WO1991016434A2 - BACILLUS THURINGIENSIS cryIF AND cryIX GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS - Google Patents

Abstract

Two purified and isolated cryI-type genes were obtained from a novel B.t. strain. One gene, designated cryIF (SEQ ID NO:1), has a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1. The 134 kDa crystal protein, designated CryIF (SEQ ID NO:2), produced by this gene is toxic to European corn borer larvae and other lepidopteran insects. The second gene, designated cryIX, produces a crystal portein of about 81 kDa, designated CryIX, that is also toxic to lepidopteran insects.

Description

BACILLUS THURINGIENSIS crylF AND crylX GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS

This material is based upon work supported by the National Science Foundation under Grant No. ISI-8700011. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the Foundation.

Field of the Invention

The present invention relates to two genes isolated from Bacillus thuringiensis (hereinafter "B.t.") encoding insecticidal crystal proteins designated CrylF and CrylX, respectively, as well as insecticidal compositions containing the proteins and plants transformed with the genes. The insecticidal compositions and transformed plants are toxic to insects of the order Lepidoptera.

Background of the Invention

B.t. is a gram-positive soil bacterium that produces crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B.t. have been shown to produce insecticidal crystal proteins. Compositions including B.t. strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms. A number of genes encoding crystal proteins have been cloned from several strains of B.t. A good overview is set forth in H. Hofte et al., Microbiol. Rev., 53, pp. 242-255 (1989), hereinafter "Hofte and hiteley (1989)." While this reference is not prior art with respect to the present invention, it provides a good overview of the genes and proteins obtained from B.t. and their uses, adopts a nomenclature and classification scheme for B.t. genes and proteins, and has an extensive bibliography.

The B.t. crystal protein is active in the insect only after ingestion. After ingestion by an insect, the alkaline pH and proteolytic enzymes in the mid-gut solubilize the crystal allowing the release of the toxic components. These toxic components disrupt the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B.t. has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.

As noted by Hofte and Whiteley (1989) , the majority of insecticidal B.t. strains are active against insects of the order Lepidoptera, i.e., caterpillar insects. These B.t. strains characteristically contain cryl-type genes that make Cryl crystal proteins. Other B.t. strains produce different classes of crystal proteins, e.g.. CryIV protein, active against insects of the order Diptera, i.e., flies and mosquitoes, or Cryll protein active against both lepidopteran and dipteran insects. In recent years, a few B.t. strains have been reported as producing a new class of crystal protein, Crylll protein, that is insecticidal to insects of the order Coleoptera, i.e. , beetles.

Lereclus et al., in Chapter 13 of Regulation of Procaryotic Development, I. Smith et al. (eds.), American Society for Microbiology, Washington, D.C., pp. 255-276 (1989), review the role, structure and molecular organization of crystal protein genes. A summary of toxin genes identified in B.t. is provided in Table 2 (p. 260) and amino acid sequence comparisons of the various crystal proteins reported in the literature are diagrammed in Figures 1 and 2. This reference is not prior art with respect to the present invention. Schnepf et al., J. Biol. Chem. , 260, pp. 6264-6272 (1985) , report the complete nucleotide sequence for a toxin gene from B.t. kurstaki HD-1 (Figure 2, pp. 6266-6267); this gene was subsequently classified as the cryΙA(a) gene by Ho^'fte and Whiteley (1989) . The published open reading frame extends 1176 amino acids and encodes a protein with a calculated M of 133,500 daltons (Da).

Wabiko et al., DNA, 5_, pp. 305-314 (1986) , describe the DNA sequence of an insecticidal toxin gene from B.t. subsp. berliner 1715, subsequently classified as cryΙA(b) by Hofte and Whiteley (1989) . The molecular mass of the protein encoded is 130,615 Da and sequential deletions indicate that the NH_-terminal 612 amino acid polypeptide is toxic.

Hofte et al., Eur. J. Biochem. , 161, pp. 273-280 (1986) , describe the cloning and nucleotide sequencing of a crystal protein gene from B.t. subsp. berliner 1715, subsequently classified as cryΙA(b) by Hofte and Whiteley (1989) . The cloned gene produces a 130 kilodalton (kDa) protein which coincides with the size of the major protein observed in the strain. A restriction map of the cloned toxin gene is presented (p. 276, Figure 3A) . Toxicity data for the cloned gene is shown in Table 1 (p. 275) . The complete nucleotide sequence for this gene is shown in Figure 3B (p. 276) . It has an open reading frame of 3466 bases which would encode a protein 1155 amino acids in length having a molecular weight of 130,533 Da. Similarities of this sequence to the previously reported sequences for the cloned crystal genes from B.t. kurstaki HD-1, B.t. kurstaki HD-73 and B.t. sotto are discussed on p. 278 and summarized in Figures 6 and 7 (pp. 279 and 280, respectively). Data identifying a minimal toxic fragment required for insecticidal activity is also presented (pp. 277 and 278, Figure 4, Table 2).

Adang et al.. Gene, 36, pp. 289-300 (1985), report the cloning and complete nucleotide sequence of a crystal protein gene harbored on the 75 kb plasmid of strain B.t subsp. kurstaki HD-73. The restriction map (Figure 2, p. 292) identifies this gene as cryΙA(c) under the current classification system of Hofte and Whiteley (1989) . The complete sequence of the gene, spanning 3537 nucleotide base pairs (bp) , coding for 1178 amino acids and potentially encoding a protein of M 133,330 Da, is shown in Figure 3 (p. 294).

Sequence comparisons of this gene and the published N-terminal sequence for the B.t. kurstaki HD-1 Dipel gene reveal only 41 base pair differences, concentrated within the last 376 base pairs of the HD-1 sequence (p. 293) . The 5' regulatory sequences are identical in both clones. A schematic of this comparison is shown in Figure 4 (p. 295) . Toxicity data against Manduca sexta for the protein made by the full length HD-73 gene are also presented (Table II, p. 296).

Brizzard et al., Nucleic Acids Res., 16 (6) , pp. 2723-2724 (1988) , describe the nucleotide sequence of crystal protein gene cryA4 (subsequently classified as crylB by Hofte and Whiteley (1989)) isolated from B.t. strain HD2. This report makes a cursory statement distinguishing the amino-terminal region of the cryIB gene from those of the cryΙA(a) , cryΙA(b) and cryΙA(c) genes. The sequence of this gene is further differentiated from the three crylA genes by virtue of its TTG translational start codon. Honee et al., Nucleic Acids Res., 16 (13) , p. 6240 (1988) , describe the complete DNA sequence for the BTVI crystal protein gene isolated from B.t. subsp. entomocidus 60.5 (termed crylC by Hofte and Whiteley (1989) ) . Extensive homology to the cryΙA(a) crystal protein gene was evident downstream from the proteolytic cleavage site. Visser et al., Mol. Gen. Genet. , 212, pp. 219-224 (1988) , report the isolation and analysis of five toxin genes belonging to four different gene families from B.t. entomocidus 60.5 (see Figure 2, p. 221). Two of these, BTIV and BTVIII, are cryΙA(a)-type genes according to the Hofte and Whiteley (1989) classification scheme. Two additional genes, BTVI and BTV, reside in opposite orientations on the same recombinant plasmid and are separated by approximately 3 kilobase (kb) of intervening DNA. BTVI is the crylC gene according to the Hofte and Whiteley (1989) classification scheme. The authors state that the restriction map for BTV closely resembles that identified for a gene isolated from B.t. strain HD68 subsp. aizawai, now termed the cryID gene (p. 222) . A fifth gene, BTVII, is also identified and its restriction map differs significantly from the other four genes described. Toxicity data against S^_ exigua, S. littoralis, H. virescens and P^ brassicae are presented for each of the isolates (see Table 1, p. 223) . BTVI is highly active against Spodoptera larvae. BTVII is toxic to P^ brassicae. The BTV gene product was inactive against all insects tested.

Sanchis et al., Mol. Microbiol. , __, pp. 393-404 (1988) , describe the isolation of recombinant clones containing two novel B.t. toxin genes from strains aizawai 7.29 (plasmids pHTA4 and pHTA6) and entomocidus 601 (pHTE4 and pHTE6) . Toxin genes on pHTA6 and pHTE6 have been subsequently classified as crylC according to the Hofte and Whiteley (1989) classification scheme. Restriction map data (see Figure 5, p. 398) indicate that the novel genes are in close proximity to each other (3 kb apart) . The restriction maps and molecular organization for the novel aizawai genes are identical to the entomocidus genes, except for a 1.4 kb insert in the upstream entomocidus gene. Toxicity data are presented for Escherichia coli (E. coli) expression constructs of these novel genes (see Table 2, p. 400 and discussion on p. 401) . The proteins produced by these crylC genes are highly toxic to S. littoralis and 1___ brassicae. No significant toxicity was demonstrated for the protein of the novel gene on plasmid pHTA4. Sanchis et al., Mol. Microbiol., __, pp. 229-238 (1989) , report the nucleotide sequence for the N-terminal coding region (2470 nucleotides) and 5' flanking region of a gene from B.t. subsp. aizawai 7.29 now classified as the crylC gene under the classification system of Hofte and Whiteley (1989) . The open reading frame encodes a polypeptide 824 amino acids long with a calculated molecular weight of 92,906 Da (see sequence. Figure 1, p. 231). Comparative analysis of this sequence to other known B.t. toxin genes indicates that for the N-terminal DNA sequence (amino acids 1-281) , the CrylC protein has only 58% identity to other PI proteins. The more homologous C-terminal region (amino acids 619-824) has less than 10% variability. The authors report the identification of five N-terminal conserved domains present in lepidopteran-active, dipteran-active and coleopteran-active endotoxins. Sanchis et al., in European Patent Application Publication No. 0 295 156, published December 14, 1988, disclose the DNA and amino acid sequences of a truncated gene, the crylC gene, and encoded crystal protein isolated from B.t. subsp. aizawai 7.29. The sequence revealed is the same as that in Sanchis et al.. Molecular Microbiol., 3_, pp. 229-238 (1989) , described above.

Schnepf et al., in U.S. Patent 4,467,036, issued August 21, 1984, disclose a hybrid recombinant plasmid capable of replication in an E. coli strain, and capable of expressing a polypeptide with the immunological properties of B.t. crystal protein, and being identifiable with a PvuII C DNA fragment probe derived from a gene, now known as cryΙA(a) , of B.t. var. kurstaki strain HD-1. The following B.t. subspecies and strains are disclosed as sources of expressible heterologous DNA: tolworthi, darmstadiensis, sotto, thuringiensis, kurstaki, HD-290, HD-120,

HD-2, HD-244, HD-73, HD-1, HD-4, HD-8, F-6, F-5 and F-9.

Generally, the sequences of B.t. genes encoding delta-endotoxin proteins active against different orders of insects are not well-conserved. Rather, the sequences of genes responsible for a given crystal phenotype and active against the same insect order are significantly more related. The homology of delta-endotoxin amino acid sequences, as well as similarities in insecticidal activity, have been used to define an ordered classification of genes encoding B.t. delta-endotoxin proteins (Hofte and Whiteley (1989)). The genes encoding the 130-138 kDa, lepidopteran-active delta- endotoxin proteins comprise the largest of these families, the cryl genes.

Within the cryl gene classification described by Hofte and Whiteley (1989) , a subranking has been established based upon further refinement of sequence relationship. Thus, the cryΙA(a) , cryΙA(b) and cryΙA(c) gene sub-families embrace the previously designated 4.5 kb, 5.3 kb and 6.6 kb PI genes, respectively. These genes originally were differentiated according to the size of a characteristic Hindlll fragment associated with the presence of the gene. The amino acid sequences of CrylA proteins are highly related (greater than 80% amino acid identity) , with most of the sequence dissimilarity relegated to a short internal variable region (Whiteley et al., Ann. Rev. Microbiol., 40, pp. 549-576 (1986)). It is believed that differences within this variable region account for the different insecticidal specificities exhibited by the proteins encoded by the cryΙA(a) , cryΙA(b) and cryΙA(c) genes.

Recently, additional genes within the cryl family have been discovered, such as the crylB gene found in B.t. subsp. thuringiensis (Brizzard et al., (1988), supra) , and the crylC and crylD genes found in subsp. aizawai (Sanchis et al., (1988), supra, Visser et al. (1988), supra, Hofte and Whiteley (1989) , European Patent Application Publication No. 0 358 557, published March 14, 1990, of Plant Genetic Systems, N.V.), and the crylE gene found in B.t. subsp. dar stadiensis (EP 0 358 557 (1990) , supra) and in B.t. subsp. kenyae (Visser et al., J. Bacteriol. , 172, pp. 6783-6788 (1990)). Other cryI-type genes are disclosed in European Patent Application Publication No. 0 367 474, published May 9, 1991, of Mycogen Corporation, in European Patent Application Publication No. 0 401 979, published December 12, 1990 of Mycogen Corporation, and in PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC. Comparisons of the sequences for these cryl-type genes to the crylA genes reveal significant sequence dissimilarities, particularly in the N-terminal protein domain. The present invention is based on the discovery of at least one additional subgroup of cryl genes. The prototype of this subgroup, which the inventors have designated cryIF, was also isolated from a B.t. subsp. aizawai strain. However, the sequence of the crylF gene and the insecticidal specificity of the CrylF protein it encodes, is distinctly different from the other cryl genes and their encoded Cryl proteins. This distinction is also true with respect to the crylC gene and its encoded protein, even though a spontaneously cured derivative of a B.t. strain containing the crylC gene was used in the isolation of the crylF gene.

In addition, the present invention includes the identification and isolation of a second gene, designated cryIX, which is located downstream from the novel crylF gene. Of the cryl-type genes discussed above, the crylX gene appears most closely related to the B.t. toxin gene disclosed in PCT International Publication No. WO 90/13651, published November 15, 1990, of Imperial Chemical Industries PLC. Data are presented hereinafter concerning the identification, cloning, sequencing and expression of these novel crylF and crylX toxin genes, as well as the insecticidal activities of the CrylF and CrylX proteins against lepidopteran larvae.

Summary of the Invention

One aspect of the present invention relates to a purified and isolated lepidopteran- active toxin gene having a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1 and hereinafter designated as the crylF gene (SEQ ID N0:1) . The crylF gene (SEQ ID N0:1) has a coding region, i.e., open reading frame, extending from nucleotide bases 478 to 3999 shown in Figure 1.

Another aspect of the present invention relates to the insecticidal protein produced by the crylF gene (SEQ ID N0:1) . The CrylF protein (SEQ ID NO:2) has the amino acid sequence, as deduced from the nucleotide sequence of the crylF gene from bases 478 to 3999, that is shown in Figure 1. The protein exhibits insecticidal activity against insects of the order Lepidoptera, in particular, Ostrinia nubilalis (European corn borer) and Spodoptera exigua (beet armyworm) .

Another aspect of the present invention relates to a purified and isolated insecticidal toxin gene hereinafter designated as the crylX

ET gene, a portion (SEQ ID NO:3) of whose nucleotide base sequence is shown in Figure 2. The present invention also relates to the insecticidal protein produced by the crylX gene and called the CrylX protein, which protein has a molecular mass of about 81 kDa. A portion (SEQ ID NO:4) of the amino acid sequence for the 81 kDa CrylX protein, as deduced from the truncated portion (SEQ ID NO:3) of the crylX gene, is also shown in Figure 2. The 81 kDa CrylX protein exhibits insecticidal activity against lepidopteran insects, such as Plutella xylostella (diamondback moth) and Ostrinia nubilalis (European corn borer) .

Still another aspect of the present invention relates to biologically pure cultures of B.t. and E_^ coli bacteria deposited with the NRRL having Accession Nos. NRRL B-18633, B-18635, and B-18805 and being designated as B.t. strains EG6345 and EG1945, and E__;_ coli strain EG1083, respectively. B.t. strains EG6345 and EG1945 carry the crylF gene and produce the insecticidal CrylF protein. E__;_ coli strain EG1083 carries the cryIX gene and produces the CrylX protein. Biologically pure cultures of other B.t. bacteria carrying the crylF gene or of B.t. strains carrying the crylX gene are also within the scope of this invention.

Yet another aspect of this invention relates to insecticidal compositions containing, in combination with an agriculturally acceptable carrier, either the CrylF or CrylX protein or fermentation cultures of a B.t. strain which has produced the CrylF protein or the CrylX protein.

TITUTE SHEET The invention also includes a method of controlling lepidopteran insects by applying to a host plant for such insects an insecticidally effective amount of the CrylF protein or the CrylX protein or of a fermentation culture of a B.t. strain that has made the CrylF protein or the CrylX protein.

Still other aspects of the present invention relate to recombinant plasmids containing the crylF gene and/or the crylX gene; biologically pure cultures of a bacterium transformed with such recombinant plasmids, the bacterium preferably being B.t. , such as the aforementioned B.t. strain EG1945; as well as plants transformed with the crylF gene and/or the crylX gene.

Brief Description of the Drawings

Figure 1 comprises Figures 1-A through 1-E and shows the partial nucleotide base sequence of DNA from a 5.7 kb fragment that contains the crylF gene inserted into plasmid pEG640. The DNA sequence begins with the 5' Sau3A cloning site and extends 4020 bp in length. The open reading frame for the crylF gene and the deduced amino acid sequence of the CrylF protein are indicated. The putative ribosome binding site (RBS) for the crylF gene is indicated on Figure 1-A. Sites for the restriction enzymes Sau3A, Ba HI and Kpnl are also indicated.

Figure 2 comprises Figures 2-A and 2-B and shows the partial nucleotide base sequence of DNA from the 5.7 kb fragment inserted into plasmid pEG640 that contains a portion of the crylX gene. The DNA sequence begins with nucleotide base position 4021 in Figure 2-A, which is immediately adjacent to and downstream from position 4020 in Figure 1-E, and extends to nucleotide base position 5649 in Figure 2-B, ending at the 3' Sau3A cloning site. The open reading frame for the truncated cryIX gene and the deduced amino acid sequence of the CrylX protein encoded by the crylX gene fragment are indicated. The putative ribosome binding site (RBS) for the crylX gene is indicated. Sites for the restriction enzymes Kpnl and Sau3A are indicated.

Figure 3 comprises a photocopy of a portion of an ethidium bromide stained agarose electrophoresis gel containing size fractionated native plasmids of B.t. subsp. aizawai strains EG6346 in the left lane and EG6345 in the right lane. The number to the right of Figure 3 indicates the approximately 45 MDa plasmid of B.t. strain EG6345 which is absent in the cured B.t. strain EG6346.

Figure 4 comprises Figures 4-A, 4-B and 4-C, which are photocopies of autoradiograms made by transferring total Hindlll-digested DNA from B.t. strains EG6346 (lane 1 of each autoradiogram) , EG6345 (lane 2 of each autoradiogram) and HD-1 (lane 3 of each autoradiogram) to nitrocellulose filters, hybridizing the filters with radioactively labeled probes and exposing the filter to X-ray film. The DNA in the autoradiogram labeled

Figure 4-A follows hybridization of the DNA to a

32 P-labeled 0.7 kb EcoRI probe from the cryΙA(a) gene of B.t. strain HD-l. The autoradiogram labeled Figure 4-B follows hybridization of the DNA to a 32P-labeled mtragenic 2.2 kb PvuII probe from the cryΙA(a) gene of B.t. strain HD-1. The autoradiogram labeled Figure 4-C follows hybridization of the DNA to a 32P-labeled plasmid pEG640 probe. The numbers to the left of Figure 4 indicate the sizes, in kb, of standard DNA fragments of phage lambda.

Figure 5 shows a restriction map of plasmid pEG640. The locations and orientations of the crylF gene and a gene designated the crylX gene are indicated by arrows. The solid black line indicates the E__ coli cloning vector pGEM™-3Z. The following letters designate the indicated restriction enzymes: B = BamHI B2 = BstEII; C = Clal; E = EcoRI; H = Hindlll; K = Kpnl; Ptl = PstI; Pv2 = PvuII; S = Sad; X = Xbal.

Figure 6, based on the same scale as Figure 5, shows a restriction map of plasmid pEG642 which was created by inserting plasmid pEG640 into a Hindlll site on the Bacillus vector pEG434. The abbreviations and other indicators referred to with respect to Figure 5 apply with respect to Figure 6. In addition, the crosshatched area of Figure 6 indicates vector pEG434 and the arrow labeled "tet" indicates the direction of transcription of the tetracycline resistance gene encoded on plasmid vector pEG434.

Figure 7 is a photocopy of a Coomassie stained SDS-polyacrylamide gel showing gradient purified crystal protein from B.t. strain EG6345 (lane 1) , B.t. strain EG6346 (lane 2) and recombinant B.t. strain EG1945 harboring the crylF gene (lane 3) . The unnumbered, extreme left lane adjacent to lane 1 contains molecular weight standards having the indicated sizes, in kDa.

Figure 8 comprises Figures 8-A, 8-B and 8-C. Figure 8-A is a photocopy of an ethidium bromide stained agarose electrophoresis gel containing size-fractionated plasmids of B.t. strains HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3) . Figure 8-B is a photocopy of an autoradiogram made by transferring the plasmids resolved by the gel shown in Figure 8-A to a nitrocellulose filter, hybridizing the filter with a 32P-labeled 2.2 kb PvuII mtragenic fragment obtained from the cryΙA(a) gene of HD-1, where lanes 1 through 3 in Figure 8-B correspond to lanes 1 through 3 of Figure 8-A. Figure 8-C is a photocopy of an autoradiogram made by transferring the plasmids resolved by the gel shown in Figure 8-A to a nitrocellulose filter, hybridizing the filter with a ³²P-labeled 0.4 kb Pstl-SacI intragenic N-terminal fragment obtained from the crylF gene, where lanes 1 through 3 of Figure 8-C correspond to lanes 1 through 3 of Figure 8-A. The numbers to the left of Figure 8-A indicate the sizes, in MDa, of various plasmids. The letter "L" to the left of Figure 8-A indicates the linear degeneration fragments from the breakdown of the larger plasmids.

Figure 9 comprises a photocopy of an autoradiogram made by transferring total DNA from B.t. strain EG6346 digested with restriction enzymes (as described below) to a nitrocellulose filter, hybridizing the filter with a 32P-labeled 0.6 kb KpnI-BamHI restriction fragment containing a portion of the crylX gene, and exposing the filter to X-ray film. The digestion of total DNA from B.t. strain EG6346 was carried out with several restriction enzymes, as follows: Asp718 (an isoschizomer of Kpnl) in lane 1, Clal in lane 2, SphI in lane 3, Asp718 (Kpnl) + SphI in lane 4, Clal + SphI in lane 5, SstI in lane 6, Asp718 (Kpnl) + SstI in lane 7, Clal + SstI in lane 8. The numbers to the left of Figure 9 indicate the sizes, in kb, of standard DNA fragments of phage lambda.

Figure 10 shows a circular restriction map of the 7.2 kb B.t.-E. coli cloning vector pEG854, originally described by Baum et al., Appl. Environ. Microbiol. 56, pp. 3420-3428 (1990) . The open box represents the pTZ19u segment of the vector that contains an a picillin resistance gene and a replication origin functional in E_^ coli. The shaded box, designated ori 43, contains a

2.8 kb replication origin region derived from a native B.t. plasmid that is function in B.t.. The solid black arrow corresponds to a chloramphenicol acetyltransferase (cat) gene that confers chloramphenicol resistance on B.t. strains transformed with pEG854 or its derivatives. Cloning vector pEG854 contains a unique Clal restriction site within the multiple cloning site, designated MCS in the Figure. Restriction sites for Xbal, Sfil, and NotI restriction endonucleases are also shown. Figure 11 shows a circular restriction map of the 11.8 kb recombinant plasmid pEG313 consisting of a 4.6 kb Clal restriction fragment isolated from total DNA of B.t. strain EG6346 inserted in the Clal site of cloning vector pEG854 (see Figure 10). The Clal sites flanking the 4.6 kb crylX-encoding fragment are indicated in bold type. An SstI restriction site, located downstream from the crylX gene, is contained within the 4.6 kb Clal restriction fragment. The orientation and approximate length of the crylX coding region is indicated by the open boxed arrow. Other annotations are as described for Figure 10.

Figure 12 shows a circular restriction map of the 2.86 kb E^_ coli cloning vector pTZ19u, used to obtain expression of the crylX gene in E. coli. A multiple cloning site region, containing unique restriction sites for AccI and SstI (in bold type) , is demarcated by unique Hindlll and EcoRI restriction sites within the lacZ' gene. Vector pTZ19u contains a beta- lactamase gene (bla) that confers resistance to ampicillin and also contains the replication region from an fl filamentous phage (fl) used for the synthesis of single-stranded DNA. The lac promoter (Plac in bold type) is positioned upstream from the multiple cloning site region. Restriction sites for Nael, Seal and Sspl restriction endonucleases are also shown. Figure 13 shows a circular restriction map of the 7.3 kb recombinant plasmid pEG314 consisting of a 4.4 kb Clal-SstI restriction fragment derived from pEG313 (see Figure 11) inserted into the AccI and SstI restriction sites of vector pTZ19u (see Figure.12). The orientation and approximate relative length of the crylX coding region is indicated by the open arrow. Other annotations are as described for Figure 12.

Figure 14 is a photocopy of a Coomassie stained 10% SDS-polyacrylamide gel showing crude (in lane 1) and gradient purified (in lane 2) CrylX crystal protein from E^ coli strain EG1083. The numbers to the left of Figure 14 indicate the sizes, in kDa, of protein molecular weight (MW) standards displayed in the leftmost (unnumbered) lane.

Detailed Description of the Preferred Embodiments The isolation and purification of the crylF gene (SEQ ID NO:l) and the lepidopteran-toxic CrylF crystal protein (SEQ ID NO:2), and the characterization of the native B.t. strain EG6345, the cured B.t. strain EG6346 derived from B.t. strain EG6345, and the recombinant B.t. strain EG1945, both of which produce the CrylF protein, are described in the Examples. The utility of recombinant B.t. strain EG1945 and of the CrylF crystal protein in insecticidal compositions and methods is also illustrated in the Examples.

Similarly, the isolation and purification of the crylX gene and the characterization of its lepidopteran-toxic CrylX crystal protein are also illustrated in the Examples. The methods and procedures described in the Examples for the crylF

TE SHEET gene and its CrylF protein are also generally applicable to the crylX gene and its insecticidal CrylX protein.

The cryl-type gene of this invention, the crylF gene (SEQ ID NO:l) , has the nucleotide base sequence shown in Figure 1. The coding region of the crylF gene extends from nucleotide base position 478 to position 3999 shown in Figure 1.

A comparison of the nucleotide base pairs of the crylF gene coding region with the corresponding coding region of the prior art cryl genes indicates significant differences between the new crylF gene and the prior art cryl genes. The crylF gene is only about 67% to about 78% homologous (positionally identical) with the cryΙA(a) , cryΙA(b) and cryΙA(c) genes and the crylB and crylC genes. There is even less ho ology with the cryll, crylll and cryIV genes, described in HSfte and Whiteley (1989) . The homology is discussed in more detail hereinafter.

The Cryl-type protein of this invention, the CrylF protein (SEQ ID NO:2) that is encoded by the crylF gene, has the amino acid sequence shown in Figure 1. In this disclosure, references to the CrylF "protein" (and to the CrylX "protein") are synonymous with its description as a "crystal protein," "protein toxin," "insecticidal protein," "delta endotoxin" or the like, unless the context indicates otherwise. The deduced size of the CrylF protein is

133,635 Da. The prior art Cryl-type proteins, encoded by the respective cryl genes, have similar deduced sizes. Despite the apparent size

SUBSTITUTE SHEET similarity, comparison of the amino acid sequence of the CrylF protein with published sequences of the other prior art Cryl-type proteins shows significant differences between them and the CrylF protein. The CrylF protein is only about 58% to about 72% identical with the other prior art Cryl- type proteins, even when considering the C-terminal regions which are more related than the N-terminal regions. The crylX gene of this invention contains approximately 2100-2200 basepairs in its coding region, of which approximately 1140 basepairs are shown for the truncated upstream portion (SEQ ID NO:3) of the crylX gene in Figure 2. The crylX gene of this invention is contained in isolated form on a DNA fragment carried on a recombinant plasmid, in E__ coli strain EG1083 which has been deposited in the NRRL under accession No. NRRL B- 18805. The CrylX protein of this invention, produced by the crylX gene, is about 81 kDa in size and exhibits insecticidal activity against insects of the order Lepidoptera. The amino acid sequence (SEQ ID NO:4) for a portion of the CrylX protein, deduced from the truncated portion of the crylX gene shown in Figure 2, is shown in Figure 2. The

380 amino acids of this initial portion (SEQ ID NO:4) of the CrylX protein shown in Figure 2 represent approximately one-half of the CrylX protein encoded by the crylX gene. The CrylF and CrylX proteins have been shown to be insecticidal against insects of the order Lepidoptera, as set forth in more detail in Examples 6 and 11, respectively.

TE SHEET The present invention is intended to cover mutants and recombinant or genetically engineered derivatives of the crylF gene and crylX gene that yield lepidopteran-toxic proteins with essentially the same properties as the respective CrylF and CrylX proteins.

The crylF gene and crylX gene are also useful as DNA hybridization probes, for discovering similar or closely related cryl-type genes in other B.t. strains. The crylF or cryIX gene, or portions or derivatives thereof, can be labeled for use as a hybridization probe, e.g., with a radioactive label, using conventional procedures. The labeled DNA hybridization probe may then be used in the manner described in the Examples.

The cryIF or crylX gene may be introduced into a variety of microorganism hosts, using procedures well known to those skilled in the art for transforming suitable hosts under conditions which allow for stable maintenance and expression of the cloned crylF or crylX gene, as the case may be. Suitable hosts that allow the cryIF and crylX genes to be expressed and the respective CrylF and CrylX proteins to be produced include Bacillus thuringiensis and other Bacillus species such as B. subtilis or B__ meg terium. E. coli or Pseudomonas fluorescens are also suitable hosts for these genes. It should be evident that genetically altered or engineered microorganisms containing the crylF gene or crylX gene can also contain other toxin genes present in the same microorganism and that these genes could concurrently produce insecticidal crystal proteins different from the CrylF and CrylX proteins.

The Bacillus and E__ coli strains described in this disclosure may be cultured using conventional growth media and standard fermentation techniques. The B.t. strains harboring the crylF gene (or the crylX gene) may be fermented, as described in the Examples, until the cultured B.t. cells reach the stage of their growth cycle when

CrylF crystal protein (or CrylX crystal protein) is formed. For sporogenous B.t. strains, fermentation is typically continued through the sporulation stage, when crystal protein is formed along with spores. The B.t. fermentation culture is then typically harvested by centrifugation, filtration or the like to separate fermentation culture solids, containing the crystal protein, from the aqueous broth portion of the culture. The B.t. strains exemplified in this disclosure are sporulating varieties (spore forming or sporogenous strains) , but the crylF gene and the crylX gene also have utility in asporogenous Bacillus strains, i.e., strains that produce the crystal protein without production of spores. It should be understood that references to "fermentation cultures" of B.t. strains (containing the crylF or crylX gene) in this disclosure are intended to cover sporulated B.t. cultures, i.e., B.t. cultures containing the CrylF or CrylX crystal protein and spores, and sporogenous Bacillus strain cultures that have produced crystal protein during the vegetative stage, as well as asporogenous Bacillus strains containing the crylF or crylX gene in which the culture has reached the growth stage where crystal protein is actually produced.

The separated fermentation solids are primarily CrylF or CrylX crystal protein, as the case may be, and B.t. spores, along with some cell debris, some intact cells, and residual fermentation medium solids. If desired, the crystal protein may be separated from the other recovered solids via conventional methods, e.g., sucrose density gradient fractionation. Highly purified CrylF or CrylX protein may be obtained by solubilizing the recovered crystal protein and then reprecipitating the protein from solution. The CrylF protein is an effective insecticidal compound against lepidopteran insects like the European cornborer, the beet armyworm, and the tobacco budworm, for example. Likewise, the CrylX protein is insecticidal to lepidopteran insect species. The CrylF protein or CrylX protein may be utilized as the active ingredient in insecticidal formulations useful for the control of lepidopteran insects. Such insecticidal formulations or compositions typically contain agriculturally acceptable carriers or adjuvants in addition to the active ingredient.

The CrylF protein or CrylX protein may be employed in insecticidal formulations in isolated or purified form, e.g., as the crystal protein itself. Alternatively, the CrylF protein or CrylX protein may be present in the recovered fermentation solids, obtained from culturing of a Bacillus strain, e.g.. Bacillus thuringiensis, or other microorganism host carrying the crylF or crylX gene and capable of producing the corresponding CrylF or CrylX protein. Preferred Bacillus hosts for the crylF gene include B.t. strain EG6345 and genetically improved B.t. strains derived from B.t. strain EG6345, such as B.t. strain EG6346. The derivative B.t. strains may be obtained via plasmid curing and/or conjugation techniques and contain the native crylF gene- containing plasmid from B.t. strain EG6345.

Genetically engineered or transformed B.t. strains or other host microorganisms, containing a recombinant plasmid that expresses the cloned crylF gene and obtained by recombinant DNA procedures, may also be used.

Examples of such transformants include B.t. strain EG1945 which contains the cloned crylF gene on a recombinant plasmid.

The recovered fermentation solids contain primarily the crystal protein and (if a sporulating B.t. host is employed) spores; cell debris and residual fermentation medium solids may also be present. The recovered fermentation solids containing the CrylF or CrylX protein may be dried, if desired, prior to incorporation into the insecticidal formulation.

The formulations or compositions of this invention containing the insecticidal CrylF or CrylX protein as the active component are applied at an insecticidally effective amount which will vary depending on such factors as, for example, the specific lepidopteran insects to be controlled, the specific plant or crop to be treated and the method of applying the insecticidally active compositions. An insecticidally effective amount of the insecticide formulation is employed in the insect control method of this invention. The insecticide compositions are made by formulating the insecticidally active component with the desired agriculturally acceptable carrier. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral) or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term

"agriculturally acceptable carrier" covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation.

The formulations containing the CrylF or CrylX protein and one or more solid or liquid adjuvants are prepared in known manners, e.g., by homogeneously mixing, blending and/or grinding the insecticidally active CrylF or CrylX protein component with suitable adjuvants using conventional formulation techniques.

The insecticidal compositions of this invention are applied to the environment of the target lepidopteran insect, typically onto the foliage of the plant or crop to be protected by conventional methods, preferably by spraying. Other application techniques, e.g., dusting, sprinkling, soaking, soil injection, seed coating, seedling coating or spraying, or the like, are also feasible. These application procedures are well known in the art.

The crylF or crylX gene or its functional equivalent, hereinafter sometimes referred to as the "toxin gene," can be introduced into a wide variety of microorganism hosts. Expression of the crylF gene results in the production of insecticidal CrylF crystal protein. Likewise, expression of the crylX gene results in production of the insecticidal CrylX protein. Suitable hosts include B.t. and other species of Bacillus, such as B^_ subtilis or B_^ megaterium, for example. Other bacterial hosts such as E__ coli and Pseudomonas fluorescens may also be used. Various procedures well known to those skilled in the art are available for introducing the crylF or crylX gene into the microorganism host under conditions which allow for stable maintenance and expression of the gene in the resulting transformants.

The transformants, i.e., host microorganisms that harbor a cloned gene in a recombinant plasmid, can be isolated in accordance with conventional ways, usually employing a selection technique, which allows growth of only those host microorganisms that contain a recombinant plasmid. The transformants then can be tested for insecticidal activity. Again, these techniques are standard procedures. Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the gene into the host, availability of expression systems, efficiency of expression, stability of the CrylF or CrylX insecticidal protein in the host, and the presence of auxiliary genetic capabilities. The cellular host containing the insecticidal cryIF or cryIX gene may be grown in any convenient nutrient medium, where expression of the crylF or crylX gene is obtained and corresponding CrylF or CrylX protein produced, typically upon sporulation. The sporulated cells containing the crystal protein may then be harvested in accordance with conventional ways, e.g., centrifugation or filtration.

The crylF and crylX genes may also be incorporated into a plant which is capable of expressing the gene and producing CrylF or CrylX protein, as the case may be, rendering the plant more resistant to insect attack. Genetic engineering of plants with the crylF or crylX gene may be accomplished by introducing the desired DNA containing the gene into plant tissues or cells, using DNA molecules of a variety of forms and origins that are well know to those skilled in plant genetic engineering. An example of a technique for introducing DNA into plant tissue is disclosed in European Patent Application Publication No. 0 289 479, published November 2, 1988, of Monsanto Company.

DNA containing the crylF or crylX gene or a modified crylF or crylX gene capable of producing the corresponding CrylF or CrylX protein may be delivered into the plant cells or tissues directly by infectious plasmids, such as Ti, the plasmid from Agrobacterium tumefaciens, viruses or microorganisms like A. tumefaciens, by the use of lysosomes or liposomes, by microinjection by mechanical methods and by other techniques familiar to those skilled in plant genetic engineering. Slight variations may be made in the crylF or crylX gene nucleotide base sequences, since the various amino acids forming the proteins encoded by the respective genes usually may be determined by more than one codon, as is well known to those skilled in the art. Moreover, there may be some variations or truncation in the coding region of the crylF and crylX nucleotide base sequences which allow expression of the gene and production of functionally equivalent forms of the corresponding CrylF and CrylX insecticidal proteins. These variations which can be determined without undue experimentation by those of ordinary skill in the art with reference to the present specification are to be considered within the scope of the appended claims, since they are fully equivalent to the specifically claimed subject matter.

The present invention will now be described in more detail with reference to the following specific, non-limiting examples. The examples relate to work which was actually done based on techniques generally known in the art and using commercially available equipment. The novel B.t. strain EG6345 and a cured derivative B.t. strain EG6346 were isolated following the procedures described in Example 1.

Example l Isolation of B.t. Strains EG6345 and EG6346 Crop dust samples were obtained from various sources throughout the U.S. and abroad, typically grain storage facilities. The crop dust samples were treated by suspending the crop dust in an aqueous buffer and heating the suspension at

60^βC for 30 min. to enrich for heat resistant spore forming Bacillus-type bacteria such as B.t. The treated dust suspensions were diluted in aqueous buffer, and the dilutions were spread on agar plates to allow each individual bacterium from the crop dust to grow into a colony on the surface of the agar plate.

After extensive screening of crop dust samples, a B.t. subsp. aizawai strain, designated B.t. strain EG6345, was isolated from a maize grain dust sample. A sporulated culture of B.t. strain EG6345 was spread for the growth of individual colonies on a nutrient salts agar plate and incubated for 3 days at 30'C. After incubation, one colony was noted on this plate which displayed a different colony morphology (i.e., shinier) than the parent B.t. strain EG6345. The colony, designated B.t. strain EG6346, was isolated as an individual colony. A sample of the isolated B.t. strain

EG6346 was further purified by streaking on an agar plate containing Spizizen's glucose peptone beef extract (SGPB) . A sample of this SGPB agar plat- culture was used for agarose gel electrophoresis analysis of plasmid DNA using the standard Gonzalez technique (Gonzalez et al., Proc. Natl. Acad. Sci. U.S.A. , ____, pp. 6951-6955 (1982)). The agarose gel electrophoretic analysis was coupled with standard plasmid curing (ie., plasmid loss) and conjugation (ie., plasmid transfer) studies. The plasmid array of the new isolate of B.t. strain EG6346 was compared to that of B.t. strain EG6345 using agarose gel electrophoresis of plasmid DNA.

The agarose gel electrophoresis analyses of plasmid DNA, coupled with the plasmid curing and conjugation studies, indicated that B.t. strain EG6345 contained two plasmids of approximately 115 MDa and 45 MDa that encoded crystal protein. B.t. strain EG6346 was identified as a spontaneously cured derivative of B.t. strain EG6345 which contained the plasmid of approximately 115 MDa, but which lacked the approximately 45 MDa plasmid.

Figure 3 is a photograph of a portion of an ethidium bromide stained agarose gel containing size-fractionated plasmids of B.t. strains EG6346 (left lane) and EG6345 (right lane) . As illustrated in Figure 3, B.t. strain EG6346 does not contain the approximately 45 MDa plasmid contained in B.t. strain EG6345. Both B.t. strain EG6345 and the cured derivative B.t. strain EG6346 produced large bipyramidal inclusions during sporulation, as detected by phase contrast microscopy of sporulated cultures. Following the Southern blot technique (E.M. Southern, J. Mol. Biol., 98, pp. 503-517 (1975)), total DNA, prepared from both B.t. strains EG6345 and EG6346, was digested with Hindlll, electrophoresed through a 0.7% agarose gel, transferred to a nitrocellulose filter and hybridized at 50°C overnight to either a 32P- labeled 0.7 kb EcoRI N-terminal probe isolated from the B.t. strain HD-1 cryΙA(a) gene or a similarly labeled 2.2 kb intragenic PvuII probe also isolated from the HD-1 cryΙA(a) gene. Digested DNA from B.t. subsp. kurstaki HD-1, which harbors the cryΙA(a) , cryΙA(b) and cryΙA(c) genes, was included as a control. The results of the Southern blot analyses are illustrated in Figure 4-A and 4-B.

Figure 4-A is the Southern blot of the agarose gel containing the total Hindlll-digested DNA from B.t. strains EG6346 (lane 1), EG6345 (lane 2) and HD-1 (lane 3) , following hybridization to the radiolabeled EcoRI probe. Figure 4-B shows the Southern blot of total Hindlll-digested DNA from the B.t. strains indicated with respect to Figure 4-A, and in the same order, following hybridization to the radiolabeled PvuII probe. As shown in Figure 4-A, the 0.7 kb EcoRI probe detected the expected 4.5, 5.3 and 6.6 kb fragments in HD-1 DNA (lane 3) corresponding to the previously described characteristic Hindlll fragments for the eryΙA(a) , cryΙA(b) and cryΙA(c) genes, respectively. This probe also detected a prominent 5.3 kb band in B.t. strain EG6345 (lane 2) which was absent in the cured derivative B.t. strain EG6346 (lane 1) . This result indicated that the 45 MDa plasmid of EG6345 harbored at least one cryΙA(b) gene. The N-terminal 0.7 kb EcoRI probe also hybridized to a 1.4 kb Hindlll fragment of unknown origin in both B.t. strains EG6345 and EG6346.

The hybridization pattern obtained with the radiolabeled intragenic PvuII probe was more complex as can be seen in Figure 4-B. This probe, as expected, also hybridized to the 4.5, 5.3 and 6.6 kb fragments in HD-1 (lane 3) confirming the presence of the respective cryΙA(a) , cryΙA(b) and cryΙA(c) genes in this strain. Internal Hindlll fragments of 1.1 kb and 0.9 kb, derived from the resident cryΙA(a) and cryΙA(b) genes in HD-1, respectively, were also detected with the PvuII probe.

The 5.3, 2.8 and 0.9 kb fragments were also detected by the PvuII probe in the DNA of B.t. strain EG6345, indicating the presence of the cryΙA(b) gene in this strain (lane 2) . These bands were not detected in B.t. strain EG6346 (lane 1) . However, the 1.4 kb Hindlll fragment, detected in both B.t. strains EG6345 and EG6346 by the EcoRI probe, was similarly detected in both strains by the PvuII probe. A faintly hybridizing 2.5 kb

Hindlll fragment was also detected with the PvuII probe in both B.t. strains EG6345 and EG6346. This band corresponds in size to the characteristic Hindlll fragment of the crylC gene detected in other B.t. subsp. aizawai strains.

The PvuII probe also hybridized to two large Hindlll fragments present in both B.t. strains EG6345 and EG6346. These fragments, approximating 8.2 and 10.4 kb in size, were not detected by the EcoRI probe in either of B.t. strains EG6345 or EG6346, nor were they observed with either probe in HD-1 DNA. The unusual size of the hybridizing fragments, along with the production of large, bipyramidal crystal protein inclusions by B.t. strain EG6346, indicated the presence of one or more novel toxin genes in B.t. strains EG6345 and EG6346.

Example 2

Isolation of the crylF Gene in E. coli

A genomic library was constructed for B.t. strain EG6346 and was screened at low stringency conditions with the intragenic 2.2 kb PvuII probe obtained from the cryΙA(a) toxin gene. DNA from B.t. strain EG6346 was chosen as the substrate DNA due to its apparent lack of crylA- type toxin genes, whose presence could potentially increase the difficulty in screening the library at low stringency with the PvuII probe.

More specifically, high molecular weight DNA, obtained from B.t. strain EG6346, was partially digested with Sau3A and size-fractionated on a 10% to 40% sucrose gradient in 100 mM NaCl- lOmM Tris hydrochloride (pH 7.4)-ImM EDTA (TE) .

Gradient fractions, containing DNA ranging in size from 5 to 10 kb, were pooled, dialyzed against TE 10:1 (pH 7.4), extracted with 2-butanol to reduce the volume and ethanol precipitated. The purified insert DNA was ligated to E^_ coli plasmid vector pGEM'-3Z digested with BamHI at a 1:2 molar ratio of vector to insert and at a final DNA concentration of 20 μg/ml, using T4 DNA ligase available from Promega Corp. Transformation of E. coli DH5 cells was based on the Hanahan procedure (Hanahan, J. Mol. Biol. , 166, pp. 557-580 (1983)) and transformed colonies were plated on agar plates of standard LB medium containing 100 jug/ml ampicillin and 50 μg/ml X-gal (5-bromo-4- chloro-3-indolyl-beta-D-galactoside) . Approximately 3.3 x 10 colonies were screened for the presence of cryl-related toxin gene sequences under low stringency conditions, using as a probe the 32P-labeled 2.2 kb PvuII intragenic fragment obtained from a cryΙA(a) gene present within B.t. strain HD-1. The low stringency conditions include hybridization conducted at 50-55^βC overnight in 3X SSC (IX SSC comprises 0.15 M NaCl, 0.015M sodium citrate), 10X Denhardt's solution (IX Denhardt's solution comprises 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone) , 200 μg/ml heparin and 0.1% SDS. The probe hybridized strongly to one E. coli recombinant colony, designated E__ coli strain EG1943, which contained an 8.4 kb recombinant plasmid, designated pEG640, that consisted of plasmid pGEM^-SZ ligated to a 5.7 kb Sau3A insert of DNA from B.t. strain EG6346.

A restriction map for the pEG640 plasmid was generated as shown in Figure 5 using those restriction enzymes indicated above in the Brief Description of the Drawings and methods well known to those skilled in the art. The relative positions of restriction sites and localization of toxin gene sequences within the map were initially accomplished by low stringency hybridization of Southern blots containing digested pEG640 DNA to the radiolabeled EcoRI and PvuII toxin gene probes as set forth above in Example 1.

Initial mapping data identified two regions on the pEG640 insert which reacted with varying intensity to the toxin gene probes. The larger region, spanning over 3 kb in length, hybridized strongly to the PvuII probe at low and high stringency hybridization conditions. The high stringency conditions are the same as the above- identified low stringency conditions, except that the temperature is increased to 65°C. The larger 3 kb region on the 5.7 kb insert of the pEG640 plasmid also reacted well with the EcoRI probe at low stringency hybridization conditions. A smaller region, positioned in close proximity to the vector, weakly hybridized to the EcoRI probe at low stringency conditions only. These data indicated the presence of two different toxin genes on the

5.7 kb insert of the pEG640 plasmid. When puri .fi.ed 32P-labeled pEG640 DNA was used to probe Hindlll genomic digests, a single 10.4 kb hybridizing band was detected in B.t. strains EG6345 and EG6346, as illustrated in lanes 1 and 2 of Figure 4-C, respectively. This

10.4 kb fragment was also detected in both B.t. strains EG6345 and EG6346 with the PvuII probe as can be seen in Figure 4-B, lanes 1 and 2, respectively. No hybridizable bands were detected in the DNA from B.t. strain HD-1, as evidenced by lane 3 in Figure 4-C, which is consistent with the absence of these novel gene sequences in this strain.

Example 3 Sequence Analyses of crylF and crylX Genes Standard dideoxy sequencing procedures (Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74, pp. 5463-5467 (1977), with Sequenase™, available from United States Biochemical Corp., were used to determine the DNA sequence of the 5.7 kb pEG640 insert from the recombinant E^ coli strain EG1943. Sequencing of the insert was initiated in both directions and on both strands from the SP6 and T7 promoters present on vector pGEM™-3Z and utilized the specific primers supplied by Promega Corp.

Preparation and denaturation of the double stranded template was also according to manufacturers⁷ directions (Promega Corp. and United States Biochemical Corp.). Subsequent 17mer oligonucleotide primers were synthesized on an Applied Biosystems, Inc. DNA synthesizer, Model 38OB.

The DNA sequence, which is flanked by Sau3A cloning sites (GATC) extends 5649 nucleotide bases in length and is shown in Figures 1 and 2. Translation of the sequence revealed the presence of two open reading frames which are separated by approximately 500 bases of non-coding DNA sequence and which are out of frame with respect to one another. The genes potentially encoded by these open reading frames have been designated crylF (SEQ ID NO:l) and crylX (SEQ ID N0:3) . Justification for this designation derives from sequence

TUTE SHEET comparisons to other toxin genes and is discussed below. The partial DNA sequence for the portion of the 5.7 kb insert of pEG640 including the crylF gene (SEQ ID N0:1) and the deduced amino acid sequence of the crystal protein encoded by the crylF gene, designated the CrylF protein (SEQ ID NO:2) , are illustrated in Figure 1. The partial, truncated DNA sequence (SEQ ID NO:3) for the portion of the 5.7 kb insert of pEG640 including the truncated crylX gene, and the deduced, truncated amino acid sequence (SEQ ID NO:4) of the crystal protein encoded by the crylX gene, designated the CrylX protein are illustrated in Figure 2. The beginning of the sequences in Figure 2 follow immediately after the end of the sequences illustrated in Figure 1, and two figures are used merely for the sake of convenience.

The crylF open reading frame, which is the larger of the two, encodes a CrylF protein consisting of 1174 amino acids and having a deduced size of 133,635 Da. The position of the crylF gene within pEG640 and its relationship to the position of the cryIX gene is schematically represented in Figure 5. An NH_-terminal methionine translational start site was identified for the crylF gene at nucleotide base position 478 of the sequence. It was immediately preceded by a putative ribosome binding site (RBS) . The crylF gene open reading frame terminates at nucleotide base position 3999. A putative promoter sequence was identified for the crylF gene 53 nucleotide bases upstream of the ribosome binding site. The nucleotide base sequence as counted from both base pairs 10 and 35 positions upstream of the methionine start is

TITUTE SHEET exactly homologous to that identified for the HD-1 cryΙA(a) gene promoter (Wong et al. , J. Biol. Chem. , 258, pp. 1960-1967 (1983)).

As indicated in Figure 2, an NH--terminal methionine codon, signifying the translational start site of the crylX open reading frame, was identified at nucleotide base position 4508. The crylX open reading frame continued an additional 1141 nucleotides, encoding 380 amino acids, and terminated with the GATC cloning site delimiting the insert DNA. The sequence presented here for the crylX gene represents an artificially truncated version of the native gene present within B.t. strain EG6346. Although a putative ribosome binding site has been identified upstream of the crylX sequence, it was not possible to identify promoter regions located 10 and 35 base pairs upstream from the methionine start for the crylX gene within the intervening DNA sequence between the crylF and crylX open reading frames by sequence inspection. Inspection of the intervening DNA sequence between the crylF and cryIX genes has identified a stem-loop termination structure at positions 4090-4132 (see Figure 2) that is nearly identical to that described downstream of the HD- 1-Dipel cryΙA(a) gene (Wong et al., J. Biol. Chem., 258, pp. 1960-1967 (1983)).

The sequence analysis program of Queen and Korn was used to compare the sequences of the cryIF and cryIX genes to the published sequences of other B.t. toxin genes (Queen et al.. Nucleic Acids Res. , 12, pp. 581-599 (1984)). The nucleotide base sequences and deduced amino acid sequences of the crylF and crylX genes were aligned with the published sequences of various delta-endotoxin genes and the results of the comparisons are tabulated in Table 1. As shown in Table 1, the amino acid sequence of the N-terminal region (amino acids 1-618) of the crylF-encoded protein differs significantly from the N-terminal region of other Cryl-type encoded proteins (about 40%-50% identity) . These sequence differences are likely responsible for the unique insecticidal activity spectrum of the CrylF protein (see Example 5 below) , since previous studies of truncated cryl genes indicate that it is the N-terminal region of the protein that determines insecticidal activity (Schnepf et al., J. Biol. Chem., 260, pp. 6273-6278 (1985); Hofte et al., Eur. J. Biochem. , 161, pp. 273-280 (1986) .

TABLE 1

Amino Acid (aa) Comparisons of the N-terminal Region of CrylF Protein

Protein Class (N-terminal region) % Similarity CryΙA(a) (aa 1-608) 51.0

CryΙA(b) (aa 1-609) 52.0

CryΙA(c) (aa 1-610) 49.0

CrylB (aa 1-637) 40.1

CrylC (aa 1-617) 48.8 CrylD (aa 1-593) 52.0

CrylE (aa 1-602) 48.1

Amino acids 1-602 of the CrylF protein were compared to the N-terminal regions of CrylA(a) (Schnepf et al., (1985), supra) , CryΙA(b) (Hofte et al., (1986), supra) , CryΙA(c) (Adang et al. , (1985), supra) , CrylB (Brizzard et al. (1988), supra) , CrylC (Hofiee et al. , (1988) , supra) , CrylD and CrylE (both in EP 0 358 557 (1990) , supra) .

The nucleotide base sequence of the entire crylF gene and the amino acid sequences of the CrylF protein were also compared to other crystal protein genes and their respectively encoded proteins. The comparisons were tabulated in Table 2.

TABLE 2

Sequence Comparisons of crylF and crylX

Genes and Encoded Proteins

With Other B.t. Genes and Proteins

crylF Cr lF

aa means amino acid. % Identity, i.e., positional identity.

Comparisons of the complete DNA sequence indicate the crylF gene was related to, but distinct from, the crylA class of toxin genes (about 76-78% identity) (Table 2). Of the three crylA genes compared to the crylF gene, crylF was most related to the HD-1 cryΙA(a) nucleotide sequence with about 78% of the nucleotides conserved between the two genes.

Table 2 indicates that the CrylF protein is significantly more related to other Cryl proteins than to the CryllA, CrylllA or CrylVD proteins. Amino acid identity ranged from about 70-72% for the CrylF protein and the CrylA, CrylC, CrylD and CrylE proteins.

The crylF gene sequence was less related to crylB (about 67%) and, as expected, much less related to dipteran and coleopteran toxin genes (cryll, crylll and cryIV genes) .

The crystal protein genes thus far disclosed in the previously cited references have been divided into four major classes and several subclasses characterized by both structural similarities and the insecticidal spectrum of the encoded crystal proteins (Hofte and Whiteley (1989) p. 242) . The four major classes, I, II, III and IV, encode lepidopteran-specific, lepidopteran- and dipteran-specific, coleopteran-specific and dipteran-specific proteins, respectively. Table 1 of Hofte and Whiteley (1989) at p. 243 lists the genes presently assigned to these four major classes.

The cryl genes can be distinguished from the other crystal protein genes by sequence homology. The amino acid sequences encoded by the cryl genes exhibit greater than 50% identity (Table 3, Hofte and Whiteley (1989) at p. 245). The amino acid sequences of three Cryl-encoded proteins (CrylA(a) , CrylA(b) and CrylA(c) ) show greater than 80% identity, and thus they are considered members of the same subgroup (CrylA) . There is ample justification for designating the novel toxin gene identified in B.t. strain EG6345 as crylF. The CrylF protein exhibits greater than 50% amino acid identity to the other Cryl proteins. More specifically, the CrylF protein is about 70-72% identical to the CrylA subgroup proteins, about 58% identical to the CrylB protein and about 70% identical to the CrylC and CrylD proteins. The CrylF protein is less related to the crystal proteins encoded by the other crystal protein gene classes cryll, crylll and crylV (see Table 2) .

However, the CrylF protein is not greater than about 80% identical to the crystal proteins encoded by the crylA subgroup of genes, and thus the crylF gene does not belong to the cryI subgroup. The CrylF protein is only somewhat related to the CrylB, CrylC, CrylD and CrylE proteins, and thus, the crylF gene is not a member of a new subgroup including any of the crylB, crylC, crylD or crylE genes.

Further substantiation of the crylF designation, i.e., its categorization as a cryl- type gene, is that the CrylB protein is about 55- 56% identical to the proteins encoded by the cryIA subgroup of genes and the CrylD protein is about 70-71% identical to the CrylA subgroup and CrylC proteins (see Hofte and Whiteley (1989) Table 3) . The crylX truncated nucleotide base sequence (SEQ ID NO:3) and the deduced amino acid sequence (SEQ ID NO: 4) were similarly compared to other toxin gene sequences, as shown in Table 2 . The crylX nucleotide base sequence is also distinct from, but related to, the other cryl genes in

Table 2, such as that of the crylB gene (about 70% identical) .

SUBSTITUTE SHEET Example 4 Expression of the Cloned crylF Gene

Studies were conducted to demonstrate the production of the CrylF protein by the crylF gene. Table 3 summarizes the relevant characteristics of the B.t. and E^ coli strains and plasmids used during these procedures. A plus ( ) indicates the presence of the designated element, activity or function and a minus (^") indicates the absence of the same. The designations s and r indicate sensitivity and resistance, respectively, to the antibiotic with which each is used. The abbreviations used in the table have the following meanings: Amp (ampicillin) ; Cry (crystalliferous) ; Tc (tetracycline) .

Table 3

Strain or Plasmid Relevant Characteristics B. thuringiensis

HD73-26 Cry EG1078 HD73-26 harboring pEG310 (cryIF~ cryIX⁺)

EG1945 HD73-26 harboring pEG642 (cryIF⁺ cryIX⁺)

EG6345 crylF crylX

EG6346 crylF + crylX+, deri,vati.ve of EG6345 cured of 45 MDa plasmid E__;_ coli

DH5«d Cry , Amp

GM2163 Cry^", Amp^s

EG1943 DH5<^ harboring pEG640(cryIF⁺ cryIX⁺) Plasmids pEG310 crylF crylX deletion mutant plasmid of pEG642 pEG434 Tc^r Bacillus vector pGEM~-3Z Amp^r E__ coli vector PEG640 Amp^r pGEM™-3Z with 5.7 kb insert (cryIF⁺ cryIX⁺ pEG642 Tc^r, E^ coli-Bacillus shuttle vector consistin pEG640 ligated into the Hindlll site of pEG434

It has been reported that E^ coli cells harboring cloned B.t. toxin genes fail to produce significant amounts of the toxin protein required 25 for critical evaluations of insecticidal activity (Donovan et al., Mol. Gen. Genet. , 214, pp. 365-372 (1988)). Returning the cloned B.t. toxin gene to a Bacillus species, and ideally to a B.t. host. maximizes toxin gene expression from its native promoter. Accordingly, the cloned crylF gene was introduced into the Cry recipient B.t. strain HD73-26, as described below. The pEG640 plasmid construct was ligated to the vector pEG434 (Mettus et al., Applied and Environ. Microbiol., 192, pp. 288-289 (1990)) at the unique Hindlll site present on both pEG640 and pEG434 and the ligation mixture used to transform E^ coli strain GM2163, which is defective for both adenine and cytosine methylation (Marinus et al., Mol. Gen. Genet., 56, pp. 1128-1134 (1983)). The resulting 11.4 kb recombinant plasmid, designated pEG642 and having a restriction map as shown in Figure 6, possessed both E__ coli and Bacillus replication origins and a selectable marker for tetracycline resistance (Tc^r) that could function in a B.t. host. Plasmid pEG642 DNA was isolated from E__ coli strain GM2163 by alkaline/SDS lysis followed by ethanol precipitation using standard procedures. Plasmid DNA was then used to transform the B.t. Cry^"" recipient strain HD73-26 by electroporation. A single Tc^r HD73-26 transformant containing pEG642, designated B.t. strain EG1945, was chosen for further study. Microscopic examination of sporulated cultures of B.t. strain EG1945 revealed the presence of crystalline inclusions (large, irregularly shaped rods and bipyramidal shapes) . Renografin density gradient purified crystal protein from B.t. strain EG1945 was used for SDS-PAGE analyses of the crylF gene product. The purified CrylF protein from the recombinant B.t. strain EG1945 was compared to similarly purified proteins obtained from the native B.t. strains EG6345 and EG6346 harboring the crylF gene. Crystal protein preparations (2.8 μg of EG6345, 0.7 μg of EG6346 and 0.70 μg of EG1945) were loaded onto a 5-20% gradient SDS-polyacrylamide gel and electrophoresed. Figure 7 is a photograph of the resulting Coomassie stained SDS-polyacrylamide gel, in which lanes 1, 2 and 3 contain proteins from native B.t. strains EG6345 and EG6346 and recombinant B.t. strain EG1945, respectively.

As indicated in lane 3, a single high molecular weight protein, approximating 135 kDa in size, was observed in recombinant B.t. strain EG1945, consistent with expression of the single crylF gene. The size of the observed protein correlates well with the predicted molecular weight of 133,635 Da deduced from the amino acid sequence. At least three distinct protein species were observed in lane 1 of Figure 7, from B.t. strain EG6345, which confirms the DNA hybridization result shown in Figure 4, verifying the presence of the cryΙA(b) , crylC and crylF genes in this strain. It is possible, however, that other similarly sized proteins encoded by additional toxin genes are also present in B.t. strain EG6345, e.g., the crylX gene.

Similarly, B.t. strain EG6346, which was used to construct the library from which crylF was cloned, contains at least two crystal proteins, the largest of which appears to co-migrate with the approximately 135 kDa recombinant crylF protein in B.t. strain EG1945. The smaller protein present in B.t. strain EG6346, also evident in B.t. strain EG6345, is believed to represent the protein encoded by the crylC gene which has been identified in each of these strains by DNA hybridization analysis with a crylC specific oligonucleotide probe.

Example 5 Plasmid Localization of the crylF Gene

To determine the location of the crylF gene in B.t. strains EG6345 and EG6346 and to compare its location to that of the cryΙA(b) gene present in B.t. strain EG6345, plasmid DNAs of B.t. strains EG6345 and EG6346 were resolved by agarose gel electrophoresis. The resulting ethidium bromide stained gel is illustrated in Figure 8-A. Plasmids from strain HD-1 (lane 1) were included as controls and were used as size standards. Lane 2 shows the plasmids from B.t. strain EG6345, while lane 3 shows the plasmids from B.t. strain EG6346. Plasmid DNAs resolved by the gel of

Figure 8-A were transferred to nitrocellulose and hybridized to either the intragenic radiolabeled 2.2 kb PvuII cryΙA(a) probe or to a crylF gene- specific probe consisting of a radiolabeled gel- purified 0.4 kb Pstl-SacI fragment isolated from the N-terminal region of the crylF gene on pEG640. Hybridizations were conducted at 65'C overnight to assure specificity of the reaction with each probe. As shown in the autoradiogram of Figure 8-B, the PvuII intragenic cryΙA(a) probe hybridized strongly to the 44 MDa plasmid present in HD-1 (lane 1) which harbors a cryΙA(b) gene. Hybridization of the PvuII probe to this plasmid was expected, since the nucleotide base sequence of the probe is highly conserved among all three crylA genes. Similarly, the PvuII probe also hybridized to the large 110 MDa plasmid in strain HD-1 containing the cryΙA(a) and cryΙA(c) toxin genes.

The PvuII probe also hybridized to the 45 MDa plasmid containing the cryΙA(b) gene present within B.t. strain EG6345 (lane 2) . Differences in the hybridization signal intensity of the PvuII probe in detecting the cryΙA(b) gene in B.t. strains HD-1 and EG6345 may be attributed to different amounts of DNA loaded onto the gel shown in Figure 8-A. Lack of a hybridization band from the PvuII probe in strain EG6346 (lane 3) was entirely consistent with the classification of this strain as a cured derivative of B.t. strain EG6345 not containing the 45 MDa plasmid. The 115 MDa plasmid present within B.t. strains EG6345 and EG6346 was weakly detected by the PvuII probe. The reduced hybridization signal observed in each of these strains, as compared to strain HD-1, may be attributed to quantitative differences in the amounts of DNA loaded, as well as to the reduced sequence homology between the PvuII probe and the novel toxin genes present on this large plasmid.

Hybridization of the crylF Pstl-SacI intragenic probe to plasmid DNAs from B.t. strains HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3) is shown in the autoradiogram of Figure 8-C. The specificity of this probe for the crylF gene is confirmed by the lack of hybridization to plasmids harboring crylA genes in B.t. strains HD-1 or EG6345, and by its hybridization to the 115 MDa plasmid present in B.t. strains EG6345 and EG6346. Both the PvuII and the Pstl-SacI probes hybridized to a low molecular weight smear, identified as "L" in Figure 8-A, which represents linear fragments of sheared larger toxin plasmids.

Example 6 Insect Toxicity of the CrylF Protein

The insecticidal activity of CrylF protein was determined against several lepidopteran larvae including Ostrinia nubilalis (European cornborer) , Spodoptera exigua (beet armyworm) , Heliothis virescens (tobacco budworm) , Heliothis zea (bollworm) and Lymantria dispar (gypsy moth) , using Renografin™ density gradient purified CrylF crystal protein from recombinant B.t. strain EG1945, which harbors the crylF gene on plasmid pEG642.

Activity was measured using a diet- surface overlay technique where the surface of an agar-based artificial diet was covered with an aliquot suspension containing CrylF protein crystals. After delivery of the aliquot to the diet surface, the diluent was allowed to evaporate, at which time one larva of the test species was placed in each cup. Each 2 ml well (cup) contained

1 ml diet having a surface area of 175 mm 2.

Bioassays were held at 28^βC for 7 days, at which time mortality was scored. Bioassays were first conducted at three doses with 1 to 10 dilutions. If the CrylF protein demonstrated sufficient activity, eight dose assays (1 to 2 dilutions) were conducted to determine LC D-.X_ΛJ values via the well- known technique of probit analysis (Daum, Bull. Entomol. Soc. Am., 16, pp. 10-15 (1970)). Each dose was tested against 32 insects. The diluent, 0.005% Triton™ X-100, served as a control treatment. All insects were tested as newly hatched first-stage larvae. The results of effective insecticidal activity are set forth in Table 4 in comparison with the results of insecticidal bioassays using other CrylA crystal proteins.

The CrylF protein exhibited the greatest toxicity to Ostrinia nubilalis larvae as indicated in Table 4. The LC_5Q value obtained is similar to LC_₀ values obtained for the purified CrylA(b) crystal protein which is highly toxic to Ostrinia nubilalis larvae. In addition, the CrylF protein was toxic to Spodoptera exigua larvae. CrylF protein was considerably more toxic to Spodoptera exigua than purified CrylA(a) and CrylA(c) crystal proteins and slightly more toxic than purified CrylA(b) crystal protein. Purified CrylF crystal protein was also toxic to Heliothis virescens, with a toxicity between that of purified CrylA(c) and CrylA(b) crystal protein. CrylF crystal protein exhibited little toxicity to Heliothis zea or Lymantria dispar at the doses tested.

Example 7 Analysis of Insecticidal Activity of CrylX Fragment The sequence of the crylX gene present on plasmid pEG642 (and likewise present on plasmid pEG640) does not encode a sufficient number of amino acids to constitute a "minimum toxic fragment" as defined by deletion analyses of crylA genes (Schnepf et al., J. Biol. Chem., 260, pp. 6273-6278 (1985)), Hofte et al., (1986) supra) . Nonetheless, to assess the contribution of crylX, if any, to the overall toxicity of the pEG642 construct, the following study was performed.

Plasmid pEG310, containing a deletion in the crylF gene, was constructed by restriction enzyme deletion from plasmid pEG642 of an N- terminal region of the crylF gene which is flanked by BstEII sites (Figure 6) . Following religation, plasmid pEG310 was introduced into the Cry~ B.t. HD73-26 recipient via electroporation, resulting in a recombinant strain designated B.t. strain EG1078. Fully sporulated cultures, containing the intact cryIX gene sequence from plasmid pEG642, but not the crylF gene which had been deleted, were assayed by the insect bioassay procedure previously described in Example 6 for toxicity against Ostrinia nubilalis. B.t. strain EG1945, containing the intact crylF gene, was the positive control.

Thirty insect larvae were assayed, at a protein dose of 4_.00 ng/550mm 2. At this dose, the

B.t. strain EG1945 was 100% toxic to larvae of

Ostrinia nubilalis. However, B.t. strain EG1078, containing the crylF deletion mutant, exhibited 0% mortality for Ostrinia nubilalis larvae. Thus, it was concluded that the sequence of the crylX gene present on plasmid pEG642 does not contribute to the observed toxicity of B.t. strain EG1945 and that the crylF gene product is the active insecticide in the strain.

Example 8

Southern Blot Analysis of the crylX Gene in B.t. Strain

EG6346

Following the Southern blot technique cited in Example 1, total DNA was obtained from

B.t. strain EG6346, digested to completion with the restriction endonucleases Asp718 (an isoschizomer of Kpnl) , Clal, SstI, and SphI both individually and in combination, electrophoresed through a 0.8% agarose gel, transferred to a nitrocellulose filter, and hybridized at 65°C overnight to a 32P- labeled 0.6 kb KpnI-BamHI probe that was isolated from pEG640 (previously described in Example 2) and that contained a portion of the crylX coding region. The positions of the Kpnl and BamHI sites flanking the crylX probe are shown in Figure 5. The results of the Southern blot analysis are shown in Figure 9. Total DNA from B.t. strain EG6346 digested with restriction endonucleases exhibited, in each instance, a single DNA fragment hybridizing to the crylX probe. Most importantly, B.t. strain EG6346 DNA digested with Clal (lane 2) yielded a 4.6 kb restriction fragment that hybridized to the probe. In addition, B.t. strain EG6346 DNA digested with both Clal and SstI (lane 8) yielded a 4.4 kb restriction fragment that was detected by the crylX probe. Since a Clal restriction site was present only 309 bp upstream from the crylX open reading frame shown in Figure 2, these results indicated that the entire crylX gene was likely to be contained on the 4.6 kb Clal restriction fragment. This assumption was shown to be correct by the fact that the CrylX protein is only 81 kDa, which corresponds to a gene of about 2.1-2.2 kb in length. In addition, the absence of an SstI site immediately upstream to or within the sequenced portion of the crylX gene displayed in Figure 2-A indicated that the SstI restriction site detected by Southern blot analysis was located downstream from crylX.

Example 9

Isolation of the Entire crylX Gene in E. coli

A genomic library was constructed from Clal-digested DNA of B.t. strain EG6346 and screened under moderate stringency conditions with the 0.6 kb KpnI-BamHI crylX probe derived from pEG640 to identify recombinant E^ coli colonies containing crylX gene sequences. More specifically, total DNA obtained from B.t. strain EG6346 was digested to completion with Clal, electrophoresed through a 0.8% agarose gel, and DNA fragments in the 4.3-5.0 kb range excised from the gel with a clean razor blade. DNA fragments within the agarose gel slice were purified using the GeneClean^® II kit and procedure available from Bio 101, Inc. of La Jolla, CA.

The E^_ coli-B.t. cloning vector pEG854, depicted as a circular restriction map in Figure 10 and described by Baum et al. , Appl. Environ. Microbiol. , 56, pp. 3420-3428 (1990) , was used to clone the crylX gene on the Clal restriction fragments. The Clal restriction fragments were ligated to Clal-digested pEG854 vector DNA pretreated with calf intestinal alkaline phosphatase to prevent self-ligation. Transformation of E__ coli HB101 cells with the ligation mixture was achieved by electroporation using the high-efficiency transformation procedure of Dower et al.. Nucleic Acids Res. , 16, pp. 6127- 6145. Transformed cells were plated on agar plates of standard LB medium containing 50 μg/ml ampicillin. Colonies were screened under moderate stringency conditions for the presence of the crylX gene sequence using the colony blot hybridization procedure outlined in Example 2. The hybridization step was performed at 65"C, rather than at 50-55"C as in Example 2, using the 0.6 kb KpnI-BamHI crylX probe described in Example 9. Filter washes were performed at 65^βC in 3X SSC, 0.1% SDS. The crylX probe hybridized strongly to one E_-_ coli recombinant colony, designated E_^ coli strain EG1082, that contained an 11.8 kb recombinant plasmid, designated pEG313, that consisted of a 4.6 kb Clal restriction fragment from B.t. strain EG6346 inserted into the Clal restriction site of cloning vector pEG854.

A circular restriction map of recombinant plasmid pEG313 is depicted in Figure 11. The orientation of the 4.6 kb Clal restriction fragment was determined by restriction endonuclease mapping using methods well known to those skilled in the art.

Example 10

Expression of the crylX Gene in E^ coli and Production of CrylX Protein To achieve expression of the crylX gene iⁿ _=___. coli and to characterize its encoded crystal protein, a 4.4. kb DNA fragment containing the cryIX gene was inserted into the E__ coli cloning vector pTZ19u, obtained from U.S. Biochemical Corporation. A circular restriction map of cloning vector pTZ19u, designated Plac in Figure 12, can be used to direct the transcription of cloned genes inserted into the multiple cloning site region demarcated by the unique Hindlll and EcoRI restriction sites within the lacZ' gene.

Accordingly, a 4.4 kb Clal-SstI restriction fragment containing the entire crylX gene, as indicated by the Southern blot analysis in Example 8, was isolated from the recombinant plasmid pEG313 (see Figure 11) and ligated to pTZ19u DNA digested with AccI and SstI, two restriction endonucleases with cleavage sites within the multiple cloning site region of the cloning vector. Note that the AccI restriction site is compatible with that of Clal, thereby allowing for efficient ligation of the cryIX gene fragment and orienting the crylX gene in the same direction as the lac promoter. The ligation mixture was used to transform E^ coli DHStj cells as described in Example 2. Using the X-gal screening procedure, a recombinant E__ coli colony, designated EG1083, was recovered that contained a 7.3 kb recombinant plasmid, designated pEG314, that consisted of a 4.4 kb Clal-SstI restriction fragment derived from pEG313 inserted into the AccI and SstI sites of vector pTZ19u. A circular restriction map of recombinant plasmid pEG314, containing the crylX gene inserted downstream from the lac promoter of pTZ19u, is depicted in Figure 13.

E. coli strain EG1083, containing pEG314 which carried the crylX gene, was grown in Luria broth containing 50 μg/ml ampicillin and ImM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37°C overnight. IPTG is an inducer of the lac promoter and is commonly used to optimize transcription from that promoter in E^ coli. After overnight growth, cells were examined by phase- contrast microscopy. E__ coli strain EG1083 cells, but not

cells containing pTZ19u, contained multiple phase-bright inclusions. Subsequent lysis of the recombinant cells with lysozyme released the large inclusions, some of which appeared rhomboid in shape. The inclusions were purified from E^ coli strain EG1083 by Renografin™ density gradient centrifugation and examined by SDS-polyacrylamide gel electrophoresis. Figure 14 is a photocopy of the resulting Coomassie-stained 10% SDS-polyacrylamide gel, in which lanes 1 and 2 contain CrylX crystal protein from E__;_ coli strain EG1083 before and after Renografin™ density gradient centrifugation, respectively. Protein molecular weight standards are displayed in the leftmost lane. Based on these standards, the CrylX crystal protein migrates with an apparent molecular mass of 81 kDa.

Example 11 Insect Toxicity of the CrylX Protein

The insecticidal activity of the 81 kDa CrylX protein was determined against lepidopteran species, using Renografin™ density gradient purified CrylX crystal protein from recombinant E. coli strain EG1083, which harbors the crylX gene on plasmid pEG314.

Activity was measured using a diet- surface overlay technique where the surface of an agar-based artificial diet was covered with an aqueous suspension containing CrylX protein crystals. Insect larvae were placed on the diet surface after the diluent had evaporated and held at 28^βC for seven days, at which time mortality was scored.

In this bioassay screening, procedure, the purified CrylX protein exhibited insecticidal activity against larvae of Plutella xylostella (diamondback moth) . In another bioassay screening procedure, using a cell paste of E_^ coli strain EG1083 that had produced CrylX protein instead of the purified CrylX protein, insecticidal activity was exhibited 5 against larvae of Ostrinia nubilalis (European corn borer) .

To assure the availability of materials to those interested members of the public upon issuance of a patent on the present application 10 deposits of the following microorganisms were made prior to the filing of present application with the ARS Patent Collection, Agricultural Research Culture Collection, Northern Regional Research Laboratory (NRRL) , 1815 North University Street, 15 Peoria, Illinois 61064, as indicated in the following Table 5:

Table 5

Bacterial Strain NRRL Accession No. Date of Deposit

B.thuringiensis HD73-26 B-18508 June 12, 1989 B.thuringiensis EG6345 B-18633 March 27, 1990 B.thuringiensis EG1945 B-18635 March 27, 1990 E__;_ coli EG1943 B-18634 March 27, 1990 E. coli EG1083 B-18805 March 29, 1991

These microorganism deposits were made 25 under the provisions of the "Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure". The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification as indicating the scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Gawron-Burke, Cynthia

Chambers, Judith A. Gonzalez Jr. , Jose M.

(ii) TITLE OF INVENTION: BACILLUS THURINGIENSIS crylF and crylX GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Panitch Schwarze et al. c/o A.S. Nadel

(B) STREET: 1601 Market Street, 36th Floor

(C) CITY: Philadelphia

(D) STATE: PA

(E) COUNTRY: U.S.A.

(F) ZIP: 19103

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

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

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/510327

(B) FILING DATE: 16-APR-1990

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Egolf, Christopher

(B) REGISTRATION NUMBER: 27633

(C) REFERENCE/DOCKET NUMBER: 7205-27 Ul

(2) INFORMATION FOR SEQ ID NO:l: ^*

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4020 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 478..4002

SUBSTITUTE SHEET

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GATCTTCAAA TGAGAAAATA AGGGTATTCC GTATGGGATG CCTTTATTTT GGTTGGGAAG 6

AAGGATTAAA AATCAAAAAT GTAAATCAGA TATAGTCCAG ATAATTTTTT AAAGAGTGTA 12

« GTATATTAAA AATAATGTTC TTATAACATA TATGTTGATT TTAAGAAAAT ATTTTGTTTA 18

AGAATTCAAT CCATATGAGT ATAAAAAGTT AAAAGGCCCA AAAATAAGTT AAGGGAAATC 24

AACTCTTTAA TACAAAAGTT TATCTCAGGA ATTCTCAACT ATGGATAGCA GGAAGAGAAG 30

TAAGCACACT ATTAACATAT TAGGTCTATT TAAATTAAGG GCATATAGTG ATATTTTATA 36

AGATTGGTTG CACTTTGTGC ATTTTTTCAT AAGATGAGTC ATATGTTTTA CATTGTAATA 420

CAGTAAGAGG TTTTAGTTTT AAAGAACAAC TATTATGATG AAATGTGGAG GGAACAT 477

ATG GAG AAT AAT ATT CAA AAT CAA TGC GTA CCT TAC AAT TGT TTA AAT 525 Met Glu Asn Asn Ile Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15

AAT CCT GAA GTA GAA ATA TTA AAT GAA GAA AGA AGT ACT GGC AGA TTA 573 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30

CCG TTA GAT ATA TCC TTA TCG CTT ACA CGT TTC CTT TTG AGT GAA TTT 621 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45

GTT CCA GGT GTG GGA GTT GCG TTT GGA TTA TTT GAT TTA ATA TGG GGT 669 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60

TTT ATA ACT CCT TCT GAT TGG AGC TTA TTT CTT TTA CAG ATT GAA CAA 717 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gin Ile Glu Gin 65 70 75 80

TTG ATT GAG CAA AGA ATA GAA ACA TTG GAA AGG AAC CGG GCA ATT ACT 765 Leu lie Glu Gin Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr

85 90 95

ACA TTA CGA GGG TTA GCA GAT AGC TAT GAA ATT TAT ATT GAA GCA CTA 813 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110

_*AGA GAG TGG GAA GCA AAT CCT AAT AAT GCA CAA TTA AGG GAA GAT GTG 861 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val 115 120 125

CGT ATT CGA TTT GCT AAT ACA GAC GAC GCT TTA ATA ACA GCA ATA AAT 909 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140

AAT TTT ACA CTT ACA AGT TTT GAA ATC CCT CTT TTA TCG GTC TAT GTT 957 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160

CAA GCG GCG AAT TTA CAT TTA T.CA CTA TTA AGA GAC GCT GTA TCG TTT 1005 Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe

165 170 175 GGG CAG GGT TGG GGA CTG GAT ATA GCT ACT GTT AAT AAT CAT TAT AAT 1053 Gly Gin Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190

AGA TTA ATA AAT CTT ATT CAT AGA TAT ACG AAA CAT TGT TTG GAC ACA 1101 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205

TAC AAT CAA GGA TTA GAA AAC TTA AGA GGT ACT AAT ACT CGA CAA TGG 1149 Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp 210 215 220

GCA AGA TTC AAT CAG TTT AGG AGA GAT TTA ACA CTT ACT GTA TTA GAT 1197 Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

ATC GTT GCT CTT TTT CCG AAC TAC GAT GTT AGA ACA TAT CCA ATT CAA 1245 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gin

245 250 255

ACG TCA TCC CAA TTA ACA AGG GAA ATT TAT ACA AGT TCA GTA ATT GAG 1293 Thr Ser Ser Gin Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270

GAT TCT CCA GTT TCT GCT AAT ATA CCT AAT GGT TTT AAT AGG GCG GAA 1341 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285

TTT GGA GTT AGA CCG CCC CAT CTT ATG GAC TTT ATG AAT TCT TTG TTT 1389 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

GTA ACT GCA GAG ACT GTT AGA AGT CAA ACT GTG TGG GGA GGA CAC TTA 1437 Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

GTT AGT TCA CGA AAT ACG GCT GGT AAC CGT ATA AAT TTC CCT AGT TAC 1485 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr

325 330 335

GGG GTC TTC AAT CCT GGT GGC GCC ATT TGG ATT GCA GAT GAG GAT CCA 1533 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350

CGT CCT TTT TAT CGG ACA TTA TCA GAT CCT GTT TTT GTC CGA GGA GGA 1581 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365

TTT GGG AAT CCT CAT TAT GTA CTG GGG CTT AGG GGA GTA GCA TTT CAA 1629 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

CAA ACT GGT ACG AAC CAC ACC CGA ACA TTT AGA AAT AGT GGG ACC ATA 167 Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400

GAT TCT CTA GAT GAA ATC CCA CCT CAG GAT AAT AGT GGG GCA CCT TGG 172 Asp Ser Leu Asp Glu Ile Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp

405 410 415 AAT GAT TAT AGT CAT GTA TTA AAT CAT GTT ACA TTT GTA CGA TGG CCA 177 Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430

GGT GAG ATT TCA GGA AGT GAT TCA TGG AGA GCT CCA ATG TTT TCT TGG 182 * Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445

_ ACG CAC CGT AGT GCA ACC CCT ACA AAT ACA ATT GAT CCG GAG AGG ATT 186 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460

ACT CAA ATA CCA TTG GTA AAA GCA CAT ACA CTT CAG TCA GGT ACT ACT 191 Thr Gin Ile Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly Thr Thr 465 470 475 480

GTT GTA AGA GGG CCC GGG TTT ACG GGA GGA GAT ATT CTT CGA CGA ACA 196 Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr

485 490 495

AGT GGA GGA CCA TTT GCT TAT ACT ATT GTT AAT ATA AAT GGG CAA TTA 201 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gin Leu 500 505 510

CCC CAA AGG TAT CGT GCA AGA ATA CGC TAT GCC TCT ACT ACA AAT CTA 206 Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525

AGA ATT TAC GTA ACG GTT GCA GGT GAA CGG ATT TTT GCT GGT CAA TTT 210 Arg Ile Tyr Val Thr Val Ala Gly Glu Arg lie Phe Ala Gly Gin Phe 530 535 540

AAC AAA ACA ATG GAT .. C GGT GAC CCA TTA ACA TTC CAA TCT TTT AGT 215 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser 545 550 555 560

TAC GCA ACT ATT AAT ACA GCT TTT ACA TTC CCA ATG AGC CAG AGT AGT 220 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gin Ser Ser

565 570 575

TTC ACA GTA GGT GCT GAT ACT TTT AGT TCA GGG AAT GAA GTT TAT ATA 225 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590

GAC AGA TTT GAA TTG ATT CCA GTT ACT GCA ACA TTT GAA GCA GAA TAT 230 "Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

^♦GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCG CTG TTT ACT TCT ATA 234 Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr Ser Ile 610 615 620

AAC CAA ATA GGG ATA AAA ACA GAT GTG ACG GAT TAT CAT ATT GAT CAA 2397 Asn Gin Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gin 625 630 635 640

GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA TTT TGT CTG GAT GAA 2445 Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu

645 650 655 AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA CTC AGT GAT 249 Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

GAG CGG AAT TTA CTT CAA GAT CCA AAC TTC AAA GGC ATC AAT AGG CAA 254 Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Lys Gly Ile Asn Arg Gin

675 680 685

CTA GAC CGT GGT TGG AGA GGA AGT ACG GAT ATT ACC ATC CAA AGA GGA 258 Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gin Arg Gly 690 695 700

GAT GAC GTA TTC AAA GAA AAT TAT GTC ACA CTA CCA GGT ACC TTT GAT 263 Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 715 720

GAG TGC TAT CCA ACG TAT TTA TAT CAA AAA ATA GAT GAG TCG AAA TTA 268 Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu

725 730 735

AAA CCC TAT ACT CGT TAT CAA TTA AGA GGG TAT ATC GAG GAT AGT CAA 273 Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr Ile Glu Asp Ser Gin 740 745 750

GAC TTA GAA ATC TAT TTG ATC CGC TAT AAT GCA AAA CAC GAA ACA GTA 278 Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

AAT GTG CTA GGT ACG GGT TCT TTA TGG CCG CTT TCA GTC CAA AGT CCA 282 Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gin Ser Pro 770 775 780

ATC AGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC CTT GAA TGG 287 Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp 785 790 795 800

AAT CCT GAT CTA GAT TGT TCC TGC AGA GAC GGG GAA AAA TGT GCA CAT 292 Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His

805 810 815

CAT TCG CAT CAT TTC TCC TTG GAC ATT GAT GTT GGA TGT ACA GAC TTA 297 His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu 820 825 830

AAT GAG GAC TTA GAT GTA TGG GTG ATA TTC AAG ATT AAG ACG CAA GAT 302 Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp

835 840 845

GGC CAT GCA AGA CTA GGA AAT CTA GAG TTT CTC GAA GAG AAA CCA TTA 306 Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu 850 855 860

GTC GGG GAA GCA CTA GCT CGT GTG AAA AGA GCA GAG AAA AAA TGG AGA 31 Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 865 870 875 880

GAT AAA CGT GAA AAA TTG GAA TTG GAA ACA AAT ATT GTT TAT AAA GAG 316 Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn lie Val Tyr Lys Glu

885 890 895 GCA AAA GAA TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA TAT GAT CAA 32 Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Gin 900 905 910

TTA CAA GCG GAT ACG AAT ATT GCC ATG ATT CAT GCG GCA GAT AAA CGT 32 Leu Gin Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg 915 920 925

GTT CAT AGA ATT CGG GAA GCG TAT CTT CCA GAG TTA TCT GTG ATT CCG 33 Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro 930 935 940

GGT GTA AAT GTA GAC ATT TTC GAA GAA TTA AAA GGG CGT ATT TTC ACT 33 Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr 945 950 955 960

GCA TTC TTC CTA TAT GAT GCG AGA AAT GTC ATT AAA AAC GGT GAT TTC 34 Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe

965 970 975

AAT AAT GGC TTA TCA TGC TGG AAC GTG AAA GGG CAT GTA GAT GTA GAA 345 Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu 980 985 990

GAA CAA AAC AAC CAC CGT TCG GTC CTT GTT GTT CCG GAA TGG GAA GCA 350 Glu Gin Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala 995 1000 1005

GAA GTG TCA CAA GAA GTT CGT GTC TGT CCG GGT CGT GGC TAT ATC CTT 354 Glu Val Ser Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu 1010 1015 1020

CGT GTC ACA GCG TAC AAG GAG GGA TAT GGA GAA GGT TGC GTA ACC ATT 359 Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile 1025 1030 1035 1040

CAT GAG ATC GAG AAC AAT ACA GAC GAA CTG AAG TTT AGC AAC TGC GTA 364 His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val

1045 1050 1055

GAA GAG GAA GTC TAT CCA AAC AAC ACG GTA ACG TGT AAT GAT TAT ACT 369 Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr 1060 1065 1070

GCA AAT CAA GAA GAA TAC GGG GGT GCG TAC ACT TCC CGT AAT CGT GGA 374 Ala Asn Gin Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly 1075 1080 1085

TAT GAC GAA ACT TAT GGA AGC AAT TCT TCT GTA CCA GCT GAT TAT GCG 378 Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala 1090 1095 1100

TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA GAC AAT CCT 383 Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Asp Asn Pro 1105 1110 1115 1120

TGT GAA TCT AAC AGA GGA TAT GGG GAT TAC ACA CCA CTA CCA GCT GGC 388

Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly

1125 1130 1135 TAT GTG ACA AAA GAA TTA GAG TAC TTC CCA GAA ACC GAT AAG GTA TGG 393 Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp 1140 1145 1150

ATT GAG ATC GGA GAA ACG GAA GGA ACA TTC ATC GTG GAC AGC GTG GAA 398 Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu 1155 1160 1165 TTA CTC CTT ATG GAG GAA TAGTCTCATA CAAAATTAGT T 402

Leu Leu Leu Met Glu Glu

1170 117

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1174 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Glu Asn Asn Ile Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15

Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 ^* 30

Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60

Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gin Ile Glu Gin 65 70 75 80

Leu Ile Glu Gin Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala lie Thr

85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val 115 120 125

Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala lie Asn 130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160

Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe

165 170 175

Gly Gin Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205

Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp 210 _. 215 220

Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gin

245 250 - 255

Thr Ser Ser Gin Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270

Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr

325 330 335

Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350

Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400

Asp Ser Leu Asp Glu Ile Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430

Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460

Thr Gin Ile Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly Thr Thr 465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr

485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gin Leu 500 505 510 Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525

Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gin Phe 530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser 545 550 555 560

Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gin Ser Ser

565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590

Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

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

Asn Gin Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gin 625 630 635 640

Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu

645 650 655

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Lys Gly Ile Asn Arg Gin 675 680 685

Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp lie Thr Ile Gin Arg Gly 690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu

725 730 735

Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr Ile Glu Asp Ser Gin 740 745 750

Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gin Ser Pro 770 775 780

Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp 785 790 795 800

Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His

805 810 815

His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu 820 825 830 Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp 835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu 850 855 860

Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 865 870 875 880

Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu

885 890 895

Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Gin

900 905 910

Leu Gin Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg 915 920 925

Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro 930 935 940

Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr 945 950 955 960

Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe

965 970 975

Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu 980 985 990

Glu Gin Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala 995 1000 1005

Glu Val Ser Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu 1010 1015 1020

Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile 1025 1030 1035 1040

His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val

1045 1050 1055

Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr 1060 1065 1070

Ala Asn Gin Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly 1075 1080 1085

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

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

Cys Glu Ser Asn Arg Gly Tyr Gly Asp T"^*r Thr Pro Leu Pro Ala Gly

1125 1130 1135

Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu 1155 1160 1165

Leu Leu Leu Met Glu Glu 1170

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1629 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 488..1629

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TTAAATATCG TTTTCAAATC AATTGCCTAA GAGCATCATT ACAAATAGAT AAGTAATTTG 6

TTGTAATGAA AAACGGACAT CACCTCCATT GAAACGGTGA GATGTCCGTT TTACTATGTT 12

ATTTTCTAGT AATACATATG TACAGAGCAA CTTAATTAAG CAGAGATATT TTCCCCTATC 18

GATGAAAATA TCTCTGCTTT TTCTTTCTTT ATTCGGTATA TGCTTTACTT GTAATTGAAA 24

ATAAAGCACT AATAAGAGTA TTTATAGGTG TTTGAAGTTA TTTCAGTTTA TTTTTAAAGG 30

AGGTTTAAAA ACGTTAGAAA GTTATTAAGG AATAATACTT ATTAGTAAAT TCCACATATA 36

TTTTATAATT AATTATGAAA TATATGTATA AATTGAAAAT GCTTTATTTG ACATTACAGC 42

TAAGTATAAT TTTGTATGAA TAAAATTATA TCTGAAAATT AAATAATATT ACAGTGGAGG 48

GATTAAT ATG AAA CTA AAG AAT CCA GAT AAG CAT CAA AGT TTT TCT AGC 52 Met Lys Leu Lys Asn Pro Asp Lys His Gin Ser Phe Ser Ser 1 5 10

AAT GCG AAA GTA GAT AAA ATC TCT ACG GAT TCA CTA AAA AAT GAA ACA 57 Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr 15 20 25 30

GAT ATA GAA TTA CAA AAC ATT AAT CAT GAA GAT TGT TTG AAA ATA TCT 62 Asp Ile Glu Leu Gin Asn Ile Asn His Glu Asp Cys Leu Lys Ile Ser

35 40 45

GAG TAT GAA AAT GTA GAG CCG TTT GTT AGT GCA TCA ACA ATT CAA ACA 67 Glu Tyr Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr Ile Gin Thr 50 55 60

GGT ATT AGT ATT GCG GGT AAA ATA CTT GGC ACC CTA GGC GTT CCT TTT 72 Gly Ile Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Val Pro Phe 65 70 75

GCA GGA CAA GTA GCT AGT CTT TAT AGT TTT ATC TTA GGT GAG CTA TGG 76 Ala Gly Gin Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp 80 85 90

_ CCT AAG GGG AAA AAT CAA TGG GAA ATC TTT ATG GAA CAT GTA GAA GAG 81 Pro Lys Gly Lys Asn Gin Trp Glu Ile Phe Met Glu His Val Glu Glu 95 100 105 110

ATT ATT AAT CAA AAA ATA TCA ACT TAT GCA AGA AAT AAA GCA CTT ACA 86 Ile Ile Asn Gin Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr

115 120 125

GAC TTG AAA GGA TTA GGA GAT GCC TTA GCT GTC TAC CAT GAA TCG CTT 91 Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu 130 135 140

GAA AGT TGG GTT GGA AAT CGT AAG AAC ACA AGG GCT AGG AGT GTT GTC 96 Glu Ser Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val 145 150 155

AAG AGC CAA TAT ATC GCA TTA GAA TTG ATG TTC GTT CAG AAA CTA CCT 100 Lys Ser Gin Tyr lie Ala Leu Glu Leu Met Phe Val Gin Lys Leu Pro 160 165 170

TCT TTT GCA GTG TCT GGA GAG GAG GTA CCA TTA TTA CCG ATA TAT GCC 105 Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala 175 180 185 190

CAA GCT GCA AAT TTA CAT TTG TTG CTA TTA AGA GAT GCA TCT ATT TTT 110 Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe

195 200 205

GGA AAA GAG TGG GGA TTA TCA TCT TCA GAA ATT TCA ACA TTT TAT AAC 115 Gly Lys Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr Asn 210 215 220

CGT CAA GTC GAA CGA GCA GGA GAT TAT TCC GAC CAT TGT GTG AAA TGG 120 Arg Gin Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp 225 230 235

TAT AGT ACA GGT CTA AAT AAC TTG AGG GGT ACA AAT GCC GAA AGC TGG 1249 Tyr Ser Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp 240 245 250

^■ GTT CGT TAT AAT CAA TTT CGT AAA GAT ATG ACA TTA ATG GTA CTT GAT 1297 Val Arg Tyr Asn Gin Phe Arg Lys Asp Met Thr Leu Met Val Leu Asp 255 260 265 270

TTA GTC GCA CTA TTC CCA AGC TAT GAT ACA CTT GTA TAT CCA ATT AAA 1345 Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys

275 280 285

ACT ACT TCT CAA CTT ACA AGA GAA GTA TAT ACA GAC GCA ATT GGG ACA 1393 Thr Thr Ser Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr 290 295 300

GTA CAT CCG AAT GCA AGT TTT GCA AGT ACG ACT TGG TAT AAT AAT AAT 1441 Val His Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn 305 310 315

GCC CCT TCG TTC TCT ACC ATA GAG TCT GCT GTT GTT CGA AAC CCG CAT 14 Ala Pro Ser Phe Ser Thr Ile Glu Ser Ala Val Val Arg Asn Pro His 320 325 330

CTA CTC GAT TTT CTA GAA CAA GTT ACA ATT TAC AGC TTA TTA AGT AGG 15 Leu Leu Asp Phe Leu Glu Gin Val Thr Ile Tyr Ser Leu Leu Ser Arg 335 340 345 350

TGG AGT AAC ACT CAG TAT ATG AAT ATG TGG GGA GGA CAT AGA CTT GAA 15 Trp Ser Asn Thr Gin Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu

355 360 365

TTC CGA ACA ATC GGA GGA ATG TTA AAT ACC TCA ACA CAA GGA TC 16

Phe Arg Thr Ile Gly Gly Met Leu Asn Thr Ser Thr Gin Gly 370 375 380

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 380 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Lys Leu Lys Asn Pro Asp Lys His Gin Ser Phe Ser Ser Asn Ala 1 5 10 15

Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile 20 25 30

Glu Leu Gin Asn Ile Asn His Glu Asp Cys Leu Lys Ile Ser Glu Tyr 35 40 45

Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr Ile Gin Thr Gly Ile 50 55 60

Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Val Pro Phe Ala Gly 65 70 75 80

Gin Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp Pro Lys

85 90 95

Gly Lys Asn Gin Trp Glu Ile Phe Met Glu His Val Glu Glu Ile Ile 100 105 110

Asn Gin Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu 115 120 125

Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu Glu Ser 130 135 140

Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val Lys Ser 145 150 155 160 Gin Tyr Ile Ala Leu Glu Leu Met Phe Val Gin Lys Leu Pro Ser Phe

165 170 175

Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gin Ala 180 185 190

Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys 195 200 205

Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr Asn Arg Gin 210 215 220

Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser 225 230 235 240

Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg

245 250 255

Tyr Asn Gin Phe Arg Lys Asp Met Thr Leu Met Val Leu Asp Leu Val 260 265 270

Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys Thr Thr 275 280 285

Ser Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His 290 295 300

Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro 305 310 315 320

Ser Phe Ser Thr Ile Glu Ser Ala Val Val Arg Asn Pro His Leu Leu

325 330 335

Asp Phe Leu Glu Gin Val Thr Ile Tyr Ser Leu Leu Ser Arg Trp Ser 340 345 350

Asn Thr Gin Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu Phe Arg 355 360 365

Thr Ile Gly Gly Met Leu Asn Thr Ser Thr Gin Gly 370 375 380

HEET

Claims

1. A purified and isolated crylF gene characterized by a nucleotide base sequence coding for the amino acid sequence illustrated in Figure 1

•5 (SEQ ID NO:2) .

2. A purified and isolated crylF gene according to claim 1 further characterized in that the gene has a coding region extending from nucleotide bases 478 to 3999 in the nucleotide base 0 sequence illustrated in Figure 1 (SEQ ID N0:1).

3. A lepidopteran-toxic protein characterized in that the protein is produced by the gene of claim 1 or claim 2.

4. A recombinant plasmid characterized 15 in that the recombinant plasmid contains the gene of claim 1 or claim 2.

5. A biologically pure culture of a bacterium characterized in that the bacterium is transformed with the recombinant plasmid of claim

20 4.

6. The bacterium of claim 5 further characterized in that the bacterium is E_^_ coli.

7. The E^ coli bacterium of claim 6 further characterized in that the bacterium is

25 deposited with the NRRL with accession number NRRL B-18634.

TITUTE SHEET

8. The bacterium of claim 5 further characterized in that the bacterium is Bacillus thuringiensis.

9. The Bacillus thuringiensis bacterium of claim 8 further characterized in that the bacterium is deposited with the NRRL with accession number NRRL B-18635.

10. An insecticide composition characterized in that the composition contains the protein of claim 3 and an agriculturally acceptable carrier.

11. An insecticide composition characterized in that the composition contains the bacterium of claim 5, a lepidopteran-toxic protein produced by such bacterium, and an agriculturally acceptable carrier.

12. A lepidopteran-toxic protein characterized by the protein being characteristic of that made by the Bacillus thuringiensis bacterium of claim 9 and having the amino acid sequence illustrated in Figure 1 (SEQ ID NO:2).

13. An insecticide composition characterized in that the composition contains the lepidopteran-toxic protein of claim 12, in combination with an agriculturally acceptable carrier.

TE SHEET

14. The insecticide composition of claim 13 further characterized in that the lepidopteran- toxic protein is contained in a Bacillus thuringiensis bacterium.

15. A method of controlling lepidopteran insects characterized by applying to a host plant for such insects an insecticidally effective amount of the lepidopteran-toxic protein of claim 3.

16. The method of claim 15 further characterized in that the lepidopteran-toxic protein is contained in a Bacillus thuringiensis bacterium.

17. A method of controlling lepidopteran insects characterized by applying to a host plant for such insects an insecticidally effective amount of the lepidopteran-toxic protein of claim 12.

18. The method of claim 17 further characterized in that the lepidopteran-toxic protein is contained in a Bacillus thuringiensis bacterium.

19. A plant characterized in that the plant is transformed with the gene of claim 1 or claim 2.

20. The crylF gene of claim 2 further characterized in that the gene or a portion thereof is labeled for use as a hybridization probe.

UBSTITUTE SHEET

21. A biologically pure culture of a Bacillus thuringiensis bacterium characterized in that the culture is deposited with the NRRL with accession number NRRL B-18633.

22. A purified and isolated crylX gene characterized by the gene being characteristic of that contained in E__;_ coli strain EG1083 deposited with the NRRL with accession number NRRL B-18805.

23. A lepidopteran-toxic protein characterized in that the protein is produced by the gene of claim 22 and has a molecular mass of about 81 kDa.

24. A recombinant plasmid characterized in that the recombinant plasmid contains the gene of claim 22.

25. A biologically pure culture of a bacterium characterized in that the bacterium is transformed with the recombinant plasmid of claim 24.

26. The bacterium of claim 25 further characterized in that the bacterium is selected from the group consisting of E^ coli and Bacillus thuringiensis.

27. An E^ coli bacterium according to claim 26 further characterized in that the bacterium is deposited with the NRRL with accession number NRRL B-18805.

TUTE SHEET - 80 -

28. An insecticide composition characterized in that the composition contains the protein of claim 23 and an agriculturally acceptable carrier.

5 29. An insecticide composition characterized in that the composition contains the bacterium of claim 25, an insecticidal protein produced by such bacterium, and an agriculturally acceptable carrier.

10 30. A method of controlling lepidopteran insects characterized by applying to a host plant for such insects an insecticidally effective amount of the lepidopteran-toxic protein of claim 23.

31. The method of claim 30 further 15 characterized in that the lepidopteran-toxic protein is contained in a Bacillus thuringiensis bacterium.

32. A plant characterized in that the plant is transformed with the gene of claim 22.

20 33. The crylX gene of claim 22 characterized in that the gene or a portion thereof is labeled for use as a hybridization probe.

UBSTITUTE SHEET