AU632335B2 - Novel bacillus thuringiensis isolates active against lepidopteran pests, and genes encoding lepidopteran-active toxins - Google Patents

Description

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632335 S F Ref: 132614 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class 0c *0 e- o 0 0 0 00a 000 00 Complete Specification Lodged: Accepted: Published: f:c-,o 83 Ly iuperand i3 on and is ccrr~ct for p: :i2 Priority: Related Art: 'i Name and Address of Applicant: CMM-WW iWTV. mimm 0 Mycogen Corporation 5451 Oberlin Drive San Diego California 92121 UNITED STATES OF AMERICA 0 0 0 r 0 00 4. 0 Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Novel Bacillus thuringiensis Isolates Active Against Lepidopteran Pests, and Genes Encoding Lepidopteran-Active Toxins The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 j

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111 4-1, 1.-1 MA39.C1 Abstract of the Disclosure Novel Bacillus thurindiensis genes encoding toxins which are active against lepidopteran insects have been cloned from novel lepidopteran-active B.

thurindiensis microbes. The DNA encoding the B. thurinmprsis toxins can be used to transform various prokaryotic and eukaryotic microbes to express the B.

thurinaiensis toxins. These recombinant microbes can be used to control lepidopteran. insects in various environents.

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DESCRIPTION

NOVEL BACILLUS THURINGIENSIS ISOLATES ACTIVE AGAINST LEPIDOPTERAN PESTS, AND GENES ENCODING NOVEL LEPIDOPTERAN-ACTIVE TOXINS o o a o o Background of the Invention *The most widely used microbial pesticides are derived from the bacterium Bacillus thurindensis. This bacterial agent is used to control a wide range of leaf-eating caterpillars and beetles, as well as mosquitos. Bacillus thuringiensis produces a proteinaceous parasporal body or crystal which is toxic upon ingestion by a susceptible insect host. For example, B. thuringiensis subsp. kurstaki HD- 1 produces a crystal inclusion consisting of a biotoxin called a delta toxin which is toxic to the larvae of a number of lepidopteran insects. The cloning, sequencing, and expression of this B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H.E. and Whitely, H.R.

[1981] Proc. Natl. Acad. Sci. USA 78:2893-2897; Schnepf et 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.

Brief Summary of the Invention The subject invention concerns novel Bacillus thuringiensis isolates designated B.t. PS81A2 and PS81RR1 which have activity against all lepidopteran pests tested.

-4 2 Also disclosed and claimed are novel toxin genes which express toxins toxic to lepidopteran insects. These toxin genes can be transferred to suitable hosts via a plasmid vector.

Specifically, the invention comprises novel B.t. isolates denoted B.t. PS81A2 and PS81RR1, mutants thereof, and novel delta endotoxin genes derived from these B.t.

isolates which encode proteins which are active against lepidopteran pests. More specifically, the gene in B.t. PS81A2 encodes a 133,601 dalton endoxin, whereas the gene in B.t. PS81RR1 encodes a 133,367 dalton endotoxin.

Table 1 discloses the DNA encoding the novel toxin expressed by PS81A2. Table 2 discloses the amino acid sequence of the novel toxin expressed by PS81A2. Table 3 is a composite of Tables 1 and 2. Table 4 discloses the DNA encoding the novel toxin expressed by PS81RR1. Table 5 discloses the amino acid sequence of the novel toxin expressed by PS81RR1. Table 6 is a composite of Tables 4 and According to a first embodiment of this invention, there is provided a process for controlling lepidopteran insect pests which comprises contacting said insect pests with an insect-controlling effective amount of Bacillus thuringiensis PS81A2, having the identifying characteristics of NRRL B-18457, or Bacillus thuringiensis PS81RR1, having the identifying characteristics of NRRL B-18458, or mutants thereof which retain the characteristics of the parent strains.

According to a second embodiment of this invention, there is provided a process for controlling soil-inhabiting insect pests of the order Lepidopteran which comprises preparing a bait granule comprising Bacillus thuringiensis PS81A2 or PS81RR1, or mutants thereof which retain the characteristics of the parent i strains, or spores or crystals of Bacillus thuringiensis PS81A2 or PS81RR1; and placing said bait granule on or in the soil.

According to a third embodiment of this invention, there is provided a composition of matter comprising Bacillus thuringiensis PS81A2 or PS81RR1, or mutants thereof :which retain the characteristics of the parent strains, or spores or crystals of Bacillus thuringiensis PS81A2 or PS81RR1 in association with an insecticide carrier, wherein said mutants are asporogenous mutants and/or phage resistant mutants.

According to a fourth embodiment of this invention, there is provided a composition of matter comprising Bacillus thuringiensis PS81A2 or PS81RRi, or mutants thereof which retain the characteristics of the parent strains, in association with formulation ingredients applied as a seed coating, wherein said mutants are asporogenous mutants and/or phage resistant mutants.

According to a fifth embodiment of this invention, there is provided Bacillus thuringiensis PS81A2, having the identifying characteristics of NRRL B-18457, or 132814rt 2A mutants thereof which retain the characteristics of the parent strain, having activity against insect pests of the order Lepidoptera.

According to a sixth embodiment of this invention, there is provided Bacillus thuringiensis PS81RR1, having the identifying characteristics of NRRL B-18458, or mutants thereof which retain the characteristics of the parent strain, having activity against insect pests of the order Lepidoptera.

According to a seventh embodiment of this invention, there is provided asporogenous and/or phage resistant mutants of Bacillus thuringiensis PS81A2 or Bacillus thuringiensis PS81RR1 which retain the characteristics of the parent strains.

According to an eighth embodiment of this invention, there is provided DNA encoding a Bacillus thuringiensis toxin having the amino acid sequences shown in Table 2 or Table I According to a ninth embodiment of this invention, there is provided toxin active I against lepidopteran insects having the amino acid sequence shown in Table 2 or Table iI 15 and mutants thereof which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained to some degree.

According to a tenth embodiment of this invention, there is provided a recombinant DNA transfer vector comprising DNA having all or part of the nucleotide sequence which codes for the amino acid sequence shown in Table 2 or Table According to an eleventh embodiment of this invention, there is provided a bacterial host transformed to express a Bacillus thuringiensis toxin having the amino acid sequence shown in Table 2 or Table According to a twelfth embodiment of this invention, there is provided a method for controlling lepidopteran insects which comprises administering to said insects or to the environment of said insects a microorganism transformed to express a Bacillus thuringiensis toxin having the amino acid sequence shown in Table 2 or Table 5, wherein said microorganism is a species of Pseudomonas, Azotobacter, Erwinia, Serratia, Klebsiella, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Alcaligenes, Bacillus, or Streptomyces.

According to a thirteenth embodiment of this invention, there is provided an insecticidal composition comprising insecticide containing substantially intact, treated cells having prolonged pesticidal activity when applied to the environment of a target pest, wherein said insecticide is a polypeptide toxic to lepidopteran insects, is intracellular, and is produced as a result of expression of a transformed microbe capable of expressing the Bacillus thuringiensis toxin having the amino acid sequence shown in Table 2 or Table According to a fourteenth embodiment of this invention, there is provided treated, substantially intact unicellular microorganism cells containing an intracellular toxin, which toxin is a result of expression of a Bacillus thuringiensis toxin gene toxic to lepidopteran insects which codes for a polypeptide toxin having the amino acid sequence shown in A 132B14rt I I I r Table 2 or Table 5, wherein said cells are treated under conditions which prolong the insecticidal activity when said cells are applied to the environment of a target insect.

Brief Description of the Drawings Figure 1 shows agarose gel electrophoresis of plasmid preparations from B.t.

PS81A2, B.t. PS81RR1, and B.t. HD-1.

Detailed Disclosure of the Invention The novel toxin genes of the subject invention were obtained from novel lepidopteran-active B. thuringiensis isolates designated PS81A2 and PS81RR1.

Characteristics of B.t. PS81A2 and PS81RR1 Colony morphology Large colony, dull surface, typical B.t.

Vegetative cell morphology typical B.t.

Flagellar serotype 7, aizawai.

Intracellular inclusions sporulating cells produce a bipyramidal crystal.

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,i I 3 MA39.C1 Plasmid preparations agarose gel electrophoresis of plasmid preparations distinguishes B.t. PS81A2 and PS81RR1 from B.t. HD-1 and other B.t. isolates. See Figure 1.

Alkali-soluble proteins B.t. PS81A2 and PS81RR1 produce 133,601 and 133,367 dalton proteins, respectively.

Unique toxins the 133,601 amd 133,367 dalton toxins are different from any previously identified.

Activity B.t. PS81A2 and PS81RR1 both kill all Lepidoptera tested (Trichoplusia i, Spodoptera exiua, and Plutella xvlostella).

9" 10 Bioassay procedures: 'Spodoptera exigua--dilutions are prepared of a spore and crystal pellet, mixed with USDA Insect Diet (Technical Bulletin 1528, U.S. Department of Agriculture) and poured into small plastic trays. Neonate Spodoptera exigua larvae are placed on the diet mixture and held at 25 C. Mortality is ,,recorded after six days.

Other insects dilutions and diet are prepared in the same manner as for the Spodoptera exigua bioassay. Fourth instar larvae are used, and mortality is recorded after' eight days.

B. thuringiensis PS81A2, NRRL B-18457, and B. thuriniensis PS81RR1, NRRL B-18458, and mutants thereof, can be cultured using standard known media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t.

spores and crystals can be formulated into a wettable powder, a liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers and other components to facilitate handling and

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I 4 MA39.C1 application for particular target pests. The formulation and application procedures are all well known in the art and are used with commercial strains of B. thuringiensis (HD-1) active against Lepidoptera, caterpillars. B.t.

PS81A2 and B.t. PS81RR1, and mutants thereof, can be used to control lepidopteran pests.

A subculture of B.t. PS81A2 and PS81RR1 and the E. coli hosts harboring the toxin genes of the invention, were deposited in the permanent collection of the Northern Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA. The accession numbers and deposit dates are as follows: .e *Subculture Accession Number Deposit Date 07 PS81A2 NRRL B-18457 MarchA, 1989 S* B.t. PS81RR1 NRRL B-18458 Marcht14, 1989 E coli(NM522)(pMYC389) NRRL B-18448 February 24, 1989 E. coli (NM522)(pMYC390) NRRL B-18449 February 24, 1989 The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 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 deposi 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

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them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 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 deposits 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 be irrevocably removed upon the granting of a.patent disclosing them.

The toxin genes of the subject invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable hosts, Pseudomonas, the microbes can be applied to the situs of lepidopteran insects where they will proliferate and be ingested by the insects.

The result is a control of the unwanted insects. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s). The resulting product retains the toxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy ti.l "phytcsphere" (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 i MA39.C1 &oft*# 0 04 o 44 *00 0 40 4444 IIt 0 4 a 4 4t 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 Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomvces Rhizobium, Rhodopseudomonas Methylophilius, Agrobacterium. Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, genera Saccharomvces, Cryptococcus. Kluyveromvces, Sporobolomvces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphdre bacterial species as Pseudomonas svringae. Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris. Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. alutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C.

laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomvces roseus, S. odorus, Kluvveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing a B.t. gene expressing a toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the toxin gene, the toxin gene under their regulatory control apd a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

~1 7 MA39.C1 The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the toxin, where expression of the toxin will only occur after release into the environment. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a toxin, but upon release into the environment, expression would 010 begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium .eoo in the environment would allow for expression of the toxin. For translational o* "initiation, a ribosomal binding site and an initiation codon will be present.

Various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, as well as by D. o employing sequences, which enhance the stability of the messenger RNA. The transcriptional and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. A hydrophobic .o "leader" sequence may be employed at the amino terminus of the translated polypeptide sequence in order to promote secretion of the protein across the at inner membrane.

In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either or 3' of the promoter, the ribosomal binding site, the nitiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for MA39.C1 0 a r 0000 0 *9 0* I I

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transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selection of those hosts which have been modified or transformed. The marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as for modifying the host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment.

Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well a§ the gene providing for the competitive advantage. Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene :il r MA39.C1 MA39.C1 *o r..

o 10 44 o 4* a 44 4 4 providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.

A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Various transcriptional regulatory regions include the regions associated with the rp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for example, U.S. Patent Nos. 4,332,898, 4,342,832 and 4,356,270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host.

The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host. A large number of plasmids are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the like. See for example, Olson et al., (1982) J. Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, and U.S. Patent Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t. gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously.

i- I~ MA39.C1 Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for pesticidal activity.

Suitable host cells, where the pesticide-containing cells will be treated to prolong.the activity of the toxin in the cell when the then treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce Sr substances toxic to higher organisms could be used, where the toxin is unstable Sor the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zvmomonas, Serratia Aeromonas, Vibrio.

SC Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, and S Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and 4 C C Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomvces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomvces, and the like.

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 j 5845/3 11 MA39.C1 for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomvces sp., and Sporobolomvces sp.; phylloplane S organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., Streptomvces sp., and the like. Specific organisms include Pseudomonas aeruinosa, Pseudomonas fluorescens, Saccharomvces cerevisiae Bacillus thuriniensis, Escherichia coli, SVBacillus suatilis Streptomvces lividans and the like.

The cell will usually be intact and be substantially in the proliferative form 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 in protecting the toxin.

SExamples 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 include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti- 2 infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, and Helly's fixative (See: Humason, Gretchen Animal Tissue Techniques, W.H. Freeman and Company, 1967); or a combination of physical 12 MA39.C1 (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 animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like.

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 inactivation 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 10 processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin.

S. 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 may be formulated in a variety of ways. They 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.

The pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be 13 MA39.C1 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 104 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 lepidopteran pest(s), plants, soil or water, by spraying, dusting, sprinkling, or the like.

Mutants of PS81A2 and PS81RR1 can be made by procedures well known J 10 in the art. For example, an asporogenous mutant can be obtained through t ethylmethane sulfonate (EMS) mutagenesis of PS81A2 and PS81RR1. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures Swell 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 S-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 fixation process that will yield a protected, encapsulated toxin protein.

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 C. The plates 25 are incuba 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 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 miu Lyi miu stan ca eietfe ysreigaprgnu i: i:; mutats n shke laskmedi an selctig thse utans tht ae stll ntac an 132614rt i% 14 MA39.C1 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 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 plated 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"C for 24 hours.

Following are examples which illustrate procedures, including the best o ,mode, 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 B.t. PS81A2 and PS81RR1 A subculture of B.t. PS81A2 and PS81RR1, 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/1

SKH

2 P0 4 3.4 g/1

K

2

HPO

4 4.35 g/1 Salt Solution 5.0 ml/1 CaC1 2 Solution 5.0 ml/I MA39.C1 Salts Solution (100 ml) MgSO 4 .7HzO 2.46 g MnSO 4

.H

2 0 0.04 g ZnSO 4 .7H 2 0 0.28 g FeSO 4 .7H 2 0 0.40 g CaC12 Solution (100 ml) CaC1 2 .2H 2 0 3.66 g pH 7.2 The salts solution and CaCI 2 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, e.g., centrifugation.

Example 2 Cloning of Novel Toxin Gene From Isolate PS81A2 and Transformation into Escherichia coli Total cellular DNA was prepared from B.t. cells grown to a low optical density (ODo 00 The cells were recovered by centrifugation and protoplasted in TES buffer (30 mM Tris-C1, 10 mM ethylenediaminetetraacetic acid [EDTA], 50 mM NaC1, pH 8.0) containing 20% sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by addition of sodium dodecyl sulfate (SDS) to a final concentration of The cellular material was precipitated 16 MA39.C1 overnight at 40C in 100 mM (final concentration) neutral potassium chloride.

The supernate was extracted twice with phenol/chloroform The DNA was precipitated with ethanol and purified by isopycnic banding on a cesium gradient.

Total cellular DNA from PS81A2 and B.t.k. HD-1 was digested with EcoRI and separated by electrophoresis on a 0.8% Agarose-TAE-buffered gel.

A Southern blot of the gel was probed with the NsiI to Nsil fragment of the toxin gene contained in plasmid pM3,130-7 of NRRL B-18332 and the NsiI to KpnI fragment of the "4.5 Kb class" toxin gene (Kronstad and Whitely [1986] Gene USA 43:29-40). These two fragments were combined and used as the 10 probe. Results show that hybridizing fragments of PS81A2 are distinct from those of HD-1. Specifically, a 3.0 Kb hybridizing band in PS81A2 was detected o: instead of the 3.8 Kb and 1.8 Kb hybridizing bands seen in HD-1.

S° Two hundred micrograms of PS81A2 total cellular DNA was digested with EcoRI and separated by electrophoresis on a preparative 0.8% Agarose-TAE gel.

The 2.5 Kb to 3.5 Kb region of the gel was cut out and the DNA from it was electroeluted and concentrated using an ELUTIPm-d (Schleicher and Schuell, Keene, NH) ion exchange column. The isolated EcoRI fragments were ligated to LAMBDA ZAP m EcoRI arms (Stratagene Cloning Systems, La Jolla, CA) .o and packaged using Gigapak GOLDm (Stratagene) extracts. The packaged recombinant phage were plated with E. coli strain BB4 (Stratagene) to give high plaque density. The plaques were screened by standard nucleic acid hybridization 0 procedure with radiolabeled robe. The plaques that hybridized were purified and re-screened at a lower plaque density. The resulting purified phage were grown with R408 M13 helper phage (Stratagene) and the recombinant BlueScripts (Stratagene) plasmid was automatically excised and p4ckaged. The "phagemid" was re-infected in XL1-Blue E. coli cells (Stratagene) as part of the automatic excision process. The infected XLl-Blue cells were screened for ampicillin resistance and the resulting colonies were analyzed by standard 17 MA39.C1 miniprep procedure to find the desired plasmid. The plasmid, designated pM6,31-1, contains an approximate 3.0 Kb EcoRI insert and was sequenced using Stratagene's T7 and T3 primers plus a set of existing B.t. endotoxin gene oligonucleotide primers. About 1.8 Kb of the toxin gene was sequenced, and data analysis comparing PS81A2 to other cloned B.t. endotoxin genes showed that the PS81A2 sequence was unique. A synthetic nligonucleotide (CAGATCCACGAGGCTrATCTCCAGAACTAC) was consti-cted to one of the regions in the PS81A2 sequence that was least homologous relative to other exiting B.t. endotoxin genes.

10 PS81A2 total cellular DNA partially digested with Sau3A and fractionated by electrophoresis into a mixture of 9-23 Kb fragments on a 0.6% agarose-TAE t Io gel was ligated into Lambda DASH T M (Stratagene). The packaged phage at a Shigh titer were plated on P2392 E. coli cells (Stratagene) and screened using the radiolabeled synthetic oligonucleotide (aforementioned) as a nucleic acid hybridization probe. Hybridizing plaques were rescreened at a lower plaque density. A single purified hybridizing plaque was used to infect P2392 E. coli cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures. Preparative amounts of recombinant phage DNA were digested with SalI (to release the inserted DNA from lambda arms) and separated by electrophoresis on a 0.6% Agarose-TAE gel. The large Sfragments, electroeluted and concentrated as described above, were ligated to Sail-digested and dephosphorylated pUC19 (NEB). The ligation mixture was introduced by transformation into E. coli DH5(alpha) competent cells (BRL) and plated on LB agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)rD-galactoside (XGAL). White colonies (with insertions in the (Beta)-galactosidase gene of pUC19) were subjected to standard miniprep procedures to isolate the plasmid, designated pM4,122-3. The full length toxin gene was sequenced by using oligonucleotide 18 MA39.C1 primers made to the "4.5 Kb class" toxin gene and by "walking" with primers made to the sequence of PS81A2.

The plasmid pM4,122-3 contains about 15 Kb of PS81A2 DNA including the 3.522 Kb which encodes the 133,601 dalton endotoxin. The ORF of the PS81A2 toxin gene was isolated from pM4,122-3 and subcloned into the Bacillus shuttle vector pBClac as a 5.5 Kb blunt-ended DraII fragment. E. cob NM522 cells were transformed and plated on LB agar supplemented with ampicillin.

The resulting colonies were analyzed by standard miniprep procedures to isolate plasmids that contained the insert. The desired plasmid, pMYC389, contains the 10 coding sequence of the PS81A2 toxin gene.

Sf* Example 3 Cloning of Novel Toxin Gene From Isolate PS81RR1 and 0 Transformation into Escherichia coli Total cellular DNA was prepared from B.t. cells grown to a low optical density (OD 600 The cells were recovered by centrifugation and protoplasted in TES buffer (30 mM Tris-C1, 10 mM ethylenediaminetetraacetic acid [EDTA], 50 mM NaCI, pH 8.0) containing 20% sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by addition of sodium dodecyl sulfate (SDS) to a final concentration of The cellular material was precipitated overnight at 40C in 100 mM (final concentration) neutral potassium chloride.

The supernate was extracted twice with phenol/chloroform The DNA was precipitated with ethanol and purified by isopycnic banding on a cesium chloride gradient.

Total cellular DNA from PS81RR1 and B.t.k. HD-1 was digested with EcoRI and separated by electrophoresis on a 0.8% Agarose-TAE-buffered gel.

i A Southern blot of the gel was probed with the NsiI to NsiI fragment of the toxin gene contained in plasmid pM3,130-7 of NRRL B-18332 and the Nsil to KInI fragment of the "4.5 Kb class" toxin gene (Kronstad and Whitely [1986] I2 MA39.C1 10 0~ 0#0

I

Gene USA 43:29-40). These two fragments were combined and used as the probe. Results show that hybridizing fragments of PS81RR1 are distinct from those of HD-1. Specifically, a 2.3 Kb hybridizing band in PS81RR1 was detected instead of the 3.8 Kb and 1.8 Kb hybridizing bands seen in HD-1.

Two hundred micrograms of PS81RR1 total cellular DNA was digested with EcoRI and separated by electrophoresis on a preparative 0.8% Agarose- TAE gel. The 2.2 Kb to 2.4 Kb region of the gel was cut out and the DNA from it was electroeluted and concentrated using an ELUTIPT-d (Schleicher and Schuell, Keene, NH) ion exchange column. The isolated EcoRI fragments were ligated to LAMBDA ZAP T EcoRI arms (Stratagene Cloning Systems, La Jolla, CA) and packaged using Gigapak GOLD" m (Stratagene) extracts. The packaged recombinant phage were plated with E. coli strain BB4 (Stratagene) to give high plaque density. The plaques were screened by standard nucleic acid hybridization procedure with radiolabeled probe. The plaques that hybridized were purified and re-screened at a lower plaque density. The resulting purified phage were grown with R408 M13 helper phage (Stratagene) and the recombinant BlueScript T (Stratageae) plasmid was automatically excised and packaged. The "phagemid" was re-infected in XL1-Blue E. coli cells (Stratagene) as part of the automatic excision process. The infected XL1-Blue cells were screened for ampicillin resistance and the resulting colonies were analyzed by standard miniprep procedure to find the desired plasmid. The plasmid, designated pM3,31-3, contains an approximate 2.3 Kb EcoRI insert and was sequenced using Stratagene's T7 and T3 primers plus a set of existing B.t. endotoxin oligonucleotide primers. About 600 bp of the toxin gene was sequenced, and data anaysis comparing PS81RR1 to other cloned B.t. endotoxin genes showed that the PS81RR1 sequence was unique. A synthetic oligonucleotide (CGTGGATATGGTGAATCITATG) was constructed to one of the regions in MA39.C1 the PS81RR1 sequence that was least homologous relative to other existing B.t.

endotoxin genes.

PS81RR1 total cellular DNA partially digested with Sau3A and fractionated by electrophoresis into a mixture of 9-23 Kb fragments on a 0.6% agarose-TAE gel was ligated into Lambda GEM lM-11 (PROMEGA). The packaged phage at a high titer were plated on P2392 E. coli cells (Stratagene) and screened using the radiolabeled synthetic oligonucleotide (aforementioned) as a nucleic acid hybridization probe. Hybridizing plaques were rescreened at a lower plaque density. A single purified hybridizing plaque was used to infect 10 P2392 E. coli cells in liquid culture for preparation of phage for DNA isolation.

DNA was isolated by standard procedures. Preparative amounts of recombinant phage DNA were digested with Sal, to release the inserted DNA from lambda arms, and separated by electrophoresis on a 0.6% Agarose-TAE gel. The large fragments, electroeluted and concentrated as described above, were ligated to SalI-digested and dephosphorylated pUC19 (NEB). The ligation mixture was introduced by transformation into E. coli DH5(alpha) competent cells (BRL) and plated on LB agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL). White colonies (with insertions in the (Beta)-galactosidase gene of pUC19) were subjected to standard miniprep procedures to isolate the plasmid, designated pM1,RR1-A. The full length toxin gene was sequenced by using oligonucleotide primers made to the "4.5 Kb class" toxin gene and by "walking" with primers made to the sequence of PS81RR1.

The plasmid pM1,RR1-A contains about 13 Kb of PS81RR1 DNA including the 3.540 Kb which encodes the 133,367 dalton endotoxin. The ORF of the PS81RR1 toxin gene was isolated from pM1,RR1-A on a 3.8 Kb NdeI fragment and ligated into the Bacillus shuttle vector pBClac. E. coli NM522 cells were transformed and the resulting colonies were analyzed by standard 34 3T 4 MA39.C1 JTable 4 (continued 21 MA39.C1 miniprep procedures to isolate plasmids that contained the correct insert. The desired plasmid, pMYC390, contains the coding sequence of the PS81RR1 toxin gene.

The above cloning procedures were conducted using standard procedures unless otherwise noted.

The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. Also, methods for the use of lambda bacteriophage as a cloning vehicle, the preparation of lambda DNA, in vitro packaging, and transfection of recombinant DNA, are well 10 known in .he art. These procedures are all described in Maniatis, Fritsch, S. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold S, Spring Harbor Laboratory, New York. Thus, it is within the skill of those in the 0o CI genetic engineering art to extract DNA from microbial cells, perform restriction enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, prepare plasmid DNA, electrophorese proteins, and. sequence DNA.

The restriction enzymes disclosed herein can be purchased from Bethesda Research Laboratories, Gaithersburg, MD, New England Biolabs, Beverly, MA, or Boehringer-Mannheim, Indianapolis, IN. The enzymes are used according to the instructions provided by the supplier.

Plasmid pMYC386 containing the B.t. toxin genes, can be removed from the transformed host microbes by use of standard well-known procedures. For example, E. coli NRRL B-18449 can be subjected to cleared lysate isopycnic density gradient prc,-edures, and the like, to recover pMYC386.

Example 4 Insertion of Toxin Genes Into Plants The novel genes coding for the novel insecticidal toxins, as disclosed herein, can be inserted into plant cells using the Ti plasmid from Agrobacter pEND4K (Klee, Yanofsky, M.F. and Nester, E.W. [1985] Bio/Technology 3:637-642). This plasmid can replicate both in plant cells and in bacteria and has multiple cloning sites for passenger genes. The toxin gene, for example, can be inserted into the BamHI site of pEND4K, propagated in E coli, and transformed into appropriate plant cells.

S 10 Example 5 Cloning of Novel B. thuriniensis Genes Into Baculoviruses 22 MA39.C1 tumefaiens. Plante e ees of the inven be ca n be cloned into baculoviruses such (Zambryski, Joos, Gentello, Leemans, Van Montague, M. and as Autorapha, J 1983] Cell 32:1033-1043). A particularolyhedrosis viruseful vctor in thPlasmids regard is pEND4K (Klee, Yanofsky, M.F. and Nester, E.W. [1985] Bio/Technology 3:637-642).constructed that contain the AcNPV genome cloned into e and in bacteria and has vector such as pUC8. The AcNPV genome is modified so that the coding region of the polyhedrin gene is removed and a unique cloning site for passenger genes. The toin gene, for example, can be insert placed into the BamHI site polyhedrin propagated inr. Examples of such vectransfors armed pGP-B6874, described by Pennock et al. (Pennock, Shoemaker, C. and Miller, L.K [1984] Mol. Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, Summers, M.D. and Fraser, M.J. [1983] Mol Cell. Biol.

3:2156-2165). The gene coding for the novel protein toxin of the invention can be modified with Ba linkers at appropriate plantregions both upstream andcells.

Exdownstream from the coding region and insis Genes Into th e passenger site of one •of the AcNPV vectors.

As disclosed previously, the nucleotide sequences encoding the novel B.t.

The noveloxin genes are sh o f the invention can be deduclned aino aculoviruses suchs as Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids can be are shown in Tables 2 and S constructed that contain the AcNPV genome cloned into a commercial cloni.g v e tor such as pUC8. The AcNPV genome is modified so that the amino acid sequence of a protein is ofe polyhterminedri by the nucleotide sequenced a unique clning site for a passengergene is placed directly behind the polyhedrin promoter. Examples of such vectors are I pGP-B6874, described by Pennock et al. (Pennock, Shoemaker, C. and J t Miller, L.K. [1984] Mol. Cell. Biol. 4:399-406), and pAC380, described by Smith I et al. (Smith, Summers, M.D. and Fraser, M.J. [1983] Mol Cell. Biol.

3:2156-2165). The gene coding for the novel protein toxin of the invention can I be modified with BamHI linkers at appropriate regons both upstream and downstream from the coding region and inserted into the passenger site of one S of the AcNPV vectors.

As disclosed previously, the nucleotide sequences encoding the novel B.t.

toxin genes are shown in Tables 1 and 4. The deduced amino acid sequences are shown in Tables 2 and It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA Because of the redundancy i MA39.C1 of the genetic code, more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the genetic code can be depicted as follows: Phenylalanine (Phe) TTK 0 8 *0 00 o 0 0 *4 0s 0 5 00 0 *s 0 Leucine (Leu) Isoleucine (Ile) Methionine (Met) Valine (Val) Serine (Ser) Proline (Pro) Threonine (Thr) Alanine (Ala) Tyrosine (Tyr) Termination signal

XTY

ATM

ATG

GTL

QRS

CCL

ACL

GCL

TAK

TAJ

Histidine (His) CAK Glutamine (Gln) CAJ Asparagine (Asn) AAK Lysine (Lys) AAJ Aspartic acid (Asp) GAK Glutamic acid (Glu) GAJ Cysteine (Cys) TGK Tryptophan (Trp) TGG Arginine (Arg) WGZ Glycine (Gly) GGL Key: Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of mRNA, having a 5'-end on the left and a 3'-end on the right. All DNA sequences given herein are those of the strand whose sequence correspond to the mRNA sequence, with thymine substituted for uracil. The letters stand for the purine or pyrimidine bases forming the deoxynucleotide sequence.

A adenine G guanine C cytosine T thymine X T or C if Y is A or G X C if Y is C or T Y A, G, C or T ifXi; C Y A or G if X is T F- 24 MA39.C1 W C or Aif Z is Aor G W- C if Z is C or T Z A, G, C or T if W is C Z AorGif W isA QR TC if S is A, G, C or T; alternatively QR AG if S is T or C J A or G K =T or C L A, T, C or G 10 M=A, C or T i 0* The above shows that the novel amino acid sequences of the B.t. toxins 4 can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein. Accordingly, the subject invention includes such equiva.nt nucleotide sequences. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T. and Kezdy, F.J. [1984] Science 2'.3:249-255). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained to some degree.

4 i" 4 1 1 1 MA39.Cl Table 1 20 30 1ATGGAGAATA ATATTGAAAA TCAATGCATA 61 GAGATATTAG GGATTGAAAG GTCAAATAGT 121 AGTCGTCTGC TCGTTTCCCG AATTCCACTA 40 CCTTACAATT GTTTAAATAA AACGTAGCAG CAGAAATCGG GGGGATTTTA TACTTGGCTT

TCCTGAAGTA

CTTGGGGCTT

GTTTGATGTA

181 ATATGGGGGG CTATAGGTCC TTCACAATGG GATATATTTT TAGAGCAAAT TGAGCTATTG 241 ATCGGCCAAA GAATAGAGGA ATTCGCTAGG AATCAGGCAA TTTCTAGATT ACAAGGGCTA 310

AGCAATCTTT

CCAGCATTAA

GCTATTCCTC

GCTGCAAATT

TTTGATGTAG

320 330 340 ACCGAATTTA CACAAATGCT TTTAAAAACT GAGAAGAGAT GCGTATTCAA TTTAATGACA TTTTTTCAGT TCAAGGTTAT GAAATTCCTC TACATTTATC GGTTTTGAGA GATGTTTCAG CAACAATCAA TAGTCGTTAT AATGATTTAA 350 360 GGGAAGTAGA TCCTAC7AAT TGAACAGTGC TCTTACAACA TTTTATCAGT ATATGTTCAA TGTTTGGACA ACGTTGGGGA CTAGGCTTAT TGGCGAATAT 610 620 ACTGATTATG CTGTACGTTG GTACGAGGAT GGGCAAGATT ATTATTTCTT TTTTCCAAAA TTAACGCGGG AAGTATATAC AGTTTCGAGA GTATTGAAAA 910 920 AATATAATTA TTGACACTGA ACTTCTCATT TTACCGGTAG AACGCAGAAC C'!TCGAAC AGAACACTAT CAGACCCTTT 630 6.40 650 GTATAATACG GGGTTAAATC GTTTACCACG TAATAGGTTT AGAAGAGAGT TAACAATATC TTACGATTCT AGATTATATC CAATTCCGAC AGATCCGGTA ATTAATATAA CTGATTATAG TTCAGCTATT AGAAGTCCCC ATCTTATGGA 660 TA AT GA A G AG TAT TAGAT

AATCTATCAA

A GTTA CC CCA

TTTCTTAAAT

930 1 fTAATTAGA

TTCGCAAGTG

TATT OCT CCT

CTTCCGAAGA

940

GGCGTTCACT

AT AA GCTCCC

AGCACTTTTC

TCCGATAATA

CCAAATAATG

1240 950 960 ATTGGGCGGG GCATCGTGTA CTCAATACGG GATAACTGCA CAGGTCTTAA TCTATTTTAT TTATGCCAAC ATTAGGAATA GTGAAGTTCT ATATAGAAGG 1141 AATGTAGTGC AGGGGGTAGG ATTCATTCAA 1220 1230 1201 AGAGGAACAG TAGATTCTCT TGATGAGTTG CCAATTGACG 1261 TATAGTCATA GATTAAGTCA CGTTACATTA ACCAGGTCGT 1250

GTGAGAATTC

TATATAA TA C 1260

ATTAGTTGGA

TAATATAACT

1321 AGCTTGCCAA CATTTGTTTG 1381 GATGTAATTA CACAAATACC 1441 GTCAGAGGCC CAGGATTTAC 1510 1520 1501 CTAAGTATGA GTCTTAATTT 1561 TATGCTGCTT CTCAAACAAT 1621 CAAGGATTCC CTAGTACTAT 1681 GCAGAATTTC CTGTAGGCAT 1741 AATAATCCAG GTAGACAAAC GACACATCAC AGTGCTACTG ATCGAAATAT AATCTATCCG ATTGGTAAAA TCATTCTCCC TTACTTCAGG TACCTCTGTA AGGAGGGGAT ATCATCCGAA CTAACGTTAA TGGTAATGTA 1530

TAGTAATACA

GGTCATGAGA

GAGTGCAAAT

TA GT ACAT CT

GTTTCACTTA

1540 1550

TCATTACAGC

GTAAATGTTG

GGGTCTTTGA

GGCAGTCAAA

GATAGAATTG

GGTATCGCGT GAGAGTTCGT GAGGGAGTAC TACTTTTGAT CATCTCAATC ATTTAGATTT CTGCTGGAAT AAGTATAAGT AATTTATCCC AGTTGATGCA 1810 1820 1830 18.4 1850 1801 ACATTTGAAG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATTC 1861 TCTTCCAATC AAATCGAGTT AAAAACAGAT GTGACGGATT ATCATATTGA 1921 AATTTAGTAG ATTGTTTATC CGATGAATTT TGTCTGGATG AAAAGCGAGA 1981 AAAGTCAAAC ATGCGAAGCG ACTCAGTGAT GAGCGGAATT TACTTCAAGA 2041 AGAGGGATCA ATAGGCAACC AGACCGTGGC TGGAGAGGAA GTACGGATAT 1860

GCTGTTTACT

TCAAGTATCC

AT T T C CGAG

TCCAAACTTC

TACCATCCAA

2110

GGAGGAGATG

TATCCAACGT

CAATTAA GAG

GCAAAACACG

AGTCCAATTG

2120 2130 ACGTATTCAA AGAGAATTAC ATTTGTATCA AAAAATAGAT GGTATATCGA AGATAGTCAA AAACAGTAAA TGTACCAGGT GAAGGTGTGG AGAACCGAAT 2140 2150 2160 GTCACACTAC CAGGTACCTT TGATGAGTGC GAGTCGAAAT TAAAAGCCTA TAACCGTTAC GACTTAGAAA TCTATTTAAT TCGCTACAAT ACGGGTTCCT TATGGCCGCT TTCAGTCGAA CGGTGTGTGC CACACCTTGA ATGGAATCCT MA39.Cl Table 1 (continued) o 0 0 3 *0 *0 0 o ot *0 0 0 S40 9* 0 000 2410 2420 2430 GATTTAGATT GTTCCTGCAG AGACGGGGAA TTGGACATTG ATGTTGGATG CACAGACTTG AAGATTAAGA CGC-AGGAAGG TTATGCAAGA CCATTAATTG GAGAAGCACT GTCTCGTGTG CGGGAAAAAC TACAATTGGA AACAAAACGA 2710 2720 2730 GCTTTATTCG TAGATTCTCA ATATGATAGA CATGCGGCAG ATAGACTTGT TCATCAGATC ATTCCAGGAA TAAATGTGGT GATTTTTGAA TCCCTATATG ATGCG.AGAAA TGTCATTAAA TGGAACGTGA AAGGGCATGT AGATGTAGTA 3010 3020 3030 GTCCCGGAAT GGGAAGCAGA AGTGTCACAA ATCCTCCGTG TTACAGCGTA CAAAGAGGGA ATCGAGAACA ATACAGACGA ACTAAAATTT ACGGATACAG GAACGTGTAA TGATTATACT TCATGTAATT CCCGTAATAT CAGATATGAG 3310 3320 3330 GTTAATTACA AACCGACTTA CGAAGAAGAA TGTGAATATG ACAGAGGGTA TGTGAATTAT GAATTAGAGT ACTTCCCAGA AACCGATAAG AAGTTTATTG TAGACAATGT CGAATTACTC 2440

AAATGTGCAC

CAAGAGGATC

TTAGGAAATC

AAGAGAGCGG

G TA TATA CA G 2740 2750 2760 TTACAAGCAG ATACAAACAT TGGTATGATT CACGAGGCTT ATCTTCCAGA ACTACCTTTC GAATTAGAAA ACCGTATTTC TACTGCATTA AATGGCGATT TCAATAATGG CTTATCATGC GAACAAAACA ACCACCGTTC GGTCCTTGTT 3040 3050 3060 ACAATTCGTG TCTGTCCGGG GCGTGGCTAT TATGGAGAAG GTTGCGTAAC CATCCATGAG AAAAACTGTG AAGAAGAGGA AGTGTATCCA GCACACCAAG GTACAGCAGG ATCCACAGAT GATGCATATG AAATGAATAC TACAGCATCT 2450 2460 ATCATTCCCA TCATTTCTCC TAGGCGTGTG GGTTGTATTC TGGAATTTAT CGAAGAGAAA AAAAAAAATG GAGAGACAAA AGGCAAAAGA AGCTGTGGAT 0 0*0 0 0 00~ 0 00 *0 0 0 0004 00

C

000 0 3340 3350 AGGTATACAG ATGTACAAGG CGACCAGTAC CAGCTGGTTA GTATGGATTG AGATCGGAGA CTTATGGAGG AA 3360 A GAT AAT CAT

TGTGACAAAA

AACGGAAGGG

I I 27 M AA39.C1 Table 2 10 1 Met Giu Asn Asn lie Giu Asn Gin Cys ILie Pro Tyr Asn Cys Leu 16 Asn Asn Pro Giu VaL Giu Ie Leu Gly Ilie Giu Ara Ser Asn Ser 31 Asn Val Ala Ala Giu Ie Gly Leu Giy Leu Ser Ara Leu Leu Val 46 Ser Ara tIe Pro Leu Gly Asp Phe Ilie Leu GLy Leu Phe Asp Vat 61 Ile Trp Gly Ala Ile Gly Pro Ser Gin Trp Asp lie Phe Leu Glu 76 Gin lie Giu Leu Leu Ilie GLy Gin Ara Ile Gtu Giu Phe Ala Arg 91 Asn Gin Ala Ile Ser Ara Leu Gin GLy Leu Ser Asn Leu Tyr Arg 106 Ilie Tyr Thr Asn Ala Phe Lys Asn Trp Giu Vat Asp Pro Thr Asn 121 Pro Ala Leu Arg Giu Giu Met Ar Ilie Gin Phe Asn Asp Met Asn 136 Ser Ala Leu Thr Thr Ala ILe Pro Leu Phe Ser Vai Gin GLy Tyr 151 Giu Ilie Pro Leu Leu Ser Vat Tyr Vat Gin Ala Ala Asn Leu His 166 Leu Ser Vat Leu Arg Asp Vat Ser Vat Phe GLy G~n Ara Trp GLy 181 Phe Asp Vat Ala Thr Ilie Asn Ser Arg Tyr Asn Asp Leu Thr Ara 196 Leu Ilie Gty Giu Tyr Thr Asp Tyr Ala Vat Ara Trp Tyr Asn Thr 211 GLy Leu Asn Ara Leu Pro Ara Asn GLu Gty Vat Ara Gly Trp Ala 226 Ar'g Phe Asn Ara Phe Ara Ara Giu Leu Thr lie Ser Vat Leu Asp S'241 lie ILie Ser Phe Phe Gin Asn Tyr Asp Ser Ara Leu Tyr Pro ILie a256 Pro Thr lie Tyr Gin Leu Thr Arg Gku Vat Tyr Thr Asp Pro Val a271 1Lie Asn lie Thr Asp Tyr Ara Vat Thr Pro Ser Phe Giu Ser ILie ,286 Giu Asn Ser Ala Ile Ara Ser Pro His Leu Met Asp Phe Leu Asn a 301 Asn I te Ile Ilie Asp Thr Asp Leu Ilie Ara Gly Val His Tyr Trp 316 Ala Giy His Ara Vat Thr Ser His Phe Thr GLy Ser Ser Gin Vat t331 Ilie Ser Ser Pro Gin Tyr GLy lie Thr Ala Asn Ala Giu Pro Ser 346 Ara Thr Ilie Ala Pro Ser Thr Phe Pro Giy Leu Asn Leu Phe Tyr 361 Ara Thr Leu Ser Asp Pro Phe Phe Arg Ara Ser Asp Asn Ilie Met 376 Pro Thr Leu Gly Ile Asn Vat Vat Gin GLy Vat GLy Phe ILie Gin 391 Pro Asn Asn GLy Giu Vat Leu Tyr Ara Ara Ara Gly Thr Vat Asp cc-. 406 Ser Leu Asp GLu Leu Pro lie Asp Gly Giu Asn Ser Leu Vat Gly 421 Tyr Ser His Ara Leu Ser His Vat Thr Leu Thr Ara Ser Leu Tyr 436 Asn Thr Asn I te Thr Ser Leu Pro Thr Phe Vat Trp Thr His His 451 Ser Ala Thr Asp Ara Asn tIe Ile Tyr Pro Asp Vat ILie Thr Gtn 466 Ilie Pro Leu Vat Lys Ser Phe Ser Leu Thr Ser GLy Thr Ser Vat 481 Vat Arg Gly Pro GLy Phe Thr GLy GLy Asp Ilie lie Ara Thr Asn 496 Vat Asn GLy Asn Vat Leu Ser Met Ser Leu Asn Phe Ser Asn Thr 511 Ser Leu Gin Arg Tyr Ara Vai Ara Vat Ara Tyr Ala Ala Ser Gin 526 Thr Met Vai Met Ara Vat Asn Vat GLy GLy Ser Thr Thr Phe Asp 541 Gin Giy Phe Pro Ser Thr Met Ser Ala Asn GLy Ser Leu Thr Ser 556 Gin Ser Phe Ara Phe Ala Giu Phe Pro Vat Gly ILe Ser Thr Ser 571 GLy Ser Gin Thr Ala GLy ILie Ser ILie Ser Asn Asn Pro Gly Ara 586 Gin Thr Phe His Leu Asp Ar-g lie Giu Phe Ilie Pro Vat Asp Ala 601 Thr Rh e Gtu Ala C! tu Tyr Asp Leu Giu Ara Ala Gin Lys Ala Vat 616 Asn Ser Leu Phe Thr Ser Ser Asn G~n Ilie Giu Leu Lys Thr Asp 631 Vat Thr Asp 7 yr His lie Asp Gin Vat Ser Asn Leu Vat Asp Cys 646 Leu Ser A'sp Gtu Phe Cys Leu Asp Giu tLys Ara Giu Leu Ser Giu 661 Lys Vat Lys His Ala Lys Ara Leu Ser Asp Giu Ara Asn Leu Leu 676 Gin Asp Pro Asn Phe Ara Gly ILie Asn Ar-g Gin Pro Asp Ara GLy 691 Trp Ara Gty Ser Thr Asp lie Thr tIe Gin Gly GLy Asp Asp Vat 706 Phe Lys Gtu Asn Tyr Vat Thr Leu Pro Gly Thr Phe Asp Glu Cys 721 Tyr Pro Thr Tyr Leu Tyr Gin Lys Ilie Asp Giu Ser Lys Leu Lys 736 Ala Tyr Asn Ara Tyr Gin Leu Ara GLy Tyr tIe Giu Asp Ser Gin 751 Asp Leu Gtu lie Tyr Leu Ilie Ara Tyr Asn Ala Lys His Giu Thr 766 Vat Asn Vat Pro Gly Thr Giy Ser Leu Trp Pro Leu Ser Vat GLu 781 Ser Pro Ilie GLy Arg Cys Gly Glu Pro Asn Ara Cys Vat Pro His 796 Lets GIu Trp Asn Pro Asp Lets Asp Cys Ser Cys Ara Asp Gly Giu 811 Lys Cys Ala His His Ser His His Phe Ser Lets Asp Ilie Asp Vat p 28 Table 2 (continued) MA39.Cl 826 Gty Cys Thr Asp Leu Gtn Gtu Asp Leu Gty Val Trp Vat Vat Phe ti t t 4* 4 44 4 tt t tt 4 4 4 £44.4 841 Lys Ilie Lys Thr 856 Phe Ile Gtu Glu 871 Lys Arg Ala GLu 886 Leu Gtu Thr Lys 901 Ala Leu Phe Vat 916 Asn Ile Gty Met 931 His Gtu Ala Tyr 946 Vat Val Ile Phe 961 Ser Leu Tyr Asp 976 Asn Gly Leu Ser 991 Gtu Gin Asn Asn 1006 Ala Giu Vat Ser 1021 Ie Leu Arg Val 1036 Vat Thr Ile His 1051 Lys Asn Cys Gtu 1066 Cys Asn Asp Tyr Gin GLu Lys Pro Lys Lys Arg Vat Asp Ser Ile His Leu Pro Gtu Gtu Ala Arg Cys Trp His Arg Gln Thr Gty Tyr Leu lie Trp Arg Tyr Thr Gin Tyr Ala Ala Glu Leu Leu Giu Asn Vat Asn Vat Sar Vat lie Arg Asp Lys Giu Ala Asp Arg Asp Arg Pro Phe Asn Arg Ilie Lys Lys Giy Leu Vat Vat Cys Gtu Giy Asn Thr Tyr Pro Giy Thr Arg GLu Lys Leu Gin Lys Giu Ala Vat Asp Leu Gin Ala Asp Thr Leu Vat His Gin Ile lie Pro Giy lie Asn Ilie Ser Thr Ala Leu Asn Giy Asp Phe Asn His Vat Asp Vat Vai Vat Pro Giu Trp Giu Pro Giy Arg Giy Tyr Tyr Gty Giu Gly Cys Asp Gtu Leu Lys Phe Thr Asp Thr Gly Thr Aia Gty Ser Thr Asp Asp Ala Tyr Gtu Met Thr Tyr GiU GLu Gtu Cys Giu Tyr Asp Arg GLy Tyr Vai Thr Lys Ala Arg Leu Gly Asn Leu GLu Gty Giu Ala Leu Ser Arg Vat Thr Ala Tyr Lys G~u Ile Giu Asn G~u Gtu GLu Vat Thr Ala His Gin 1081 Ser Cys 1096 Asn Thr 1111 Arg Tyr 1126 Gly Tyr 1141 GLu Leu 1156 Gly Gtu 1171 Leu Met Asn Ser Arg Thr Ala Ser Thr Asp Vat Vat Asn Tyr Asn Ilie Vat Asn Gin GLy Arg Pro Arg Tyr Tyr Lys Asp Asn Vat Pro G~u Tyr Phe Pro Giu Thr Giu Gly Lys Phe Glu GLU Thr Asp Lys Vat Trp Ilie Giu lie tIe Vat Asp Asn Vat GLu Leu Leu f t 4 MA39.C1 Table 3 .Met GLu Asn Asn Ile Giu Asn 15 Gin Cys Ilie Pro Tyr Asn Cys Leu Asn Asn Pro GLu Vat ATG GAG MAT MAT ATT GMA MT CAA TGC ATA CCT TAC AAT TGT TTA AAT MAT COT GAA GTA Gtu Ile Leu GLy lie Giu Arg Ser Asn Ser Asn V at Ala Ala Glu Ile GLy GAG ATA TTA GGG ATT GMA AGG TCA MAT AGT MAC GTA GCA GOA GAA ATC GGC Leu Gly Leu TTG GGG OTT Ser Arg Leu Leu Vat Ser Arg Ile Pro Leu Gly Asp Phe Ile Leu Gty Leu Phe Asp Vat AGT CGT CTG CTC GTT TCC CGA ATT CCA CTA GGG OAT TTT ATA CTT GGC TTO TTT GAT GTA Ile Trp Gly Ala Ilie Giy Pro Ser Gin Trp Asp lie Phe Leu Giu ATA TGG GGG GOT ATA GGT CCT TCA CAA TGG GAT ATA TTT TTA GAG Gin Ile Oiu Leu Leu CAA ATT GAG CTA TTG 497444 .4,4 4 4 44 4 4* 4 4 4 4 44~ 4~r 44 t 4 4 4 4 44 I 4 444 C ILie Giy Gin Arg ILie Giu Giu Phe Ala Arg ATC GGC CAA AGA ATA GAG GMA TTC GOT AGG Asn Gin Ala Ile Ser Arg Leu Gin GLy Leu AAT CAG GOA ATT TCT AGA TTA CAA GGG CTA Ser Asn Leu Tyr Arg Ile Tyr Thr Asn Ala Phe Lys Asn Trp Giu Vat Asp AO AT OTT TAC CGA ATT TAC ACA MAT GCT TTT AMA AAC TGG GAA GTA OAT 120 Pro Thr Asn CCT ACT AAT 125 Pro Ala Leu Arg Giu CCA OCA TTA AGA GAA 145 Ate le Pro Leu Phe GOT ATT OCT OTT TTT Giu Met Arg Ilie Gin Phe Asn Asp Met Asn Ser Ala Leu Thr Thr GAG ATG CGT ATT CAA TTT AAT GAC ATG AAC AGT GCT OTT ACA ACA 160 Tyr Vat Gin Ser Vat Gin Oly Tyr Giu Ile Pro Leu Leu Ser Vai TCA GTT CAA GGT TAT GAA ATT CCT CTT TTA TCA GTA TAT GTT CAA Ala Ala Asn Leu His Leu Ser Vat Leu Arg Asp Vat Ser Vat Phe GLy Gin GCT GOA AAT TTA CAT TTA TCG OTT TTG AGA OAT OTT TCA GTG TTT GGA CAA 180 Arg Trp Oty COT TOG OGA 200 Oty GLu Tyr GGC GAA TAT Phe Asp Vat Ala Thr lie Asn Ser TTT GAT OTA GOA ACA ATC MAT AOT Arg Tyr Asn Asp COT TAT AAT OAT 195 Leu Thr Arg Leu le TTA ACT AGO CTT ATT e :)eu z!)220 Thr Asp Tyr Ala Vat Arg Trp Tyr Asn Thr Giy Leu Asn Arg Leu Pro Arg Asn OLu GLy ACT OAT TAT GOT OTA COT TOO TAT AAT ACO 000 TTA AAT COT TTA CCA COT AAT GAA 000 Vat Arg Gly Trp Ala Arg Phe OTA CGA OGA TOO GOA AGA TTT 230 Asn Arg Phe Arg Arg AAT AGO TTT AGA AGA 235 Olu Leu Thr Ile Ser Vat Leu GAG TTA ACA ATA TCA OTA TTA 255 Tyr PrL Ile Pro Thr Ilie Tyr TAT CCA AT COG ACA ATC TAT 245 Ile Ilie Ser Phe Phe ATT ATT TCT TTT TTC 250 Gin Asn Tyr Asp Ser Arg Leu CAA AAT TAC OAT TCT AGA TTA 265 Leu Thr Arg Giu Vat Tyr TTA ACG COG GAA GTA TAT 270 Thr Asp Pro Va Ile Asn Ile ACA OAT COG GTA ATT AAT ATA 275 Thr Asp Tyr Arg Vat ACT OAT TAT AGA OTT 280 Thr Pro ACC CCA 94 42 activity against insect pests of the order Lepidoptera.

11. Bacillur thunnai4,M nloom"4 MA39.Cl Table 3 (continued) 285 290 295 300 Set Phe Glu Ser Ile Gtu Asn Ser Ala Ile .Arg Ser Pro His Leu Met Asp Phe Leu Ron ACT TTC GAG ACT ATT OAR AAT TCA GOT ATT AGA ACT COO CAT OTT ATO CAT TTC TTA RAT 305 310 315 320 Ron Ile Ile lie Asp Thr Asp Lou Ile Arg Gly Vat His Tyr Trp Ala GLy His Arg Vat ART ATA ATT ATT GAC ACT CAT TTA ATT AGA COC OTT CAC TAT TOO COO COG CAT CGT GTA 325 330 335 340 Thr Set His Phe Thr Oty Ser Ser Gin Vat Ile Ser Ser Pro Gin Tyr Gly lie Thr Ala ACT TCT CAT TTT ACC GOT ROT TCG CAR GTG ATA AGO TCC CCT CAR TAC 000 ATA ACT GOAR 345 350 355 360 Rsn Ala Otu Pro Sor Arg Thr lie Ala Pro Ser Thr Phe Pro Gly Leu Asn Lo-u Phe Tyr ARC GOR A CCO ROT CGA ACT RTT GOT CCT AGO ACT TTT CCA GOT CTT AAT CTA TTT TAT S 5365 370 375 380 Rrg Thr Leu Set Asp Pro Phe Phe Rrg Arg Ser As:; Asn lie Met Pro Thr Lou Gty Ile S 5AGA RCA CTA TCR GAC CCT TTC TTC COR RGA -,CC SA T RAT ATT ATO CCA RCA TTA GOA ATA 385 390 395 400 Rn Vat Vat Gin Gty Vat Oty Phe Ilie Gin Pro Aon Ron Gty Gtu Vol Lou Tyr Arg Arg ART OTA GTO CR0 000 GTA OGA TTC ATT CAR COR ART RAT GOT OAR OTT CTA TAT AGA AGO 405 410 415 420 Arg Oty Thr Vat Asp Ser Leu Asp Oiu Lau Pro lie Asp Gty Giu Ron Ser Lau Vat Oty AGA GOR ACA GTA GAT TOT CTT OAT GAO TTG COR ATT GAC GOT GAO ART TCA TTR OTT OA 425 430 435 440 Tyr Ser His Arg Lou Set His Vat Thr Lou Thr Rrg Set Lou Tyr Ron Thr Ron Ile Thr 'TAT ROT CAT AGA TTA ROT CRC OTT RCA TTR ACC AGO TCO TTA TAT ART ACT ART ATA ACT 450 455 460 Ser Lou Pro Thr Phe Vat Trp Thr His His Son Als Thr Asp Arg Ron ILe Ile Tyr Pro *AGO TTG CC RCA TTT OTT TOG RCA CAT CRC ROT OC7 ACT OAT CGR ART ATA ATC TAT CCG 465 470 475 480 Asp Vat Ile Thr Gin lie Pro Leu Vai Lys Ser Pho Ser Lou Thr Ser Oty Thr Ser Vai GAT OTA ATT RCA OAR ATA CR TTG OTA AAA TCA TTC TCC OTT ACT TCR GOT ACC TCT GTA *485 490 495 500 Vat Ang Giy Pro Oly Phe Thr GLy Gly A sp :Ie ile Arg Thr Ron Vol Ron Gly Ron Vat 070 AGA 000 CR GOA TTT RCA OGA 000 OAT ATC ATC COR ACT ARC OTT RAT GOT ART GTA 505 510 515 520 Lou Set Hot Son Lou Ron Pho Sot Ron Thr Son Lou Gin Anrg Tyr Arg Vat Arg Vol Ang OTA ROT ATO AOT OTT ART TTT ROT RAT ACA 7CR TTA CR0 COG TAT COO GTG AGA OTT COT 525 t530 535 540 Tyr Rio Ata Ser Gin Thr Met Vat Met Arg Vol Ron Vol Gly Gly Set Thr Thr Phe Asp TAT GOT GOT TOT OAR RCA ATO OTO ATO AGA GTA ART OTT GOA 00O ROT ACT ACT TTT OAT 545 550 555 560 Gin Oly Phe Pro Set Thr Meot Set Rlo Ron Gly Sot Lou Thr Sot Gin Set Phe Arg PIho OAR GOR TTC OCT ROT ACT ATO ROT OR RAT 000 TOT TTG RCA TOT CAR TOR TTT AGA TTT 565 570 575 sao Ala Olu Phe Pro Vat Oly ILe Sen Thr Son Gly Son Gin Thr Rlo Gty le Set lie Set GOR OA TTT COT GTA 000 ATT ROT RCA TOT COCT CAA ACT OT GOA ATA ROT ATA ACT -14 31 MA39.C1 Table 3_(continued) 585 590 595 600 Asn Asn Pro GLy Arg Gin Thr 'he His Leu Asp Ars Ile Giu Phe Ilie Pro Vat Asp Ala AAT AAT CCA GOT AGA CAA ACG TTT CAC TTA GAT AGA ATT GAA TTT ATC CCA GTT OAT GOA 605 610 615 620 Th- Phe Giu Ata GiU Tyr Asp Leu Giu Arg Ata Gin Lys Ala Vat Asn Set Leu Phe Thr ACA TTT GMA GOA GAA TAT GAT TTA GAA AGA GOA CAA AAG 000 070 AAT TOG CTG TTT ACT 625 630 635 640 Ser Set Asn Gin Ile Glu Leu Lye Thr Asp VaL Thr Asp Tyr His lie Asp Gin Val Set TCT TOO MAT CMA ATO GAG TTA AAA ACA OAT 070 ACG GAT TAT CAT ATT GAT CAA GTA TOO 645 650 655 660 Asn Leu VaL Asp Cys Leu Set Asp Giu Phe Cys Leu Asp Glu Lys Arg Giu Leu Set Giu MAT TTA GTA OAT TOT TTA TCC GAT GAA TTT TOT CTG GAT GAA AAG CGA GAA TTG TOO GAG 665 670 675 680 Lys Val Lye His Ala Lys Arg Leu Ser Asp Giu Arg Asn Leu Leu G Inr Asp Pro Asn Phe AA OTO AAA OAT 000 MOG OGA O70 AGT OAT GAG 000 AAT TTA OTT CAA OAT OCA AAO TTO 685 690 695 700 Arg GLy Ilie Asn At; Gin Pro Asp At; Giy Trp Arg Giy Set Thr Asp lie Thr Ile Gin AGA 000 ATO MAT AGO CMA OCA GAO CGT 000 TOG AGA OGA AGT ACG OAT ATT ACC ATC CAA 705 710 715 720 Giy Giy Asp Asp VaL Phe Lye Oiu Asn Tyr Vai Thr Lau Pro Gty Tht Phe Asp Giu Cys GGA OGA OAT GAC OTA TTC AMA GAO MAT TAC 070 ACA OTA OCA GOT ACC TTT OAT GAO TOO 725 730 735 740 Tyr Pro Thr Tyr Lau Tyr Gin Lye Ilie Asp Oiu Set Lye Leu Lye Ala Tyr Asn Ar; Tyr TAT OOA ACO TAT 770 TAT CAA AAA ATA OAT GAG TOG AAA TTA AAA 000 TAT AAO COT TAO 745 750 755 760 Gin Lou Arg Oiy Tyr lie GiU Asp Set Gin Asp Leu G W lie Tyr Leu Ilie Arg Tyr Aen CAA TTA AGA 000 TAT ATO OAA OAT AGT CAA GAO TTA GMA ATO TAT TTA ATT 000 TAO AAT 765 770 775 780 Aia Lye His Oiu Thr Val Asn Vat Pro Gly Tht Giy Set Leu Trp Pro Leu Set Vai Giu OCA AAA CAC GMA ACA GTA MT OTA OCA GOT ACO 007 TOO TTA TOO 000 OTT TOA 070 GAA 785 790 795 B00 Set Pro Ile Oiy Arg Cys Oiy Oiu Pro Aen At; Cys Vat Pro His Leu Oiu Trp, Asn Pro AGT OCA ATT OGA AGO TOT OGA GMA COO AAT COO TOT 070 OCA CAC OTT OAA TOG AAT OCT 805 810 815 820 Asp Leu Asp Cys Sct Cys Arg Asp Oiy Giu Lye Cys Aie His His Ser His His Phe Set OAT TTA OAT TOT 7CC TOO AGA GAO 000 OAA AMA TOT GOA OAT OAT TOO CAT OAT TTO TOO 82-5 830 3 8.40 Leu Asp Ile Asp Val Oly Cys Tht Asp Leu Gin Oiu Asp Leu Oly Vai Trp Vat Vai Phe TTG GAO ATT OAT OTT OGA TOO ACA GAO TTO CAA GAG OAT OTA 000 070 TOO OTT GTA TTO 845 850 855 860 Lys Ile Lye Thr Gin Oiu GLy Tyr Ala Ars Leu Gly Asn Leu G1. Ph. le Gtu GI. Lye MAG ATT AAO ACC CAG GMA GOT TAT OCA AGA TTA OGA AAT OTO GAA TTT ATO OAA GAG AAA 865 870 875 880 Pro Leu Ile Oty Glu Ake Leu Set Arg Vat Lys At; Ale Giu Lye Lys Trp Arg Asp Lye OCA TTA ATT OGA GAA OCA OTG T0! COT OTG AAG AGA 000 GAA AAA AAA TGG AGA GAO AAA 32 MA39.Cl Table 3 (continued) 885 890 895 900 Ars Gtu Lys Leu Gin LeLJ Gtu Thr Lys Arg Vat Tyr Thr Gtu Ala Lys Gtu ALa Vat Asp COG GAA AAA CTA CMA TTG GAA ACA AAA CGA GTA TAT ACA GAG GCA AAA GAA OCT GTG CAT 905 910 915 920 Al a I au Phle Vat Asp Ser Gin Tyr Asp Arg Leu Gin Ata Asp Thr Asn Ile Oty Met Ite OCT TTA TTC GTA OAT TOT CAA TAT GAT AGA TTA CMA OCA GAT ACA MAC ATT GOT ATO ATT 925 930 935 940 His Ata Ala Asp Arg Leti Vet His Gin Ile His Gtu Ala Tyr Leu Pro Gtu Leu Pro Pile CAT 000 OCA OAT AGA OTT OTT OAT CAG ATO CAC GAG OCT TAT OTT OCA OAA OTA OCT TTO 945 950 955 960 Ile Pro Oty ILie Asn Vat Vet Ile Phle Oiu Oiu Leu Giu Asn Arg Ilie Ser Thr Ate Leu ATT OCA GOA ATA MAT OTO OTO ATT TTT GMA GMA TTA GMA MOA COT ATT TOT ACT OCA TTA 965 970 975 980 Ser Leu Tyr Asp Ate Arg Aen Val Ilie Lye Aen Gly Asp Phle Asn Aen G ty Leu Ser Cys TOO OTA TAT OAT 000 AGA MAT OTC ATT AMA MT 000 OAT TTO AAT MAT 000 TTA TOA TOO 4985 990 995 1000 Trp Asn Vet Lys Oty His Vat Asp Vat Vat Otu Gin Asn Asn His Arg Sen Vat Leu Val TOO AAC OTO AAA 000 OAT OTA OAT OTA OTA GMA CAA AAO AAC CAC COT TCO OTC OTT OTT 1005 1010 1015 1020 Vat Pro Otu Trp Gtu Atea Oiu Vat Ser Gtn Thr Ilie Arg Vat Cys Pro Oty Arg Oty Tyr OTC COO GMA TOO GMA OOA GAA OTO TOA CMA AOA ATT COT OTC TOT 000 000 COT 000 TAT 1025 1030 1035 1040 lie Leu Arg Vat Thr Ala Tyr Lye Gtu Oty Tyr Oty Giu Oty Cys Vat Thr Ile His Otu ATO OTO COT OTT ACA 000 TAO AMA GAG OGA TAT OGA GMA GOT TOO OTA ACC ATC CAT GAO 1045 1050 1055 1060 le Gtu Asn Asn Thr Asp Otu Leu Lye Pile Lye Asn Cys Gtu Giu Otu Otu Vat Tyr Pro ATO GAO AAO MAT ACA GAO OAA OTA AMA TTT AMA MO TOT GAA OAA GAO GMA OTO TAT OCA 1065 1070 1075 1080 Thr Asp Thn Oty Thn Cys Aen Asp Tyr Thr Ata His Gin Oty Thr Ata Oty Sen Thn Asp AOO OAT ACA OA ACO TOT MAT OAT TAT ACT OCA CAC CAA GOT ACA GOA OGA TOO ACA OAT 1085 1090 1095 1100 Sen Cys Aen Sen Arg Asn Ile Arg Tyn Oiu Asp Ata Tyr Giu Met Aen Thr Thn Ata Ser TOA TOT MAT TOO COT AAT ATO AGA TAT GAG OAT OOA TAT GMA ATO AAT ACT ACA OCA TOT 1105 1110 1115 1120 Vat Aen Tyr Lye Pro Thn Tyn Oiu Oiu Oiu Ang Tyr Thr Asp Vet Gin Oty Asp Asn His OTT AAT TAO MA COO ACT TAO OAA GAA OAA AGO TAT ACA OAT GTA CAA GOA OAT AAT OAT 1125 1130 1135 1140 Cys Otu Tyr Asp Ang Oty Tyr Vat Aen Tyr Arg Pro Vat Pro Ate Oty Tyr Vat Thr Lys TOT OAA TAT GAO AGA 000 TAT OTO AAT TAT OGA OCA GTA OCA OCT GOT TAT OTG ACA AMA 1145 1150 1155 1160 Oiu Leu Giu Tyr Phle Pro Gtu Thr Asp Lye Vat Trio Ilie u Ile GLy OtU Tht Otu Oty GAA TTA GAO TAO TTC OOA OAA ACC OAT AAO OTA TOO ATT GAr, ATO OGA OAA ACO GMA 000 1165 1170 Lye Phle lie Vat Asp Asn Vet Otu Leu Leu Leu Met Otu G AAG TTT ATT OTA GAO AAT OTC GAA TTA CTC OTT ATO GAO OAA 33 Table 4 20 30 40 ATGGAGATAA TGAATAATCA GAATCAATGC GTTCCTTATA ATTGA.AMAT TAGAAGGAGA AAGAATAGAA ACTGOOTTACA TCGCTAACGC AATTTCTGTT GAGTGAATTT GTCCCAGGTG ATTGATTTAA TATGGGGGTT TGTGGGTCCC TCTCAATGGG GAACAGTTAA TTAACCAAAG AATAGAGGAA TTCGCTAGGA MA39.Cl 50 ACTGTTTGAA TGATCCGACA CCCCAATAGA TATTTCCTTG CTGGGTTTGT ATTAGGTTTA ATGCATTTCT TGTGCAAATT ACCkAGCAAT TTCTAGATTA 310 320 330 340 350 301 GAAGGGCTAA GCAACCTTTA TCAAATTTAC GCAGAA0-,TT TTAGAGAGTG 361 CCTACTAATC CAGCATTAAC AGAAGAGATG CGTATTCAGT TCAATGACAT 421 CTTACAACCG CTATTCCTCT TTTTACAGTT CAAAATTATC AAGTACCTCT 481 TATGTTCAAG CTGCAAATTT ACATTTATCG GTTTTGAGAG ATGTTTCAGT 541 CGTTGGGGAT TTGATGTAGC AACAATCAAT AGTCGTTATA ATGATTTAAC 360

GGAAGCAGAT

GAACAGTGCT

TCTATCAGjA

GTTTGGACAA

TAGGCTTATT

4*eqeq

C

4 C ~q 9* 9 9 4 99 .9 r *499 *4 4 *49 f 610 620 630 6,40 650 660 601 GGCACCTATA CAGATTATGC TGTACGCTGG TATAATACGG GATTAGAACG TGTATGGGGA 661 CCGGATTCTA GAGATTGGGT AAGGTATAAT CAATTTAGAA GAGAGCTAAC ACTAACTGTA 721 TTAGATATCG TTTCTCTGTT CCCGAACTAT GATAGTAGAA CGTATCCAAT TCGAACAGTT 781 TCCCAATTAA CTAGAGAAAT TTATACAAAC CCAGTATTAG AAAATTTTGA TGGTAGTTTT 841 CGTGGAATGG CTCAGAGAAT AGAACAGAAT ATTAGGCAAC CACATCTTAT GGATCTCCTT 910 901 AATAGTATAA 961 ATAACAGCTT 1021 ATGGGAAATG 920 930 940 CCATTTATAC TGATGTGCAT AGAGGCTTTA CTCCTGTCGG TTTTGCGGGG CCAGAATTTA CTGCTCCACC CGTACTGATC TCAACTACTG 950 960 ATTATTGGTC AGGACATCAA CTTTTCCTAG ATATGGAACC GTTTGGGGAT TTTTAGAACA GCCCAAATAA TCAGAACCTG CAGCCGATTT ACCTTCTACT 44cr 44Cc 44*-c

C

4 4! 1 4 4 (ftc ft I 4 444 4 1081 TTATCTTCAC CTCTTTACAG 1141 TTTGTCCTTG ATGGAACGGA 1210 1220 1201 ATATACAGAC AAAGGGGAAC 1261 GTGCCAGCAC GTGCGGGATT 1321 GCTGGAGCAG TTTACACCTT 1381 TTCTCTAACC TAATTCCTTC 1441 CTTGG.CTCTG GGACCTCTGT AAGAATTATA CTTGGTTCAG ATTTTCTTTT GCCTCCCTAA 1230 1240 1250 1260 GGTCGATTCA CTAGATGTAA TACCGCCACA GGATAATAGT TAGT CAT CGA GA GAG CT CCA

ATCACAAATC

TGTTAAAGGA

TTAAGTCATG TTACAATGCT GAGCCAAGCA ACGTTTTCTT GGCGACATCG ACACAGATAC CTTTAACAAA CCAGGATTTA CAGGAGGAGA

TAGTGCTGAA

GTCTATTAAT

TATTCTTCGA

1510 1520 1530

AGAACTTCAC

AGATATCGCG

GACGGAAGAC

CAGTCCGGAA

CTGGCCAGAT TTCAACCTTA AGAGTC TAAGAATTCG CTACGCTTCT ACTACO CTATTAATCA GGGGAATTTT TCAGCA GC7TTAGGAC TGCAGGTTTT ACTAC7 1741 AGTATATTTA CGTTAAGTGC TCATGTCTTC AATTCA 1810 1820 1830 1801 ATTGAATTTG TTCCGGCAG.A AGTAACATTT GAGGCG -1540 1550 1560 ~ACTA TTACTGCACC ATTATCACAA LAATT TACAATTCCA TACATCA.TT .ACTA TGAGTAGTGG GGGTAATTTA 'CCGT TTAACTTTTC AAATGGATCA GGCA ATGALAGTTTA TATAGATCGA 1840 1850 1860 ;GAAT ATGATTTAGA AAGAGCGCAA CTAG GATTAAAAAC AAATGTGACG TGTT TATCCGGTGA ATTCTGTCTG GCGA AGCGACTCAG TGATGAGCGG 1861 GAGGCGGTGA ATGCTCTGTT 1921 GACTATCATA TTGATCAAGT 1981 GATGAAAAGA GAGAATTGTC TA CTT CTTC C GTCCfLATCTA

CGAGAAAGTC

AATCAA

GTCGAA

AAACAT

2041 AATTTACTTC AAGACCCAAA CTTCAGAGGC ATCAATAGAC AACCAGACCG TGGCTGGAGA 2110 2120 2130 GGCAGTACGG ATATTACCAT CCAAGGAGGA CTACCGGGTA CCTTTAATGA GTGTTATCCT AAATTAAAAG CCTATACCCG TTACCAATTA GAAATCTATT TAATTCGCTA CAATACAAAA TCCTTATGGC CGCTTTCAGT CGAAA.ATCCA 2140 2150 2160 GATGACGTAT TCAAAGAGAA TTACGTCACA ACGTATCTGT ATCAAAAAAT AGATGAGTCG AGAGGGTACA TCGAGGATAG TCAAGACTTA CACGAAACAG TAAATGTGCC AGGTACGGGT ATTGGAAAGT GCGGAGAACC AAATCGATGC 94 34 Table 4 (continued MA39.C1 2410 2420 GCACCACAAC TTGAATGGAA GCACATCACT CCCATCATTT AACTTAGGTG TATGGGTGAT AATCTAGAGT TTCTCGAAGA GCGGAGAAGA AGTGGAGAGA 2710 2720 AAAGAGGCAA AAGAATCTGT GCGGATACCG ACATCGCGAT GCATATCTTC CAGAGTTATC GAGGGACGTA TTTTCACAGC GATTTCAATA ATGGCTTATC 2430

TCCTGATCTA

CT CC 7T S AC

ATTCAAAATT

C AAA CCATT7A CAAAC SAGA S 2440 2450 2460 GATTGTTCCT GCAGAGACGG GGAAAAATGT ATTGATATTG GATGTACAGA TTTAAATGAG AAGACGCAAG ATGGTCACGC AAGACTAGGT GTAGGCGAAT CGTTAGCACG CGTGAAGAGA AAATTGCAAG TGGAAACAAA TATCGTTTAT 2730 2740 2750 2760 AGATGCTTTA TTTGTGAACT CTCAATATGA TAGATTACAA t~ V girt V Ct it t t GATT CAT 5CG

TGTAATTCCG

CTACTCTTTA

ATS CTGG AA C 3010 3020 3030 3001 A.ACAACCACC GTTCGC7'CT TGTTGTCCCG 3061 CGTGTCTGTC CAGGTCGTSG CTATATCCTA 3121 GAAGGTTGCG TAACGATTCA TGAGATCGAA 3181 TGTGTAGAAG AGGAAGTATA TCCAAACAAC 3241 CAAGAAGAAT ACGGGGGTGC GTACACTTCT GCAGATAAAC GCGTTCATCG AATTCGAGAA SSTGTCAATG CGSGCATTTT TGAAGAATTA TATGATGCUA GAAATGTCAT TAAAAATGGC GTGAAAGGGC ATGTAGATGT ASAAGAACAA 3040 3050 3060 GAATGGGAAG CAGAGGTGTC ACAAGAGGTT CGTGTTACAG CGTACAAAGA GSATATGGA GACAATACAG ACGAACTGAA ATTCAGCAAC ACGGTAACGT GTAATGATTA TACTGCAAAT CGTAATCGTG GATATGGTGA ATCTTATGAA 3310

AGTAATTCTT

AGAAAAGASA

SC TTAT ST GA SGGGAAA CGG 3320 3330 3340 CCATACCAGC TGAGTATGCG CCAGTTTATG ATCCTTGTGA ATCTAACAGA GSATATGGGG CA.AAAGAATT AGAGTACTTC CCAGAAACCG AAGGAACATT CATCGTGGAT AGCGTGGAAT 3350 3360 AGGAASCATA TATASATGGA ATTACACGCC ACTACCAGCT ATAAGGTATG GATTGAGATC TACTCCTTAT GGAGGAA* segment 1-

C

MA39.C1 Table 10 115: 1 Met Giu Ile Met Asn Asn Gin Asn Gin Cys Vet Pro Tyr Asn Cyi; 16 Leu Asn Asp Pro Thr I te Gu Ilie Leu Gtu Gly Giu Arg Ilie GLU 31 Thr GLy Tyr Thr Pro Ilie Asp Ilie Ser Leu Ser Leu Thr Gin Phe 46 Lau Leu Ser GLo Phe Vat Pro Gly Ala Giy Phe Vet Leo Gly Leu 61 Ile Asp Leu Ilie Trp Gly Phe Vat Gly Pro Ser Gin Ti-p Asp Ala 76 Phe Leu Vat Gin Ilie Gbu Gin Leu Ilie Asn Gin Arg Ile Giu Cbo 91 Phe Ala Ars Asn Gin Ala 'Lie Ser Arg Leu Giu Giy Leu Ser Asn 106 Leu Tyr Gin ILie Tyr Aia Giu Aie Phe Arg Giu Ti-p Giu Ala Asp 121 Pro Thi- Asn Pro Aia Leu Thi- GLo Glu Met Arg Ilie Gin Phi Asn 136 Asp Met Asn Ser Ala Leu Thi- Thi- Ale Ilie Pro Leu Phe Thr Vat 151 Gin Asn Tyr Gin Vat Pro Leu Leu SEr Vel Tyr Vat Gin Ala Ale 166 Asn Leu His Leu Ser Val Leu Arg Asp Vat Ser Vai Phe Gly 1Gin 181 Arg Ti-p GLy Phe Asp Val Ala Th- Ilie Asn Ser Arg Tyr Asn Asp 196 Leu Thi- Arg Leu ILie GLy Thi- Tyr Thi- Asp Tyr Ale Vat Arg Ti-p 211 Tyr Asi, Thi- GLy Leo GLu Arg Vei Ti-p Giy Pro Asp Ser Arg Asp 226 Ti-p Vet Arg Tyr Asn Gin Phe Arg Arg Giu Leu Thi- Leu Thi- Val 241 Leu Asp ILie Vet Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thi- Tyr 256 Pro lie Arg Thi- Vat Ser Gin Leu Thi- Arg Giu Ilie Tyr Thi- Asn 271 Pro Vat Leu Giu Asn Phe Asp Giy Ser Phe Arg Giy Met Aia Gin 286 Arg lie Gtu Gin Asn lie Arg Gin Pro His Leu Met Asp Leu Leu 301 Asn Se Ilie Th- Ilie Tyr Thi- Asp Val His Arg Giy Phe Asn Tyr 316 Ti-p Ser GLy His Gin Ilie Thr Ala Ser Pro Vat GLy Phe Aia GLy 331 Pro Gtu Phe Thi- Phe Pro Arg Tyr Giy Thi- Met Giy Asn Ala Ala 346 Pro Pro Vat Leo ILie Ser Thi- Thi- Gly LeU Giy lie Phe Arg Thi- 361 Leo Ser Ser Pro Leu Tyr Arg Arg lie lie Leu Gly Ser GLy Pro 376 Asn Asn Gin Asn Leu Phe Val Leu Asp Gly Thi- Gbu Phe Ser Phe 391 Ale Ser Leu Thi- Ala Asp Leu Pro Ser Thi- lie Tyr Arg Gin Arg 406 Gly Thi- Vat Asp Ser Leu Asp Vat Ilie Pro Pro GIn Asp Asn Ser 421 Vet Pro Ala Arg Ala Gly Phe Ser His Arg Leu Ser His Vat Thi- 4436 Met Leu Ser Gin Ala Ala GLy Aia Val Tyr Thi- Leu Arg Ala Pro 451 Thr Phe Ser Ti-p Arg His Arg Ser Ala Gtu Phe Ser Asn Leu ILe I466 Pro Ser Ser Gin Ilie Thi- Gin Ilie Pro Leu Thr Lys Sec ILe Asn 481 Leu GLy Ser Gly Thr Ser Vat Vat Lys GLy Pro GLy Phe Thi- GLy 496 GLy Asp lie Leu Arg Arg Thi- Ser Pro Gly Gin lie Ser Thi- Leu 511 Arg Vat Thi- lie Thi- Ala Pro Leu Ser Gin Arg Tyr Arg Val Arg V526 1L~e Arg Tyr Ala Ser Thi- Thi- Asn Leu Gin Phe His Thr Ser ILe 541 Asp Gly Arg Pro Ilie Asn GIn GLy Asn Phe Ser Ala Thr Met Ser 556 Ser GLy GLy Asn Leu GIn Ser GLy Ser Phe Arg Thi- Aie GLy Phe 571 Thr Thi- Pro Phe Asn Phe Ser Asn GLy Ser Ser Ile Phe Thr Leo I'586 Ser Ala His Vat Phe Asn Ser Gly Asn GLu Val Tyr Ile Asp Arg V601 Ile Glu Plie Vat Pro Ale GLu Val Thi- Phe Gio Ala Gbu Tyr Asp 616 Leu Gbu Arg Ala Gin Gbu Ale Vat Asn Ala Leu Phe Thi- Ser Ser 631 Asn Gin Leo GLy Leu Lys Thi- Asn Vat Thr Asp Tyr His I e Asp 646 Gin Vat Ser Asn Leu Val Glu Cys Leo Ser GLY Glu Phe Cys Leo 661 Asp Gbu Lys Arg Gbu Leo Ser Gbu Lys Vat Lys His Ala Lys Arg 676 Leo Ser Asp Giu Arg Asn Leu Leu GIn Asp Pro Asn Phe Arg GLy 691 Ilie Asn Arg Gin Pro Asp Arg Gly Ti-p Acg GLy Ser Thr Asp Ilie 706 Th- Ilie Gin Gly Gty Asp Asp Vat Phe Lys GiU Asn Tyr Vat Thi- 721 Leu Pro GLy Thi- Phe Asn Giu Cys Tyr Pro Thi- Tyr Leu Tyr Gin 736 Lys lie Asp Gbu Ser Lys Leu Lys Ala Tyr Thi- Acg Tyr Gin Leo 751 Arg GLy Tyr lie Giu Asp Ser Gin Asp Leo Gbu Ile Tyr Leo lie 766 Acg Tyr Asn Thi- Lys His G~u Thi- Val Asn Val Pro Giy Thi- GLy 781 Sec Leo Ti-p Pro Leo Ser Vat Gbu Asn Pro Ile Gly Lys Cys GLy 796 Gtu Pro Asn Acg Cys Ala Pro Gin Leo Glu Ti-p Asn Pro Asp Leo 811 Asp Cys Ser Cys Arg Asp Gly Gbu Lys Cys Ala His His Ser His 36 MA.39.C1 Table 5 (continued) 826 His Phe Ser Lau Asp Ile Asp ILie Gly Cys Thr Asp Lau Asn GLu 841 Asn Lau Giy Vat Ti-p Vat I e Phe Lys ILie Lys Thi- Gin Asp Gly 856 His Ala Ai-g Lau Giy Asn Leu Giu Phe Leu Giu Giu Lys Proa Leu 871 Vat Gly Giu Ser Leu Ala Arg Vat Lys Arg Ala Giu Lys Lys Ti-p 886 Arg Asp Lys Arg Gtu Lys Leu Gin Vat Giu Thi- Asn Ilie Vat Tyr 901 Lys Gtu Ala Lys Giu Ser Vat Asp Ala Lau Phe Vat Asn Ser Gin 916 Tyr Asp Arg Lau Gin Ala Asp Thr Asp Ilie Ala Met lie His Ala 931 ALa Asp Lys Ar-g Vat His Arg Ilie Arg Giu Ala Tyr Leu Pr-a Giu 946 Leu Ser Vat Ile Proa Gty Vat Asn ALa GLy ILie Phe Giu Giu Leu 961 Gtu GLy Ar Ilie Phe Thi- Ala Tyr Ser Leu Tyr Asp Ala Arg Asn 976 Vat ILie Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cyis Trp Asn 991 Vat Lys GLy His Vat Asp Vat Giu GLU Gin Asn Asn His Arg Ser 1006 Vat Leu Vat Vat Proa Glu Ti-p Giu Ala GiU Vat Ser Gin Giu Vai 1021 Arg Vat Cys Pro Giy Arg Gly Tyr Ilie Leu Arg Vat Thi- Ala Tyr 1036 Lys Giu Giy Tyr GLy Giu Giy Cys Vat Thi- lie His Giu Ilie Giu 1051 Asp Asn Thi- Asp Giu Lau Lys Phe Ser Asn Cys Vat Giu Giu Giu 1066 Vat Tyr Pro Asn Asn Thi- Vat Thi- Cys Asn Asp Tyr Thr Ala Asn 1081 Gin Gtu Gtu Tyr Gty GLy Ala Tyr- Thr Ser A iq Asn Arg GLy Tyr 1096 GLy Giu Ser Tyr Giu Ser Asn Ser Ser Ilie Pro Ala Giu Tyr Ala 1111 Pro Vat Tyr GLU Gtu Ala Tyr Ile Asp Gly Arg Lys Giu Asn Pro 1126 Cys Giu Ser Asn Arg GLy Tyr Gty Asp Tyr Thi- Pr-o Leu Pro Ala I1141 Gly Tyr Vat Thi- Lys Giu Leu Giu Tyr Phe Pro Giu Thr Asp Lys 1156 Val Ti-p Ilie GLu ILie Gly GLu Thi- Giu GLy Thr Phe Ilie Vat Asp 1171 Ser Vat Giu Leu Leu Leu Met Giu Giu Fragment1- L I 37 MA39.C1 Table 6 10 15 Met GilJ Ile Met Asn Asn Gin Asn GIn Cys Vat Pro Tyr Asn Cys Leu Asn Asp Pro Thr ATG GAG ATA ATO MAT MAT CAG MAT CAA TGC GTT CCT TAT MAC TOT TTG MAT GAT CCG ACA 30 35 lie Glu Ilie Leu Glu Gty Giu Arg Ile Giu Thr Oiy Tyr Thr Pro Ile Asp Ile Ser Leu ATT GMA ATA TTA GMA GGA GMA AGA ATA GMA ACT GGT TAC ACC CCA ATA OAT ATT TCC TTG 50 55 Ser Leu Thr Gin Phe Leu Leu Ser Giu Phe Vat Pro Gly Ata GLy Phe Vat Leu Giy Leu TCG CTA ACG CMA TTT CTG TTG AGT GMA TTT GTC CCA GGT GCT GGG TTT GTA TTA GGT TTA 70 75 le Asp Leu Ilie Trp Giy Phe Vat Giy Pro Ser Gin Trp Asp Aia Phe Leu Vat Gin Ile ATT GAT TTA ATA TOG GGG TTT OTO GOT CCC TCT CMA TOG OAT GCA TTT CTT GTG CMA ATT 90 95 100 OiU Gin Leu Ilie Asn Gin Arg Ile OiU Giu P~e Aie Arg Asn Gin Aia Ilie Ser Arg Leu GMA CAG TTA ATT MAC CMA AGA ATA GAO GMA TTC OCT AGO MAC CMA OCA ATT TCT AGA TTA 105 110 115 120 Giu Giy Leu Ser Asn Leu Tyr Gin le 7yi Aie Giu Ate Phe Arg Oiu Trp Oiu Ale Asp GMA COG CTA AGC MAC CTT TAT CMA ATT TAC G' A GMA OCT TTT AGA GAG TOG GMA GCA CkT 125 130 135 140 Pro Thr Asn Pro Aia Leu Thr Giu Giu Met Arg lie Gin Phe Asn Asp ML. Asn Ser Ate CCT ACT MAT CCA GCA TTA ACA GMA GAG ATO COT ATT CAG TTC MAT GAC ATO MAC ACT OCT 145 150 55 160 Le'! Thr Thr Aie lie Pro Leu Phe Thr Vat .in Asn Tyr Gin Vat Pro Leu Leu Ser Vai CTT ACA ACC OCT ATT CCT CTT TTT ACA OTT CMA MT TAT CMA GTA CCT CTT CTA TCA GTA 165 170 175 180 Tyr Vai Gin Aie Aie Asn Leu His Leij Ser Vai Leu Arg Asp Vai Ser Vat Phe Oiy Gin TAT OTT CMA OCT OCA MAT TTA CAT TTA TCG OTT TTG AGA OAT OTT TCA OTG TTT OGA CMA 185 190 195 200 Arg Trp Ciy Phe Asp Vai Ate Thr le Aen Ser Arg Tyr Asn Asp LeLJ Thr Arg Leu I Le COT TOG 01A TTT OAT OTA OCA ACA ATC MAT ACT COT TAT MAT OAT TTA ACT AGO CTT ATT 205 210 215 220 oiy Thr Tyr Thr Asp Tyr Ate Vat Arg Trp Tyr Asn Thr Gly Leu Giu Arg Vat Trp G iy GOC ACC TAT ACA OAT TAT OCT OTA COC TOG TAT MAT ACO OGA TTA GMA COT OTA TOG OGA 'V225 230 235 240 Pro Asp Ser Arg Asp 7 rp Vat Arg Tyr Asn Gin Phe Arg Arg Giu Leu Thr Leu Thr Vai CCG OAT TCT AGA CAT TOO OTA AOG TAT MAT CAA TTT AGA AGA GAG CTA ACA CTA ACT OTA 245 250 255 260 Leu Asp ILie Vat Ser Leu Phe Pro Aen Tyr Asp Ser Arg Thr Tyr Pro lie Arg Thr Vat TTA OAT ATC OTT TCT CTO TTC CCG MAC TAT OAT ACT AGA ACO TAT CCA ATT CCA ACA OTT 265 270 275 280 Ser Gin Leu Thr Arg Giu Ile Tyr Thr Asn Pro Vat Leu Oiu Asn Phe Asp Oty Ser Phe TCC CMA TTA ACT AGA GMA ATT TAT ACA AAC CCA GTA TTA GMA MT TTT OAT GOT ACT TTT Table 6 (continued) 285 290 -195 300 Arg Gly Met Ala Gtn Arg lie Gtu Gin Asn lie Arg Gin Pro His Leu Met Asp Lau Leu CGT GGA ATG O!T CAG AGA ATA GMA CAG MAT ATT AGG CAA CCA CAT CTT ATG GAT CTC OTT 305 310 315 320 4Asn Ser Ile Thr lie Tyr Thr Asp Vat His Arg GLy Phe Asn Tyr Trp Ser Gty His Gin MAT AGT ATA ACC ATT TAT ACT GAT GTG CAT AGA GGC TTT MAT TAT TGG TCA GGA CAT CAA 325 330 335 340 3lie Thr Ate Ser Pro Vat Gly Phe Ale GLy Pro Glu Phe Thr Phe Pro Arg Tyr GLy Thr ATA ACA GOT TCT CCT GTC GGT TTT GCG GGG COX GMA TTT ACT TTT COT AGA TAT GGA ACC 345 350 355 360 Met Oty Asn Ala Ala Pro Pro Vat Leu lie Ser Thr Thr Giy Leu Gly lie Phe Arg Thr ATG GA MAT GOT GOT CCA CCC GTA CTG ATC TCA ACT ACT GGT TTG GG ATT TTT AGA ACA 365 370 375 380 Leu Ser Ser Pro Leu Tyr Arg Arg Ilie Ilie Leu Giy Ser Gty Pro Asn Asn Gin Asn Leu TTA TCT TCA CCT OTT TAC AGA AGA ATT ATA OTT GOT TCA GOC CCA MAT MAT CAG MAC CTG 385 390 395 400 Phe Vat Leu Asp Gly Thr Giu Phe Ser Phe ALn Ser Leu Thr Ala Asp Leu Pro Ser Thr rTT OTC CTT GAT OGA ACG GMA TTT TCT TTT 0CC TCC CTA ACA 0CC OAT TTA CCT TCT ACT 405 410 415 420 lie Tyr Arg Gin Arg Oty Thr Vat Asp Ser Leu Asp Vet Ile Pro Pro Gin Asp Asn Ser AT A TAC AGA CMA AGO GGA ACO OTC OAT TCA CTA GAT OTA ATA CCG CCA CAG OAT AA AOT 425 430 435 440 Vat Pro Ate Arg Ale Oty Phe Ser His Arg Leu Ser His Vat Thr Met Leu Ser Gin Ale GTO CCA OCA COT OCO OGA TTT AGT CAT COA TTA AOT CAT OTT ACA ATG CTG AGC CMA OCA 450 455 460 Ate GLy Ate Vat Tyr Thr Lau Arg Ate Pro Thr Phe Sf.-r Trp Arg His Arg Ser Ala Otu GOT OGA GCA OTT TAC ACC TTG AGA GOT CCA ACG TTT rCT TOG CGA CAT COT AGT GOT GMA 465 470 475 480 Phe Ser Aen Leu lie Pro 5cr Ser Gin lie Thr Gin lie Pro Leu Thr Lye Ser lie Asn 7TC TCT MAC CTA ATT CC-T TCA TCA CMA ATC ACA CAG ATA CCT TTA ACA AAG TCT ATT AAT 485 490 495 500 Leu Oty Ser oty Thr Ser Vat 'dat Lye Gly Pro Oty Phe Thr Gty oly Asp lie Leu Arg OTT OGC TCT 000 ACC TOT OTT CTT AAA OGA CCA GG TTT ACA OGA GGA OAT ATT OTT CGA 505 510 515 520 Arg Thr Ser Pro Gty Gin lie Ser Thr Leu Arg Vat Tnr Ile Thr Ate Pro Leu Ser Gin AGA ACT TCA COT GOC CAG ATT TCA ACC TTA AGA OTG ACT ATT ACT GOA CCA TTA TCA CMA 525 530 535 540 Are Tyr Arg Vat Arg ILe Arg Tyr Ata Ser Thr Thr Asn Lau Gtn Phe His Thr Ser lie AGA TAT COC OTA AGA ATT COC TAC GOT TCT ACT ACA MAT TTA CMA TTC CAT ACA TCA ATT 545 550 555 'D Asp OLy Arg Pro .te Aen Gin Gty Asn Phe Sur Ala Thr Met Scr Ser Oly Oty Aen Leu GAC GGA AGA CCT ATT AAT CAL G000 MT TTT TCA GOA ACT ATO AGT AGT 000 GOT AAT TTA 565 570 575 080 Gin Ser OLy Ser Phe Arg Thr Ate Gty Phe Thr Thr 1-ro Phe Aen Phe Ser Aen GLy Ser CAG TCC GGA AGC TTT AGO ACT GCA GOT TTT ACT ACT CCG TTT MAC TTT TCA MAT OGA TCA 39 MA39.Cl Table 6 (continued) 585 590 595 600 Ser Ile Phe Thr LeU Set Ala His Vat Phe Asn Ser Gty Asn Giu Vat Tyr Ile Asp Arg AGT ATA TTT ACG TTA AGT GCT CAT GTC TTC MAT TCA GGC MAT GAA GTT TAT ATA GAT CGA 605 610 615 620 Ile Giu Phe Vat Pro Ala Giu Vat Thr Phe Giu Ala Stu Tyr Asp Leu Stu Arg Ala Gin ATT GMA TTT OTT CCG GCA GMA OTA ACA TTT GAG GCG GMA TAT GAT TTA GMA AGA GCG CMA 625 630 635 Oiu Ala Vai Asn Ala Leu Phe Thr Ser Set Asn Gin Leu Oly Leu Lys Thr Asn Vai Thr GAG GCG GTG M.T OCT CTG TTT ACT TCT TCC MAT CMA CTA GOA TTA AAA ACA MAT OTG ACO 6.45 650 655 660 Asp Tyr His Ile A Gin Vat Ser Asn Lew Vat Stu Cys Leu Ser Sly Siu Phe Cys Lau f S~AC TAT CAT ATT GAT CMA OTO TCC MAT CTA OTC GMA TOT TTA TCC GOT GMA TTC TOT CTG 6.65 670 675 6.80 Asp Giu Lys Arg Giu Leu Set Gtu Lye Vat Lys His Ala Lys Arg Leu Ser Asp Giu Atg OAT GMA MG AGA GMA TTG TCC GAG MAA GTC AAA CAT OCO MAG COA CTC AOT OAT GAG CG 685 690 695 700 Asn Leu Lew Gin Asp Pro Aen Phe Arg Siy Ilie Aen Arg Gin Pro Asp Arg Sly Trp Arg MAT TTA CTT CMA GAC CCA MAC TTC AGA GOC ATC MAT AGA CMA CCA SAC COT GOC TOG AGA 705 710 715 720 Oly Set Thr Asp Ilie Tht Ile Gin Gly Gty Asp Asp Val Phe Lye Oiu Asn Tyr Vat Thr GOC AGT ACO OAT ATT ACC ATC CMA OGA OGA OAT GAC OTA TTC MAA GAG MAT TAC GTC ACA 725 730 735 740 t Leu Pro Siy Thr Phe Aen Oiu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Oiu Set CTA CCG GOT ACC TTT MAT GAG TGT TAT CCT ACG TAT CTO TAT CMA AAA ATA OAT GAG TCG 745 735)0 755 760 4 Lys Lewi Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Sly Tyr le Giu Asp Set Gin Asp Leu 4, M AA TTA MAA GCC TAT ACC COT TAC CMA TTA AGA GGG TAC ATC GAG GAT AGT CMA GAC TTA 765 770 775 780 Giu Ilie Tyr Leu Ilie Atg Tyr Aen Yhr Lye His Giu Thr Vat Aen Vai Pro Oty Thr Giy GMA ATC TAT TTA ATT COC TAC MAT ACA MA CAC GMA ACA OTA MAT OTO CCA GOT ACO GOT 785 790 79oo0 Set Leu Trp Pro Lew Ser Vai Gitu Aen Pro lie Gly Lye Cys Sly Giu Pro Aen Arg Cys TCC TTA TG-, CCG CTT TCA GTC GMA MT CCA ATT OGA MAG TOC OGA GMA CCA MAT COA TOC 805 810 5 15 Ala Pro Gin Lewi Giu 7tp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Giu Lye Cys OCA CCA CMA CTT GMA TOO MAT CC! OAT CTA OAT TOT TCC TOC AGA GAC 000 GM MA TOT 825 530 835 8.40 Ale His His Set His His Phe Set Lew Asp Ile Asp Ile Oty Cys Tht Asp Leu Asn Giu OCA CAT CAC TCC CAT CAT TTC TCC TTG GAC ATT GAT ATT OGA TOT ACA OAT TTA MAT GAG 845 850 855 860 Asn Lewi Gly Vat Trp Vat Ile Phe Lys Ile Lye Tht Gin Asp Sly His Ala Arg Lew Oty MAC TTA GOT GTA TOG GTG ATA TTC AAA ATT MAG ACG CAA OAT OGT CAC OCA AGA CTA COT 865 870 C775 880 Aen Leu Giu Phe Lew Gtu Giu Lye Pro Leu Vat Sly Oiu Set Leu Ate Arg Vat Lys Atg MAT CTA GAO TTT CTC GMA GAG AAA CCA TTA GTA GOC GMA TCG TTA GCA CGC OTG MOG AGA MA39.C1 Table 6 (continued) 885 890 895 900 Ata Glu Lys Lys Trp Arg Asp Lys Arg Oiu Lys Leu Gin Vat Glu Thr Asn Ite Vat Tyr GCG GAG AAG MAG TOO AGA GAC AAA CGA GAG AAA TTG CMA GTG GMA ACA AAT ATC OTT TAT 905 910 915 920 Lys GLU Ala Lys Olu Ser Vat Asp Ala Leu Phe Vat Asn Ser Gin Tyr Asp Arg Leu Gin AAA GAG GCA AAA GMA TCT GTA GAT OCT TTA TTT GTG MAC TCT CMA TAT GAT AGA TTA CMA 925 930 935 940 Ala Asp Thr Asp Ilie Ala Mat Ile His Ala Ala Asp Lys Arg Vat His Arg Ile Arg Giu GOOG GAT ACC GAC ATC GCT ATG ATT CAT GCG GCA GAT MAA CGC GTT CAT COA ATT CGA GMA 945 950 955 960 Ala Tyr- Leu Pro Oiu Leu Ser Vat Ile Pro Gly Vat Asn Ala Gly ike Phe Gtu Gtu Leu G:A TAT CTT CCA GAG TTA TCT GTA ATT CCG GGT GTC M.T GCG GGC ATT TTT GMA GMA TTA 965 970 975 980 GLu Gly Arg Ile Phe Thr Ala Tyr Sar Leu Tyr Asp Ala Arg Asn Vat Ilie Lys Asn Oty GAG GGA CGT ATT TTC ACA GCC TAC TCT TTA TAT GAT GCG AGA MAT GTC ATT MAA MAT GGC 985 990 995 1000 Asp Pha Aan Asn Gly Leu Sar Cya Trp Asn Vat Lys Gly His Vat Asp Vat Glu Gtu Gin GAT TTC MAT MkT GGC TTA TCA TOC TGG MAC GTG AM. GGG CAT GTA GAT GTA GMA GM CMA 1005 1010 1015 1020 Asn Aan His Arg Ser Vat Leu Vat Vat Pro Giu Trp Gtu Ala Otu Vat Ser Gin Giu Vat MAC MAC CAC COT TCG OTT CTT OTT OTC CCG GMA TG GMA OCA GAG OTO TCA CAA GAG GTT 1025 103C 1035 1040 Arg Vat Cys Pro Oty Arg Oty Tyr ike Leu Arg Vat Thr Ate Tyr Lys GiU Oty Tyr Gly COT GTC TOT CCA GOT COT GGC TAT ATC CTA CGT OTT ACA GCG TAC AAA GAG GGA TAT GGA 10.45 1050 1055 1060 Giu GLy Cys Vat 'hr Ile His Gtu Ilie GLU Asp Asn Thr Asp Giu Leu Lya Phe Ser Asn GMA GGT TGC GTA ACG ATT CAT GAG ATC GMA GAC MAT ACA GAC GMA CTG AAA TTC AGC MAC 1065 1070 1075 1080 Cys Vat Giu Giu Giu Vat Tyr Pro Asn Asn Thr Vat Thr Cys Asn Asp Tyr Thr Ate Asn TOT GTA GMA GAG GMA GTA TAT CCA MAC MAC ACO OTA ACO TOT MAT OAT TAT ACT OCA MAT 1085 1090 1095 1100 Gin Giu Giu Tyr Giy Gly Ate Tyr Thr Ser Arg Aen Arg GLy Tyr Oty Giu Ser Tyr Giu CAA GMA GM TAC 000 GOT GCO TAC ACT TCT COT AAT COT OGA TAT GOT GMA TCT TAT GMA 1105 1110 1115 1120 Ser Aen Ser Ser Ike Pro Ale Otu Tyr Ate Pro Vat Tyr Giu Gtu Ate Tyr ike Asp GLy AGT MAT TCT TCC ATA CCA OCT GAG TAT OCO CCA OTT TAT GAG GMA OCA TAT ATA OAT OGA 1125 1130 1135 1140 Arg Lys Gtu Asn Pro Cys Otu Ser Aen Arg Gly Tyr Oty Asp Tyr Thr Pro Leu Pro Ale AGA AAA GAG MAT CCT TO2T GMA TCT MAC AGA OGA TAT 000 OAT TAC ACO CCA CTA CCA OCT 1145 1150 1155 116,0 Oty Tyr Val Thr Lys Gtu Leu Otu Tyr Phe Pro Gtu Thr Asp Lye Vat Trp ILe Otu ike GOT "*AT OTG ACA AM AA TTA GAG TAC TTC CCA GMA ACC GAT MAG OTA TOG ATT GAG ATC 1165 1170 1175 Gty Gtu Thr Oiu Gty Thr Phe Ile Vat Asp Car Vat Otu Lwu Leu Leu Mat Oiu Oiu 000 GMA ACO GMA GA ACA TTC ATC GTO OAT AGC GTO GMA TTA CTC CTT ATO GAG GAA

Claims (18)

1. A process for controlling lepidopteran insect pests which comprises contacting said insect pests with an insect-controlling effective amount of Bacillus thuringiensis PS81A2, having the identifying characteristics of NRRL B-18457, or Bacillus thuringiensis PS81RR1, having the identifying characteristics of NRRL B-18458, or mutants thereof which retain the characteristics of the parent strains.

2. The process, according to claim 1, wherein said mutants are asporogenous mutants and/or phage resistant mutants.

3. The process, according to claim 1, wherein said insect pest is contacted with an insect-controlling effective amount of Bacillus thuringiensis PS81A2 or PS81RR1, by incorporating said Bacillus thuringiensis PS81A2 or PS81RR1 into a bait granule and placing said granule on or in the soil when planting seed of a plant upon which plant insect pest is known to feed.

4. A process for controlling soil-inhabiting insect pests of the order Lepidoptera which comprises preparing a bait granule comprising Bacillus thuringiensis PS81A2 or PS81RR1, or mutants thereof which retain the characteristics of the parent strains, or spores or crystals of Bacillus thuringiensis PS81A2 or PS81RR1; and placing said bait granule on or in the soil.

5. The process, according to claim 4, wherein said bait granule is applied at the same time corn seed is planted in the soil.

6. The process, according to claims 1 or 4, wherein substantially intact Bacillus thuringiensis PS81A2 or PS81RR1 cells, or mutants thereof which retain the characteristics of the parent strains, are treated to prolong the pestidical activity when the substantially intact cells are applied to the environment of a target pest.

7. A composition of matter comprising Bacillus thuringiensis PS81A2 or PS81RR1, or mutants thereof which retain the characteristics of the parent strains, or spores or crystals of Bacillus thuringiensis PS81A2 or PS81RR1, in association with an insecticide carrier, wherein said mutants are asporogenous mutants and/or phage resistant mutants.

8. The composition of matter according to claim 7, wherein said carrier comprises phagostimulants or attractants.

9. A composition of matter comprising Bacillus thuringiensis PS81A2 or PS81RR1, or mutants thereof which retain the characteristics of the parent strains, in association with formulation ingredients applied as a seed coating, wherein said mutants are asporogenous mutants and/or phage resistant mutants. Bacillus thuringiensis PS81A2, having the identifying characteristics of NRRL B-18457, or mutants thereof which retain the characteristics of the parent strain, having S 132814rt A 42 activity against insect pests of the order Lepidoptera.

11. Bacillus thuringiensis PS81RR1, having the identifying characteristics of NRRL B-18458, or mutants thereof which retain the characteristics of the parent strain, having activity against insect pests of the order Lepidoptera.

12. Asporogenous and/or phage resistant mutants of Bacillus thuringiensis PS81A2 or Bacillus thuringiensis PS81RR1 which retain the characteristics of the parent strains. I 11 Va 32614r r 2;i i 43 MA39.C1 1 13. DNA encoding a Bacillus thuringiensis toxin having the amino acid 2 sequences shown in Table 2 or Table 1 14. DNA, according to claim 13, having the nucleotide sequences shown 2 in Table 1 or Table 4, respectively. 1 15. Toxin active against lepidopteran insects having the amino acid 2 sequence shown in Table 2 or Table 5, and mutants thereof which do not alter 3 the protein secondary structure, or if the structure is altered, the biological 4 activity is retained to some degree. 1 16. A recombinant DNA transfer vector comprising DNA having all or 2 part of the nucleotide sequence which codes for the amino acid sequence shown 3 in Table 2 or Table 1 17. The DNA transfer vector, according to claim 16, transferred to and 2 replicated in a prokaryotic or eukaryotic host. 1 18. A bacterial host transformed to express a Bacillus thuringiensis toxin 2 having the amino acid sequence shown in Table 2 or Table S 1 19. Escherichia coli, according to claim 18, transformed with a plasmid 2 vector containing the Bacillus thuringiensis toxin gene encoding the Bacillus 3 thuringiensis toxin having the amino acid sequence shown in Table 2 or Table 1 20. Escherichia coli (NM522)(pMYC389), having the identifying 2 characteristics of NRRL B-18448, or Escherichia coli (NM522)(pMYC390), i 4i 0 0 0) 44 MA39.C1 3 having the identifying characteristics of NRRL B-18449, hosts according to claim 4 18. 1 21. A microorganism according to claim 18, which is a species of 2 Pseudomonas, Azotobacter Erwinia Serratia, Klebsiella Rhizobium, 3 Rhodopseudomonas, Methylophilius. Agrobacterium, Acetobacter, Alcaligenes, 4 Bacillus, or Streptomvces. 1 22. A microorganism according to claim 21, wherein said microorganism o 2 is pigmented and phylloplane adherent. oQ *o 1 23. A method for controlling lepidopteran insects which comprises 2 administering to said insects or to the environment of said insects a 3 microorganism according to claim 21. 1 24. A method according to claim 23, wherein said administration is to 2 the rhizosphere. 23 1 25. A method according to claim 24, wherein said administration is to 2 the phylloplane. 1 26. A method according to claim 23, wherein said administration is to a 2 body of water. 1 27. An insecticidal composition comprising insecticide containing 2 substantially intact, treated cells having prolonged pesticidal activity when applied 3 to the environment of a target pest, wherein said insecticide is a polypeptide 4 toxic to lepidopteran insects, is intracellular, and is produced as a result of MA39.C1 expression of a transformer' ,icrobe capable of expressing the Bacillus 6 thuringiensis toxin having the amino acid sequence shown in Table 2 or Table 1 28. The insecticidal composition, according to claim 27, wherein said 2 treated cells are treated by chemical or physical means to prolong their 3 insecticidal activity in the environment. 1 29. The insecticidal composition, according to claim 28,.wherein said 2 cells are prokaryotes or lower eukaryotes. t 1 30. The insecticidal composition, according to claim 29, wherein said 2 prokaryotic cells are selected from the group consisting of Enterobacteriaceae, 3 Bacillaceae, Rhizobiaceae, Spirillaceae, Lactobacillaceae, Pseudomonadaceae, 4 Azotobacteraceae, Nitrobacteraceae, and Actinomycetales. 1 31. The insecticidal composition, according to claim 29, wherein said 2 lower eukaryotic cells are selected from the group consisting of Phycomycetes, 3 Ascomycetes, and Basidiomycetes. 1 32. The insecticidal composition, according to claim 27, wherein said cell 2 is a pigmented bacterium, yeast, or fungus. 1 33. Treated, substantially intact unicellular microorganism cells containing 2 an intracellular toxin, which toxin is a result of expression of a Bacillus 3 thuringiensis toxin gene toxic to lepidopteran insects which codes for a 4 polyp-ptide toxin having the amino acid sequence shown in Table 2 or Table wherein said cells are treated under conditions which prolong the insecticidal 6 activity when said cells are applied to the environment of a target insect. S1'i i t 46

34. The cells according to claim 33, wherein the cells are treated by chemical or physical means to prolong their insecticidal activity in the environment. The cells according to claim 33, wherein said microorganism is Pseudomonas and said toxin is a Bacillus thuringiensis toxin having the amino acid sequence shown in Table 2 or Table

36. Pseudomonas cells according to claim 35, wherein said cells are treated with iodine.

37. The cells, according to claim 33, which are Pseudomonasfluorescens.

38. Plasmid denoted pMYC389 or pMYC390, according to claim 17.

39. A composition of matter comprising Bacillus thuringiensis PS81A2 or PS81RR1, or mutants thereof which retain the characteristics of the parent strains, spores or crystals of Bacillus thuringiensis PS81A2 or PS81RR1, in association with an insecticide carrier, wherein said mutants are asporogenous mutants and/or phage resistant mutants, which composition is substantially as hereinbefore described with reference to Example 1. A process for controlling lepidopteran insect pests which comprises contacting S said insect pests with an insect-controlling effective amount of the composition of claim 39.

41. A recombinant DNA transfer vector comprising DNA having all or part of the nucleotide sequence which codes for the amino acid sequence shown in Table 2 or Table 5, which vector is substantially as hereinbefore described with reference to Example 2 or Example 3.

42. A process for preparing a recombinant DNA transfer vector comprising DNA Ii having all or part of the nucleotide sequence which codes for the amino acid sequence S 25 shown in Table 2 or Table 5, which process is substantially as hereinbefore described with n |reference to Example 2 or Example 3. S"'i 43. A bacterial host transformed to express a Bacillus thuringiensis toxin having the amino acid sequence shown in Table 2 or Table 5, which host is substantially as hereinbefore described with reference to Example 2 or Example 3. Dated this TWENTY-THIRD day of OCTOBER 1992 Mycogen Corporation Patent Attorneys for the Applicant SPRUSON FERGUSON ir i14rt 1I Yajs^'' t i