<i>BACILLUS THURINGIENSIS</i> HYBRID TOXINS

Abstract: <i>Bacillus thuringiensis</i> hybrid toxin fragment comprising structural domains I, II and III in this order starting from the N-terminal, wherein the domains are derived from at least two different Cry protein, domain I is domain I of any <i>Bacillus thuringiensis</i> Cry protein or a part of said domain or a peptide substantially similar to said domain, domain II is domain II or Cry 1 Ia or a part of said domain or a peptide substantially similar to said domain, and domain III is domain III of Cry1Ba or a part of said domain or a peptide substantially similar domain.

Description: Bacillus thuringiensis hybrid toxins 1 .0. Field of the invention The present invention relates to hybrid toxin fragments, and toxins comprising them, derived from Bacillus thuringiensis msecticidal crystal proteins 1 .1 . Description of the related art Bacillus thuringiensis (hereinafter B.t.) is a gram-positive bacterium that produces msecticidal crystal proteins during sporulation The crystal (Cry) proteins form a large (over 1 60 and growing) family of homologous proteins with unique specificities Each protein is active against only one or a few insect species Most reported proteins are active against lepidopterans, with a smaller number showing activity against diptera or coleoptera as reviewed in Schnepf et al , 1 998) While initially the Cry proteins were classified according to their activity against one of the abovementioned insect orders (Hofte and Whiteley, 1 989), the more recent, now commonly accepted classification is based on ammo acid homology (Cπckmore et al , 1 998) The mode of actions of crystal proteins has been partially elucidated After ingestion by the insect larvae, crystals dissolve in the midgut environment, releasing the proteins as protoxins of 70-1 40 kDa The solubi zed protoxins are subsequently processed ( "activated ") by midgut proteases, resulting in a protease-resistant fragment of about 65 kDa, which is the active toxin. The toxin binds to receptors on epithelial cells of the insect midgut and penetrates the membrane. This eventually leads to lysis of the cells and death of the larvae. The activity range of a particular delta-endotoxin is to a large extent determined by the occurrence of receptors on the midgut cells of the insect, although solubilization efficiency and proteolytic activition are also factors involved. The importance of binding to receptors is further examplified by the decrease in binding occuring in many instances of resistance to Cry proteins (Ferre et al., 1 995). Structure determination by X-ray cristallography has shown that three different Cry proteins, and probably all Cry proteins, share a common three domain-structure ( Li et al., 1 991 ; Grochulski et al., 1 995; Morse et al., 1 998) . If projected on Cry1 sequences, domain I runs from about amino acid residue 28 to 260, domain II from about 260 to 460 and domain III from about 460 to 620. Since the various toxins have different lengths, the borders of the domains can be defined only approximately. A person skilled in this art will be able to find the borders for the various toxins by comparing the amino acid sequences. The N-terminal domain I consists of 7 α-helices and is considered to be inserting (partially) into the target membrane, and forming part of the pore that eventually kills the insect gut epithelial cells. Both domain II and the C- terminal domain III are very variable, and have been shown to determine activity against specific insects. Although it is not yet clear how these domains individually or acting together may determine specificity, there is strong evidence that both can be involved in binding to (putative) receptors (Lee et al., 1 995; Dean et al., 1 996; de Maagd et al., 1 996b; de Maagd et al., 1 999) . Exchange of domain III between toxins by in vivo recombination of their encoding genes may not only alter specificity of a toxin, but can also result in a hybrid toxin with superior toxicity for certain insects (Bosch et al. , 1 994; de Maagd et a/., 1 996a). Intl. Pat. Appl. Publ. No. WO 95/06730 discloses the construction of a hybrid delta- endotoxin consisting of domains I and II of Cryl E, and domain III and protoxin-specific fragment of Cryl C. In bioassays, this hybrid as purified after production in E. coli is active against Manduca sexta, Spodoptera exigua, and Mamestra brassicae. When expressed in and purified from a recombinant Bacillus thuringiensis strain, the 1 E/1 C- hybrid was 1 .5 times as active as the most active natural toxin against S. exigua, Cryl C. The abovementioned patent application also describes a hybrid delta-endotoxin consisting of domains I and II of Cryl Ab, and domain III of Cryl C . When purified from a recombinant Bacillus thuringiensis strain, this Cry1 Ab/Cry1 C-hybrid was approximately 6.6 times as toxic as Cry1 C (de Maagd et al., 1 996a). Intl. Pat. Appl. Publ. No. WO 98/22595 discloses a number of hybrid toxins consisting of different combinations of fragments of Cryl Ab, Cryl Ac, Cryl C or Cryl F, of which some have improved acitivity against important pest larvae and/or an extended activity spectrum. Producing succesfull hybrids is not easy. From the prior art it appears that only a very limited number of hybrids have an improved activity or a broader host-range specificity. In addition there are many possibilities for recombinantly-engineered crystal proteins, in view of the big number of different crystal proteins (more than 1 60). Further, the effect of the hybrids produced is not predictable. 2.0 Summary of the invention Proteins of the Cry3 (Herrnstadt et al. , 1 986; McPherson et al., 1 988), Cry7 (Lambert et a/., 1 992) and Cry8 (Sato et a/., 1 994) classes were found to be active against insects of the order Coleoptera (beetles). Cry3A is the singlemost active protein for the important potato pest Colorado potato beetle (CPB) , Leptinotarsa decemlineata (Say). It is known that resistance to the endotoxins may occur. Accordingly , resistance to Cry3A may occur in CPB in response to its exposure to Cry3A in transgenic potato. Actually, resistant CPB has already been disclosed . Therefore, it is desirable to have an alternative or replacement for Cry3A. The present inventors have found specific hybrid crystal proteins which have a high activity against CPB and therefore can be used as a replacement for Cry3A. The hybrids of the invention comprise domains derived from Cryl la and Cryl Ba. Cry1 proteins are generally active against lepidopterans (larvae of moths and butterflies) . Cryl B and Cryl l have been shown to also have some activity against coleopterans, among which CPB, although their toxicity for CPB is much lower than that of Cry3A (Tailor et al. , 1 992; Bradley et al., 1 995) . The toxicity of the hybrid crystal proteins of the invention against CPB, as compared to the parental proteins Cryl Ba and Cryl la, is surprisingly higher. Additionally a number of the hybrids of the invention have retained the high activity against some Lepidopterans, making them toxins with meaningful dual activity for Coleopterans and Lepidopterans. According to the present invention there is provided a B.t. hybrid toxin fragment comprising structural domains I, 11 and 111 in this order starting from the N-terminal, wherein the domains are derived from at least two different Cry proteins, domain I is domain I of any of B. t. Cry protein or a part of said domain or a peptide substantially similar to said domain , domain II is domain II of Cry1 la or a part of said domain or a peptide substantially similar to said domain, and domain III is domain 111 of Cryl Ba or a part of said domain or a peptide substantially similar to said domain. Preferred is a fragment which comprises domain I of Cry l la or Cry l Ba or a part of said domain or a peptide substantially similar to said domain, domain II of Cryl la or a part of said domain or a peptide substantially similar to said domain, and domain III of Cryl Ba or a part of said domain or a peptide substantially similar to said domain. The term " or a peptide substantially similar to said domain" should be understood to mean a peptide having an amino acid sequence which is at least 85 % similar to the sequence of the domain. It is preferred that the degree of similarity is at least 90% . In the context of the present invention, two amino acid sequences with at least 85 % or 90% similarity to each other have at least 85% or 90% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 6 gaps with the proviso that in respect of the gaps a total not more than 1 5 amino acid residues are affected. For the purpose of the present invention conservative replacements may be made between amino acids with the following groups: 1 . Serine and Threonine; 2. Glutamic acid and Aspartic acid; 3. Arginine and Lysine 4. Asparagine and Glutamine 5. Isoleucine, Leucine, Valine, and Methionine; 6. Phenylalanine, Tyrosine, and Tryptophan 7. Alanine and Glycine By "or a part of said domain" is meant a peptide comprised by the said domain and having at least 80% of the consecutive sequence thereof . It is most particularly preferred that the toxin fragment according to the invention comprises either: 1 . An amino acid sequence from about amino acid 20 to about amino acid 641 in SEQ ID NO :2 or 2. An amino acid sequence from about amino acid 20 to about amino acid 632 in SEQ ID N0:4. The invention also includes a hybrid toxin comprising the above disclosed fragment or a toxin at least 85 % similar to such a hybrid toxin which has substantially similar insecticidal activity. The hybrid toxin comprises the three structural domains as defined above and generally may comprise a pro-toxin segment at the carboxyl end, which is not toxic and is thought to be important for crystal formation . SEQ ID NO :2 and 4 comprise such pro-toxin segment. Further, the hybrid toxin may comprise a lead sequence, being amino acids 1 -1 9 in SEQ ID NO:2 and 4. The invention still further includes pure proteins which are at least 90% identical to the toxin fragments or hybrid toxins according to the invention. The invention still further includes recombinant DNA comprising a sequence encoding a protein having an amino acid sequence of the above disclosed toxins or fragments thereof . In a preferred embodiment the invention provides recombinant DNA comprising the sequence as shown in SEQ ID NO: 1 or 3 or DNA similar thereto encoding a substantially similar protein. In a more preferred embodiment the invention provides recombinant DNA comprising the sequence from about nucleotide 1 70 to about 1 929 in SEQ ID NO: 1 or from about nucleotide 1 47 to about 1 896 in SEQ I D NO :3. By similar DNA is meant a test sequence which is capable of hybridizing to the inventive recombinant sequence. When the test and inventive sequences are double stranded the nucleic acid constituting the test sequence preferably has a T m within 20 ° C of that of the inventive sequence. In the case that the test and inventive sequences are mixed together and denantured simultaneously, The T m values of the sequences are preferably within 1 0°C of each other. More preferably the hybridization is performed under stringent conditions, with either the test or inventive DNA preferaby being supported. Thus either a denatured test or inventive sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 50 and 70 °C in double strength citrate buffered saline containing 0.1 % SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SSC containing 0.1 % SDS. Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the test and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing 0.1 % SDS. The recombinant DNA may further encode a protein having herbicide resistance, plant growth-promoting, anti-fungal , anti-bacterial, anti-viral and/or anti-nematode properties. In the case that the DNA is to be introduced into a heterologous organism it may be modified to remove known mRNA instability motifs (such as AT rich regions) and polyadenylation signals, and/or codons which are preferred by the organism into which the recombinant DNA is to be inserted may be used so that expression of the thus modified DNA in the said organism yields substantially similar protein to that obtained by expression of the unmodified recombinant DNA in the organism in which the protein components of the hybrid toxin or toxin fragments are endogenous. The invention still further includes a DNA sequence which is complementary to one which hybridizes under stringent conditions with the recombinant DNA according to the invention. Also included in the present invention are: a vector containing such a recombinant (or complementary thereto) DNA sequence; a plant or micro-organism which includes, and enables expression of such DNA, plants transformed with such DNA; the progeny of such plants which contain the DNA stably incorporated and hereditable in a Mende an manner, an/or the seeds of such plants and such progeny. The invention still further includes protein derived from expression of the said DNA, and msecticidal protein produced by expression of the recombinant DNA within plants transformed therewith The invention still further includes an msecticidal composition containing one or more of the toxin fragments or toxins comprising them according to the invention; a process for combatting insects which comprises exposing them to such fragments or toxins or compositions, and an extraction process for obtaining msecticidal proteins from organic material containing them comprising submitting the material to maceration and solvent extraction The invention will be further apparent from the following description, which describes the production of Bt hybrid toxin fragments according to the invention, taken in conjunction with the associated drawings and sequence listings A person skilled in the art may use different methods to construct the hybrid toxin genes of the invention Domain encoding regions may be exchanged between two homologous, though different genes by exchanging fragments through restriction enzyme digestion and subsequent gation, if the same restriction enzyme recognition sites are present at the same position in the two genes If suitable restriction enzyme recognition sites are not available, new sites can be created at homologous positions in the two genes by various methods of site-directed mutagenesis, as described in the examples below for construction of a common Rsrll-site using DNA oligomers SEQ ID: 9 and SEQ ID: 1 0. Alternatively, fragments that are to be combined in a hybrid toxin gene may be produced by PCR-mediated amplification using the original genes as templates, taking care that the ends of the fragments contain compatible restriction enzyme digestion sites. Furthermore, a hybrid toxin encoding DNA fragment may be produced by in vitro recombination between overlapping, partially homologous DNA fragments as performed in so-called DNA shuffling experiment (Crameπ et al, 1 998; Zhao et al., 1 998) . Also, hybrid toxin encoding DNA fragments may be produced by in vivo recombination between two homologous DNA fragments that have been cloned in tandem on a single plasmid (Bosch et al ., 1 994), followed by screening for the desired recombination events. 3.0 Brief description of the drawings Figure 1 shows the sequence of the relevant part of the cry lBa (top) and cry lla (bottom) genes, respectively, aligned with the respective oligomers ( SEQ ID NO:9 and 10) used for mutagenesis in order to produce a common RsrW restriction enzyme recognition site in both genes. Mutated nucleotides are indicated by asterisks and the newly produced RsrW recognition sites are underlined Figure 2 shows both the cry lla gene (solid bar) as well as the cry lBa fragment (open bar) presented as they occur in expression vectors pSN1 8 and pSN 17, respectively Both genes are inserted in the pKK233-2 derived expression vector for E coli, pBD1 2 For construction of pSN 1 8 the 3' part of the cry lBa gene, encoding the C- termmal part of the protoxin-specific fragment, was replaced by the corresponding part of the cry l Ca gene (base pairs 2038-3627 , dashed bar) The positions of the common Nco\ and Mun\ restriction sites, as well as of the Λsrll sites deπved by mutagenesis, and the SstXI-site used for exchange of the 3'end of the crylBa gene are indicated, with nucleotide position numbers below the respective genes. Figure 3 shows in a scheme the construction of hybrid gene SN 1 5 from cry lBa (pSN 1 7) and crylla (pSN1 8), and the subsequent construction of hybrid gene SN 1 9 from crylBa (pSN17) and pSN 1 5. Figure 4 shows the alignment of crylBa and cry! la nucleotide sequences, as present in plasmids pSN1 7 and pSN 1 8, respectively, around the junctions between the domain II and III (A) and between domain I and II (B). Nucleotide sequences as present in the hybrid genes of SN1 5 and pSN 1 9 (A) or of pSN 1 9 alone (B) are given in capitals, while identical nucleotides in the DNA that is not present in SN 1 5 and SN 1 9 is represented by dots. The position of the common RsrW and Mun\ recognition sites are underlined . The site of crossover in the hybrids is represented by the shift of the fully written out nucleotide sequence from top strand to bottom strand in this recognition sites. The am o acid sequence of the resulting hybrid proteins is given in three-letter code below each alignment. 4.0 Brief description of the sequence identifiers SEQ ID NO : 1 shows the nucleotide sequence of the hybrid protoxm gene SN 1 5. SEQ ID NO :2 shows the am o acid sequence of the protein encoded by the gene SN 1 5 shown in SEQ ID NO: 1 . SEQ ID NO :3 shows the nucleotide sequence of the hybrid protoxm gene SN 1 9 SEQ ID N0 :4 shows the amino acid sequence of the protein encoded by the gene SN 1 9 shown in SEQ ID NO:3 SEQ ID NO:5 shows the nucleotide sequence of the modified cry lBa gene as used in expression vector pSN 1 7. SEQ ID f\IO:6 shows the amino acid sequence of the protein encoded by the crylBa gene shown in SEQ ID NO:5. SEQ ID NO :7 shows the nucleotide sequence of the modified crylla gene as used in expression vector pSN 1 8. SEQ ID NO:8 shows the amino acid sequence of the protein encoded by the cry lla gene shown in SEQ ID NO :7. SEQ ID IMO:9 shows the nucleotide sequence of the oligomer used for mutagenesis of the cry lBa gene. SEQ ID NO:10 shows the nucleotide sequence of the oligomer used for mutagenesis of the cry lla gene. 5.0 Examples 5.1 DNA manipulations All recombinant DNA techniques are as described by Ausubel et al. ( 1 997) . Mutagenesis, restriction enzyme digestion and ligation are performed according the instructions of the manufacturers. DNA sequencing is performed by the dideoxytriphosphate methode with fluorescent dyes attached to the dideoxynucleotides. Analysis is automated by using an Applied Biosystems 370A nucleotide sequence analyzer. 5.2 Expression vectors All Cry protein expression vectors are based on pBD 1 2, a derivative of pKK233-2 (Bosch et al., 1 994) . For expression of Cry3Aa protein, the full cry3Aa gene is cloned into pBD 1 2, giving expression plasmid pMH 1 0. For cloning purposes both cry Ba as well as cry lla are mutagenized in order to contain a Λ/col-site overlapping with the start codon. For production of Cryl Ba protein, a Nco\-BstX\ (bases 1 - 1 977) fragment o f crylCa in pBD150 (Bosch eta/., 1994) is replaced by the corresponding fragment of crylBa (bases 1-2037), resulting in crylBa expression vector pMH19. pMH19 therefor contains the 5'active toxin encoding part of CrylBa and 3' protoxin specific part of Cry1 Ca. Cry1 la expression vector pBD172 contains the full cry la gene with the Spe\- site (base 2180) fused to the Spel-site in the polylinker of pBluescript SK". 5.3 Mutagenesis of crylBa and crylla In order to be able to directly exchange the domain III encoding regions between crylBa and crylla, a new common restriction enzyme recognition site is made in both genes by site directed mutagenesis. Complementary mutagenic oligonucleotide pairs are used to create unique ftsrll-sites at positions 1464 and 1488 of crylBa (pMH19) and crylla (pBD172), respectively (see Fig. 1), using the using the QuickChange™ kit (Stratagene). These mutations do not change the encoded proteins. This results in two new expression plasmids, pSN17 (CrylBa, Seq. ID NO: 5) and pSN18 (Crylla, Seq. ID NO: 7), respectively (Fig.2). The unique RsrW restriction sites at the border of the domain II and domain III encoding regions, together with the common at the border between the domain I and domain II encoding regions allowed simple swapping of domain encoding regions between the two genes. 5.4 Construction of hybrid toxins Fig.3 schematically shows the construction of two novel hybrid toxins. Both pSN18 and pSN17 are digested with Λ/col and RsrW. Subsequently, the 1488 base fragment of pSN18, containing the domain I and II encoding fragment of crylla is ligated into the corresponding sites of the pSN17-derived fragment containing the 3'portion of crylBa. This results in plasmid pSN15 encoding a 1 la/1 la/1 Ba-hybrid (Seq. ID NO:1 ). The nucleotide sequence of the cross-over region with the encoded amino acid sequence is shown in Fig. 4A. Subsequently a Nco\-Mun\ (base 1 -896) fragment encoding domain I of Cryl la from pSN 1 5 is replaced by the corresponding fragment encoding domain I of Cryl Ba, derived from pSN17. This results in 1 Ba/1 la/1 Ba-hybπd encoding plasmid pSN 1 9 (Seq. ID. NO:3) . The nucleotide sequence of the crossover region with the encoded ammo acid sequence is shown in Fig . 4B. 5.5 Protein isolation and insect bioassays. For large-scale production, all parental and hybrid protoxins are expressed in E. coli strain XL- 1 and extracted as described earlier (Bosch et al., 1 994). Solubilized protoxins are dialyzed overnight in 25 mM NaHC0 3 , 1 00 mM NaCI, pH 10. Protein concentrations are estimated by SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis). To test toxicity to Colorado potato beetle (CPB), leaflets of greenhouse grown potato cu tivar Desiree are dipped in toxin dilutions in water containing 0.01 % Tween-20 After drying of the leaves to the air they are transferred to petπ dishes and 1 0 neonate CPB larvae are placed on each leaf . After incubation for two days at 28°C, the leaves are replaced by fresh leaves dipped in identical protoxm dilutions. Mortality is scored after 4 days. LC 50 (concentration with 50% mortality) and 95% fiducial limits are determined by Probit analysis of results from three or more independent experiments, using the PoloPC computer program (Russel et al , 1 977) 5.6 Toxicity of wild type and hybrid proteins to Colorado potato beetle Table 1 shows the toxicity of the hybrid proteins SN 1 5 and SN 1 9 against Colorado potato beetle (CPB), as compared to the parental proteins Cry1 Ba and Cry1 la, and to the most CPB-active natural toxin available, Cry3A 1 la/1 la/1 Ba-hybπd SN1 5 shows slightly higher toxicity (lower LC50) than its best parental toxin, Cry1 la, on a per weight basis When considering that the molecular weight of the SN 1 5 protein is considerably larger than that of Cry1 la, it follows that SN1 5 performs even better on a per mol basis. 1 Ba/1 la/1 Ba-hybrid protein SN 1 9 is even considerably more toxic than SN 1 5. Table 1 . Toxicity of wild type and hybrid protoxins to Colorado potato beetles. Concentration in microgram per milliliter of dipping solution. LC 50 : concentration which leads to 50% mortality; b Approximate molecular weight in kiloDaltons. c Relative toxicity on molar basis in percents, with toxicity of Cry3A set at 1 00% . Although the present invention has been particularly described for the production of SN 1 5 and SN 1 9 hybrid toxins, the skilled scientist will appreciate that other hybrid toxins with improved insecticidal characteristics may be produced according to the invention. Hybrid toxins containing the combination of Cryl la domain II and Cryl Ba domain III , with various combinations of domain I and C-terminal extensions may be made. Moreover, the gene encoding SN 1 5 , SN 1 9 and/or other hybrids may be transferred into strains of B.t. and/or integrated into the chromosome of strains of B.t., of other bacteria or be introduced into plant genomes to provide for in situ insecticidal activity within the plant per se. In this regard, it is particularly preferred that the recombinant DNA encoding the toxins is modified in that sequences which are detrimental to high level expression in plants are removed and in that codons which are preferred by the plant are used. This should lead to production in the plant of a substantially similar protein to that obtained by expression of the unmodified recombinant DNA in the organism in which the protein components of the hybrid toxins or toxin fragments are endogenous. 6.0 References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Intl. Pat. Appl. Publ. No. WO 95/06730, published 9 March 1 995 Intl. Pat. Appl. Publ. No. WO 98/22595 , published 28 May 1 998 Ausubei, F. et al. ( 1 997) Current Protocols in Molecular Biology. John Wiley & Sons. Bosch, D., Schipper, B., van der Kleij, H., de Maagd, R. and Stiekema, W. ( 1 994) Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/technology, 12, 91 5-91 8. Bradley, D. , Harkey, M.A., Kim, M.K., Biever, K.D. and Bauer, L.S. ( 1 995) The insectidal CrylB crystal protein of Bacillus thuringiensis ssp. thuringiensis has dual specificity to Coleopteran and Lepidopteran larvae. Journal of Invertebrate Pathology, 65, 1 62-1 73. Crameri, A . , S . A. Raillard, E. Bermudez, and W. P. C . Stemmer. 1 998. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature. 391 (6664):288-291 . Crickmore, N. , Zeigler, D.R., Feitelson, J., Schnepf, E., VanRie, J., Lereclus, D., Baum, J. and Dean, D.H . ( 1 998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and molecular biology reviews, 62, 807&. de Maagd, R.A., Bakker, P.L., Masson, L., Adang, M.J., Sangadala, S., Stiekema, W. and Bosch, D. (1 999) Domain III of the Bacillus thuringiensis delta-endotoxin Cry1 Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N. Molecular microbiology, 31 , 463-471 . de Maagd, R.A., Kwa, M.S.G ., van der Klei, H., Yamamoto, T., Schipper, B., Vlak, J.M., Stiekema, W.J. and Bosch, D. ( 1 996a) Domain III substitution in Bacillus thuringiensis delta-endotoxin CrylA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Applied & Environmental Microbiology, 62, 1 537-1 543. de Maagd, R.A., van der Klei, H ., Bakker, P.L., Stiekema, W.J. and Bosch, D . ( 1 996b) Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots. Applied & Environmental Microbiology, 62, 2753-2757. Dean, D.H., Rajamohan, F., Lee, M.K., Wu, S.J., Chen, X.J. , Alcantara, E. and Hussain, S.R. (1 996) Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site directed mutagenesis: a minireview. Gene, 179, 1 1 1 -1 1 7. Ferre, J ., Escriche, B., Bel, Y. and van Rie, J. ( 1 995 ) Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins. FEMS Microbiology Lett. , 1 32, 1 -7. Grochulski, P., Masson, L. , Borisova, S., Pusztai-Carey, M. , Schwartz, J.L., Brousseau, R. and Cygler, M. ( 1 995) Bacillus thuringiensis CrylA(a) insecticidal toxin - crystal structure and channel formation. Journal of Molecular Biology, 254, 447-464. Herrnstadt, C, Soares, G.G., Wilcox, E.R. and Edwards, D.L. (1 986) A new strain of Bacillus thuringiensis with activity against coleopteran insects. Biotechnology, 4, 305-308. Hofte, H. and Whiteley, H.R. (1 989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. , 53, 242-255. Lambert, B., Hofte, H., Annys, K., Jansens, S., Soetaert, P. and Peferoen, M. (1 992) Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae. Applied And Environmental Microbiology, 58, 2536- 2542. Lee, M.K. , Young, B.A. and Dean, D .H. ( 1 995) Domain III exchanges of Bacillus thuringiensis Cry1 A toxins affect binding to different Gyspy Moth midgut receptors. Biochemical and Biophysical Research Communications, 216, 306- 31 2. Li, J., Carroll, J. and Ellar, D.J. (1 991 ) Crystal structure of insectidicidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature (London), 353, 81 5-821 . McPherson, S., Perlak, F., Fuchs, R., Marrone, P., Lavrik, P. and Fischhoff, D. (1 988) Characterization of the coleopteran-specific protein gene of Bacillus thuringiensis var. tenebrionis. Biotechnology, 6, 61 -66. Morse, R.J., Powell, G., Ramalingam, V., Yamamoto, T. and Stroud, R.M. ( 1 998) Crystal structure of Cry2Aa from Bacillus thuringiensis at 2.2 Angstroms: structural basis of dual specificity. In lizuka, T. (ed .) Vllth International Colloquium on Invertebrate Pathology and Microbial Control and IVth International Conference on Bacillus thringiensis. , Sapporo, pp. 1 -2. Russel, R.M., Robertson, J.L. and Savin, N.E. ( 1 977) POLO: a new computer program for Probit analysis. ESA Bulletin, 23, 209-21 3. Sato, R., Takeuchi, K., Ogiwara, K., Minami, M., Kaji, Y., Suzuki, N., Hori, H. and Asano, S. ( 1 994) Cloning, heterologous expression, and localization of a novel crystal protein gene from bacillus-thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects. Curr Microbiol, 28, 1 5-1 9. Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R. and Dean, D.H. ( 1 998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and molecular biology reviews, 62, 775-806. Tailor, R., Tippet, J., Gibb, G., Pells, S., Pike, D., Jordan, L. and Ely, S. (1 992) Identification and characterization of a novel Bacillus thuringiensis delta- endotoxin entomocidal to coleopteran and lepidopteran larvae. Mol. Microbiol. , 6, 1 21 1 - 1 21 7. Zhao, H . M., L. Giver, Z. X. Shao, J. A. Affholter, and F. H . Arnold . 1 998. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature biotechnology. 16(3):258-261 . SEQUENCE LISTING <110> CPRO-DLO <120> Bacillus thuringiensis hybrid toxins <130> E158870 <140> <141> <160> 10 <170> Patentln Ver. 2.1 <210> 1 <211> 3651 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: hybrid protoxin gene SN15 <400> 1 atgaaactaa agaatcaaga taagcatcaa agtttttcta gcaatgcgaa agtagataaa 60 atccctacgg attcactaaa aaatgaaaca gatatagaat tacaaaacat taatcatgaa 120 gattgtttga aaatgtctga gtatgaaaat gtagagccgt ttgttagtgc atcaacaatt 180 caaacaggta ttggtattgc gggtaaaata cttggtaccc taggcgttcc ttttgcagga 240 caagtagcta gtctttatag ttttatctta ggtgagctat ggcctaaggg gaaaaarcaa 300 tgggaaatct ttatggaaca tgtagaagag attattaatc aaaaaatatc aacttatgca 360 agaaataaag cacttacaga cttgaaagga ttaggagatg ccttagctgt ctaccargat 420 tcgcttgaaa gttgggttgg aaatcgtaat aacacaaggg ctaggagtgt tgtcaagagc 480 caatatatcg cattagaatt gatgttcgtt cagaaactac cttcttttgc agtgtcrgga 540 gaggaggtac cattattacc gatatatgcc caagctgcaa atttacattt gttgctatta 600 agagatgcat ctatttttgg aaaagagtgg ggattatcat cttcagaaat ttcaacattt 660 tataaccgtc aagtcgaacg agcaggagat tattcctacc attgtgtgaa atggtatagc 720 acaggtctaa ataacttgag gggtacaaat gccgaaagtt gggtacgata taatcaattc 780 cgtagagaca tgactttaat ggtactagat ttagtggcac tatttccaag ctatgataca 840 caaatgtatc caattaaaac tacagcccaa cttacaagag aagtatatac agacgcaatt 900 gggacagtac atccgcatcc aagttttaca agtacgactt ggtataataa taatgcacct 960 tcgttctctg ccatagaggc tgctgttgtt cgaaacccgc atctactcga ttttctagaa 1020 caagttacaa tttacagctt attaagtcga tggagtaaca ctcagtatat gaatac cgg 1080 ggaggacata aactagaatt ccgaacaaca ggaggaacgt taaatatccc aacacaagga 1140 cctactaata cttctattaa tcctgtaaca ttaccgttca cttctcgaga cgtctatagg 1200 actgaatcat tggcagggct gaatctattt ttaactcaac ctgttaatgg agtacctagg 1260 gttgattttc attggaaatt cgtcacacat ccgatcgcat ctgataattt ctattaccca 1320 gggcatgctg gaattgggac gcaattacag gatccagaaa atgaattacc acctgaagca 1380 acaggacagc caaattatga atcttatagt catagattat ctcatatagg actcatttca 1440 gcatcacatg tgaaagcatc ggtatattct tggacgcatc gtagtgcgga ccgtacgaat 1500 acgattggac caaatagaat cacccaaatc ccaatggtaa aagcatccga acttcctcaa 1560 ggtaccactg ttgttagagg accaggattt actggtgggg atattcttcg aagaacgaat 1620 accggtggat ttggaccgat aagagtaact gttaacggac cattaacaca aagatatcgt 1680 ataggattcc gctatgcttc aactgtagat tttgatttct ttgtatcacg tggaggtact 1740 actgtaaara attttagatt cctacgtaca atgaacagtg gagacgaact aaaatacgga 1800 aattttgtga gacgtgcttt tactacacct tttactttta cacaaattca agatataatt 1860 cgaacgtcta ctcaaggcct tagtggaaat ggggaagtgt atatagataa aattgaaatt 1920 attccagtta ctgcaacctt cgaagcagaa tatgatttag aaagagcgca agaggcggtg 1980 aatgctctgt ttactaatac gaatccaaga agattgaaaa cagatgtgac agattatcat 2040 attgatcaag tatccaattt agtggattgt ttatcagatg aattttgtct ggatgaaaag 2100 cgagaattgt ccgagaaagt caaacatgcg aagcgactca gtgatgagcg gaatttactt 2160 caagatccaa acttcagagg gatcaataga caaccagacc gtggctggag aggaagtaca 2220 gatattacca tccaaggagg agatgacgta ttcaaagaga attacgtcac actaccgggt 2280 accgttgatg agtgctatcc aacgtattta tatcagaaaa tagatgagtc gaaattaaaa 2340 gcttataccc gttatgaatt aagagggtat atcgaagata gtcaagactt agaaatctat 2400 ttgatccgtt acaatgcaaa acacgaaata gtaaatgtgc caggcacggg ttccttatgg 2460 ccgctttcag cccaaagtcc aatcggaaag tgtggagaac cgaatcgatg cgcgccacac 2520 ctcgaatgga atcctgatct agattgttcc tgcagagacg gggaaaaatg tgcacatcat 2580 tcccatcatt tcaccttgga tattgatgtt ggatgtacag acttaaatga ggacttaggt 2640 gtatgggtga tattcaagat taagacgcaa gatggccatg caagactagg gaatctagag 2700 trtctcgaag agaaaccatt attaggggaa gcactagctc gtgtgaaaag agcggagaag 2760 aagtggagag acaaacgaga gaaactgcag ttggaaacaa atattgttta taaagaggca 2820 aaagaatctg tagatgcttt atttgtaaac tctcaatatg atagattaca agtggatacg 2880 aacatcgcga tgattcatgc ggcagataaa cgcgttcata gaatccggga agcgtatctg 2940 ccagagttgt ctgtgattcc aggtgtcaat gcggccattt tcgaagaatt agagggacgt 3000 atttttacag cgtattcctt atatgatgcg agaaatgtca ttaaaaatgg cgatttcaat 3060 aatggcttat tatgctggaa cgtgaaaggt catgtagatg tagaagagca aaacaaccac 3120 cgttcggtcc ttgttatccc agaatgggag gcagaagtgt cacaagaggt tcgtgtctgt 3180 ccaggtcgtg gctatatcct tcgtgtcaca gcatataaag agggatatgg agagggctgc 3240 gtaacgatcc atgagatcga agacaataca gacgaactga aattcagcaa ctgtgtagaa 3300 gaggaagtaύ atccaaacaa cacagraacg tgtaataatt atactgggac tcaagaagaa 3360 tatgagggta cgtacacttc tcgtaatcaa. ggatatgacg aagcctatgg taataaccct 3420 tccgtaccag ctgattacgc ttcagtctat gaagaaaaat cgtatacaga tggacgaaga 3480 gagaatcctt gtgaatctaa cagaggctat ggggattaca caccactacc ggctggttat 3540 gtaacaaagg atttagagta cttcccagag accgataagg tatggattga gatcggagaa 3600 acagaaggaa cattcatcgt ggatagcgtg gaattactcc ttatggagga a 3651 <210> 2 <211> 1217 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: protein encoded by the gene S 15 <400> 2 Met Lys Leu Lys Asn Gin Asp Lys His Gin Ser Phe Ser Ser Asn Ala 1 5 10 15 Lys Val Asp Lys lie Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp lie 20 25 30 Glu Leu Gin Asn lie Asn His Glu Asp Cys Leu Lys Met Ser Glu Tyr 35 40 45 Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr lie Gin Thr Gly lie 50 55 60 Gly lie Ala Gly Lys lie Leu Gly Thr Leu Gly Val Pro Phe Ala Gly 65 " 70 75 80 Gin Val Ala Ser Leu Tyr Ser Phe lie Leu Gly Glu Leu Trp Pro Lys 85 90 95 Gly Lys Asn Gin Trp Glu lie Phe Met Glu His Val Glu Glu lie lie 100 105 110 Asn Gin Lys lie 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 Asp Ser Leu Glu Ser 130 135 140 Trp Val Gly Asn Arg Asn Asn Thr Arg Ala Arg Ser Val Val Lys Ser 145 150 155 160 Gin Tyr lie 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 lie Tyr Ala Gin Ala 180 185 190 Ala Asn Leu His Leu Leu Leu Leu A.rg Asp Ala Ser lie Phe Gly Lys 195 200 205 Glu Trp Gly Leu Ser Ser Ser Glu lie Ser Thr Phe Tyr Asn Arg Gin 210 215 220 Val Glu Arg Ala Gly Asp Tyr Ser Tyr His Cys Val Lys Trp Tyr Ser 225 230 235 240 ?hr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg 245 250 255 Tyr A.sn Gin Phe Arg Arg Asp Met Thr Leu Met Val Leu Asp Leu Val 260 265 270 Ala Leu Phe Pro Ser Tyr Asp Thr Gin Met Tyr Pro lie Lys Thr Thr 275 280 285 Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala lie Gly Thr Val His 290 295 300 Pro His Pro Ser Phe Thr Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro 305 310 315 320 Ser Phe Ser Ala lie Glu Ala Ala Val Val Arg Asn Pro His Leu Leu 325 330 335 Asp Phe Leu Glu Gin Val Thr lie Tyr Ser Leu Leu Ser Arg Trp Ser 340 345 350 Asn Thr Gin Tyr Met Asn Met Trp Gly Gly His Lys Leu Glu Phe Arg 355 360 365 Thr lie Gly Gly Thr Leu Asn lie Ser Thr Gin Gly Ser Thr Asn Thr 370 375 380 Ser lie Asn Pro Val Thr Leu Pro Phe Thr Ser Arg Asp Val Tyr Arg 385 390 395 400 Thr Glu Ser Leu Ala Gly Leu Asn Leu Phe Leu Thr Gin Pro Val Asn 405 410 415 Gly Val Pro Arg Val Asp Phe His Trp Lys Phe Val Thr His Pro lie 420 425 430 Ala Ser Asp Asn Phe Tyr Tyr Pro Gly Tyr Ala Gly lie Gly Thr Gin 435 440 445 Leu Gin Asp Ser Glu Asn Glu Leu Pro Pro Glu Ala Thr Gly Gin Pro 450 455 460 Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His lie Gly Leu lie Ser 465 470 475 430 Ala Ser His Val Lys Ala Ser Val Tyr Ser Trp Thr His Arg Ser Ala 485 490 495 Asp Arg Thr Asn Thr He Gly Pro Asn Arg He Thr Gin He Pro Met 500 505 510 Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val A.rg Gly Pro 515 520 525 Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr Asn Thr Gly Gly Phe 530 535 540 Gly Pre He Arg Val Thr Val Asn Gly Pro Leu Thr Gin Arg Tyr Arg 545 550 555 560 He Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe Phe Val Ser 565 570 575 Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg Thr Met Asn 580 585 590 Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg Ala Phe Thr 595 600 605 Thr Pro Phe Thr Phe Thr Gin He Gin Asp He He Arg Thr Ser He 610 615 620 Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr He Asp Lys He Glu He 625 630 635 640 He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala 645 650 655 Gin Glu Ala Val Asn Ala Leu Phe Thr Asn Thr Asn Pro Arg Arg Leu 660 665 670 Lys Thr A.sp Val Thr Asp Tyr His He Asp Gin Val Ser A.sn Leu Val 675 680 685 Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys A.rg Glu Leu Ser 590 695 700 Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu 705 710 715 720 Gin Asa Pro Asn Phe Arg Gly He Asn Arg Gin Pro Asp Arg Gly Trp 725 730 735 Arc Gly Ser Thr Asp He Thr He Gin Gly Gly Asp Asp Val Phe Lys 740 745 750 Glu Asn Tvr Val Thr Leu Pro Gly Thr Val Asp Glu Cys Tyr Pro Thr 755 760 765 Tvr Leu Tyr Gin Lys He Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg 770 775 780 Tyr Glu Leu Arg Gly Tyr He Glu Asp Ser Gin Asp Leu Glu He Tyr 785 790 795 800 Leu He Arg Tyr Asn Ala Lys His Glu He Val Asn Val Pro Gly Thr 805 810 815 Gly Ser Leu Trp Pro Leu Ser Ala Gin Ser Pro He Gly Lys Cys Gly 820 825 830 Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp 835 840 845 Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe 850 855 860 Thr Leu Asp He Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 865 870 875 880 Val Trp Val He Phe Lys He Lys Thr Gin Asp Gly His Ala Arg Leu 885 890 895 Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu 900 905 910 Ala Arg Val Lys Arg .Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys 915 920 925 Leu Gin Leu Glu Thr Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val 930 935 940 Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Arg Leu Gin Val Asp Thr 945 950 955 960 Asn He Ala Met He His Ala Ala Asp Lys Arg Val His Arg He Arg 965 970 975 Glu Ala Tyr Leu Pro Glu Leu Ser Val He Pro Gly Val Asn Ala Ala 980 985 990 He Phe Glu Glu Leu Glu Gly Arg He Phe Thr Ala Tyr Ser Leu Tyr 995 1000 1005 Asp Ala Arg Asn Val He Lys Asn Gly Asp Phe Asn Asn Gly Leu Leu 1010 1015 1020 Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gin Asn Asn His 1025 1030 1035 1040 Arg Ser Val Leu Val He Pro Glu Trp Glu Ala Glu Val Ser Gin Glu 1045 1050 1055 Val Arg Val Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr Ala Tyr 1060 1065 1070 Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr He His Glu He Glu Asp 1075 1080 1085 Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr 1090 1095 1100 Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gin Glu Glu 1105 1110 1115 1120 Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Gin Gly Tyr Asp Glu Ala Tyr 1125 1130 1135 Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu 1140 1145 1150 Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg 1155 1160 1165 Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp 1170 H75 1180 Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp He Glu He Gly Glu 1185 H90 1195 1200 Thr Glu Gly Thr Phe He Val Asp Ser Val Glu Leu Leu Leu Met Glu 1205 1210 1215 Glu <210> 3 <211> 3624 <212> DNA <213> Artificial Sequence <220> <223> Description of .Artificial Sequence : hybrid protoxin gene SN19 <400> 3 atggcttcaa ataggaaaaa tgagaatgaa attataaatg ctgtatcgaa tcattccgca 60 caaatggatc tattaccaga tgctcgtatt gaggatagct tgtgtatagc cgaggggaac 120 aatatcgatc catttgttag cgcatcaaca gtccaaacgg gtattaacat agctggtaga 180 atactaggcg tattgggcgt accgtttgct ggacaactag ctagttttta tagttttctt 240 gttggtgaat tatggccccg cggcagagat cagtgggaaa ttttcctaga acatgtcgaa 300 caacttataa atcaacaaat aacagaaaa. gctaggaata cggctcttgc tcgattacaa 360 ggtttaggag attccttcag agcctatcaa cagtcacttg aagattggct agaaaaccgt 420 gatgatgcaa gaacgagaag tgttctttat acccaatata tagctttaga acttgatttt 480 cttaatgcga tgccgctttt cgcaattaga aaccaagaag ttccattatt gatggtatat 540 gctcaagctg caaatttaca cctattatra ttgagagatg cctctctttt tggtagtgaa 600 tttgggctta catcgcagga aattcaacgc tattatgagc gccaagtgga acgaacgaga 660 gattattccg actattgcgt agaatggtat aatacaggtc taaatagctt gagagggaca 720 aatgccgcaa gttgggtacg gtataatcaa ttccgtagag atctaacgtt aggagtatta 780 gatctagtgg cactattccc aagctatgac actcgcactt atccaataaa tacgagtgct 840 cagttaacaa gagaagttta tacagacgca attgggacag tacatccgca tccaagtttt 900 acaagtacga cttggtataa taataatgca ccttcgttct ctgccataga ggctgctgtt 960 gttcgaaacc cgcatctact cgattttcta gaacaagtta caatttacag cttattaagt 1020 cgatggagta acactcagta tatgaatatg tggggaggac ataaactaga attccgaaca 1080 ataggaggaa cgttaaatat ctcaacacaa ggatctacta atacttctat taatcctgta 1140 acattaccgt tcacttctcg agacgtctat aggactgaat cattggcagg gctgaatcta 1200 tttttaactc aacctgttaa tggagtacct agggttgatt ttcattggaa attcgtcaca 1260 catccgatcg catctgataa tttctattat ccagggtatg ctggaattgg gacgcaatta 1320 caggattcag aaaatgaatt accacctgaa gcaacaggac agccaaatta tgaatcttat 1380 agtcatagat tatctcatat aggactcatt tcagcatcac atgtgaaagc atcggtatat 1440 tcttggacgc atcgtagtgc ggaccgtacg aatacgattg gaccaaatag aatcacccaa 1500 atcccaatgg taaaagcatc cgaacttccc caaggtacca ctgttgttag aggaccagga 1560 tttactggtg gggatattct tcgaagaacg aatactggtg gatttggacc gataagagta 1620 actgttaacg gaccattaac acaaagatat cgtataggat tccgctatgc ttcaactgta 1680 gattttgatt tctttgtatc acgtggaggt actactgtaa ataattttag attcctacgt 1740 acaatgaaca gtggagacga actaaaatac ggaaattttg tgagacgtgc ttttactaca 1800 ccttttactt ttacacaaat tcaagatata attcgaacgt ctattcaagg ccttagtgga 1860 aatggggaag tgcatataga taaaattgaa attattccag ttactgcaac cttcgaagca 1920 gaatatgatt tagaaagagc gcaagaggcg gtgaatgctc tgtttactaa tacgaatcca 1980 agaagattga aaacagatgt gacagatta catattgatc aagtatccaa tttagtggat 2040 tgtttatcag atgaattttg tctggatgaa aagcgagaat tgtccgagaa agtcaaacat 2100 gcgaagcgac tcagtgatga gcggaattta cttcaagatc caaacttcag agggatcaat 2160 agacaaccag accgtggctg gagaggaagt acagatatta ccatccaagg aggagatgac 2220 gtattcaaag agaattacgt cacactaccg ggtaccgttg atgagtgcta tccaacgtat 2230 t tatatcaga aaatagatga gtcgaaatta aaagcttata cccgttatga attaagaggg 2340 t atatcgaag atagtcaaga cttagaaatc tatttgatcc gttacaatgc aaaacacgaa 2400 atagtaaatg tgccaggcac gggttcctta tggccgcttt cagcccaaag tccaatcgga 2460 aagcgtggag aaccgaatcg atgcgcgcca caccttgaat ggaatcctga tctagattgt 2520 tcctgcagag acggggaaaa atgtgcacat cattcccatc atttcacctt ggatattgat 2530 gttggatgta cagacttaaa tgaggactta ggtgtatggg tgatattcaa gattaagacg 2640 caagatggcc atgcaagact agggaatcta gagtttctcg aagagaaacc attattaggg 2700 gaagcactag ctcgtgtgaa aagagcggag aagaagtgga gagacaaacg agagaaactg 2760 cagttggaaa caaatattgt ttataaagag gcaaaagaat ctgtagatgc tttatttgta 2820 aactctcaat atgatagatt acaagtggat acgaacatcg cgatgattca tgcggcagat 2880 aaacgcgttc atagaatccg ggaagcgtat ctgccagagt tgtctgtgat tccaggtgtc 2940 aatgcggcca ttttcgaaga attagaggga cgtattttta cagcgtattc cttatatgat 3000 gcgagaaatg tcattaaaaa tggcgatttc aataatggct tattatgctg gaacgtgaaa 3060 ggtcatgtag atgtagaaga gcaaaacaac caccgttcgg tccttgttat cccagaatgg 3120 gaggcagaag tgtcacaaga ggttcgtgtc tgtccaggtc gtggctatat ccttcgtgtc 3180 acagcatata aagagggata tggagagggc tgcgtaacga tccatgagat cgaagacaat 3240 acagacgaac tgaaattcag caactgtgta gaagaggaag tatatccaaa caacacagta 3300 acgtgtaata attatactgg gactcaagaa gaatatgagg gtacgtacac ttctcgtaat 3360 caaggatatg acgaagccta tggtaataac ccttccgtac cagctgatta cgcttcagtc 3420 tatgaagaaa aatcgtatac agatggacga agagagaatc cttgtgaatc taacagaggc 3480 tatggggatt acacaccact accggctggt tatgtaacaa aggatttaga gtacttccca 3540 gagaccgata aggtatggat tgagatcgga gaaacagaag gaacattcat cgtggatagc 3600 gtggaattac tccttatgga ggaa 3624 <210> 4 <21i> 1208 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: protein encoded by the gene SN19 <400> 4 Met Ala Ser Asn Arg Lys Asn Glu Asn Glu He He Asn Ala Val Ser 1 5 10 15 Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg He Glu Asp 20 25 30 Ser Leu Cys He Ala Glu Gly Asn Asn He Asp Pro Phe Val Ser Ala 35 40 45 Ser Thr Val Gin Thr Gly He Asn He Ala Gly Arg He Leu Gly Val 50 55 60 Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu He Phe Leu 85 90 95 Glu His Val Glu Gin Leu He Asn Gin Gin He Thr Glu Asn Ala Arg 100 105 110 Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg .Ala 115 120 125 Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg 130 135 140 Thr Arg Ser Val Leu Tyr Thr Gin Tyr He Ala Leu Glu Leu Asp Phe 145 150 155 160 Leu Asn Ala Met Pro Leu Phe Ala He Arg Asn Gin Glu Val Pro Leu 165 170 175 Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190 Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu He 195 200 205 Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp 210 215 220 Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr 225 230 235 240 Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr 245 250 255 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg 260 265 270 Thr Tyr Pro He Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr 275 280 285 Asp Ala He Gly Thr Val His Pro His Pro Ser Phe Thr Ser Thr Thr 290 295 300 Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala He Glu Ala Ala Val 305 310 315 320 Val Arg As. Pro His Leu Leu Asp Phe Leu Glu Gin Val Thr He Tyr 325 330 335 Ser Leu Leu Ser Arg Trp Ser Asn Thr Gin Tyr Met Asn Met Trp Gly 340 345 350 Gly His Lys Leu Glu Phe Arg Thr He Gly Gly Thr Leu Asn He Ser 355 360 365 Tnr Gin Gly Ser Thr Asn Thr Ser He Asn Pro Val Thr Leu Pro Pne 370 375 380 Tnr Ser Arg Asp Val Tyr Arg Tnr Glu Ser Leu Ala Gly Leu Asn Leu 335 390 395 400 Pne Leu Tnr Gin Pro Val Asn Gly Val Pro Arg Val Asp Phe His Trp 405 410 415 Lys Phe Val Thr His Pro He Ala Ser Asp Asn Phe Tyr Tyr Pro Gly 420 425 430 Tyr Ala Gly He Gly Thr Gin Leu Gin Asp Ser Glu Asn Glu Leu Pro 435 440 445 Pro Glu Ala Thr Gly Gin Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu 450 455 460 Ser His He Gly Leu He Ser Ala Ser His Val Lys Ala Ser Val Tyr 465 470 475 480 Ser Trp Thr His Arg Ser Ala Asp Arg Tnr Asn Thr He Gly Pro Asn 485 490 495 Arg He Thr Gin He Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly 500 505 510 Tnr Thr Val Val Arg Gly Pro Gly Pne Tnr Gly Gly Asp He Leu Arg 515 520 525 Arg Thr Asn Tnr Gly Gly Phe Gly Pro He Arg Val Thr Val Asn Gly 530 535 540 Pro Leu Tnr Gin Arg Tyr Arg He Gly Pne Arg Tyr Ala Ser Tnr Val 545 550 555 560 Asp Phe Asp Pne Pne Val Ser Arg Gly Gly Thr Thr Val Asn Asn Pne 565 570 575 Arg Pne Leu Arg Tnr Met ASP Ser Gly Asp Glu Leu Lys Tyr Gly Asn 580 585 590 ?->e Val Arg Arg Ala Pne Thr Tr.r Pro Pne Thr Pne Tnr Gin He Gin 595 600 605 Asp He He Arg Thr Ser He Gin Gly Leu Ser Gly Asn Gly Glu Val 610 615 620 Tyr He Asp Lys He Glu He He Pro Val Thr Ala Thr Phe Glu Ala 625 630 635 640 Glu Tyr Asp Leu Glu Arg Ala Gin Glu Ala Val Asn Ala Leu Phe Thr 645 650 655 Asn Thr Asn Pro Arg .Arg Leu Lys Thr Asp Val Thr Asp Tyr His He 660 665 670 Asp Gin Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu 675 680 685 A.sp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu 690 695 700 Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Arg Gly He A.sn 705 710 715 720 Arg Gin Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp He Tnr He Gin 725 730 735 Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr 740 745 750 Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser 755 760 765 Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr He Glu Asp 770 775 780 Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His Glu 785 790 795 800 He Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gin 805 810 815 Ser Pro He Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro H s Leu 820 825 830 Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys 835 840 845 Ala His His Ser His His Phe Thr Leu Asp He Asp Val Gly Cys Tnr 850 855 860 Asp Leu Asn Glu Asp Leu Gly Val Trp Val He Phe Lys He Lys Thr 865 870 875 880 Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys 885 890 895 Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys 900 905 910 Trp Arg Asp Lys Arg Glu Lys Leu Gin Leu Glu Thr Asn He Val Tyr 915 920 925 Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr 930 935 940 Asp Arg Leu Gin Val Asp Thr Asn He Ala Met He His Ala Ala Asp 945 950 955 960 Lys Arg Val His Arg He Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val 965 970 975 He Pro Gly Val Asn Ala Ala He Phe Glu Glu Leu Glu Gly Arg He 980 985 990 Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val He Lys Asn Gly 995 1000 1005 Asp Pne Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp 1010 1015 1020 Val Glu Glu Gin Asn Asn His Arg Ser Val Leu Val He Pro Glu Trp 1025 1030 1035 1040 Glu Ala Glu Val Ser Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr 1045 1050 1055 He Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1060 1065 1070 Thr He His Glu He Glu Asp Asn Thr Asp Glu Leu Lys Pne Ser Asn 1075 1080 1085 Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Tnr Cys Asn Asn 1090 1095 1100 Tyr Tnr Gly Tnr Gin Glu Glu Tyr Glu Gly Thr Tyr Tnr Ser Arg Asn 1105 1110 1115 1120 Gin Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala Asp 1125 1130 1135 Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu 1140 1145 1150 Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro 1155 1160 1165 Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu Thr Asp Lys 1170 1175 1180 Val Trp He Glu He Gly Glu Thr Glu Gly Thr Phe He Val Asp Ser 1185 1190 1195 1200 Val Glu Leu Leu Leu Met Glu Glu 1205 <210> 5 <211> 3627 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: modified crylBa gene <400> 5 atgacttcaa ataggaaaaa tgagaatgaa attataaatg ctgtatcgaa tcattccgca 60 caaatggatc tattaccaga tgctcgtatt gaggatagct tgtgtatagc cgaggggaac 120 aatatcgatc catttgttag cgcatcaaca gtccaaacgg gtattaacat agctggtaga 180 atactaggcg tattgggcgt accgtttgct ggacaactag ctagttttta tagttttctt 240 gttggtgaat tatggccccg cggcagagat cagtgggaaa ttttcctaga acatgtcgaa 300 caacttataa atcaacaaat aacagaaaat gctaggaata cggctcttgc tcgattacaa 360 ggtttaggag attccttcag agcctatcaa cagtcacttg aagattggct agaaaaccgt 420 gatgatgcaa gaacgagaag tgttctttat acccaatata tagctttaga acttgatttt 480 c taatgcga tgccgctttt cgcaattaga aaccaagaag ttccattatt gatggtatat 540 gctcaagctg caaatttaca cctattatta ttgagagatg cctctctttt tggtagtgaa 600 tttgggctta catcgcagga aattcaacgc tattatgagc gccaagtgga acgaacgaga 660 gattattccg actattgcgt agaatggtat aatacaggtc taaatagctt gagagggaca 720 aatgccgcaa gttgggtacg gtataatcaa ttccgtagag atctaacgtt aggagtatta 780 gatctagtgg cactattccc aagctatgac actcgcactt atccaataaa tacgagtgct 840 cagttaacaa gagaagttta tacagacgca attggagcaa caggggtaaa tatggcaagt 900 atgaattggt ataataataa tgcaccttcg ttctctgcca tagaggctgc ggctatccga 960 agcccgcatc tacttgattt tctagaacaa cttacaattt ttagcgcttc atcacgatgg 1020 agtaatacta ggcatatgac ttattggcgg gggcacacga ttcaatctcg gccaatagga 1080 ggcggattaa atacctcaac gcatggggct accaatactt ctattaatcc tgtaacatta 1140 cggttcgcat ctcgagacgt ttataggact gaatcatatg caggagtgct tctatgggga 1200 atttaccttg aacctattca tggtgtccct actgttaggt ttaattttac gaaccctcag 1260 aatatttctg atagaggtac cgctaactat agtcaacctt atgagtcacc tgggcttcaa 1320 ttaaaagatt cagaaactga attaccacca gaaacaacag aacgaccaaa ttatgaatct 1380 tacagtcaca ggttatctca tataggtata attttacaat ccagggtgaa tgtaccggta 1440 tattcttgga cgcatcgtag tgcggaccgt acgaatacga ttggaccaaa tagaatcacc 1500 caaatcccaa tggtaaaagc atccgaactt cctcaaggta ccactgttgt tagaggacca 1560 ggatttactg gtggggatat tcttcgaaga acgaatactg gtggatttgg accgataaga 1620 gtaactgtta acggaccatt aacacaaaga tatcgtatag gattccgcta tgcttcaact 1680 gtagattttg atttctttgt atcacgtgga ggtactactg taaataattt tagattccta 1740 cgtacaatga acagtggaga cgaactaaaa tacggaaatt ttgtgagacg tgcttttact 1800 acacctttta cttttacaca aattcaagat ataattcgaa cgtctattca aggccttagt 1860 ggaaatgggg aagtgtatat agataaaatt gaaattattc cagttactgc aaccttcgaa 1920 gcagaatatg atttagaaag agcgcaagag gcggtgaatg ctctgtttac taatacgaat 1980 ccaagaagat tgaaaacaga tgtgacagat tatcatattg atcaagtatc caatttagtg 2040 gattgtttat cagatgaatt ttgtctggat gaaaagcgag aattgtccga gaaagtcaaa 2100 catgcgaagc gactcagtga tgagcggaat ttacttcaag atccaaactt cagagggatc 2160 aatagacaac cagaccgtgg ctggagagga agtacagata ttaccatcca aggaggagat 2220 gacgtattca aagagaatta cgtcacacta ccgggtaccg ttgatgagtg ctatccaacg 2280 tatttatatc agaaaataga tgagtcgaaa ttaaaagctt atacccgtta tgaattaaga 2340 gggtatatcg aagatagtca agacttagaa atctatttga tccgttacaa tgcaaaacac 2400 gaaatagtaa atgtgccagg cacgggttcc ttatggccgc tttcagccca aagtccaatc 2460 ggaaagtgtg gagaaccgaa tcgatgcgcg ccacaccttg aatggaatcc tgatctagat 2520 tgttcctgca gagacgggga aaaatgtgca catcattccc atcatttcac cttggatatt 2580 gatgttggat gtacagactt aaatgaggac ttaggtgtat gggtgatatt caagattaag 2640 acgcaagatg gccatgcaag actagggaat ctagagtttc tcgaagagaa accattatta 2700 ggggaagcac tagctcgtgt gaaaagagcg gagaagaagt ggagagacaa acgagagaaa 2760 ctgcagttgg aaacaaatat tgtttataaa gaggcaaaag aatctgtaga tgctttattt 2820 gtaaactctc aatatgatag attacaagtg gatacgaaca tcgcgatgat tcatgcggca 2880 gataaacgcg ttcatagaat ccgggaagcg tatctgccag agttgtctgt gattccaggt 2940 gtcaatgcgg ccattttcga agaattagag ggacgtattt ttacagcgta ttccttatat 3000 gatgcgagaa atgtcattaa aaatggcgat ttcaataatg gcttattatg ctggaacgtg 3060 aaaggtcatg tagatgtaga agagcaaaac aaccaccgtt cggtccttgt tatcccagaa 3120 tgggaggcag aagtgtcaca agaggttcgt gtctgtccag gtcgtggcta tatccttcgt 3180 gtcacagcat ataaagaggg atatggagag ggctgcgtaa cgatccatga gatcgaagac 3240 aatacagacg aactgaaatt cagcaactgt gtagaagagg aagtatatcc aaacaacaca 3300 gtaacgtgta ataattatac tgggactcaa gaagaatatg agggtacgta cacttctcgt 3360 aatcaaggat atgacgaagc ctatggtaat aacccttccg taccagctga ttacgcttca 3420 gtctatgaag aaaaatcgta tacagatgga cgaagagaga atccttgtga atctaacaga 3480 ggctatgggg attacacacc actaccggct ggttatgtaa caaaggattt agagtacttc 3540 ccagagaccg ataaggtatg gattgagatc ggagaaacag aaggaacatt catcgtggat 3600 agcgtggaat tactccttat ggaggaa 3627 <210> 6 <2il> 1209 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: protein encoded by the modified crylBa gene <400> 6 Met Thr Ser Asn Arg Lys Asn Glu Asn Glu He He Asn Ala Val Ser 1 5 10 15 A.sn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg He Glu Asp 20 25 30 Ser Leu Cys He Ala Glu Gly Asn Asn He Asp Pro Phe Val Ser Ala 35 40 45 Ser Thr Val Gin Thr Gly He Asn He Ala Gly Arg He Leu Gly Val 50 55 60 Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu He Phe Leu 85 90 95 Glu His Val Glu Gin Leu He Asn Gin Gin He Thr Glu Asn Ala Arg 100 105 110 Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala 115 120 125 Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg 130 135 140 Thr Arg Ser Val Leu Tyr Thr Gin Tyr He Ala Leu Glu Leu Asp Phe 145 150 155 160 Leu Asn Ala Met Pro Leu Phe Ala He Arg Asn Gin Glu Val Pro Leu 165 170 175 Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190 Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu He 195 200 205 Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp 210 215 220 Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr 225 230 235 240 Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr 245 250 255 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg 260 265 270 Thr Tyr Pro He Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr 275 280 285 Asp Ala He Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr 290 295 300 Asn Asn Asn Ala Pro Ser Phe Ser Ala He Glu Ala Ala Ala He Arg 305 310 315 320 Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr He Phe Ser Ala 325 330 335 Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His 340 345 350 Thr He Gin Ser Arg Pro He Gly Gly Gly Leu Asn Thr Ser Thr His 355 360 365 Gly Ala Thr Asn Thr Sεr He Asn Pro Val Thr Leu Arg Phe Ala Ser 370 375 380 Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly 385 390 395 400 He Tyr Leu Glu Pro He His Gly Val Pro Thr Val Arg Phe Asn Phe 405 410 415 Thr Asn Pro Gin Asn He Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin 420 425 430 Pro Tvr Glu Ser Pro Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu 435 440 445 Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg 450 455 460 Leu Ser His He Gly He He Leu Gin Ser Arg Val Asn Val Pro Val 465 470 475 4S0 Tvr Ser Trp Thr His A.rg Ser Ala Asp Arg Thr Asn Thr He Gly Pro 435 490 495 Asn Arg He Thr Gin He Pro Met Val Lys Ala Ser Glu Leu Pro Gin 500 505 510 Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu 515 520 525 Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro He Arg Val Thr Val Asn 530 535 540 Gly Pro Leu Thr Gin Arg Tyr Arg He Gly Phe Arg Tyr Ala Ser Thr 545 550 555 560 Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn 565 570 575 Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly 580 585 590 Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin He 595 600 605 Gin Asp He He Arg Thr Ser He Gin Gly Leu Ser Gly Asn Gly Glu 610 615 620 Val Tyr He Asp Lys He Glu He He Pro Val Thr Ala Thr Phe Glu 625 630 635 640 A.la Glu Tyr A.sp Leu Glu Arg Ala Gin Glu Ala Val Asn Ala Leu Phe 645 650 655 Thr Asn Thr Asn Pro Arg Arg Leu Lys Thr Asp Val Thr Asp Tyr His 660 665 670 He Asp Gin Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys 675 680 685 Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg 690 695 700 Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Arg Gly He 705 710 715 720 Asn Arg Gin Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp He Thr He 725 730 735 Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly 740 745 750 Thr Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu 755 760 765 Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr He Glu 770 775 780 Asp Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His 785 790 795 800 Glu He Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala 805 810 815 Gin Ser Pro He Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His 820 825 830 Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys 835 840 845 Cys Ala His His Ser His His Phe Thr Leu Asp He Asp Val Gly Cys 850 855 860 Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val He Phe Lys He Lys 865 870 875 880 Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu 885 890 895 Lys Pro Leu Leu Gly Glu .Ala Leu Ala Arg Val Lys Arg Ala Glu Lys 900 905 910 Lys Trp Arg Asp Lys Arg Glu Lys Leu Gin Leu Glu Thr Asn He Val 915 920 925 Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin 930 935 940 Tyr Asp Arg Leu Gin Val Asp Thr Asn He Ala Met He His Ala Ala 945 950 955 960 Asp Lys Arg Val His Arg He Arg Glu Ala Tyr Leu Pro Glu Leu Ser 965 970 975 Val He Pro Gly Val Asn Ala Ala He Phe Glu Glu Leu Glu Gly Arg 980 985 990 He Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val He Lys Asn 995 1000 1005 Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val 1010 1015 1020 Asp Val Glu Glu Gin Asn Asn His Arg Ser Val Leu Val He Pro Glu 1025 1030 1035 1040 Trp Glu Ala Glu Val Ser Gin Glu Val Arg Val Cys Pro Gly Arg Gly 1045 1050 1055 Tyr He Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys 1060 1065 1070 Val Thr He His Glu He Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser 1075 1080 1085 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn 1090 1095 1100 Asn Tyr Thr Gly Thr Gin Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg 1105 HIO 1115 1120 Asn Gin Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala 1125 1130 1135 Asp Tyr Ala Ser Val Tyr Glu Glu Lys Sεr Tyr Thr Asp Gly Arg Arg 1140 1145 1150 Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu 1155 1160 1165 Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu Thr Asp 1170 1175 1180 Lys Val Trp He Glu He Gly Glu Thr Glu Gly Thr Phe He Val Asp 1185 H90 1195 1200 Ser Val Glu Leu Leu Leu Met Glu Glu 1205 <210> 7 <211> 2160 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: modified crylla gene <400> 7 atgaaactaa agaatcaaga taagcatcaa agtttttcta gcaatgcgaa agtagataaa 60 atctctacgg attcactaaa aaatgaaaca gatatagaat tacaaaacat taatcatgaa 120 gattgtttga aaatgtctga gtatgaaaat gtagagccgt ttgttagtgc atcaacaatt 180 caaacaggta ttggtattgc gggtaaaata cttggtaccc taggcgttcc ttttgcagga 240 caag t agcta gtctttatag ttttatctta ggtgagctat ggcctaaggg gaaaaatcaa 300 tgggaaatct ttatggaaca tgtagaagag attattaatc aaaaaatatc aacttatgca 360 agaaataaag cacttacaga cttgaaagga ttaggagatg ccttagctgt ctaccatgat 420 tcgcttgaaa gttgggttgg aaatcgtaat aacacaaggg ctaggagtgt tgtcaagagc 480 caatatatcg cattagaatt gatgttcgtt cagaaactac cttcttttgc agtgtctgga 540 gaggaggtac cattattacc gatatatgcc caagctgcaa atttacattt gttgctatta 600 agagatgcat ctatttttgg aaaagagtgg ggattatcat cttcagaaat ttcaacattt 660 tataaccgtc aagtcgaacg agcaggagat tattcctacc attgtgtgaa atggtatagc 720 acaggtctaa ataacttgag gggtacaaat gccgaaagtt gggtacgata taatcaattc 780 cgtagagaca tgactttaat ggtactagat ttagtggcac tatttccaag ctatgataca 840 caaatgtatc caattaaaac tacagcccaa cttacaagag aagtatatac agacgcaatt 900 gggacagtac atccgcatcc aagttttaca agtacgactt ggtataataa taatgcacct 960 tcgttctctg ccatagaggc tgctgttgtt cgaaacccgc atctactcga ttttctagaa 1020 caagttacaa tttacagctt attaagtcga tggagtaaca ctcagtatat gaatatgtgg 1080 ggaggacata aactagaatt ccgaacaata ggaggaacgt taaatatctc aacacaagga 1140 tctactaata cttctattaa tcctgtaaca ttaccgttca cttctcgaga cgtctatagg 1200 actgaatcat tggcagggct gaatctattt ttaactcaac ctgttaatgg agtacctagg 1260 gttgattttc attggaaatt cgtcacacat ccgatcgcat ctgataattt ctattatcca 1320 gggtatgctg gaattgggac gcaattacag gattcagaaa atgaattacc acctgaagca 1380 acaggacagc caaattatga atcttatagt catagattat ctcatatagg actcatttca 1440 gcatcacatg tgaaagcatt ggtatattct tggacgcatc gtagtgcgga ccgtacaaat 1500 acaattgagc caaatagcat tacacaaata ccattagtaa aagctttcaa tctgtcttca 1560 ggtgccgctg tagtgagagg accaggattt acaggtgggg atatccttcg aagaacgaat 1620 actggtacat ttggggatat acgagtaaat attaatccac catttgcaca aagatatcgc 1680 gtgaggattc gctatgcttc taccacagat ttacaattcc atacgtcaat taacggtaaa 1740 gctattaatc aaggtaattt ttcagcaact atgaatagag gagaggactt agactataaa 1800 acctttagaa ctgtaggctt taccactcca tttagctttt tagatgtaca aagtacattc 1860 acaataggtg cttggaactt ctcttcaggt aacgaagttt atatagatag aattgaattt 1920 gttccggtag aagtaacata tgaggcagaa tatgattttg aaaaagcgca agagaaggtt 1980 actgcactgt ttacatctac gaatccaaga ggattaaaaa cagatgtaaa ggattatcat 2040 attgaccagg tatcaaattt agtagagtct ctatcagatg aattctatct tgatgaaaag 2100 agagaattat tcgagatagt taaatacgcg aagcaactcc atattgagcg taacatgtag 2160 <210> 8 <211> 719 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: protein encoded by the modified crylla gene <400> 8 Met Lys Leu Lys Asn Gin Asp Lys His Gin Ser Phe Ser Ser Asn Ala 1 5 10 15 Lvs Val Asp Lys He Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp He 20 25 30 Glu Leu Gin Asn He Asn His Glu Asp Cys Leu Lys Met Ser Glu Tyr 35 40 45 Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr He Gin Thr Gly He 50 55 60 Gly He Ala Gly Lys He Leu Gly Thr Leu Gly Val Pro Phe Ala Gly 65 70 75 80 Gin Val Ala Ser Leu Tyr Ser Phe He Leu Gly Glu Leu Trp Pro Lys 85 90 95 Gly Lys Asn Gin Trp Glu He Phe Met Glu His Val Glu Glu He He 100 105 110 Asn Gin Lys He 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 Asp Ser Leu Glu Ser 130 135 140 Trp Val Gly Asn Arg Asn Asn Thr Arg Ala Arg Ser Val Val Lys Sεr 145 150 155 160 Gin Tyr He 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 He Tyr Ala Gin Ala 130 185 190 Ala Asn Leu His Leu Leu Leu Lεu Arg Asp Ala Ser He Phe Gly Lys 195 200 205 Glu Trp Gly Leu Ser Ser Ser Glu He Ser Thr Phe Tyr Asn Arg Gin 210 215 220 Val Glu Arg Ala Gly Asp Tyr Ser Tyr 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 Phε Arg Arg Asp Mεt Thr Leu Met Val Leu Asp Leu Val 260 265 270 Ala Leu Phe Pro Ser Tyr Asp Thr Gin Met Tyr Pro He Lys Thr Thr 275 280 285 Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala He Gly Thr Val His 290 295 300 Pro His Pro Ser Phe Thr Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro 305 310 315 320 Ser Phe Ser Ala He Glu Ala Ala Val Val Arg Asn Pro His Leu Leu 325 330 335 Asp Phe Leu Glu Gin Val Thr He Tyr Ser Leu Leu Ser Arg Trp Ser 340 345 350 Asn Thr Gin Tyr Met Asn Met Trp Gly Gly His Lys Lεu Glu Phe Arg 355 360 365 Thr He Gly Gly Thr Leu Asn He Ser Thr Gin Gly Ser Thr Asn Thr 370 375 380 Ser He Asn Pro Val Thr Leu Pro Phe Thr Ser Arg Asp Val Tyr Arg 385 390 395 400 Thr Glu Ser Lεu Ala Gly Leu Asn Leu Phe Leu Thr Gin Pro Val Asn 405 410 415 Gly Val Pro Arg Val Asp Phe His Trp Lys Phe Val Thr His Pro He 420 425 430 Ala Ser Asp Asn Phe Tyr Tyr Pro Gly Tyr Ala Gly He Gly Thr Gin 435 440 445 Lεu Gin Asp Ser Glu Asn Glu Leu Pro Pro Glu Ala Thr Gly Gin Pro 450 455 460 Asn Tyr Glu Sεr Tyr Ser His Arg Leu Ser His He Gly Leu He Ser 465 470 475 480 Ala Ser His Val Lys Ala Leu Val Tyr Ser Trp Thr His Arg Ser Ala 485 490 495 Asp Arg Thr Asn Thr He Glu Pro Asn Ser He Thr Gin He Pro Leu 500 505 510 Val Lys Ala Phe Asn Lεu Ser Ser Gly Ala Ala Val Val Arg Gly Pro 515 520 525 Gly Phe Thr Gly Gly Asp He Lεu Arg Arg Thr Asn Thr Gly Thr Phε 530 535 540 Gly Asp He Arg Val Asn He Asn Pro Pro Phe Ala Gin Arg Tyr Arg 545 550 555 560 Val Arg He Arg Tyr Ala Ser Thr Thr Asp Leu Gin Phe His Thr Ser 565 570 575 He Asn Gly Lys Ala He Asn Gin Gly Asn Phe Ser Ala Thr Mεt Asn 580 585 590 Arg Gly Glu Asp Leu Asp Tyr Lys Thr Phe Arg Thr Val Gly Phe Thr 595 600 605 Thr Pro Phe Ser Phe Leu Asp Val Gin Ser Thr Phe Thr He Gly Ala 610 615 620 Trp Asn Phε Ser Ser Gly Asn Glu Val Tyr He Asp Arg He Glu Phe 625 630 635 640 Val Pro Val Glu Val Thr Tyr Glu Ala Glu Tyr Asp Phe Glu Lys Ala 645 650 655 Gin Glu Lys Val Thr Ala Leu Phe Thr Ser Thr Asn Pro Arg Gly Lεu 660 665 670 Lys Thr Asp Val Lys Asp Tyr His He Asp Gin Val Ser Asn Leu Val 675 680 685 Glu Ser Leu Ser Asp Glu Phe Tyr Leu Asp Glu Lys Arg Glu Leu Phe 690 695 700 Glu He Val Lys Tyr Ala Lys Gin Leu His He Glu Arg Asn Mεt 705 710 715 <210> 9 <211> 37 <212> DNA <213> Artificial Sequεnce <220> <223> Description of Artificial Sequence: oligomer used for mutagenesis of the crylBa gene <400> 9 ggacgcatcg tagtgcggac cgtacgaata cgattgg 37 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligomer used for mutagenesis of the crylla genε <400> 10 ggacgcatcg tagtgcggac cgtacaaata caattg 36

Claims: CLAIMS 1 . Bacillus thuringiensis hybrid toxin fragment comprising structural domains I, II and III in this order starting from the N-terminal, wherein the domains are derived from at least two different Cry proteins, domain I is domain I of any Bacillus thuringiensis Cry protein or a part of said domain or a peptide substantially similar to said domain, domain II is domain II of Cryl la or a part of said domain or a peptide substantially similar to said domain, and domain III is domain III of Cry1 Ba or a part of said domain or a peptide substantially similar to said domain. 2. Toxin fragment according to claim 1 , wherein domain I is domain I of Cryl la or Cryl Ba or a part of said domain or a peptide substantially similar to said domain . 3. Toxin fragment according to claim 1 or 2 comprising an amino acid sequence from about amino acid 20 to about amino acid 641 as shown in SEQ ID NO:2 or an amino acid sequence from about amino acid 20 to about amino acid 632 as shown in SEQ ID NO:4. 4. A hybrid toxin comprising the fragment of any one of the preceding claims , 5. A toxin according to claim 4 comprising an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4. 6. Recombinant DNA comprising a sequence encoding a protein having an amino acid sequence of the proteins as claimed in any one of claims 1 -5. 7. Recombinant DNA according to claim 6 comprising the sequence as shown in SEQ ID NO: 1 or 3 or DNA similar thereto encoding a substantially similar protein. 8. Recombinant DNA according to claim 6 comprising the nucleotide sequence from about nucleotide 1 70 to about 1 929 in SEQ ID NO: 1 or from about 147 to about 1896 in SEQ ID N0:3. 9. Recombinant DNA according to any one of claims 6 to 8 which is modified in that known mRNA instability motifs or polyadenylation signals are removed and/or codons which are preferred by the organism into which the recombinant DNA is to be inserted are used so that expression of the thus modified DNA in the said organism yields substantially similar protein to that obtained by expression of the unmodified recombinant DNA in the organism in which the protein components of the hybrid toxin or toxin fragments are endogenous. 1 0. A DNA sequence which is complementary to one which hybridizes under stringent conditions with the DNA of any one of claims 6 to 9. 1 1 .A vector containing a DNA sequence as claimed in any one of claims 6 to 1 0. 2. A plant or micro-organism which includes, and enables expression of, the DNA of any one of claims 6-10 or the vector of claim 1 1 . 3. Plants transformed with recombinant DNA as claimed in any one of claims 6 to 10, the progeny of such plants which contain the DNA stably incorporated and hereditable in a Mendelian manner, and/or the seeds of such plants and such progeny. 14. Protein derived from expression of the DNA as claimed in any one of claims 6 to 1 0 and insecticidal protein produced by expression of the recombinant DNA within plants as claimed in claim 1 3. 1 5. An insecticidal composition containing one or more of the proteins as claimed in any one of claims 1 -5 and 1 4. 1 6. A process for combatting insects which comprises exposing them to proteins or compositions as claimed in any one of claims 1 -5 , 1 4 and 1 5 or the micro-organism of claim 1 2.

Inventors: BOSCH HENDRIK JAN, DE MAAGD RUDOLF AART, BOSCH, HENDRIK, JAN, DE MAAGD, RUDOLF, AART
Applicants: BOSCH HENDRIK JAN, MAAGD RUDOLF AART DE, PLANT RES INT BV, BOSCH, HENDRIK, JAN, DE MAAGD, RUDOLF, AART, PLANT RESEARCH INTERNATIONAL B.V.
Application year: 2000
Main classification: C12N15
IPC codes: C07K14, C12N15