AU756885B2 - Pesticidal toxins - Google Patents

Description

I

DESCRIPTION

PESTICIDAL TOXINS Background of the Invention The soil microbe Bacillus thuringiensis is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear-ingcro caJly 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 15 addition, with the use of genetic engineering techniques, new approaches for delivering these endotoxins to agricultural environments are under development, including the use of plants 0: genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, L. Kim [1988] TIBTECH 6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.

20 Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (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 6endotoxin which is toxic to the larvae of a number of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of namely israelensis and tenebrionis 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, T.L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; Beegle, (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A.M. Huger, G.A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.

Recently, new subspecies of B.t. have been identified, and genes responsible for active 6-endotoxin proteins have been isolated (H6fte, H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). H6fte and Whiteley classified B.i. crystal protein genes into 4 major classes. The classes were Cryl (Lepidoptera-specific), Cryll (Lepidoptera- and Diptera-specific), CryIII (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).

The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in 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 4,797,276 and 4,853,331 disclose B.

15 thuringiensis strain tenebrionis M-7, a.k.a. B.t. san diego) 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. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Patent No. 5,208,077 discloses coleopteran-active Bacillus thuringiensis isolates. U.S. Patent No. 5,151,363 and U.S. Patent No. 4,948,734 S 20 disclose certain isolates of B.t. which have activity against nematodes. 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. However, the discovery of new B.t. isolates and new uses of known B.t.

isolates remains an empirical, unpredictable art.

5. Coleopterans are an important group of agricultural pests which cause a very large 25 amount of damage each year. Examples of coleopteran pests include alfalfa weevils and corn rootworm.

The alfalfa weevil, Hypera postica, and the closely related Egyptian alfalfa weevil, Hypera brunneipennis, are the most important insect pests of alfalfa grown in the United States, with 2.9 million acres infested in 1984. An annual sum of 20 million dollars is spent to control these pests. The Egyptian alfalfa weevil is the predominant species in the southwestern U.S., where it undergoes aestivation hibernation) during the hot summer months. In all other respects, it is identical to the alfalfa weevil, which predominates throughout the rest of the U.S.

The larval stage is the most damaging in the weevil life cycle. By feeding at the alfalfa plant's growing tips, the larvae cause skeletonization of leaves, stunting, reduced plant growth, and, ultimately, reductions in yield. Severe infestations can ruin an entire cutting of hay. The adults, also foliar feeders, cause additional, but less significant, damage.

Approximately 9.3 million acres of U.S. corn are infested with corn rootworm species complex each year. The corn rootworm species complex includes the northern corn rootworm, S Diabrotica barberi, the southern corn rootworm, D. undecimpunctata howardi, and the western corn rootworm, D. virgifera virgifera. The soil-dwelling larvae of these Diabrotica species feed on the root of the corn plant, causing lodging. Lodging eventually reduces corn yield and often results in death of the plant. By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally affect the yield of corn per plant. In addition, adults and larvae of the genus Diabrotica to attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.

Control of corn rootworm has been partially addressed by cultivation methods, such as crop rotation and the application of high nitrogen levels to stimulate the growth of an adventitious root system. However, chemical insecticides are relied upon most heavily to guarantee the desired level of control. Insecticides are either banded onto or incorporated into the soil. The major problem i associated with the use of chemical insecticides is the development of resistance among the treated insect populations.

SBrief Summary of the Invention The subject invention concerns novel materials and methods for controlling non-mammalian pests. In a preferred embodiment, the subject invention provides materials and methods for the control of coleopteran pests. In specific embodiments, the materials and methods described herein are used to control alfalfa weevil and/or corn rootworm.

Herein disclosed are two new classes of polynucleotide sequences which encode corresponding novel classes of pesticidal proteins. One novel class of polynucleotide sequences as 25 described herein encodes toxins which have a full-length molecular weight of approximately 40-50 kDa. More typically, these toxins have a molecular weight of about 43-47 kDa. A second class of polynucleotides, which encodes pesticidal proteins of about 10-15 kDa, is also described. More typically, these toxins have a molecular weight of about 13-14 kDa. Herein described are polynucleotides which encode the 40-50 kDa and 10-15 kDa toxins, polynucleotides which encode pesticidal fragments of the full length toxins, and polynucleotide sequences which encode longer forms of these toxins which include, for example, a protoxin region. In a preferred embodiment, these toxins, including the fragments, are active against coleopteran pests.

[I:\DAYLIB\LIBA]03698.doc:mrr 4 Specific B.t. toxins useful according to the invention include toxins which can be obtained from the B.t. isolates designated as PS8OJJ1, PS149B1, and PS167H2. Of these, PS149B1 and PS167H2 are novel isolates. The subject invention also includes the use of variants of the exemplified B.t. isolates and toxins which have substantially the same coleopteran-active properties S as the specifically exemplified B.t. isolates and toxins. Such variant isolates would include, for example, mutants. Procedures for making mutants are well known in the microbiological art.

Ultraviolet light and chemical mutagens such as nitrosoguanidine are used extensively toward this end.

Disclosed herein are polynucleotide sequences which encode toxins of approximately 43-47 to kDa. These toxins can be used to control coleopteran pests, and may be particularly useful for control of corn rootworms. The genes which encode the 43-47 kDa toxins can be obtained from, for P5149B1, orPSI67H2. The toxins of approximately 13-14 kDa can also be used to control coleopteran pests. The approximately 13-14 kDa toxin, as well as the genes which encode these toxins, can also be obtained from PS8OJJI, P5149B1, or P5167H2. It has also been found that the activity of the 43-47 kDa toxins can be augmented and/or facilitated by further contacting the target pests with an approximately 13-14 kDa toxin.

The subject invention also concerns plant 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.

20 Alternatively, the B.t. isolates 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 a" °treatment of substantially intact B.t. cells, and/or recombinant cells containing the expressed toxins 0 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 25 pesticidal toxin. The toxin becomes active upon ingestion by a target insect.

According to a first embodiment of the invention, there is provided an isolated polynucleotide which encodes a toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes a pesticidal portion of SEQ ID NO. 32, DNA which encodes a pesticidal portion of SEQ ID NO. 36, and DNA which encodes a pesticidal portion of SEQ ID NO. 41, hybridises under stringent conditions with said polynucleotide or a complement thereof.

According to a second embodiment of the invention, there is provided an isolated polynucleotide which encodes a toxin active against a non-mammalian pest, wherein a nucleotide [I:\DAYL1B\LIBA]03698.doc:mrr 4a sequence selected from the group consisting of: SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 33, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with said polynucleotide or a complement thereof.

According to a third embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence which encodes a toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof.

to According to a fourth embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence which encodes a toxin active against a nonmammalian pest, wherein a portion of said nucleotide sequence can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

According to a fifth embodiment of the invention, there is provided an isolated polynucleotide S1. 1 comprising a nucleotide sequence which encodes a toxin which immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a sixth embodiment of the invention, there is provided an isolated Spolynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight 20 of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said nucleotide sequence is from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a seventh embodiment of the invention, there is provided an isolated .polynucleotide comprising a nucleotide sequence which encodes an approximately 10 to 15 kDa 2 5 PS80JJ1 toxin active against non-mammalian pests, wherein said nucleotide sequence has been optimised for expression in plants.

According to an eighth embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said nucleotide sequence is from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

According to a ninth embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight S of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said nucleotide [I:\DAYLIB\LIBA]03698.doc:mrr sequence is from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

According to a tenth embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence which encodes a toxin which immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

According to an eleventh embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleotide sequence which encodes a toxin active against a nonmammalian pest, wherein said toxin comprises an amino acid sequence which has at least about 1i 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. More preferably the degree of identity is at least about 80%, and even more preferably it is According to a twelfth embodiment of the invention, there is provided an isolated i polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

According to preferred aspects of these embodiments, the polynucleotide further encodes a 20 second toxin having a molecular weight of approximately 40 to 50 kDa active against a nonmammalian pest, said second toxin and/or its encoding DNA being as herein described.

s* According to a thirteenth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO.

25 41 or pesticidal portions thereof.

According to a fourteenth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest, wherein said toxin is encoded by a nucleotide sequence, a portion of which can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

According to a fifteenth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to kDa from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a sixteenth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an [I:\DAYLIB\LIBA03698doc:m 4c antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a seventeenth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

According to a nineteenth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS149B1 having to the identifying characteristics of NRRL B-21553.

According to a twentieth embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately to 15 kDa from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

i According to a twenty-first embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS167H2, having the identifying characteristics of NRRL B-21554.

According to a twenty-second embodiment of the invention, there is provided an isolated or 20 purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid S: sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID SNO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41.

More preferably the degree of identity is at least about 80%, and even more preferably it is Ooo* 25 According to a twenty-third embodiment of the invention, there is provided an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately to 15 kDa, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

3o According to a twenty-fourth embodiment of the invention, there is provided a biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain pesticidal activity, said culture comprising a toxin active against a non-mammalian pest and having a molecular weight of f 35p proximately 10 to 15 kDa.

[I:\DAYLIB\LIBA]03698.doc:mrr 4d According to preferred aspects of embodiments one to twelve, there are also provided recombinant DNA molecules, plasmids and vectors comprising polynucleotides according to the invention, as well as methods of transforming host cells therewith.

Thus, according to a twenty-fifth embodiment there are provided recombinant host cells and recombinant plants transformed with polynucleotides, recombinant DNA or plasmids according to the invention. Also provided is the transgenic seed from transgenic plants produced according to the invention.

According to a twenty-sixth embodiment of the invention, there is provided a recombinant host cell transformed with a polynucleotide coding for a toxin having activity against a non- 1o mammalian pest, wherein said toxin has at least one characteristic selected from the group consisting of: said toxin is encoded by a polynucleotide wherein a nucleotide sequence selected from the group consisting of DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO.

5, and DNA which encodes SEQ ID NO. 7, hybridises under stringent conditions with said IS5 polynucleotide or a complement thereof; said toxin is an approximately 10 to 15 kDa pesticidal toxin encoded by a nucleotide sequence from a Bacillus thuringiensis isolate selected from the group consisting of PS80JJ1 having the identifying characteristics of NRRL B-18679, PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B- 20 21554; said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from a Bacillus thuringiensis isolate selected from the group consisting of PS80JJ1 having the identifying characteristics of NRRL B-18679, PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554; 25 said toxin is encoded by a nucleotide sequence wherein a portion of said nucleotide sequence can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33; said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof; said toxin comprises an amino acid sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. SEQ ID NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions of SEQ ID NO. 36, and pesticidal portions of sequence ID NO. 41; and said toxin is encoded by a polynucleotide wherein a nucleotide sequence selected 7 from the group consisting of SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO.

r 35_ f, SEQ ID NO. 31, SEQ ID NO. 33, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34, SEQ [I :\DAYLIB\LIBA03698.doc:mrr ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with said polynucleotide or a complement thereof.

According to a preferred aspect the host cell is a plant cell, and according to another preferred aspect there are provided transgenic plants comprising a plurality of said transformed cells and/or regenerated from said transformed cells.

According to preferred aspects, the recombinant host cell is further transformed with a polynucleotide encoding a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, said second toxin and/or its encoding DNA being as herein described.

io According to a twenty-seventh embodiment of the invention, there is provided a method for preparing a pesticidal composition comprising a toxin active against non-mammalian pests having a molecular weight of approximately 10 to 15 kDa, said method comprising culturing a biologically pure culture or a plurality of transgenic cells according to the invention, under conditions promoting expression of said toxin. A pesticidal composition prepared by said method is also provided.

According to a twenty-eighth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin according to any one of embodiments thirteen to twenty-three, or a biologically pure culture according to embodiment twenty-four. Preferably said :pesticidal compositions further comprise agriculturally/pesticidally acceptable additives, including carriers, adjuvants, stabilising agents, rheological agents, emulsifiers, dispersants, polymers and 20 surfactants.

S. According to a twenty-ninth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against non-mammalian pests, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes SEQ 25 ID NO. 32, DNA which encodes SEQ ID NO. 36, and DNA which encodes SEQ ID NO. 41, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

According to a thirtieth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of SEQ ID NO. 29, a 10 to 15 kDa toxinencoding portion of SEQ ID NO. 30, SEQ ID NO. 31, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO.

and SEQ ID NO. 44, hybridises under stringent conditions with the nucleotide sequence Zencoding said toxin or a complement thereof.

I :\DAYLI B\LI BA]03698.doc:mrr 4f According to a thirty-first embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO.

32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof.

According to a thirty-second embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein said toxin is encoded by a nucleotide sequence, a portion of which can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

According to a thirty-third embodiment of the invention, there is provided a pesticidal o1 composition comprising an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a thirty-fourth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein 15 said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a thirty-fifth embodiment of the invention, there is provided a pesticidal S: composition comprising an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate 20 PS149B1 having the identifying characteristics of NRRL B-21553.

According to a thirty-sixth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

25 According to a thirty-seventh embodiment of the invention, there is provided a pesticidal composition comprising, an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

According to a thirty-eighth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

According to a thirty-ninth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest, wherein R said toxin comprises an amino acid sequence which has at least about 75% identity with an amino [I:\DAYLIB\LIBA]03698.doc:mrr 4g acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. More preferably the degree of identity is at least about 80%, and even more preferably it is According to a fortieth embodiment of the invention, there is provided a pesticidal composition comprising an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

According to preferred aspects of embodiments twenty-eight to forty, the pesticidal composition also comprises a second toxin having a molecular weight of approximately 40 to kDa active against a non-mammalian pest, said second toxin andlor its encoding DNA being as herein described.

According to a forty-first embodiment of the invention, there is provided a pesticidal *5 composition comprising a biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553, and o. PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain pesticidal activity, said culture comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa.

20 According to a forty-second embodiment of the invention, there is provided a composition of matter for controlling coleopterans comprising a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain activity against coleopterans, in association with an agricultural carrier appropriate for use in 9oo.

25 controlling coleopterans.

According to a forty-third embodiment of the invention, there is provided a method for controlling a non-mammalian pest comprising applying to the situs or environment of said pest a pesticidally effective amount of an isolated or purified toxin according to any one of embodiments thirteen to twenty-three, a biologically pure culture according to embodiment twenty-four, a transformed cell or transgenic plant according to embodiment twenty-five or twenty-six, or a pesticidal composition according to any one of embodiments twenty-seven to forty-one.

According to a forty-fourth embodiment of the invention, there is provided a method for controlling a coleopteran pest comprising applying to the situs or environment of said pest a pesticidally effective amount of a composition of matter according to embodiment forty-two.

[Il:\DAYLIB\LI BA]03698.doc:mrr 4h According to a forty-fifth embodiment of the invention, there is provided a method for controlling a non-mammalian pest comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a Bacillus thuringiensis isolate, or a toxin of said Bacillus thuringiensis isolate, wherein said isolate is selected from the group consisting of PS149B1, having the identifying characteristics of NRRL B-21553; and PS167H2, having the identifying characteristics of NRRL B-21554; and mutants thereof which retain pesticidal activity.

According to a forty-sixth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a purified or isolated toxin active against non-mammalian pests, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes SEQ ID NO. 32, DNA which encodes SEQ ID NO. 36, and DNA which encodes SEQ ID NO. 41, hybridises under stringent conditions with the nucleotide sequence encoding said toxin, or a complement thereof.

5 According to a forty-seventh embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a purified or isolated toxin active against non-mammalian pests, wherein a nucleotide sequence selected from the group consisting of SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30, SEQ ID NO. 31, a 10 to 2o kDa toxin-encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

According to a forty-eighth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof.

According to a forty-ninth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin is encoded by a nucleotide sequence, a portion of which can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

According to a fiftieth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or [I :\DAYLIB\LIBA]03698.doc:mrr contacting said pest with a pesticidal amount of an isolated or purified toxin active against a nonmammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

According to a fifty-first embodiment of the invention, there is provided a method for S controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

0o According to a fifty-second embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

1s5 According to a fifty-third embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of 20 NRRL B-21553.

According to a fifty-fourth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus 25 thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

According to a fifty-fifth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

According to a fifty-sixth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence which has at least [I:\DAYLJB\LIBAj03698.doc:mrr 4j about 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO.

3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. More preferably the degree of identity is at least about 80%, and even more preferably it is According to a fifty-seventh embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide io sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO.

39.

According to preferred aspects of embodiments forty-six to fifty-seven, the method also comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin active against a non-mammalian pest having a 15 molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, said second toxin and/or its encoding DNA being as herein described.

~According to a fifty-eighth embodiment of the invention, there is provided a method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a biologically pure culture of a Bacillus 20 thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553; and PS167H2 having the identifying characteristics of NRRL B- 21554, and mutants thereof which retain pesticidal activity, said culture comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa.

Brief Description of the Drawings Figure 1 shows three specific 43-47 kDa pesticidal toxins of the subject invention as well as a consensus sequence for these pesticidal toxins.

Figure 2 shows the relationship of the 14 and 45 kDa sequences of PSSOJJ1 (SEQ ID NOS. 31 and

ST

[i :\DAYLI3\LIBA]03698.doc:mrr Brief Description of the Sequences SSEQ ID NO. 1 is a 5-amino acid N-terminal sequence of the approximately 45 kDa toxin of 80JJ1.

SEQ ID NO. 2 is a 25-amino acid N-terminal sequence of the approximately 45 kDa toxin of 80JJ1.

SEQ ID NO. 3 is a 24-amino acid N-terminal sequence of the approximately 14 kDa toxin of 80JJ1.

SEQ ID NO. 4 is the N-terminal sequence of the approximately 47 kDa toxin from 149B1.

SEQ ID NO. 5 is a 50-amino acid N-terminal amino acid sequence for the purified approximately 14 kDa protein from PS 149B 1.

SEQ ID NO. 6 is the N-terminal sequence of the approximately 47 kDa toxin from 167H2.

SEQ ID NO. 7 is a 25-amino acid N-terminal sequence for the purified approximately 15 14 kDa protein from PS167H 2 SEQ ID NO. 8 is an oligonucleotide probe for the gene encoding the PS80JJ1 44.3 kDa toxin and is a forward primer for PS149B1 and PS167H2 used according to the subject invention.

SEQ ID NO. 9 is a reverse primer for PS149B1 and PS167H2 used according to the 20 subject invention.

SEQ ID NO. 10 is the nucleotide sequence of the gene encoding the approximately kDa PS80JJI toxin.

SEQ ID NO. 11 is the amino acid sequence for the approximately 45 kDa PS80JJ1 toxin.

SEQ ID NO. 12 is the partial nucleotide sequence of the gene encoding the approximately 44 kDa PS149B1 toxin.

SEQ ID NO. 13 is the partial amino acid sequence for the approximately 44 kDa SPS149B1 toxin.

SEQ ID NO. 14 is the partial nucleotide sequence of the gene encoding the approximately 44 kDa PS 167H2 toxin.

SEQ ID NO. 15 is the partial amino acid sequence for the approximately 44 kDa PS 167H2 toxin.

SEQ ID NO. 16 is a peptide sequence used in primer design according to the subject invention.

6 SEQ ID NO. 17 is a peptide sequence used in primer design according to the subject invention.

SEQ ID NO. 18 is a peptide sequence used in primer design according to the subject invention.

SEQ ID NO. 19 is a peptide sequence used in primer design according to the subject invention.

SEQ ID NO. 20 is a nucleotide sequence corresponding to the peptide of SEQ ID NO.

16.

SEQ ID NO. 21 is a nucleotide sequence corresponding to the peptide of SEQ ID NO.

17.

SEQ ID NO. 22 is a nucleotide sequence corresponding to the peptide of SEQ ID NO.

18.

SEQ ID NO. 23 is a nucleotide sequence corresponding to the peptide of SEQ ID NO.

19.

15 SEQ ID NO. 24 is a reverse primer based on the reverse complement of SEQ ID NO.

22.

SEQ ID NO. 25 is a reverse primer based on the reverse complement of SEQ ID NO.

23.

SEQ ID NO. 26 is a forward primer based on the PS80JJ1 44.3 kDa toxin.

20 SEQ ID NO. 27 is a reverse primer based on the PS80JJ1 44.3 kDa toxin.

SEQ ID NO. 28 is a generic sequence representing a new class of toxins according to the subject invention.

SEQ ID NO. 29 is an oligonucleotide probe used according to the subject invention.

SEQ ID NO. 30 is the nucleotide sequence of the entire genetic locus containing open o 25 reading frames of both the 14 and 45 kDa PS80JJ1 toxins and the flanking nucleotide sequences.

SEQ ID NO. 31 is the nucleotide sequence of the PS80JJ1 14 kDa toxin open reading frame.

SEQ ID NO. 32 is the deduced amino acid sequence of the 14 kDa toxin of PS80JJ1.

SEQ ID NO. 33 is a reverse oligonucleotide primer used according to the subject invention.

SEQ ID NO. 34 is the nucleotide sequence of the entire genetic locus containing open reading frames of both the 14 and 44 kDa PS167H2 toxins arid the flanking nucleotide sequences.

SEQ ID NO. 35 is the nucleotide sequence of the gene encoding the approximately 14 kDa PS167H2 toxin.

SEQ ID NO. 36 is the amino acid sequence for the approximately 14 kDa PS167H2 toxin.

SEQ ID NO.37 is the nucleotide sequence of the gene encoding the approximately 44 kDa PS167H2 toxin.

SEQ ID NO. 38 is the amino acid sequence for the approximately 44 kDa PS167H2 toxin.

SEQ ID NO. 39 is the nucleotide sequence of the entire genetic locus containing open reading frames of both the 14 and 44 kDa PS149B1 toxins and the flanking nucleotide sequences.

SEQ ID NO. 40 is the nucleotide sequence of the gene encoding the approximately 14 kDa o1 PS149B1 toxin.

SEQ ID NO. 41 is the amino acid sequence for the approximately 14 kDa PS149B1 toxin.

SEQ ID NO. 42 is the nucleotide sequence of the gene encoding the approximately 44 kDa PS149B1 toxin.

i" SEQ ID NO. 43 is the amino acid sequence for the approximately 44 kDa PS149B1 toxin.

15 SEQ ID NO. 44 is a maize-optimized gene sequence encoding the approximately 14 kDa toxin of 80JJ1.

SEQ ID NO. 45 is a maize-optimized gene sequence encoding the approximately 44 kDa toxin of 80JJ1.

Detailed Disclosure of the Invention 20 The subject invention concerns two new classes of polynucleotide sequences which encode novel pesticidal toxins. In one class, the toxins have a full-length molecular weight of approximately 40-50 kDa. In specific cases exemplified herein, these toxins have a molecular weight of about 43- 47 kDa. In the second class, the pesticidal toxins have a molecular weight of approximately 10-15 kDa. In specific cases exemplified herein, these toxins have a molecular weight of about 13-14 25 kDa. Certain specific toxins are exemplified herein. For toxins having a known amino acid sequence, the molecular weight is also known. Those skilled in the art will recognize that the apparent molecular weight of a protein as determined by gel electrophoresis will sometimes differ from the true molecular [I:\DAYLI B\LIBA]03698.doc:mrr weight. Therefore, reference herein to, for example, a 45 kDa protein or a 14 kDa protein is understood to refer to proteins of approximately that size even if the true molecular weight is somewhat different.

The subject invention concerns not only the polynucleotide sequences which encode these classes of toxins, but also the use of these polynucleotide sequences to produce recombinant hosts which express the toxins. In a further aspect, the subject invention concerns the combined use of an approximately 40-50 kDa toxin of the subject invention together with an approximately 10-15 kDa toxin of the subject invention to achieve highly effective control of pests, including coleopterans such as corn rootworm.

A further aspect of the subject invention concerns two novel isolates and the toxins and genes obtainable from these isolates. The novel B.t. isolates of the subject invention have been designated PS149B1 and PS167H2.

The new classes of toxins and polynucleotide sequences provided here are defined according to several parameters. One critical characteristic of the toxins described herein is 5 pesticidal activity. In a specific embodiment, these toxins have activity against coleopteran 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 within each novel class can be defined ,:'°°herein in terms of homology 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 classes of toxins 20 provided herein can also be identified based on their immunoreactivity with certain antibodies and based upon their adherence to a generic formula.

o The sequence of three approximately 45 kDa toxins are provided as SEQ ID NOS. 11, 43, and 38. Typically, the toxins in this new class have a sequence which conforms to the generic sequence presented as SEQ ID NO. 28. Typically, the toxins of this class will conform to the consensus sequence shown in Figure 1.

Typically, the toxins of the subject invention have at least one of the following characteristics: said toxin is encoded by a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 2, DNA which encodes SEQ ID NO. 4, DNA which encodes SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, DNA which encodes SEQ ID NO. 11, SEQ ID NO. 12, DNA which encodes SEQ ID NO.

13, SEQ ID NO. 14, DNA which encodes SEQ ID NO. 15, DNA which encodes [R\LIBA]03698.doc:mrr SEQ ID NO. 16, DNA which encodes SEQ ID NO. 17, DNA which encodes 1 SEQ ID NO. 18, DNA which encodes SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. SEQ ID NO. 26, SEQ ID NO. 27, DNA which encodes a pesticidal portion of SEQ ID NO. 28, SEQ ID NO. 37, DNA which encodes SEQ ID NO. 38, SEQ ID NO. 42, and DNA which encodes SEQ ID NO. 43; said toxin immunoreacts with an antibody to an approximately 40-50 kDa pesticidal toxin, or a fragment thereof, from a Bacillus thuringiensis isolate selected from the group consisting of PS80JJ1 having the identifying characteristics of NRRL B-18679, PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying S: characteristics of NRRL B-21554; S(c) said toxin is encoded by a nucleotide sequence wherein a portion of said nucleotide sequence can be amplified by PCR using a primer pair selected from 15 the group consisting of SEQ ID NOS. 20 and 24 to produce a fragment of about 495 bp, SEQ ID NOS. 20 and 25 to produce a fragment of about 594 bp, SEQ ID NOS. 21 and 24 to produce a fragment of about 471 bp, and SEQ ID NOS.

*g 21 and 25 to produce a fragment of about 580 bp; said toxin comprises a pesticidal portion of the amino acid sequence shown in 20 SEQ ID NO. 28; said toxin comprises an amino acid sequence which has at least about homology with a pesticidal portion of an amino acid sequence selected from the group consisting of SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 38, and SEQ ID NO. 43; said toxin is encoded by a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence selected from the group consisting of DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes SEQ ID NO. 32, DNA which encodes SEQ ID NO. 36, and DNA which encodes SEQ ID NO. 41; said toxin immunoreacts with an antibody to an approximately 10-15 kDa pesticidal toxin, or a fragment thereof, from a Bacillus thuringiensis isolate selected from the group consisting of PS80JJ1 having the identifying characteristics of NRRL B-18679, PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554; said toxin is encoded by a nucleotide sequence wherein a portion of said nucleotide sequence can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33; and said toxin comprises an amino acid sequence which has at least about homology with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions of SEQ ID NO. 36, and pesticidal portions of SEQ ID NO. 41.

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 S" Southern blots with 32P-labeled gene-specific probes was performed by standard methods 15 (Maniatis, E.F. Fritsch, J. Sambrook (1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, In general, hybridization and subsequent washes were carried out under stringent conditions that allowed for detection of target sequences with homology to the PS80JJ1 toxin genes. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25 C below the melting temperature (Tm) of the 20 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.

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*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*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 C)-2(number TIA base pairs) +4(nurnler GIG base pairs) (Suggs, T. Miyake, E-H. Kawashime, M.J. Johnson, K. Itakur-a, and R.B. Walflace [1981] *ICN- UCLA Symp. Dev. BioL Using Purified Genes, D.D. Brown fed.], 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 IX SSPE, 0. 1% SDS (moderate stringency wash).

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

Microorganisms useful according to the subject invention have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815S North University Stret, Peoria Illinois 61604, USA.

The culture repository numbers of the deposited strains are as follows: Culte B-c strain PS80JJI B.t. strain.PS149B1 B.t. strain PSI 67H2 10~ E. coi NM522 (pMYC23 65) E. coi NM522 (pMYC23 82) E. coli NM522 (pMYC2379) R, coli NMS22(pMYC242 1) Ycoli NM522(pMYC2427) J E. coli NM522( 1 pMYC2429) E. coli NM522(pMYC2426) Repository No.

NRRL B-1 8679 NRRL B-2 1553 NRRL B-2 1554 NRRL B-21170 NRRL B-2 1329 NRRL B-21155 NRRL B-21555 NRRL B-21672 NRRL B-21673 NRRL B-21671 Deposit Date July 17, 1990 March 28, 1996 March28, 1996 January 5, 1994 September 28, 1994 November 3, 1993 March 28, 1996 March 26, 1997 March 26, 1997 March 26, 1997 Thle PS80JJI isolate is available to the public by virtue of the issuance of U.S. Patent No.

5,151,363.

B.i. isolates PS149BI and P5167112 have been deposited under conditions that assure S that access to the cultures will be available during the pendency of this patent appliaint n 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 are 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 a deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. 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 the deposit(s). All restrictions on the availability to the public of the subject culture deposits will le irrevocably removed upon the granting of a patent disclosing them.

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

1 Table 1. Description of B.t. strains toxic to coleopterans Deposit Date Culture Crystal Approx. MW Serotype

NRRL

Description (kDa) Deposit PS80JJ1 multiple 130, 90,47, 37, 4a4b, sotto B-18679 7-17-90 attached 14 PS149BI 130,47,14 B-21553 3-28-96 S* PS167H2 70 47, 14 B-23554 3-28-96 Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. 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 claimed toxins.

It should be apparent to a person skilled in this art that genes encoding active toxins can W 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 mutagehesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction 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.t 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 15 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. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or western blotting.

20 Antibodies to the toxins disclosed herein, or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures in this art. The genes which encode these toxins can then be obtained from the microorganism.

Fragments and equivalents which retain the pesticidal 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.

A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently O fluorescent as described in International ApplicationNo. W093/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a.strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well- S known in the art, as described, for example, in Keller, MM. Manak (1987) DNA Probe s Stockton Press, New York, NY., pp- 169-170. Detection of the probe provides a means for determining in a known manner whether hybridization has occurred- Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized to using a DNA synthesizer and standard procedures These nucleotide sequences can also be used -s PCR primers to amplify genes of the subject invention.

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 comprises variant or equivalent toxins (and nucleotide So sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplifiedtoxin. Equivalent toxins will have amino acid homology with an exemplified toxin.

The amino acid identity will typically be greater than 60%, preferably be greater than 75%, mor preferably greater than 80%, more preferably greater than 90%, and can be greater than The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of thre-dimensional cofigurationwhich ultimately is resposible for the biological activity. In this regard, certain amino acid S: substitutions are acceptable and can be. expected if these substitutions are in regions which are hot critical to activity or are conservative amino acid substitutions which do not affect the threedimensional configuation of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall withi the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 2 provides a listing of examples of amino acids belonging to each class.

Table 2.

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.

Recombinant hosts. The toxin-encoding genes harbored by the isolates 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 intracellular production and maintenance of the 15 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 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 Where the B.t. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environmerit in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest.

These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, genera Pseudomonas, Erwinia, Serratia, Klebsiella, 16 Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonasfluorescens, Serratia marcescens, Acetobacterxylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A wide variety of ways 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 S* 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.

Control of coleopterans, including corn rootworm 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 ofB.t. isolates to the pests (or their location), the application of recombinant microbes to the pests (or their locations), and the 20 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.

Synthetic genes which are functionally equivalent to the 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. 5,380,831.

Control of other pests such as lepidopterans and other insects, nematodes, and mites can also be accomplished by those skilled in the art using standard techniques combined with the teachings provided herein.

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 microcapsule that is formed comprises the B.t. toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes, normally being 17 limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.

The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.

Treatment of the microbial cell, a microbe containing the B.t. toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques Sinclude treatment with aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran i 15 chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, various acids and Helly's fixative i (See: Humason, Gretchen Animal Tissue Techniques, W.H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host environment.

20 Examples of physical means are short wavelength radiation such as gamma-radiation and Xradiation, freezing, UV irradiation, lyophilization, and the like. 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.

The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of treatment should retain at least a substantial portion of the bio-availability or bioactivity of the toxin.

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

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 B.t. 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 Sseparating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert 15 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.

Formulations. Formulated bait granules containing an attractant and spores and crystals 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 S 20. 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 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). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like.

The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

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 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 19 cells/mg. These formulations 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 the isolates of 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.

A smaller percentage of the asporogenous mutants will remain intact and not lyse for extended fermentation periods; these strains are designated lysis minus Lysis minus strains can be identified by screening asporogenous mutants in shake flask media and selecting those mutants that are still intact and contain toxin crystals at the end of the fermentation. Lysis minus strains are suitable for a cell treatment process that will yield a protected, encapsulated toxin protein.

15 To prepare a phage resistant variant of said asporogenous mutant, an aliquot of the phage lysate is spread onto nutrient agar and allowed to dry. An aliquot of the phage sensitive "-bacterial strain is then plated directly over the dried lysate and allowed to dry. The plates are incubated at 30 0 C. The plates are incubated for 2 days and, at that time, numerous colonies could be seen growing on the agar. Some of these colonies are picked and subcultured onto 20 nutrient agar plates. These apparent resistant cultures are tested for resistance by cross streaking with the phage lysate. A line of the phage lysate is streaked on the plate and allowed to dry. The presumptive resistant cultures are then streaked across the phage line. Resistant bacterial cultures show no lysis anywhere in the streak across the phage line after overnight incubation at 30 0 C. The resistance to phage is then reconfirmed by plating a lawn of the resistant culture onto a nutrient agar plate. The sensitive strain is also plated in the same manner to serve as the positive control. After drying, a drop of the phage lysate is placed in the center of the plate and allowed to dry. Resistant cultures showed no lysis in the area where the phage lysate has been placed after incubation at 30 0 C for 24 hours.

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 Culturing of B.t. Isolates of the Invention A subculture of the B.t. isolates, or mutants thereof, can be used to inoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone 7.5 g/1 Glucose 1.0 g/l KHPO, 3.4 g/1

K

2 HPO, 4.35 g/1 Salt Solution 5.0 ml/1 CaCI 2 Solution 5.0 ml/1 pH 72 Salts Solution (100 ml) MgSO,*7HO 2.46 g 15 MnSO4-H,O 0.04 g ZnSO 4 -7H20 0.28 g FeSO 4 -7H,O 0.40 g o CaC, Solution (100 ml) 20 CaCIl2H 2 0 3.66 g The salts solution and CaCI2 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. spores and/or crystals, obtained in 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 Protein Purification for 45 kDa 80JJ1 Protein One gram of lyophilized powder of 80JJ1 was suspended in 40 ml of buffer containing mM Tris-Cl pH 7.8,5 mM EDTA, 100 M PMSF, 0.5 pg/ml Leupeptin, 0.7. pg/ml Pepstatin, and 40 pg/ml Bestatin. The suspension was centrifuged, and the resulting supernatant was discarded. The pellet was washed five times using 35-40 ml of the above buffer for each wash.

i The washed pellet was resuspended in 10 ml of 40% NaBr, 5 mM EDTA, 100 uM PMSF, pg/ml Leupeptin, 0.7 pg/ml Pepstatin, and 40 pg/ml Bestatin and placed on a rocker platform for 75 minutes. The NaBr suspension was centrifuged, the supernatant was removed, and the pellet was treated a second time with 40% NaBr, 5 mM EDTA, 100 pM PMSF, 0.5 pg/ml Leupeptin, 0.7 tg/ml Pepstatin, and 40 pg/ml Bestatin as above. The supernatants (40% NaBr soluble) were combined and dialyzed against 10 mM CAPS pH 10.0, 1 mM EDTA at 4°C. The dialyzed extracts were centrifuged and the resulting supernatant was removed. The pellet NaBr dialysis pellet) was suspended in 5 ml of H20 and centrifuged. The resultant supernatant was removed and discarded. The washed pellet was washed a second time in 10 ml of H20 and centrifuged as above. The washed pellet was suspended in 1.5 ml of HO and contained primarily three peptides with molecular weights of approximately 47 kDa, 45 kDa, and 15 kDa when analyzed using SDS-PAGE. At this stage of purification, the suspended 40% NaBr "dialysis pellet contained approximately 21 mg/ml of protein by Lowry assay.

15 The peptides in the pellet suspension were separated using SDS-PAGE (Laemlli, U.K.

[1970] Nature 227:680) in 15% acrylamide gels. The separated proteins were then electrophoretically blotted to a PVDF membrane (Millipore Corp.) in 10 mM CAPS pH 11.0, MeOH at 100 V constant After one hour the PVDF membrane was rinsed in water briefly and placed for 3 minutes in 0.25% Coomassie blue R-250, 50% methanol, 5% acetic acid. The 20 stained membrane was destained in 40% MeOH, 5% acetic acid. The destained membrane was air-dried at room temperature (LeGendre et al. [1989] In A Practical Guide to Protein Purification For Microsequencing, P. Matsudaira, ed., Academic Press, New York, NY). The membrane was sequenced using automated gas phase Edman degradation (Hunkapillar, M.W., R.M. Hewick, W.L. Dreyer, L.E. Hood [1983] Meth. Enzymol. 91:399).

The amino acid analysis revealed that the N-terminal sequence of the 45 kDa band was as follows: Met-Leu-Asp-Thr-Asn (SEQ ID NO. 1).

The 47 kDa band was also analyzed and the N-terminal amino acid sequence was determined to be the same 5-amino acid sequence as SEQ ID NO. 1. Therefore, the N-terminal amino acid sequences of the 47 kDa peptide and the 45 kDa peptide were identical.

This amino acid sequence also corresponds to a sequence obtained from a 45 kDa peptide obtained from PS80JJ1 spore/crystal powders, using another purification protocol, with the N-terminal sequence as follows: Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-le-Ser-Asn-Leu- Ala-Asn-Gly-Leu-Tyr-Thr-Set-Thr-Tyr-Leu-Ser-Leu (SEQ ID NO. 2).

Example 3 Purification of the 14 kDa Peptide of 0.8 ml of the white dialysis suspension (approximately 21 mg/ml) containing the 47 kDa, 45 kDa, and 15 kDa peptides, was dissolved in 10 ml of 40% NaBr, and 0.5 ml of 100 mM EDTA were added. After about 18 hours (overnight), a white opaque suspension was obtained.

This was collected by centrifugation and discarded. The supernatant was concentrated in a (Amicon Corporation) to a final volume of approximately 15 ml. The filtered volume was washed with water by filter dialysis and incubated on ice, eventually forming a milky white suspension. The suspension was centrifuged and the pellet and supernatant were separated and retained. The pellet was then suspended in 1.0 ml water (approximately 6 mg/ml).

The pellet contained substantially pure 15 kDa protein when analyzed by SDS-PAGE.

The N-terminal amino acid sequence was determined to be: Ser-Ala-Arg-Glu-Val-His- S. Ile-Glu-le-Asn-Asn-Thr-Arg-His-Thr-Leu-Gln-Leu-Glu-Ala-Lys-Thr-Lys-Leu (SEQ ID NO.

3) 3) 15 Example 4 Protein Purification and Characterization ofPS149B1 45 kDa Protein The P 1 pellet was resuspended with two volumes of deionized water per unit wet weight, ~and to this was added nine volumes of 40% aqueous sodium bromide. This and all subsequent operations were carried out on ice or at 4-6 0 C. After 30 minutes, the suspension was diluted with 36 volumes of chilled water and centrifuged at 25,000 x g for 30 minutes to give S 20 a pellet and a supernatant.

The resulting pellet was resuspended in 1-2 volumes of water and layered on a 20-40% sodium bromide gradient and centrifuged at 8,000 x g for 100 minutes. The layer banding at approximately 32% sodium bromide (the "inclusions", or INC) was recovered and dialyzed overnight against water using a dialysis membrane with a 6-8 kDa MW cut-off.

Particulate material was recovered by centrifugation at 25,000 x g, resuspended in water, and aliquoted and assayed for protein by the method of Lowry and by SDS-PAGE.

The resulting supematant was concentrated 3- to 4-fold using concentrators, then dialyzed overnight against water using a dialysis membrane with a 6-8 kDa MW cut-off. Particulate material was recovered by centrifugation at 25,000 x g, resuspended in water, and aliquoted and assayed for protein by the method of Lowry and by SDS-PAGE.

This fraction was denoted as P1.P2.

The peptides in the pellet suspension were separated using SDS-PAGE (Laemlli, U.K., supra) in 15% acrylamide gels. The separated proteins were then electrophoretically blotted to a PVDF membrane (Millipore Corp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 V constant.

After one hour the PVDF membrane was rinsed in water briefly and placed for 3 minutes in 0.25% Coomassie blue R-250, 50% methanol, 5% acetic acid. The stained membrane was destained in 40% MeOH, 5% acetic acid. The destained membrane was air-dried at room temperature (LeGendre et al., supra). The membrane was sequenced using automated gas phase Edman degradation (Hunkapillar et al., supra).

Protein analysis indicated the presence of two major polypeptides, with molecular weights of 47 kDa and 14 kDa. Molecular weights were measured against standard polypeptides of known molecular weight. This process provides only an estimate of true molecular weight.

The 47 kDa band from PS149B1 migrated on SDS-PAGE in a manner indistinguishable from the 47 kDa protein from PS80JJ1. Likewise, the 14 kDa band from PS149B1 migrated on SDS- PAGE in a manner indistinguishable from 14 kDa bands from PS 167H2 and PS80JJ1. Apart S* from these two polypeptides, which were estimated to account for 25-35% (47 kDa) and 35-55% (15 kDa) of the Coomassie staining material respectively, there may be minor bands, including those of estimated MW at 46 kDa, 130 kDa, and 70 kDa.

15 Protein analysis indicated that fraction INC contained a single polypeptide with MW of 47 kDa, and that fraction P1.P2 contained a single polypeptide with MW of 14 kDa. These polypeptides were recovered in yields greater than 50% from P1.

The N-terminal amino acid sequence for the purified 47 kDa protein from PS 149B 1 is: Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-e-Ser-Asn-His-Ala-Asn-Gly-Leu-Tyr-Ala-Ala-Thr- 20 Tyr-Leu-Ser-Leu (SEQ ID NO. 4).

The N-terminal amino acid sequence for the purified 14 kDa protein from PS149B1 is: Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp- S: Lys-Thr-Lys-Leu-Asp-Gly-Gly-Arg-Trp-Arg-Thr-Ser-Pro-Xaa-Asn-Val-Ala-Asn-Asp-Gln-Ile- Lys-Thr-Phe-Val-Ala-Glu-Ser-Asn (SEQ ID NO. Example 5 Amino Acid Sequence for 45 kDa and 14 kDa Toxins of PS167H2 The N-terminal amino acid sequence for the purified 45 kDa protein from PS 167H2 is: Met-Leu-Asp-Thr-Asn-Lys-Ie-Tyr-Glu-Ie-Ser-Asn-Tyr-Ala-Asn-Gly-Leu-His-Ala-Ala-Thr- Tyr-Leu-Ser-Leu (SEQ ID NO. 6).

The N-terminal amino acid sequence for the purified 14 kDa protein from PS 167H2 is: Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-As-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp- Lys-Thr-Lys-Leu (SEQ ID NO. 7).

These amino acid sequences can be compared to the sequence obtained for the 47 kDa peptide obtained from 80JJl spore/crystal powders with the N-terminal sequence (SEQ ID NO.

24 1) and to the sequence obtained for the 14 kDa peptide obtained from 80JJ1 spore/crystal powders with the N-terminal sequence (SEQ ID NO. 3).

Clearly, the 45-47 kDa proteins are highly related and probably represent one gene family, and the 14 kDa proteins are highly related and probably represent another gene family.

Example 6 Molecular Cloning. Expression. and DNA Sequence Analysis of a Novel 6- Endotoxin Gene from Bacillus thuringiensis Strain Total cellular DNA was prepared from Bacillus thuringiensis cells grown to an optical density, at 600 nm, of 1.0. Cells were pelleted by centrifugation and resuspended in protoplast buffer (20 mg/ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 25 mM SEDTA). After incubation at 37"C for 1 hour, protoplasts were lysed by two cycles of freezing and thawing. Nine volumes of a solution of 0.1 M NaC1, 0.1% SDS, 0.1 M Tris-CI were added to complete lysis. The cleared lysate was extracted twice with phenol:chloroform Nucleic acids were precipitated with two volumes of ethanol and pelleted by centrifugation. The pellet 15 was resuspended in TE buffer and RNase was added to a final concentration of 50 Pg/ml. After incubation at 37C for 1 hour, the solution was extracted once each with phenol:chloroform S* and TE-saturated chloroform. DNA was precipitated from the aqueous phase by the addition of one-tenth volume of 3 M NaOAc and two volumes of ethanol. DNA was pelleted by centrifugation, washed with 70% ethanol, dried, and resuspended in TE buffer.

20 An oligonucleotide probe for the gene encoding the PS80JJ1 45 kDa toxin was designed from N-terminal peptide sequence data. The sequence of the 29-base oligonucleotide probe was: 5'-ATG YTW GAT ACW AAT AAA GTW TAT GAA AT-3' (SEQ ID NO. 8) This oligonucleotide was mixed at four positions as shown. This probe was radiolabeled with "P and used in standard condition hybridization of Southern blots of PS80JJ1 total cellular DNA digested with various restriction endonucleases. Representative autoradiographic data from these experiments showing the sizes of DNA restriction fragments containing sequence homology to the 443 kDa toxin oligonucleotide probe of SEQ ID NO. 8 are presented in Table Table 3. RFLP of PS80JJl cellular DNA fragments on Southern blots that hybridized under standard conditions with the 44.3 kDa toxin gene oligonucleotide probe (SEQ ID NO. 8) Restriction Enzyme Approximate Fragment Size (kbp) EcoRI HindIII 8.3 KpnI 7.4 PstI 11.5 XbaI 9.1 These DNA fragments identified in these analyses contain all or a segment of the PS80JJ kDa toxin gene. The approximate sizes of the hybridizing DNA fragments in Table 3 are in reasonable agreement with the sizes of a subset of the PS80JJ1 fragments hybridizing with a PS80JJ1 45 kDa toxin subgene probe used in separate experiments, as predicted (see Table 4, below).

A gene library was constructed from PS80JJ1 DNA partially digested with Sau3AI.

Partial restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 9.3 to 23 kbp in size were excised from the gel, electroeluted from the gel slice, purified on an 20 Elutip-D ion exchange column (Schleicher and Schuell, Keene, NH), and recovered by ethanol precipitation. The Sau3AI inserts were ligated into BamHI-digested LambdaGem-11 (Promega, Madison, WI). Recombinant phage were packaged and plated on E. coli KW251 cells. Plaques were screened by hybridization with the oligonucleotide probe described above. Hybridizing phage were plaque-purified and used to infect liquid cultures ofE. coli KW251 cells for isolation of DNA by standard procedures (Maniatis et al., supra).

Southern blot analysis revealed that one of the recombinant phage isolates contained an approximately 4.8 kbp XbaI-SacI band that hybridized to the PS80JJ1 toxin gene probe. The SacI site flanking the PS80JJI toxin gene is a phage vector cloning site, while the flanking XbaI site is located within the PS80JJ1 DNA insert. This DNA restriction fragment was subcloned by standard methods into pBluescript S/K (Stratagene, San Diego, CA) for sequence analysis.

The resultant plasmid was designated pMYC2421. The DNA insert was also subcloned into pHTBluell (an E. colilB. thuringiensis shuttle vector comprised of pBluescript S/K [Stratagene, La Jolla, CA] and the replication origin from a resident B.t. plasmid Lereclus et al. (1989) FEMS Microbiology Letters 60:211-218]) to yield pMYC2420.

An oligonucleotide probe for the gene encoding the PS80JJI 14 kDa toxin was designed 0 from N-terminal peptide sequence data. The sequence of the 28-base oligonucleotide probe was: GW GAA GTW CAT ATW GAA ATW AAT AAT AC 3' (SEQ ID NO. 29). This oligonucleotide was mixed at four positions as shown. The probe was radiolabelled with 32 P and used in standard condition hybridizations of Southern blots of PS80JJ1 total cellular and pMYC2421 DNA digested with various restriction endonucleases. These RFLP mapping experiments demonstrated that the gene encoding the 14 kDa toxin is located on the same genomic EcoRI fragment that contains the N-terminal coding sequence for the 44.3 kDa toxin.

To test expression of the PS80JJ1 toxin genes in pMYC2420 was transformed into the acrystalliferous (Cry-) B.t. host, CryB Aronson, Purdue University, West Lafayette, IN), by electroporation. Expression of both the approximately 14 and 44.3 kDa PS80JJ1 toxins encoded by pMYC2420 was demonstrated by SDS-PAGE analysis. Toxin crystal preparations from the recombinant CryB[pMYC2420] strain, MR536, were assayed and found to be active S. against western corn rootworm.

15 The PS80JJ1 toxin genes encoded by pMYC2421 were sequenced using the ABI373 automated sequencing system and associated software. The sequence of the entire genetic locus containing both open reading frames and flanking nucleotide sequences is shown in SEQ ID NO.

The termination codon of the 14 kDa toxin gene is 121 base pairs upstream from the initiation codon of the 44.3 kDa toxin gene (Figure The PS80JJ1 14 kDa toxin open reading frame nucleotide sequence (SEQ ID NO. 31), the 44.3 kDa toxin open reading frame nucleotide sequence (SEQ ID NO. 10), and the respective deduced amino acid sequences (SEQ ID NO. 32 o. and SEQ ID NO. 11) are novel compared to other toxin genes encoding pesticidal proteins.

SThus, the nucleotide sequence encoding the 14 kDa toxin of PS80JJ1 is shbwn in SEQ ID NO. 31. The deduced amino acid sequence of the 14 kDa toxin of PS80JJ1 is shown in SEQ ID NO. 32. The nucleotide sequences encoding both the 14 and 45 kDa toxins of PS80JJ1, as well as the flanking sequences, are shown in SEQ ID NO. 30. The relationship of these sequences is shown in Figure 2.

A subculture of E. coli NM522 containing plasmid pMYC2421 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, IL 61604 USA on March 28, 1996. The accession number is NRRL B-21555.

Example 7 RFLP and PCR Analysis of Additional Novel 6-Endotoxin Genes from Bacillus thuringiensis Strains PS 149B 1 and PS 167H2 Two additional strains active against corn rootworm, PS149B1 and PS167H2, also produce parasporal protein crystals comprised in part ofpolypeptides approximately 14 and kDa in size. Southern hybridization and partial DNA sequence analysis were used to examine the relatedness of these toxins to the 80JJ1 toxins. DNA was extracted from these B.t. strains as described above, and standard Southern hybridizations were performed using the 14 kDa toxin oligonucleotide probe (SEQ ID NO. 29) and an approximately 800 bp PCR fragment of the 80JJ1 44.3 kDa toxin gene-encoding sequence. Representative RFLP data from these experiments showing the sizes of DNA restriction fragments containing sequence homology to the 44.3 kDa toxin are presented in Table 4. Representative RFLP data from these experiments showing the sizes of DNA restriction fragments containing sequence homology to the approximately 14 kDa toxin are presented in Table Table 4. RFLP ofPS80JJ1, PS149B1, and PS167H2 cellular DNA fragments on Southern blots that hybridized with the approximately 800 bp PS80JJ1 44.3 kDa toxin subgene probe under standard conditions Strain PS80JJ1 PS149B1 PS167H2 20 Restriction enzyme Approximate fragment size (kbp) EcoRI 6.4 5.7 2.6 13 2.8 0.6 HindII 8.2 6.2 4.4 KpnI 7.8 10.0 11.5 PstI 12.0 9.2 9.2 8.2 XbaI 9.4 10.9 10.9 SacI 17.5 15.5 11.1 13.1 10.5 6.3 SI I I I I I L I m 28 Each of the three strains exhibited unique RFLP patterns. The hybridizing DNA fragments from PS 149B 1 or PS 167H2 contain all or part of toxin genes with sequence homology to the PS80JJ1 44.3 kDa toxin.

Table 5. Restriction fragment length polymorphisms of PS80JJI, PS149B1, and PS 167H2 cellular DNA fragments on Southern blots that hybridized with the PS80JJ 14 kDa toxin oligonucleotide probe under standard conditions 0 0 00..

0 Strain PS80JJ1 PS149B1 PS167H2 Restriction enzyme Approximate fragment size (kbp) EcoRI 5.6 2.7 2.7 HindIII 7.1 6.0 4.7 Xbal 8.4 11.2 11.2 Each of the three strains exhibited unique RFLP patterns. The hybridizing DNA fragments from PS149B 1 or PS 167H2 contain all or part of toxin genes with sequence homology to the PS80JJ1 14 kDa toxin gene.

Portions of the toxin genes in PS149B1 or PS167H2 were amplified by PCR using 20 forward and reverse oligonucleotide primer pairs designed based on the PS80JJI 44.3 kDa toxin gene sequence. For PS149B1, the following primer pair was used: Forward: YTW GAT ACW AAT AAA GTW TAT GAA AT-3' (SEQ ID NO. 8) Reverse: 5'-GGA TTA TCT ATC TCT GAG TGT TCT TG-3' (SEQ ID NO. 9) For PS167H2, the same primer pair was used. These PCR-derived fragments were sequenced using the ABI373 automated sequencing system and associated software. The partial gene and peptide sequences obtained are shown in SEQ ID NO. 12-15. These sequences contain portions of the nucleotide coding sequences and peptide sequences for novel corn rootworm-active toxins present in B.t. strains PS149B1 or PS167H2.

Example 8 Molecular Cloning and DNA Sequence Analysis of Novel 6-Endotoxin Genes from Bacillus thuringiensis Strains PS 149B1 and PS167H2 Total cellular DNA was extracted from strains PS149B1 and PS 167H2 as described for PS80JJ1. Gene libraries of size-fractionated Sau3A partial restriction fragments were constructed in Lambda-Gem 11 for each respective strain as previously described. Recombinant phage were packaged and plated on E. coli KW251 cells. Plaques were screened by hybridization with the oligonucleotide probe specific for the 44 kDa toxin gene. Hybridizing phage were plaque-purified and used to infect liquid cultures ofE. coli KW251 cells for isolation of DNA by standard procedures (Maniatis et al., supra).

For PS 167H2, Southern blot analysis revealed that one of the recombinant phage isolates contained an approximately 4.0 to 4.4 kbp HindIII band that hybridized to the PS80JJI 44 kDa .toxin gene 5' oligonucleotide probe (SEQ ID NO. This DNA restriction fragment was subcloned by standard methods into pBluescript S/K (Stratgene, San Diego, CA) for sequence analysis. The fragment was also subcloned into the high copy number shuttle vector, pHT370 15 Arantes, D. Lereclus [1991] Gene 108:115-119) for expression analyses in Bacillus thuringiensis (see below). The resultant recombinant, high copy number bifunctional plasmid was designated pMYC2427.

The PS167H2 toxin genes encoded by pMYC2427 were sequenced using the ABI automated sequencing system and associated software. The sequence of the entire genetic locus 20 containing both open reading frames and flanking nucleotide sequences is shown in SEQ ID NO.

34. The termination codon of the 14 kDa toxin gene is 107 base pairs upstream from the initiation codon of the 44 kDa toxin gene. The PS167H2 14 kDa toxin coding sequence (SEQ i ID NO. 35), the 44 kDa toxin coding sequence (SEQ ID NO. 37), and the respective deduced amino acid sequences, SEQ IDNO. 36 and SEQ IDNO. 38, are novel compared to other known toxin genes encoding pesticidal proteins. The toxin genes are arranged in a similar manner to, and have some homology with, the PS80JJ1 14 and 44 kDa toxins.

A subculture ofE. coli NM522 containing plasmid pMYC2427 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Illinois 61604 USA on 26 March 1997. The accession number is NRRL B-21672.

For PS149B1, Southern blot analysis using the PS80JJ1 44 kDa oligonucleotide 5' probe (SEQ ID NO. 8) demonstrated hybridization of an approximately 5.9 kbp ClaI DNA fragment.

Complete ClaI digests ofPS149B1 genomic DNA were size fractionated on agarose gels and cloned into pHTBlueII. The fragment was also subcloned into the high copy number shuttle vector, pHT370 (Arantes, D. Lereclus [1991] Gene 108:115-119) for expression analyses in Bacillus thuringiensis (see below). The resultant recombinant, high copy number bifunctional plasmid was designated pMYC2429.

The PS149B1 toxin genes encoded by pMYC2429 were sequenced using the ABI automated sequencing system and associated software. The sequence of the entire genetic locus containing both open reading frames and flanking nucleotide sequences is shown in SEQ ID NO.

39. The termination codon of the 14 kDa toxin gene is 108 base pairs upstream from the initiation codon of the 44 kDa toxin gene. The PS149B1 14 kDa toxin coding sequence (SEQ ID NO. 40), the 44 kDa toxin coding sequence (SEQ ID NO. 42), and the respective deduced amino acid sequences, SEQ ID NO. 41 and SEQ ID NO. 43, are novel compared to other known toxin genes encoding pesticidal proteins. The toxin genes are arranged in a similar manner as, and have some homology with, the PS80JJI and PS167H2 14 and 44 kDa toxins. Together, these three toxin operons comprise a new family of pesticidal toxins.

A subculture of E. coli NM522 containing plasmid pMYC2429 was deposited in the 15 permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Illinois 61604 USA on 26 March 1997. The accession number is NRRL B-21673.

Example 9 PCR Amplification for Identification and Cloning Novel Corn Rootworm-Active 20 Toxin The DNA and peptide sequences of the three novel approximately 45 kDa corn rootworm-active toxins from PS80JJ1, PS149B1, and PS167H2 (SEQ ID NOS. 12-15) were aligned with the Genetics Computer Group sequence analysis program Pileup using a gap weight of 3.00 and a gap length weight of 0.10. The sequence alignments were used to identify conserved peptide sequences to which oligonucleotide primers were designed that were likely to hybridize to genes encoding members of this novel toxin family. Such primers can be used in PCR to amplify diagnostic DNA fragments for these and related toxin genes. Numerous primer designs to various sequences are possible, four of which are described here to provide an example. These peptide sequences are: Asp-Ie-Asp-Asp-Tyr-Asn-Leu (SEQ ID NO. 16) Trp-Phe-Leu-Phe-Pro-Ile-Asp (SEQ ID NO. 17) Gln-Ile-Lys-Thr-Thr-Pro-Tyr-Tyr (SEQ ID NO. 18) Tyr-Glu-Trp-Gly-Thr-Glu (SEQ ID NO. 19).

The corresponding nucleotide sequences are: 5'-GATATWGATGAYTAYAAYTTR-3' (SEQ ID NO. O 5'-TGGTITTTRTTTCCWATWGAY-3' (SEQ ID NO. 21) 5'-CAAATHAAAACWACWCCATATTAT-3' (SEQ ID NO. 22) 5'-TAYGARTGGGGHACAGAA-3' (SEQ ID NO.-23).

Forward primers for polymerase amplification in thermocycle reactions were designed based on the nucleotide sequences of SEQ ID NOS. 20 and 21.

Reverse primers were designed based on the reverse complement of SEQ ID NOS. 22 and 23: 5'-ATAATATGGWGTWGTTrTDAITrG-3' (SEQ ID NO. 24) ,o 5'-TTCTGTDCCCAYTCRTA-3' (SEQ ID NO. These primers can be used in combination to amplify DNA fragments of the following sizes (Table 6) that identify genes encoding novel corn rootworm toxins.

Table 6. Predicted sizes of diagnostic DNA fragments (base pairs) amplifiable with primers specific for novel corn rootworm-active toxins is Primer pair (SEQ ID NO.) DNA fragment size (bp) 20 24 495 594 21 24 471 21 25 580 to Similarly, entire genes encoding novel corn rootworm-active toxins can be isolated by polymerase amplification in thermocycle reactions using primers designed based on DNA sequences flanking the open reading frames. For the PS80JJ1 44.3 kDa toxin, one such primer pair was designed, synthesized, and used to amplify a diagnostic 1613 bp DNA fragment that included the entire toxin coding sequence. These primers are: is Forward: 5'-CTCAAAGCGGATCAGGAG-3' (SEQ ID NO. 26) Reverse: 5'-GCGTATTCGGATATGCTTGG-3' (SEQ ID NO. 27).

For PCR amplification of the PS80JJI 14 kDa toxin, the oligonucleotide coding for the N-terminal peptide sequence (SEQ ID NO. 29) can be used in combination with various reverse oligonucleotide primers based on the sequences in the PS80JJ1 toxin gene locus. One such 3o reverse primer has the following sequence: CATGAGATITATCTCCTGATCCGC 3' (SEQ ID NO. 33).

When used in standard PCR reactions, this primer pair amplified a diagnostic 1390 bp DNA Sfragment that includes the entire 14 kDa toxin coding sequence and some 3' flanking sequences corresponding to the 121 base intergenic spacer and a portion of the 44.3 kDa toxin gene. When used in combination with the 14 kDa forward primer, PCR will generate a diagnostic 322 base pair DNA fragment.

Example 10 Bioassay of Protein A preparation of the insoluble fraction from the dialyzed NaBr extract of 80JJ1 containing the 47 kDa, 45 kDa, and 15 kDa peptides was bioassayed against Western corn rootworm and found to exhibit significant toxin activity.

S.

0O S 000 S 0@ S 0

S

S. *e

S

0* 0 .:1 Example 11 Bioassay of Protein The purified protein fractions from PS149B1 were bioassayed against western corn rootworm and found to exhibit significant toxin activity when combined. In fact, the combination restored activity to that noted in the original preparation The following bioassay data set presents percent mortality and demonstrates this effect.

2 Concentration (jg/cm 2 300 100 33.3 11.1 3.7 1.2 0.4 P1 88, 100, 94 94, 50, 63 19, 19, 44 13, 56, 25 0, 50, 0 13, 43, 12 6. 12.6 Table 7.

INC

19 31 38 12 0 0 25 P1.P2 13 38 13 31 31 12 19 INC P1.P2 100 94 13 13 19 6 i Example 12 Clone Dose-Response Bioassays The PS80JJ1 toxin operon was also subcloned from pMYC2421 into pHT370 for direct comparison ofbioactivity with the recombinant toxins cloned from PS149B1 and PS167H2. The resultant recombinant, high copy number bifunctional plasmid was designated pMYC2426.

33 A subculture ofE. coli NM522 containing plasmid pMYC2426 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Illinois 61604 USA on 26 March 1997. The accession number is NRRL B-21671.

To test expression of the PS80JJ1, PS149B1 and PS167H2 toxin genes in B.t., pMYC2426, pMYC2427 and pMYC2429 were separately transformed into the acrystalliferous (Cry-) B. t. host, CryB Aronson, Purdue University, West Lafayette, IN), by electroporation.

The recombinant strains were designated MR543 (CryB [pMYC2426]), MR544 (CryB [pMYC2427]) and MR546 (CryB [pMYC2429]), respectively Expression of both the approximately 14 and 44 kDa toxins was demonstrated by SDS-PAGE analysis for each recombinant strain.

Toxin crystal preparations from the recombinant strains were assayed against western corn rootworm. Their diet was amended with sorbic acid and SIGMA pen-strep-ampho-B. The 0: material was top-loaded at a rate of 50 il of suspension per cm 2 diet surface area. Bioassays 15 were run with neonate Western corn rootworm larvae for 4 days at approximately Percentage mortality and top-load LC estimates for the clones (pellets) are set forth in Table 8.

Table 8.

20 Percentage mortality at given protein concentration (pg/cm 2 Samnle 50 5 MR543 pellet 44 19 9 MR544 pellet 72 32 21 MR546 pellet 52 32 21 dH20 7 Example 13 Insertion and Expression of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with genes encoding the insecticidal toxin. The transformed plants are resistant to attack by the target pest.

The novel corn rootworm-active genes described here can be optimized for expression in other organisms. Maize optimized gene sequences encoding the 14 and 44 kDa PS80JJ1 toxins are disclosed in SEQ ID NO. 44 and SEQ ID NO. 45, respectively.

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- 20 46; and An et al. (1985) EMBO J. 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 ;0 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 SAgrobacteria. 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, roots, but also protoplasts or suspensioncultivated 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 15 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.

The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the S. normal maimer and crossed with plants that have the same transformed hereditary factors or 20 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, which is hereby incorporated by reference. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about of the full length toxin. Methods for creating synthetic B.t. genes for use in plants are known in the art.

Example 14 Cloning of B.t. Genes Into Insect Viruses A number of viruses are known to infect insects. These viruses include, for example, baculoviruses and entomopoxviruses. In one embodiment of the subject invention, genes encoding the insecticidal toxins, as described herein, can be placed within the genome of the Sinsect virus, thus enhancing the pathogenicity of the virus. Methods for constructing insect viruses which comprise B.t. toxin genes are well known and readily practiced by those skilled in the art These procedures are described, for example, in Merryweather et al. (Merryweather, U. Weyer, M.P.G. Harris, M. Hirst, T. Booth, R.D. Possee (1990) J. Gen. Virol. 71:1535- 1544) and Martens et al. (Martens, G. Honee, D. Zuidema, J.W.M. van Lent, B. Visser, J.M. Vlak (1990) Appl. EnvironmentalMicrobiol. 56(9):2764-2770).

S 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.

EDITORIAL NOTE NO 23125/01 Sequence listing pages 37 to 66 is part of the description.

The claims pages are to follow.

SEQUENCE LISTING O GENERAL INFORMATION:

APPLICANT:

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 (ii) TITLE OF INVENTION: Pesticidal Toxins (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Saliwanchik, Lloyd Saliwanchik STREET: 2421 N.W. 41st Street, Suite A-1 CITY: Gainesville S: STATE: FL COUNTRY: USA ZIP: 32606-6669 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.25 o* (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER:

US

FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 08/633,993 FILING DATE: 19-APR-1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Sanders, Jay M.

REGISTRATION NUMBER: 39,355 REFERENCE/DOCKET NUMBER: MA-703C1 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 352-375-8100 TELEFAX: 352-372-5800 INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECUL-E TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Leu Asp Thr Asn 1 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lieu Asp Thr Asn Lys Val Tyr Glu le Ser Asn Leu Ala Asn Gly 1 5 10 Leu Tyr Tbr Ser Tbr Tyr Leu Ser Lieu -20 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (iji) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn Thx Arg His Thr ieU 1 5 10 1 Gin Lieu Glu Ala Lys Tlir Lys Lieu INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRMNDEDKESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Leu Asp Thr Asn Lys Val Tyr Giu Ile Ser Asfl His Ala Asn Gly 1 5 10 Leu Tyr Ala Ala Thr Tyr Leu Ser Lieu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 50 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID Ser Ala Arg Giu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 10 Leu Gin Lieu Giu Asp Lys Thr Lys Lieu Asp Gly Gly Arg Trp Arg Thr 25 316 *:Ser Pro Xaa Asn. Val Ala Asn Asp Gin Ile Lys Thr Phe Val A-la Giu Ser Asn INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Leu Asp Thr Asn Lys* Ile Tyr Giu Ile Ser Asn Tyr Ala Asn Gly 1 510 1 Leu His Ala Ala Thr Tyr Lieu Ser Lieu INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 1 5 10 Leu Gln Leu Glu Asp Lys Thr Lys Leu INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATGNTNGATA CNAATAAAGT NTATGAAAT INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGATTATCTA TCTCTGAGTG TTCTTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1158 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 41 (xi) SEQUENCE DESCRIPTION: SEQ ID ATOTTAGATA CTAATAAAGT TTATGAAATA AGCAATCTTG

ACTTATTTAA

GATCATTACA

AGCTATGGAG

ACTTATTCTT

ATAATACAAA

GTACGCCTAA

CAAACAATTC

TATTCAGAAA

GTACCTTGTA

CCATATTATA

TTACTTCCAC

ACAACTATTA

GTACCAGAAG

GTTGAATATA

AATCCAACTA

TATCGATATA

TACACTCTTA

TATGAAGAAG

CATTATTTTA

GTCTTGATGA

ATTTAAAATG

CTAATAATTG

CAACAAACTC

GTGATAATGG

CTGATGAATT

AACTCCCACA

CCGGAAATAT

TTATGGTAAA

TTTTTAAAAA

ATCAAAAAAG

T1'AATACAGT

TAGGAGGAGG

GCACTGAAAC

ATCAACCAAT

ACGGTACAGA

CTTC'TTATCC

TAGAAGAAAT

AAAAATAA

TTCAGGTGTT

GTT'FTTATTT

TAAAGTTTGG

TGTACAAAAA

AAAGGTCTTA

TCCAGAGAAT

AAAACCTAAA

AAATCCTAAA

TGATTCAAAA

ATATAAATAC

ATCATATGAT

AGGATTGCAA

TACAGAAGAC

CAAAATAATG

GAATTCTATA

AATTAAGATA

AAATCATAAA

AACAAAAATA

AGTTTAATGA

CCTATTGATA

AATGTTAAAA

TGGCAAATAA

ACAGCAGGAG

TCTAACCAAC

ATAGATGAAA

ACAACTCCTC

ATAGATAAAA

TGGAATCTAG

TATGAATGGG

ATTAATATAG

ATAAAAACAC

ACGAAATATC

GGACTTCTTA

ATGGACATAG

GAAGCATTAT

CCTAAGCATA

CTAATGGATT

GTAAAAAGGA

ATAATCAATA

ATGATAAAAT

A.AGCTAA-AGA

TAGGTCAATC

AATGGAATTT

AATTAAAAGA

AATTAATGGG

ACACTCAAAT

CAAAAGGAAG

GTACAGAAAA

ATTCAGGAAT

AATTAACTGA

AAGAACACTC

TTTATACTTC

AAACTTCAGA

TACTTCTCAC

CACTTATAAA

ATATACATCA

TGAAGATATT

TATTATTACA

AAATGTTTCA

TTCTTCATAT

TCTTGGAATA

AACTCCTGTA

TCATCCTGAA

ATGGACATTA

TAAAACTACT

TAATGTATCT

AAATCAAAAA

GAAATTTGAA

AGAATTAAAA

AGAGATAGAT

TTTAGAATTA

TCATGATACT

AAACCATTCG

ATTGAAAAAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1158 INFORMATION FOR SEQ ID NO:1l: SEQUENCE CHARACTERISTICS: LENGTH: 385 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala Asn Gly 1 5 10 42 Leu Leu Tyr Thr Ser Thr Tyr Leu Ser Asp Asp Ser Gly Val Ser Leu 25 Met Leu Asn Thr 4 4 *4 4 4 4 4 4 4 4*4* *4 4 4@.

44 0 Asp Giy Giu Leu 145 Tyr Giy Lys Lys Gin 225 Thr Met Thr Ile *Ser Phe Asn Tyr Ser Val Asn 130 Pro Ser Trp Asn Tyr 210 Lys Thr Lys Gin Met 290 LyE Prc Cys Ser Ser Giy Ser Gin Giu Thr Thr 195 Trp Arg Ile Phe Leu 275 Thr Lys Ile *Lys *Ser Tyr i100 Gin Asn Lys Thr Len 180 Gini Asn Ser Ile Giu' 260 Thr4 Lys Asr Asp Val Thr Ile Ser Gin Pro Gly 165 Vai Ile Len Tyr A*sn 245 Vai G3m Tyr Giu Asn Asp Asn Ile 40 Gin Asp Tyr Asp Ile Tyr Ile Asn Thr Leu Ser Lys Trp Asn Ile Leu Gin Lys 150 Asn Pro Lys Ala Asp 230 Thr Pro4 Giu Gin4 Asr Ser Gin Giy Trp 135 Ile Ile Cys Thr Lys 215 Tyr Val Glm [seu Giu 295 Val Lys Asn Asp Lys Ile Asn Vai Ser *Val Ser Ile 120 Asn Asp Asn Ile Thr 200 Gly Giu Giy Val Lys' 280 His Gin Asp 105 Vai Len Gin Pro Met 185 Pro Ser rrp Lieu Giy 265 V'al Ser Lys 90 Asn Arg Thr Lys Lys 170 Vai Tyr Asn Gly Gin 250 Gly Giu Gin Trp Gly ILeu Pro Leu 155 Thr Asn Tyr Val Thr 235 Ile Gly Tyr Ile Gin Lys Thr Val 140 Lys Thr Asp Ile Ser 220 Gin Asn Thr Ser Asp2 300 Ile Vai Asp 125 Gin Asp Pro Ser Phe 205 [Leu [Lys Ile 3mu Lhr ksn Lys Len 110 Glu Thr His Gin Lys.

190 Lys Leu Asn Asp Asp 270 Gin Pro Ala Thr Phe Ile Pro Leu 175 Ile Lys Pro Gin Ser 255 Ile Thr Thr Lys M~a Pro Gin 160 Met Asp His Lys 240 Giy Lys Lys Asn Trp Gly Phe Ala Gin Pro -Met Asn Ser Ile Giy Leu Len Ile Tyr Thr Ser teu diu Leu 305 310' 315 320 43 Tyr Arg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp Ile Giu Th2 Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Lys Gi.1u A la 340 34 350 Leu Leu Leu Leu Thr Asn His. Ser Tyr Giu Giu Val Glu Giu Ile Thr 355 360 365 Lys Ile Pro Lys His Thr Leu Ile.Lys Leu Lys Lys His Tyr Phe Lys 370 375 380 Lys 385 INFOR:MATION FOR SEQ ID NO.:12: SEQUENCE CHARCTERISTICS: LENGTH: 834 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE -DESCRIPTION: SEQ ID NO:12:

C.

9

C.

C

C

C. C

*C

C.

C. C .9

C.

C C C.

CCC...

GGACTATAI

CAGCAACTTA

AATGGATG

ATATTGATGA

0UATATTTA

TTACAAGCTA

AAAATAAMT

TTTCGACTTA

AkATGGTZTM

CATATGTAAI'

CAAGCTCTTG

CATTGATACG

3%ATTTAACTT CTGThcAAAC AAAGATTATC

CCAAATATIC

ATGGPIGGA

.CATTAMTCC

CAAATTAAAA CTILCTCC7LTA GGAAGTAZATG TAGCTTTACG GAAATAGC

AAAAAACAAC

GGAATGAAAT

TTGATATAC

TTTAAGTTTA GATATVCiG GTGTTAGTTT AA.TGAATAAA TTATAA~CTTA AAATGGTTTT TGCAGCAAAT

AATTGTAAAG

T.LCTTCAACA

AATTCAATAC

ACAAAGTGAT

AATGG.AAAILG

TTTAACTGATr

GAATCCTCAA

AATTCAACTT

CCACAAAAALC

ACCAACTGGA ANAhTAGMA TTGTATTATG

GTAIATGATC

TTATAT 'TTA AAAAAATATC TCCALATGAA

AALAAAATCAT

AATTATAAAT

ACATTAGGAT

AGAAGTAGGT

GGAGGTACAG

TATTTCCTAT

TTTGGAA.TGT

AAAAATGGCA

TC!TTAACAGC

ATAATCCCAA

CTATAATAGA

ATGGAA.CATC

CAAATATAGA

AATATTGGCA

).TACTTATGA

TGATGATGAT

TAA!TAATGAT

AATAAAAGCT

AGGAACCGGT

TCAACAATGG

TACAAAATTA

TCCTCAATTA

TAAAAATACT

ACGA3CAGTA

ATGGGGCACA

120 180 240 300 360 420 460 540 600 660 720 780 TTCAAATCAA TATAGATTCA ATGAAATAAA AACACAACTA.

AATGWAAAT TAAAATAGA ATATAGTCAT

GAAACTAAATATGAATA

INFORMATION FOR SEQ ID NO: 13: SEQUENCE

CHARACTERISTICS:

LENGTH: 278 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO0:13: Gly Leu Tyr Ala Ala Thr Tyr Leu Ser Leu C.

S

S. S S S

S.

0 0.@SS@ 0 SO 00 0 9

S

*5 S 4* *0 90S9S0

S

9* 9 0 0* ~0 *9 000S S. 000S

*SOS

0 5.

@5 5

S

5#@SS 0 1 Leu Met Phe Lieu Ala Asn 50 Ser Thr 65 Asfl Gly Ala Gly Ser Asn Gin Lieu 130 Lys Tyr 145 Met Gly Asp Lys Tyr Gin His Gilu 210 Asn Phe 35 Asn Tyr Ser Thr Asn 115 Pro Ser Trp Asn Tyr 195 Lys Lys 20 Pro Cys Ser Ser Gly 100 Pro Gin Pro Thr Thr 180 Trp Lys 5 Asn Ile Lys.

Ser Tyr Gin Asn Lys Thr Leu 165 Gin Gin Ser Asp Asp Val Thr 70 Val Al a Gin Pro Gly 150 Val Ile Arg Tyr Asp Asp Trp 55 Asn Ile Leu Gin Ile 135 Asn Pro Lys Ala Thr 215 Asp Asp 40 Asn.

Ser Gin Gly Trp 120 Ile Ile Cys Thr Val 200 Tyr Ile 25 Gin Val Ile Ser Lieu 105 Asn Asp Asp Ile Thr 185 Gly Giu Asp Tyr Asn Gin Asp 90 Ile lieu Th~r Asn Met 170 Pro Ser Trp Asp Asp Ile Asn Lys 75 Asn Arg Thr Lys Gly 155 Val Tyr Asn Giy Asp Tyr Ile Asp Trp Gly Lieu Ser Leu 140 Thr Asn Tyr Val Thr 220 Ser Asn Thr Lys Gin Lys Thr Val 125 Lys Ser Asp Ile Ala 205 Glu Gly lieu Ser Ile Ile Val Asp 110 Gin Asp Pro Pro lieu 190 Lieu Ile Val1 Lys Tyr Asn Lys lieu Glu Thr Tyr Gin Asn 175 Lys Arg Asp Ser Trp Al a Val Ala Thr Ser Ile Pro Lieu 160 Ile Lys Pro Gin Lys Thr 225 Thr Ile le'Asn Thr lieu Gly Phe Gin Ile Asn Ile Asp Ser 230 235 240 Gly Met Lys Phe Asp Ile Pro Giu Vai Gly Gly Gly Thr Asp Giu Ile 245 250 255 Lys Thr Gin Leu Asn Giu Giu Leu Lys Ile Glu Tyr Ser His Giu Thr 260 265 270 Lys Ile Met Giu Lys Tyr 275 INFORMATION FOR SEQ ID NO:i4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 829 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: :ACATGCAGCA ACTTATTTAA GTTTAGATGA TTCAGGTGIr AGTITAATGA ATAAAAATGA TGATGATATT GATGACTATA ATTTAAGGTG GT-171I'TATTT CCTATTGATG ATAATCAATA 120 TATTATTACA AGCTACGCAG CGAAIAATTG TAAGGTTTGG AATGTTAATA ATGATAAAAT 180 AAATGTTTCA ACTTATTCTT CAACAAACTC GATACAGAAA TGGCAAATAA AAGCTAATGC 240 o. 0 00 TTCTTCGTAT GTAATACAAA GTAATAATGG GAAAGTTCTA ACAGCAGGAA CCGGTCAATC 300 oo.*TCTTGGATTP. ATACGTTTAA CGGATGAATC ACCAGATAAT CCCAATCAAC AATGGAATTT 360 AACTCCTGTA CAAACAATC AACTCCCACC AAAACCTACA ATAGATACAA AGT]TAA)AAGA 420 o o:TrACCCCAAA TATTCACAAA CTGGCAATAT AGACAAGGGA ACACCTCCTC AATTAATGGG 480 *ATGGACATTA .ATACCTTGTA TTA7GGTAAA TGATCCCAAT ATAGATAAAL ACACTCAAAT 540 CAAAACTACT CCATATTATA TTTTAAAAAA ATATCAATAT TGGCAACAAG CAGTAGGAAG 600 TAATGTAGCT TTACGTCCGC ATGAAAAAAA ATCATATGCT TATGAGTGGG GTACAGAAAT 660 AGATCAAAAA ACAACTATCA TTAATACATT AGGATTTCAG ATTAATATAG ATI'CGGGAAT 720

GAAA

T

TrrrAT ATACCAGAAG TAGGTGGAGG TACAGATGAA ATAAAAACAC AATIAAACGA 780 AGAATTAAAA ATAGAATATA GCCGTGAAAC CAAAATAATG GAAAAATAT 829 INFORMATION FOR SEQ ID NO:iS: SEQUENCE CH1ARACTERISTICS: (Al LENGTH: 276 amino acids 46 TYPE: amino acid STRANDEDIESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID His Ala Ala Thr Tyr Leu Ser Len Asp Asp Ser Gly Val Ser Leu Met 1 Asn Phe Asn Tyr 65 Ser Thr Asn Pro Ser 145 Trp Asn Tyr Lys Thr 225 Lys Pro Cys so Ser Ser Gly Pro Pro 130 Gin Thr Thr Trp Lys 210 Ile Asn Ile Lys Ser Tyr Gin Asn 115 Lys Thr Len Gin Gin 195 Ser Ile Asp Asp Val Thr Val Ser 100 Gin Pro Gly Ile Ile 180 Gin Tyr Asn 5 Asp Asp Trp Asn Ile 85 Leu Gin Thr Asn Pro Lys Ala Ala Thr Pro 245 Asp Asn Asn Ser 70 Gin Gly Trp Ile Ile 150 Cys Thr Val Tyr Leu 230 Ile Gin Val 55 Ile Ser Leu Asn.

Asp 135 Asp Ile Thr Gly Gin 215 Gly Asp Tyr 40 Asn Gin Asn Ile Len 120 Thr Lys Met Pro Ser 200 Trp Phe Asp 25 Ile Asn Lys Asn Arg 105 Tbr Lys Gly Val Tyr 185 Asn Gly Gin Tyr Ile Asp Trp 'Gly 90 Len Pro Len Thir Asn 170 Tyr Vai Thr Ile Asn Thr Lys Gin 75 Lys Thr Vai Lys Pro 155 Asp Ile Ala Gin Asn 235 Len Ser Ile Ile Val1 Asp Gin Asp 140 Pro Pro Len Len Ile 220 le Arg Tyr Asn Lys Len Giu Thr 125 Tyr Gin Asn Lys Arg 205 Asp Asp Trp Ala Vai Ala Tbhr Ser 110 Ile Pro Len Ile Lys 190 Pro Gin Ser Phe Al a Ser Asn Al a Pro Gin Lys Met Asp 175 Tyr His Lys Gly Leu Asn Thr Ala Gly Asp.

Leu Tyr Gly 160 Lys Gin Gin Thr Met 240 Lys Phe Asp Ile Gin Val Gly Gly Giy Thr Asp Giu le'Lys Thr 250 2 5 47 Gin Leu Asn Glu Giu Leu Lys Ile Giu Tyr Ser Arg Giu Thr Lys Ile 260. 265 270 Met Glu Lys Tyr 275 INFORMATION FOR SEQ ID NO:lG: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO.-16: Asp Ile Asp Asp Tyr Asn Leu 0 0 .0 INFORMATION FOR SEQ ID NO:17: o o .o SEQUENCE CHARACTERISTICS: oo LENGTH: 7 Amino acids TYPE: amino acid 010 STRANDEDNESS.- single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SE Q ID NO: 17: Tip Phe Leu Phe Pro Ile Asp 2) INFORMATION FOR SEQ ID NO: 18: U SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Gin Ile Lys Thr Thr Pro Tyr Tyr I1. -5 INFORMATION FOR SEQ ID NO: 19: 48 SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Tyr Glu Trp Gly Thr Glu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID GATATNGATG ANTAYAAYTT N 21 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TGGTTTTTNT TTCCNATNGA N 21 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CAAATNAAAA CNACNCCATA TTAT 24 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) M4OLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TANGANTGGG GNACAGAA 18 INFORMATION FOR SEQ ID NO:24: Ci) SEQUENCE CHARACTERISTICS: (A)'LENGTH; 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATAATATGGN GTNGTT1'TNA TTTG 24 INFORMATION FOR'SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleip acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID 1:2 TTCTGTNCCC CANTC1NTA 18 INFORMATION FOR SEQ ID NO: 26: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTCAAAGCGG ATOAGGAG INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27-: GCGTATI'CGG ATATGCTTGG INFORMATION FOR SEQ ID NO:28; SEQUENCE CHARACTERISTICS:.

LENGTH: 386 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE 'DESCRIPTION: SEQ ID NO:28: Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Thr Tyr Leu Ser Leu Asp.

20 25 Me t Xaa Lys Xaa Asp Xaa Asp Ile Asp Asp 40 Lieu Phe Pro Ile Asp Xaa Xaa Gin Tyr le so Asn Asn Cys Lys Val Trp Asn Val Xaa Asn.

70 Thr Tyr Ser Ser Thr Asn Ser Xaa 'Gln Lys 90 Xaa Ser ser Tyr Xaa Ile Gin Ser Xaa Asn 100 105 Gly Xaa Gly Gln Xaa Leu Gly Xaa Xaa Arg 115 120 Xaa-Xaa Xaa Gly Val Ser Leu Xaa Trp Ser Tyr Xaa le Asn Val le Lys Ala Val Leu Thr 110 Asp Glu Xaa 125 Xaa Lieu Phe Ala Ser s0 Xaa Ala Xaa 0 0. 0 0 Xaa Asn Xaa Asn Gin Gin Trp 130 135 Leu Pro Xaa Lys Pro Xaa Ile 145 150 Tyr Ser Xaa Thr Gly Asn Ile 165 Gly Trp Thr Leu Xaa Pro Cys 180 Lys Asn Thr Gin Ile Lys Thr 195 2 Xaa Tyr Trp Xaa Xaa Ala Xaa G 210 215 Xaa Lys Xaa Ser Tyr Xaa Tyr G 225 230 Thr Thr Ile Ile Asn Thr Xaa G 245 Met Lys Phe Xaa Xaa Pro Glu Vi 260 Thr Gin Leti Xaa Glu Glu Leu Lj 2752 Ile Met-Xaa Lys Tyr Xaa Xaa Xz 290 295 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xz 305 310 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa 325 XaaXaa Xaa Xaa Xaa Xaa Xaa Xa 340 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa 355 36 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa 370 375 Xaa Xaa 385 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid 51 Asn Leu Thr Xaa Val 140 A.sp Xaa Lys Leu Lys 155 (aa Xaa Xaa Thr Xaa 170 [le Met Val Asn Asp .185 .hr Pro Tyr Tyr Ile :00 ly Ser Asn Val Xaa 220 iu Trp Giy Thr Glu 235 ly Xaa Gin Ile Asn 250 1l Gly Gly Gly Thr 2 265 rs Xaa Glu Tyr Ser X~ 30 2 ia Xaa Xaa Xaa Xaa X 300 La Xaa Xaa Xaa Xaa X 315 La Xaa Xaa Xaa Xaa X 330 .a Xaa Xaa Xaa Xaa X 345 .a Xaa Xaa Xaa Xaa Xi 0 3 a Xaa Xaa Xaa Xaa Xi 380 Gin Thr Ile Asp Xaa Pro Pro Gin Leu 175 Xaa Xaa le 190 Xaa Lys Lys 205 [eu Xaa Pro (aa Xaa Gin Ele Asp Ser C 255 :Aa Xaa le 270 :aa Giu TbrL aa Xaa Xaa X aa-Xaa Xaa X 3aa Xaa Xaa Xi 335 aa Xaa Xaa Xi 350 aa Xaa Xaa Xi aa Xaa Xaa X2 Gin Xaa 160 Met Asp Tyr !is fly lys Ys aa aa STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GNGAAGTHCA TATNGAAATN' AATAATAC 28 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2015 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoatic) (xi) SEQUENCE DESCRIPTION: SEQ ID ATI'AATTTTA TGGAGGTTGA TATTTATGTC AGCTCGCGAA GTACACATTG TAAAACACGT CATACATTAC AATTAGAGGA TAAAACTAAA CTTAGCGGCG AACATCACCT ACAAATGTTG CTCGTGATAC AATTAAAACA TTEG TAGCAG TTI'TATGACA GGAGTAGAAG GTATTATATA TTITAGTGTA AACGGAGACG TTTACATTTT GACAATCCTT ATATAGGTI'C TAATAAATGT GATGGTTCTT TGAATATGAA GTrATTACTC AAAGCGGATC AtJGAGATAAA

TCTCATGTGA

TCAGACATA TCTTTACGAT TATAAGGAAA ATTTATAAAA ACTGTATTT AC AMPI* T AATA.TTTAT TTTTTGGTAT TTTCTAATAT GAAATATGAA ATTAATAAAA AAGGTGATAA AAATTATG;TT TCTTGCTAAT G.GATTATATA cATCAACTrA AATGAGTAAA AAGGATGAAG

ATATTGATGA

TGATAATAAT CANTATATTA. TTACAAGCTA TAAAAATGAT AAAATA AATG TTTCAACTTA AATAAAAGCT AAAGATTCTT

CATATATAAT

AGGAGTAGGT CAATCTCTTG GAATAGTACG CCAACAATGG AATTTAACTC CTGTACAAAC TGAAAAATTA AAAGATCATC CTGAATATTC TCCTCAATTA ATGGGATGGA CATTAGTACC AGATACTAAT AAAGTTTATG TTTAAGTCTT GATGATTCAG TTACAATTTA AAATGGTTT TGGAGCTAALT AATTGTAAAG TTCTTCAACA AACTCTGTAC ACAAAGTGAT AATGGAAAGG CCTAACTGAT GAATTTCCAG AATTCAACTC CCACAAAAAC AGAAACCGGA AATATAAATC TTGTATTATG

GTAAATGATT

AAATAAACAA

GTAGATGGCG

AATCACATGG

CAGAAATTAG

CTGATAAACC

CATATALCTAT

TTACTAAAAT

TTATAAAAAT

AAATAAGCAA

GTGTTAGTTT

TAITTCCTAT

TTTGGAATGT

AAAAATGGCA

TCTTAACAGC

AGAATTCTAA

CTAAAATAGA

CTAAAACAAC

CAAAAATAGA

120 180 240 300 36~0 420 480 540 600 660 720 780 840 900 960.

1020 1080 53 cAAATTAAAA cTAcTccATA TTATATTTTT AAAAAATATA AATACTGCAA 1140

TAAAAACC

TCTAGCAAAA

ATGGGGTACA

GGAAGTA7ATG TATCTTTAT GAAAAAAATC .AAAAAACAAC TCCAC-ATCAA AAAAGATCAT -TATAGATJXA GGAATGAAAT AACACAATTA ACTGAAGAAT ATATCAAGAA

CACTCAGAGA

TCTTATTTAT

ACTTCITTAG

CATAGAAACT

TCAGATCATG

ATTATTAC1T.

CTCACAAACC

GCATACACTT ATAAAATTGA -TGACTGATTA

ATATCTCTCG

AAAAGATTCC

TAACGGAATG

ATATCTGCCT 'rIGGACAGAC TAGCTcrAT TCCGGCAATC CCGAATACGC TITrTGATAA

TTGAAGTACC

TAAAAGTTGA

TAGATAATCC

AATTATATCG

ATACTTACAC

ATTICGTATGA

AAAAACATTA

AAAAGGTTr

GAACATTAGG

TTCTCCCCTT

ATTTTTGTAG

CCGATGTCTT

TA'rrATTAAT

AGAAGTAGGA

ATATAGCACT

AACTAATCAA

ATATAACGGT

TCTrlACTTCT

AGAAGTAGAA

TTTTAAAAAA

GGTGCAAAAA

CTG'rrAAATC

GGAGAGTTTG

CTG7TTGCA

GTTCAATGAT

ACAGTAGGAT

GGAGGTACAG

GAAACCAAAA

CCAATGAATT

ACAGAAATTA

TATCCAAATC

GAAATAACAA

TAAAAAACAT

TAGTGGGATA

AAAAAGTTTA

TCCTTTTTTG

GGATTTTAAT

ATTGTTTAAT

TGCAAATTAA

AAGACATAAA

TAATGACGAA

CTATAGGACT

AGATAATGGA

ATAAAGAAGC

AAATACCTAA

AATATATAAA

TGAAAAAAGC

TTGATAAAAT

ACCATATGCA

CCAAGCATAT

ATTTTCACAkC 1200 1320 1380 1440 1500 1580 1620 1680 1740 1800 1860 .1920 1980 GAATTGGCTA CTGTGCGGTA TCCTGTCTC!C TTTAT 2015 iFOMATION FOR SEQ ID NO:31: SEQUENCE

CHIARACTERISTICS:

LEN~GTH: 360 base pairs TYPE: nucleic acid sTR1WIEDESS: single TOPOLOGY: linear (ii) MOLECULE TYPE- DNA (genoaiic) (Xi) SEQUENCE DESCRIPTION: SEQ ID 210:31:* ATGTCAGCTC GCGAAGTACA, CATTGAAATA AACAATAAAA

CACGTCATAC

GAGGATAAMA CrAAACTTAG CGGCGGTAGA TGGCGAACAT

CACCTACAAA

GATACAATTA A7AACATTTGT AGCAGAATCA CATGGTTf TA TGACAGGAGT ATATATTTTA GTTAACGG AGACGCAGAA ATTAGTTTAC

ATTTTGACAA

GGTICTAATA AATGTGATGG TTCTTC!TGAT AAACCTGAAT

ATGAAGTITAT

GGATCAGGAG ATAAATCTCA TGTGACATAT ACTATTCAGA-

CAGTATCTTT

ATTACAATTA

TGTTGCTC!GT

AGAAGGTATT

TCCTTATATA

TACTCAAAGC

ACG ATTATAA 120 180 240 300 360 0 INFORMATION FOR SEQ ID NO: 32: Wi SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO.32: Met Ser Ala Arg Giu Val His Ile GJlu Ile Asn 1 5 10 Thr Leu Gin Leu Giu Asp Lys Thr Lys Leu Ser 20 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile 35 40 Glu Ser His Giy Phe Met Thr Gly. Val Glu Gly s0 55 Val Asn Giy Asp Ala Glu Ile Ser Leu His Phe 65 70 is5 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys 85 90 Ile Thr Gin Ser GlySer Gly Asp Lys Ser His 100 105 Gin Thr Val Ser Leu Arg Leu 115 12) INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHLARACTERISTICS: LENGTH: 2 4 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CATGAGATTT ATCTCCTGAT CCGC INFORMATION FOR -SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 2230 base pairs TYPE: nucleic acid Asn Lys Gly Gly Lys Thr le Ile Asp Asn Pro Glu Val Thr Thr Arg His Arg Tip AMg Phe Val Ala Tyr Phe Ser Pro Tyr Ile Tyr Glu Val Tyr Thr le 110 STRANDEDNESS: single.

TOPOLOGY: linear (ii).MOLECULE TYPE:- DNA (genomtic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ACTATGACAA TGATTATGAC TGCTGATGAA

TTAGCTTTAT

CCATCAAATA TAACAGGAGA TAAAAGTAAA CATACATIAT ATI'CAAATAA AAGATCAAGC AAATGGG GTTGTTTTTG2 TCAGGGGCTG AAGAATCAAG TAAGTTTATT GATGTATATr CTrAAATATT ATCAATTTAT AAAAGTAGCA ATrGATTrTG AATTTTAATA ATcPLTGACAA TATAGGGATA TTTAATTTTG AATAATGAAA ATGATTAATA AAAAATTTAA

TTTGTAIAAT

2AATACCAGG ATATTCTAAA rTACTAATAT AATTGGAGAT !LTCCCCCTCT TAATCGTATr TCCTTCTGA AGATAGTAAC aTATTAATGA AGATTTTATT TACACGAAA TTTTTEATTA e 9* 0 *0**00 0. GAATCGCA7iT

ATTAATCGAG

TGTCAGCACG

TGAAGTACAC

AAGATAAAAC

AAAACTTGAT

ATCAAATTAA

AACATTTGTA

TATATTATAG

TATAAATGGA

GTTCTAATAA

ATATGATGGG

GATCAGGA AA TCAATCTCAT ATAATTCATA

I.AAAP.ATATT

CTAATATAAT

TCATAAATAT

TTAGATACTA

ATAAAATTTA

TATTTAAGTT

TAGATGATTC

GACTATAATT TAAGGTGGTr TATGTGTAAT AAATTTTAAT ATTGATGTrAA

ATAATAAGAC

GGTGGTAGAT GGCGAACATC GCAGAATCAC ATGGTrI'TAT GAAGCAGAAA

TTAGTTTATA

CATTCCAATA

AAAATCAATA

GTTACGTATA

CTATTCAAAC

TTTTTTTACG

AAAATACCAIA

TTTAATAATA

AAATTATAAG

TGAAATAAGT

AAMIATGCTA

AGGTGTTAGT

TTAATGAATA

TTTATTTCCT

ATTGATGATA

'1

I

I

C

*1

'-I

'I

LTGTTI'ATT

rTTATGGAGG

LGGTCALTACAL

,cC ACAAAT ~aCAGGTACA -rTTGACAAT

VAWTTATT

-TATCTTCA

AAAATTTr

TTTGAAAATT

TTGATATTTA

TTACAATTAG

GTTGCTAATG

GAAGGTACTA

CCTTATTCAG

ACCCAAGGAG

CGATATGGGA

T1N3GTATrT 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1L500

TACGCAGCGA

TATTCTTCAA

ATACAAAGTA

CGTTTAACGG

ACAhTTCAAC

TCACAAACW'G

ATAATIGTAA GGTTTGGAAT

GTTAATAATG;

CAAACTCGAT ACAGAAATGG

CAAATAAAAG

ATAATGGGAA AGTTCTAACA~ GCAS3GAACCG ATGAATCACC AGATAATCCC

AATCAACAAT

TCCCACCA ACCTACAATA GATACAAAGT GCAATATAGA cAAGGGAACA

CCTCCTCAAT

AAAAGGTGAT-AAATATTATG-

ATGGATTACA

TGCAGCAACT

AAAATGATGA

TGATATTGAT

ATCAATATAT

TATTACAAGC

ATAAAATAAA TGTTTCAACT CTAATGCTTC

TTCGTATGTA

GTCAATCTCT

TGGATTAATA

GGAATTTAAC TCCTGTACAA TAAAAGATTA

CCCCAAATAT

TAATGGGATG

GACATTAATA

56

GATAAA-AACA

CCTTGTATTA TGGTAAATGA

TCCAAATATA

W TATATAm

TAAAAAAATA

CGTCCGCATG

A~AAAAAAATC

ACTATCATTA ATACATTAG CCAGAAGTAG

'TGGAGGTAC

GAATATAGCC

GTGAAACOAA

CCAACTGATC

AATCAATGAA

CGATATAA

GTTCGGA)IAT

AATGTGAccT

CIEATCCAGA

AGAG

AAGAAATAAC

TATTTTTAAA

ACATAATTAT

AATGAAAGAAL

AAJTCTTTTAT

TACTTCCTTG

TCAATAT7GG CAACAAGCAG ATATGCTAT GAGTGGGGTA ATFITCAGATT

AATATAGATT

AGATGAAATA

AAAACACAAT

AATAATGGAA

AAATATCAGG

TrCTATAGGA

TTCCTCACTA

TAGTGTAATG

AAAATTCAAA

TCATCAACAA GCTCTATTAC AAATATrCCC 1AAAATATCAC ATTTTGATAG CTTITTTAAAA GAAACTTTAA TACAATrAAAA CTCAAATCAA AACTACTCCA TAGGAAGTAA TGTAGCTTrA CAGAAATAGA TCAAAAAACA CGGGAATGAA ATrlTGATATA TAAACGAAGA

ATTAAAAATA

AAtAATCAGA

GATAGALTAAT

TTACTTCTrT

AGAATIATAT

CTCAGATAA TGATACTTAC TICTTACAAA

TCATTCATAT

TGAAAAAATT

AAAAAAATAT

AmAAGATG

TTCAAAGTA

GAGGAATATT

TTCTTATAAG

1560 1680 1740 1800 1860 1920 1980 2040 .23.00 2160 2220 2230 0# 4

S..

4 INpoIyAATION FOR SEQ ID NO: SEQUENCE cokRACTERISTICS: LENGTH: 372 base pairs TYPE: nucleic acid STRA14DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: ]DNA (genoinic) (xi) SEQUENCE DESCRIP'TION: SZ.Q ID ATGTCAGC&C GTGAAGTACA cATTGJATGTA AATAATAAGA maGGTCPTc

ATTACAATTA

GAAGATAAAA CAAAACTTGA TGGTGGTAGA TGGCGAACAT CACCTACAAIL TGT1'GCTAAT GATCARATTA 3AAACATTTGT AGCAGAATCA CATGGTTTTA TGACAGGTAC

P.GAG=CT

ATATATT3ATA GTATAAATGG; AGAAGCWAAA ATTAGTTThT ATTTTGACAA

TCCTTATTCA

GGTTCTAATA AATATGATGG GC-ATTCCAAT AAAAATCAAT ATGAAGTTAT

TACCCAAGGA

GATCAGGAA ATCAATCTCA TGTTACGTAT ACTATTCAAA CTGTATCTTC

ACGATATGGG

AATAATTCALT

AA

120

ISO

240 300.

360 372 INFORMATILO1 FOR SEQ ID NO:36: SEQUENCE

CHAPJACTERISTICS:.

57 LENGTH: 123 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:36: (ii) (xi) Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr Leu Gin Thr Ser Pro 35 Leu Giu Asp Lys Tkr Lys Leu Asp Gly Gly Arg Trp Arg 25 Thr Asn Val Ala Asn Asp Gin Ile Lys Thr Phe Val Ala 40 0S 0@ S 000 00 S

OS

00 000**e 0 S. 00 S S 0 @5 S 0* 0* 0 0~S*

S

0000 *6 S 0*

S.

0005 0 005s 0 @0 5* 0 0 0 Giu Ser so Ile Asn 65 Gly Ser Ile Thr His Gly Phe Met Thr Gly 55 le Ser Thr Giu Giy Thr Ile Tyr Tyr Ser Giy Giu Ala Asn Lys Tyr .85 Gin Gly Gly 100 Glu 70 Leu Tyr Phe 75 Asp Asn Pro Tyr Ser PA5p Gly His Ser Gly Asn Ser Asn 90 Gin Ser 105 Lys Asn Gin Tyr Glu Val His Val Thr Tyr Thr Ile 110 Gin Thr Val 115 Ser Ser Arg Tyr Gly Asn Asn Ser 120 INFORMATION FOR SEQ ID NO:37-: SEQUENCE

CHARACTERISTICS:

LENGTH: 1152 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (i)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ATGTTAGATA, CTAATAAAAT TTATGAAATA AGTAATTATG CTAATGGATT

ACATGCAGCA

ACTTATTTAA GTTTAGATGA TTCAGGTGTT AGTI'TAATGA. ATAAAAATGA

TGATGATAT

GATGACTATA ATrTAAGGTG GTTTTTATTT CCTATTGATG ATAATCAATA

TATTATTACA

AGCTACGCAG OGAATAATTG TAAGGTTTIGG AATGTTAATA ATGATAAAAT

AAATGTTTCA

ACTTATTCTT CAACAAACTC GATACAGAAA. TGGCAAATAA AAGCTAATGC TTCTTCGTAT GTAATACAAA GTAATAATGG GAAAGTTCTA ACAGCAGGAA CCGGTCAATC TCTTGGATTA 120 180 240 300 360 58 ATACGTTTAA COGATGAATC ACCAGATAAT CCCAATCAAC AATGGAATrI CAAACAATTC AACTCCCACC AAAACCTACA ATAGATACAA AGTTAAAAGA TATTCACAAA CTGGCAATAT AGACAAGGGA ACACCTCCTC AA'rrAATGGG ATACCTTGTA TTATGGTAAA TGATCCAAAT ATAGATAAAA ACACTCAAAT CCATATTATA T TTTAAAAAA ATATCAATAT TGGCAACAAG CAGTAGGAAG TTACGTCCGC ATGAAAAAAA ATCATATGCT TATGAGTGGG GTACAGAAAT ACAACTATCA TTAATACATT AGGATTTCAG ATTAATATAG ATTCGGGAAT ATAccAGAAG TAGGTGGAGG-TACAGATGAA ATAAAAACAC AATTAAACGA ATAGAATATA GCCGTGAAAC CAAAATAATG GAAAAATATC AGGAACAATC AATCCAACTG ATCAATCAAT GAATTCTATA GGATTCCTCA CTATTACTTC TATCGATATA ATGGTTCGGA AATTAGTGTA ATGAAAATTC AAACTTCAGA TACAATGTGA CCTCTTATCC AGATCATCAA CAAGCTCTAT TACTTCTTAC TATGAAGAAG TAGAAGAAAT AACAAATATT CCCAAAATAT CACTGAAAAA TATTATTTTT AA *AACTCcTGTA

*TTACCCCAAA

ATGGACATTA

CAAAACTACT

TAATGTAGCT

AGATCAAAAA

GAAATTrGAT

AGAATTAAAA

AGAGATAGAT

TTTAGAATTA

TAATGATACT

AAATCATTCA

ATTAAAAAAA

420 480 540 600 660 720 780 840 900 960 1020.

1080 1140 1152 INFORMATION FOR SEQ ID NO:38: Wi SEQUENCE CHARACTERISTICS: LENGTH: 383 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Leu Asp Thr Asn Lys Ile Tyr Glu Ile Ser Asn Tyr Ala Asn Gly 1 5 10 Leu His Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 25 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Arg Trp Phe 40 Leu Phe Pro Ile Asp Asp Asn Gin Tyr Ile Ile Thr Ser Tyr Ala Ala 55 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 70 75 Thr Tyr Ala Ser Gly Thr Ser Ser Gly Ser Tyr 100 Gin Thr Val Ser Asn Ile Leu Ser Gin Gly Ile Ser Leu 59 Gin Lys 90 Asn Asn 105 li e Arg Trp Gin Gly Lys Leu Thr Ile Val1 Asp Lys Leu 110 Giu Ala Thr Ser Asn Al a Pro 115 120 125 Asp Leu 145 Tyr Asn 130 Pro Ser Pro Pro Gin Asn Gin Lys Pro Thr Gly Gin Thr 150 Asn Trp 135 Ile Ile Asn Leu Asp Thr Asp Lys Thr Lys Giy Pro Leu 155 Thr Va.

14' Ly Prc 165 170 Gly Trp Thr Leu Ile Pro Cys Lys Gin Asn Tyr 210 Giu Lys 225 Thr Thx Met Lys Thr Gin Ile Met 290 Gin Ser 305 Tyr Arg Asp Asn Leu Leu Thr 195 Trp Lys Ile Phe Leu 275 Giu Met Tyr Asp Leu 180 Gin Gin Ser Tyr Ile Asn 245 Asp Ile 260 Asn Giu Lys Tyr Asn Ser Asn Giy 325 Thr Tyr 340 Leu Thr Lys Ala Al1 a 230 Thr Pro Glu Gin Ile 310 Ser Asn Asn Thr Val 215 Tyr Leu Giu Leu Giu 295 Gly Giu Val H~is Ile -Thr 200 Giy Giu Giy Val Lys 280 Gin Phe Ile Thr 360 Met 185 Pro Ser Tip Phe Gly 265 Ile Ser Leu Ser Ser 345 Tyr Val Tyr Asn Giy Gin 250 Giy Giu Giu Thr Vail 330 Tyr] GiuC Asi Tyl Val Thr 235 Ile Gly Tyr Ile Ile 315 4et ?ro 3lu 1 Asr Sle *Ala 220 Giu Asn Thr Ser Asp 300 Thr Lys Asp Val I Gin 0 s Asp Pro Pro Leu 205 Leu Ile Ile Asp( Arg C 285 Asn I Ser L Ile G His G 3 Giu G 365 Th: Gir.

Asn 190 Lys Axrg k*sp ksp flu ;iu ~ro ,eu in In lu rle Pro Leu 175 Ile Lys Pro Gin Ser 255 Ile Thr Thr2 Glu Thr 335 Gin 7 Ile '1 Gin Lys 160 Met Asp, Tyr His Lys 240 Giy Lys Lys %sp.

jeu ~er lia 'hr 355 Asn Ile Pro Lys Ile Ser Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 INFORMATION FOR SEQ ID NO:-3 9: SEQUENCE CHARACTERISTICS: LENGTH 2132 base pairs (B3) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GTATTTCAGG GGGTGAAGAT TCAAGTAAGT. TTATTGATGT ATAT1'ATCCT TTTGAAGATA GTAATTTIAA ATATTATCAA TTTATAAAAG TAGCAATTGA ~TTGATATT AATGAAGATT TTATTAATTT TAATAATCAT GACAATATAG GGATATTTAA

TTTAATTTGT

TTTTGTTACA

TTATTATGTT TATAAATAA TGAAA.ATGAT AAATTGAATG CATATATTAA ATITATGTCA GCACGTGAAG ATTAGAAGAT AAAACAAAAC.

TAATGATCAA ATTAAAACAT TACTATATAT TATAGTATAA TGCAGGTTCT AATAAATATG AGGAGGATCA GGAAATCAAT TGGGCATAAA TCATAACAAA TATTTTCTAA TATAAATTAC ATTATGTrAG ATACTAATAA GCAACTTATT TAAGTTTAGA ATI'GATGATT ATAACTTAAA.

ACAAGCTATG CAGCAAATAA TCGACTTrATT CTTCAACAAA TATGTAATAC AAAGTGATAA TTGATACGTT tAACTGATGA2 GTACAAACAA TTCAACTI'CC2 AAATATTCAC CAACTGGAAA GAATAAAAA2

TCGAGTATGI

TACACATTG1

TTGATGGTGG

TTGTAGCAGA

ATGGAGAAGC

ATGGACATTC

CTCATGTTAC

TAATTTTTTA

AAATATATTA

AGTTTATGAA

TGATTCAGGT

ATGGT1'TTTA

TTGTAAAGTT

TTCAATACAA

rGGAAAAGTC k.TCCTCAAAT

!LCAAAAACCT

PATAGATAAT

EATAATAAATT TTAATTTTAT LTGTAAATAAT -AAGACAGGTC TAGATGGCGA ACATCACCTA ATCAAATGGT TTTATGACAG AGAAATTAGT TTATATTTTG CAATAAATCT CAATATGAAA GTATACTATT CAAACCACAT CGAAAATACC AAAAAATAAA ATAATAA AAT TATAAGAAAA ATAAGCAATC ATGCTAATGG.

GTTAI3TTTAA TGAATAAAAA TTTCCTATTG ATGATGATCA.

TGGAATGTTA ATAATGATAA AAATGGCAAA TAAAAGCTAA' TTAACAGCAG GAACCGGTCA AATCCCAATC AACAATGGAA ATAATAGATA CAAAATTAAA 2 GGAACATCTC CTCAATTAAT(

CGAAATTTTT

TATTTTTTGA

GGAGGTI'GAT

ATACATTACA

CAAATGTTGC

GTACAGAAGG

ACAATCCTT

TTATTACCCA

CCTCACGATA

TATTTrlTTGG 3GTGATAAAG

B.CTATATGCA

PGATGATGAT

%TATATTATT

k.ATAAATGTT rGG1TTCTTCA kGCTCTTGGA

.TTAACTTCT

LGATTATCCC

;GGATGGACA

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 TiFAGTACCTT GTATTATGGT- AAATGATCCA AATATAGATA AAAATACTCA AATTAAPLACT 18 1380 61 TA ITGGCAAC ACTCCATATT ATATTTTAAA AAAATATCAA GAGCAGTAGG AAGTAATGTA GGGGCACAGA AATAGATCAA

GCTI'ACGTC

AAAACAACAA

GATATACCAG

AAAATAGAAT

GATAATCCAA

TrATATAGAT

ACTTATAATG

TCATATGAAX3

AAATATTATT

ATCT1'TAATT

GTAATATCTG

CACATGAAAA AAAATCATAT TTATAAATAC ATTAGGATT AAGTAGGTGG AGGTACAGAT ATAGTCATGA AACTAAAATA CTGATCAATC AATGAATTCT ATAATGGCTC AGAAA ITCGT TI'ACTTCTTA TCCAAATCAT AAGTAGAAGA

AATAACAAAT

TTrAAATATT

GAAATTAGAA

ATTTGTAAGA

TAATCGTAT

TACGTGAAAT TGGT1'TCGCT

ACTTATGAAT

CAAATCAATA

GAAATAAAAA

ATGGAAAAAT

ATAGGATTTC

ATAATGCAAA

CAACAAGCTT

ATTCCTAAAA

ATTATCTAAA

TTATTTGTAT

TCAATATCTA

TAGATTCAGG

CACAACTAAA

ATCAAGAACA

TTACTAITAC

TrCAAACCTC-

TATTACTTCT

GTACACTAAA

ACAAAACGAA

TAATTTTTAT

ATCTCATCTC

AATGAAA=r TGAAGAA'rrA

ATCTGAAATA

TTCCTTAGAA

AGATAATGAT

TACAAATCAT

AAAATTAAAA

AGATAAII'TA

ACAATATAAA

ATGTATTACA

1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2132 TGCGTAATAC cTTCTTGTTC TGCTTCTACA AG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTrH: 372 base pairs TYPE: nucleic acid STRANDEDNESS: single- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genontic) (xi) SEQUENCE -DESCRIPTION: SEQ ID ATGTCAGCAC GTGAAGTACA CATTGATGTA AATAATAAGA

CAGGTCATAC

GAAGATAAAA CAAAACTTGA TGGTGGTAGA TGGCGAACAT

CACCTACAAA

GATCAAATTA AAACATITGT AGCAGAATCA AATGGTTTTA

TGACAGGTAC

ATATATTATA GTATAAATGG AGAAGCAGAA ATTAGTTTAT

ATTTTGACAA

GGTTCTAATA AATATGATGG ACATITCCAAT AAATCTCAAT ATGAAATTAT GGATCAGGAA ATCAATCTCA TGTTACGTAT ACTATTCAAA

CCACATCCTC

CATAAATCAT AA

ATTACAATTA

TGTTGCTAAT

AGAAGC-TACT

TCCTTTTGCA

TACCCAAGGA

ACGATATGGG

INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 123 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:41: Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His (i i) (xi) Met 1 Thr Leu Gin Leu Glu Asp Lys Thr Lys Leu Asp Giy Gly Arg Trp Arg Asp Gin Ile Lys Thr Phe Val Ala 0 00 Thr Ser Pro Thr Giu Ser Asn Gly Ile Asn Gly Giu 65 Gly Ser Asn Lys le Thr Gln Gly 100 Gin Thr Thr Ser 11s Asn Val Ala Asn Phe Met Thr Gly 55 Ala Giu Ile Ser Thr Glu Giy Thr Ile Leu Tyr Phe Asp Asn 75 Tyr Tyr Pro Phe Ala Tyr Giu Ile 70 Tyr Asp Gly 5cr Gly His 5cr Giy Asn Gin Asn 90 Ser Lys Scr Gin Thr Ile His Val Thr Tyr 110 Ser Arg Tyr Giy 120 His Lys INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS:.

LENGTH: 1152 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: ATGTTAGATA CTAATAAAGT TTATGAAATA AGCAATCATG CTAATGGACT ATATGCAGCA ACTTATTTAA GTTI'AGATGA TTCAGGTGTIT AGTTTAATGA ATAAAAATGA TGATGATAT GATGATTATA ACTTAAAATG GITTTTATTT CCTATTGATG ATGATCAATA TATTATTACA AGCTATGCAG CAAATAATI'G TAAAGTTTGG AATGTTAATA ATGATAAAAT AAATGTTTCG ACTTATTCTT CAACAAATTC AATACAAAAA TGGCAAATAA AAGCTAATGG TTCTTCATAT GTAATACAAA GTGATAATGG AAAAGTCTTA AC.AGCAGGAA CCGGTCAAGC TCTTGGATTG 120 180 240 300 360O 63 ATACGTrTAA CTGATGAATC CTCAP.ATAAT CCCAATCAAC AATCGAATTT AACTTCTGTA CAAACAATTC AACTTCCACA AAAACCTATA ATAGATACAA AATTAAAAGA TTATCCCAAA TAITCACCAA CTfGGAAATAT AGATAATGGA ACATCTCCTC AATTAATGGG ATGGACATTA GTACCTTGTA TTATGGTAAA TGATCCAAAT ATAGATAAAA ATACTCAAAT TAAAACTACT CCATATTATA TTTTAAAAAA ATATCAATAT TGGCAACGAG CAGTAGGAAG TAATGTAGCT TTACGTCCAC ATGAAAAAAA ATCATATACT TATGAATGGG GCACAGAAAT AGATCAAAAA ACAACAA'17A TAAATACATT AGGATTTCAA ATCAATATAG ATTCAGGAAT GAAATI"IGAT ATACCAGAAG TAGGTGGAGG TACAGATGAA ATAAAAACAC AACTAAATGA AGAATTAAAA ATAGAATATA GTCATGAAAC TAAAATAATG GAAAAATATC AAGAACAATC TGAAATAGAT AATCCAACTG ATCAATCAAT GAATTCTATA GGATTTCTTA CTATTACTTC CTTAGAATTA TATAGATATA ATGGCTCAGA AATTCGTATA ATGCAAATTC AAACCTCAGA TAATGATACT TATAATGTTA CTTCTI'ATCC AAATCATCAA CAAGCTTTAT TACTTCTTAC AAATCATTCA TATGAAGAAG TAGAAGAAAT AACAAATATT CCTAAAAGTA CACTAAAAAA ATTAAAAAAA TATA'Frrrr AA 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1152 0 INFORMATION FOR SEQ ID NO:43: Wi SEQUENCE CHARACTERISTICS: LENGTH: 383 amino acid~s TYPE: amino acid STRAN'DEDNESS: single TOPOLOGY: linear (iMOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Met Leu Asp Thr Asn Lys Val Tyr Glu Ile 1 5 10 Leu Tyr Ala A la Thr Tyr Leu Ser Leu Asp 25 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp 40 Leu Phe Pro Ile Asp Asp Asp Gin Tyr Ile 55 Asn Asn Cys Lys Val Trp Asn Val Asn Asn 70 Ser Asn His Ala Asn Gly Asp 5cr Gly Val Ser Leu Tyr Asn Leu Lys Trp Phe Ile Thr Ser Tyr Ala Ala Asp Lys le Asn Val Ser 75 64 Thr Tyr Ser Ser Thr Asn Ser Ile Gin Lys Trp Gin Ile Lys Ala Asn 90 Gly Ser Ser Tyr-Val Ile Gin Ser Asp Asp Giy Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gin Ala Leu Giy Leu Ile Arg Leu Thr Asp Giu Ser Ser 115 120 125 Asn Asn Pro Asn Gin Gin Trp Asn Leu Thr Ser Val Gin Thr Ile Gin 130 135 140 Leu Pro Gin Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 .:Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gin ILeu Met Giy Trp Thr Leu Val Pro Cys Ile Met Vai Asn Asp Pro Asn Ile Asp 185 190 *Lys Asn Thr Gin Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 *Gin Tyr Trp Gin Arg Ala Val Giy Ser Asn Vai Ala Leu Arg Pro His *.210 215 220 Giu Lys Lys Ser Tyr Thr Tyr Giu Trp Gly Thr Giu Ile Asp Gin Lys 225 230 235 240 Thr Thr Ile Ile Asn. Thr Leu Giy Phe Gin Ile Asn Ile Asp Ser Giy 0..245 250 255 .*Met Lys Phe Asp Ile Pro Giu Val Giy Gly Giy Thr Asp Giu Ile Lys 260 265 270 Tbr Gin Leu Asn Giu Giu Leu Lys Ile Giu Tyr Ser His Giu Thr Lys 275 280 285 Ile Met Giu Lys Tyr Gin Giu Gin Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gin Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Giu Leu 305 310 315 320 Tyr Arg Tyr Asn Giy Ser Giu Ile Arg Ile Met Gin Ile Gin Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gin Gin Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Giu Giu Vai Giu Giu Ile Thr 355 .360 36.5 Asn Ilie Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 360 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: line ar (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:.

ATGTCCGCCC GCGAGGTGCA CATCGAGATC AACAACAAGA CCCGCCACAC CCTCCAGCTC GAGGACAAGA CCAAGCTCTC CGGGGCGG TGGCGCACCT CCCCGACCAA CGTGGCCCGC GACACCATCA, AGACGTTCGT GGCGGAGTCC CACGGCTTCA TGACCGGCGT CGAGGGCATC ATCTACTTCT -CCGTGAACGG CGACGCCGAG ATCTCCCTCC ACTTCGACAA CCCGTACATC GGCTCCAACA AGTGCGACGG CTCCTCCGAC AAGCCCGAGT ACGAGGTGAT CACCCAGTCC GGCTCCGGCG ACAAGTCCCA CGTGACCTAC ACCATCCAGA CCGTGTCCCT CCGCCTCTGA a. a a a a a. a IFl~ORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1158 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID ATGCTCGACA CCAACAAGGT GTZACGAA3ATC TCCAACCTCG CCAACGGCCT ACCTACCTCT -CCCTCGACGA CTCCGGCGTG TCCCTCATGT CCAAGAAGGA GACGACTACA ACCTCAAGTG GTTCCTCTTC CCGATCGACA ACAACCAGTA TCCTACGGCG CCAACAACTG CAAGGTGTGG AACGTGAAGA ACGACAAGAT ACCTACTCCT CCACCAACTC CGTGCAGAAG TGGCAiGATCA AGGCCAAGGA ATCATCCAGT CCGACAACGG CAAGGTGCTC ACCGCGGGCG TGGGCCAGTC GTGCGCCTCA CCGACGAGT' CCCGGAGAAC TCCAACCAGC AATGGAACCT CAGACCATCC AGCTCCCGCA GAAGCCGAAG ATCGACGAGA AGCTCAAGGA TACTCCGAGA CCGGCAACAT CAACCCGAAG ACCACCCCGC AGCTCALTGGG GTGCCGTGCA TCATGGTGAA CGACTCCAAG ATCGACAAGA ACACCCAGAT 120 180 240 300 360 120 180 240 300 360 420 480 540 600

CTACACCTCC

CGAGGACATC

CATCATCACC

CAACGTGTCC

CTCCTCCTAC

CCTCGGCATC

CACCCCGGTG

CCACCCGGAG

CTGGACCCTC

CAAGACC&CC

66 CCGTACTACA TCT~cAAGAA ATACAAGTAC TGGAACCTCG CTCCTCCCGC ACCAGAAGCG CAGCTACGAC TACGAGTGGG ACCACCATCA TCAACACCGT GGGCCTGCAG ATCAACATCG GTGCCGGAGG TGGGCGGCGG CACCGAGGAC ATCAAGACCC GTGGAGTACT CCACCGAGAC CAAGATCATG ACCAAGTACC AACCCGACCA ACC-AGCCGAT GAACTCCATC GGCCTCCTCA TACCGCTACA ACGGCACCGA GATCAAGATC ATGGACATCG TACACCCTCA CCTCCTACCC GAACCACAAG GAGGCGCTGC TACGAGGAGG TGGAGGAGAT CACCAAGATC CCGAAGCACA CACTACTTCA AGAAGTGA CCAAGGGCTC CAACGTGTCC GCACCGAGAA GAACCAGAAG ACTCGGGGAT GAAGTTCGAG AGCTCACCGA GGAGCTGAAG AGGAGCACTC CGAGATCGAC TCTACACCTC CCTCGAGC!TG AGACCTCCGA CCACGACACC TGCTGCTGAC CAACCACTCC CCCTCATCAA GCTCAAGAAG 660 720 780 840 900 960 1020 1080 1140 1158

Claims (378)

1. An isolated polynucleotide which encodes a toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes a pesticidal portion of SEQ ID NO. 32, DNA which encodes a pesticidal portion of SEQ ID NO. 36, and DNA which encodes a pesticidal portion of SEQ ID NO. 41, hybridises under stringent conditions with said polynucleotide or a complement thereof.

2. An isolated polynucleotide which encodes a toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of: SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 33, a 10 to 15 kDa toxin- encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with said polynucleotide or a complement thereof.

3. The isolated polynucleotide according to claim 1 or claim 2, wherein DNA which encodes SEQ ID NO. 3 hybridises with said polynucleotide or a complement thereof.

4. The isolated polynucleotide according to claim 1 or claim 2, wherein DNA which encodes SEQ ID NO. 5 hybridises with said polynucleotide or a complement thereof.

5. The isolated polynucleotide according to claim 1 or claim 2, wherein DNA which encodes SSEQ ID NO. 7 hybridises with said polynucleotide or a complement thereof. 20 6. The isolated polynucleotide according to claim 1 or claim 2, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 32 hybridises with said polynucleotide or a complement thereof.

7. The isolated polynucleotide according to claim 1 or claim 2, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 36 hybridises with said polynucleotide or a complement thereof.

8. The isolated polynucleotide according to claim 1 or claim 2, wherein DNA which encodes 25 a pesticidal portion of SEQ ID NO. 41 hybridises with said polynucleotide or a complement thereof.

9. The isolated polynucleotide according to claim 1 or claim 2, wherein SEQ ID NO. 29 hybridises with said polynucleotide or a complement thereof.

10. The isolated polynucleotide according to claim 1 or claim 2, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30 hybridises with said polynucleotide or a complement thereof.

11. The isolated polynucleotide according to claim 1 or claim 2, wherein SEQ ID NO. 31 hybridises with said polynucleotide or a complement thereof.

12. The isolated polynucleotide according to claim 1 or claim 2, wherein SEQ ID NO. 33 hybridises with said polynucleotide or a complement thereof.

13. The isolated polynucleotide according to claim 1 or claim 2, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34 hybridises with said polynucleotide or a complement thereof.

14. The isolated polynucleotide according to claim 1 or claim 2, wherein SEQ ID NO. bridises with said polynucleotide or a complement thereof. C04229#2 68 The isolated polynucleotide according to claim 1 or claim 2, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39 hybridises with said polynucleotide or a complement thereof.

16. The isolated polynucleotide according to claim 1 or claim hybridises with said polynucleotide or a complement thereof.

17. The isolated polynucleotide according to claim 1 or claim hybridises with said polynucleotide or a complement thereof.

18. The isolated polynucleotide according to claim 1 or claim 2, comprises a 10 to 15 kDa toxin-encoding portion of SEQ ID NO.

19. The isolated polynucleotide according to claim 1 or claim 2, comprises SEQ ID NO. 31. The isolated polynucleotide according to claim 1 or claim 2, comprises 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34.

21. The isolated polynucleotide according to claim 1 or claim 2, comprises SEQ ID NO.

22. The isolated polynucleotide according to claim 1 or claim 2, comprises a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39.

23. The isolated polynucleotide according to claim 1 or claim 2, comprises SEQ ID NO.

24. The isolated polynucleotide according to claim 1 or claim 2, comprises SEQ ID NO. 44. 2, wherein SEQ ID NO. 2, wherein SEQ ID NO. 44 wherein said polynucleotide wherein said polynucleotide wherein said polynucleotide wherein said polynucleotide wherein said polynucleotide wherein said polynucleotide wherein said polynucleotide 0 0 00 0 0 00 0. 0000 0 0 0000 0 0.0

25. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof.

26. The isolated polynucleotide according to claim 25, wherein said amino acid sequence comprises SEQ ID NO. 32 or a pesticidal portion thereof.

27. The isolated polynucleotide according to claim 25, wherein said amino acid sequence comprises SEQ ID NO. 36 or a pesticidal portion thereof.

28. The isolated polynucleotide according to claim 25, wherein said amino acid sequence comprises SEQ ID NO. 41 or a pesticidal portion thereof.

29. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin active against a non-mammalian pest, wherein a portion of said nucleotide sequence can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

30. The isolated polynucleotide according to any one of claims 1 to 29, wherein the toxin has a molecular weight of approximately 10 to 15 kDa.

31. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin which immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus Tp Jringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. C04229#2 69

32. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said nucleotide sequence is from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

33. An isolated polynucleotide comprising a nucleotide sequence which encodes an approximately 10 to 15 kDa PS80JJ1 toxin active against non-mammalian pests, wherein said nucleotide sequence has been optimised for expression in plants.

34. The isolated polynucleotide according to claim 32 or claim 33, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 32 or a pesticidal portion thereof.

35. The isolated polynucleotide, according to claim 32, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO. 31.

36. The isolated polynucleotide, according to claim 33, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO. 44.

37. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said nucleotide sequence is from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

38. The isolated polynucleotide according to claim 37, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 41 or a pesticidal portion thereof. 20 39. The isolated polynucleotide, according to claim 37, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin which immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus thuringiensis PS149B1 having the identifying characteristics of NRRL B-21553. 25 41. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said nucleotide sequence is from Bacillus thuringiensis isolate PS167H2 having the y: identifying characteristics of NRRL B-21554.

42. The isolated polynucleotide according to claim 41, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 36 or a pesticidal portion thereof.

43. The isolated polynucleotide, according to claim 41, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO.

44. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin which immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of TRX~.EQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal .40 pi ton of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. C04229#2

46. The isolated polynucleotide according to claim 45, wherein said nucleotide sequence encodes a toxin which comprises an amino acid sequence which has at least about 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41.

47. The isolated polynucleotide according to claim 45, wherein said nucleotide sequence encodes a toxin which comprises an amino acid sequence which has at least about 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal o1 portion of sequence ID NO. 41.

48. An isolated polynucleotide comprising a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

49. The isolated polynucleotide according to any one of claims 1 to 48, which further encodes a second toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 2; DNA which encodes SEQ ID NO. 4; DNA which encodes SEQ ID NO. 6; SEQ ID NO. 8; SEQ ID NO. 10; DNA which encodes a 20 pesticidal portion of SEQ ID NO. 11; SEQ ID NO. 12; DNA which encodes SEQ ID NO. 13; SEQ ID NO. 14; DNA which encodes SEQ ID NO. 15; DNA which encodes SEQ ID NO. 16; DNA which encodes SEQ ID NO. 17; DNA which encodes SEQ ID NO. 18; DNA which encodes SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ ID NO. 24; SEQ ID NO. SEQ ID NO. 26; SEQ ID NO. 27; DNA which encodes a pesticidal portion of SEQ ID NO. 28; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 30; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 34; SEQ ID NO. 37; DNA which encodes a pesticidal portion of SEQ ID NO. 38; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 39; SEQ ID NO. 40; SEQ ID NO. 42; DNA which encodes a pesticidal portion of SEQ ID NO. 43; and SEQ ID NO. 45; hybridises under stringent conditions with said polynucleotide or a complement thereof.

50. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 8 hybridises under stringent conditions with said polynucleotide or a complement thereof.

51. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 10 hybridises under stringent conditions with said polynucleotide or a complement thereof.

52. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 12 hybridises under stringent conditions with said polynucleotide or a complement thereof.

53. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 14 hybridises under stringent conditions with said polynucleotide or a complement thereof.

54. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 20 hybridises !.under stringent conditions with said polynucleotide or a complement thereof. C04229#2 71 The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof. S56. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof.

57. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof.

58. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof.

59. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof.

61. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. under stringent conditions with said polynucleotide or a complement thereof.

62. The isolated polynucleotide according to claim 49, wherein a 40 to 21 hybridises 22 hybridises 23 hybridises 24 hybridises 25 hybridises 26 hybridises 27 hybridises 50 kDa toxin- S. 55. Se 0 0 S eeg S. 05 S. S encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with said polynucleotide or a complement thereof.

63. The isolated polynucleotide according to claim 49, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with said polynucleotide or a 20 complement thereof.

64. The isolated polynucleotide according to claim 49, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with said polynucleotide or a complement thereof.

65. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 42 hybridises 25 under stringent conditions with said polynucleotide or a complement thereof.

66. The isolated polynucleotide according to claim 49, wherein SEQ ID NO. 45 hybridises under stringent conditions with said polynucleotide or a complement thereof.

67. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 2 hybridises under stringent conditions with said polynucleotide or a complement thereof.

68. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 4 hybridises under stringent conditions with said polynucleotide or a complement thereof.

69. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 6 hybridises under stringent conditions with said polynucleotide or a complement thereof. The isolated polynucleotide according to claim 49, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 11 hybridises under stringent conditions with said polynucleotide or a complement thereof.

71. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 13 hybridises under stringent conditions with said polynucleotide or a complement thereof. R 72. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID N. 15 hybridises under stringent conditions with said polynucleotide or a complement thereof. C04229#2 (T -0

73. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 16 hybridises under stringent conditions with said polynucleotide or a complement thereof.

74. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 17 hybridises under stringent conditions with said polynucleotide or a complement thereof.

75. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 18 hybridises under stringent conditions with said polynucleotide or a complement thereof.

76. The isolated polynucleotide according to claim 49, wherein DNA which encodes SEQ ID NO. 19 hybridises under stringent conditions with said polynucleotide or a complement thereof.

77. The isolated polynucleotide according to claim 49, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 28 hybridises under stringent conditions with said polynucleotide or a complement thereof.

78. The isolated polynucleotide according to claim 49, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 38 hybridises under stringent conditions with said polynucleotide or a complement thereof.

79. The isolated polynucleotide according to claim 49, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 43 hybridises under stringent conditions with said polynucleotide or a complement thereof.

80. The isolated polynucleotide according to claim 49, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 20 81. The isolated polynucleotide according to claim 49, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 37.

82. The isolated polynucleotide according to claim 49, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 42.

83. The isolated polynucleotide according to claim 49, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO.

84. The isolated polynucleotide according to claim 49, wherein said second toxin comprises S* the amino acid sequence set forth in SEQ ID NO. 11 or a pesticidal portion thereof.

85. The isolated polynucleotide according to claim 49, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 38 or a pesticidal portion thereof. 30 86. The isolated polynucleotide according to claim 49, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 43 or a pesticidal portion thereof.

87. The isolated polynucleotide according to claim 49, wherein said second toxin comprises the consensus sequence shown in Figure 1.

88. The isolated polynucleotide according to any one of claims 49 to 87, wherein said second toxin has a molecular weight of approximately 40 to 50 kDa.

89. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin active against a non-mammalian pest, wherein a portion of the nucleotide sequence ^s==.encoding said second toxin can be amplified by PCR using the primer pair selected from the following C04229#2 73 SEQ ID NOs. 20 and 24 to produce a fragment of about 495 bp; SEQ ID NOs. 20 and to produce a fragment of about 594 bp; SEQ ID NOs. 21 and 24 to produce a fragment of about 471 bp; and SEQ ID NOs. 21 and 25 to produce a fragment of about 580 bp. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

91. The isolated polynucleotide according to any one of claims 1 to 48, further encoding a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non- mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

92. The isolated polynucleotide according to any one of claims 1 to 48, further encoding an approximately 40 to 50 kDa PS80JJ1 toxin active against non-mammalian pests, wherein the nucleotide sequence encoding said second toxin has been optimised for expression in plants.

93. The isolated polynucleotide according to claim 91 or claim 92, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 11 or a pesticidal portion thereof.

94. The isolated polynucleotide, according to claim 91, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO.

95. The isolated polynucleotide according to claim 92, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 20 96. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

97. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non- mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. i98. The isolated polynucleotide according to claim 97, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 43 or a pesticidal portion thereof.

99. The isolated polynucleotide according to claim 97, wherein said nucleotide sequence 30 encoding said second toxin comprises the sequence shown in SEQ ID NO. 42.

100. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

101. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non- mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

102. The isolated polynucleotide according to claim 101, wherein said second toxin >'^|Bjnprises the amino acid sequence shown in SEQ ID NO. 38 or a pesticidal portion thereof. C04229#2 74

103. The isolated polynucleotide according to claim 101, wherein said nucleotide sequence encoding said second toxin comprises the sequence shown in SEQ ID NO. 37.

104. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43.

105. The isolated polynucleotide according to any one of claim 1 to 48, further encoding a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 80% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43.

106. The isolated polynucleotide according to any one of claims 1 to 48, further encoding a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 90% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43.

107. An isolated polynucleotide encoding a first toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, and a second toxin having a molecular weight of approximately 40 to 50 kDa wherein said isolated polynucleotide comprises at least pesticidal-encoding portions of the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39. 20 108. The isolated polynucleotide according to any one of claims 1 to 107, wherein said pest is an insect.

109. The isolated polynucleotide according to claim 108, wherein said insect is a coleopteran.

110. The isolated polynucleotide according to claim 108, wherein said insect is a lepidopteran. S 25 111. The isolated polynucleotide according to any one of claims 1 to 107, wherein said pest is a mite.

112. The isolated polynucleotide according to any one of claims 1 to 107, wherein said pest is .corn rootworm.

113. The isolated polynucleotide according to any one of claims 1 to 107, wherein said pest is 30 western corn rootworm.

114. An isolated polynucleotide encoding a toxin having a molecular weight of approximately to 15 kDa active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the Examples.

115. An isolated polynucleotide encoding a first toxin having a molecular weight approximately 10 to 15 kDa active against a non-mammalian pest, and a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the Examples.

116. An isolated or purified toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of DNA which encodes SEQ ID NO. 3, DNA which ncodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes SEQ ID NO. 32, C04229#2 DNA which encodes SEQ ID NO. 36, and DNA which encodes SEQ ID NO. 41, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

117. An isolated or purified toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30, SEQ ID NO. 31, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

118. The isolated or purified toxin according to claim 116 or claim 117, wherein DNA which 1 o encodes SEQ ID NO. 3 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

119. The isolated or purified toxin according to claim 116 or claim 117, wherein DNA which encodes SEQ ID NO. 5 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

120. The isolated or purified toxin according to claim 116 or claim 117, wherein DNA which encodes SEQ ID NO. 7, hybridises under stringent conditions with the nucleotide sequence encoding said toxin, or a complement thereof.

121. The isolated or purified toxin according to claim 116 or claim 117, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 32 hybridises under stringent conditions with the nucleotide sequence encoding said toxin, or a complement thereof.

122. The isolated or purified toxin according to claim 116 or claim 117, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 36 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

123. The isolated or purified toxin according to claim 116 or claim 117, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 41 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

124. The isolated or purified toxin according to claim 116 or claim 117, wherein SEQ ID NO. 29 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. :o 30 125. The isolated or purified toxin according to claim 116 or claim 117, wherein a 10 to kDa toxin-encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

126. The isolated or purified toxin according to claim 116 or claim 117, wherein SEQ ID NO. 31 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

127. The isolated or purified toxin according to claim 116 or claim 117, wherein SEQ ID NO. 33 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. C04229#2 76

128. The isolated or purified toxin according to claim 116 or claim 117, wherein a 10 to kDa toxin-encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

129. The isolated or purified toxin according to claim 116 or claim 117, wherein SEQ ID NO. 35 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

130. The isolated or purified toxin according to claim 116 or claim 117, wherein a 10 to kDa toxin-encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

131. The isolated or purified toxin according to claim 116 or claim 117, wherein SEQ ID NO. hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

132. The isolated or purified toxin according to claim 116 or claim 117, wherein SEQ ID NO. 44, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

133. The isolated or purified toxin according to claim 116 or claim 117, wherein said nucleotide sequence encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO.

134. The isolated or purified toxin according to claim 116 or claim 117, wherein said nucleotide sequence encoding the toxin is SEQ ID NO. 31. 20 135. The isolated or purified toxin according to claim 116 or claim 117, wherein said nucleotide sequence encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34.

136. The isolated or purified toxin according to claim 116 or claim 117, wherein said S" nucleotide sequence encoding the toxin is SEQ ID NO.

137. The isolated or purified toxin according to claim 116 or claim 117, wherein said nucleotide sequence encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39.

138. The isolated or purified toxin according to claim 116 or claim 117, wherein said nucleotide sequence encoding the toxin is SEQ ID NO.

139. The isolated or purified toxin according to claim 116 or claim 117, wherein said ::':nucleotide sequence encoding the toxin is SEQ ID NO. 44. 30 140. An isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41 or pesticidal portions thereof.

141. The isolated or purified toxin according to claim 140, wherein said amino acid sequence comprises SEQ ID NO. 32 or a pesticidal portion thereof.

142. The isolated or purified toxin according to claim 140, wherein said amino acid sequence comprises SEQ ID NO. 36 or a pesticidal portion thereof.

143. The isolated or purified toxin according to claim 140, wherein said amino acid sequence comprises SEQ ID NO. 41 or a pesticidal portion thereof. ~144. The isolated or purified toxin according to any one of claims 116 to 143, wherein the oxin has a molecular weight of approximately 10 to 15 kDa. I- P C04229#2

145. An isolated or purified toxin active against a non-mammalian pest, wherein said toxin is encoded by a nucleotide sequence, a portion of which can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

146. An isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

147. The isolated or purified toxin according to claim 146, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 32 or a pesticidal portion thereof. 148 The isolated or purified toxin according to claim 146, wherein said toxin is encoded by the nucleotide sequence shown in SEQ ID NO. 31.

149. An isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

150. An isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

151. The isolated or purified toxin according to claim 150, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 41 or a pesticidal portion thereof.

152. The isolated or purified toxin according to claim 150, wherein said toxin is encoded by 20 the nucleotide sequence shown in SEQ ID NO.

153. An isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

154. An isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

155. The isolated or purified toxin according to claim 154, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 36 or a pesticidal portion thereof.

156. The isolated or purified toxin according to claim 154, wherein said toxin is encoded by 30 the nucleotide sequence shown in SEQ ID NO.

157. An isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS167H2, having the identifying characteristics of NRRL B-21554.

158. An isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41.

159. The isolated or purified toxin according to claim 158 which has at least about identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID C04229#2 78 NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41.

160. The isolated or purified toxin according to claim 158 which has at least about identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41.

161. An isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

162. The isolated or purified toxin according to any one of claims 116 to pest is an insect.

163. The isolated or purified toxin according to claim 162, wherein coleopteran.

164. The isolated or purified toxin according to claim 162, wherein lepidopteran.

165. The isolated or purified toxin according to any one of claims 116 to pest is a mite.

166. The isolated or purified toxin according to any one of claims 116 to 20 pest is corn rootworm.

167. The isolated or purified toxin according to any one of claims 116 to pest is western corn rootworm. 161, wherein said said insect is a said insect is a 161, wherein said 161, wherein said 161, wherein said 0 0 0000.. 0 00 0 **0 *00 00 0 .0 0 00000 00 0 *00

168. An isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples.

169. A biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain pesticidal activity, said culture comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa.

170. The biologically pure culture according to claim 169, wherein said Bacillus thuringiensis isolate is PS149B1, having the identifying characteristics of NRRL B-21553.

171. The biologically pure culture according to claim 169, wherein said Bacillus thuringiensis isolate is PS167H2 having the identifying characteristics of NRRL B-21554.

172. A biologically pure culture of a Bacillus thuringiensis isolate comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples.

173. A recombinant DNA molecule comprising a polynucleotide according to any one of claims 1 to 115. C04229#2 79

174. The recombinant DNA molecule according to claim 173, further comprising appropriate regulatory regions for transformation of a desired host.

175. The recombinant DNA molecule according to claim 174, wherein said host is selected from bacteria, fungi or plants.

176. The recombinant DNA molecule according to claim 175, wherein said bacteria are selected from a genus from the group comprising Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes.

177. The recombinant DNA molecule according to claim 176, wherein said bacteria are selected from the group comprising Pseudomonas fluorescens, Pseudomonas syringae, Serratia marcescens, Xanthomonas campestris, Rhizobium meliloti, Rhodopseudomonas spheroides, Agrobacterium tumefaciens, Acetobacter xylinum, Azotobacter vinlandii, and Alcaligenes entrophus.

178. The recombinant DNA molecule according to claim 175, wherein said fungi are selected from a genus from the group comprising Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

179. The recombinant DNA molecule according to claim 178, wherein said fungi are selected from the group comprising Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Cryptococcus albidus, C. diffluens, C. laurentii, Kluyveromyces veronae, Sporobolomyces roseus, S. odorus, Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, and Aureobasidium pullulans. 20 180. The recombinant DNA molecule according to any one of claims 174 to 179, wherein said host is pigmented. .0 181. The recombinant DNA molecule according to claim 175, wherein said plant is a vegetable or field crop.

182. The recombinant DNA molecule according to claim 181, wherein said plant is selected 25 from the group comprising maize, alfalfa, and cucurbits.

183. The recombinant DNA molecule according to claim 181, wherein said plant is maize.

184. The recombinant DNA molecule according to claim 183, wherein the nucleotide sequence encoding the toxin having a molecular weight of approximately 10 to 15 kDa is SEQ ID NO. 0. 44. 30 185. The recombinant DNA molecule according to claim 183 or claim 184, further comprising a nucleotide sequence having the sequence set forth in SEQ ID NO. 45, encoding a toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest.

186. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence comprising SEQ ID NO.

187. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence comprising SEQ ID NO. 31.

188. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence comprising SEQ ID NO. 34. S189. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said /7 i polynucleotide includes a nucleotide sequence comprising SEQ ID NO. C04229#2

190. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence comprising SEQ ID NO. 39.

191. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence comprising SEQ ID NO.

192. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence comprising SEQ ID NO. 44.

193. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 32 or a pesticidal portion thereof.

194. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 36 or a pesticidal portion thereof.

195. The recombinant DNA molecule according to any one of claims 173 to 183, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 41 or a pesticidal portion thereof.

196. A recombinant DNA molecule comprising a nucleotide sequence encoding a toxin having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples.

197. A recombinant DNA molecule comprising a nucleotide sequence encoding a first toxin having a molecular weight of approximately 10 to 15 kDa, and a nucleotide sequence encoding a second toxin having a molecular weight of approximately 40 to 50 kDa substantially as hereinbefore described with reference to any one of the Examples.

198. A plasmid comprising a polynucleotide according to any one of claims 1 to 115, or a recombinant DNA molecule according to any one of claims 173 to 197.

199. The plasmid according to claim sequence comprising SEQ ID NO.

200. The plasmid according to claim sequence comprising SEQ ID NO. 31.

201. The plasmid according to claim sequence comprising SEQ ID NO. 34.

202. The plasmid according to claim sequence comprising SEQ ID NO.

203. The plasmid according to claim sequence comprising SEQ ID NO. 39.

204. The plasmid according to claim sequence comprising SEQ ID NO.

205. The plasmid according to claim sequence comprising SEQ ID NO. 44. 198, wherein said polynucleotide includes a nucleotide 198, wherein said polynucleotide includes a nucleotide 198, wherein said polynucleotide includes a nucleotide 198, wherein said polynucleotide includes a nucleotide 198, wherein said polynucleotide includes a nucleotide 198, wherein said polynucleotide includes a nucleotide 198, wherein said polynucleotide includes a nucleotide C04229#2 81

206. The plasmid according to claim 198, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 32 or a pesticidal portion thereof..

207. The plasmid according to claim 198, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 36 or a pesticidal portion thereof.

208. The plasmid according to claim 198, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 41 or a pesticidal portion thereof.

209. A plasmid comprising a polynucleotide encoding a toxin having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples.

210. A plasmid comprising a nucleotide sequence encoding a first toxin having a molecular weight of approximately 10 to 15 kDa, and a nucleotide sequence encoding a second toxin having a molecular weight of approximately 40 to 50 kDa, substantially as hereinbefore described with reference to any one of the Examples.

211. A vector comprising a polynucleotide according to any one of claims 1 to 115, a recombinant DNA molecule according to any one of claims 173 to 197, or a plasmid according to any one of claims 198 to 210. 9 9r 9 9 Se 9 9 9 *99. 9 0 5595 59 9 5 20 212. The vector according to claim 211, wherein sequence comprising SEQ ID NO.

213. The vector according to claim 211, wherein sequence comprising SEQ ID NO. 31.

214. The vector according to claim 211, wherein 25 sequence comprising SEQ ID NO. 34.

215. The vector according to claim 211, wherein sequence comprising SEQ ID NO.

216. The vector according to claim 211, wherein sequence comprising SEQ ID NO. 39. 30 217. The vector according to claim 211, wherein sequence comprising SEQ ID NO.

218. The vector according to claim 211, wherein sequence comprising SEQ ID NO. 44. polynucleotide polynucleotide polynucleotide polynucleotide polynucleotide polynucleotide polynucleotide includes includes includes includes includes includes includes nucleotide nucleotide nucleotide nucleotide nucleotide nucleotide nucleotide

219. The vector according to claim 211, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 32 or a pesticidal portion thereof.

220. The vector according to claim 211, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 36 or a pesticidal portion thereof. C04229#2 82

221. The vector according to claim 211, wherein said polynucleotide includes a nucleotide sequence which encodes an amino acid sequence comprising SEQ ID NO. 41 or a pesticidal portion thereof.

222. The vector according to any one of claims 211 to 221, wherein said vector is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

223. The vector according to any one of claims 211 to 221, wherein said vector is a virus.

224. A vector comprising a polynucleotide encoding a toxin having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples.

225. A vector comprising a nucleotide sequence encoding a first toxin having a molecular weight of approximately 10 to 15 kDa, and a nucleotide sequence encoding a second toxin having a molecular weight of approximately 40 to 50 kDa, substantially as hereinbefore described with reference to any one of the Examples.

226. A method for transforming a host cell with a nucleotide sequence encoding a toxin having a molecular weight of approximately 10 to 15 kDa and active against a non-mammalian pest, said method comprising contacting said host cell with a polynucleotide according to any one of claims 1 to 115, a recombinant DNA molecule according to any one of claims 173 to 197, a plasmid according to any one of claims 198 to 210, or a vector according to any one of claims 211 to 225. 4 S S S 5 S S 4 4 0* S'r

227. The method according to claim 226, wherein said polynucleotide includes 20 sequence comprising SEQ ID NO. 31.

228. The method according to claim 226, wherein said polynucleotide includes sequence comprising SEQ ID NO.

229. The method according to claim 226, wherein said polynucleotide includes sequence comprising SEQ ID NO. 25 230. The method according to claim 226, wherein said polynucleotide includes sequence which encodes an amino acid sequence comprising SEQ ID NO. 32.

231. The method according to claim 226, wherein said polynucleotide includes sequence which encodes an amino acid sequence comprising SEQ ID NO. 36.

232. The method according to claim 226, wherein said polynucleotide includes sequence which encodes an amino acid sequence comprising SEQ ID NO. 41. a nucleotide a nucleotide a nucleotide a nucleotide a nucleotide a nucleotide

233. The method according to any one of claims 226 to 232, wherein the method comprises transfection of the host cell via a vector according to any one of claims 211 to 225.

234. The method according to any one of claims 226 to 232, wherein the method comprises physical means of transformation selected from the group comprising microinjection, electroporation or biolistics.

235. The method according to any one of claims 226 to 234, wherein said host is selected from bacteria, fungi or plants.

236. The method according to claim 235, wherein said bacteria are selected from a genus from the group comprising Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, C04229#2 83 Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes.

237. The method according to claim 235, wherein said bacteria are selected from the group comprising Pseudomonas fluorescens, Pseudomonas syringae, Serratia marcescens, Xanthomonas campestris, Rhizobium meliloti, Rhodopseudomonas spheroides, Agrobacterium tumefaciens, Acetobacter xylinum, Azotobacter vinlandii, and Alcaligenes entrophus.

238. The method according to claim 235, wherein said fungi are selected from a genus from the group comprising Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

239. The method according to claim 238, wherein said fungi are selected from the group comprising Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Cryptococcus albidus, C. diffluens, C. laurentii, Kluyveromyces veronae, Sporobolomyces roseus, S. odorus, Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, and Aureobasidium pullulans.

240. The method according to any one of claims 235 to 239, wherein said host is pigmented.

241. The method according to claim 235, wherein said plant is a vegetable or field crop.

242. The method according to claim 241, wherein said plant is selected from the group comprising maize, alfalfa, and cucurbits.

243. The method according to claim 241, wherein said plant is maize. i. 244. The method according to claim 243, wherein the nucleotide sequence encoding the toxin 20 having a molecular weight of approximately 10 to 15 kDa is SEQ ID NO. 44.

245. The method according to claim 243 or claim 244, wherein the polynucleotide further comprises a nucleotide sequence having the sequence set forth in SEQ ID NO. 45, encoding a toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest.

246. A method for transforming a host cell with a nucleotide sequence encoding a toxin 25 having a molecular weight of approximately 10 to 15 kDa and active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the examples.

247. A method for transforming a host cell with a nucleotide sequence encoding a first toxin having a molecular weight of approximately 10 to 15 kDa and active against a non-mammalian pest, and a nucleotide sequence encoding a second toxin having a molecular weight of approximately 30 to 50 kDa and active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the examples.

248. A host bacterial or fungal cell transformed by a method according to any one of claims 226 to 240, 246 or 247.

249. A host plant cell transformed by a method according to any one of claims 226 to 235 or 241 to 247.

250. A method of producing a plant transformed with a nucleotide sequence encoding a toxin having a molecular weight of approximately 10 to 15 kDa, said method comprising a method according to any one of claims 235 or 241 to 247, further comprising re-generation of whole plants from the transformed plant cells.

251. A transgenic plant prepared by a method according to claim 250. C04229#2 84

252. A transgenic plant according to claim 251, which also expresses a toxin having a molecular weight of approximately 40 to 50 kDa.

253. A plant comprising a plurality of cells according to claim 249.

254. Transgenic seed from a plant according to any one of claims 251 to 253.

255. A recombinant host cell containing a polynucleotide according to any one of claims 1 to 115, a recombinant DNA molecule according to any one of claims 173 to 197, or a plasmid according to any one of claims 198 to 210.

256. A recombinant host cell transformed with a polynucleotide coding for a toxin having activity against a non-mammalian pest, wherein said toxin has at least one characteristic selected from the group consisting of: said toxin is encoded by a polynucleotide wherein a nucleotide sequence selected from the group consisting of DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. and DNA which encodes SEQ ID NO. 7, hybridises under stringent conditions with said polynucleotide or a complement thereof; said toxin is an approximately 10 to 15 kDa pesticidal toxin encoded by a Snucleotide sequence from a Bacillus thuringiensis isolate selected from the group consisting of SPS80JJ1 having the identifying characteristics of NRRL 8-18679, PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B- 21554; 20 said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from a Bacillus thuringiensis isolate selected from the group consisting of PS80JJ1 having the identifying characteristics of NRRL B-18679, PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B- 21554; S 25 said toxin is encoded by a nucleotide sequence wherein a portion of said nucleotide sequence can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33; said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof; said toxin comprises an amino acid sequence which has at least about identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions of SEQ ID NO. 36, and pesticidal portions of sequence ID NO. 41; and said toxin is encoded by a polynucleotide wherein a nucleotide sequence selected from the group consisting of SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. SEQ ID NO. 31, SEQ ID NO. 33, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with said polynucleotide or a complement thereof.

257. The recombinant host cell according to claim 256, wherein DNA which encodes SEQ ID 4 ZO 3 hybridises under stringent conditions with said polynucleotide or a complement thereof. C04229#2

258. The recombinant host cell according to claim 256, wherein DNA which encodes SEQ ID NO. 5 hybridises under stringent conditions with said polynucleotide or a complement thereof.

259. The recombinant host cell according to claim 256, wherein DNA which encodes SEQ ID NO. 7 hybridises under stringent conditions with said polynucleotide or a complement thereof.

260. The recombinant host cell according to claim 256, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 32 hybridises under stringent conditions with said polynucleotide or a complement thereof.

261. The recombinant host cell according to claim 256, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 36 hybridises under stringent conditions with said polynucleotide or a complement thereof.

262. The recombinant host cell according to claim 256, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 41 hybridises under stringent conditions with said polynucleotide or a complement thereof.

263. The recombinant host cell according to claim 256, wherein SEQ ID NO. 29 hybridises under stringent conditions with said polynucleotide or a complement thereof.

264. The recombinant host cell according to claim 256, wherein a 10 to 15 kDa toxin- encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with said polynucleotide or a complement thereof.

265. The recombinant host cell according to claim 256, wherein SEQ ID NO. 31 hybridises under stringent conditions with said polynucleotide or a complement thereof.

266. The recombinant host cell according to claim 256, wherein SEQ ID NO. 33 hybridises under stringent conditions with said polynucleotide or a complement thereof.

267. The recombinant host cell according to claim 256, wherein a 10 to 15 kDa toxin- encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with said polynucleotide or a 25 complement thereof.

268. The recombinant host cell according to claim 256, wherein SEQ ID NO. 35 hybridises under stringent conditions with said polynucleotide or a complement thereof.

269. The recombinant host cell according to claim 256, wherein a 10 to 15 kDa toxin- encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with said polynucleotide or a complement thereof.

270. The recombinant host cell according to claim 256, wherein SEQ ID NO. 40 hybridises under stringent conditions with said polynucleotide or a complement thereof.

271. The recombinant host cell according to claim 256, wherein SEQ ID NO. 44 hybridises under stringent conditions with said polynucleotide or a complement thereof.

272. The recombinant host cell according to claim 256, wherein the nucleotide sequence encoding said toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO.

273. The recombinant host cell according to claim 256, wherein the nucleotide sequence encoding said toxin is SEQ ID NO. 31.

274. The recombinant host cell according to claim 256, wherein the nucleotide sequence oding said toxin is SEQ ID NO. 34. C04229#2 86

275. The recombinant host cell according to claim 256, wherein the nucleotide sequence encoding said toxin is SEQ ID NO.

276. The recombinant host cell according to claim 256, wherein the nucleotide sequence encoding said toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39.

277. The recombinant host cell according to claim 256, wherein the nucleotide sequence encoding said toxin is SEQ ID NO.

278. The recombinant host cell according to claim 256, wherein the nucleotide sequence encoding said toxin is SEQ ID NO. 44.

279. The recombinant host cell according to claim 256, wherein said amino acid sequence comprises SEQ ID NO. 32 or a pesticidal portion thereof.

280. The recombinant host cell according to claim 256, wherein said amino acid sequence comprises SEQ ID NO. 36 or a pesticidal portion thereof.

281. The recombinant host cell according to claim 256, wherein said amino acid sequence comprises SEQ ID NO. 41 or a pesticidal portion thereof.

282. The recombinant host cell according to claim 256, wherein a portion of said nucleotide sequence can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

283. The recombinant host cell according to any one of claims 256 to 282, wherein the toxin has a molecular weight of approximately 10 to 15 kDa.

284. The recombinant host cell according to claim 256, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

285. The recombinant host cell according to claim 256, wherein said toxin has a molecular weight of approximately 10 to 15 kDa, and is encoded by a nucleotide sequence from Bacillus thuringiensis isolate PS80JJ1, having the identifying characteristics of NRRL B-18679.

286. The recombinant host cell according to claim 256, wherein said polynucleotide encodes an approximately 10 to 15 kDa PS80JJ1 toxin active against non-mammalian pests, wherein said nucleotide sequence has been optimised for expression in plants.

287. The recombinant host cell according to claim 285 or claim 286, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 32 or a pesticidal portion thereof.

288. The recombinant host cell according to claim 285, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO. 31.

289. The recombinant host cell according to claim 286, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO. 44.

290. The recombinant host cell according to claim 256, wherein said toxin is encoded by a nucleotide sequence from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553.

291. The recombinant host cell according to claim 290, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 41 or a pesticidal portion thereof.

292. The recombinant host cell according to claim 290, wherein said nucleotide sequence rccomprises the sequence shown in SEQ ID NO. C04229#2 87

293. The recombinant host cell according to claim 256, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa pesticidal toxin from Bacillus thuringiensis PS149B1 having the identifying characteristics of NRRL B-21553.

294. The recombinant host cell according to claim 256, wherein said toxin is encoded by a nucleotide sequence from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554.

295. The recombinant host cell according to claim 294, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 36 or a pesticidal portion thereof.

296. The recombinant host cell according to claim 294, wherein said nucleotide sequence comprises the sequence shown in SEQ ID NO.

297. The recombinant host cell according to claim 256, wherein said toxin comprises an amino acid sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions of SEQ ID NO. 36, and pesticidal portions of sequence ID NO. 41.

298. The recombinant host cell according to claim 256, wherein said toxin comprises an amino acid sequence which has at least about 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions of SEQ ID NO. 36, and pesticidal portions of sequence ID NO. 41.

299. The recombinant host cell according to claim 256, wherein said toxin has a molecular weight of approximately 10 to 15 kDa and comprises an amino acid sequence which has at least about 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, o "SEQ ID NO. 5, SEQ ID NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions of SEQ ID NO. 36, and pesticidal portions of sequence ID NO. 41.

300. The recombinant host cell according to claim 256, wherein said toxin comprises a o: 25 pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

301. The recombinant host cell according to any one of claims 256 to 300, which is further. transformed with a polynucleotide coding for a second toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 2; DNA which encodes SEQ ID NO. 4; DNA which encodes SEQ ID NO. 6; SEQ ID NO. 8; SEQ ID NO. 10; DNA which encodes a pesticidal portion of SEQ ID NO. 11; SEQ ID NO. 12; DNA which encodes SEQ ID NO. 13; SEQ ID NO. 14; DNA which encodes SEQ ID NO. 15; DNA which encodes SEQ ID NO. 16; DNA which encodes SEQ ID NO. 17; DNA which encodes SEQ ID NO. 18; DNA which encodes SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ ID NO. 24; SEQ ID NO. 25; SEQ ID NO. 26; SEQ ID NO. 27; DNA which encodes a pesticidal portion of SEQ ID NO. 28; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 30; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 34; SEQ ID NO. 37; DNA which encodes a pesticidal portion of SEQ ID NO. 38; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 39; SEQ ID NO. 40; SEQ ID NO. 42; DNA which encodes a pesticidal portion of SEQ ID NO. 43; and SEQ ID NO. 45; hybridises C04229#2 88 under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

302. The recombinant host cell according to claim 301, wherein SEQ ID NO. 8 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

303. The recombinant host cell according to claim 301, wherein SEQ ID NO. 10 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

304. The recombinant host cell according to claim 301, wherein SEQ ID NO. 12 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

305. The recombinant host cell according to claim 301, wherein the second nucleotide sequence hybridises with SEQ ID NO. 14 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

306. The recombinant host cell according to claim 301, wherein the second nucleotide sequence hybridises with SEQ ID NO. 20 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

307. The recombinant host cell according to claim 301, wherein SEQ ID NO. 21 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

308. The recombinant host cell according to claim 301, wherein SEQ ID NO. 22 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

309. The recombinant host cell according to claim 301, wherein SEQ ID NO. 23 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement Sthereof.

310. The recombinant host cell according to claim 301, wherein SEQ ID NO. 24 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

311. The recombinant host cell according to claim 301, wherein SEQ ID NO. 25 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

312. The recombinant host cell according to claim 301, wherein SEQ ID NO. 26 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

313. The recombinant host cell according to claim 301, wherein SEQ ID NO. 27 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof. C04229#2

314. The recombinant host cell according to claim 301, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

315. The recombinant host cell according to claim 301, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

316. The recombinant host cell according to claim 301, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

317. The recombinant host cell according to claim 301, wherein SEQ ID NO. 42 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

318. The recombinant host cell according to claim 301, wherein SEQ ID NO. 45 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

319. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 2 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

320. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 4 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

321. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 6 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof. S 25 322. The recombinant host cell according to claim 301, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 11 hybridises under stringent conditions with the nucleotide S"sequence encoding said second toxin or a complement thereof. N.323. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 13 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

324. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 15 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

325. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 16 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

326. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 17 hybridises under stringent conditions with the nucleotide sequence encoding said second Stoxin or a complement thereof. C04229#2

327. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 18 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

328. The recombinant host cell according to claim 301, wherein DNA which encodes SEQ ID NO. 19 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

329. The recombinant host cell according to claim 301, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 28 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

330. The recombinant host cell according to claim 301, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 38 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof.

331. The recombinant host cell according to claim 301, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 43 hybridises under stringent conditions with the nucleotide sequence encoding said second toxin or a complement thereof. S S S S *C S S S S S S..

332. The recombinant host cell according encoding said second toxin is SEQ ID NO.

333. The recombinant host cell according encoding said second toxin is SEQ ID NO. 37.

334. The recombinant host cell according encoding said second toxin is SEQ ID NO. 42.

335. The recombinant host cell according encoding said second toxin is SEQ ID NO. to claim 301, wherein the nucleotide sequence to claim 301, wherein the nucleotide sequence to claim 301, wherein the nucleotide sequence to claim 301, wherein the nucleotide sequence

336. The recombinant host cell according to claim 301, wherein said 25 amino acid sequence set forth in SEQ ID NO. 11 or a pesticidal portion thereof.

337. The recombinant host cell according to claim 301, wherein said amino acid sequence set forth in SEQ ID NO. 38 or a pesticidal portion thereof.

338. The recombinant host cell according to claim 301, wherein said amino acid sequence set forth in SEQ ID NO. 43 or a pesticidal portion thereof. second toxin has the second toxin has the second toxin has the

339. The recombinant host cell according to claim 301, wherein said second toxin comprises the consensus sequence shown in Figure 1.

340. The recombinant host cell according to any one of claims 301 to 339, wherein said second toxin has a molecular weight of approximately 40 to 50 kDa.

341. The recombinant host cell according to any one of claims 256 to 300, further transformed with a polynucleotide coding for a second toxin active against a non-mammalian pest, wherein a portion of the nucleotide sequence encoding said second toxin can be amplified by PCR using the primer pair selected from the following group: SEQ ID NOs. 20 and 24 to produce a fragment of about 495 bp; SEQ ID NOs. 20 and to produce a fragment of about 594 bp; SEQ ID NOs. 21 and 24 to produce a fragment of about 471 4^bp; and SEQ ID NOs. 21 and 25 to produce a fragment of about 580 bp. C04229#2 91

342. The recombinant host cell according to any one of claims 256 to 300, further transformed with a polynucleotide coding for a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, wherein the nucleotide sequence encoding said second toxin is from a Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679.

343. The recombinant host cell according to claim 342, wherein the toxin encoded by second nucleotide sequence comprises the amino acid sequence shown in SEQ ID NO. 11 or a pesticidal portion thereof.

344. The recombinant host cell according to claim 342, wherein said second nucleotide sequence comprises the sequence shown in SEQ ID NO.

345. The recombinant host cell according to any one of claims 256 to 300, further transformed to express a second toxin having a molecular weight of approximately 40 to 50 kDa PS80JJ1 toxin active against non-mammalian pests, wherein the nucleotide sequence encoding said second toxin has been optimised for expression in plants.

346. The recombinant host cell according to claim 345, wherein the polynucleotide encoding said second toxin comprises the sequence shown in SEQ ID NO.

347. The recombinant host cell according to any one of claims 256 to 300, further transformed with a polynucleotide coding for a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, wherein the nucleotide sequence 20 encoding said second toxin is from a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554.

348. The recombinant host cell according to any one of claims 256 to 300, further transformed with a polynucleotide coding for a second toxin active against a non-mammalian pest 25 having an amino acid sequence which has at least about 75% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43.

349. The recombinant host cell according to any one of claims 256 to 300, further transformed with a polynucleotide coding for a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 80% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43.

350. The recombinant host cell according to any one of claims 256 to 300, further transformed with a polynucleotide coding for a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 90% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43.

351. A recombinant host cell transformed with a polynucleotide coding for a first toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, and a V ucleotide coding for a second toxin having a molecular weight of approximately 40 to 50 kDa C04229#2 92 wherein said toxins are encoded by at least pesticidal-encoding portions of a nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39.

352. The recombinant host cell according to any one of claims 256 to 351, wherein said pest is an insect.

353. The recombinant host cell according to claim 352, wherein said insect is a coleopteran.

354. The recombinant host cell according to claim 352, wherein said insect is a lepidopteran.

355. The recombinant host cell according to any one of claims 256 to 351, wherein said pest is a mite.

356. The recombinant host cell according to any one of claims 256 to 351, wherein said pest is corn rootworm.

357. The recombinant host cell according to any one of claims 256 to 351, wherein said pest is western corn rootworm.

358. A recombinant host cell transformed with a nucleotide sequence which encodes a toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the Examples.

359. A recombinant host cell transformed with a nucleotide sequence which encodes a first toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, :and with a nucleotide sequence which encodes a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the Examples. S* 360. The recombinant host cell according to any one of claims 243 to 344, wherein said recombinant host cell is a plant cell.

361. A transgenic plant regenerated from a recombinant plant cell according to claim 360.

362. A transgenic plant comprising a plurality of cells according to claim 360. 25 363. The transgenic plant according to claim 361 or claim 362, wherein said plant is a vegetable or field crop.

364. The transgenic plant according to claim 363, wherein said plant is selected from the group comprising maize, alfalfa, and cucurbits.

365. The transgenic plant according to claim 363, wherein said plant is maize.

366. The transgenic plant according to claim 365, wherein the nucleotide sequence encoding the toxin having a molecular weight of approximately 10 to 15 kDa is SEQ ID NO. 44.

367. The transgenic plant according to claim 365 or claim 366, also comprising a nucleotide sequence having the sequence set forth in SEQ ID NO. 45, encoding a toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest.

368. A transgenic plant comprising a nucleotide sequence encoding a toxin having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the examples.

369. A transgenic plant comprising a nucleotide sequence encoding a first toxin having a molecular weight of approximately 10 to 15 kDa, and a nucleotide sequence encoding a second toxin C04229#2 93 having a molecular weight of approximately 40 to 50 kDa, substantially as hereinbefore described with reference to any one of the examples.

370. A method for preparing a pesticidal composition comprising a toxin active against non- mammalian pests having a molecular weight of approximately 10 to 15 kDa, said method comprising culturing a biologically pure culture according to any one of claims 169 to 172, or a plurality of transgenic cells according to any one of claims 248, 249, or 255 to 360, under conditions promoting expression of said toxin.

371. The method according to claim 370, wherein said composition also comprises a toxin active against non-mammalian pests having a molecular weight of approximately 40 to 50 kDa.

372. The method according to claim 370 or 371, wherein said cultured cells are treated so as to release said toxin, or toxins from said cells.

373. The method according to claim 372, further comprising isolating or purifying at least said toxin having a molecular weight of approximately 10 to 15 kDa from said treated cell culture.

374. The method according to claim 373, wherein said toxin having a molecular weight of approximately 40 to 50 kDa is also isolated/purified from said treated cell culture together with said toxin having a molecular weight of approximately 10 to 15 kDa.

375. A method for preparing a pesticidal composition comprising a toxin active against non- S mammalian pests having a molecular weight of approximately 10 to 15 kDa, said method being substantially as hereinbefore described with reference to any one of the examples.

376. A method for preparing a pesticidal composition comprising a first toxin active against S non-mammalian pests having a molecular weight of approximately 10 to 15 kDa, and a second toxin active against non-mammalian pests having a molecular weight of approximately 40 to 50 kDa said method being substantially as hereinbefore described with reference to any one of the examples.

377. A pesticidal composition prepared by a method according to any one of claims 355 to 25 357. '378. A pesticidal composition comprising an isolated or purified toxin according to any one of claims 116 to 168, or a biologically pure culture according to any one ofclaims 169 to 172. *379. A pesticidal composition according to claim 377 or claim 378, further comprising agriculturally/pesticidally acceptable additives, including carriers, adjuvants, stabilising agents, rheological agents, emulsifiers, dispersants, polymers and surfactants.

380. A pesticidal composition comprising an isolated or purified toxin active against non- mammalian pests, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes SEQ ID NO. 32, DNA which encodes SEQ ID NO. 36, and DNA which encodes SEQ ID NO. 41, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

381. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein a nucleotide sequence selected from the group consisting of SEQ ID NO. 9, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30, SEQ ID NO. 31, a 10 to 15 kDa toxin- oding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin-encoding portion of SEQ C04229#2 94 ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

382. The pesticidal composition according to claim 380 or claim 381, wherein DNA which encodes SEQ ID NO. 3 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

383. The pesticidal composition according to claim 380 or claim 381, wherein DNA which encodes SEQ ID NO. 5 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof,

384. The pesticidal composition according to claim 380 or claim 381, wherein DNA which encodes SEQ ID NO. 7 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

385. The pesticidal composition according to claim 380 or claim 381, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 32 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

386. The pesticidal composition according to claim 380 or claim 381, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 36 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

387. The pesticidal composition according to claim 380 or claim 381, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 41 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

388. The pesticidal composition according to claim 380 or claim 381, wherein SEQ ID NO. 29 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

389. The pesticidal composition according to claim 380 or claim 381, wherein a 10 to 15 kDa S 25 toxin-encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

390. The pesticidal composition according to claim 380 or claim 381, wherein SEQ ID NO. 31 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

391. The pesticidal composition according to claim 380 or claim 381, wherein SEQ ID NO. 33 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

392. The pesticidal composition according to claim 380 or claim 381, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

393. The pesticidal composition according to claim 380 or claim 381, wherein SEQ ID NO. hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. C04229#2

394. The pesticidal composition according to claim 380 or claim 381, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

395. The pesticidal composition according to claim 380 or claim 381, wherein SEQ ID NO. hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

396. The pesticidal composition according to claim 380 or claim 381, wherein SEQ ID NO. 44 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof.

397. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO.

398. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is SEQ ID NO. 31.

399. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34.

400. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is SEQ ID NO.

401. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39.

402. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is SEQ ID NO.

403. The pesticidal composition according to claim 380 or claim 381, wherein said nucleotide sequence encoding the toxin is SEQ ID NO. 44.

404. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof.

405. The pesticidal composition according to claim 404, wherein said amino acid sequence comprises SEQ ID NO. 32 or a pesticidal portion thereof.

406. The pesticidal composition according to claim 404, wherein said amino acid sequence comprises SEQ ID NO. 36 or a pesticidal portion thereof.

407. The pesticidal composition according to claim 404, wherein said amino acid sequence comprises SEQ ID NO. 41 or a pesticidal portion thereof.

408. The pesticidal composition according to any one of claims 380 to 407, wherein the toxin has a molecular weight of approximately 10 to 15 kDa.

409. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein said toxin is encoded by a nucleotide sequence, a portion of which can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33.

410. A pesticidal composition comprising an isolated or purified toxin active against a non- ammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus 40 ingiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. C04229#2

411. The pesticidal composition according to claim 410, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 32 or a pesticidal portion thereof.

412. The pesticidal composition according to claim 410, wherein said toxin is encoded by the nucleotide sequence shown in SEQ ID NO. 31.

413. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-

18679. 414. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. 415. The pesticidal composition according to claim 414, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 41 or a pesticidal portion thereof. 416. The pesticidal composition according to claim 414, wherein said toxin is encoded by the nucleotide sequence shown in SEQ ID NO. 417. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-

21553. 20 418. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest and having a molecular weight of approximately 10 to 15 kDa from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. 419. The pesticidal composition according to claim 418, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 36 or a pesticidal portion thereof. .oo 25 420. The pesticidal composition according to claim 418, wherein said toxin is encoded by the nucleotide sequence shown in SEQ ID NO. 421. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-

21554. 422. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest, wherein said toxin comprises an amino acid sequence which has at least about identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. 423. The pesticidal composition according to claim 422 which has at least about 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. C04229#2 97 424. The pesticidal composition according to claim 422 which has at least about 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. 425. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest and having a molecular weight of approximately 10 to 15 kDa, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39. 426. A pesticidal composition comprising an isolated or purified toxin active against a non- mammalian pest and having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples. 427. The pesticidal composition according to any one of claims 380 to 426, also comprising a second toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 2; DNA which encodes SEQ ID NO. 4; DNA which encodes SEQ ID NO. 6; SEQ ID NO. 8; SEQ ID NO. 10; DNA which encodes a pesticidal portion of SEQ ID NO. 11; SEQ ID NO. 12; DNA which encodes SEQ ID NO. 13; SEQ ID NO. 14; DNA which encodes SEQ ID NO. 15; DNA which encodes SEQ ID NO. 16; DNA which encodes SEQ ID NO. 17; DNA which encodes SEQ ID NO. 18; DNA which encodes SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ ID NO. 24; SEQ ID NO. 25; SEQ ID NO. 20 26; SEQ ID NO. 27; DNA which encodes a pesticidal portion of SEQ ID NO. 28; a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 30; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 34; SEQ ID NO. 37; DNA which encodes a pesticidal portion of SEQ ID NO. 38; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 39; SEQ ID NO. 40; SEQ ID NO. 42; DNA which encodes a pesticidal portion of SEQ ID NO. 43; and SEQ ID NO. 45; hybridises under stringent conditions with the polynucleotide 25 encoding said second toxin or a complement thereof. i 428. The pesticidal composition according to claim 427, wherein SEQ ID NO. 8 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 429. The pesticidal composition according to claim 427, wherein SEQ ID NO. 10 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 430. The pesticidal composition according to claim 427, wherein SEQ ID NO. 12 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 431. The pesticidal composition according to claim 427, wherein SEQ ID NO. 14 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 432. The pesticidal composition according to claim 427, wherein SEQ ID NO. 20 hybridises Te nder stringent conditions with the polynucleotide encoding said second toxin or a complement C04229#2 433. The pesticidal under stringent conditions thereof. 434. The pesticidal under stringent conditions thereof. 435. The pesticidal under stringent conditions thereof. 436. The pesticidal under stringent conditions thereof. 437. The pesticidal under stringent conditions thereof. 438. The pesticidal under stringent conditions thereof. 439. The pesticidal under stringent conditions thereof. composition according to claim 427, wherein SEQ ID NO. 21 hybridises with the polynucleotide encoding said second toxin or a complement composition according to claim 427, wherein SEQ ID NO. 22 hybridises with the polynucleotide encoding said second toxin or a complement composition according to claim 427, wherein SEQ ID NO. 23 hybridises with the polynucleotide encoding said second toxin or a complement composition according to claim 427, wherein SEQ ID NO. 24 hybridises with the polynucleotide encoding said second toxin or a complement composition according to claim 427, wherein SEQ ID NO. 25 hybridises with the polynucleotide encoding said second toxin or a complement composition according to claim 427, wherein SEQ ID NO. 26 hybridises with the polynucleotide encoding said second toxin or a complement composition according to claim 427, wherein SEQ ID NO. 27 hybridises with the polynucleotide encoding said second toxin or a complement 0 000 S S S S S. 5 S S S 0 5 S S S *5 S 0 **S 440. The pesticidal composition according to claim 427, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 25 441. The pesticidal composition according to claim 427, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 442. The pesticidal composition according to claim 427, wherein a 40 to 50 kDa toxin- encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 443. The pesticidal composition according to claim 427, wherein SEQ ID NO. 42 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 444. The pesticidal composition according to claim 427, wherein SEQ ID NO. 45 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 445. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 2 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a ,complement thereof. C04229#2 99 446. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 4 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 447. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 6 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 448. The pesticidal composition according to claim 427, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 11 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 449. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 13 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 450. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 15 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 451. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 16 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 452. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID 20 NO. 17 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. S453. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 18 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a °complement thereof. 454. The pesticidal composition according to claim 427, wherein DNA which encodes SEQ ID NO. 19 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 455. The pesticidal composition according to claim 427, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 28 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 456. The pesticidal composition according to claim 427, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 38 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 457. The pesticidal composition according to claim 427, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 43 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 458. The pesticidal composition according to claim 427, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 459. The pesticidal composition according to claim 427, wherein the nucleotide sequence f 40 ding said second toxin is SEQ ID NO. 37. C04229#2 a a a a. a. a a a a a a a a I vTJ -'7 100 460. The pesticidal composition according to claim 427, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 42. 461. The pesticidal composition according to claim 427, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 462. The pesticidal composition according to claim 427, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 11 or a pesticidal portion thereof. 463. The pesticidal composition according to claim 427, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 38 or a pesticidal portion thereof. 464. The pesticidal composition according to claim 427, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 43 or a pesticidal portion thereof. 465. The pesticidal composition according to claim 427, wherein said second toxin comprises the consensus sequence shown in Figure 1. 466. The pesticidal composition according to any one of claims 427 to 465, wherein said second toxin has a molecular weight of approximately 40 to 50 kDa. 467. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin active against a non-mammalian pest, wherein a portion of the nucleotide sequence encoding said second toxin can be amplified by PCR using the primer pair selected from the following group: SEQ ID NOs. 20 and 24 to produce a fragment of about 495 bp; SEQ ID NOs. 20 and 20 to produce a fragment of about 594 bp; SEQ ID NOs. 21 and 24 to produce a fragment of about 471 bp; and SEQ ID NOs. 21 and 25 to produce a fragment of about 580 bp. 468. The pesticidal composition according to any one of claims 380 to 426, further encoding a second toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. 25 469. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non- mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. 470. The pesticidal composition according to claim 469, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 11 or a pesticidal portion thereof. 471. The pesticidal composition according to claim 469, wherein said second toxin is encoded by SEQ ID NO. 472. The pesticidal composition according to claim 469, wherein the nucleotide sequence encoding said second toxin comprises the sequence shown in SEQ ID NO. 35 473. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. 474. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non- .1' C04229#2 mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. 475. The pesticidal composition according to claim 474, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 43 or a pesticidal portion thereof. 476. The pesticidal composition according to claim 474, wherein said nucleotide sequence encoding said second toxin comprises the sequence shown in SEQ ID NO. 42. 477. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. 478. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin having a molecular weight of approximately 40 to 50 kDa active against a non- mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. 479. The pesticidal composition according to claim 478, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 38 or a pesticidal portion thereof. 480. The pesticidal composition, according to claim 478, wherein said nucleotide sequence encoding said second toxin comprises the sequence shown in SEQ ID NO. 37. 481. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin active against a non-mammalian pest having an amino acid sequence which has at 20 least about 75% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43. 482. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 80% identity with an amino acid sequence selected from the group consisting of: SEQ ID 25 NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43. 483. The pesticidal composition according to any one of claims 380 to 426, further comprising a second toxin active against a non-mammalian pest having an amino acid sequence which has at least about 90% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43. 484. A pesticidal composition comprising a first toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, and a second toxin having a molecular weight of approximately 40 to 50 kDa wherein the polynucleotide encoding said toxins comprises at least pesticidal-encoding portions of the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39. 485. A pesticidal composition comprising a biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain pesticidal activity, said culture comprising a toxin active against a non-mammalian pest T nd having a molecular weight of approximately 10 to 15 kDa. C04229#2 486. The pesticidal composition according to claim 485, wherein said Bacillus thuringiensis isolate is PS149B1 having the identifying characteristics of NRRL B-21553. 487. The pesticidal composition according to claim 485, wherein said Bacillus thuringiensis isolate is PS167H2 having the identifying characteristics of NRRL B-21554. 488. The pesticidal composition according to any one of claims 377 to 487, wherein said pest is an insect. 489. The pesticidal composition according to claim 488, wherein said insect is a coleopteran. 490. The pesticidal composition according to claim 488, wherein said insect is a lepidopteran. 491. The pesticidal composition according to any one of claims 377 to 487, wherein said pest is a mite. 492. The pesticidal composition according to any one of claims 377 to 487, wherein said pest is corn rootworm. 493. The pesticidal composition according to any one of claims 377 to 487, wherein said pest is western corn rootworm. 494. A pesticidal composition comprising a toxin having a molecular weight of approximately to 15 kDa active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the Examples. 495. A pesticidal composition comprising a first toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, and a second toxin having a 20 molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, substantially as hereinbefore described with reference to any one of the Examples. 496. A pesticidal composition comprising a biologically pure culture of a Bacillus thuringiensis isolate comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the S 25 *Examples. 497. A composition of matter for controlling coleopterans comprising a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL o B-21553, and PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain activity against coleopterans, in association with an agricultural carrier appropriate for use in controlling coleopterans. 498. A composition of matter for controlling coleopterans, substantially as hereinbefore described with reference to any one of the Examples. 499. A method for controlling a non-mammalian pest comprising applying to the situs or environment of said pest a pesticidally effective amount of an isolated or purified toxin according to any one of claims 116 to 168, a biologically pure culture according to any one of claims 169 to 172, a transformed cell according to any one of claims 248, 249 or 255 to 360, a transgenic plant according to any one of claims 251 to 253 or 361 to 369, or a pesticidal composition according to any one of claims 377 to 496. C04229#2 500. A method for controlling a coleopteran pest comprising applying to the situs or environment of said pest a pesticidally effective amount of a composition of matter according to claim 497 or claim 498. 501. A method for controlling a non-mammalian pest comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a Bacillus thuringiensis isolate, or a toxin of said Bacillus thuringiensis isolate, wherein said isolate is selected from the group consisting of PS149B1, having the identifying characteristics of NRRL B-21553; and PS167H2, having the identifying characteristics of NRRL B-21554; and mutants thereof which retain pesticidal activity. 502. The method according to claim 501, wherein said Bacillus thuringiensis isolate is PS149B1 having the identifying characteristics of NRRL B-21553. 503. The method according to claim 501, wherein said Bacillus thuringiensis isolate is PS167H2 having the identifying characteristics of NRRL B-21554. 504. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a purified or isolated toxin active against non-mammalian pests, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID NO. 7, DNA which encodes SEQ ID NO. 32, DNA which encodes SEQ ID NO. 36, and DNA which encodes SEQ ID NO. 41, hybridises under stringent conditions with the nucleotide C OO° 20 sequence encoding said toxin, or a complement thereof. 505. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a purified or isolated toxin active against non-mammalian pests, wherein a nucleotide sequence selected from the group consisting of SEQ ID NO. 29, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 30, SEQ ID NO. 25 31, a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34, SEQ ID NO. 35, a 10 to 15 kDa toxin- encoding portion of SEQ ID NO. 39, SEQ ID NO. 40, and SEQ ID NO. 44, hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 506. The method according to claim 504 or claim 505, wherein DNA which encodes SEQ ID 00°.o NO. 3 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 507. The method according to claim 504 or claim 505, wherein DNA which encodes SEQ ID NO. 5 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 508. The method according to claim 504 or claim 505, wherein DNA which encodes SEQ ID NO. 7 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 509. The method according to claim 504 or claim 505, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 32 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. C04229#2 510. The method according to claim 504 or claim 505, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 36 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 511. The method according to claim 504 or claim 505, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 41 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 512. The method according to claim 504 or claim 505, wherein SEQ ID NO. 29 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 513. The method according to claim 504 or claim 505, wherein a 10 to 15 kDa toxin-encoding l0 portion of SEQ ID NO. 30 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 514. The method according to claim 504 or claim 505, wherein SEQ ID NO. 31 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 515. The method according to claim 504 or claim 505, wherein SEQ ID NO. 33 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 516. The method according to claim 504 or claim 505, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 517. The method according to claim 504 or claim 505, wherein SEQ ID NO. 35 hybridises 20 under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 518. The method according to claim 504 or claim 505, wherein a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 519. The method according to claim 504 or claim 505, wherein SEQ ID NO. 40 hybridises 25 under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. 520. The method according to claim 504 or claim 505, wherein SEQ ID NO. 44 hybridises under stringent conditions with the nucleotide sequence encoding said toxin or a complement thereof. *c 521. The method according to claim 504 or claim 505, wherein said encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 522. The method according to claim 504 or claim 505, wherein said encoding the toxin is SEQ ID NO. 31. 523. The method according to claim 504 or claim 505, wherein said encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 34. 524. The method according to claim 504 or claim 505, wherein said encoding the toxin is SEQ ID NO. 525. The method according to claim 504 or claim 505, wherein said encoding the toxin is a 10 to 15 kDa toxin-encoding portion of SEQ ID NO. 39. 526. The method according to claim 504 or claim 505, wherein said .encoding the toxin is SEQ ID NO. nucleotide sequence nucleotide sequence nucleotide sequence nucleotide sequence nucleotide sequence nucleotide sequence C04229#2 527. The method according to claim 504 or claim 505, wherein said nucleotide sequence encoding the toxin is SEQ ID NO. 44. 528. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 36, and SEQ ID NO. 41, or pesticidal portions thereof. 529. The method according to claim 528, wherein said amino acid sequence comprises SEQ ID NO. 32 or a pesticidal portion thereof. 530. The method according to claim 528, wherein said amino acid sequence comprises SEQ ID NO. 36 or a pesticidal portion thereof. 531. The method according to claim 528, wherein said amino acid sequence comprises SEQ ID NO. 41 or a pesticidal portion thereof. 532. The method according to any one of claims 499 to 531, wherein the toxin has a molecular weight of approximately 10 to 15 kDa. *533. A method for controlling a non-mammalian pest which comprises applying to the situs or *0 0 environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin is encoded by a nucleotide sequence, V.0. a portion of which can be amplified by PCR using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 20 33. "00 534. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to kDa from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B- oO 25 18679. 535. The method according to claim 534, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 32 or a pesticidal portion thereof. 536. The method according to claim 534, wherein said toxin is encoded by the nucleotide sequence shown in SEQ ID NO. 31. 537. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. 538. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to kDa from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B- C04229#2 539. The method according to claim 538, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 41 or a pesticidal portion thereof. 540. The method according to claim 538, wherein said toxin is encoded by the nucleotide sequence shown in SEQ ID NO. 541. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. 542. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to kDa from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B- 21554. 543. The method according to claim 542, wherein said toxin comprises the amino acid sequence shown in SEQ ID NO. 36 or a pesticidal portion thereof. sequence shown in SEQ ID NO. 545. A method for controlling a non-mammalian pest which comprises applying to the situs or 20 environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin immunoreacts with an antibody to an approximately 10 to 15 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. 546. A method for controlling a non-mammalian pest which comprises applying to the situs or 25 environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest, wherein said toxin comprises an amino acid sequence :which has at least about 75% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. 547. The method according to claim 546 which has at least about 80% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. 548. The method according to claim 546 which has at least about 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, a pesticidal portion of SEQ ID NO. 32, a pesticidal portion of SEQ ID NO. 36, and a pesticidal portion of sequence ID NO. 41. 549. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified ST 0 toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to C04229#2 107 kDa, wherein said toxin comprises a pesticidal portion of an amino acid sequence encoded by the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39. 550. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of an isolated or purified toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to kDa, substantially as hereinbefore described. 551. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin active against a non-mammalian pest, wherein a nucleotide sequence selected from the group consisting of: DNA which encodes SEQ ID NO. 2; DNA which encodes SEQ ID NO. 4; DNA which encodes SEQ ID NO. 6; SEQ ID NO. 8; SEQ ID NO. 10; DNA which encodes a pesticidal portion of SEQ ID NO. 11; SEQ ID NO. 12; DNA which encodes SEQ ID NO. 13; SEQ ID NO. 14; DNA which encodes SEQ ID NO. 15; DNA which encodes SEQ ID NO. 16; DNA which encodes SEQ ID NO. 17; DNA which encodes SEQ ID NO. 18; DNA which encodes SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ ID NO. 24; SEQ ID NO. SEQ ID NO. 26; SEQ ID NO. 27; DNA which encodes a pesticidal portion of SEQ ID NO. 28; a 40 to kDa toxin-encoding portion of SEQ ID NO. 30; a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 34; SEQ ID NO. 37; DNA which encodes a pesticidal portion of SEQ ID NO. 38; a 40 to 50 kDa 20 toxin-encoding portion of SEQ ID NO. 39; SEQ ID NO. 40; SEQ ID NO. 42; DNA which encodes a pesticidal portion of SEQ ID NO. 43; and SEQ ID NO. 45; hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 552. The method according to claim 551, wherein SEQ ID NO. 8 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 25 553. The method according to claim 551, wherein SEQ ID NO. 10 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 554. The method according to claim 551, wherein SEQ ID NO. 12 hybridises under stringent ;conditions with the polynucleotide encoding said second toxin or a complement thereof. 555. The method according to claim 551, wherein SEQ ID NO. 14 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 556. The method according to claim 551, wherein SEQ ID NO. 20 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 557. The method according to claim 551, wherein SEQ ID NO. 21 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 558. The method according to claim 551, wherein SEQ ID NO. 22 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 559. The method according to claim 551, wherein SEQ ID NO. 23 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 560. The method according to claim 551, wherein SEQ ID NO. 24 hybridises under stringent 4 onditions with the polynucleotide encoding said second toxin or a complement thereof. C04229#2 561. The method according to claim 551, wherein SEQ ID NO. 25 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 562. The method according to claim 551, wherein SEQ ID NO. 26 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 563. The method according to claim 551, wherein SEQ ID NO. 27 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 564. The method according to claim 551, wherein a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 30 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 565. The method according to claim 551, wherein a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 34 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 566. The method according to claim 551, wherein a 40 to 50 kDa toxin-encoding portion of SEQ ID NO. 39 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 567. The method according to claim 551, wherein SEQ ID NO. 42 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 568. The method according to claim 551, wherein SEQ ID NO. 45 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 20 569. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 2 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 570. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 4 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a 25 complement thereof. 571. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 6 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 572. The method according to claim 551, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 11 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 573. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 13 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 574. The method according to claim 551, wherein DNA which encodes SEQ ID NO. hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 575. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 16 .,ST hybridises under stringent conditions with the polynucleotide encoding said second toxin or a l -(complement thereof. C04229#2 109 576. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 17 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 577. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 18 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 578. The method according to claim 551, wherein DNA which encodes SEQ ID NO. 19 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 579. The method according to claim 551, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 28 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 580. The method according to claim 551, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 38 hybridises under stringent conditions with the polynucleotide encoding said second toxin or a complement thereof. 581. The method according to claim 551, wherein DNA which encodes a pesticidal portion of SEQ ID NO. 43 hybridises under stringent conditions with the polynucleotide encoding said second I toxin or a complement thereof. 582. The method according to claim 551, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 583. The method according to claim 551, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 37. 584. The method according to claim 551, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 42. i 25 585. The method according to claim 551, wherein the nucleotide sequence encoding said second toxin is SEQ ID NO. 586. The method according to claim 551, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 11 or a pesticidal portion thereof. 0.. 587. The method according to claim 551, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 38 or a pesticidal portion thereof. 588. The method according to claim 551, wherein said second toxin comprises the amino acid sequence set forth in SEQ ID NO. 43 or a pesticidal portion thereof. 589. The method according to claim 551, wherein said second toxin comprises the consensus sequence shown in Figure 1. 590. The method according to any one of claims 551 to 589, wherein said second toxin has a molecular weight of approximately 40 to 50 kDa. 591. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin active against a non-mammalian pest, wherein a portion of the nucleotide C04229#2 110 sequence encoding said second toxin can be amplified by PCR using the primer pair selected from the following group: SEQ ID NOs. 20 and 24 to produce a fragment of about 495 bp; SEQ ID NOs. 20 and to produce a fragment of about 594 bp; SEQ ID NOs. 21 and 24 to produce a fragment of about 471 bp; and SEQ ID NOs. 21 and 25 to produce a fragment of about 580 bp. 592. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. 593. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS80JJ1 having the identifying characteristics of NRRL B-18679. 594. The method according to claim 593, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 11 or a pesticidal portion thereof. 595. The method according to claim 593, wherein the nucleotide sequence encoding said S second toxin comprises the sequence shown in SEQ ID NO. 596. The method according to claim 593, wherein the nucleotide sequence encoding said 20 second toxin comprises the sequence shown in SEQ ID NO. 597. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. 25 598. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second .isolated or purified toxin having a molecular weight of approximately 40 to 50 kDa active against a non-mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS149B1 having the identifying characteristics of NRRL B-21553. 599. The method according to claim 598, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 43 or a pesticidal portion thereof. 600. The method according to claim 598, wherein the nucleotide sequence encoding said second toxin comprises the sequence shown in SEQ ID NO. 42. 601. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin which immunoreacts with an antibody to a 40 to 50 kDa toxin from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. 602. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second 'i isolated or purified toxin having a molecular weight of approximately 40 to 50 kDa active against a C04229#2 non-mammalian pest, wherein the nucleotide sequence encoding said second toxin is from Bacillus thuringiensis isolate PS167H2 having the identifying characteristics of NRRL B-21554. 603. The method according to claim 602, wherein said second toxin comprises the amino acid sequence shown in SEQ ID NO. 38 or a pesticidal portion thereof. 604. The method according to claim 602, wherein said nucleotide sequence encoding said second toxin comprises the sequence shown in SEQ ID NO. 37. 605. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin active against a non-mammalian pest having an amino acid sequence which io has at least about 75% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43. 606. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin active against a non-mammalian pest having an amino acid sequence which has at least about 80% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43. oo s 607. The method according to any one of claims 504 to 550, also comprising applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a second isolated or purified toxin active against a non-mammalian pest having an amino acid sequence which S 20 has at least about 90% identity with an amino acid sequence selected from the group consisting of: .i SEQ ID NO. 11; SEQ ID NO. 13; SEQ ID NO. 15; SEQ ID NO. 38; and SEQ ID NO. 43. 608. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a first isolated or purified toxin having a molecular weight of approximately 10 to 15 kDa active against a non-mammalian pest, 25 and a second isolated or purified toxin having a molecular weight of approximately 40 to 50 kDa =wherein the polynucleotide encoding said toxins comprises at least pesticidal-encoding portions of the nucleotide sequence selected from the group comprising SEQ ID NO. 30, SEQ ID NO. 34, and SEQ ID NO. 39. 609. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a first isolated or purified toxin having a molecular weight of between about 10 kDa and 15 kDa active against a non- mammalian pest, and a second isolated or purified toxin having a molecular weight of between about kDa and 50 kDa active against a non-mammalian pest, substantially as hereinbefore described. 610. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a biologically pure culture of a Bacillus thuringiensis isolate selected from the group consisting of PS149B1 having the identifying characteristics of NRRL B-21553; and PS167H2 having the identifying characteristics of NRRL B-21554, and mutants thereof which retain pesticidal activity, said culture comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa. /1 C04229#2 112 611. The method according to claim 610, wherein said Bacillus thuringiensis isolate is PS149B1 having the identifying characteristics of NRRL B-21553. 612. The method according to claim 610, wherein said Bacillus thuringiensis isolate is PS167H2 having the identifying characteristics of NRRL B-21554. 613. A method for controlling a non-mammalian pest which comprises applying to the situs or environment of said pest, or contacting said pest with a pesticidal amount of a Bacillus thuringiensis isolate comprising a toxin active against a non-mammalian pest and having a molecular weight of approximately 10 to 15 kDa, substantially as hereinbefore described with reference to any one of the Examples. 614. The method according to any one of claims 499 to 613, wherein said pest is an insect. 615. The method according to claim 614, wherein said insect is a coleopteran. 616. The method according to claim 614, wherein said insect is a lepidopteran. 617. The method according to any one of claims 499 to 613, wherein said pest is a mite. 618. The method according to any one of claims 499 to 613, wherein said pest is corn rootworm. 619. The method according to any one of claims 499 to 613, wherein said pest is western corn rootworm. *620. A method for controlling a non-mammalian pest, comprising contacting said pest with a S* virus according to claim 223, wherein said virus is virulent towards said pest. 20 Dated 14 March, 2001 Mycogen Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0oo* C04229#2