Bacillus thuringiensis strains and their insecticidal proteins

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to four novel strains of Bacillus thuringiensis (the "BTS02617A strain", the "BTS02618A strain", the "BTS02654B strain" and the "BTS02652E strain"), each of which produces crystallized proteins (the "BTS02617A crystal proteins", the "BTS02618A crystal proteins", the "BTS02654B crystal proteins" and the "BTS02652E crystal proteins", respectively) which are packaged in crystals (the "BTS02617A crystals", the "BTS02618A crystals", the "BTS02654B crystal" and the "BTS02652E crystals", respectively) during sporulation. The BTS02617A, BTS02618A, BTS02654B and BTS02652E strains were deposited under the provisions of the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms--Collection Laboratorium voor Microbiologie Belgium ("BCCM-LMG"), R.U.G., K. Ledeganckstraat 35, B-9000 Gent.
This invention also relates to an insecticide composition that is active against Lepidoptera and that comprises the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, as such, or preferably the BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals, crystal proteins or the active component(s) thereof as an active ingredient.
This invention further relates to a gene (the "bTS02618A gene"), which is present in the genome of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains and which encodes an insecticidal protein (the "BTS02618A protoxin") that is found in the BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals. The BTS02618A protoxin is the protein that is produced by the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains before being packaged into their respective BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals.
This invention still further relates to a toxin (the "BTS02618A toxin") which can be obtained (e.g., by trypsin digestion) from the BTS02618A protoxin. The BTS02618A toxin is an insecticidally active protein which can be liberated from the BTS02617A crystals, the BTS02618A crystals, the BTS02654B crystals, and the BTS02652E crystals, which are produced by the BTS02617A strain, the BTS02618A strain, the BTS02654B strain and the BTS02652E strain, respectively. This toxin and its protoxin have a high activity against a wide range of lepidopteran insects, particularly against Noctuidae, especially against Spodoptera and Agrotis spp., but also against other important lepidopteran insects such as Pyralidae, particularly the European corn borer, Ostrinia nubilalis, Gelechiidae such as Phthorimaea operculella and Yponomeutidae such as Plutella xylostella. Furthermore, the BTS02618A protein is the first Bt protein with significant activity towards Agrotis segetum. This new characteristic of the BTS02618A protoxin and toxin ("(pro)toxin"), i.e., the combination of activity against different economically important Lepidopteran insect families such as Noctuidae, Yponomeutidae, Gelechiidae and Pyralidae, makes this (pro) toxin an ideally suited compound for combatting a wide range of insect pests by contacting these insects with the (pro)toxin, e.g., by spraying or by expressing the bTS02618A gene in plant-associated bacteria or in plants. The BTS02618A toxin is believed to represent the smallest portion of the BTS02618A protoxin which is insecticidally effective against Lepidoptera.
This invention also relates to transformed Bacillus thuringiensis strains, containing DNA sequences encoding a BTS02618A protein or variants thereof having substantially the same insecticidal activity.
This invention yet further relates to a chimeric gene that can be used to transform a plant cell and that contains the following operably linked DNA fragments:
1) a part of the bTS02618A gene (the "insecticidally effective bTS02618A gene part") encoding an insecticidally effective portion of the BTS02618A protoxin, preferably a truncated part of the bTS02618A gene (the "truncated bTS02618A gene") encoding just the BTS02618A toxin;
2) a promoter suitable for transcription of the insecticidally effective bTS02618A gene part in a plant cell; and
3) suitable 3' end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618A gene part in a plant cell. This chimeric gene is hereinafter generally referred to as the "bTS02618A chimeric gene".
This invention also relates to:
1) a cell (the "transformed plant cell") of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene; and
2) a plant (the "transformed plant") which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene, and which is resistant to Lepidoptera.
This invention still further relates to:
1) a microbial organism, such as B. thuringiensis or Pseudomonas spp., the genome of which is transformed with all or part of the bTS02618A gene; and
2) a microbial spore, containing a genome which is transformed with all or parts of the bTS02618A gene.
Another embodiment of the present invention relates to artificially made bTS02618A genes which encode BTS02618A proteins, and to proteins which are more protease resistant than native Bt proteins, more preferably the native BTS02618A protein. A particular example of a protein that is more protease resistant is the BTS02618Aa protein. Furthermore, the present invention also relates to a DNA sequence encoding the BTS02618Aa protein.
Yet another embodiment of the present invention relates to a chimeric gene that can be used to transform a plant cell and that contains:
1) a DNA sequence encoding an insecticidally effective portion of the BTS02618Aa protoxin, preferably a truncated part of the bTS02618Aa gene (the "truncated bTS02618Aa gene") encoding just the BTS02618Aa toxin;
2) a promoter suitable for transcription of the insecticidally effective bTS02618Aa gene part in a plant cell; and
3) suitable 3' end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618Aa gene part in a plant cell. This chimeric gene is hereinafter generally referred to as the "bTS02618Aa chimeric gene".
This invention further relates to:
1) a cell (the "transformed plant cell") of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618Aa gene part, preferably the bTS02618Aa chimeric gene; and
2) a plant (the "transformed plant") which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618Aa gene part, preferably the bTS0261Aa chimeric gene, and which is resistant to Lepidoptera.
This invention still further relates to:
1) a microbial organism, such as B. thurinciensis or Pseudomonas spp., the genome of which is transformed with all or part of a DNA sequence encoding the BTS02618Aa protein; and
2) a microbial spore, containing a genome which is transformed with all or part of the bTS02618Aa gene.
Yet another embodiment of the present invention relates to insecticidal compositions that are active against Lepidoptera and that comprise a more protease resistant Bt protein, more particularly the BTS02618Aa protein or a variant thereof which has substantially the same insecticidal activity.
2. Description of the Prior Art
B. thurinciensis ("Bt") is a Gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae. These crystal proteins and corresponding genes have been classified based on their structure and insecticidal spectrum (Hofte and Whiteley, 1989). The four major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), and Diptera-specific (cryIV) genes.
The fact that conventional submerged fermentation techniques can be used to produce Bt spores on a large scale makes Bt bacteria commercially attractive as a source of insecticidal compositions.
Gene fragments from some Bt strains, encoding insecticidal proteins, have heretofore been identified and integrated into plant genomes in order to render the plants insect-resistant. However, obtaining expression of such Bt gene fragments in plants is not a straightforward process. In order to achieve optimal expression of an insecticidal protein in plant cells, it has been found necessary to engineer each Bt gene fragment in a specific way so that it encodes a part of a Bt protoxin that retains substantial toxicity against its target insects (European patent application ("EPA") 86/300,291.1 and 88/402,115.5; U.S. patent application Ser. No. 821,582, filed Jan. 22, 1986).
SUMMARY OF THE INVENTION
In accordance with this invention, four novel Bt strains, i.e., the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains, are provided. The BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals and crystal proteins, the BTS02618A protoxin and toxin produced by the strains during sporulation, and insecticidally effective portions of the BTS02618A protoxin, as well as equivalents of these crystals, crystal proteins, protoxin, toxin and insecticidally effective protoxin portions, each possess insecticidal activity and can therefore be formulated into insecticidal compositions against Lepidoptera in general, and particularly against Noctuidae, such as Agrotis spp. (cutworms such as Agrotis ipsilon and Agrotis segetum), Mamestra spp. (e.g., the cabbage moth, Mamestra brassica) and Spodoptera spp. (armyworms, such as Spodoptera exigua, Spodoptera frugiterda, Spodoptera littoralis and Spodoptera litura), against Pyralidae (e.g., the European corn borer, Ostrinia nubilalis), against Gelechiidae such as Phthorimaea operculella and Yponomeutidae (such as Plutella xylostella) which are major pests of various economically important crops, such as corn, cotton and many vegetables such as Brassicas.
Also in accordance with this invention, a plant cell genome is transformed with the insecticidally effective bTS02618A gene part, preferably the truncated bTS02618A gene, or an equivalent thereof such as a modified, synthetic bTS02618A gene. It is preferred that this transformation be carried out with the bTS02618A chimeric gene. The resulting transformed plant cell can be used to produce transformed plants, seeds of transformed plants and plant cell cultures consisting essentially of the transformed cells. The transformed cells in some or all of the tissues of the transformed plants: 1) contain the insecticidally effective bTS02618A gene part as a stable insert in their genome, and 2) express the insecticidally effective bTS02618A gene part by producing an insecticidally effective portion of its BTS02618A protoxin, preferably its BTS02618A toxin, thereby rendering the plant resistant to Lepidoptera. The transformed plant cells of this invention can also be used to produce, for recovery, such insecticidal Bt proteins.
Further in accordance with this invention, a process is provided for rendering a plant resistant to Lepidoptera by transforming the plant cell genome with the insecticidally effective bTS02618A gene part, preferably the truncated bTS02618A gene, or an equivalent thereof. In this regard, it is preferred that the plant cell be transformed with the bTS02618A chimeric gene.
Yet further in accordance with this invention, there are provided the BTS02618A protoxin, the insecticidally effective portions of such protoxin and the BTS02618A toxin, as well as functional parts of the BTS02618A toxin, as well as the bTS02618A gene, the insecticidally effective bTS02618A gene part, the truncated bTS02618A gene and the chimeric bTS02618A gene, as well as their equivalents.
Also in accordance with this invention, a DNA sequence, either natural or artificial, encoding the BTS02618A protoxin or insecticidally effective portions thereof, such as the toxin, is provided.
Also in accordance with this invention are provided an insecticidal composition against Lepidoptera, particularly Noctuidae, Pyralidae, Gelechiidae and Yponomeutidae, and a method for controlling Lepidoptera, particularly Noctuidae, Pyralidae, Gelechiidae and Yponomeutidae, with the insecticidal composition, wherein the insecticidal composition comprises the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, crystals and/or crystal proteins or the BTS02618A protoxin, toxin and/or insecticidally effective protoxin portions or their equivalents.
Also in accordance with this invention, bacteria, particularly E. coli and Bacillus thuringiensis, transformed to express a DNA encoding the BTS02618A protein variant, such as the BTS02618Aa protein or more improved protease resistant Bt proteins are provided.
Furthermore, in accordance with this invention, an artificial DNA sequence encoding the BTS02618A protein, as well as new forms of Bt proteins with improved protease resistance, more particularly the BTS02618Aa or the modified BTS02618A protein are provided, and DNA sequences encoding these new proteins. Further provided are plant cells expressing an artificial DNA sequence encoding the BTS02618A toxin or Bt toxins with improved protease resistance, more preferably the BTS02618Aa toxin.
Also provided is an insecticidal composition, comprising as an active ingredient the BTS02618Aa protein, or a variant thereof with substantially the same insecticidal activity. Also provided is a method to combat Lepidopteran insects by contacting these insects with Bt proteins having improved protease resistance, more preferably the BTS02618Aa protein or a variant thereof.
More specifically provided are new Bt proteins, preferably Lepidoptera active Bt proteins, having substantially the same insecticidal activity as the native Bt protein, but characterized in their resistance to further proteolytic cleavage of the about 60 to 70 kD toxin form. Such new Bt proteins have inactivated internal protease cleavage sites, so that these proteins have increased stability while retaining substantially the same insecticidal activity. Thus, these new Bt proteins are not readily cleaved into smaller proteolytic fragments which lower their insecticidal activity upon prolonged incubation in the presence of proteases.

BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 represents Southern blot analysis of Alul-digested total DNA of Bt strain HD127 (lane 1), the BTS02618A strain (lane 2), Bt strain BTS02459 (containing crylA(c); 81k, crylc en crylE, lane 3), and Bt strain BTS02480E (containing the same genes as HD-127, lane 4), using a mixture of DNA-probes for cryl crystal protein genes, including the crylG probe (SEQ ID NO:1). Each band corresponds to a particular crystal protein gene. With these probes, the BTS02618A strain is found to contain the crylA(b) gene and a novel gene, which is the bTS02618A gene, identified by an Alul fragment of approximately 530 bp, hybridizing to the crylG probe of SEQ ID NO:1. The names of the recognized cryl genes are indicated, as well as the size of some fragments. The bTS02618A

DETAILED DESCRIPTION OF THE INVENTION
The BTS02618A protoxin of this invention can be isolated in a conventional manner from the BTS02617A strain, deposited on July, 2 at the BCCM-LMG under accession number LMG P-12592, the BTS02618A strain, deposited on Jul. 2, 1992 at the BCCM-LMG under accession number LMG P-12593, the BTS02654B strain, deposited on Jul. 2, 1992 at the BCCM-LMG under accession number LMG P-12594, or the BTS02652E strain deposited on Mar. 1, 1993 at the BCCM-LMG under accession number LMG P-13493. For example, the BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals can be isolated from sporulated cultures of their respective strain (Mahillon and Delcour, 1984), and then, the BTS02618A protoxin can be isolated from the crystals according to the method of Hofte et al. (1986). The protoxins can be used to prepare monoclonal or polyclonal antibodies specific for the protoxin in a conventional manner (Hofte et al., 1988). The BTS02618A toxin can be obtained by protease (e.g., trypsin) digestion of the BTS02618A protoxin.
The bTS02618A gene can be isolated in a conventional manner. The bTS02618A gene can be identified in the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, using the procedure described in U.S. patent application Ser. No. 821,582, filed Jan. 22, 1986, and in EPA 86/300,291.1 and 88/402,115.5 (which are incorporated herein by reference). The bTS02618A gene was identified by: digesting total DNA from one of the above strains with restriction enzymes; size fractionating the DNA fragments, so produced, into DNA fractions of 5 to 10 Kb; ligating these fractions to cloning vectors; screening the E. coli, transformed with the cloning vectors, with a DNA probe that was constructed from a region of the cryIG gene (Smulevitch et al., 1991; Gleave et al., 1992).
The term "bTS02618A gene" as used herein includes a DNA sequence encoding the BTS02618A protoxin or toxin or functionally equivalent variants thereof. Indeed, because of the degeneracy of the genetic code, some amino acid codons can be replaced with others without changing the amino acid sequence of the protein. Furthermore, some amino acids can be substituted by other equivalent amino acids without significantly changing the insecticidal activity of the protein. Also, changes in amino acid composition in regions of the molecule, different from those responsible for binding and toxicity are less likely to cause a difference in insecticidal activity of the protein. Such equivalents of the gene include DNA sequences hybridizing to the DNA sequence of the BTS02618A toxin or protoxin of SEQ ID. No. 4 and encoding a protein with the same insecticidal characteristics as the BTS02618A (pro)toxin, of this invention. In this context, the term "hybridization" refers to conventional hybridization conditions, most preferably stringent hybridization conditions.
The "BTS02618A protein" is a general term for the BTS02618A protoxin and variants or mutants thereof with substantially the same insecticidal activity; for example, the BTS02618A or BTS02618Aa toxins.
As used herein, the term "more or improved protease resistant protein" means that the Bt protein fragment resulting from protease cleavage of the native protoxin does not result in a substantial loss of insecticidal activity due to the further cleavage of the insecticidally active toxin part of the protein. It is preferable that the insecticidally active toxin part of the protein be about 60 to 70 kD and more particularly that the further cleavage is at the N-terminal part of the toxin. It is also preferred that the protein is insecticidal for Lepidoptera.
A preferred example of an "alternative form" of the bTS02618A gene is the artificial bTS02618A gene of SEQ ID. No. 6, encoding a BTS02618A toxin. A further preferred example of an artificial bTS02618A gene is illustrated in the DNA sequence of SEQ ID. No. 8, for reasons of clarity further named "the bTS02618Aa gene", encoding a (similar but different) protein with an insecticidal activity substantially similar to the BTS02618A protein.
Of course, the present invention is not limited to the particular preferred embodiments described herein as "alternative variants or forms." In fact, any other DNA sequences differing in their codon usage but encoding the same protein or a similar protein with substantially the same insecticidal activity, can be constructed by the person skilled in the art. In some prokaryotic and eucaryotic expression systems, for example, changing the codon usage to that of the host cell can increase gene expression (Bennetzen & Hall, 1982; Itakura, 1977). Moreover, since many Bt genes are known to have no bias towards eucaryotic codons, and to have very AT-rich genes it is sometimes beneficial to change the codon usage (Adang et al., 1985, Schnepf et al., 1985). To accomplish this codon usage tables which are available in the literature (Wada et al., 1990; Murray et al., 1989) and in the major DNA sequence databanks (e.g., EMBL at Heidelberg, Germany) are often referred to by the person skilled in the art. Accordingly, synthetic DNA sequences can be constructed so that the same or substantially the same proteins may be produced. See, for example, Cohen et al., 1973.
The term "substantially the same", when referring to a protein, is meant to include a protein that differs in some amino acids, or has some amino acids added (e.g., a fusion protein, see Vaeck et al., 1987) or deleted (e.g., N- or C-terminal truncation), while retaining the protein's insecticidal activity. It is generally known to those skilled in the art that general amino acid replacements in many parts of a polypeptide chain may be made without seriously modifying the activity of the polypeptide (Watson et al Molecular Biology of the Gene (1987) 226-227.
The term "functional parts of the BTS02618A toxin" as used herein means any part(s) or domain(s) of the toxin with a specific structure that can be transferred to another (Bt) protein for providing a new hybrid protein with at least one functional characteristic (e.g., the binding and/or toxicity characteristics) of the BTS02618A toxin (Ge et al., 1991). Such parts can form an essential feature of the hybrid Bt protein with the binding and/or toxicity characteristics of the BTS02618A protein. Such a hybrid protein can have an enlarged host range, an improved toxicity and/or can be used in a strategy to prevent insect resistance development (European Patent Publication ("EP") 408 403; Visser et al., 1993).
The "BTS02618Aa toxin", as used herein, refers to a new form of the BTS02618A toxin, differing in some amino acids from the native BTS02618A toxin. Indeed, the BTS02618A protoxin has been found to be digested by proteases into an about 69 kD protein and about 55 kD protein, the latter having substantially lower insecticidal activity. The longer the protease digestion, the more of the about 55 kD protein was formed. This 55 kD protein was found to be cleaved by digestion at the Arginine at amino acid position 164 shown in SEQ ID. No. 4. Thus, in the BTS02618Aa toxin, the Argentine at this position was replaced with a Lysine, the 43 N-terminal amino acids was replaced by amino acids Met-Ala, and the C-terminal end was truncated up to amino acid 666 (in SEQ ID. No. 4). Similar to other Bt toxins, the C-terminal end of the BTS02618Aa toxin can be further truncated to the minimum toxic fragment (up to amino acid 658 in SEQ ID. No. 4).
In another form of the BTS02618A protein, "the BTS02618Ab protein", this Arginine has been substituted with an Alanine. Both the BTS02618Aa/b proteins are less susceptible to proteases and still have substantially the same insecticidal activity. The part C-terminal from the toxic fragment of both the BTS02618Aa and BTS02618Ab protoxins is 100% identical to the C-terminal part of the BTS02618A protoxin.
The "bTS02618Aa gene" and the "bTS02618Ab gene", as used herein, refer to DNA sequences encoding respectively the BTS02618Aa and BTS02618Ab proteins. It is evident that several DNA sequences can be devised once the amino acid sequence of the BTS02618Aa and BTS02618Ab proteins are known. Such other DNA sequences include synthetic or semi-synthetic DNA sequences that have been changed in order to inactivate certain sites in the gene, e.g., by selectively inactivating certain cryptic regulatory or processing elements present in the native sequence as described in PCT publications WO 91/16432 and WO 93/09218, or by adapting the overall codon usage to that of a more related host organism, preferably that of the host organism, in which expression is desired.
Such a modification of the BTS02618A protein can also be achieved by deleting the Arginine at amino acid position 123, or by replacing this amino acid by another amino acid provided that the insecticidal activity of the new BTS02618A protein is not substantially changed. Other amino acids surrounding the protease cleavage site can also be altered such that the insecticidal activity is not substantially changed.
The new proteins can be tested in routine bio-assays to compare their toxicity with that of the native BTS02618A protein. The overall toxicity parameters of such proteins should be similar to those of the native proteins.
Due to the retention of their insecticidal activity, such new proteins are very useful for combatting important pest insects. Their improved resistance for protease activity makes them the toxins of choice for combatting insects, e.g., by expressing a DNA sequence encoding such proteins in a foreign host, such as bacteria or plants. Small modifications to a DNA sequence such as described above are routinely made by PCR-mediated mutagenesis (Ho et al.,1989, White et al., 1989).
The above variants show that indeed modifications can be made to the BTS02618A protein without causing any substantial changes to the insecticidal activity. Besides a deletion of up to 43 amino acids at the N-terminus, and a major deletion of C-terminal amino acids, also some internally located amino acids can be replaced by others while retaining substantially the same insecticidal activity of the BTS02618A toxin.
Similarly, the CryIB protoxin (Brizzard & Whiteley, 1988), and a naturally occurring variant thereof (EP publication 408 403) has been found to be cleaved into an about 69 kD toxin and a smaller about 55 kD toxin by protease activity. Also for this toxin, modification of the Arginine at amino acid positions 144 and 146 (relative to the start codon) in the sequence of Brizzard & Whiteley (1988) or the sequence of EP 408 403 can increase the stability of the protein in the insect gut. Indeed, prolonged protease treatment of the CryIB protoxin, either obtained from Bt strain 4412 or expressed in E. coli, resulted in an about 55 kD protein with an N-terminal end starting at amino acid position 145 (Thr-Arg-Ser-Val-Leu-) and another about 55 kD protein starting at position 147 (Ser-Val-Leu-Tyr-Thr-). Modifying the Arginine amino acids at positions 144 and 146 leads to a more stable toxin form, which is still toxic. This modification can be incorporated into a natural or synthetic DNA sequence encoding the CryIB protein or variants thereof such as the Bt14 toxin in EP 358 557, by techniques well known in the art, so that a more stable CryIB protein is produced. Such a CryIB protein can be used together with the BTS02618A or BTS02618Aa protein and the CryIAb protein in combatting Lepidopteran insects, particularly Ostrinia nubilalis, by expressing DNA sequences encoding these proteins in a host cell, particularly a plant cell. So the modification of one or more amino acids is useful in other Bt proteins, particularly anti-Lepidoptera Bt proteins, that are also further cleaved by proteases.
Furthermore, the 5 to 10 Kb fragments, prepared from total DNA of the BTS02617A or BTS02618A or BTS02654B or BTS02652E strain, can be ligated in suitable expression vectors and transformed in E. coli, and the clones can then be screened by conventional colony immunoprobing methods (French et al., 1986) for expression of the toxin with monoclonal or polyclonal antibodies raised against the BTS02618A toxin.
Also, the 5 to 10 Kb fragments, prepared from total DNA of the BTS02617A or BTS02618A or BTS02654B or BTS02652E strain, can be ligated in suitable Bt shuttle vectors (Lereclus et al., 1992) and transformed in a crystal minus Bt-mutant. The clones are then screened for production of crystals (detected by microscopy) or crystal proteins (detected by SDS-PAGE).
The so-identified bTS02618A gene was sequenced in a conventional manner (Maxam and Gilbert, 1980) to obtain the DNA sequence. Hybridization in Southern blots and sequence comparison indicated that this gene is different from previously described genes encoding protoxins and toxins with activity against Lepidoptera (Hofte and Whiteley, 1989).
An insecticidally effective part of the bTS02618A gene, encoding an insecticidally effective portion of its protoxin, and a truncated part of the gene, encoding just its toxin, can be made in a conventional manner after sequence analysis of the gene. The amino acid sequence of the BTS02618A protoxin and toxin was determined from the DNA sequence of the bTS02618A gene and the truncated bTS02618A gene. By "an insecticidally effective part" or "a part" of the bTS02618A gene is meant a DNA sequence encoding a polypeptide which has fewer amino acids than the BTS02618A protoxin but which is Still toxic to Lepidoptera.
In order to express all or an insecticidally effective part of the bTS02618A gene or an equivalent gene in E. coli, in other Bt strains and in plants, suitable restriction sites can be introduced, flanking each gene or gene part. This can be done by site-directed mutagenesis, using well-known procedures (Stanssens et al., 1989; White et al., 1989). In order to obtain improved expression in plants, it may be preferred to modify the codon usage of the bTS02618A gene or insecticidally effective bTS02618A gene part to form an equivalent, modified or artificial gene or gene part in accordance with PCT publications WO 91/16432 and WO 93/09218; EP 0,358,962 and EP 0,359,472. For obtaining enhanced expression in monocot plants such as corn, a monocot intron also can be added to the bTS02618A chimeric gene, and the DNA sequence of the bTS02618A gene part can be further changed in a translationally neutral manner, to modify possibly inhibiting DNA sequences present in the gene part by means of site-directed intron insertion and/or by introducing changes to the codon usage, e.g., adapting the codon usage to that most preferred by the specific plant (Murray et al., 1989) without changing significantly the encoded amino acid sequence.
Preferred examples of modified bTS02618A genes are shown in SEQ ID. Nos. 6 and 8, illustrating DNA sequences encoding the BTS02618A toxin and a variant thereof. These DNA sequences have an overall modified codon usage, which has been adapted to that of plants, particularly monocots such as corn. The DNA of SEQ ID. No. 6 encodes exactly the same toxin as the native bTS02618A gene, but yields higher expression levels in plants, particularly monocots such as corn, due to the adaptation of its codon usage to that of the plant host cells.
Furthermore, the BTS02618Aa toxin was found to bind to a receptor different from the CryIAb toxin receptor population in Ostrinia nubilalis gut membranes. This indicates that the BTS02618A toxin has a unique receptor in its susceptible insects. The broad spectrum, the binding to a different receptor and the low homology with other Bt toxins indicates that the BTS02618A toxin represents a new class of Bt toxins. Since the BTS02618A toxin apparently recognizes a different target site, it can prove to be especially useful for preventing the development of insect resistance, or for combatting insects resistant to other Bt toxins. Particularly the combined expression of the bTS02618A gene with other Bt genes encoding non-competitively binding toxins (as described in EP 408 403) in one host is interesting for preventing resistance development, preferably the combined expression of CryIAb and BTS02618A proteins.
Because of the broad spectrum of susceptible pest insects, the BTS02618A toxin and its variants are extremely useful for transforming plants, e.g., monocots such as corn and vegetables such as Brassicas, to protect these plants from insect damage.
The insecticidally effective bTS02618A gene part or its equivalent, preferably the bTS02618A chimeric gene, encoding an insecticidally effective portion of the BTS02618A protoxin, can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that is insect-resistant. In this regard, a disarmed Ti-plasmid, containing the insecticidally effective bTS02618A gene part, in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0,116,718, EP 0,270,822, PCT publication WO 84/02,913 and European Patent Application ("EPA") 87/400,544.0 (which are also incorporated herein by reference), and in Gould et al. (1991). Preferred Ti-plasmid vectors each contain the insecticidally effective bTS02618A gene part between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0,233,247), pollen mediated transformation (as described, for example in EP 0,270,356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example in EP 0,067,553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example in U.S. Pat. No. 4,536,475), and other methods such as the recently described methods for transforming certain lines of corn (From et al., 1990; Gordon-Kamm et al., 1990) and rice (Shimamoto et al., 1989; Datta et al., 1990) and the recently described method for transforming monocots generally (PCT publication WO 92/09696).
The resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective bTS02618A gene part in other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain the insecticidally effective bTS02618A gene part as a stable genomic insert. Cells of the transformed plant can be cultured in a conventional manner to produce the insecticidally effective portion of the BTS02618A protoxin, preferably the BTS02618A toxin, which can be recovered for use in conventional insecticide compositions against Lepidoptera (U.S. patent application Ser. No. 821,582; EPA 86/300291.1.).
The insecticidally effective bTS02618A gene part, preferably the truncated bTS02618A gene, is inserted in a plant cell genome so that the inserted gene is downstream (i.e., 3') of, and under the control of, a promoter which can direct the expression of the gene part in the plant cell. This is preferably accomplished by inserting the bTS02618A chimeric gene in the plant cell genome. Preferred promoters include: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus of isolates CM 1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1978); and the TR1' promoter and the TR2' promoter (the "TR1' promoter" and "TR2' promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby the inserted bTS02618A gene part is expressed only in cells of the specific tissue(s) or organ(s). For example, the insecticidally effective bTS02618A gene part could be selectively expressed in the leaves of a plant (e.g., corn, cotton) by placing the insecticidally effective gene part under the control of a light-inducible promoter such as the promoter of the ribulose-1,5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant such as pea as disclosed in U.S. patent application Ser. No. 821,582 and EPA 86/300,291.1. Another alternative is to use a promoter whose expression is inducible (e.g., by temperature or chemical factors).
The insecticidally effective bTS02618A gene part is inserted in the plant genome so that the inserted gene part is upstream (i.e., 5') of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the bTS02618A chimeric gene in the plant cell genome. Preferred polyadenylation and transcript formation signals include those of the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3'-untranslated DNA sequences in transformed plant cells.
The insecticidally effective bTS02618A gene part can optionally be inserted in the plant genome as a hybrid gene (EPA 86/300,291.1; Vaeck et al., 1987) under the control of the same promoter as a selectable marker gene, such as the neo gene (EP 0,242,236) encoding kanamycin resistance, so that the plant expresses a fusion protein.
All or part of the bTS02618A gene, encoding an anti-lepidopteran protein, can also be used to transform other bacteria, such as a B. thuringiensis which has insecticidal activity against Lepidoptera or Coleoptera. Thereby, a transformed Bt strain can be produced which is useful for combatting a wide spectrum of lepidopteran and coleopteran insect pests or for combatting additional lepidopteran insect pests. Transformation of bacteria with all or part of the bTS02618A gene, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electroporation techniques as described in Mahillon et al. (1989) and in PCT Patent publication WO 90/06999.
Alternatively, mutants of the BTSO2618A, BTS02617A, BTS02654B and BTS02652E strains can be obtained by treating these strains with mutagenic agents such as nitrosoguanidine or with UV light; techniques which are well known to those skilled in the art. Also, asporogenous mutants can be obtained by treatment with ethylmethane sulfonate. Such mutants can be screened for improved characteristics (such as suitability for large-scale fermentation and the like), while retaining substantially the same insecticidal activity.
The BTS02617A, BTS02618A, BTS02654B or BTS02652E strain also can be transformed with all or an insecticidally effective part of one or more foreign Bt genes such as: the bt18 gene (EP 0,358,557) or another Bt gene coding for an anti-Lepidoptera protein; and the bt109P gene (PCT publication WO 91/16433), coding for an anti-Coleoptera protein. Thereby, a transformed Bt strain can be produced which is useful for combatting an even greater variety of insect pests (e.g., Coleoptera and/or additional Lepidoptera).
Transformation of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain with all or part of a foreign Bt gene, incorporated in a conventional cloning vector, can be carried out in a well known manner, preferably using conventional electroporation techniques (Chassy et al., 1988) or other methods, e.g., as described by Lereclus et al. (1992).
Each of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strains can be fermented by conventional methods (Dulmage, 1981; Bernhard and Utz, 1993) to provide high yields of cells. Under appropriate conditions which are well understood (Dulmage, 1981), the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains each sporulate to produce crystal proteins containing the BTS02168A protoxin in high yields.
An insecticidal, particularly anti-lepidopteran, composition of this invention can be formulated in a conventional manner using the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain or preferably their respective crystals, crystal proteins or the BTS02168A protoxin, toxin or insecticidally effective protoxin portion as an active ingredient, together with suitable carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, 1993). This insecticide composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc. The concentration of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, crystals, crystal proteins, or the BTS02618A protoxin, toxin or insecticidally effective protoxin portions in such a composition will depend upon the nature of the formulation and its intended mode of use. Generally, an insecticide composition of this invention can be used to protect a field for 2 to 4 weeks against Lepidoptera with each application of the composition. For more extended protection (e.g., for a whole growing season), additional amounts of the composition should be applied periodically.
A method for controlling insects, particularly Lepidoptera, in accordance with this invention preferably comprises applying (e.g., spraying), to a locus (area) to be protected, an insecticidal amount of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, spores, crystals, crystal proteins or the BTS02168A protoxin, toxin or insecticidally effective protoxin portions, preferably the BTS2168A toxin. The locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown.
To obtain the BTS02618A protoxin or toxin, cells of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain can be grown in a conventional manner on a suitable culture medium and then lysed using conventional means such as enzymatic degradation or detergents or the like. The protoxin can then be separated and purified by standard techniques such as chromatography, extraction, electrophoresis, or the like. The toxin can then be obtained by trypsin digestion of the protoxin.
The BTS02617A, BTS02618A, BTS02654B or BTS02652E cells can also be harvested and then applied intact, either alive or dead, preferably dried, to the locus to be protected. In this regard, it is preferred that a purified BTS02617A, BTS02618A, BTS02654B or BTS02652E strain (either alive or dead) be used, particularly a cell mass that is 90.0 to 99.9% of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain.
The BTS02617A, BTS02618A, BTS02654B, or BTS02652E cells, crystals or crystal proteins or the BTS02618A protoxin, toxin, or insecticidally effective protoxin portion can be formulated in an insecticidal composition in a variety of ways, using any number of conventional additives, wet or dry, depending upon the particular use. Additives can include wetting agents, detergents, stabilizers, adhering agents, spreading agents and extenders. Examples of such a composition include pastes, dusting powders, wettable powders, granules, baits and aerosol sprays. Other Bt cells, crystals, crystal proteins, protoxins, toxins, and insecticidally effective protoxin portions and other insecticides, as well as fungicides, biocides, herbicides and fertilizers, can be employed along with the BTS02617A, BTS02618A, BTS02654B or BTS02652E cells, crystals or crystal proteins or the BTS02618A protoxin, toxin or insecticidally effective protoxin portions to provide additional advantages or benefits. Such an insecticidal composition can be prepared in a conventional manner, and the amount of the BTS02617A, BTS02618A, BTS02654B or BTS02652E cells, crystals or crystal proteins or the BTS02618A protoxin, toxin or insecticidally effective protoxin portion employed depends upon a variety of factors, such as the insect pest targeted, the composition used, the type of area to which the composition is to be applied, and the prevailing weather conditions. Generally, the concentration of the BTS02618A protoxin, insecticidally effective protoxin portions or toxin will be at least about 0.1% by weight of the formulation to about 100% by weight of the formulation, more often from about 0.15% to about 0.8% by weight of the formulation.
In practice, some insects can be fed the BTS02618A protoxin, toxin, insecticidally effective protoxin portion or mixtures thereof in the protected area, that is in the area where such protoxin, toxin and/or insecticidally effective protoxin portion has been applied. Alternatively, some insects can be fed intact and alive cells of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain or transformants thereof, so that the insects ingest some of the strain's protoxin and suffer death or damage.
For the purpose of combatting insects by contacting them with the BTS02618A protein, e.g., in the form of transformed plants or insecticidal formulations and the like, any of the above described variants of the BTS02618A protein with substantially the same insecticidal activity can be used, preferably the BTS02618Aa and BTS02618Ab proteins. Furthermore, any of the above-described methods for transforming plants and bacteria can also be utilized to combat insects with the BTS02618Aa or BTS02618Ab proteins or other more protease resistant protein variants of the BTS02618A protein in lieu of the native BTS02618A protein.
The following Examples illustrate the invention. The sequence listing referred to in the Examples is as follows:
Sequence Listing
SEQ ID No. 1--Nucleotide sequence of the DNA probe used to isolate the bTS02618A gene. This probe is derived from part of the cryIG DNA sequence and is complementary to nucleotides 2732-2750 of the DNA sequence described by Smulevitch et al. (1991).
SEQ ID No. 2--The 5' partial nucleotide sequence of the bTS02618A gene, comprising the presumptive translation initiation codon at nucleotide position 195-197.
SEQ ID No. 3--The 3' partial nucleotide sequence of the bTS02618A gene (N: unknown nucleotide), comprising the presumptive translational stop codon at nucleotide position 1146-1148.
SEQ ID No. 4--The nucleotide sequence of the bTS02618A gene and the translated amino acid sequence of the BTS02618A protoxin. The open reading frame of the protoxin reaches from nucleotide 668 to nucleotide 4141. The translation initiation codon is at nucleotide position 668-670, the translation stop codon is at nucleotide position 4139-4141.
SEQ ID. No. 5--The amino acid sequence of the BTS02618A protein. The sequence of the about 69 kD BTS02618A toxin stretches from amino acid 44 to amino acid 658.
SEQ ID. No. 6--The nucleotide sequence of a modified truncated bTS02618A gene, and the translated amino acid sequence of the BTS02618A toxin.
SEQ ID. No. 7--The translated amino acid sequence of the modified bTS02618A toxin gene. Although only the toxin part is shown here, the full length protein is 100% identical in amino acid sequence to the BTS02618A protein (SEQ ID. No.5).
SEQ ID. No. 8--The nucleotide sequence of the modified bTS02618Aa toxin gene, and the translated amino acid sequence of the BTS02618Aa toxin. Besides N- and C-terminal amino acid deletions and the addition of an Alanine codon after the N-terminal Methionine codon, the BTS02618Aa toxin only differs from the BTS02618A toxin in amino acid number 123 (Arg codon has been changed into a Lys codon).
SEQ ID. No. 9--The amino acid sequence of the BTS02618Aa toxin. The BTS02618Aa protoxin is 100% identical to the BTS02618A protoxin in its part C-terminal from the toxin fragment.
Unless otherwise stated in the Examples, all procedures for making and manipulating recombinant DNA are carried out by the standardized procedures described in Sambrook et al., Molecular Cloning--A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, N.Y. (1989).
EXAMPLE 1
Characterization of the BTS02617A, BTS02618A, BTS02654B and BTS02652E Strains.
The BTS02617A, the BTS02618A and the BTS02654B strain were isolated from grain dust sampled in Cadlan, province of Bicol, The Philippines and were deposited at the BCCM-LMG on Jul. 2, 1992 under accession Nos. LMG P-12592, LMG P-12593 and LMG P-12594, respectively. Strain BTS02652E was also isolated from Philippine grain dust, and was deposited at the BCCM-LMG on Mar., 1, 1993 under accession No. LMG P-13493.
Each strain can be cultivated on conventional standard media, preferably T.sub.3 medium (tryptone 3 g/l, tryptose 2 g/l, yeast extract 1.5 g/l, 5 mg MnCl.sub.2, 0.05 M Na.sub.2 PO.sub.4, pH 6.8 and 1.5% agar), preferably at 28.degree. C. For long term storage, it is preferred to mix an equal volume of a spore-crystal suspension with an equal volume of 50% glycerol and store this at -70.degree. C. or lyophilize a spore-crystal suspension. For sporulation, growth on T.sub.3 medium is preferred for 48 hours at 28.degree. C., followed by storage at 4.degree. C. During its vegetative phase, each of the strains can also grow under facultative anaerobic conditions, but sporulation only occurs under aerobic conditions.
Sterilization of each strain occurs by autoclave treatment at 120.degree. C. (1 bar pressure) for 20 minutes. Such treatment totally inactivates the spores and the BTS02617A, BTS02618A, BTS02654B, and BTS02652E protoxins. UV radiation (254 nm) also inactivates the spores.
After cultivating on Nutrient Agar ("NA", Difco Laboratories, Detroit, Mich., USA) for one day, colonies of each of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains form opaque white colonies with irregular edges. Cells of each strain (Gram positive rods of 1.7-2.4.times.5.6-7.7 .mu.m) sporulate after 48 hrs cultivation at 28.degree. C. on T.sub.3 agar. The crystal proteins produced during sporulation are packaged in crystals of the BTS02617A, BTS02618A, BTS02654B, and BTS02652E strains. Quite remarkably, the crystal remains attached to the spore after sporulation.
The Bt serotype of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains was determined to be serotype tolworthi H9 of all these strains which was determined by conventional serotyping methods as conducted by the WHO Collaborating Center for Entomopathogenic Bacillus.
EXAMPLE 2
Insecticidal Activity of the BTS02617A, BTS02618A, BTS02654B and BTS02652E Strains, the BTS02618A Protoxin and the BTS02618Aa and BTS02618Ab Toxins or Protoxins Against Noctuidae ssp., Gelechiidae spp., Yponomeutidae spp. and Pyralidae spp.
Toxicity assays were performed on neonate larvae (for Plutella xylostella, third instar larvae were used) fed on an artificial diet layered with spore-crystal mixtures from one of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains or the BTS02618A protoxin or toxin or the BTS02618Aa and BTS02618Ab toxins or protoxins. The artificial diet was dispensed in wells of Costar 24-well plates. Formaldehyde was omitted from the diet. 50 .mu.l of a sample dilution was applied on the surface of the diet and dried in a laminar air flow. For LC.sub.50 assays, the dilutions were made in a PBS-BSA buffer, and five dilutions were applied. Two larvae were placed in each well and 24 larvae were used per sample dilution. Dead and living M. brassica, S. fruagiperda, H. virescens, O. nubilalis, Plutella xylostella and S. exigua larvae were counted on the fifth day, and dead and living A. ipsilon, A. segetum and S. littoralis larvae were counted on the sixth day. The LC.sub.50 and LC.sub.95 values (the concentrations required to kill respectively 50% or 95% of the insects tested, expressed in number of spore-crystals/cm.sup.2 or ng (pro) toxin/cm.sup.2) were calculated using Probit-analysis (Finney, 1971), and the results are set forth below.
The potato moth, Phthorimaea operculella, was tested by the following assay: disks, cut from potato tubers, were dipped in solutions of varying concentrations of BTS0216Aa protein. Three of such disks, which were allowed to dry, were placed in a tray with 20 Phthorimaea larvae. Mortality was recorded after 4 to 5 days for each concentration applied.
______________________________________Spodoptera littoralis Experiment/Strain LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________Experiment 1 BTS02618A 2.4 7.7 1.5-3.4 3.2 HD127.sup.c 2.5 168 1.2-7.4 1.0 Experiment 2 BTS02618A 1.1 4 0.8-1.6 3.0 HD127 21.2 133.7 14.4-31.9 2.0______________________________________ .sup.a 10.sup.5 sporecrystals per cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values .sup.c from the Howard Dulmage collection, housed at the Northern Region Research Center, 1815 North University, Peoria, Ill, USA. The curator is Dr. L. Nakamura.
Experiments with purified BTS02618A protoxin also show a significant toxicity of this protoxin against S. littoralis larvae.
______________________________________Spodoptera exigua 1. Crystal/spore mixtures Experiment/Strain LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________Experiment 1 BTS02618A 1.4 7.9 0.48-3.9 2.2 HD127 8.2 163.5 5.1-15.7 1.3 Experiment 2 BTS02618A 1.2 3.56 0.91-1.57 3.5 BTS02617A 0.79 2.12 0.61-1.03 3.81 HD127 3.5 44.2 1.36-11.5* 1.5 Florbac 4.1 53.9 1.5-17.0* 1.47 BTS00170U.sup.c 5.1 46.5 1.83-24.4* 1.71 Experiment 3 Javelin.sup.d 23.12 195.7 14.6-56.7 1.77 Experiment 4 BTS02618A 1.07 2.91 0.83-1.39 3.8 BTS02617A 0.87 4.7 0.59-1.21 2.22 HD127 4.7 56.9 1.85-18.7* 1.52 Florbac.sup.e 2.53 48.1 0.79-6.71* 1.29 BTS00170U 1.94 56.3 0.55-5.4* 1.12______________________________________ .sup.a 10.sup.5 sporecrystals per cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values, values marked with * are 90% fiducial limits of LC.sub.50 values .sup.c PCT patent publication WO 90/06999 .sup.d strain isolated from Javelin .RTM. (Sandoz, Lichtstrasse, Basel, Switzerland) .sup.e strain from Florbac .RTM. (Novo Nordisk, Novo Alle, Bagsv.ae butted.rd, Denmark)
______________________________________2. Toxin/protoxin assays. ICP LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________BTS02618A Protoxin 26.6 100.6 20.9-33.9 2.8 CryIC Toxin 68.9 313.2 50.5-94.1 2.5 CryID Toxin 118.6 870.6 82.7-170.0 1.9______________________________________ .sup.a ng/cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values
Mamestra brassica
______________________________________1. Crystal/spore mixtures. Experiment/Strain LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________HD127 37.8 297.6 17.8-91.1 1.8 BTS02618A 8.6 59.6 6.0-12.2 1.9 BTS02617A 5.2 25.8 3.7-7.1 2.4 BTS02652E 12.9 44.2 9.7-17.2 3.0 BTS02654B 14.2 60.5 10.8-19.9 2.6______________________________________ .sup.a 10.sup.5 sporecrystals per cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values
______________________________________2. Protoxin assays. ICP LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________BTS02618A Protoxin 25.3 125.1 19.3-33.2 2.4 CryIC Protoxin 22.0 62.9 16.3-29.6 3.6 CryIA (b) Protoxin 162.4 7169 93.2-283.1 1.0______________________________________ .sup.a ng/cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values
Agrotis ipsilon
______________________________________1. Crystal/spore mixtures. Strain mortality.sup.a genes.sup.b______________________________________Btgall..sup.c 1/20 cryIF, cryIG, cryII, 81k HD127.sup.d 2/20 cryIAa, cryIAb, cryIC, cryID, cryII, 81k BTS02618A 16/20.sup.e cryIAb, cryII, bTS02618A Buffer 1/20 none______________________________________ .sup.a number of 1st instar larvae killed after 6 days (10.sup.7 sporecrystals per cm.sup.2) .sup.b genes known to be present in these strains .sup.c Btgall. as described by Smulevitch et al (1991) .sup.d HD127 is available at the Howard Dulmage Collection (NRRC, see above) .sup.e surviving larvae show severe growthinhibition
______________________________________STRAIN LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________BTS02618A 84.4 207.9 65.9-109.6 4.2 HD127 >250 BTS02617A 53.4 261.0 27.7-112.3 2.4______________________________________ .sup.a 10.sup.6 spores/cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values
______________________________________2. Toxin/protoxin assay. ICP LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________CryIAc Toxin >1350 BTS02618A Protoxin 212.2 1973 168.1-267.9 1.7______________________________________ .sup.a ng/cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values
Since MacIntosh et al. (1990) described some activity of the CryIAc toxin towards A. ipsilon, purified CryIAc toxin was tested on this insect for comparison but did not cause any significant mortality of A. ipsilon.
Heliothis virescens
______________________________________1. Crystal/spore mixture. Experiment/Strain LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________BTS02617A 1.69 14.99 0.67-2.89 1.73 BTS02618A 2.71 25.4 0.88-6.99 1.69 BTS00170U.sup.c 15.1 398.7 8.3-41.2 1.15 Dipel.sup.d 2.99 14.11 1.25-7.76 2.45______________________________________ .sup.a 10.sup.3 sporecrystals per cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values .sup.c PCT patent publication WO 90/06999 .sup.d strain isolated from Dipel .TM. (Abbott Laboratories, North Chicago, Ill., USA)
______________________________________2. Toxin/protoxin assay. ICP LC.sub.50.sup.a FL.sub.min-max.sup.b LC.sub.95.sup.a Slope______________________________________BTS02618A Protoxin 31.6 20-50 182.7 2.1 CryIAb Toxin 7.2 4.9-10.5 169.1 1.2______________________________________ .sup.a ng/cm2 .sup.b 95% fiducial limits of LC.sub.50 values
Ostrinia nubilalis
______________________________________1. Crystal/spore mixtures. Experiment/Strain LC.sub.50.sup.a LC.sub.95.sup.a FL.sub.min-max.sup.b Slope______________________________________BTS02617A 4.92 12.49 2.45-6.81 4.0 BTS02618A 6.17 39.7 2.93-9.74 2.0 Dipel.sup.c >30______________________________________ .sup.a 10.sup.5 sporecrystals per cm.sup.2 .sup.b 95% fiducial limits of LC.sub.50 values .sup.c strain isolated from Dipel .TM. (Abbott Laboratories)
______________________________________2. Purified protoxin assay ICP 100% Mortality.sup.a______________________________________CryIAb Toxin 1350 CryIB Toxin 1350 BTS02618A Protoxin 100______________________________________ .sup.a concentration at which 100% mortality was observed (in ng/cm.sup.2
The purified BTS02618A protoxin also showed a significant toxicity to Ostrinia nubilalis larvae, as compared with the CryI toxins that are most active against Ostrinia.
Plutella xylostella
Plutella xylostella larvae also showed significant mortality after application of purified BTS02618A toxin to their artificial diet in several experiments.
Spodoptera frugiperda
Crystal/spore mixtures of a bTS02618A gene-transformed crystal-minus Bt strain (Mahillon et al., 1989) were also found to significantly inhibit larval growth of S. frugiperda larvae in insect feeding trials.
Agrotis segetum
The cutworm Agrotis segetum was also found to be susceptible to the BTS02618Aa toxin. This variant of the BTS02618A protein killed 50% of the Agrotis larvae at a concentration of BTS02618Aa toxin of 980 ng/cm.sup.2 (=LC.sub.50). In comparative assays, all other CryI toxins tested (CryIAb, CryIAc, CryIAa, CryIB, CryIC, CryID, CryIE (Hofte & Whiteley 1989; EP 358 557)) were found to have an LC.sub.50 of more than 15.000 ng/cm.sup.2 for this insect. Agrotis segetum is an important pest insect on various crops.
Phthorimaea operculella
Also the potato tubermoth, Phthorimaea operculella, was found to be susceptible to the BTS02618Aa toxin. Larvae which ingested the BTS02618A toxin showed a significantly higher mortality rate than control larvae.
Furthermore, the BTS02618Aa toxin was tested on several insects and was found to have substantially the same insecticidal activity as the BTS02618A protein. Indeed, bio-assays were conducted with Heliothis virescens, Mamestra brassicae, Ostrinia nubilalis, Spodoptera exigua and Spodoptera littoralis, and these showed only minor differences in LC.sub.50 values when compared to the BTS02618A protein. This shows that the new BTS02618Aa toxin does not differ substantially in insecticidal activity from the native form.
Also, an Alanine mutant of the BTS02618A toxin and protoxin, the BTS02618Ab toxin and the BTS02618Ab protoxin, were tested on Ostrinia nubilalis and were found to be substantially as toxic as the BTS02618Aa toxin or protoxin.
At the same time, the BTS02618Aa toxin was found to be non-toxic to the tested Coleopteran insects: Leptinotarsa decemlineata and Diabrotica undecimpunctata howardi were not affected by the BTS02618Aa toxin. These insects were tested in diet application assays well known in the art. See, for example, Rupar et al., 1991.
In conclusion, the strains of this invention and the BTS02618A protein of this invention and its variants have a strong insecticidal activity against a broad range of insects that are not susceptible to any single currently available Bt protein and have an activity against at least three Spodoptera spp. and against other Noctuidae, such as A. ipsilon, A. segetum, M. brassica and H. virescens, as well as against Pyralidae, such as O. nubilalis, Gelechiidae such as P. operculella and Yponomeutidae such as Plutella xylostella. These results are summarized and compared with results for other Cryl genes (Van Frankenhuyzen, 1993) in Table 1 which shows the unique range of insects susceptible to the BTS02618A protein.
The same spectrum applies for the BTS02618Aa and BTS02618Ab toxins. So these new toxins can also be used for combatting insects, and they have the added advantage that they are more stable, due to their lower susceptibility to protease activity, since almost no about 55 kD protein is formed.
EXAMPLE 3
Identification of the bTS02618A Gene
The bTS02618A gene was identified in the BTS02618A strain by Southern blot analysis (FIG. 1) of AluI digested total DNA of the strain using, as a DNA probe, the DNA sequence of the cryIG gene (Gleave et al., 1992) of SEQ ID No. 1 and using standard hybridization conditions. Partial DNA sequences of the bTS02618A gene, showing its 5' and 3' end portions, are shown in SEQ ID Nos. 2 and 3, respectively, and the full DNA sequence of the bTS02618A gene and the full amino acid sequence of the BTS02618A protein are shown in SEQ ID No. 4.
The partial sequences of SEQ ID Nos. 2 and 3 allow the bTS02618A gene to be recognized in the BTS02617A, BTS02654B and BTS02652E strains and allow the construction of probes to identify and isolate the full gene sequence in these and other Bt strains. The translation initiation codon of the bTS02618A gene is identified at nucleotide position 195-197 in SEQ ID No. 2, corresponding to nucleotide position 668-670 in SEQ ID No.4. The translation stop codon is identified at nucleotide position 1146-1148 in SEQ ID No. 3, corresponding to nucleotide position 4139-4141 in SEQ ID No. 4.
The bTS02618A gene was also identified in the BTS02617A, BTS02654B and BTS02652E strains by using the DNA sequence of SEQ ID No. 1 as a probe, as well as other DNA probes of conserved DNA fragments in cryI genes.
The full length bTS02618A gene was found to encode a 129.9 kD protoxin. A comparison of the amino acid sequence with other known CryI proteins showed that the C-terminal part (C-terminal of conserved sequence block 5) was homologous with CryIG (88%). The best homology for the N-terminal part (the toxin) was found with the CryIB toxin, but this was found to be less than 50% (homology is expressed as the number of perfect matches divided by the number of amino acids of the longest fragment).
The smallest insecticidal protein is believed to be a 69 kD (615 amino acids) protein stretching from amino acid number 44 to amino acid number 658 in SEQ ID No. 4. A smaller tryptic fragment of 55 kD (494 amino acids), stretching from amino acid number 165 to amino acid number 658 in SEQ ID No. 4, still has insecticidal activity towards S. exigua, but this activity is significantly reduced. Thus, a truncated bTS02618A gene or an equivalent truncated gene preferably encodes the 69 kD protein of the BTS02618A protdxin of SEQ ID No.4 as described above.
EXAMPLE 4
Cloning and Expression of the bTS02618A Gene
In order to isolate the bTS02618A gene, total DNA from the BTS02618A strain was prepared and partially digested with Sau3A. The digested DNA was size fractionated on a sucrose gradient and fragments ranging from 7 Kb to 10 Kb were ligated to the BamH1-digested and BAP-treated cloning vector pUC19 (Yannisch-Perron et al., 1985). Recombinant E.coli clones containing the vector were then screened with the cryIG DNA probe of SEQ ID No. 1 which is described in Example 3, to identify clones containing the bTS02618A gene.
The so-identified DNA fragments were then sequenced according to Maxam and Gilbert (1980). Partial sequences of the bTS02618A gene are shown in SEQ ID Nos. 2 and 3, and a full sequence of the bTS02618A gene and the BTS02618A protein is shown in SEQ ID No. 4. Based on the DNA sequence analysis, the gene is cut with appropriate restriction enzymes to give the truncated bTS02618A gene encoding the BTS02618A toxin. Expression of the gene in E. coli was induced using standard procedures (Sambrook et al., 1989, supra).
The bTS02618A gene was also introduced by routine procedures into a crystal-minus Bt 1715 berliner strain and a Bt HD-1 kurstaki strain (the production strain of Dipel.TM. (Abbott Laboratories)) under the control of its own bacterial promoter, using an appropriate shuttle vector (Mahillon et al., 1988).
Spore-crystal mixtures of 2 transformants of Bt strain kurstaki HD-1 (containing the bTS02618A gene), the parental Bt kustaki HD-1 strain, the wild-type BTS02618A strain, the Bt 1715 berliner crystal-minus strain and one transformant of Bt 1715 berliner crystal-minus (containing the bTS02618A gene) were bioassayed on beet armyworm (Spodoptera exigua). Bioassays were performed as described in Example 2. The transformed Bt 1715 berliner crystal-minus (containing bTS02618A) was highly toxic to S. exigua (100% mortality at 4.times.10.sup.4 spore-crystals per square cm of diet agar) while the Bt 1715 berliner crystal-minus was not toxic. The Bt kurstaki HD-1 (bTS02618A) transformants were (on average) 22 times (LC.sub.50 -level) to 76 times (LC.sub.95 -level) as toxic as the parental HD-1 (Table 2).
Similarly, the BTS02618Aa protein or other variants of the BTS02618A protein can be transferred to and expressed in a Bt strain by any method available in the art (Baum et al., 1991; Gamel & Piot, 1992; Lecadet et al., 1992), provided the vector used is compatible with the Bt host strain, and is stably maintained in the bacterial host. It is known that plasmid vectors having replicon homology with the host strain are not suitable vectors (Gamel & Piot, 1992).
EXAMPLE 5
Insertion of the bTS02618A Gene and the Truncated bTS02618A Gene in E. coli and Insertion of the Truncated bTS02618A Gene in Plants.
In order to express the bTS02618A gene and the truncated bTS02618A gene of Example 4 in E. coli and in plants, different gene cassettes are made in E. coli according to the procedure described in EPA 86/300291.1 and EPA 88/402115.5.
To allow significant expression in plants, cassettes containing a) the truncated gene or b) a hybrid gene that is a fusion of i) the truncated gene and ii) the neo gene are each: inserted between the T-DNA border sequences of intermediate plant expression vectors as described in EPA 86/300291.1; fused to transcript formation and polyadenylation signals in the plant expression vectors; placed under the control of the constitutive promoter from cauliflower mosaic virus driving the 35S3 transcript (Hull and Howell, 1987) or the 2' promoter from the TR-DNA of the octopine Ti-plasmid (Velten et al., 1984); and fused to 3' end transcript formation and polyadenylation signals of the octopine synthase gene (Gielen et al., 1984).
Using standard procedures (Deblaere et al., 1985), the intermediate plant expression vectors, containing the truncated bTS02618A gene, are transferred into the Agrobacterium strain C58C1Rif.sup.R (U.S. patent application Ser. No. 821,582; EPA 86/300,291.1) carrying the disarmed Ti-plasmid pGV2260 (Vaeck et al., 1987). Selection for spectinomycin resistance yields cointegrated plasmids, consisting of pGV2260 and the respective intermediate plant expression vectors. Each of these recombinant Agrobacterium strains is then used to transform different cotton plants so that the truncated bTS02618A gene is contained in, and expressed by, different plant cells.
EXAMPLE 6
Expression of the Truncated bTS02618A Gene in Plants.
The insecticidal activity against Lepidoptera of the expression products of the truncated bTS02618A gene in leaves of transformed plants, generated from the transformed plant cells of Example 5, is evaluated by recording the growth rate and mortality of Agrotis and Spodoptera spp. larvae fed on these leaves. These results are compared with the growth rate of larvae fed leaves from untransformed plants. Toxicity assays against Agrotis and Spodoptera spp. are performed as described in EP 0,358,557, U.S. patent application Ser. No. 821,582 and EPA 86/300,291.1.
A significantly higher mortality rate is obtained among larvae fed on leaves of transformed plants containing the truncated bTS02618A gene and the truncated bTS02618A-neo hybrid gene than among larvae fed the leaves of untransformed plants. The transformed plants are also found to resist Ostrinia nubilalis. Mamestra brassica, Heliothis virescens and Plutella xylostella attack by their expression of the BTS02618A protein.
Needless to say, this invention is not limited to the BTS02617A strain (BCCM-LMG P-12592), the BTS02618A strain (BCCM-LMG P-12593), the BTS02654B strain (BCCM-LMG P-12594) and the BTS02652E (BCCM-LMG P-13493) strain. Rather, the invention also includes any mutant or variant of the BTS02617A, BTS02618A, BTS02654B, and BTS02652E strain which produces crystals, crystal proteins, protoxin or toxin having substantially the same properties, particularly anti-Lepidoptera properties, quite particularly anti-Noctuidae, anti-Yponomeutidae, anti-Gelechiidae and anti-Pyralidae properties, especially anti-Spodoptera, anti-Plutella, anti-Ostrinia, anti-Mamestra; anti-Heliothis, anti-Phthorimaea and anti-Agrotis properties, as the respective BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals or crystal proteins, or the BTS02618A protoxin or toxin. This invention also includes the bTS02618A gene and any insecticidally effective parts thereof, like the truncated bTS02618A gene. In this regard, the term "bTS02618A gene" as used herein means the gene isolated from the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain and hybridizing to the nucleotide sequence of SEQ ID No. 1 and any equivalent gene encoding a protoxin having substantially the same amino acid sequence and insecticidal activity as the BTS02618A protoxin and preferably containing the partial nucleotide sequences shown in SEQ ID Nos. 2 and 3, or the full sequence shown in SEQ ID No. 4.
This invention also is not limited to cotton plants transformed with the truncated bTS02618A gene. It includes any plant, such as tomato, tobacco, rapeseed, alfalfa, sunflower, lettuce, potato, corn, rice, soybean, Brassica species, sugar beet and other legumes and vegetables, transformed with an insecticidally effective part of the bTS02618A gene or an equivalent gene such as the bTS02618Aa gene.
Methods for transforming corn cells and regenerating transgenic plants have been described (D'Halluin et al., 1992; From et al., 1990; Gould et al., 1991; Koziel et al., 1993; Omirulleh et al.,1993; Spencer et al., 1992; Klein et al., 1992; Walters et al., 1992). Vectors for transforming corn cells contain chimeric genes encoding a bTS02618A protein or variant thereof (e.g., BTS02618Aa), comprising suitable promoters such as derivatives of the 35S promoter or a promoter from a maize gene, preferably a constitutively expressed maize gene; and suitable 3' end formation sequences such as those of natural maize genes or those derived from the 35S, gene7 or octopine synthase genes (See, Detailed Description and Mogen et al 1990; Wu et al 1993). Increased expression may be obtained when an intron is introduced in the chimeric gene construct (e.g. a natural maize gene intron such as the AdhI intron (see Callis et tl., 1987; Maas et al., 1991)), provided the intron is correctly spliced in the corn cells. Enhancer elements can also be provided in the promoter sequence (e.g., Omirulleh et al., 1993). Transgenic corn plants are grown on selective media by inclusion of well known selectable marker genes such as herbicide resistance genes or antibiotic resistance genes as chimeric genes in the transforming DNA. Transgenic corn plants are selected for their transformed phenotype by means of bio-assays on Ostrinia nubilalis larvae. These larvae quickly stop feeding on the bTS02618Aa-transformed corn and cause no major damage to the corn plants.
This invention is not limited to the use of Agrobacterium tumefaciens Ti-plasmids for transforming plant cells with an insecticidally effective bTS02618A gene part. Other known techniques for plant cell transformations, such as by means of liposomes, by electroporation or by vector systems based on plant viruses or pollen, can be used for transforming monocotyledons and dicotyledons with such a gene part.
Furthermore, DNA sequences other than those present naturally in the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains and encoding the BTS02618A protoxin and toxin can be used for transforming plants and bacteria. In this regard, the natural DNA sequence of these genes can be modified by: 1) replacing some codons with others that code either for the same or different, preferably the same, amino acids; 2) deleting or adding some codons; and/or 3) reciprocal recombination as described by Ge et al. (1991); provided that such modifications do not substantially alter the properties, particularly the insecticidal properties, especially anti-lepidoptera properties, of the encoded, insecticidally effective portions of the BTS02618A protoxin (e.g., toxin). For example, an artificial bTS02618A gene or gene part of this invention, as described above, having a modified codon usage, could be used in certain circumstances instead of a natural insecticidally effective bTS02618A gene part in a bTS02618A chimeric gene of this invention for transforming plants.
Also, other DNA recombinants containing all or part of the bTS02618A gene in association with other foreign DNA, particularly the DNA of vectors suitable for transforming plants and microorganisms other than E. coli, are encompassed by this invention. In this regard, this invention is not limited to the specific plasmids containing the bTS02618A gene, or parts thereof, that were heretofore described, but rather, this invention encompasses any DNA recombinants containing DNA sequences that are their equivalent. Further, the invention relates to all DNA recombinants that include all or part of the bTS02618A gene and that are suitable for transforming microorganisms (e.g., plant associated bacteria such as other Bacillus thuringiensis strains, Bacillus subtilis, Pseudomonas, and Xanthomonas or yeasts such as Streptomyces cerevisiae) under conditions which enable all or part of the gene to be expressed and to be recoverable from said microorganisms or to be transferred to a plant cell.
EXAMPLE 7
Construction of an Artificial bTS02618A Gene, Encoding the BTS02618A Toxin.
Based on the amino acid sequence of the BTS02618A toxin, an artificial DNA sequence encoding substantially the same protein was designed. At first, a DNA sequence for the artificial BTS02618A toxin gene was designed, using corn-preferred codons (Murray et al., 1989). During the design of the artificial gene, TA and CG doublets at codon position 2 and 3 were avoided. The artificial gene was also corrected for local high GC-content (GC stretches of more than 5 bp were avoided). Also, suitable restriction sites were incorporated throughout the gene. So the final gene did not always use the most preferred corn codons. The artificial gene was synthesized on an Applied Biosystems 380B DNA synthesizer using standard cyanoethyl phoshoramidite chemistry. The oligonucleotides were gel purified and assembled into full length fragments using known techniques. See also, the method of Davies et al. (1991). The artificial toxin gene also carried a deletion of codon 2 to codon 43 of the BTS02618A coding sequence, and codon 44 is preceded by an ATG (start) and GCT (Ala) codon, to create a suitable translation initiation context as proposed by Joshi (1987). The C-terminal end of the artificial bTS02618A toxin gene contained some codons in addition to the determined minimal toxic gene fragment, because of the presence of a suitable maize translational stop context in this further C-terminal part.
The chimeric gene construct containing the artificial bTS02618A gene is introduced into corn cells as described above. Most of the corn plants that are regenerated from these cells and that are identified as transformed are insecticidal because of expression of the bTS02618A gene. Northern and Southern analysis of some selected transgenic corn plants show the stable integration of the transgene and the presence of readily detectable levels of BTS02618A mRNA expression. These plants also show good insect control, and the degree of insecticidal activity is linked to the quantity of the Bt protein present in the tissues, as determined by ELISA.
It is believed that any method known for transforming corn so that it expresses the BTS02618A protein at sufficient levels can be used to develop insect-resistant corn. In this regard, it may preferred to express at least 2 non-competitively binding Bt proteins, such as CryIAb and BTS02618A in one plant to prevent the development of insect resistance.
EXAMPLE 8
Design of a new variant of the BTS02618A protein
To prevent further proteolytic cleavage of the BTS02618A about 69 kD toxin, a new variant of the BTS02618A protein was made. In one new variant of this protein, the Arg at amino acid position 123 in SEQ ID. No. 6 was replaced with a Lys (the BTS02618Aa protein). In another variant, the Arg at position 123 in SEQ ID. No. 6 was replaced by Ala (the BTS02618Ab protein). These proteins were found to be more resistant to protease treatment (i.e., the proteins yielded no about 55 kD protein) and insect assays confirmed that their toxicity was retained. The amino acid sequence of the BTS02618Aa toxin is shown in SEQ ID. No. 9.
Other examples having the amino acid sequence changes around the protease cleavage site are made and are also found to have more resistance to protease activity, while retaining their insecticidal activity.
Also, an artificial gene encoding a BTS02618Aa toxin fragment was designed. Besides N- and C-terminal deletions and the addition of a Met and an Ala codon at positions 1 and 2 (as for the DNA of SEQ ID. No. 6), this gene differs in one codon from the synthetic gene of SEQ ID. No. 6: the Arg codon (CGC) was replaced by a Lys (AAG) codon. The other nucleotides were the same as for the artificial bTS02618A gene of SEQ ID. No. 6. Such a modification was made by PCR-mediated mutagenesis, starting from the bTS02618A artificial gene, using the appropriate primers. Essentially, a PCR-generated and restriction enzyme-digested fragment having the mutated codon at position 123 was inserted into the corresponding site of the digested bTS02618A gene of SEQ ID. No. 6 to give the DNA of SEQ ID. No. 8.
Corn plants are also transformed with the bTS02618Aa gene, following the procedures described above. Selected transformed corn plants expressing the bTS02618Aa gene are insecticidal for Ostrinia nubilalis larvae. It is believed that any method described in the Detailed Description can be used for expressing the above Bt genes in transformed corn plants, either alone or in combination with other Bt genes. A particularly preferred candidate is a DNA sequence encoding the CryIAb protein. Following routine procedures, appropriate lines having desired qualities can be selected between the obtained regenerants.
EXAMPLE 9
Binding of BTS02618A Toxin to Insect Gut Membranes.
The BTS02618Aa toxin was found not to inhibit binding of the CryIAb toxin to midgut membrane vesicles of Ostrinia nubilalis.
In this experimental setup, the proteins used were: the Lysine mutant of BTS02618A (BTS02618Aa), non-biotinylated; CryIAb, non-biotinylated; and biotinylated (and biologically active) CryIAb . All ICPs used were trypsin resistant toxins. The following combinations were tested:
Biotinylated CryIAb.times.no competitor;
Biotinylated CryIAb.times.1000-fold excess of CryIAb toxin;
Biotinylated CryIAb.times.1000-fold excess of BTS02618Aa toxin.
For these experiments, 10 ng biotinylated CryIAb, with or without an excess of an unlabeled crystal protein, was mixed with 10 microgram brush border membrane vesicles derived from larval midguts of Ostrinia nubilalis. These vesicles were prepared according to the method of Wolfersberger et al. (1987). These mixtures were made in PBS (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, pH 7.4) containing 0.1% BSA. The mixtures were incubated during 1 hour at room temperature and were then centrifuged for 10 minutes. After washing the pellet in 500 microliter PBS-0.1% BSA, the pellet was centrifuged again and dissolved in sample buffer for SDS-PAGE. The samples were run on a 10% polyacrylamide gel. The gel was blotted at room temperature during two hours on a semi-dry blotting apparatus (LKB Novablot; the blotting buffer used was: 39 mM glycine, 48 mM Tris, 0.0375% (w/v) sodium dodecyl sulphate, 20% methanol). The membrane was blocked for at least 2 hours in TBS (10 mM Tris, 150 mM NaCl, pH 7,6) with 0.1% BSA, followed by incubation with a streptavidin-peroxidase conjugate, diluted 1/1000 in TBS-0.1% BSA for 45 minutes. The membrane was washed for 4 times 5 minutes and once for 15 minutes with TBS-0.2% Tween 20. Between the wash steps, the blot was thoroughly washed under the tap. The membrane was incubated in ECL reagent (Amersham) for 1 minute and was then exposed to X-ray film.
For the biotinylated CryIAb, a band corresponding to bound toxin was observed on the X-ray film. When biotinylated CryIAb toxin was incubated in the presence of excess CryIAb toxin, no band was observed on the film: as expected, the excess unlabeled toxin had displaced the labeled toxin. For the biotinylated CryIAb toxin in the presence of an excess of the BTS02618Aa toxin, a band corresponding to bound biotinylated CryIAb was seen: unlabeled BTS02618Aa toxin was apparently unable to compete with CryIAb for binding to the vesicles, indicating that BTS02618Aa binds to another receptor other than CryIAb in Ostrinia nubilalis.
In a similar setup, unlabeled CryIAb toxin did not compete for the receptors of biotin-labeled and biologically active BTS02618Aa toxin, while such competition was observed with an excess of unlabeled BTS02618Aa toxin.
Thus, the BTS02618A protein recognizes a different receptor site in Ostrinia midgut membranes, and can be used in a strategy to delay or prevent the development of insect resistance or to combat insects resistant to the CryIAb toxin, e.g., by expressing the CryIAb and the BTS02618A toxin in a plant. Since both toxins are highly active against a group of major insect pests and apparently recognize different receptor molecules, their use in transgenic plants such as corn and vegetables, provides a supplemental advantage. Corn plants can be transformed with the CryIAb and bTS02618Aa gene with any method available in the art, such as crossing plants expressing either toxin, or any of the methods described in EP publication number 408 403.
TABLE 1______________________________________Activity of CryI proteins towards several lepidopteran insect pests: + and - indicates the presence or absence of insecticidal activity, +/- indicates low activity (according to Van Frankenhuyzen (1993)), NA indicates no data available, the protein BTS02618A is abbreviated as 2618A (data of Van Frankenhuyzen (1993) and this invention (for A. ipsilon and 2618A)). 2618A IAb IAc IB IC IF______________________________________S. exigua + +/- - - + + S. littoralis + - - - + NA H. virescens + + + - +/- + A. ipsilon + NA - NA NA NA O. nubilalis + + + NA NA + P. xylostella + + + + + NA M. brassica + + - - + NA______________________________________
TABLE 2______________________________________LC.sub.50 -LC.sub.95 assays with spore-crystal mixtures of recombinant Bt's. Tests were performed as described in the text. Values indicate the number of spore-crystals .times. 10.sup.6 per square cm of dietagar. Strain LC.sub.50 LC.sub.95 Fl95min-max Slope______________________________________Bt kurstaki HD-1 8.9 91.2 3.9-15.4 1.6 .+-. 0.4 HD-1/1 0.4 1.7 0.1-0.6 2.5 .+-. 0.7 (bTS02618A) HD-1/2 0.4 0.93 0.2-0.5 4.3 .+-. 1.2 (bTS02618A) BTS02618A 1.5 4.3 0.9-2.1 3.6 .+-. 0.9______________________________________
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__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 9 - - - - (2) INFORMATION FOR SEQ ID NO: 1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..19 (D) OTHER INFORMATION: - #/function= "for isolating bTS02618A gene from - # its containing strain" /note= - #"the probe is a part of the coding DNA strand of - # the cryIG gene (Smulevitch et al. (1991)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1: - - TTCTGTACTA TTGATTGTA - # - # - # 19 - - - - (2) INFORMATION FOR SEQ ID NO: 2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1561 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis (B) STRAIN: BTS02618A - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..1561 (D) OTHER INFORMATION: - #/note= "contains the translation initiation - #codon of the bTS02618A gene" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: - - AAAAAGAAAT AGGAATAAAT ACTATCCATT TTTTCAAGAA ATATTTTTTT AT -#TAGAAAGG 60 - - AATCTTTCTT ACACGGGAAA ATCCTAAGAT TGAGAGTAAA GATATATATA TA -#TAAATACA 120 - - ATAAAGAGTT TGTCAGGATT TTTGAAAGAT ATGATATGAA CATGCACTAG AT -#TTATAGTA 180 - - TAGGAGGAAA AAGTATGAAT CGAAATAATC AAAATGAATA TGAAATTATT GA -#TGCCCCCC 240 - - ATTGTGGGTG TCCATCAGAT GACGATGTGA GGTATCCTTT GGCAAGTGAC CC -#AAATGCAG 300 - - CGTTACAAAA TATGAACTAT AAAGATTACT TACAAATGAC AGATGAGGAC TA -#CACTGATT 360 - - CTTATATAAA TCCTAGTTTA TCTATTAGTG GTAGAGATGC AGTTCAGACT GC -#GCTTACTG 420 - - TTGTTGGGAG AATACTCGGG GCTTTAGGTG TTCCGTTTTC TGGACAAATA GT -#GAGTTTTT 480 - - ATCAATTCCT TTTAAATACA CTGTGGCCAG TTAATGATAC AGCTATATGG GA -#AGCTTTCA 540 - - TGCGACAGGT GGAGGAACTT GTCAATCAAC AAATAACAGA ATTTGCAAGA AA -#TCAGGCAC 600 - - TTGCAAGATT GCAAGGATTA GGAGACTCTT TTAATGTATA TCAACGTTCC CT -#TCAAAATT 660 - - GGTTGGCTGA TCGAAATGAT ACACGAAATT TAAGTGTTGT TCGTGCTNAA TT -#TATAGCTT 720 - - TAGACCTTGA TTTTGTTAAT GCTATTCCAT TGTTTGCAGT AAATGGACAG CA -#GGTTCCAT 780 - - TACTGTCAGT ATATGCACAA GCTGTGAATT TACATTTGTT ATTATTAAAA GA -#TGCATCTC 840 - - TTTTTGGAGA AGGATGGGGA TTCACACAGG GGGAAATTTC CACATATTAT GA -#CCGTCAAT 900 - - TGGAACTAAC CGCTAAGTAC ACTAATTACT GTGAAACTTG GTATAATACA GG -#TTTAGATC 960 - - GTTTAAGAGG AACAAATACT GAAAGTTGGT TAAGATATCA TCAATTCCGT AG -#AGAAATGA 1020 - - CTTTAGTGGT ATTAGATGTT GTGGCGCTAT TTCCATATTA TGATGTACGA CT -#TTATCCAA 1080 - - CGGGATCAAA CCCACAGCTT ACACGTGAGG TATATACAGA TCCGATTGTA TT -#TAATCCAC 1140 - - CAGCTAATGT TGGACTTTGC CGACGTTGGG GTACTAATCC CTATAATACT TT -#TTCTGAGC 1200 - - TCGAAAATGC CTTCATTCGC CCACCACATC TTTTTGATAG GCTGAATAGC TT -#AACAATCA 1260 - - GCAGTAATCG ATTTCCAGTT TCATCTAATT TTATGGATTA TTGGTCAGGA CA -#TACGTTAC 1320 - - GCCGTAGTTA TCTGAACGAT TCAGCAGTAC AAGAAGATAG TTATGGCCTA AT -#TACAACCA 1380 - - CAAGAGCAAC AATTAATCCC GGAGTTGATG GAACAAACCG CATAGAGTCA AC -#GGCAGTAG 1440 - - ATTTTCGTTC TGCATTGATA GGTATATATG GCGTGAATAG AGCTTCTTTT GT -#CCCAGGAG 1500 - - GCTTGTTTAA TGGTACGACT TCTCCTGCTA ATGGAGGATG TAGAGATCTC TA -#TGATACAA 1560 - - A - # - # - # 1561 - - - - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1554 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis (B) STRAIN: BTS02618A - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1146..1148 (D) OTHER INFORMATION: - #/function= "Presumed translational stop codo - #n of bTS02618A gene" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3: - - AAAATTATCC AACATACATT TATCAAAAAG TAGATGCATC GGTGTTAAAG CC -#TTATACAC 60 - - GCTATAGACT AGATGGATTT GTGAAGNGTA GTCAAGATTT AGAAATTGAT CT -#CATCCACC 120 - - ATCATAAAGT CCATCTTGTA AAAAATGTAC CAGATAATTT AGTATCTGAT AC -#TTACTCAG 180 - - ATGGTTCTTG CAGCGGAATC AACCGTTGTG ATGAACAGCA TCAGGTAGAT AT -#GCAGCTAG 240 - - ATGCGGAGCA TCATCCAATG GATTGCTGTG AAGCGGCTCA AACACATGAG TT -#TTCTTCCT 300 - - ATATTAATAC AGGGGATCTA AATGCAAGTG TAGATCAGGG CATTTGGGTT GT -#ATTAAAAG 360 - - TTCGAACAAC AGATGGGTAT GCGACGTTAG GAAATCTTGA ATTGGTAGAG GT -#TGGGCCAT 420 - - TATCGGGTGA ATCTCTAGAA CGGGAACAAA GAGATAATGC GAAATGGAAT GC -#AGAGCTAG 480 - - GAAGAAAACG TGCAGAAATA GATCGTGTGT ATTTAGCTGC GAAACAAGCA AT -#TAATCATC 540 - - TGTTTGTAGA CTATCAAGAT CAACAATTAA ATCCAGAAAT TGGGCTAGCA GA -#AATTAATG 600 - - AAGCTTCAAA TCTTGTAGAG TCAATTTCGG GTGTATATAG TGATACACTA TT -#ACAGATTC 660 - - CTGGGATTAA CTACGAAATT TACACAGAGT TATCCGATCG CTTACAACAA GC -#ATCGTATC 720 - - TGTATACGTC TAGAAATGCG GTGCAAAATG GAGACTTTAA CAGTGGTCTA GA -#TAGTTGGA 780 - - ATACAACTAT GGATGCATCG GTTCAGCAAG ATGGCAATAT GCATTTCTTA GT -#TCTTTCGC 840 - - ATTGGGATGC ACAAGTTTCC CAACAATTGA GAGTAAATCC GAATTGTAAG TA -#TGTCTTAC 900 - - GTGTGACAGC AAGAAAAGTA GGAGGCGGAG ATGGATACGT CACAATCCGA GA -#TGGCGCTC 960 - - ATCACCAAGA AACTCTTACA TTTAATGCAT GTGACTACGA TGTAAATGGT AC -#GTATGTCA 1020 - - ATGACAATTC GTATATAACA GAAGAAGTGG TATTCTACCC AGAGACAAAA CA -#TATGTGGG 1080 - - TAGAGGTGAG TGAATCCGAA GGTTCATTCT ATATAGACAG TATTGAGTTT AT -#TGAAACAC 1140 - - AAGAGTAGAA GAGGGGGATC CTAACGTATA GCAACTATGA GAGGATACTC CG -#TACAAACA 1200 - - AAGATTAAAA AAAGGTAAAA TGAATAGAAC CCCCTACTGG TAGAAGGACC GA -#TAGGGGGT 1260 - - TCTTACATGA AAAAATGTAG CTGTTTACTA AGGTGTATAA AAAACAGCAT AT -#CTGATAGA 1320 - - AAAAAGTGAG TACCTTATAA AGAAAGAATT CCATTCACAG TTTCGGTATC AT -#ATAAATAA 1380 - - TGATAGGGGT ATCCTTCTTA TTTACATTAT TTTTCGCAAT TATCTCGACG TT -#CTTCTTTC 1440 - - CGCTCACAAT GATGATGATC ATGACAACAA TCGCGTCCAT AGCGAACTCT TT -#CGATATTA 1500 - - ATAATATCTA AACTCGTGTA GCAGTCATTT CCATTTTTTT TGATCCAGTA AA - #TA 1554 - - - - (2) INFORMATION FOR SEQ ID NO: 4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4344 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 668..4141 - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..4344 (D) OTHER INFORMATION: - #/note= "encompasses entire sequence of SEQ - #ID NO (SID) 2: from nt position 474 to 2034 in SID - #4" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..4344 (D) OTHER INFORMATION: - #/note= "also encompasses part of the seque - #nce of SID 3: from nt position 2994 to 4344 in - #SID 4" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..4344 (D) OTHER INFORMATION: - #/note= "SID 3 shows additional nucleotides, - # located 3' from the sequence shown in SID 4 - #(1352-1554 in SID 4)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4: - - GAATTCGAGC TCGGTACCTT TTCAGTGTAT CGTTTCCCTT CCATCAGGTT TT -#CAAATTGA 60 - - AAAGCCGAAT GATTTGAAAC TTGTTTACGA TGTAAGTCAT TTGTCTATGA CG -#AAAGATAC 120 - - GTGTAAAAAA CGTATTGAGA TTGATGAATG TGGACAAGTA GAAATTGACT TA -#CAAGTATT 180 - - AAAGATTAAG GGTGTCCTTT CTTTTATCGG AAATTTCTCT ATTGAACCTA TT -#CTGTGTGA 240 - - AAACATGTAT ACAACGGTTG ATAGAGATCC GTCTATTTCC TTAAGTTTCC AA -#GATACGGT 300 - - ATATGTGGAC CATATTTTAA AATATAGCGT CCAACAACTA CCATATTATG TA -#ATTGATGG 360 - - TGATCATATT CAAGTACGTG ATTTACAAAT CAAACTGATG AAAGAGAATC CG -#CAATCTGC 420 - - TCAAGTATCA GGTTTGTTTT GTTTTGTATA TGAGTAAGAA CCGAAGGTTT GT -#AAAAAAGA 480 - - AATAGGAATA AATACTATCC ATTTTTTCAA GAAATATTTT TTTATTAGAA AG -#GAATCTTT 540 - - CTTACACGGG AAAATCCTAA GATTGAGAGT AAAGATATAT ATATATAAAT AC -#AATAAAGA 600 - - GTTTGTCAGG ATTTTTGAAA GATATGATAT GAACATGCAC TAGATTTATA GT -#ATAGGAGG 660 - - AAAAAGT ATG AAT CGA AAT AAT CAA AAT GAA TAT - #GAA ATT ATT GAT GCC 709 Met Asn Arg Asn Asn Gln - #Asn Glu Tyr Glu Ile Ile Asp Ala 1 - # 5 - # 10 - - CCC CAT TGT GGG TGT CCA TCA GAT GAC GAT GT - #G AGG TAT CCT TTG GCA 757 Pro His Cys Gly Cys Pro Ser Asp Asp Asp Va - #l Arg Tyr Pro Leu Ala 15 - # 20 - # 25 - # 30 - - AGT GAC CCA AAT GCA GCG TTA CAA AAT ATG AA - #C TAT AAA GAT TAC TTA 805 Ser Asp Pro Asn Ala Ala Leu Gln Asn Met As - #n Tyr Lys Asp Tyr Leu 35 - # 40 - # 45 - - CAA ATG ACA GAT GAG GAC TAC ACT GAT TCT TA - #T ATA AAT CCT AGT TTA 853 Gln Met Thr Asp Glu Asp Tyr Thr Asp Ser Ty - #r Ile Asn Pro Ser Leu 50 - # 55 - # 60 - - TCT ATT AGT GGT AGA GAT GCA GTT CAG ACT GC - #G CTT ACT GTT GTT GGG 901 Ser Ile Ser Gly Arg Asp Ala Val Gln Thr Al - #a Leu Thr Val Val Gly 65 - # 70 - # 75 - - AGA ATA CTC GGG GCT TTA GGT GTT CCG TTT TC - #T GGA CAA ATA GTG AGT 949 Arg Ile Leu Gly Ala Leu Gly Val Pro Phe Se - #r Gly Gln Ile Val Ser 80 - # 85 - # 90 - - TTT TAT CAA TTC CTT TTA AAT ACA CTG TGG CC - #A GTT AAT GAT ACA GCT 997 Phe Tyr Gln Phe Leu Leu Asn Thr Leu Trp Pr - #o Val Asn Asp Thr Ala 95 - #100 - #105 - #110 - - ATA TGG GAA GCT TTC ATG CGA CAG GTG GAG GA - #A CTT GTC AAT CAA CAA 1045 Ile Trp Glu Ala Phe Met Arg Gln Val Glu Gl - #u Leu Val Asn Gln Gln 115 - # 120 - # 125 - - ATA ACA GAA TTT GCA AGA AAT CAG GCA CTT GC - #A AGA TTG CAA GGA TTA 1093 Ile Thr Glu Phe Ala Arg Asn Gln Ala Leu Al - #a Arg Leu Gln Gly Leu 130 - # 135 - # 140 - - GGA GAC TCT TTT AAT GTA TAT CAA CGT TCC CT - #T CAA AAT TGG TTG GCT 1141 Gly Asp Ser Phe Asn Val Tyr Gln Arg Ser Le - #u Gln Asn Trp Leu Ala 145 - # 150 - # 155 - - GAT CGA AAT GAT ACA CGA AAT TTA AGT GTT GT - #T CGT GCT CAA TTT ATA 1189 Asp Arg Asn Asp Thr Arg Asn Leu Ser Val Va - #l Arg Ala Gln Phe Ile 160 - # 165 - # 170 - - GCT TTA GAC CTT GAT TTT GTT AAT GCT ATT CC - #A TTG TTT GCA GTA AAT 1237 Ala Leu Asp Leu Asp Phe Val Asn Ala Ile Pr - #o Leu Phe Ala Val Asn 175 1 - #80 1 - #85 1 -#90 - - GGA CAG CAG GTT CCA TTA CTG TCA GTA TAT GC - #A CAA GCT GTG AATTTA 1285 Gly Gln Gln Val Pro Leu Leu Ser Val Tyr Al - #a Gln Ala Val Asn Leu 195 - # 200 - # 205 - - CAT TTG TTA TTA TTA AAA GAT GCA TCT CTT TT - #T GGA GAA GGA TGG GGA 1333 His Leu Leu Leu Leu Lys Asp Ala Ser Leu Ph - #e Gly Glu Gly Trp Gly 210 - # 215 - # 220 - - TTC ACA CAG GGG GAA ATT TCC ACA TAT TAT GA - #C CGT CAA TTG GAA CTA 1381 Phe Thr Gln Gly Glu Ile Ser Thr Tyr Tyr As - #p Arg Gln Leu Glu Leu 225 - # 230 - # 235 - - ACC GCT AAG TAC ACT AAT TAC TGT GAA ACT TG - #G TAT AAT ACA GGT TTA 1429 Thr Ala Lys Tyr Thr Asn Tyr Cys Glu Thr Tr - #p Tyr Asn Thr Gly Leu 240 - # 245 - # 250 - - GAT CGT TTA AGA GGA ACA AAT ACT GAA AGT TG - #G TTA AGA TAT CAT CAA 1477 Asp Arg Leu Arg Gly Thr Asn Thr Glu Ser Tr - #p Leu Arg Tyr His Gln 255 2 - #60 2 - #65 2 -#70 - - TTC CGT AGA GAA ATG ACT TTA GTG GTA TTA GA - #T GTT GTG GCG CTATTT 1525 Phe Arg Arg Glu Met Thr Leu Val Val Leu As - #p Val Val Ala Leu Phe 275 - # 280 - # 285 - - CCA TAT TAT GAT GTA CGA CTT TAT CCA ACG GG - #A TCA AAC CCA CAG CTT 1573 Pro Tyr Tyr Asp Val Arg Leu Tyr Pro Thr Gl - #y Ser Asn Pro Gln Leu 290 - # 295 - # 300 - - ACA CGT GAG GTA TAT ACA GAT CCG ATT GTA TT - #T AAT CCA CCA GCT AAT 1621 Thr Arg Glu Val Tyr Thr Asp Pro Ile Val Ph - #e Asn Pro Pro Ala Asn 305 - # 310 - # 315 - - GTT GGA CTT TGC CGA CGT TGG GGT ACT AAT CC - #C TAT AAT ACT TTT TCT 1669 Val Gly Leu Cys Arg Arg Trp Gly Thr Asn Pr - #o Tyr Asn Thr Phe Ser 320 - # 325 - # 330 - - GAG CTC GAA AAT GCC TTC ATT CGC CCA CCA CA - #T CTT TTT GAT AGG CTG 1717 Glu Leu Glu Asn Ala Phe Ile Arg Pro Pro Hi - #s Leu Phe Asp Arg Leu 335 3 - #40 3 - #45 3 -#50 - - AAT AGC TTA ACA ATC AGC AGT AAT CGA TTT CC - #A GTT TCA TCT AATTTT 1765 Asn Ser Leu Thr Ile Ser Ser Asn Arg Phe Pr - #o Val Ser Ser Asn Phe 355 - # 360 - # 365 - - ATG GAT TAT TGG TCA GGA CAT ACG TTA CGC CG - #T AGT TAT CTG AAC GAT 1813 Met Asp Tyr Trp Ser Gly His Thr Leu Arg Ar - #g Ser Tyr Leu Asn Asp 370 - # 375 - # 380 - - TCA GCA GTA CAA GAA GAT AGT TAT GGC CTA AT - #T ACA ACC ACA AGA GCA 1861 Ser Ala Val Gln Glu Asp Ser Tyr Gly Leu Il - #e Thr Thr Thr Arg Ala 385 - # 390 - # 395 - - ACA ATT AAT CCC GGA GTT GAT GGA ACA AAC CG - #C ATA GAG TCA ACG GCA 1909 Thr Ile Asn Pro Gly Val Asp Gly Thr Asn Ar - #g Ile Glu Ser Thr Ala 400 - # 405 - # 410 - - GTA GAT TTT CGT TCT GCA TTG ATA GGT ATA TA - #T GGC GTG AAT AGA GCT 1957 Val Asp Phe Arg Ser Ala Leu Ile Gly Ile Ty - #r Gly Val Asn Arg Ala 415 4 - #20 4 - #25 4 -#30 - - TCT TTT GTC CCA GGA GGC TTG TTT AAT GGT AC - #G ACT TCT CCT GCTAAT 2005 Ser Phe Val Pro Gly Gly Leu Phe Asn Gly Th - #r Thr Ser Pro Ala Asn 435 - # 440 - # 445 - - GGA GGA TGT AGA GAT CTC TAT GAT ACA AAT GA - #T GAA TTA CCA CCA GAT 2053 Gly Gly Cys Arg Asp Leu Tyr Asp Thr Asn As - #p Glu Leu Pro Pro Asp 450 - # 455 - # 460 - - GAA AGT ACC GGA AGT TCA ACC CAT AGA CTA TC - #T CAT GTT ACC TTT TTT 2101 Glu Ser Thr Gly Ser Ser Thr His Arg Leu Se - #r His Val Thr Phe Phe 465 - # 470 - # 475 - - AGC TTT CAA ACT AAT CAG GCT GGA TCT ATA GC - #T AAT GCA GGA AGT GTA 2149 Ser Phe Gln Thr Asn Gln Ala Gly Ser Ile Al - #a Asn Ala Gly Ser Val 480 - # 485 - # 490 - - CCT ACT TAT GTT TGG ACC CGT CGT GAT GTG GA - #C CTT AAT AAT ACG ATT 2197 Pro Thr Tyr Val Trp Thr Arg Arg Asp Val As - #p Leu Asn Asn Thr Ile 495 5 - #00 5 - #05 5 -#10 - - ACC CCA AAT AGA ATT ACA CAA TTA CCA TTG GT - #A AAG GCA TCT GCACCT 2245 Thr Pro Asn Arg Ile Thr Gln Leu Pro Leu Va - #l Lys Ala Ser Ala Pro 515 - # 520 - # 525 - - GTT TCG GGT ACT ACG GTC TTA AAA GGT CCA GG - #A TTT ACA GGA GGG GGT 2293 Val Ser Gly Thr Thr Val Leu Lys Gly Pro Gl - #y Phe Thr Gly Gly Gly 530 - # 535 - # 540 - - ATA CTC CGA AGA ACA ACT AAT GGC ACA TTT GG - #A ACG TTA AGA GTA ACG 2341 Ile Leu Arg Arg Thr Thr Asn Gly Thr Phe Gl - #y Thr Leu Arg Val Thr 545 - # 550 - # 555 - - GTT AAT TCA CCA TTA ACA CAA CAA TAT CGC CT - #A AGA GTT CGT TTT GCC 2389 Val Asn Ser Pro Leu Thr Gln Gln Tyr Arg Le - #u Arg Val Arg Phe Ala 560 - # 565 - # 570 - - TCA ACA GGA AAT TTC AGT ATA AGG GTA CTC CG - #T GGA GGG GTT TCT ATC 2437 Ser Thr Gly Asn Phe Ser Ile Arg Val Leu Ar - #g Gly Gly Val Ser Ile 575 5 - #80 5 - #85 5 -#90 - - GGT GAT GTT AGA TTA GGG AGC ACA ATG AAC AG - #A GGG CAG GAA CTAACT 2485 Gly Asp Val Arg Leu Gly Ser Thr Met Asn Ar - #g Gly Gln Glu Leu Thr 595 - # 600 - # 605 - - TAC GAA TCC TTT TTC ACA AGA GAG TTT ACT AC - #T ACT GGT CCG TTC AAT 2533 Tyr Glu Ser Phe Phe Thr Arg Glu Phe Thr Th - #r Thr Gly Pro Phe Asn 610 - # 615 - # 620 - - CCG CCT TTT ACA TTT ACA CAA GCT CAA GAG AT - #T CTA ACA GTG AAT GCA 2581 Pro Pro Phe Thr Phe Thr Gln Ala Gln Glu Il - #e Leu Thr Val Asn Ala 625 - # 630 - # 635 - - GAA GGT GTT AGC ACC GGT GGT GAA TAT TAT AT - #A GAT AGA ATT GAA ATT 2629 Glu Gly Val Ser Thr Gly Gly Glu Tyr Tyr Il - #e Asp Arg Ile Glu Ile 640 - # 645 - # 650 - - GTC CCT GTG AAT CCG GCA CGA GAA GCG GAA GA - #G GAT TTA GAA GCG GCG 2677 Val Pro Val Asn Pro Ala Arg Glu Ala Glu Gl - #u Asp Leu Glu Ala Ala 655 6 - #60 6 - #65 6 -#70 - - AAG AAA GCG GTG GCG AGC TTG TTT ACA CGT AC - #A AGG GAC GGA TTACAG 2725 Lys Lys Ala Val Ala Ser Leu Phe Thr Arg Th - #r Arg Asp Gly Leu Gln 675 - # 680 - # 685 - - GTA AAT GTG ACA GAT TAT CAA GTG GAC CAA GC - #G GCA AAT TTA GTG TCA 2773 Val Asn Val Thr Asp Tyr Gln Val Asp Gln Al - #a Ala Asn Leu Val Ser 690 - # 695 - # 700 - - TGC TTA TCC GAT GAA CAA TAT GGG CAT GAC AA - #A AAG ATG TTA TTG GAA 2821 Cys Leu Ser Asp Glu Gln Tyr Gly His Asp Ly - #s Lys Met Leu Leu Glu 705 - # 710 - # 715 - - GCG GTA AGA GCG GCA AAA CGC CTC AGC CGC GA - #A CGC AAC TTA CTT CAA 2869 Ala Val Arg Ala Ala Lys Arg Leu Ser Arg Gl - #u Arg Asn Leu Leu Gln 720 - # 725 - # 730 - - GAT CCA GAT TTT AAT ACA ATC AAT AGT ACA GA - #A GAG AAT GGC TGG AAG 2917 Asp Pro Asp Phe Asn Thr Ile Asn Ser Thr Gl - #u Glu Asn Gly Trp Lys 735 7 - #40 7 - #45 7 -#50 - - GCA AGT AAC GGT GTT ACT ATT AGC GAG GGC GG - #T CCA TTC TTT AAAGGT 2965 Ala Ser Asn Gly Val Thr Ile Ser Glu Gly Gl - #y Pro Phe Phe Lys Gly 755 - # 760 - # 765 - - CGT GCA CTT CAG TTA GCA AGC GCA AGA GAA AA - #T TAT CCA ACA TAC ATT 3013 Arg Ala Leu Gln Leu Ala Ser Ala Arg Glu As - #n Tyr Pro Thr Tyr Ile 770 - # 775 - # 780 - - TAT CAA AAA GTA GAT GCA TCG GTG TTA AAG CC - #T TAT ACA CGC TAT AGA 3061 Tyr Gln Lys Val Asp Ala Ser Val Leu Lys Pr - #o Tyr Thr Arg Tyr Arg 785 - # 790 - # 795 - - CTA GAT GGA TTT GTG AAG AGT AGT CAA GAT TT - #A GAA ATT GAT CTC ATC 3109 Leu Asp Gly Phe Val Lys Ser Ser Gln Asp Le - #u Glu Ile Asp Leu Ile 800 - # 805 - # 810 - - CAC CAT CAT AAA GTC CAT CTT GTA AAA AAT GT - #A CCA GAT AAT TTA GTA 3157 His His His Lys Val His Leu Val Lys Asn Va - #l Pro Asp Asn Leu Val 815 8 - #20 8 - #25 8 -#30 - - TCT GAT ACT TAC TCA GAT GGT TCT TGC AGC GG - #A ATC AAC CGT TGTGAT 3205 Ser Asp Thr Tyr Ser Asp Gly Ser Cys Ser Gl - #y Ile Asn Arg Cys Asp 835 - # 840 - # 845 - - GAA CAG CAT CAG GTA GAT ATG CAG CTA GAT GC - #G GAG CAT CAT CCA ATG 3253 Glu Gln His Gln Val Asp Met Gln Leu Asp Al - #a Glu His His Pro Met 850 - # 855 - # 860 - - GAT TGC TGT GAA GCG GCT CAA ACA CAT GAG TT - #T TCT TCC TAT ATT AAT 3301 Asp Cys Cys Glu Ala Ala Gln Thr His Glu Ph - #e Ser Ser Tyr Ile Asn 865 - # 870 - # 875 - - ACA GGG GAT CTA AAT GCA AGT GTA GAT CAG GG - #C ATT TGG GTT GTA TTA 3349 Thr Gly Asp Leu Asn Ala Ser Val Asp Gln Gl - #y Ile Trp Val Val Leu 880 - # 885 - # 890 - - AAA GTT CGA ACA ACA GAT GGG TAT GCG ACG TT - #A GGA AAT CTT GAA TTG 3397 Lys Val Arg Thr Thr Asp Gly Tyr Ala Thr Le - #u Gly Asn Leu Glu Leu 895 9 - #00 9 - #05 9 -#10 - - GTA GAG GTT GGG CCA TTA TCG GGT GAA TCT CT - #A GAA CGG GAA CAAAGA 3445 Val Glu Val Gly Pro Leu Ser Gly Glu Ser Le - #u Glu Arg Glu Gln Arg 915 - # 920 - # 925 - - GAT AAT GCG AAA TGG AAT GCA GAG CTA GGA AG - #A AAA CGT GCA GAA ATA 3493 Asp Asn Ala Lys Trp Asn Ala Glu Leu Gly Ar - #g Lys Arg Ala Glu Ile 930 - # 935 - # 940 - - GAT CGT GTG TAT TTA GCT GCG AAA CAA GCA AT - #T AAT CAT CTG TTT GTA 3541 Asp Arg Val Tyr Leu Ala Ala Lys Gln Ala Il - #e Asn His Leu Phe Val 945 - # 950 - # 955 - - GAC TAT CAA GAT CAA CAA TTA AAT CCA GAA AT - #T GGG CTA GCA GAA ATT 3589 Asp Tyr Gln Asp Gln Gln Leu Asn Pro Glu Il - #e Gly Leu Ala Glu Ile 960 - # 965 - # 970 - - AAT GAA GCT TCA AAT CTT GTA GAG TCA ATT TC - #G GGT GTA TAT AGT GAT 3637 Asn Glu Ala Ser Asn Leu Val Glu Ser Ile Se - #r Gly Val Tyr Ser Asp 975 9 - #80 9 - #85 9 -#90 - - ACA CTA TTA CAG ATT CCT GGG ATT AAC TAC GA - #A ATT TAC ACA GAGTTA 3685 Thr Leu Leu Gln Ile Pro Gly Ile Asn Tyr Gl - #u Ile Tyr Thr Glu Leu 995 - # 1000 - # 1005 - - TCC GAT CGC TTA CAA CAA GCA TCG TAT CTG TA - #T ACG TCT AGA AAT GCG 3733 Ser Asp Arg Leu Gln Gln Ala Ser Tyr Leu Ty - #r Thr Ser Arg Asn Ala 1010 - # 1015 - # 1020 - - GTG CAA AAT GGA GAC TTT AAC AGT GGT CTA GA - #T AGT TGG AAT ACA ACT 3781 Val Gln Asn Gly Asp Phe Asn Ser Gly Leu As - #p Ser Trp Asn Thr Thr 1025 - # 1030 - # 1035 - - ATG GAT GCA TCG GTT CAG CAA GAT GGC AAT AT - #G CAT TTC TTA GTT CTT 3829 Met Asp Ala Ser Val Gln Gln Asp Gly Asn Me - #t His Phe Leu Val Leu 1040 - # 1045 - # 1050 - - TCG CAT TGG GAT GCA CAA GTT TCC CAA CAA TT - #G AGA GTA AAT CCG AAT 3877 Ser His Trp Asp Ala Gln Val Ser Gln Gln Le - #u Arg Val Asn Pro Asn 1055 1060 - # 1065 - # 1070 - - TGT AAG TAT GTC TTA CGT GTG ACA GCA AGA AA - #A GTA GGA GGC GGA GAT 3925 Cys Lys Tyr Val Leu Arg Val Thr Ala Arg Ly - #s Val Gly Gly Gly Asp 1075 - # 1080 - # 1085 - - GGA TAC GTC ACA ATC CGA GAT GGC GCT CAT CA - #C CAA GAA ACT CTT ACA 3973 Gly Tyr Val Thr Ile Arg Asp Gly Ala His Hi - #s Gln Glu Thr Leu Thr 1090 - # 1095 - # 1100 - - TTT AAT GCA TGT GAC TAC GAT GTA AAT GGT AC - #G TAT GTC AAT GAC AAT 4021 Phe Asn Ala Cys Asp Tyr Asp Val Asn Gly Th - #r Tyr Val Asn Asp Asn 1105 - # 1110 - # 1115 - - TCG TAT ATA ACA GAA GAA GTG GTA TTC TAC CC - #A GAG ACA AAA CAT ATG 4069 Ser Tyr Ile Thr Glu Glu Val Val Phe Tyr Pr - #o Glu Thr Lys His Met 1120 - # 1125 - # 1130 - - TGG GTA GAG GTG AGT GAA TCC GAA GGT TCA TT - #C TAT ATA GAC AGT ATT 4117 Trp Val Glu Val Ser Glu Ser Glu Gly Ser Ph - #e Tyr Ile Asp Ser Ile 1135 1140 - # 1145 - # 1150 - - GAG TTT ATT GAA ACA CAA GAG TAGAAGAGGG GGATCCTAA - #C GTATAGCAAC 4168 Glu Phe Ile Glu Thr Gln Glu 1155 - - TATGAGAGGA TACTCCGTAC AAACAAAGAT TAAAAAAAGG TAAAATGAAT AG -#AACCCCCT 4228 - - ACTGGTAGAA GGACCGATAG GGGGTTCTTA CATGAAAAAA TGTAGCTGTT TA -#CTAAGGTG 4288 - - TATAAAAAAC AGCATATCTG ATAGAAAAAA GTGAGTACCT TATAAAGAAA GA - #ATTC 4344 - - - - (2) INFORMATION FOR SEQ ID NO: 5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1157 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5: - - Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Il - #e Ile Asp Ala Pro His 1 5 - # 10 - # 15 - - Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Ty - #r Pro Leu Ala Ser Asp 20 - # 25 - # 30 - - Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Ly - #s Asp Tyr Leu Gln Met 35 - # 40 - # 45 - - Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile As - #n Pro Ser Leu Ser Ile 50 - # 55 - # 60 - - Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Th - #r Val Val Gly Arg Ile 65 - # 70 - # 75 - # 80 - - Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gl - #n Ile Val Ser Phe Tyr 85 - # 90 - # 95 - - Gln Phe Leu Leu Asn Thr Leu Trp Pro Val As - #n Asp Thr Ala Ile Trp 100 - # 105 - # 110 - - Glu Ala Phe Met Arg Gln Val Glu Glu Leu Va - #l Asn Gln Gln Ile Thr 115 - # 120 - # 125 - - Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Le - #u Gln Gly Leu Gly Asp 130 - # 135 - # 140 - - Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln As - #n Trp Leu Ala Asp Arg 145 1 - #50 1 - #55 1 -#60 - - Asn Asp Thr Arg Asn Leu Ser Val Val Arg Al - #a Gln Phe Ile AlaLeu 165 - # 170 - # 175 - - Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Ph - #e Ala Val Asn Gly Gln 180 - # 185 - # 190 - - Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Al - #a Val Asn Leu His Leu 195 - # 200 - # 205 - - Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Gl - #u Gly Trp Gly Phe Thr 210 - # 215 - # 220 - - Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gl - #n Leu Glu Leu Thr Ala 225 2 - #30 2 - #35 2 -#40 - - Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr As - #n Thr Gly Leu AspArg 245 - # 250 - # 255 - - Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Ar - #g Tyr His Gln Phe Arg 260 - # 265 - # 270 - - Arg Glu Met Thr Leu Val Val Leu Asp Val Va - #l Ala Leu Phe Pro Tyr 275 - # 280 - # 285 - - Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser As - #n Pro Gln Leu Thr Arg 290 - # 295 - # 300 - - Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pr - #o Pro Ala Asn Val Gly 305 3 - #10 3 - #15 3 -#20 - - Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr As - #n Thr Phe Ser GluLeu 325 - # 330 - # 335 - - Glu Asn Ala Phe Ile Arg Pro Pro His Leu Ph - #e Asp Arg Leu Asn Ser 340 - # 345 - # 350 - - Leu Thr Ile Ser Ser Asn Arg Phe Pro Val Se - #r Ser Asn Phe Met Asp 355 - # 360 - # 365 - - Tyr Trp Ser Gly His Thr Leu Arg Arg Ser Ty - #r Leu Asn Asp Ser Ala 370 - # 375 - # 380 - - Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr Th - #r Thr Arg Ala Thr Ile 385 3 - #90 3 - #95 4 -#00 - - Asn Pro Gly Val Asp Gly Thr Asn Arg Ile Gl - #u Ser Thr Ala ValAsp 405 - # 410 - # 415 - - Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Va - #l Asn Arg Ala Ser Phe 420 - # 425 - # 430 - - Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Se - #r Pro Ala Asn Gly Gly 435 - # 440 - # 445 - - Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Le - #u Pro Pro Asp Glu Ser 450 - # 455 - # 460 - - Thr Gly Ser Ser Thr His Arg Leu Ser His Va - #l Thr Phe Phe Ser Phe 465 4 - #70 4 - #75 4 -#80 - - Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn Al - #a Gly Ser Val ProThr 485 - # 490 - # 495 - - Tyr Val Trp Thr Arg Arg Asp Val Asp Leu As - #n Asn Thr Ile Thr Pro 500 - # 505 - # 510 - - Asn Arg Ile Thr Gln Leu Pro Leu Val Lys Al - #a Ser Ala Pro Val Ser 515 - # 520 - # 525 - - Gly Thr Thr Val Leu Lys Gly Pro Gly Phe Th - #r Gly Gly Gly Ile Leu 530 - # 535 - # 540 - - Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Le - #u Arg Val Thr Val Asn 545 5 - #50 5 - #55 5 -#60 - - Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg Va - #l Arg Phe Ala SerThr 565 - # 570 - # 575 - - Gly Asn Phe Ser Ile Arg Val Leu Arg Gly Gl - #y Val Ser Ile Gly Asp 580 - # 585 - # 590 - - Val Arg Leu Gly Ser Thr Met Asn Arg Gly Gl - #n Glu Leu Thr Tyr Glu 595 - # 600 - # 605 - - Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gl - #y Pro Phe Asn Pro Pro 610 - # 615 - # 620 - - Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Th - #r Val Asn Ala Glu Gly 625 6 - #30 6 - #35 6 -#40 - - Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Ar - #g Ile Glu Ile ValPro 645 - # 650 - # 655 - - Val Asn Pro Ala Arg Glu Ala Glu Glu Asp Le - #u Glu Ala Ala Lys Lys 660 - # 665 - # 670 - - Ala Val Ala Ser Leu Phe Thr Arg Thr Arg As - #p Gly Leu Gln Val Asn 675 - # 680 - # 685 - - Val Thr Asp Tyr Gln Val Asp Gln Ala Ala As - #n Leu Val Ser Cys Leu 690 - # 695 - # 700 - - Ser Asp Glu Gln Tyr Gly His Asp Lys Lys Me - #t Leu Leu Glu Ala Val 705 7 - #10 7 - #15 7 -#20 - - Arg Ala Ala Lys Arg Leu Ser Arg Glu Arg As - #n Leu Leu Gln AspPro 725 - # 730 - # 735 - - Asp Phe Asn Thr Ile Asn Ser Thr Glu Glu As - #n Gly Trp Lys Ala Ser 740 - # 745 - # 750 - - Asn Gly Val Thr Ile Ser Glu Gly Gly Pro Ph - #e Phe Lys Gly Arg Ala 755 - # 760 - # 765 - - Leu Gln Leu Ala Ser Ala Arg Glu Asn Tyr Pr - #o Thr Tyr Ile Tyr Gln 770 - # 775 - # 780 - - Lys Val Asp Ala Ser Val Leu Lys Pro Tyr Th - #r Arg Tyr Arg Leu Asp 785 7 - #90 7 - #95 8 -#00 - - Gly Phe Val Lys Ser Ser Gln Asp Leu Glu Il - #e Asp Leu Ile HisHis 805 - # 810 - # 815 - - His Lys Val His Leu Val Lys Asn Val Pro As - #p Asn Leu Val Ser Asp 820 - # 825 - # 830 - - Thr Tyr Ser Asp Gly Ser Cys Ser Gly Ile As - #n Arg Cys Asp Glu Gln 835 - # 840 - # 845 - - His Gln Val Asp Met Gln Leu Asp Ala Glu Hi - #s His Pro Met Asp Cys 850 - # 855 - # 860 - - Cys Glu Ala Ala Gln Thr His Glu Phe Ser Se - #r Tyr Ile Asn Thr Gly 865 8 - #70 8 - #75 8 -#80 - - Asp Leu Asn Ala Ser Val Asp Gln Gly Ile Tr - #p Val Val Leu LysVal 885 - # 890 - # 895 - - Arg Thr Thr Asp Gly Tyr Ala Thr Leu Gly As - #n Leu Glu Leu Val Glu 900 - # 905 - # 910 - - Val Gly Pro Leu Ser Gly Glu Ser Leu Glu Ar - #g Glu Gln Arg Asp Asn 915 - # 920 - # 925 - - Ala Lys Trp Asn Ala Glu Leu Gly Arg Lys Ar - #g Ala Glu Ile Asp Arg 930 - # 935 - # 940 - - Val Tyr Leu Ala Ala Lys Gln Ala Ile Asn Hi - #s Leu Phe Val Asp Tyr 945 9 - #50 9 - #55 9 -#60 - - Gln Asp Gln Gln Leu Asn Pro Glu Ile Gly Le - #u Ala Glu Ile AsnGlu 965 - # 970 - # 975 - - Ala Ser Asn Leu Val Glu Ser Ile Ser Gly Va - #l Tyr Ser Asp Thr Leu 980 - # 985 - # 990 - - Leu Gln Ile Pro Gly Ile Asn Tyr Glu Ile Ty - #r Thr Glu Leu Ser Asp 995 - # 1000 - # 1005 - - Arg Leu Gln Gln Ala Ser Tyr Leu Tyr Thr Se - #r Arg Asn Ala Val Gln 1010 - # 1015 - # 1020 - - Asn Gly Asp Phe Asn Ser Gly Leu Asp Ser Tr - #p Asn Thr Thr Met Asp 1025 1030 - # 1035 - # 1040 - - Ala Ser Val Gln Gln Asp Gly Asn Met His Ph - #e Leu Val Leu Ser His 1045 - # 1050 - # 1055 - - Trp Asp Ala Gln Val Ser Gln Gln Leu Arg Va - #l Asn Pro Asn Cys Lys 1060 - # 1065 - # 1070 - - Tyr Val Leu Arg Val Thr Ala Arg Lys Val Gl - #y Gly Gly Asp Gly Tyr 1075 - # 1080 - # 1085 - - Val Thr Ile Arg Asp Gly Ala His His Gln Gl - #u Thr Leu Thr Phe Asn 1090 - # 1095 - # 1100 - - Ala Cys Asp Tyr Asp Val Asn Gly Thr Tyr Va - #l Asn Asp Asn Ser Tyr 1105 1110 - # 1115 - # 1120 - - Ile Thr Glu Glu Val Val Phe Tyr Pro Glu Th - #r Lys His Met Trp Val 1125 - # 1130 - # 1135 - - Glu Val Ser Glu Ser Glu Gly Ser Phe Tyr Il - #e Asp Ser Ile Glu Phe 1140 - # 1145 - # 1150 - - Ile Glu Thr Gln Glu 1155 - - - - (2) INFORMATION FOR SEQ ID NO: 6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1897 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 13..1887 - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 13..1887 (D) OTHER INFORMATION: - #/note= "artificial DNA sequence of the bTS02 - #618A gene, encoding the BTS02618A protein" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6: - - GGTACCAAAA CC ATG GCT GAC TAC CTG CAG ATG ACC - # GAC GAG GAC TAC 48 Met Ala As - #p Tyr Leu Gln Met Thr Asp Glu Asp Tyr 1 - # 5 - # 10 - - ACC GAC AGC TAC ATC AAC CCC AGC CTG AGC AT - #C AGC GGT CGC GAC GCC 96 Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Il - #e Ser Gly Arg Asp Ala 15 - # 20 - # 25 - - GTG CAG ACC GCT CTG ACC GTG GTG GGT CGC AT - #C CTG GGT GCC CTG GGC 144 Val Gln Thr Ala Leu Thr Val Val Gly Arg Il - #e Leu Gly Ala Leu Gly 30 - # 35 - # 40 - - GTG CCC TTC AGC GGT CAG ATC GTG AGC TTC TA - #C CAG TTC CTG CTG AAC 192 Val Pro Phe Ser Gly Gln Ile Val Ser Phe Ty - #r Gln Phe Leu Leu Asn 45 - # 50 - # 55 - # 60 - - ACC CTG TGG CCA GTG AAC GAC ACC GCC ATC TG - #G GAA GCT TTC ATG CGC 240 Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Tr - #p Glu Ala Phe Met Arg 65 - # 70 - # 75 - - CAG GTG GAG GAG CTG GTG AAC CAG CAG ATC AC - #C GAG TTC GCT CGC AAC 288 Gln Val Glu Glu Leu Val Asn Gln Gln Ile Th - #r Glu Phe Ala Arg Asn 80 - # 85 - # 90 - - CAG GCC CTG GCT CGC CTG CAG GGC CTG GGC GA - #C AGC TTC AAC GTG TAC 336 Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly As - #p Ser Phe Asn Val Tyr 95 - # 100 - # 105 - - CAG CGC AGC CTG CAG AAC TGG CTG GCC GAC CG - #C AAC GAC ACC CGC AAC 384 Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Ar - #g Asn Asp Thr Arg Asn 110 - # 115 - # 120 - - CTG AGC GTG GTG AGG GCC CAG TTC ATC GCC CT - #G GAC CTG GAC TTC GTG 432 Leu Ser Val Val Arg Ala Gln Phe Ile Ala Le - #u Asp Leu Asp Phe Val 125 1 - #30 1 - #35 1 -#40 - - AAC GCC ATC CCC CTG TTC GCC GTG AAC GGC CA - #G CAG GTG CCC CTGCTG 480 Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gl - #n Gln Val Pro Leu Leu 145 - # 150 - # 155 - - AGC GTG TAC GCC CAG GCC GTG AAC CTG CAC CT - #G CTG CTG CTG AAG GAT 528 Ser Val Tyr Ala Gln Ala Val Asn Leu His Le - #u Leu Leu Leu Lys Asp 160 - # 165 - # 170 - - GCA TCC CTG TTC GGC GAG GGC TGG GGC TTC AC - #C CAG GGC GAG ATC AGC 576 Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Th - #r Gln Gly Glu Ile Ser 175 - # 180 - # 185 - - ACC TAC TAC GAC CGC CAG CTC GAG CTG ACC GC - #C AAG TAC ACC AAC TAC 624 Thr Tyr Tyr Asp Arg Gln Leu Glu Leu Thr Al - #a Lys Tyr Thr Asn Tyr 190 - # 195 - # 200 - - TGC GAG ACC TGG TAC AAC ACC GGT CTG GAC CG - #C CTG AGG GGC ACC AAC 672 Cys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Ar - #g Leu Arg Gly Thr Asn 205 2 - #10 2 - #15 2 -#20 - - ACC GAG AGC TGG CTG CGC TAC CAC CAG TTC CG - #C AGG GAG ATG ACCCTG 720 Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Ar - #g Arg Glu Met Thr Leu 225 - # 230 - # 235 - - GTG GTG CTG GAC GTG GTG GCC CTG TTC CCC TA - #C TAC GAC GTG CGC CTG 768 Val Val Leu Asp Val Val Ala Leu Phe Pro Ty - #r Tyr Asp Val Arg Leu 240 - # 245 - # 250 - - TAC CCC ACC GGC AGC AAC CCC CAG CTG ACA CG - #T GAG GTG TAC ACC GAC 816 Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Ar - #g Glu Val Tyr Thr Asp 255 - # 260 - # 265 - - CCC ATC GTG TTC AAC CCA CCA GCC AAC GTG GG - #C CTG TGC CGC AGG TGG 864 Pro Ile Val Phe Asn Pro Pro Ala Asn Val Gl - #y Leu Cys Arg Arg Trp 270 - # 275 - # 280 - - GGC ACC AAC CCC TAC AAC ACC TTC AGC GAG CT - #G GAG AAC GCC TTC ATC 912 Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Le - #u Glu Asn Ala Phe Ile 285 2 - #90 2 - #95 3 -#00 - - AGG CCA CCC CAC CTG TTC GAC CGC CTG AAC AG - #C CTG ACC ATC AGCAGC 960 Arg Pro Pro His Leu Phe Asp Arg Leu Asn Se - #r Leu Thr Ile Ser Ser 305 - # 310 - # 315 - - AAT CGA TTC CCC GTG AGC AGC AAC TTC ATG GA - #C TAC TGG AGC GGT CAC 1008 Asn Arg Phe Pro Val Ser Ser Asn Phe Met As - #p Tyr Trp Ser Gly His 320 - # 325 - # 330 - - ACC CTG CGC AGG AGC TAC CTG AAC GAC AGC GC - #C GTG CAG GAG GAC AGC 1056 Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Al - #a Val Gln Glu Asp Ser 335 - # 340 - # 345 - - TAC GGC CTG ATC ACC ACC ACC AGG GCC ACC AT - #C AAC CCA GGC GTG GAC 1104 Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Il - #e Asn Pro Gly Val Asp 350 - # 355 - # 360 - - GGC ACC AAC CGC ATC GAG AGC ACC GCT GTG GA - #C TTC CGC AGC GCT CTG 1152 Gly Thr Asn Arg Ile Glu Ser Thr Ala Val As - #p Phe Arg Ser Ala Leu 365 3 - #70 3 - #75 3 -#80 - - ATC GGC ATC TAC GGC GTG AAC AGG GCC AGC TT - #C GTG CCA GGT GGCCTG 1200 Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Ph - #e Val Pro Gly Gly Leu 385 - # 390 - # 395 - - TTC AAC GGC ACC ACC AGC CCA GCC AAC GGT GG - #C TGC CGA GAT CTG TAC 1248 Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gl - #y Cys Arg Asp Leu Tyr 400 - # 405 - # 410 - - GAC ACC AAC GAC GAG CTG CCA CCC GAC GAG AG - #C ACC GGC AGC AGC ACC 1296 Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Se - #r Thr Gly Ser Ser Thr 415 - # 420 - # 425 - - CAC CGC CTG AGC CAC GTC ACC TTC TTC AGC TT - #C CAG ACC AAC CAG GCT 1344 His Arg Leu Ser His Val Thr Phe Phe Ser Ph - #e Gln Thr Asn Gln Ala 430 - # 435 - # 440 - - GGC AGC ATC GCC AAC GCT GGC AGC GTG CCC AC - #C TAC GTG TGG ACC AGG 1392 Gly Ser Ile Ala Asn Ala Gly Ser Val Pro Th - #r Tyr Val Trp Thr Arg 445 4 - #50 4 - #55 4 -#60 - - AGG GAC GTG GAC CTG AAC AAC ACC ATC ACC CC - #C AAC CGC ATC ACCCAG 1440 Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pr - #o Asn Arg Ile Thr Gln 465 - # 470 - # 475 - - CTG CCC CTG GTG AAG GCC AGC GCT CCC GTG AG - #C GGC ACC ACC GTG CTG 1488 Leu Pro Leu Val Lys Ala Ser Ala Pro Val Se - #r Gly Thr Thr Val Leu 480 - # 485 - # 490 - - AAG GGT CCA GGC TTC ACC GGT GGC GGT ATA CT - #G CGC AGG ACC ACC AAC 1536 Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Le - #u Arg Arg Thr Thr Asn 495 - # 500 - # 505 - - GGC ACC TTC GGC ACC CTG CGC GTG ACC GTG AA - #T TCC CCA CTG ACC CAG 1584 Gly Thr Phe Gly Thr Leu Arg Val Thr Val As - #n Ser Pro Leu Thr Gln 510 - # 515 - # 520 - - CAG TAC CGC CTG CGC GTG CGC TTC GCC AGC AC - #C GGC AAC TTC AGC ATC 1632 Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Th - #r Gly Asn Phe Ser Ile 525 5 - #30 5 - #35 5 -#40 - - CGC GTG CTG AGG GGT GGC GTG AGC ATC GGC GA - #C GTG CGC CTG GGCAGC 1680 Arg Val Leu Arg Gly Gly Val Ser Ile Gly As - #p Val Arg Leu Gly Ser 545 - # 550 - # 555 - - ACC ATG AAC AGG GGC CAG GAG CTG ACC TAC GA - #G AGC TTC TTC ACC CGC 1728 Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Gl - #u Ser Phe Phe Thr Arg 560 - # 565 - # 570 - - GAG TTC ACC ACC ACC GGT CCC TTC AAC CCA CC - #C TTC ACC TTC ACC CAG 1776 Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pr - #o Phe Thr Phe Thr Gln 575 - # 580 - # 585 - - GCC CAG GAG ATC CTG ACC GTG AAC GCC GAG GG - #C GTG AGC ACC GGT GGC 1824 Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gl - #y Val Ser Thr Gly Gly 590 - # 595 - # 600 - - GAG TAC TAC ATC GAC CGC ATC GAG ATC GTG CC - #C GTG AAC CCA GCT CGC 1872 Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pr - #o Val Asn Pro Ala Arg 605 6 - #10 6 - #15 6 -#20 - - GAG GCC GAG GAG GAC TGAGGCTAGC - # - # 1897 Glu Ala Glu Glu Asp 625 - - - - (2) INFORMATION FOR SEQ ID NO: 7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 625 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7: - - Met Ala Asp Tyr Leu Gln Met Thr Asp Glu As - #p Tyr Thr Asp Ser Tyr 1 5 - # 10 - # 15 - - Ile Asn Pro Ser Leu Ser Ile Ser Gly Arg As - #p Ala Val Gln Thr Ala 20 - # 25 - # 30 - - Leu Thr Val Val Gly Arg Ile Leu Gly Ala Le - #u Gly Val Pro Phe Ser 35 - # 40 - # 45 - - Gly Gln Ile Val Ser Phe Tyr Gln Phe Leu Le - #u Asn Thr Leu Trp Pro 50 - # 55 - # 60 - - Val Asn Asp Thr Ala Ile Trp Glu Ala Phe Me - #t Arg Gln Val Glu Glu 65 - # 70 - # 75 - # 80 - - Leu Val Asn Gln Gln Ile Thr Glu Phe Ala Ar - #g Asn Gln Ala Leu Ala 85 - # 90 - # 95 - - Arg Leu Gln Gly Leu Gly Asp Ser Phe Asn Va - #l Tyr Gln Arg Ser Leu 100 - # 105 - # 110 - - Gln Asn Trp Leu Ala Asp Arg Asn Asp Thr Ar - #g Asn Leu Ser Val Val 115 - # 120 - # 125 - - Arg Ala Gln Phe Ile Ala Leu Asp Leu Asp Ph - #e Val Asn Ala Ile Pro 130 - # 135 - # 140 - - Leu Phe Ala Val Asn Gly Gln Gln Val Pro Le - #u Leu Ser Val Tyr Ala 145 1 - #50 1 - #55 1 -#60 - - Gln Ala Val Asn Leu His Leu Leu Leu Leu Ly - #s Asp Ala Ser LeuPhe 165 - # 170 - # 175 - - Gly Glu Gly Trp Gly Phe Thr Gln Gly Glu Il - #e Ser Thr Tyr Tyr Asp 180 - # 185 - # 190 - - Arg Gln Leu Glu Leu Thr Ala Lys Tyr Thr As - #n Tyr Cys Glu Thr Trp 195 - # 200 - # 205 - - Tyr Asn Thr Gly Leu Asp Arg Leu Arg Gly Th - #r Asn Thr Glu Ser Trp 210 - # 215 - # 220 - - Leu Arg Tyr His Gln Phe Arg Arg Glu Met Th - #r Leu Val Val Leu Asp 225 2 - #30 2 - #35 2 -#40 - - Val Val Ala Leu Phe Pro Tyr Tyr Asp Val Ar - #g Leu Tyr Pro ThrGly 245 - # 250 - # 255 - - Ser Asn Pro Gln Leu Thr Arg Glu Val Tyr Th - #r Asp Pro Ile Val Phe 260 - # 265 - # 270 - - Asn Pro Pro Ala Asn Val Gly Leu Cys Arg Ar - #g Trp Gly Thr Asn Pro 275 - # 280 - # 285 - - Tyr Asn Thr Phe Ser Glu Leu Glu Asn Ala Ph - #e Ile Arg Pro Pro His 290 - # 295 - # 300 - - Leu Phe Asp Arg Leu Asn Ser Leu Thr Ile Se - #r Ser Asn Arg Phe Pro 305 3 - #10 3 - #15 3 -#20 - - Val Ser Ser Asn Phe Met Asp Tyr Trp Ser Gl - #y His Thr Leu ArgArg 325 - # 330 - # 335 - - Ser Tyr Leu Asn Asp Ser Ala Val Gln Glu As - #p Ser Tyr Gly Leu Ile 340 - # 345 - # 350 - - Thr Thr Thr Arg Ala Thr Ile Asn Pro Gly Va - #l Asp Gly Thr Asn Arg 355 - # 360 - # 365 - - Ile Glu Ser Thr Ala Val Asp Phe Arg Ser Al - #a Leu Ile Gly Ile Tyr 370 - # 375 - # 380 - - Gly Val Asn Arg Ala Ser Phe Val Pro Gly Gl - #y Leu Phe Asn Gly Thr 385 3 - #90 3 - #95 4 -#00 - - Thr Ser Pro Ala Asn Gly Gly Cys Arg Asp Le - #u Tyr Asp Thr AsnAsp 405 - # 410 - # 415 - - Glu Leu Pro Pro Asp Glu Ser Thr Gly Ser Se - #r Thr His Arg Leu Ser 420 - # 425 - # 430 - - His Val Thr Phe Phe Ser Phe Gln Thr Asn Gl - #n Ala Gly Ser Ile Ala 435 - # 440 - # 445 - - Asn Ala Gly Ser Val Pro Thr Tyr Val Trp Th - #r Arg Arg Asp Val Asp 450 - # 455 - # 460 - - Leu Asn Asn Thr Ile Thr Pro Asn Arg Ile Th - #r Gln Leu Pro Leu Val 465 4 - #70 4 - #75 4 -#80 - - Lys Ala Ser Ala Pro Val Ser Gly Thr Thr Va - #l Leu Lys Gly ProGly 485 - # 490 - # 495 - - Phe Thr Gly Gly Gly Ile Leu Arg Arg Thr Th - #r Asn Gly Thr Phe Gly 500 - # 505 - # 510 - - Thr Leu Arg Val Thr Val Asn Ser Pro Leu Th - #r Gln Gln Tyr Arg Leu 515 - # 520 - # 525 - - Arg Val Arg Phe Ala Ser Thr Gly Asn Phe Se - #r Ile Arg Val Leu Arg 530 - # 535 - # 540 - - Gly Gly Val Ser Ile Gly Asp Val Arg Leu Gl - #y Ser Thr Met Asn Arg 545 5 - #50 5 - #55 5 -#60 - - Gly Gln Glu Leu Thr Tyr Glu Ser Phe Phe Th - #r Arg Glu Phe ThrThr 565 - # 570 - # 575 - - Thr Gly Pro Phe Asn Pro Pro Phe Thr Phe Th - #r Gln Ala Gln Glu Ile 580 - # 585 - # 590 - - Leu Thr Val Asn Ala Glu Gly Val Ser Thr Gl - #y Gly Glu Tyr Tyr Ile 595 - # 600 - # 605 - - Asp Arg Ile Glu Ile Val Pro Val Asn Pro Al - #a Arg Glu Ala Glu Glu 610 - # 615 - # 620 - - Asp 625 - - - - (2) INFORMATION FOR SEQ ID NO: 8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1897 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 13..1887 - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 13..1887 (D) OTHER INFORMATION: - #/note= "DNA sequence of bTS02618Aa gene enco - #ding the BTS02618Aa protein" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8: - - GGTACCAAAA CC ATG GCT GAC TAC CTG CAG ATG ACC - # GAC GAG GAC TAC 48 Met Ala As - #p Tyr Leu Gln Met Thr Asp Glu Asp Tyr 1 - # 5 - # 10 - - ACC GAC AGC TAC ATC AAC CCC AGC CTG AGC AT - #C AGC GGT CGC GAC GCC 96 Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Il - #e Ser Gly Arg Asp Ala 15 - # 20 - # 25 - - GTG CAG ACC GCT CTG ACC GTG GTG GGT CGC AT - #C CTG GGT GCC CTG GGC 144 Val Gln Thr Ala Leu Thr Val Val Gly Arg Il - #e Leu Gly Ala Leu Gly 30 - # 35 - # 40 - - GTG CCC TTC AGC GGT CAG ATC GTG AGC TTC TA - #C CAG TTC CTG CTG AAC 192 Val Pro Phe Ser Gly Gln Ile Val Ser Phe Ty - #r Gln Phe Leu Leu Asn 45 - # 50 - # 55 - # 60 - - ACC CTG TGG CCA GTG AAC GAC ACC GCC ATC TG - #G GAA GCT TTC ATG CGC 240 Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Tr - #p Glu Ala Phe Met Arg 65 - # 70 - # 75 - - CAG GTG GAG GAG CTG GTG AAC CAG CAG ATC AC - #C GAG TTC GCT CGC AAC 288 Gln Val Glu Glu Leu Val Asn Gln Gln Ile Th - #r Glu Phe Ala Arg Asn 80 - # 85 - # 90 - - CAG GCC CTG GCT CGC CTG CAG GGC CTG GGC GA - #C AGC TTC AAC GTG TAC 336 Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly As - #p Ser Phe Asn Val Tyr 95 - # 100 - # 105 - - CAG CGC AGC CTG CAG AAC TGG CTG GCC GAC CG - #C AAC GAC ACC AAG AAC 384 Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Ar - #g Asn Asp Thr Lys Asn 110 - # 115 - # 120 - - CTG AGC GTG GTG AGG GCC CAG TTC ATC GCC CT - #G GAC CTG GAC TTC GTG 432 Leu Ser Val Val Arg Ala Gln Phe Ile Ala Le - #u Asp Leu Asp Phe Val 125 1 - #30 1 - #35 1 -#40 - - AAC GCC ATC CCC CTG TTC GCC GTG AAC GGC CA - #G CAG GTG CCC CTGCTG 480 Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gl - #n Gln Val Pro Leu Leu 145 - # 150 - # 155 - - AGC GTG TAC GCC CAG GCC GTG AAC CTG CAC CT - #G CTG CTG CTG AAG GAT 528 Ser Val Tyr Ala Gln Ala Val Asn Leu His Le - #u Leu Leu Leu Lys Asp 160 - # 165 - # 170 - - GCA TCC CTG TTC GGC GAG GGC TGG GGC TTC AC - #C CAG GGC GAG ATC AGC 576 Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Th - #r Gln Gly Glu Ile Ser 175 - # 180 - # 185 - - ACC TAC TAC GAC CGC CAG CTC GAG CTG ACC GC - #C AAG TAC ACC AAC TAC 624 Thr Tyr Tyr Asp Arg Gln Leu Glu Leu Thr Al - #a Lys Tyr Thr Asn Tyr 190 - # 195 - # 200 - - TGC GAG ACC TGG TAC AAC ACC GGT CTG GAC CG - #C CTG AGG GGC ACC AAC 672 Cys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Ar - #g Leu Arg Gly Thr Asn 205 2 - #10 2 - #15 2 -#20 - - ACC GAG AGC TGG CTG CGC TAC CAC CAG TTC CG - #C AGG GAG ATG ACCCTG 720 Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Ar - #g Arg Glu Met Thr Leu 225 - # 230 - # 235 - - GTG GTG CTG GAC GTG GTG GCC CTG TTC CCC TA - #C TAC GAC GTG CGC CTG 768 Val Val Leu Asp Val Val Ala Leu Phe Pro Ty - #r Tyr Asp Val Arg Leu 240 - # 245 - # 250 - - TAC CCC ACC GGC AGC AAC CCC CAG CTG ACA CG - #T GAG GTG TAC ACC GAC 816 Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Ar - #g Glu Val Tyr Thr Asp 255 - # 260 - # 265 - - CCC ATC GTG TTC AAC CCA CCA GCC AAC GTG GG - #C CTG TGC CGC AGG TGG 864 Pro Ile Val Phe Asn Pro Pro Ala Asn Val Gl - #y Leu Cys Arg Arg Trp 270 - # 275 - # 280 - - GGC ACC AAC CCC TAC AAC ACC TTC AGC GAG CT - #G GAG AAC GCC TTC ATC 912 Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Le - #u Glu Asn Ala Phe Ile 285 2 - #90 2 - #95 3 -#00 - - AGG CCA CCC CAC CTG TTC GAC CGC CTG AAC AG - #C CTG ACC ATC AGCAGC 960 Arg Pro Pro His Leu Phe Asp Arg Leu Asn Se - #r Leu Thr Ile Ser Ser 305 - # 310 - # 315 - - AAT CGA TTC CCC GTG AGC AGC AAC TTC ATG GA - #C TAC TGG AGC GGT CAC 1008 Asn Arg Phe Pro Val Ser Ser Asn Phe Met As - #p Tyr Trp Ser Gly His 320 - # 325 - # 330 - - ACC CTG CGC AGG AGC TAC CTG AAC GAC AGC GC - #C GTG CAG GAG GAC AGC 1056 Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Al - #a Val Gln Glu Asp Ser 335 - # 340 - # 345 - - TAC GGC CTG ATC ACC ACC ACC AGG GCC ACC AT - #C AAC CCA GGC GTG GAC 1104 Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Il - #e Asn Pro Gly Val Asp 350 - # 355 - # 360 - - GGC ACC AAC CGC ATC GAG AGC ACC GCT GTG GA - #C TTC CGC AGC GCT CTG 1152 Gly Thr Asn Arg Ile Glu Ser Thr Ala Val As - #p Phe Arg Ser Ala Leu 365 3 - #70 3 - #75 3 -#80 - - ATC GGC ATC TAC GGC GTG AAC AGG GCC AGC TT - #C GTG CCA GGT GGCCTG 1200 Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Ph - #e Val Pro Gly Gly Leu 385 - # 390 - # 395 - - TTC AAC GGC ACC ACC AGC CCA GCC AAC GGT GG - #C TGC CGA GAT CTG TAC 1248 Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gl - #y Cys Arg Asp Leu Tyr 400 - # 405 - # 410 - - GAC ACC AAC GAC GAG CTG CCA CCC GAC GAG AG - #C ACC GGC AGC AGC ACC 1296 Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Se - #r Thr Gly Ser Ser Thr 415 - # 420 - # 425 - - CAC CGC CTG AGC CAC GTC ACC TTC TTC AGC TT - #C CAG ACC AAC CAG GCT 1344 His Arg Leu Ser His Val Thr Phe Phe Ser Ph - #e Gln Thr Asn Gln Ala 430 - # 435 - # 440 - - GGC AGC ATC GCC AAC GCT GGC AGC GTG CCC AC - #C TAC GTG TGG ACC AGG 1392 Gly Ser Ile Ala Asn Ala Gly Ser Val Pro Th - #r Tyr Val Trp Thr Arg 445 4 - #50 4 - #55 4 -#60 - - AGG GAC GTG GAC CTG AAC AAC ACC ATC ACC CC - #C AAC CGC ATC ACCCAG 1440 Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pr - #o Asn Arg Ile Thr Gln 465 - # 470 - # 475 - - CTG CCC CTG GTG AAG GCC AGC GCT CCC GTG AG - #C GGC ACC ACC GTG CTG 1488 Leu Pro Leu Val Lys Ala Ser Ala Pro Val Se - #r Gly Thr Thr Val Leu 480 - # 485 - # 490 - - AAG GGT CCA GGC TTC ACC GGT GGC GGT ATA CT - #G CGC AGG ACC ACC AAC 1536 Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Le - #u Arg Arg Thr Thr Asn 495 - # 500 - # 505 - - GGC ACC TTC GGC ACC CTG CGC GTG ACC GTG AA - #T TCC CCA CTG ACC CAG 1584 Gly Thr Phe Gly Thr Leu Arg Val Thr Val As - #n Ser Pro Leu Thr Gln 510 - # 515 - # 520 - - CAG TAC CGC CTG CGC GTG CGC TTC GCC AGC AC - #C GGC AAC TTC AGC ATC 1632 Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Th - #r Gly Asn Phe Ser Ile 525 5 - #30 5 - #35 5 -#40 - - CGC GTG CTG AGG GGT GGC GTG AGC ATC GGC GA - #C GTG CGC CTG GGCAGC 1680 Arg Val Leu Arg Gly Gly Val Ser Ile Gly As - #p Val Arg Leu Gly Ser 545 - # 550 - # 555 - - ACC ATG AAC AGG GGC CAG GAG CTG ACC TAC GA - #G AGC TTC TTC ACC CGC 1728 Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Gl - #u Ser Phe Phe Thr Arg 560 - # 565 - # 570 - - GAG TTC ACC ACC ACC GGT CCC TTC AAC CCA CC - #C TTC ACC TTC ACC CAG 1776 Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pr - #o Phe Thr Phe Thr Gln 575 - # 580 - # 585 - - GCC CAG GAG ATC CTG ACC GTG AAC GCC GAG GG - #C GTG AGC ACC GGT GGC 1824 Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gl - #y Val Ser Thr Gly Gly 590 - # 595 - # 600 - - GAG TAC TAC ATC GAC CGC ATC GAG ATC GTG CC - #C GTG AAC CCA GCT CGC 1872 Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pr - #o Val Asn Pro Ala Arg 605 6 - #10 6 - #15 6 -#20 - - GAG GCC GAG GAG GAC TGAGGCTAGC - # - # 1897 Glu Ala Glu Glu Asp 625 - - - - (2) INFORMATION FOR SEQ ID NO: 9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 625 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9: - - Met Ala Asp Tyr Leu Gln Met Thr Asp Glu As - #p Tyr Thr Asp Ser Tyr 1 5 - # 10 - # 15 - - Ile Asn Pro Ser Leu Ser Ile Ser Gly Arg As - #p Ala Val Gln Thr Ala 20 - # 25 - # 30 - - Leu Thr Val Val Gly Arg Ile Leu Gly Ala Le - #u Gly Val Pro Phe Ser 35 - # 40 - # 45 - - Gly Gln Ile Val Ser Phe Tyr Gln Phe Leu Le - #u Asn Thr Leu Trp Pro 50 - # 55 - # 60 - - Val Asn Asp Thr Ala Ile Trp Glu Ala Phe Me - #t Arg Gln Val Glu Glu 65 - # 70 - # 75 - # 80 - - Leu Val Asn Gln Gln Ile Thr Glu Phe Ala Ar - #g Asn Gln Ala Leu Ala 85 - # 90 - # 95 - - Arg Leu Gln Gly Leu Gly Asp Ser Phe Asn Va - #l Tyr Gln Arg Ser Leu 100 - # 105 - # 110 - - Gln Asn Trp Leu Ala Asp Arg Asn Asp Thr Ly - #s Asn Leu Ser Val Val 115 - # 120 - # 125 - - Arg Ala Gln Phe Ile Ala Leu Asp Leu Asp Ph - #e Val Asn Ala Ile Pro 130 - # 135 - # 140 - - Leu Phe Ala Val Asn Gly Gln Gln Val Pro Le - #u Leu Ser Val Tyr Ala 145 1 - #50 1 - #55 1 -#60 - - Gln Ala Val Asn Leu His Leu Leu Leu Leu Ly - #s Asp Ala Ser LeuPhe 165 - # 170 - # 175 - - Gly Glu Gly Trp Gly Phe Thr Gln Gly Glu Il - #e Ser Thr Tyr Tyr Asp 180 - # 185 - # 190 - - Arg Gln Leu Glu Leu Thr Ala Lys Tyr Thr As - #n Tyr Cys Glu Thr Trp 195 - # 200 - # 205 - - Tyr Asn Thr Gly Leu Asp Arg Leu Arg Gly Th - #r Asn Thr Glu Ser Trp 210 - # 215 - # 220 - - Leu Arg Tyr His Gln Phe Arg Arg Glu Met Th - #r Leu Val Val Leu Asp 225 2 - #30 2 - #35 2 -#40 - - Val Val Ala Leu Phe Pro Tyr Tyr Asp Val Ar - #g Leu Tyr Pro ThrGly 245 - # 250 - # 255 - - Ser Asn Pro Gln Leu Thr Arg Glu Val Tyr Th - #r Asp Pro Ile Val Phe 260 - # 265 - # 270 - - Asn Pro Pro Ala Asn Val Gly Leu Cys Arg Ar - #g Trp Gly Thr Asn Pro 275 - # 280 - # 285 - - Tyr Asn Thr Phe Ser Glu Leu Glu Asn Ala Ph - #e Ile Arg Pro Pro His 290 - # 295 - # 300 - - Leu Phe Asp Arg Leu Asn Ser Leu Thr Ile Se - #r Ser Asn Arg Phe Pro 305 3 - #10 3 - #15 3 -#20 - - Val Ser Ser Asn Phe Met Asp Tyr Trp Ser Gl - #y His Thr Leu ArgArg 325 - # 330 - # 335 - - Ser Tyr Leu Asn Asp Ser Ala Val Gln Glu As - #p Ser Tyr Gly Leu Ile 340 - # 345 - # 350 - - Thr Thr Thr Arg Ala Thr Ile Asn Pro Gly Va - #l Asp Gly Thr Asn Arg 355 - # 360 - # 365 - - Ile Glu Ser Thr Ala Val Asp Phe Arg Ser Al - #a Leu Ile Gly Ile Tyr 370 - # 375 - # 380 - - Gly Val Asn Arg Ala Ser Phe Val Pro Gly Gl - #y Leu Phe Asn Gly Thr 385 3 - #90 3 - #95 4 -#00 - - Thr Ser Pro Ala Asn Gly Gly Cys Arg Asp Le - #u Tyr Asp Thr AsnAsp 405 - # 410 - # 415 - - Glu Leu Pro Pro Asp Glu Ser Thr Gly Ser Se - #r Thr His Arg Leu Ser 420 - # 425 - # 430 - - His Val Thr Phe Phe Ser Phe Gln Thr Asn Gl - #n Ala Gly Ser Ile Ala 435 - # 440 - # 445 - - Asn Ala Gly Ser Val Pro Thr Tyr Val Trp Th - #r Arg Arg Asp Val Asp 450 - # 455 - # 460 - - Leu Asn Asn Thr Ile Thr Pro Asn Arg Ile Th - #r Gln Leu Pro Leu Val 465 4 - #70 4 - #75 4 -#80 - - Lys Ala Ser Ala Pro Val Ser Gly Thr Thr Va - #l Leu Lys Gly ProGly 485 - # 490 - # 495 - - Phe Thr Gly Gly Gly Ile Leu Arg Arg Thr Th - #r Asn Gly Thr Phe Gly 500 - # 505 - # 510 - - Thr Leu Arg Val Thr Val Asn Ser Pro Leu Th - #r Gln Gln Tyr Arg Leu 515 - # 520 - # 525 - - Arg Val Arg Phe Ala Ser Thr Gly Asn Phe Se - #r Ile Arg Val Leu Arg 530 - # 535 - # 540 - - Gly Gly Val Ser Ile Gly Asp Val Arg Leu Gl - #y Ser Thr Met Asn Arg 545 5 - #50 5 - #55 5 -#60 - - Gly Gln Glu Leu Thr Tyr Glu Ser Phe Phe Th - #r Arg Glu Phe ThrThr 565 - # 570 - # 575 - - Thr Gly Pro Phe Asn Pro Pro Phe Thr Phe Th - #r Gln Ala Gln Glu Ile 580 - # 585 - # 590 - - Leu Thr Val Asn Ala Glu Gly Val Ser Thr Gl - #y Gly Glu Tyr Tyr Ile 595 - # 600 - # 605 - - Asp Arg Ile Glu Ile Val Pro Val Asn Pro Al - #a Arg Glu Ala Glu Glu 610 - # 615 - # 620 - - Asp 625__________________________________________________________________________

Number	Date	Country	Kind
93400949	Apr 1993	GBX
PCT/EP93/01820	Jul 1993	WOX

Number	Date	Country
0358557	Mar 1990	EPX
0408403	Jan 1991	EPX
0440581	Jan 1991	EPX
0498537	Jan 1992	EPX
9006999	Jun 1990	WOX
9304587	Mar 1993	WOX

Bacillus thuringiensis strains and their insecticidal proteins

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