sequence of which is disclosed therein.

Sequence information for a lepidopteran-active toxin from HD525 and the sequence of a lepidopteran-active toxin from HD573 are disclosed in WO 98/00546. Those toxins are not Cry2-type toxins. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review.

However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art. U.S. Patent No. 5,506,099 describes methods for identifying unknown B.t. isolates. U.S. Patent No. 5,204,237 describes specific and universal probes for the isolation of B.t. toxin genes. These patents, however, do not describe the probes and primers of the subject invention. Brief Summary of the Invention The subject invention concerns materials and methods useful in the control of nonmammalian pests and, particularly, plant pests. In a specific embodiment, the subject invention provides new toxins useful for the control of lepidopterans. A preferred embodiment of the subject invention further provides nucleotide sequences which encode the novel lepidopteranactive toxins of the subject invention.

The subject invention further provides nucleotide sequences and methods useful in the identification and characterization of novel genes which encode pesticidal toxins. In one embodiment, the subject invention concerns unique nucleotide sequences which are useful as primers in PCR techniques. The primers produce characteristic gene fragments which can be used in the identification and isolation of novel toxin genes. A further aspect of the subject invention is the use of the disclosed nucleotide sequences as probes to detect genes encoding B.t.

toxins which are active against lepidopterans.

Further aspects of the subject invention include other novel genes and toxins identified using the methods and nucleotide sequences disclosed herein, in addition to the novel genes and toxins specifically disclosed herein. The genes thus identified encode toxins active against lepidopterans. Similarly, the isolates capable of producing these toxins have activity against these pests. Thus, the subject invention further provides new Bacillus thuringiensis isolates having pesticidal activities which are found with the primers and probes according to the subject invention.

In one embodiment of the subject invention, B.t. isolates can be cultivated under conditions resulting in high multiplication of the microbe. After treating the microbe to provide single-stranded genomic nucleic acid, the DNA can be contacted with the primers of the invention and subjected to PCR amplification. Characteristic fragments of toxin-encoding genes are amplified by the procedure, thus identifying the presence of the toxin-encoding gene(s).

In a preferred embodiment,-the subject invention concerns plants cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by the target pests. Such transformation of plants can be accomplished using techniques well known to those skilled in the art and would typically involve modification of the gene to optimize expression of the toxin in plants. In addition, the toxins of the subject invention may be chimeric toxins produced by combining portions of multiple toxins.** As an alternative to the transformation of plants, the B.t. isolates and toxins of the subject invention, or recombinant microbes expressing the toxins described herein, can be used to control pests. In this regard, the invention includes the treatment of substantially intact B.t.

cells, and/or recombinant cells containing the expressed toxins of the invention, treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active upon ingestion by a target insect.

Brief Description of the Sequences SEQ ID NO. 1 is a forward primer useful according to the subject invention.

SEQ ID NO. 2 is a reverse primer useful according to the subject invention.

SEQ ID NO. 3 is a nucleotide sequence which encodes the 192M4 toxin.

SEQ ID NO. 4 is the predicted amino acid sequence of the 192M4 toxin.

SEQ ID NO. S is a nucleotide sequence which encodes the HD573 toxin.

SEQ ID NO. 6 is the predicted amino acid sequence of the HD573 toxin.

SEQ ID NO. 7 is a nucleotide sequence which encodes the HD525 toxin.

SEQ ID NO. 8 is the predicted amino acid sequence of the HD525 toxin.

SEQ ID NO. 9 is a nucleotide sequence which encodes the 8612 toxin.

SEQ ID NO. 10 is the predicted amino acid sequence of the 8612 toxin.

Detailed Disclosure of the Invention The subject invention concerns materials and methods for the control of non-mammalian pests. In specific embodiments, the subject invention pertains to new Bacillus thuringiensis toxins, and genes encoding toxins, which have activity against lepidopterans. The subject invention concerns not only the polynucleotide sequences which encode these toxins, but also the use of these polynucleotide sequences to produce recombinant hosts which express the toxins. The subject invention further concerns novel nucleotide sequences that are useful as primers and probes for Bacillus thuringiensis genes that encode pesticidal toxins, especially lepidopteran-active toxins. The subject invention still further concerns novel methods for identifying and characterizing B.t. isolates, toxins, and genes with useful properties. The new toxins and polynucleotide sequences provided here are defined according to several parameters. One critical characteristic of the toxins described herein is pesticidal activity. In a specific embodiment, these toxins have activity against lepidopteran pests. The toxins and genes of the subject invention can be further defined by their amino acid and nucleotide sequences. The sequences of the molecules can be defined in terms of homology or identity to certain exemplified sequences as well as in terms of the ability to hybridize with, or..

be amplified by, certain exemplified probes and primers. The toxins provided herein can also be identified based on their immunoreactivity with certain antibodies.

Methods have been developed for making useful chimeric toxins by combining portions of B.t. crystal proteins. The portions which are combined need not, themselves, be pesticidal so long as the combination of portions creates a chimeric protein which is pesticidal. This can be done using restriction enzymes, as described in, for example, European Patent 0 228 838; Ge, N.L. Shivarova, D.H. Dean (1989) Proc. Natl. Acad. Sci. USA 86:4037-4041; Ge, A.Z., D. Rivers, R. Milne, D.H. Dean (1991) J. Biol. Chem. 266:17954-17958; Schnepf, K.

Tomczak, J.P. Ortega, H.R. Whiteley (1990) J. Biol. Chem. 265:20923-20930; Honee, D.

Convents, J. Van Rie, S. Jansens, M. Peferoen, 3. Visser (1991) Mol. Microbiol. 5:2799-2806.

Alternatively, recombination using cellular recombination mechanisms can be used to achieve similar results. See, for example, Caramori, A.M. Albertini, A. Galizzi (1991) Gene 98:37- 44; Widner, H.R. Whiteley (1990) J. Bacteriol. 172:2826-2832; Bosch, B. Schipper, H. van der Kliej, R.A. de Maagd, WJ. Stickema (1994) Biotechnology 12:915-918. A number of other methods are known in the art by which such chimeric DNAs can be made. The subject 6, invention is meant to include chinmeric proteins that utilize the novel sequences identified in the subject application.

With the teachings provided herein, one skilled in the art could readily produce and use the various toxins and polynucleotide sequences described herein.

B.I. isolates useful according to the subject invention have been deposited in the peirnanent collection of the Agricultural Research Service Patent Culture Collection

(NRRL),

Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA.

The culture repository numbers of the B. t. strains are as follows: Table 1.

B.I. Isolate Repository No. Deposit Date PS86I2 NRRL B-21957 March 12, 1998 PS192M4 NRRLB1- 18932 December 27. 1991 Table 2.

Source Isolate E coli Strain Plasmid Repository No. Deposit Date PS I92M4 MR908 pMYC2586 NR.RL B-21631 October 17, 1996 HD573 MR909 pMfYC2587 NRRL B-21632 October 17, 1996 HD525 MR910 WMC2588 NRRL B-21633 October 17. 1996 Cultures have been deposited under conditions that assure that access to the cultures is available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1. 14 and 35 U.S.C. 122. The deposits will be 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.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the-Deposit of Microorganisms, they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture(s). The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of a deposit. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

The isolates HD525 and HD573 are available fromm the USDA-ARS NRRL Culture Collection, Peoria, Illinois.

Following is a table which provides characteristics of certain isolates useful according to the subject invention.

Table 3. Description of native B.t. strains Strain Inclusion Type H-Serotype SDS-PAGE protein profile 192M4 Amorphic 4a4b, sotto 130, 68 8612 Bipyramidal 8 130, 30, 15 HD525 Bipyramidal with ORT not motile 130 HD573 Bipyramidal not motile 140. 130. Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the novel toxins specifically exemplified herein. Chimeric genes and toxins, produced by combining portions from more than one B.t. toxin or gene, may also be utilized according to the teachings of the subject invention. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the exemplified toxins.

It should be apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be 8 obtained using a variety of restr-iction enzymes. Proteases may be used to directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can be derived from B.tI. isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example. antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other B.t. toxins.

Fragments and equivalents which retain the Desticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition.

Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention also relates to variants or equivalents of novel genes and toxins having the same or similar pesticidal activity of the exemplified novel toxins. Equivalent toxins will have amino acid homology with a novel exemplified toxin. These equivalent genes and toxins will typically have greater than 60% identity with the sequences specifically exemplified herein; preferably, there will be more than 75% identity, more preferably greater than 80%, most preferably greater than 90%, and the identity can be greater than 95%. The amino acid homology will be highest in critical regions of the toxin which accounit for biological activity or are involved in the determ ination. of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, conservative substitutions whereby an amino acid of one class is replaced. with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 4 provides a listing of examples of amino acids belonging to each class.

9 Table 4.

Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic Asp, Glu Basic Lys, Arg, His In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.

The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions, which are described above. Although novel crystal proteins are specifically exemplified herein, isolates for use according to the subject invention can be grown under conditions that facilitate the secretion of toxins. Thus, the supernatant from these cultures can be used to obtain toxins according to the subject invention. Thus, the subject invention is not limited to crystal proteins; useful soluble proteins are also contemplated.

As used herein, reference to "isolated" polynucleotides and/or "purified" toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to "isolated and purified" signifies the involvement of the "hand of man" as described herein. Chimeric toxins and genes also involve the "hand of man." The use of oligonucleotide probes provides a method for identifying the toxins and genes of the subject invention, and additional novel genes and toxins. Probes provide a rapid method for identifying toxin-encoding genes. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures, for example.

Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the production and maintenance of'the pesticide. With suitable microbial hosts, Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is a control of the pest. Alternatively, the microbe hosting the toxin gene can be killed and treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.

A wide variety of methods are available for introducing a B-t. gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in United States Patent No. 5,135,867, which is incorporated herein by reference.

Altemnatively, a plant transformed to express a toxin of the subject invention can be used to contact the target pest with the toxin. Synthetic genes which are functionally equivalent to the novel toxins of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,83 1.

Treatment of cells. As mentioned above, B.t. or recombinant cells expressing a B.t.

toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide 0 microcapsule that is formed comprises the B.i. toxin within a cellular structure that has been stabilized and will protect the toxin when the inicrocapsule is applied to the environment of the target pest. Methods for treatment of microbial cells are disclosed in United States Patent Nos.

4,695,455 and 4,695,462, which are incorporated herein by reference.*0 Growth of cells. The cellular host containing the B.t. insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene.

These cells may then be harvested in accordance with conventional ways. Alternatively, the: cells can be treated prior to harvesting.

The cells of the invention can be cultured using standard art media and fermentation: techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the .00 art. Any B.t. spores and crystals can be recovered employing well-known techniques and used as a conventional 8-endotoxin B.t. preparation. For example, the spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art. Alternately, the supernatant from the fermentation process can be used to obtain toxins according to the present invention. Soluble, secreted toxins are then isolated and purified employing well-known techniques.

Methods and formulations for control of ests. Control of lepidopterans using the isolates, toxins, and genes of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of B. I isolates to the pests (or their location), the application of recombinant microbes to the pests (or their locations), and the transformation of plants with genes which encode the pesticidal toxins of the subject invention. Recombinant microbes may be, for example, a E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.

Formulated bait granules containing an attractant and toxins of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of B.t. cells may be employed as liquids, wettable powders, granules, or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least about 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 102 to about 104 cells/mg. These formulations that contain cells will be administered at about 50 mg (liquid or.

dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, soil and foliage, by spraying, dusting, sprinkling, or the like. Mutants. Mutants of novel isolates obtainable according to the invention can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

Polvnucleotide probes. Hybridization may be used to test whether two pieces of DNA are complementary in their base sequences., It is this hybridization mechanism which facilitates the use of probes of the subject invcntion to readily detect and characterize DNA sequences of interest. The probes may be RNA or DNA. The probe will normally have at least about bases, more usually at least about 18 bases, and may have up to about 50 bases or more, usually not having more than about 200 bases if the probe is made synthetically. However, longer 12 probes can readily be utilized, and such probes can be, for example, several kilobases in length.

The probes may be labeled utilizing techniques which are well known to those skilled in this art.

One approach for the use of the subject invention as probes entails first identifying by Southern blot analysis of a gene bank of the B.t. isolate all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the probable activity of many new B.t. isolates, and of the individual endotoxin gene products expressed by a given B.t. isolate. Such a probe analysis provides a rapid method for identifying potentially commercially valuable insecticidal endotoxin genes within the multifarious subspecies of B.t.

The particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied. The nucleotide segments of the subject invention which are used as probes can be synthesized by use of DNA synthesizers using standard procedures. In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels.

Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under stringent conditions by techniques well known in the art, as described, for example, in Keller, M.M. Manak (1987) DNA Probes, Stockton Press, New York, NY., pp. 169-170. As used herein "stringent" conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by standard methods (Maniatis et In general, hybridization and subsequent washes were carried out under stringent conditions that allowed for detection of target sequences with homology to the exemplified toxin genes. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, K.A. Jacobs, T.H.

Eickbush, P.T. Cherbas, and F.C. Kafatos [1983] Methods ofEnzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285).

Tm=81.5 C+16.6 Log[Na+]+0.41(%G+C)-0.61(%formamide)-600/length of duplex in base pairs.

13 Washes are typically carried out as follows: Twice at room temperature for 15 minutes in IX SSPE, 0.1% SDS (low stringency wash).

Once at Tm-20 0 C for 15 minutes in 0.2X SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at 10-20 0 C below the melting temperature (Tm) of the hybrid in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula: Tm (0 C)=2(number T/A base pairs) +4(number G/C base pairs) (Suggs, T. Miyake, E.H. Kawashime, M.J. Johnson, K. Itakura, and R.B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D.D. Brown Academic Press, New York, 23:683-693).

Washes were typically carried out as follows: Twice at room temperature for 15 minutes IX SSPE, 0.1% SDS (low stringency wash).

Once at the hybridization temperature for 15 minutes in X SSPE, 0.1% SDS (moderate stringency wash).

PCR technology. The DNA sequences of the subject invention can be used as primers* for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5' end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 Generia CuGturing Methods for B.t. Isolates Referred to Herein A subculture ofB.t. isolates, or mutants thereof, can be used to inoculate the following peptone, glucose, salts medium: Bacto Peptone 7.5 g/l Glucose 1.0 g/l 14 KHPO, 3.4 g/1 KHPO 4.35 g/l Salt Solution 5.0 ml/1 CaCI, Solution 5.0 ml/1 pH 7.2 Salts Solution (100 ml) MgSO 4 -7H,O 2.46 g MnSO 4 -H20 0.04 g ZnSO 4 -7H20 0.28 g FeSO,-7H,O 0.40g CaC12 Solution (100 ml) 3.66 g The salts solution and CaC1, solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30°C on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art. The B.t. toxins obtainable with the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, centrifugation. Example 2-Identification of Genes Encoding Novel Lenidopteran-Active Bacillus thuringiensis Toxins A DNA-based polymerase chain reaction (PCR) technique was used for the identification and classification of novel toxin genes in B.t. strains. Two PCR primers useful for the identification of toxin genes (Forward 1 and Reverse 1) were designed. These primers contain degenerate codons in the nucleotide positions designated by ambiguity codes, and have restriction sites incorporated into the 5' ends to enable molecular cloning of novel, amplified DNA fragments. The sequences of these oligonucleotides are: Forward 1: GGCCACTAGT AAAAAGGAGA TAACCATGAA TAATGTATTG AATARYGGAA T 3' (SEQ ID NO. 1) Reverse 1: GGCCCTCGAG GGTACCCAAA CCTTAATAAA GTGGTGRAAK ATTAGTTGG -3' (SEQ ID NO. 2) Primers were synthesized using an Applied Biosystems model 381A DNA synthesizer.

Toxin genes were then amplified from genomic B.t. DNA templates with these primers by standard PCR protocols (Perkin-Elmer) as follows: DNA templates for PCR were prepared from B.t. cells grown for 18 hours on agar plates. A loopful of cells were resuspended in TE buffer containing 50 gg/ml proteinase K and incubated at 55 °C for 15 minutes. The cell suspensions were then boiled for 15 minutes. Cellular debris was pelleted in a microfuge, and the supernatant containing the DNA was transferred to a clean tube. Ten pl of this crude genomic DNA template was then used in a 100 pl PCR reaction mixture comprised of 50 mM KCI, 10 mM Tris-CI (pH 1.5 mM MgCI,, 200 pM each dNTP, 0.1-1 IM each primer, and 2.5 units of Taq DNA polymerase.

Example 3 Restriction Fragment Length Polymorphism (RFLP) Analysis of Bacillus thuringiensis Toxin Genes *o PCR amplification using primer pair 1 (Forward I and Reverse 1)is expected to yield DNA fragments approximately 1900 base pairs in length from B.t. toxin genes related to the crv2 subfamily. Amplified gene sequences were discriminated from one another, and from known genes, by comparing the sizes of DNA restriction fragments generated by digestion of the PCR products with, for example, BgffI, HincI, Scal, or HinFI (Table Briefly, approximately 0.25 l g DNA from a PCR reaction was digested with a given restriction enzyme and electrophoresed on an agarose or polyacrylamide gel. The gel was then stained with cthidium bromide and DNA restriction fragments were visualized by illumination with UV light at 260- 280 nm. The sizes of the restriction fragments were determined by their electrophoretic mobility relative to standard DNA fragments of known sizes. In some strains the number of fragments suggested the presence of more than one amplified toxin gene.

Table 5. Sizes of restriction fragments obtained by digestion of PCR-amplified

DNA

B.t. toxin gene Restriction (GenBank Accession enzyme Approximate DNA fragment size (base pairs) Number) or source strain cry2Aal (M31738) BgIII 616, 1333 cry2Aal (M3 1738) Scal 937, 1012 c-iy2Aal (M3 1738) HinFI 51, 223, 340, 363, 375.,597 crv2Ab I (M23724) HindI 815. 1134 ci-y2Abl (M23724) HinFI 51, 105, 112,223, 263, 363, 832 cry2Ac (X57252) Scal 185.,1731 cry2Ac (X57252) HinFI 112,223, 244, 293, 360, 684 PS I92M4 BgII 616.,1339 PS I92M4 HincII 813, 1135 PS 192M4 ScaI 943, 1012 PS I92M4 HinFI 51,112,175,188,223,261,269,340363,597, 1161 HD573 Hind~ 813, 1135 HD573 ScaI 185, 1734 HD573 HinFI 112,223,244,261,293,360363687,1161 HiD525 Hincfl 813, 1135 HD525 ScaI 185, 1734 HD525 HinFI 51,112,223,244,261,293,360,363,687,1161 PS8612 Hindl 793,1109 PS8612 HinFI 51.112.263.341.1135 Genes from strains with unique restriction fragment polymorphisms were cloned into pBluescript SK (Stratagene, San Diego, CA) and transformed into E. coli NM522 for further study. Subcultures of recombinant E. coli strains harboring these plasmids encoding these new toxins were deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Illinois 61604 on October 17, 1996.

ExaMple 4 DNA Seuence Analysis of Novel Toxin Genes DNA templates for automated sequencing were amplified by PCR using vector primers.

These DNA templates were sequenced using Applied Biosystems (Foster City, CA) automated sequencing methodologies. Novel toxin gene sequences (SEQ ID NOs. 3, 5, 7, and 9) and their respective predicted polypeptide sequences (SEQ ID NOs. 4, 6, 8, and 10) are listed in Table 6, below.

Table 6.

Source Strain Nucleotide SEQ ID NO. Peptide SEQ ID NO.

192M4 3 4 HD573 5 6 HD525 7 8 8612 9 Example 5 Heterologous Expression of Novel B.t. Toxins in Pseudomonasflorescens The toxin genes listed above were engineered into plasmid vectors by standard DNA cloning methods, and transformed into Pseudomonasfluorescens. Recombinant bacterial strains were grown in shake flasks for production of toxin for expression and quantitative bioassay against a variety of lepidopteran insect pests.

Example 6 Activity of Novel B.t. Toxins Against Heliothis virescens (Fabricius) and Helicoverpa zea (Boddie) Suspensions of powders containing recombinant clones according to the subject invention were prepared by individually mixing powder samples with distilled water and agitating vigorously. Suspensions were mixed with toasted soy flour artificial diet at a rate of 6 mL suspension plus 54 mL diet, yielding a concentration of 100 pg toxin/mL finished diet.

After vortexing, this mixture was poured into plastic trays with compartmentalized 3 ml wells (Nutrend Container Corporation, Jacksonville, FL). A water blank containing no recombinant toxin served as the control. First instar larvae (USDA-ARS, Stoneville, MS) were placed singly into the diet mixture. Wells were then sealed with "MYLAR" sheeting (ClearLam Packaging, IL) using a tacking iron, and several pinh-oles were made in each well to provide gas exchange.

Larvae were held at 25°C in a 14:10 (light:dark) holding room. Mortality was recorded after six days.

Table 7. H. virescens larval mortality with toxins in diet incorporation bioassays Source Strain Percent Mortality 192M4 87 HD573 HD525 17 water control 8 Table 8. H. zea larval mortality with toxins in diet incorporation bioassays Source Strain Percent Mortality 192M4 19 HD525 21 water control 8 Example 7 Activity of Novel B.t. Toxins Against Ostrinia nubilalis (Huebner) Test suspensions were prepared in 0.5 ml or 1 ml volumes by mixing powder samples with distilled water. Test suspensions were held in sterile-packaged 12 x 75 mm polypropylene tubes with snap cap Elkay Laboratory Products). Tubes were placed in hot block Fisher Scientific Hot Block) prewarmed to 34-35 C approximately 15 minutes (or less) prior to dispensation of the diet. The test suspensions were vortexed for a few seconds just prior to the addition of the diet to the 12 x 75 mm tube. To the 0.5 ml or 1 ml volumes was added 1 or 2 ml diet, respectively. The diet was measured and squirted into the tube by means of a 3 ml or ml syringe with rubber tip plunger. The tube with the test suspension and diet was vortexed for 5-10 seconds or until visibly mixed. The toxin/diet suspension was then dispensed into a prelabeled 96-well assay tray.

Diet was dispensed into the 96-well assay tray by means of a rcpeatc-: pipettr vith a 1.25 ml capacity pipet tip at a 4 setting for approximately 100 pl per well.

Larvae were infested one per well and sealed with waxy adhesive covering by heat treatment with iron (Oliver Products, MI). Bioassays were held at 26-28 0 C, and data were collected in 7 days.

19 Table 9. 0. nubilalis larval mortality with toxins in diet incorporation bioassays Source Strain Percent Mortality 192M4 HD573 water control 8 Example 8 Insertion of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with genes encoding the insecticidal toxin of the subject invention. The transformed plants are resistant to attack by the target pest.

Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary.

If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1- 46; and An et al. (1985) EMBOJ. 4:277-287.

Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

A large number of techniques are available for inserting DNA into a plant host cell.

Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which* are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, meristematic tissue, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

In biolistics transformation, plasmid DNA or linear DNA can be employed.

The transformed cells are regenerated into morphologically normal plants in the usual manner. If a transformation event involves a germ line cell, the inserted DNA and corresponding phenotypic trait(s) will be transmitted to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Patent No.

5,380,831. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about 80% of the full length toxin. Methods for creating synthetic B.t. genes for use in plants are known in the art.

All of the U.S. patents cited herein are hereby incorporated by reference.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

*go@ 0 0 *o0 o@ **o Page(s) 9 -1 are claims pages they appear after the sequence listing SEQUENCE LISTING GENERAL INFORMATION: APPLICANT INFORMATION: Applicant Name(s) MYCOGEN CORPORATION Street address: 5501 Oberlin Drive City: San Diego State/Province: California Country: US Postal code/Zip: 92121 Phone number: (619) 453-8030 Fax number: (619)453-6991 Telex number: (ii) TITLE OF INVENTION: Toxins Active Against Pests (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Saliwanchik, Lloyd Saliwanchik STREET: 2421 N.W. 41st Street, Suite A-i CITY: Gainesville STATE: Florida COUNTRY: USA ZIP: 32606 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn o (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: o FILING DATE: CLASSIFICATION: (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Sanders, Jay M.

REGISTRATION NUMBER: 39,355 REFERENCE/DOCKET NUMBER: MA-709 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (352) 375-8100 TELEFAX: (352) 372-5800 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 23 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:!: GGCCACTAGT AAAAAGGAGA. TAACCATGAA TAATGTA'G AATARYGGAA T INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: nucleic acid STRANDEflNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGC'CCTCGAG GGTACCCAAA CCTTAATAAA GTGGTGRAAK ATTAGTTGG INFORMATION FOR SEQ ID NO:3: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1908 base pairs TYPE: nucleic acid STRANDEflNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: p p. ATGAATAATG TATTGAATAG TGGAAGAACA ACTATTTGTA

CACGATCCAT

TGGAAAAGAA

CTGCTAAAGA

ATATTTCCTA

CTAAATCAAA

CAAGCGAATA

CCTGTTCCTT

TTACCCCAGT

GCCAATATGC

TCAGCAGCAA

TTAGTTTTGA

CAGATCATAG

AAGTGGGGAG

GTGGTAGTAC

GACTTIAATAC

TAAGGGAGTT

TATCAATAAC

TCCAGATACA

ATCTTTCTT

CACTACGTAC

ACATAAATCA

TTTATATGTA

TCTAATTGGA

AAATCTAATG

AGACACCCTT

TAATCAACAA

TTCTTCAGTT

AGGATACCAG

TATTAGAGAT

GTATCGAGAC

TTAGATACCA

GCTCCTGTAG

AAAAGGATAT

CAAGATATTT

GATCGTGTAA

GTAGATAATT

AATACAATGC

TTGTTATTAT

GTTATTCTTA

TACCTGAGAA

ATGCGTATAA

TCCAAGAAGA

TCGGAACTGT

TGAGTGAATT

TAAGAGAGAC

ATGCAGAATT

TTTTA.AACCC

AGCAATTATT

TACCTTTATT

ATGCAGATGA

ATTATACAAG

TGTAGTGGCT

ATGGATGGAG

GTCTAGTTFI'

ATGGGGGTTA

AGAACAATTC

GGAAGGGCTC

TACTCAAAAC

TCTAAATAGA

TGCACAGGCA

ATGGGGCATT

AGATTATTCT

24 AATTATI'GTA TAAATACGTA TCAAACTGCG TTTAGAGGGT TAAACACCCG TTTACACGAT ATGTTAGAAT TTAGAACATA TAZTGTTTTTA TTGTTTAAAT ATCAGAGTCT GGACCACAGC AGACACAATC CAAGTTAATT CGAATTATAT CCTAATATTG GTGGTTTACC AATTATAGCG GAGGAGTTTC AATTGCAGCA CGGTCCTCCC GGTACAGATC GAGAGGGCGT ACTTCAGGTT TAAGGTGTGG CCAGArFATT TTATCCGTAA ACAAGACCGT TACACTATAA GGATTACGAG CTTATATGGT GAAAATGGTA CTATGATTCA ATACATGCCA CTCAAGTGAA CAAGGTGATT CCTTAAGATT AATGGAAATA GTTACAATCT GTTACTATAA ACGGAAGAGT GGAGTCATTG ATAATGGAGC GATAATACTA ATGTACCGTT CTTATGAATA

TTATGTTTGT

TATGGTATCT

ATTTACTGCA

ATTATCTGGT

GGGTAGTACT

ATCTGGTCTC

TCCTTTATCA

TGCTACCTCT

TGCTTTTCCT

TATTTCTGGG

CCAAATAAGA

ATCTGTGCAT

TTTGGCACCG

TAATCAAACT

TGAACAAAGT

TTrATTTAAGG

TTATACTGTT

TCGTTTTTCA

AGATATAAAC

TCCAACTAAT

AATGTATTTG AATATGTATC TCTGGCGCTA A TTrATATGC

CAAAACTGGC

ATTAGTGGTA

ACAATTCATT

ATAGGGGCGA

ACACCATTTG

ACGACTTGGC

TTTCAGCTC

GTTCCT'rTAG

AATATAGAAA

AACAGAAAAA

GAAGATTATA

CGAACATTTA

AACACGACAG

GTATCTrrCTC

CCAAATGTTA

GATATTAATA

GGGACATTAA

CTTCCACCAC

CATTT'rTATA

ATAGGCTTTC

CATTGAACAG

CTAATCTCAA

TTAGAAGTTG

AGACAGAATC

GTGGAAATTC

TTATTAGAAA

GTCCTTCGGG

ATAATATCTA

CAGGATTTAC

TTTCTGAAAA

CTCGTTATAC

TAGGAAATTC

ATACAAATAT

TCGGTAATGT

GTTCTGGAAC

TTATTAA

CATTTGGTCA

TAGTGGTAGT

TTCTCTTrC TACTAccTTC

TGCCAGGGTT

TCACAACTTT

GCTGGATI'CA

CTTCCAAATA

AAACTATTTC

CGAAGATCTA

AACACCTGGT

TGCCGCTCAT

TATATCACCA

ATTTGGAAAT

GCTTAGAGGG

CACTATTCGA

AAATAACGAT

AGTAGCAAGT

TCAATTTGAG

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1908 a. a a a a a a a.

a. .a 0 a 0~ a. a IN'FORM~ATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 635 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Asn Asn Val Leu Asn Ser Gly Ara- Thr Thr Ile Cys Asn Ala Tyr 1 5 10 Asn Thr Tvr Val Ile Thr Val Gin Ser 145 Leu Phe Leu Arg Asn 225 MetI Ser Va IlE Val Giy Phe Giu Asn Gin 130 Ile Pro Aa Asn k1sp 210 eu lie 1Val Gin -Ala *Ser Pro Gin Ala 115 Val Thr Gin Gin Ala 195 Tyr] Tyr Glu I Trp S Ala Gil Pro Leu Ser Phe 100 Glu Asp Ser Phe Ala 180 ksp eu 3mn The ~er LHis Glu Val Ile Gly Leu Leu Asn Ser Gin Ala Glu Arg Thr Arg 245 Leu 3Asp Trp Val Gly 70 Ser Asn Glu Phe Val 150 Ile Asn Trp Asn Ala 230 Thr Phe I Pro Phe Ser Phe Glu Hi 25 Met Giu Trp Lys Arg Th s Lys Ser Le .1 r

GI~

Lys Thr Gin Gly Leu 135 Asn Gln M4et Gly ?he 'yr qs 40 IThr Arg Asn Arg Leu 120 Asn Thr Gly His Ile 200 Thr Arg Met Val Ile Leu Leu 105 Gin Pro Met Tyr Leu 185 Ser A.rg Gly Phe Leu Met 90 Asn Al a Thr Gln Gin 170 Ser Ala Asp Leu Leu 250 *Ser Phe *Ser Glu 75 Gin Asp Thr Asp Asn Ile Gin Asn 140 Gin Leu 155 Leu Leu Phe Ile Ala Thr Tyr Ser 220 Asn Thr 235 Asn Val As Le Lei lE Thr Arg 125 Pro Phe Leu Arg Leu 205 Asn krg ?he tal p His u Leu a Trp Leu Leu 110 Glu Val Leu Leu Asp1 190 Arg Leu I Ser S 270 Ser Lys Gly Arg Asp Phe Pro Asn Pro 175 Val rhr Uys I 2 'yr V er G Asn Leu Lys Leu Glu Aro Asn ILeu A.rg 1.60 Leu Ile 'r le ~sp al1 ly *see *OeO 0 0 Tyr Gin Ser Leu Met 265

I

Ala Asn Leu 275 Tyr Ala Ser Gly Gly Pro Gin Gin Thr Gin Ser Phe 285 Thr Ala Gin Asn Trp Pro Phe 26 Leu 290 Tyr Ser Leu Phe Gin Val Asn Ser 300 295 Asn 305 Pro Ser Ala Leu Glu 385 Thr Ser Leu Ile Tyr 465 Glu Thr Phe Gin Tyr 2 545 Asn Ala Thr Ser 370 Gly Ser Asn Val Arg 450 Met Asn lie lie 3er ~sn Ile SIle Arg Asn 355 Thr Val Gly Tyr Ile 435 Asn Val Gly Ser Ser 515 Asn Leu Leu Ser Gly 310 Gly Gly Leu 325 Val Asn Tyr 340 Leu Asn His Pro Phe Val Ala Thr Ser 390 Leu Arg Cys 405 Phe Pro Asp 420 Arg Asn Glu Ile Glu Ser Ser Val His 470 Tht Met Ile 485 Pro Ile His 500 Glu Lys Phe Thr Thr Ala Tyr Leu Arg 550 Ile Ser Gly Asn Arg Leu Ser Thr Pro Ser Asn Arg 375 Thr Glv Tyr Asp Pro 455 Asn His Ala ly krg 535 lal Gly Gly Phe 360 Ser Thr Ala Phe Leu 440 Ser Arg Leu Thr Asn 520 Tyr Ser 315 Thr Phe 320 Ser Thr Thr Ile His Gi 345 Asn Trp Trp Phe lIle 425 Thr Gly Lys Ala Gin 505 Gln rhr Ser 330 Val Ser Ser Gly Cys Ser Thr Val 365 Leu Asp Ser Gly 380 Gin Thr Giu Ser 395 Pro Phe Ser Ala 410 Arg Asn Ile Ser Arg Pro Leu His 445 Thr Pro Gly Gly 460 Asn Asn Ile Tyr 475 Pro Giu Asp Tyr 490 Val Asn Asn Gin Gly Asp Ser Leu 525 Leu Arg Giy Asn 540 Leu Gly Asn Ser 555 Ser Leu Asi 335 Leu Ile Gl) 350 Leu Thr Phe Arg Gly 430 Tyr Leu Ala Thr Thr 510 Arg ly Chr Pro Asp Gin Giv 415 Val Asn Arg Ala Gly 495 Arg Phe Asn Ile 2 Pro Arg Ile 400 Asn Pro Gln Ala His 480 Phe rhr lu Ser k.rg

S

0*6@

S

*SSOSS

0 *055

S

*00@SO

S

0 Val Thr Ile Asn Arg Val Tyr Thr Val Pro Asn Val Asn Thr Asn 575 Ile Asn Asn Asp GJly Val Ile Asp Asn Gly Ala Arg Phe Ser Asp Ile 580 585 590 Asn Ile Gly Asn Val Val Ala Ser Asp Asn Thr Asm Val Pro Leu Asp 595 600 605 Ile Asn Gly Thr Leu Ser Ser Gly Thr Gin Phe Glu Leu Met Asn Ile 610 615 620 Met Phe Val Pro Thr Asn. Leu Pro Pro Leu Tyr 625 630 635 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1872 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID ATGAATAATG TATTGAATAG

CGGAAGAAAT

CATGATCCAT

TTAGTTTTGA.

TGGAAAAGA

CTGATCATAG

CTATTAAAGA

AAGTAGGGAG

ATTTI'TCCTA

GTGGTAGTAT

ATAAATCAAA

GGCITAATGC

CAAGCGAATG

TGGCAGAGTT

CCTGTI'CCTT TAGCAATAAT TTACCACAGT

TCCAGATACA

GCCAATTTAC

ATCTTTCTIT

TCAGCAGCA

CAGTACGC

AATTA-GTA.

TAAATACGTA

ATGTTAGAAT

TTAGAACATA

TTATTTAAAT

ATCAAAGCCT

GGTCCAACAC

AATCATTTAC

ACATAAATCA

TTTATATGTA

ACTACTTGTC

TTAAATACCA

GCCCCTATTG

TCTTGTTGGA

AAAAGGATAC

AGATTAATG

AGACACCCTT

TAATCGACAA

TGATTCAGTI'

AGGCTATCAA

TATTAGAGAT

ATATAGAGAT

TCAAACTGCA

TATGTI'TTA

TCTAGTATCT

AGCACATAAC

CAAGAGATTT

GGTCGTGTAA

GTAGATAATT

AATACATTGC

CTGTTATTAT

GTCATCCTTA

CACCTGAGA

TTTAGAGGTT

AATGTATTTG

TCCGGCGCTA

TGGCCAT

ATGCACATAA

TAGAAAAAGA.

TGGGAACTGT

TGAGTGAGTT

TAAGAGCGAC

ATGCAGAATT

TTTTAAACCC

AGCAATTATT

TACC'ITTAT-r

ATGCAGATGA

ATTTCACAAG.

TAAACACTCG

AATATGTCTC

ATTTATATGC

TATATTCTCT

TGTAGTTOCT

ATGGAAAGA

GGGTAGTTT-r

ACAGAATTTA

AGAACAATTC

GGCAG.GTCT-T

TAATCAAAAC

TCTAAGTAGA

TGCACAGGCA

ATGGGGCATT

AGATTACTCT

TTTACACGA:I;

TATCTGGTCG

GAGTGGTAGT

rTTCCAAG~-r 120 180 240 300 360 420 480 540 600 660 720 780 840 900 0 AATTCTAATr ATGTATTAAA TG-GTTTGAGT GGTGCTAGGA CCACCATTAC TTTCTCTAAT

ATTGGTGGTC

AGAGGTGGAG

TCCACACTTT

GATCGGGAGG

TTACGATTTA

CGTAATATTT

TTTAATGAAA

AGAAAAAATA

GACTATACAG

ACGTTTATTT

ACAACGGCTC

TCTTCAATAG

GTTAATACTA

AATATCGGTA

TTAACGGCA

CCACTTTATT

TTCCCGGTTC

TGTCATCTAG

TCAATCCTr

GCGTTGCCAC

GCATTTTTTC

CTGGTGTTGT

TAAGAGATAT

ATATCTATGA

GATTTACCGT

CCGAAAAATA

GATACACACT

GAAGTTCCAC

CCACAAATAA

ATGTAGTGGC

ATCCACAATT

AA

TACCACAACT

CCGCATAGGT

ACAAACACCG

CTCTACAAAC

AGCTCGTGGT

TGGGACTATT

AGGAACGACA

CACTCATGA.A

ATCTCCAATA

TGGTAATCAG

TAGAGGGAAT

AATTCGAGTT

TGATGGAGTA

AAGTGCTAAT

TGAGCTTATG

CAAACATTGC ATTTTGCGAG

CAAGCTAATC

TTTATTAGAA

TGGCAATCAG

ALATTCGAACT

AGCAACGCAG

GCAGTCGCTA

AATGGTACTA

CATGCCACTC

GGTGATTCCT

GGAAATAGTT

ACTATAAACG

CTTGATAATG

ACTAATGTAC

AATATTATGT

TTAATCAAAA

GTTGGCTAGA

GAGCCTTTGA

TTTTCCCAGA

ATTTAGCAAG

GCCTTGTAAC

TGATTCATTT

AAGTAAATAA

TGAGATTTGA

ACAATCTTTA

GTAGAGTTTA

GAGCTCGTTT

CATTAGATAT

TTGTTCCAAC

GATTAATTAT

CTTTAACATT

TTCTGGTACA

GACAACTTTA

TTATTT'TATC

ACCTCTACAC

AGTGCATAAC

AGCGCCAAAT

TCAAATTCGA

GCTAAGCA-AC

TTTAAGAGTA

TACTGCAALAT

TTCAGATATT

ACAAGTGACA

TAATCCTTCA

1020 1080 11410 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1872 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 623 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY. linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met..Asn-Asn Val -Leu-An Ser fSly Arg Asn Thr Thr Cys His Ala His 1 5 10 Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His Lys Ser Leu Asn 25 Thr Ile Glu Lys Glu TrD Lys Glu Trp Lys Arg Thr Asp His Ser Leu 40 Tyr Val Ala Pro Ile Val Gly Thr Val Gly Ser Phe Leu Leu Lys Lys 55 Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Giu Leu Gin Asn Leu 70 75 Ile Phe Pro Ser Giy Ser Ile Asp Leu Met Gin Giu Ile Leu Arg Ala 90 Thr Giu Gin Phe Ile Asn Gin Arg Leu Asn Ala Asp Thr Leu Giy Arg 100 105 110 Val Asn Ala Giu Leu Ala Glv Leu Gin Ala Asn Val Ala Giu Phe Asn 115 120 125 Arg Gin Val Asp Asn Phe Leu Asn Pro Asn Gin Asn Pro Val Pro Leu 130 135 140 9999 Ala Ile Ile Asp Ser Val Asn Thr Leu Gin Gin Leu Phe Leu Ser Aro 145 150 155 160 Leu Pro Gin Phe Gin Ile Gin Gly Tyr Gin Leu Leu Leu Leu Pro Leu 999999 165 170 175 Phe Ala Gin Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val Ile 180 185 190 Leu Asn Ala Asp Giu Trp Gly Ile Ser Ala Ala Thr Val Arg Thr Tyr 195 200 205 Arg Asp His Leu Arg Asn Phe Thr Arg Asp Tyr Ser Asn Tyr Cys Ile 210 215 220 Asn Thr Tyr Gin Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp 225 230 235 240 Met Leu Giu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Giu Tyr Val 245 250 255 Ser Ile Trp Ser Leu Phe Lys Tyr Gin Ser Leu Leu Val Ser Ser Gly 260 265 270 Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Thr Gln Ser Phe Thr Ala 275 280 285 His Asn Trp Pro Phe Leu Tyr Ser Leu Phe G'n Val Asn Ser Asn Tyr 290 295 300 Val Leu Asn Gly Leu Ser Gly Al-a Arg Tr Thr Ile Thr Phe Ser Asn 305 310 315 320 Ile Gly Gly Leu Pro Gly Ser Thr Thr Thr Gin Thr Leu His Phe Ala 325 330 335 Arg Asn Thr Val 385 Leu Asn Al a Thr Ile 465 Asp Asn Ser Gly Ser 545 Val Phe Val Leu Ile Leu Pro 370 Al a Arg Tyr Asp Thr 450 Tvr Tyr Gin Leu Asn 530 Ser Asn Ser Pro Met 610 Tyr 340 Gin Ile Ser Se r Ile 420 Aia Val1 Thr Gly Arg 500 Phe Asn Ile Thr Ile 580 Asp Ile Gly Asn Trp 375 Trp Ser Ile Leu Leu 455 Asn Val Ile Ser Asn 535 Thr Asn Gly Val Val 615 Val Ile 360 Leu Gin Al a Ser His 440 Vai Gly Ser Ser Asn 520 Leu Ile Asp Asn Thr 600 Pro Ser Ser Arg Thr Leu Phe Ser Gly Thr 380 Gly Ala Phe 395 Gly Asn Ser 410 Val Val Glv Asn Giu Ile Val His Asn 460 Met Ile His -475 Ile His Ala 490 Lys Tyr Giy Thr Ala Arg Leu Arg Val 540 Gly Arg Val 555 Vai Leu Asp 570 Val Ala Ser Asn Gly Asn Asn Pro Ser 620 Ile Gly 350 Asn Pro 365 Asp Arg Giu Thr Asn Phe Thr le 430 Arg Asp 445 Arg Lys Leu Ala Thr Gin Asn Gin 510 Tyr Thr 525 Ser Ser Tyr Thr Asn Gly Ala Asn 590 Pro Gin 605 Pro Leu Gin Ala Leu Gin Glu Giy Thr Leu 400 Phe Pro 415S Ser Asn Ile Gly Asn Asri Pro Asn 480 Val Asn 495 Gly Asp Leu Arg Ile Giy Aia Asn 560 Ala Arg 575 Thr Asn Phe Giu Tyr 31 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1902 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGAATAATG TATTGAATAG TGGAAGAAAT ACTATTTGTG ATGCGTATAA

CATGATCCAT

TGGAAAAAAG'

CTGTTAAAGA

ATATTTCCTA

CTAAATCAAA

CAAGCAAATG

GCTGTTCCTT

TTATCCCAGT

GCCAATTTAC

TCAGCAGCAA

AATTATTGTA

ATGTTAGAAT

TTGTAAAT

GGACCACAGC

CAAGTTAATT

CCTAATATTG

AATTATAGTG

AATTGCAACA

GGTTCAGATC

TTAGTTTTCA

ATAATCATAG

AATTGGGGAG

GTGGCAGTAC

AACTTAATAC

TAGAAGAGTT

TATCAATAAC

TCCAGATGCA

ATCTTTCTTT

CATTACGTAC

TAGATACGTA

TTAGAACATA

ATCAAAGTCT

AGACCCAATT

CGAATTATGT

GTGGCTTACC

GAGGAATTAC:

CGATATCGCC

GACAGGGCGT

ACATAAATCA

TI'TATATGTA

CC-TTATTGGA

AAATCTAATG

AGACACTCTT

TAATCGACAA

TTCTTCAGTT

AGGATACCAA

TATTAGAGAT

GTATCAAAAT

TCAAACTGCG

TATGTTI'TTA

TCTAGTATCT

AI-rTACTI-A

ATTATCCGGC

TGGTTCTACT

ATCTGCGTAGI

ACCTTTGTCA

TACTACCGTI'

TTAGATACCA TACAAAAAGA GATCCTATTG TTGGAACTGT AAACGGATAC TGAGTGAATT GAAGATATTT TAAGAGAGAC TCCCGTGTAA ATGCGGAATT GTAGATAATT TTTTGAACCC

AATACAATGC

CTGTTATTAT

GTI'ATTCTTA

CACCTGAGAA

TTTAGAGGTT

AATGTATTTG

TCTGGCGCTA

CAAGACTGGC

TTTAGTGGGG

ACAACTCAAG

ATAGGGGGTT

ACGTCATTTG

ACAAATTGGC

AGCAATTATT

TACCTTTATT

ATGCAGAAGA

ATTATACAAG

TAAACACCCG

AATATGTATC

ATTTATATGC

CATTTTATA

CTAGTCTTTT

CATTACTTGC

CTAATTTTAA

TTIAGAATTTG

AAACAGAGTC

TGTAGTGGT"1

ATGGATGGAG

GGCTAGTTTT7

ACGGAATTTA

AGAAAAATTC

GACAGGGCTG

TAACCGAAAC

TCTAAATAGA

TGCACAGGCA

ATGGGGCAT'

AGATTACTCT

TTTACACGAT

TATCTGGTCG

AAGTGGTAGT

TTCTCTTTTC

TACTACCTT

TGCAAGGGTT

TCAAAATTTT

GCTAGATTCG

CTTTGAGACA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260

S

ACTTCAGGTIT TAAGGTGTGG TGCTTTTACA. CCTCGTGGTA ATTCGAACTA TTACCCTGGT

TATTTTATCC

CCGTTATACT

AGAGCTTATA

GGTACTATGA

GCCACTCAAG

GATTCCTTAA

AATAGTTACA

ATAAACGGTA

AATGATAACG

TCTGATGTAC

GTAATATI'TC

ATAACGAAAA

TGGTATCTGT

TTCATTTAGC

TGAATAATCA

GA'ITTGAACA

ATCTTTATTT

GAGTTTATAC

GAGCTCGTTT

CATTAGATAT

)32.

TGGTGTTTCT

TTAG'ITCTTA

AAGGAATATA GAAAGCCCTT GCATAACAAA AAAAATAACA GCCGGAAGAT

AATACAGGAT

AACGCGAACA TTTATTTCCG AAGCAACACG ACAGCTCGTT AAGAGTATCT TCAATAGGA-A TGCTTCAAAT

GTTAATACTA

TTCAGATATT

AATATCGGTA

AAATGTAACA

TTAAACTCCG

GAAATGAAGA

CAGGAACACC

TTTATGCAGT

TTACTATATC

AAAAATTTGG

ATACCCTTAG

ATTCCACTAT

CTACAAATAA

ATGTAGTAGC

GTACTCAATT

CTTAAAAAGA

TGGTGGAGCA

TCATGAAAAT

ACCGATAAT

AAATCAAAGT

AGGGAATGGA

TCGAGTTACT

CGATGGAGTT

AAGTAGTAAT

TGATCTTATG

1320 1380 1440 1500 1560 1620 -1680 1.740 1800 1860 1902 AATATTATGC TTGTACCAAC TAATCTTCCA CCACTTTATT AA INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHAR.ACTERISTICS: LENGTH: 633 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: %00% Met Asn Asn Val Leu Asn Ser Gly Arg Asn Thr Ile Cys Asp Ala Tyr His Asn Val Val Val Asp Pro Phe Gin His Lys Thr Ile Gin Tyr Val Asp Lys Glu Trp, Pro Ile Val Met Glu Gly Thr Lys Lys Asp Ser Leu Asp His. Ser Leu Leu Lys Lys Val Ala Ser Leu Gly Ser Leu Ile Ile Lys Arg Ile Leu Ser Giu Thr Asn Leu Met Giu Asp Leu Arg Asn Ile Leu Arg Phe Pro Ser Gly Ser Leu Asn Thr Giu Lys Gin Lys Leu 105 Asn Thr Asp Thr Leu Ser Arg 110 Val Asn Ala Glu Leu Thr Gly Leu Gin Ala Asn Val Glu Giu Phe Asn 115 120 125 Arg Ser 145 Leu Phe Leu Gin Asp) 225 Met Ser Al a Thr Asn 305 Pro Al1 a Gly Leu Gin 385 Gin 130 Ile Ser Ala Asn Asn 210 Thr Leu Ile Asn Ser 290 Tyr Asn Al a Ser Ser 370 Asn Ser Gin Al a Giu Arg Thr Arg 245 Leu Al a Trp Ser Gly 325 Asn Asn Phe Leu 135 Asn Gin Leu Gly Tyr 215 Phe Tyr Lys Giy Phe 295 Phe Pro Ser Asn Arg 375 Pro Met Tyr Leu 185 Ser Arg Gly Phe Gin 265 Gly Tyr Gly Ser Gly 345 Asn Trp Al a Phe Leu Arg Leu 205 Asri Arg Phe Val1 Thr 285 Gin Phe Gin Gly Ile 365 Gly Leu Arg 160 Leu Ile Tyr Ile Asp 240 Val Gly Phe Ser Phe 320 Leu Gly Pro Ara .9 Giy Vai Thr Thr Val Tbr Asn Trp Gin Thr Giu Ser Phe Giu Thr Thr Ser Gly Leu Arg Cys Gly Ala Phe Thr Pro Arg Gly Asn Ser Asn Tyr Leu Asn Val 465 OWv Ser Ser As n Leu 545 Ile Asn Giv Val1 Val1 Tyr Arg Ile 450 Ser Thr Pro Giu Thr 330 7hyr Asn Asp Asn Thr 610 Pro Gly 420 Giu Ser His Ile His 500 Phe Al a Arg Arg Val 580 Val Asn Asn Phe Leu Ser Lys 470 Leu Thr Asn Ty r Ser 550 Tyr Asp Ser Gly Pro Arg Arg 440 Thr Asn Pro Val Ser 520 Leu Ile Al a Gly Asn 600 Gin Leu 410 Ile Leu Giy Ile Asp 490 Asn Ser Giy Asn Asn 570 Arg Asp Asp Gly Tyr Aia 460 Al a Thr Thr Arg Gly 540 Thr Asn Ser Pro Met 620 4 Leu Lvs Tyr Giu Thr 495 Phe Gin Tvr Val Thr 575 Asn Ile Met

S

INFORMATION FOR SEQ-ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1902 base pairs TYPE: nucieic acid STRANI)EDNESS: single TOPOLOGY: iinear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGAATAATG TATTGAATAA TGGAAGAAAT ACTATTTGTG

CATGATCCAT

TGGAAAAAAG

CTGTTAAAGA

ATATTTCCTA

CTAAATCAAA

CAAGCAAATG

GCTGTTCCTT

TTATCCCAGT

GCCAATATAC

TCAGCAGCAA

AATTATTGTA

ATGTTAGAAT

TTGTTTAAAT

GGACCACAGC

TTAGTTTTCA

ATAATCATAG

AATTGGGGAG

GTGGCAGTAC

AACTTAATAC

TAGAAGAGTT

TATCAATAAC

TCCAGATGCA

ATCTTTCTTA

CATTACGTAC

ACATAAATCA

TTTATATGTA

CCTTATTGGA

AAATCTAATG

AGACACTCTT

TAATCGACAA

TTCTTCAGTT

AGGATACCAA

TATTAGAGAT

GTATCAAAAT

TAGATACGTA TCAAACTGCG TTAGAACATA TATGTTTTTA ATCAAAGTCT TCTAGTATCT AGACCCAATT A TTT ACTTCA

TTAGATACCA

GATCCTATTG

AAACGGATAC

GAAGATATTT

TCCCGTGTAA

GTAGATAATT

AATACAATGC

CTGTTATTAT

GTTATTCTTA

CACCTGAGAA

TTTAGAGGTT

AATGTATTTG

TCTGGCGCTA

CAAGACTGGC

TTTAGTGGGG

ACAACTCAAG

ATAGGGGGTT

ACGTCATTI'G

ACAAATTGGC

CCTCGTGGTA

TTAGTTCTTA

GAAAGCCCTT

AAAAATAACA

AATACAGGAT

ATGCGTATAA

TACAAAAAGA

TTGGAACTGT

TGAGTGAATT

TAAGAGAGAC

ATGCGGAATT

TTTTGAACCC

AGCAATTATT

TACCTTTATT

ATGCAGAAGA

ATI'ATACAAG

TAAACACCCG

AATATGTATC

ATTTATATGC

CATTTTTATA

CTAGTCTTTT

CATTACTTGC

CTAATTTTAA

TTAGAAGTTG

AAACAGAGTC

ATTCGAACTA

GAAATGAAGA

CAGGAACACC

TTTATGCAGT

TTACTATATC

AAAAATT-TGG

ATACCCTTAG

TGTAGTGGTT

ATGGATGGAG

GGCTAGTTr'r

ACGGAATTTA

AGAAAAATTC

GACAGGGCTG

TAACCGAAAC

TCTAAATAGA

TGCACAGGCA

ATGGGGCATT

AGATTACTCT

TATACACGAT

TATCTGGTCG

AAGTGGTAGT

TTCTCTTTTC

TACTACCTTT

TGCAAGGGTT

TCAAAATTTT

GCTAGATTCG

CTTTGAGACA

TTACCCTGGT

CTTAAAAAGA

TGGTGGAGCA

TCATGAAAAT

ACCGATACAT

AAATCAAGGT

AGGGAATGGA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 a. a a a a a.

a. *a a.

CAAGTTAATT CGAATTATGT ATTATCCGGC

CCTA.ATATTG

AATIATAGTG

AATTGCAACA

GGTTCAGATC

ACTTCAGGTT

TATTI'TATCC

CCGTTATACT

AGAGCTTATA

GGTACTATGA

GCCACTCAAG

GATTCCTTAA

GTGGCTTACC

GAGGAATTAC

CGATATCGCC

GACAGGGCGT

TAAGGTGTGG

GTAATATTTC

ATAACGAAAA

TGGTATCTGT

TTCATTTAGC

TGGTTCTACT

ATCTGGTAGT

ACCTTTGTCA

TACTACCGTT

TGCTTTTACA

TGGTGTTTCT

AAGGAATATA

GCATAACAAA

GCCGGAAGAT

TGAATAATCA AACGCGAACA TTTATTTCCG GATTTGAACA AAGCAACACG ACAGCTCGTT 36 AATAGTTACA ATCTTTATTT AAGAGTATCT TCAATAGGAA ATTCCACTAT TCGAGTTAT ATAAACGGTA GAGTTTATAC TGCTTCA.AAT GTTAATACTA CTACAAATAA

CGATGGAGTT

AATGATAACG GAGCTCGTTT TTCAGATATT AATATCGGTA ATGTAGTAGC AAGTAGTAT TCTGATGTAC CATTAGATAT AAATGTAACA T AAACTCCG GTACTCAATT

TGATCTTATG

AATATTATGC TTGTACCAAC TAATATTTCA CCACTTTATT AA 1680 1740 1800 1860 1902 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 633 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xiJ) SEQUENCE DESCRIPTION: SEQ ID Met Asn Asn Val Leu Asn Asn Gly Arg Asn Thr Ile Cys Asp Ala Tyr Asn Val Val Val Thr Ile Gin Lys Asp Pro Phe

S

S

S

S.

*SS* S

S

Phe Gin His Lys Giu Trp Met Lys Lys Asp Ser Leu Asp His Ser Leu Leu Lys Lys Tyr Val Leu Gly Asp Pro Ile Val Thr Val Ala Ser Ser Leu Ile Lys Arg Ile Leu Leu Arg Asn Ile Phe Pro Ser Ser Thr Asn Leu Giu Asp Ile Leu Arg Giu Thr Glu Lys Val Asn Ala 115 Leu Asn Gin Lys Asn Thr Asp Thr Giu Leu Thr Gly Ala Asn Val Leu Ser Arg 110 Glu Phe Asn Val Pro Leu Arg Gin 130 Ser Ile 145 Val Asp Asn Phe Pro Asn Ara Asn 140 Thr Ser Ser Val 150 Asn Thr Met Gin Gin Leu 155 Phe Leu Asn Leu Ser Gin Phe Gin Met Gin Gly Tyr Gin Leu Leu Leu Leu Pro Leu 165 Phe Leu Gin Asp 225 Met Ser Al a Thr Asn 305 Pro Al a Gly Leu Gin 385 Thr Tyr Al a Asn Asn 210 Thr Leu Ile Asn Ser 290 Tyr Asn Al a Ser Ser 370 Gly Ser Tyr Gin Ala 195 His Tyr Giu Trp Leu 275 Gin Val Ile Arg Asn 355 Thr Val Gly Pro Ala 180 Glu Leu Gin Phe Ser 260 Tyr Asp Leu Gly Val 340 Phe Ser Thr Leu Gly 420 Ala Glu Arg Thr Arg 245 Leu Ala Trp Ser Gly 325 Asn Asn Phe Thr Arg 405 Tyr Asn Trp Asn Ala 230 Thr Phe Ser Pro Gly 310 Leu Tyr Gin Val Val 390 Cys Phe Ile Gly Tyr 215 Phe Lys Gly Phe 295 Phe Pro Ser Asn Arg 375 Thr Gly Ile His Leu 185 Ile Ser 200 Thr Arg Arg Gly Met Phe T'yr Gin 265 Ser Gly 280 Leu Tyr Ser Gly Gly Ser Gly Gly 345 Phe Asn 360 Ser Trp, Asn Trp Ala Phe Arg Asn 425 170 Ser Al a Asp Leu Leu 250 Ser Pro Ser Al a Thr 330 Ile Cys Leu Gin Thr 410 Ile Tyr Ala Tyr Asn 235 Asn Leu Gin Leu Ser 315 Thr Thr Asn Asp Thr 395 Pro Ser 175 Ile Thr Ser 220 Thr Val Leu Gin Phe 300 Leu Thr Ser Thr Ser 380 Glu Arg Gly Arg *Leu 205 *Asn Arg Phe Val Thr 285 Gin Phe Gin Gly Ile 365 Gly Ser Gly Val Asp 190 Arg Tyr Ile Giu Ser 270 Gin Val Thr Al a Ser 350 Ser Ser Phe Asn Ser 430 Val1 Thr Cys His Tyr 255 Ser Leu Asn.

Thr Leu 335 Ile Pro Asp Glu Ser 415 Leu Ile

TV-

Ile Asp 240 Val1 Gly Phe Ser Phe 320 Leu Gly Pro Arg Thr 400 Asn Val 0 Leu Arg Asn Giu Asp Leu Lys Arg Pro Leu Tyr Tyr Asn Giu Lys Arg 435 440 445 38 Asn Ile Giu Ser Pro Ser Gly Tbr Pro Gly Gly Ala Arg Ala Tvr Met 455 Lys Lys 470 Leu Ala Thr Gin Asn Gin Tyr Thr 535 Ser Ser 550 Tyr Thr Asp Asn Ser Ser Gly Thr 615 460 Asn Asn Ile Tyr Ala Val His Giu Asn 475 480 Pro Giu Asp Asn Thr Gly Phe Thr Ile 490 495 Val Asn Asn Gin Thr Arg Thr Phe Ile 505 510 Gly Asp Ser Leu Arg Phe Giu Gin Ser 520 525 Leu Arg Gly Asn Gly Asn Ser Tyr Asn 540 Ile Giy Asn Ser Thr Ile Arg Val. Thr 555 560 Ala Ser Asn Val Asn Thr Thr Thr Asn 570 575 Gly Ala Arg Phe Ser Asp Ile Asn Ile 585 590 Asn Ser Asp Val Pro Leu Asp Ile Asn 600 605 Gin Phe Asp Leu Met Asn Ile Met Leu 620 Pro Thr Asn Ile Pro Leu Tyr

Claims (54)

11. The polynucleotide sequence, according to claim 5, wherein said polynucleotide 2 sequence comprises the nucleotide sequence of SEQ ID NO. 3. 1

12. The polynucleotide-sequence, according to claim 5, wherein said polynucleotide 2 sequence comprises the nucleotide sequence of SEQ ID NO. 9 or a fragment thereof. 1

13. The polynucleotide sequence, according to claim 5, wherein said polynucleotide 2 sequencecomprises the nucleotide sequence of SEQ ID NO. 9. 1 14. A polynucleotide sequence which encodes a lepidopteran-active toxin, wherein said 2 toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 6. 3 SEQ ID NO. 8, and fragments thereof. 1 15. The polynucleotide sequence, according to claim 14, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 6 or a fragment thereof. 1 16. The polynucleotide sequence, according to claim 14, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 8 or a fragment thereof. 1 17. The polynucleotide sequence according to claim 14, wherein said polynucleotide 2 sequence comprises the nucleotide sequence of SEQ ID NO. 7 or a fragment thereof. *0@6 S 1 18. The polynucleotide sequence, according to claim 14, wherein said polynucleotide 2 sequence comprises the nucleotide sequence of SEQ ID NO. 7 or a fragment thereof. 1 19. A lepidopteran-active toxin from a Bacillus thuringiensis isolate wherein said 2 isolate is selected from the group consisting of PS8612 and PS192M4. 1 20. The lepidopteran-active toxin, according to claim 19, wherein said isolate is PS8612. 1 21. The lepidopteran-active toxin, according to claim 19, wherein said isolate is 2 PS192M4. 1 22. A lepidopteran-active toxin wherein said toxin comprises an amino acid sequence 2 selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8. SEQ ID 3 NO. 10, and fragments thereof. 1 23. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 4 or a fragment thereof. 1 24. The lepidopteran-active toxin, according to claim 22. wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 4. 1 25. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 6 or a fragment thereof. 1 26. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 6. 1 27. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 8 or a fragment thereof. 1 28. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 8. 1 29. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 10 or a fragment thereof. 1 30. The lepidopteran-active toxin, according to claim 22, wherein said toxin comprises 2 the amino acid sequence of SEQ ID NO. 1 31. A transformed host which expresses a polynucleotide sequence encoding a 2 lepidopteran-active toxin vwherein said toxin comprises an amino acid sequence selected from 3 the group consisting of SEQ ID NO. 4, SEQ ID NO. 10, SEQ ID NO. 6, SEQ ID NO. 8, and 4 fragments thereof. '32. The host, according to claim 31, wherein said host is a plant or a plant cell. 42

33. An oligonucleotide primer selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2.

34. A method for controlling a lepidopteran pest wherein said method comprises contacting said pest with a toxin from a Bacillus thuringiensis isolate.selected from the group consisting of PS8612 and PS192M4. The polynucleotide sequence according to claim 34. wherein said isolate is PS86I2.

36. The polynucleotide sequence, according to claim 34, wherein said isolate is PS192M4.

37. A method for controlling a lepidopteran pest wherein said method comprises contacting said pest with a toxin comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 10, SEQ ID NO. 6, SEQ.ID NO. 8, and fragments thereof.

38. The method according to claim 37, wherein said lepdiopteran pest is an Ostrinia nubilalis.

39. The method according to claim 37, wherein said lepdiopteran pest is a Heliothis virescens. The method according to claim 37, wherein said lepdiopteran pest is a Helicoverpa zea.

41. An isolated polynucleotide sequence which encodes a lepidopteran-active toxin from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573.

42. An isolated polynucleotide sequence which encodes a lepidopteran-active toxin, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, and lepidopteran-toxic fragments thereof.

43. An isolated polynucleotide sequence which encodes a lepidopteran-active toxin, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

44. An isolated polynucleotide sequence which encodes a lepidopteran-active toxin, wherein said polynucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, and lepidopteran-toxic polypeptide-encoding fragments thereof. An isolated polynucleotide sequence which encodes a lepidopteran-active toxin, wherein said polynucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7.

46. An isolated polynucleotide sequence which encodes a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, said polynucleotide being substantially as hereinbefore described with reference to any one of the examples.

47. A vector comprising a polynucleotide sequence according to any one of claims 41 to 46.

48. A vector comprising a polynucleotide sequence which encodes a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, said vector being substantially as hereinbefore described with reference to any one of the examples.

49. A method for transforming a host, said method comprising contacting said host with a polynucleotide sequence according to any one of claims 41 to 46, or a vector according to claim 47 or claim 48, under conditions promoting uptake of said polynucleotide or vector by said host. A method according to claim 49, wherein said host is a plant or a plant cell.

51. A method according to claim 49, wherein said host is a bacterial cell.

52. A method for transforming a host to contain a polynucleotide sequence which encodes a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, said method being substantially as hereinbefore described with reference to any one of the examples.

53. A host transformed by a method according to any one of claims 49 to 52.

54. A transformed host comprising a polynucleotide according to any one of claims 41 to 46. A04104# 44 A transformed host comprising a polynucleotide sequence encoding a lepidopteran-active toxin, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, and lepidopteran-toxic fragments thereof.

56. A transformed host comprising a polynucleotide sequence encoding a lepidopteran-active toxin, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

57. A transformed host comprising a polynucleotide sequence encoding a lepidopteran-active toxin wherein said a nucleotide sequence selected from the group o1 consisting of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, and lepidopteran-toxic polypeptide-encoding fragments thereof.

58. A transformed host comprising a polynucleotide sequence encoding a lepidopteran-active toxin wherein said a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7.

59. A host according to any one of claims 53 to 58, wherein said host is a plant or a plant cell. A host according to any one of claims 53 to 58, wherein said host is a bacterial cell.

61. A transformed host comprising a polynucleotide sequence which encodes a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, said host being substantially as hereinbefore described with reference to any one of the examples.

62. A host according to any one of claims 53 to 61, wherein said host expresses said polynucleotide sequence.

63. A method for producing a lepidopteran-active toxin, comprising culturing a host according to claim 62 under conditions promoting production of said protein.

64. A method for recombinantly producing a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, said method being substantially as hereinbefore described, with reference to any one of the examples. A lepidopteran-active toxin, prepared by a method according to claim 63 or claim 64.

66. An isolated lepidopteran-active toxin from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573.

67. An isolated lepidopteran-active toxin, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, and lepidopteran-toxic fragments thereof. A04104#

68. An isolated lepidopteran-active toxin, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

69. An isolated lepidopteran-active toxin, wherein said toxin is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID and SEQ ID NO.7, and lepidopteran-toxic polypeptide-encoding fragments thereof. An isolated lepidopteran-active toxin, wherein said toxin is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID and SEQ ID NO.7. lo 71. An isolated lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, substantially as hereinbefore described with reference to any one of the examples.

72. A composition for controlling a lepidopteran pest comprising a toxin according to any one of claims 65 to 71.

73. A composition according to claim 72, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

74. A composition according to claim 72 or claim 73, wherein said toxin is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7. A composition for controlling a lepidopteran pest comprising a host according to claim 62.

76. A composition according to claim 75, wherein said host contains a lepidopteran-active toxin which comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

77. A composition according to claim 75 or claim 76, wherein said toxin is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7.

78. A composition for controlling a lepidopteran pest comprising a lepidopteran- active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, substantially as hereinbefore described, with reference to any one of the examples.

79. A method for controlling a lepidopteran pest wherein said method comprises applying to the environment of said pest, or contacting said pest with, a toxin according to any one of claims 65 to 71, or a composition according to any one of claims 72 to 74 or 78. A method according to claim 79, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8. A04104# 46

81. A method according to claim 79 or claim 80, wherein said toxin is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID and SEQ ID NO.7.

82. A method according to any one of claims 79 to 81, wherein said toxin is produced by a plant and said protein is present in said plant.

83. A method for controlling a lepidopteran pest wherein said method comprises applying to the environment of said pest, or contacting said pest with, a host according to claim 60 or a composition according to any one of claims 75 to 77.

84. A method according to claim 83, wherein said host contains a toxin comprising an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8. A method according to claim 83 or claim 84, wherein said toxin is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.3, SEQ ID and SEQ ID NO.7.

86. A method according to any one of claims 79 to 85, wherein said lepidopteran pest is Ostrinia nubilalis.

87. A method according to any one of claims 79 to 85, wherein said lepidopteran pest is Heliothis virescens.

88. A method according to any one of claims 79 to 85, wherein said lepidopteran pest is Helicoverpa zea.

89. A method for controlling a lepidopteran pest comprising applying to the environment of said pest, or contacting said pest with, a toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, or a transformed cell, plant, culture or composition comprising said toxin, said method being substantially as hereinbefore described, with reference to any one of the examples. A polynucleotide sequence according to any one of claims 41 to 46, when used for recombinant production of a toxin for the control of a lepidopteran pest.

91. A polynucleotide sequence which encodes a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, when used for recombinant production of a toxin for the control of a lepidopteran pest substantially as hereinbefore described with reference to any one of the examples.

92. A transformed host according to claim 62, when used for recombinant production of a toxin for the control of a lepidopteran pest.

93. A transformed host comprising a polynucleotide sequence which encodes a lepidopteran-active toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, when used for recombinant production of a toxin for the control of a lepidopteran pest substantially as hereinbefore described with reference to any one of the examples. A04104# 47

94. A toxin according to any one of claims 65 to 71, when used for controlling a lepidopteran pest. A host according to claim 62, when used for controlling a lepidopteran pest.

96. A composition according to any one of claims 72 to 78, when used for controlling a lepidopteran pest.

97. A toxin obtainable from a Bacillus thuringiensis isolate selected from PS192M4, HD525 or HD573, or a transformed host, a culture or a composition comprising said toxin, when used for controlling a lepidopteran pest substantially as hereinbefore described, with reference to any one of the examples. Dated 19 December, 2001 Mycogen Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S A04104#