Protein toxins active against lepidopteran pests

BACKGROUND OF THE INVENTION
The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. These crystalline proteins can be proforms of .delta.-endotoxins which are highly toxic to pests and specific in their toxic activity. Certain B.t. endotoxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B.thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B.thuringiensis var. kurstaki HD-1 produces a delta endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, subspecies of B.t., namely israelensis and san diego (a.k.a. B.t. tenebrionis, a.k.a. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent 96:500-508, describe a B.t. isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.
Recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into 4 major classes. The classes were CryI (Lepidoptera-specific),CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported. (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275).
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B.thuringiensis var. san diego (a.k.a. B.t. tenebrionis, a.k.a. M-7) which can be used to control coleopteranpests in various environments. U.S. Pat. No. 5,164,180 discloses a B.t. isolate, PS81A2, which is active against lepidopteran pests. U.S. Pat. No. 5,151,363 discloses certain isolates of B.t. which have activity against nematodes. Many other patents have issued for new B.t. isolates and new uses of B.t. isolates. The discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns novel Bacillus thuringiensis isolates which have activity against lepidopteran pests.
Specifically, the invention comprises novel B.t. isolates and mutants thereof, and novel delta endotoxin genes obtainable from these B.t. isolates which encode proteins which are active against lepidopteran pests.

BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows the one-letter amino acid sequence of the Generic Formula (SEQ ID NO. 27). Numbering is for convenience and approximate location only. In the Generic Formula, the N-terminal half of the molecule is comprised of residue nos. 1-638. The terminal half is comprised of residues 639-1213. Wherein
______________________________________A = ala G = gly M = met S = serC = cys H = his N = asn T = thrD = asp I = ile P = pro V = valE = glu K = lys Q = gln W = trpF = phe L = leu R = arg Y = tyrk = K or Rz = G, S, D, or Nj = E, Q, R, or Kx = G, S, D, N, E, Q, R, or Ku = C, P, T, or Ab = M, I, L, V, or Fo = C, P, T, A, M, I, L, V, or F______________________________________ - = any naturally occurring amino acid . = any naturally occurring amino acid or complete omission thereof.

BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 is the nucleotide sequence of the gene 81A2.
SEQ ID NO. 2 is the amino acid sequence of the toxin 81A2.
SEQ ID NO. 3 is the nucleotide sequence of the gene 91C2.
SEQ ID NO. 4 is the amino acid sequence of the toxin 91C2.
SEQ ID NO. 5 is a radiolabeled oligonucleotide probe used in RFLP analysis as described in Example 3.
SEQ ID NO. 6 is a forward oligonucleotide primer used to amplify gene 91C2 according to the subject invention.
SEQ ID NO. 7 is a reverse oligonucleotide primer used to amplify gene 91C2 according to the subject invention.
SEQ ID NO. 8 is a synthetic oligonucleotide probe used to identify gene 91C2 according to the subject invention.
SEQ ID NO. 9 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 10 is a nucleotide probe according to the subject invention.
SEQ ID NO. 11 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 12 is a nucleotide probe according to the subject invention.
SEQ ID NO. 13 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 14 is a nucleotide probe according to the subject invention.
SEQ ID NO. 15 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 16 is a nucleotide probe according to the subject invention.
SEQ ID NO. 17 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 18 is a nucleotide probe according to the subject invention.
SEQ ID NO. 19 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 20 is a nucleotide probe according to the subject invention.
SEQ ID NO. 21 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 22 is a nucleotide probe according to the subject invention.
SEQ ID NO. 23 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 24 is a nucleotide probe according to the subject invention.
SEQ ID NO. 25 is the peptide sequence encoded by probes for CryIF genes.
SEQ ID NO. 26 is a nucleotide probe according to the subject invention.
SEQ ID NO. 27 is the Generic Formula according to the subject invention.
DETAILED DISCLOSURE OF THE INVENTION
The subject invention concerns isolates of Bacillus thuringiensis having anti-lepidopteran activity. These isolates comprise genes which code for .delta.-endotoxins, which toxins are responsible for the observed anti-lepidopteran activity. Thus, the subject invention concerns anti-lepidopteran B.t. isolates, anti-lepidopteran B.t. toxins, and genes which encode these toxins. Further embodiments of the subject invention concern recombinant hosts transformed with genes encoding the anti-lepidopteran B.t. toxins.. The subject invention further concerns methods for controlling lepidopterans, said methods comprising the use of the isolates, toxins, genes, and recombinant hosts of the subject invention.
Specifically exemplified herein are the isolates designated B.t. PS81T1, B.t. PS53C2, B.t. PS31F4, B.t. PS86V1, B.t. PS 8612, B.t. PS73E, B.t. PS81K, B.t. PS83E2, B.t. PS81E, B.t. PS81Z3, B.t. PS53B5, B.t. PS83R, B.t. PS53B2, Bt. PS83N2, B.t. PS81B5, B.t. PS86W1, and B.t. PS93C2. Also specifically exemplified is the toxin designated 91C2 and the gene which encodes this toxin. The 91C2 gene is a CryIF gene. CryIF is a subclass of genes within the lepidopteran-active CryI class of B.t. genes. The discovery described in the subject application enables a person skilled in the art to identify other CryIF toxins (and genes coding for these toxins) having anti-lepidopteran activity. The toxins of the subject invention are characterized as being active against lepidopterans and having one or more of the following characteristics:
1. A high degree of amino acid homology with toxin 91C2.
2. A nucleotide sequence encoding the toxin wherein the nucleotide sequence hybridizes with probes or genes disclosed herein.
3. A nucleotide sequence encoding the toxin wherein the nucleotide sequence can be amplified by PCR using primers disclosed herein.
4. An amino acid sequence which conforms to the Generic Formula presented herein.
5. Immunoreactivity to an antibody raised to toxin 91 C2.
Bacillus thuringiensis isolates useful according to the subject invention have the following characteristics in their biologically pure form:
TABLE 1______________________________________Taxonomic characterization of the B.t. isolates of the subject inventionApprox. ToxinIsolate Crystal Type MW (kD) Serotype Activity______________________________________PS81T1 bipyramid 130 aizawai LepidopteraPS53C2 bipyramid 130, 60 kurstaki LepidopteraPS31F4 bipyramid 130, 60 kurstaki LepidopteraPS86V1 bipyramid 130 galleriae LepidopteraPS86I2 bipyramid 130 morrisoni LepidopteraPS73E bipyramid 130 aizawai LepidopteraPS81K bipyramid 130 aizawai LepidopteraPS83E2 amorphic 130 aizawai LepidopteraPS81E bipyramid 130 aizawai LepidopteraPS81Z3 bipyramid 130 aizawai LepidopteraPS53B5 bipyramid 130, 60 kenyae LepidopteraPS83R bipyramid 130 aizawai LepidopteraPS53B2 bipyramid 130, 60 kenyae LepidopteraPS83N2 bipyramid 130, 60 sotto/kenyae LepidopteraPS81B5 amorphic 130 aizawai LepidopteraPS86W1 bipyramid 130 galleriae LepidopteraPS91C2 bipyramid 130 morrisoni Lepidoptera______________________________________
B.t. isolates useful according to the subject invention have been deposited. Also deposited are recombinant microbes comprising the B.t. genes of interest. The cultures have been deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Ill. 61604 USA.
______________________________________Culture Accession Number Deposit Date______________________________________Bacillus thuringiensis PS81IA NRRL B-18484 April 19, 1989Bacillus thuringiensis PS91C2 NRRL B-18931 December 27, 1991E. Coli NM522 (pMYC2361) NRRL B-21016N December 17, 1992______________________________________
The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14and 35 U.S.C. .sctn.122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
Toxins and genes. The toxins and genes according to the subject invention include not only the full length sequences disclosed herein but also fragments of these sequences, longer sequences, and fusion proteins, which retain the characteristic pesticidal activity of the toxins specifically exemplified herein.
One aspect of the subject invention concerns the discovery of a generic chemical formula hereinafter referred to as the Generic Formula (SEQ ID NO.27), which can be used to identify toxins having activity against lepidopterans. The Generic Formula describes toxin proteins having molecular weights of about 130 kDa.
The Generic Formula is shown in FIG. 1 designated by a one-letter amino acid sequence. The Sequence Listing provided herein according to the PatentIn format utilizes the three-letter amino acid code and has no provision for showing a choice between two amino acids at a given position. Therefore, within the PatentIn Sequence Listing, "Xaa" is used to denote points of variation within a sequence, but the single letter code of FIG. 1 should be referred to for the specific amino acid substitutions which are acceptable at a given location in the sequence.
Further guidance for characterizing the lepidopteran toxins of the subject invention is provided in Tables 2 and 3, which demonstrate the relatedness among toxins within the known CryI subclasses of lepidopteran toxins. These tables show a numeric score for the best matching alignment between two proteins that reflects: (1) positive scores for exact matches, (2) positive or negative scores reflecting the likelihood (or not) of one amino acid substituting for another in a related protein, and (3) negative scores for the introduction of gaps. A protein sequence aligned to itself will have the highest possible-score, i.e., all exact matches and no gaps. However, an unrelated protein or a randomly generated sequence will typically have a low positive score. Related sequences have scores between the random background score and the perfect match score.
The sequence comparisons reported herein were made using the algorithm of Smith and Waterman ([1981] Advances in Applied Mathematics 2:482-489), implemented as the program "Bestfit" in the GCG Sequence Analysis Software Package Version 7, April 1991. The sequences were compared with default parameter values (comparison: Swagappep.Cmp, Gap: 3.0, length weight: 0.1). The program output value is referred to as the Quality score.
Tables 2 and 3 show the pairwise alignment scores between the indicated amino acids of the CryI toxin proteins. Table 4 shows the amino acids compared from the proteins of interest.
Table 2 shows the scores prior to adjustment for random sequence scores. Note that for each subclass, the highest alignment score is always with another toxin protein from the same subclass. For example, the highest alignment score with CryIA(a), aside from itself, is with CryIA(d). Furthermore, CryIA(a) scores highest with all three other CryIA toxin proteins. In a similar manner, other CryI toxins score highest with other members of the same subclass. Of particular relevance to the subject invention is the fact that the CryIF toxin proteins score highest with each other.
Table 3 shows the same analysis after subtraction of the average score of 50 alignments of random shuffles of the column sequences with the row sequences. Note that in Table 3 the same relationships hold as in Table 2, i.e., toxin proteins score highest with other members of the same subclass. Again, the two CryIF toxin proteins score highest with each other. Examination of the adjusted alignment scores for members of the same subclass reveals that CryI subclasses can be defined as those proteins with adjusted alignment scores of about 450 or greater.
Thus, certain toxins of the subject invention can be defined as those which have lepidopteran activity and have an alignment value of 450-500 or greater with CryIF(a) or CryIF(b) (91C2). As used herein, the term "alignment value" refers to the adjusted scores obtained above and used to create the scores reported in Table 3.
TABLE 2__________________________________________________________________________Raw quality scores CryIA(a) CryIA(b) CryIA(c) CryIA(d) CryIB CryIC CryID CryIE(a) CryIE(b) CryIF(a) CryIF(b)__________________________________________________________________________ (91C2)CryIA(a) 911 819 706 857 426 519 533 536 535 532 557CryIA(b) 912 785 790 428 512 540 547 546 543 565CryIA(c) 914 679 390 482 508 512 533 502 501CryIA(d) 911 422 514 539 549 538 539 559CryIB 954 428 410 408 375 421 434CryIC 926 525 547 480 494 495CryID 888 505 499 507 497CryIE(a) 902 722 494 487CryIE(b) 899 480 477CryIF(a) 902 803CryIF(b) (91C2) 900__________________________________________________________________________
TABLE 3__________________________________________________________________________Net quality scores CryIA(a) CryIA(b) CryIA(c) CryIA(d) CryIB CryIC CryID CryIE(a) CryIE(b) CryIF(a) CryIF(b)__________________________________________________________________________ (91C2)CryIA(a) 724 633 520 671 240 332 352 351 350 349 373CryIA(b) 726 600 606 241 327 359 362 360 360 383CryIA(c) 728 493 204 295 327 328 347 319 317CryIA(d) 727 236 328 357 363 353 356 377CryIB 763 240 229 223 189 235 249CryIC 738 343 361 293 309 309CryID 710 325 319 328 318CryIE(a) 717 538 310 304CryIE(b) 713 296 294CryIF(a) 719 620CryIF(b) (91C2) 713__________________________________________________________________________
TABLE 4______________________________________Protein Amino acids compared______________________________________CryIA(a) 1-607CryIA(b) 1-608CryIA(c) 1-609CryIA(d) 1-607CryIB 1-636CryIC 1-617CryID 1-592CryIE(a) 1-601CryIE(b) 1-599CryIF(a) 1-601CryIF(b) (91C2) 1-600______________________________________
Toxins of the subject invention are specifically exemplified herein by the toxin encoded by the gene designated 91C2. Since this toxin is merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention further comprises variant toxins (and nucleotide sequences coding for variant toxins) having the same, or essentially the same, biological activity against lepidopterans of 91C2. These equivalent toxins will have amino acid homology with 91C2. This amino acid homology will typically be greater than 75%, preferably be greater than 90%, and most preferably be greater than 95%. The amino acid homology will be highest in certain critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be readily made in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 5 provides a listing of examples of amino acids belonging to each class.
TABLE 5______________________________________Class of Amino Acid Examples of Amino Acids______________________________________Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, TrpUncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, GlnAcidic Asp, GluBasic Lys, Arg, His______________________________________
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.
The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions, which are described above.
It should be apparent to a person skilled in this art that genes encoding lepidopteran-active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene machine. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences which code for the same toxins or which code for equivalent toxins having lepidopteran activity. Variations of these genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which code for active fragments may be obtained using a variety of other restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.
Equivalent toxins and/or genes encoding these equivalent toxins can also be located from B.t. isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other B.t. toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures in this art. The genes coding for these toxins can then be obtained from the microorganism.
A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Patent Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Detection of the probe provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention.
The nucleotide segments which are used as probes according to the invention can be synthesized by use of DNA synthesizers using standard procedures. In the use of labeled nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include .sup.32 P, .sup.125 I, .sup.35 S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Preferably, hybridization is conducted under stringent conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1989) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting.
Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid; a certain degree of mismatch can be tolerated. Therefore, the probes of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
The known methods include, but are not limited to:
(1) synthesizing chemically or otherwise an artificial sequence which is a mutation, insertion or deletion of the known sequence;
(2) using a probe of the present invention to obtain via hybridization a new sequence or a mutation, insertion or deletion of the probe sequence; and
(3) mutating, inserting or deleting a test sequence in vitro or in vivo.
It is important to note that the mutational, insertional, and deletional variants generated from a given probe may be more or less efficient than the original probe. Notwithstanding such differences in efficiency, these variants are within the scope of the present invention.
Thus, mutational, insertional, and deletional variants of the disclosed sequences can be readily prepared by methods which are well known to those skilled in the art. These variants can be used in the same manner as the instant probes so long as the variants have substantial sequence homology with the probes. As used herein, substantial sequence homology refers to homology which is sufficient to enable the variant to function in the same capacity as the original probe. Preferably, this homology is greater than 50%; more preferably, this homology is greater than 75%; and most preferably, this homology is greater than 90%. The degree of homology needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are designed to improve the function of the sequence or otherwise provide a methodological advantage.
Specific nucleotide probes useful according to the subject invention in the rapid identification of CryIF class toxin genes include:
(i) DNA coding for a peptide sequence "Ser Thr Gly Arg Leu Pro Leu Asp" (SEQ ID NO. 9). A specific example of such a probe is "AGTACWGGMA GRTTACCRTT RGAY" (SEQ ID NO. 10);
(ii) DNA coding for a peptide sequence "Glu Asp Ser Pro Val Ser Ala Asn" (SEQ ID NO. 11). A specific example of such a probe is "GARGATTCWC CAGTWTCWGC WAAT" (SEQ ID NO. 12);
(iii) DNA coding for a peptide sequence "Asn Gly Phe Asn Arg Ala Glu Phe Gly Val" (SEQ ID NO. 13). A specific example of such a probe is "AATGGWTTTA ATAGTGCTGAATTTGGGAGTW" (SEQ ID NO. 14);
(iv) DNA coding for a peptide sequence "Val Thr Ala Glu Thr Val Arg Ser Gln Thr" (SEQ ID NO. 15). A specific example of such a probe is "GTAACWGCAG ARACWGTWAG WAGTCAAACW" (SEQ ID NO. 16);
(v) DNA coding for a peptide sequence "Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu" (SEQ ID NO. 17). A specific example of such a probe is "GTMTTYAATC CWGGWGGMGCMATWTGGATWGCWGATGARG AT" (SEQ ID NO. 18);
(vi) DNA coding for a peptide sequence "Val Arg Gly Gly Phe Gly" (SEQ ID NO. 19). A specific example of such a probe is "GTMMGAGGWG GWTTTGGR" (SEQ ID NO. 20);
(vii) DNA coding for a peptide sequence "Gly Thr Asn His Thr Arg Thr" (SEQ ID NO. 21). A specific example of such a probe is "GGWACRAAYC AYACMMGAAC W" (SEQ ID NO. 22);
(viii) DNA coding for a peptide sequence "Val Arg Trp Pro Gly Glu Ile" (SEQ ID NO. 23). A specific example of such a probe is "GTWMGATGGC CWGGWGARAT W" (SEQ ID NO. 24);
(ix) DNA coding for a peptide sequence "Ser Asp Ser Trp Arg Ala" (SEQ ID NO. 25). A specific example of such a probe is "AGTGATTCWT GGAGAGCW" (SEQ ID NO. 26).
Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the amino acid sequences of the B.t. toxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the subject invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art.
Recombinant hosts. The toxin-encoding genes harbored by the isolates of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable microbial hosts, e.g., Pseudomonas, live microbes can be applied to the situs of lepidopterans where they will proliferate and be ingested by the pest. The result is a control of this pest. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.
Where the B.t. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. For example, microorganism hosts can be selected which are known to occupy the soil. These microorganisms are selected so as to be capable of successfully competing in the soil with the wild-type microorganisms. It is also important that they provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
A large number of microorganisms are known to inhabit the rhizosphere (the soil surrounding plant roots). These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes and Clostridium; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium; microalgae, e.g., families Cyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae, Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromycesveronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A wide variety of ways are available for introducing a B.t. gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.
Treatment of cells. As mentioned above, B.t. or recombinant cells expressing a B.t. toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the B.t. toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.
The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.
Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host's environment. In one preferred embodiment, acids can be used to stabilize the cells. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.
The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of treatment should retain a substantial portion of the bio-availability or bioactivity of the toxin.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Growth of cells. The cellular host containing the B.t. insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The B.t. cells of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art.
Formulations. Formulated bait granules containing an attractant and spores and crystals of the B.t. isolates, or recombinant microbes comprising the gene(s) obtainable from the B.t. isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle.
As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 10.sup.2 to about 10.sup.4 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the lepidopteran, e.g., soil, by spraying, dusting, sprinkling, or the like.
Mutants. Mutants of the novel isolates of the invention can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of a novel isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
A smaller percentage of the asporogenous mutants will remain intact and not lyse for extended fermentation periods; these strains are designated lysis minus (-). Lysis minus strains can be identified by screening asporogenous mutants in shake flask media and selecting those mutants that are still intact and contain toxin crystals at the end of the fermentation. Lysis minus strains are suitable for a cell fixation process that will yield a protected, encapsulated toxin protein.
To prepare a phage resistant variant of said asporogenous mutant, an aliquot of the phage lysate is spread onto nutrient agar and allowed to dry. An aliquot of the phage sensitive bacterial strain is then plated directly over the dried lysate and allowed to dry. The plates are incubated at 30.degree. C. The plates are incubated for 2 days and, at that time, numerous colonies could be seen growing on the agar. Some of these colonies are picked and subcultured onto nutrient agar plates. These apparent resistant cultures are tested for resistance by cross streaking with the phage lysate. A line of the phage lysate is streaked on the plate and allowed to dry. The presumptive resistant cultures are then streaked across the phage line. Resistant bacterial cultures show no lysis anywhere in the streak across the phage line after overnight incubation at 30.degree. C. The resistance to phage is then reconfirmed by plating a lawn of the resistant culture onto a nutrient agar plate. The sensitive strain is also plated in the same manner to serve as the positive control. After drying, a drop of the phage lysate is plated in the center of the plate and allowed to dry. Resistant cultures showed no lysis in the area where the phage lysate has been placed after incubation at 30.degree. C. for 24 hours.
Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLE 1
Culturing of the B.t. Isolates of the Invention
A subculture of a novel B.t. isolate, or mutants thereof, can be used to inoculate the following medium, a peptone, glucose, salts medium.
______________________________________Bacto Peptone 7.5 g/lGlucose 1.0 g/lKH.sub.2 PO.sub.4 3.4 g/lK.sub.2 HPO.sub.4 4.35 g/lSalt Solution 5.0 ml/lCaCl.sub.2 Solution 5.0 ml/l______________________________________
Salts Solution (100 ml)
______________________________________ MgSO.sub.4 .multidot.7H.sub.2 O 2.46 g MnSO.sub.4 .multidot.H.sub.2 O 0.04 g ZnSO.sub.4 .multidot.7H.sub.2 O 0.28 g FeSO.sub.4 .multidot.7H.sub.2 O 0.40 g______________________________________
CaCl.sub.2 Solution (100 ml)
______________________________________ CaCl.sub.2 .multidot.2H.sub.2 O 3.66 g pH 7.2______________________________________
The salts solution and CaCl.sub.2 solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30 .degree. C. on a rotary shaker at 200 rpm for 64 hr.
The above procedure can be readily scaled up to large fermentors by procedures well known in the art.
The B.t. spores and/or crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.
EXAMPLE 2
Activity of B.t. Isolates Against Lepidopterans
The following strains have been tested for anti-lepidopteran activity with the following results:
TABLE 6______________________________________Bioassay results % MortalityStrain Trichoplusia ni Spodoptera exigua______________________________________PS81T1 96,8PS53C2 100, 100PS31F4 100, 100PS86V1 100PS86I2 100, 92PS73E 100, 100PS81K 100, 100PS83E2 100, 100PS81E 100, 92PS81Z3 100PS53B5 100PS83R 100PS53B2 100PS83N2 100PS81B5 100, 100PS86W1 100PS91C2 100, 100PS81A2 100, 100______________________________________
Spodoptera exigua bioassay procedure. B.t. cultures were harvested and resuspended in sterile deionized water. Fixed volumes of each culture were incorporated into USDA Insect Diet (Technical Bulletin 1528, U.S. Department of Agriculture, 1976). Twenty-four neonate S. exigua were exposed to the diet for 6 days. Mortality readings were taken at this time.
Trichoplusia ni bioassay procedure. B.t. cultures were harvested and resuspended in sterile deionized water. Fixed volumes of each culture were top loaded onto USDA Insect Diet. Trays were infested with neonate T. ni. After 6 days mortality was determined.
EXAMPLE 3
Characterization of Toxin Genes by RFLP Analysis
Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cells grown to an optical density, at 600 mn, of 1.0. The cells were recovered by centrifugation, and protoplasts were prepared in TES buffer (30 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl, pH=8.0) containing 20% sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a final concentration of 4%. The cellular material was precipitated overnight at 4.degree. C. in 100 mM (final concentration) neutral potassium chloride. The supernate was extracted twice with phenol/chloroform(1:1). The DNA was precipitated with ethanol and purified by isopycnic banding on a cesium chloride-ethidium bromide gradient.
Total cellular DNA isolated from B.t. cells was digested with a restriction endonuclease and separated by electrophoresison a 0.8% (w/v) agarose-TAE (50 mM Tris-HCl, 20 mM NaOAc, 2.5 mM EDTA, pH=8.0) buffered gel. A Southern blot of the gel was hybridized with the [.sup.32 P]-radiolabeled oligonucleotide probe, ATGATTCATGCGGCAGATA (SEQ ID NO. 5), and then washed to remove unbound radioactivity. The blot was exposed to KODAK X-OMAT.TM. film using standard autoradiography techniques. The results are an array of hybridizing bands (fingerprint) which correspond to toxin genes or toxin gene fragments. This type of characterization is known as Restriction Fragment Length Polymorphism (RFLP) analysis which classifies each isolate by a distinct DNA fingerprint.
TABLE 7______________________________________DNA fingerprints for B.t. isolates of the subject inventionIsolate Hybridizing HindIII Fragments (Kb)______________________________________PS81T1 1.13, 3.0, 9.4PS53C2 1.052, 5.8, 6.6PS31F4 5.5, 8.0PS86V1 5.5, 6.0, 6.6PS86I2 5.0, 6.6, 7.5, 12PS73E 1.052, 1.13, 3.0, 8.5PS81K 3.2, 7.5, 9.4, 13PS83E2 3.2, 8.5, 12PS81E 1.13, 3.2, 9.4PS81Z3 1.13, 3.0, 8.5PS53B5 1.13, 3.0, 7.5PS83R 1.13, 3.0, 8.5, 12PS53B2 1.052, 1.13, 3.0, 7.5PS83N2 5.5PS81B5 8.0, 13PS86W1 5.5, 6.6PS91C2 1.13, 3.0, 6.0, 7.5, 8.5PS81A2 13, 16______________________________________
TABLE 8______________________________________Hybridizing HindIII fragments of B.t. isolatesof the subject inventionIsolate Novel Hybridizing HindIII Fragments (.about.Kb)______________________________________PS91C2 3.0, 6.0, 7.5PS83E2 3.2PS86I2 5.0PS31F4 5.5, 8.0PS53C2 5.8PS81T1 9.4______________________________________
EXAMPLE 4
Molecular Cloning and Expression of a Novel CryIF Toxin Gene from Bacillus thuringiensis Strain PS91C2
Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cells grown to an optical density, at 600 nm, of 1.0. Cells were pelleted by centrifugation and resuspended in protoplast buffer (20 mg/ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After incubation at 37.degree. C. for 1 hour, protoplasts were lysed by two cycles of freezing and thawing. Nine volumes of a solution of 0.1 M NaCl, 0.1% SDS, 0.1 M Tris-Cl were added to complete lysis. The cleared lysate was extracted twice with phenol:chloroform (1:1). Nucleic acids were precipitated with two volumes of ethanol and pelleted by centrifugation. The pellet was resuspended in TE buffer and RNase was added to a final concentration of 50 .mu.g/ml. After incubation at 37.degree. C. for 1 hour, the solution was extracted once each with phenol:chloroform (1:1) and TE-saturated chloroform. DNA was precipitated from the aqueous phase by the addition of one-tenth volume of 3 M NaOAc and two volumes of ethanol. DNA was pelleted by centrifugation, washed with 70% ethanol, dried, and resuspended in TE buffer.
A 1.58 kbp fragment of the novel 130 kDa toxin gene was obtained by polymerase chain reaction (PCR) amplificationfrom PS91C2 cellular DNA using the following primers: forward 5'-GAGTGGGAAGCAGATCTTAATAATGCACAATTAAGG-3' (SEQ ID NO. 6) and reverse 5'-ATAC(C or T)CGATCGATATGATA(G or A)TCCGT-3' (SEQ ID NO. 7). This DNA fragment was cloned into pBluescript S/K (Stratagene, La Jolla, Calif.) and the DNA sequence determined by dideoxynucleotide sequencing methodology (Sanger et al. [1977] Proc. Natl. Acad. Sci USA 74:5463-5467) using Sequenase (U.S. Biochemicals, Cleveland, Ohio). DNA sequences unique to the CryIF gene were identified by computer comparison with other CryI genes. An oligonucleotide probe with the following sequence was synthesized: 5'-CCCAATGTGAATGTACTTTGCGC-3' (SEQ ID NO. 8). This probe was radiolabeled with .sup.32 P and used in standard hybridizations of Southern blots of PS91 C2 total cellular DNA. Hybridizing bands included an approximately 7.5 kbp HindIII fragment.
A gene library was constructed from PS91C2 DNA partially digested with NdeII. Partial restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 9.3 to 23 kbp in size were excised from the gel, electroeluted from the gel slice, purified on an Elutip D ion exchange column (Schleicher and Schuell, Keene, N.H.), and recovered by ethanol precipitation. The NdeII inserts were ligated into BamHI-digested LambdaGem-11 (Promega, Madison, Wis.). Recombinant phage were packaged and plated on E. coli KW251 cells. Plaques were screened by hybridization with each of the respective probes described above. Hybridizing phage were plaque-purified and used to infect liquid cultures of E. coli KW251 cells for isolation of DNA by standard procedures (Maniatis et al., supra).
For subcloning the gene encoding the 130 kDa CryIF toxin, preparative amounts of phage DNA were digested with Sau3A and electrophoresed on agarose gel. The approximately 8 kbp band containing the toxin gene was excised from the gel, electroeluted from the gel slice, and purified by ion exchange chromatography as described above. The purified DNA insert was ligated into an XhoI-digested pHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised of pBluescript S/K (Stratagene) and the replication origin from a resident B.t. plasmid (D. Lereclus et al. [1989] FEMS Microbiol. Lett. 60:211-218). The ligation mix was used to transform frozen, competent E. coli NM522 cells (ATCC 47000). .beta.-galactosidase transformants were screened by restriction digestion of alkaline lysate plasmid minipreps as above. The desired plasmid construct, pMYC2361, contains a toxin gene that is novel compared to other toxin genes containing insecticidal proteins.
pMYC2361 was introduced into the acrystalliferous (Cry.sup.-) B.t. host, CryB (A. Aronson, Purdue University, West Lafayette, Ind.) by electroporation. Expression of the 130 kDa toxin was demonstrated by SDS-PAGE analysis. NaBr-purified crystals were prepared (Pfannenstiel, M. A. et al. [1984] FEMS Microbiol. Lett. 21:39) for determination of toxicity of the cloned gene product to Plutella xylostella by the screening method described in Example 3. The LC.sub.50 for the CryIF toxin against P. xylostella was determined to be 5 .mu.g toxin/ml diet.
EXAMPLE 5
Insertion of Toxin Genes Into Plants
One aspect of the subject invention is the transformation of plants with genes encoding a lepidopteran toxin. The transformed plants are resistant to attack by lepidopterans.
Genes encoding lepidopteran-active toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.
The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 0 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J. 4:277-287.
Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, or electroporation as well as other possible methods. If agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into agrobacteria (Holsterset al. [1978] Mol. Gen. Genet. 163:181-187). The agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
EXAMPLE 6
Cloning of Novel B.t. Genes Into Insect Viruses
A number of viruses are known to infect insects. These viruses include, for example, baculoviruses and entomopoxviruses. In one embodiment of the subject invention, lepidopteran-active genes, as described herein, can be placed with the genome of the insect virus, thus enhancing the pathogenicity of the virus. Methods for constructing insect viruses which comprise B.t. toxin genes are well known and readily practiced by those skilled in the art. These procedures are described, for example, in Merryweather et al. (Merryweather, A. T., U. Weyer, M. P. G. Harris, M. Hirst, T. Booth, R. D. Possee [1990] J. Gen. Virol. 71:1535-1544) and Martens et al. (Martens, J. W. M., G. Honee, D. Zuidema, J. W. M. van Lent, B. Visser, J. M. Vlak [1990] Appl. Environmental Microbiol. 56(9):2764-2770).
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 27- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 3522 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE:#thuringiensisORGANISM: Bacillus (B) STRAIN: aizawai#PS81A2 (C) INDIVIDUAL ISOLATE:- (vii) IMMEDIATE SOURCE:#- 11 (tm) Library of August Sick (B) CLONE: 81A2- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- ATGGAGAATA ATATTGAAAA TCAATGCATA CCTTACAATT GTTTAAATAA TC - #CTGAAGTA 60- GAGATATTAG GGATTGAAAG GTCAAATAGT AACGTAGCAG CAGAAATCGG CT - #TGGGGCTT 120- AGTCGTCTGC TCGTTTCCCG AATTCCACTA GGGGATTTTA TACTTGGCTT GT - #TTGATGTA 180- ATATGGGGGG CTATAGGTCC TTCACAATGG GATATATTTT TAGAGCAAAT TG - #AGCTATTG 240- ATCGGCCAAA GAATAGAGGA ATTCGCTAGG AATCAGGCAA TTTCTAGATT AC - #AAGGGCTA 300- AGCAATCTTT ACCGAATTTA CACAAATGCT TTTAAAAACT GGGAAGTAGA TC - #CTACTAAT 360- CCAGCATTAA GAGAAGAGAT GCGTATTCAA TTTAATGACA TGAACAGTGC TC - #TTACAACA 420- GCTATTCCTC TTTTTTCAGT TCAAGGTTAT GAAATTCCTC TTTTATCAGT AT - #ATGTTCAA 480- GCTGCAAATT TACATTTATC GGTTTTGAGA GATGTTTCAG TGTTTGGACA AC - #GTTGGGGA 540- TTTGATGTAG CAACAATCAA TAGTCGTTAT AATGATTTAA CTAGGCTTAT TG - #GCGAATAT 600- ACTGATTATG CTGTACGTTG GTATAATACG GGGTTAAATC GTTTACCACG TA - #ATGAAGGG 660- GTACGAGGAT GGGCAAGATT TAATAGGTTT AGAAGAGAGT TAACAATATC AG - #TATTAGAT 720- ATTATTTCTT TTTTCCAAAA TTACGATTCT AGATTATATC CAATTCCGAC AA - #TCTATCAA 780- TTAACGCGGG AAGTATATAC AGATCCGGTA ATTAATATAA CTGATTATAG AG - #TTACCCCA 840- AGTTTCGAGA GTATTGAAAA TTCAGCTATT AGAAGTCCCC ATCTTATGGA TT - #TCTTAAAT 900- AATATAATTA TTGACACTGA TTTAATTAGA GGCGTTCACT ATTGGGCGGG GC - #ATCGTGTA 960- ACTTCTCATT TTACCGGTAG TTCGCAAGTG ATAAGCTCCC CTCAATACGG GA - #TAACTGCA1020- AACGCAGAAC CGAGTCGAAC TATTGCTCCT AGCACTTTTC CAGGTCTTAA TC - #TATTTTAT1080- AGAACACTAT CAGACCCTTT CTTCCGAAGA TCCGATAATA TTATGCCAAC AT - #TAGGAATA1140- AATGTAGTGC AGGGGGTAGG ATTCATTCAA CCAAATAATG GTGAAGTTCT AT - #ATAGAAGG1200- AGAGGAACAG TAGATTCTCT TGATGAGTTG CCAATTGACG GTGAGAATTC AT - #TAGTTGGA1260- TATAGTCATA GATTAAGTCA CGTTACATTA ACCAGGTCGT TATATAATAC TA - #ATATAACT1320- AGCTTGCCAA CATTTGTTTG GACACATCAC AGTGCTACTG ATCGAAATAT AA - #TCTATCCG1380- GATGTAATTA CACAAATACC ATTGGTAAAA TCATTCTCCC TTACTTCAGG TA - #CCTCTGTA1440- GTCAGAGGCC CAGGATTTAC AGGAGGGGAT ATCATCCGAA CTAACGTTAA TG - #GTAATGTA1500- CTAAGTATGA GTCTTAATTT TAGTAATACA TCATTACAGC GGTATCGCGT GA - #GAGTTCGT1560- TATGCTGCTT CTCAAACAAT GGTCATGAGA GTAAATGTTG GAGGGAGTAC TA - #CTTTTGAT1620- CAAGGATTCC CTAGTACTAT GAGTGCAAAT GGGTCTTTGA CATCTCAATC AT - #TTAGATTT1680- GCAGAATTTC CTGTAGGCAT TAGTACATCT GGCAGTCAAA CTGCTGGAAT AA - #GTATAAGT1740- AATAATCCAG GTAGACAAAC GTTTCACTTA GATAGAATTG AATTTATCCC AG - #TTGATGCA1800- ACATTTGAAG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATTC GC - #TGTTTACT1860- TCTTCCAATC AAATCGAGTT AAAAACAGAT GTGACGGATT ATCATATTGA TC - #AAGTATCC1920- AATTTAGTAG ATTGTTTATC CGATGAATTT TGTCTGGATG AAAAGCGAGA AT - #TGTCCGAG1980- AAAGTCAAAC ATGCGAAGCG ACTCAGTGAT GAGCGGAATT TACTTCAAGA TC - #CAAACTTC2040- AGAGGGATCA ATAGGCAACC AGACCGTGGC TGGAGAGGAA GTACGGATAT TA - #CCATCCAA2100- GGAGGAGATG ACGTATTCAA AGAGAATTAC GTCACACTAC CAGGTACCTT TG - #ATGAGTGC2160- TATCCAACGT ATTTGTATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TA - #ACCGTTAC2220- CAATTAAGAG GGTATATCGA AGATAGTCAA GACTTAGAAA TCTATTTAAT TC - #GCTACAAT2280- GCAAAACACG AAACAGTAAA TGTACCAGGT ACGGGTTCCT TATGGCCGCT TT - #CAGTCGAA2340- AGTCCAATTG GAAGGTGTGG AGAACCGAAT CGGTGTGTGC CACACCTTGA AT - #GGAATCCT2400- GATTTAGATT GTTCCTGCAG AGACGGGGAA AAATGTGCAC ATCATTCCCA TC - #ATTTCTCC2460- TTGGACATTG ATGTTGGATG CACAGACTTG CAAGAGGATC TAGGCGTGTG GG - #TTGTATTC2520- AAGATTAAGA CGCAGGAAGG TTATGCAAGA TTAGGAAATC TGGAATTTAT CG - #AAGAGAAA2580- CCATTAATTG GAGAAGCACT GTCTCGTGTG AAGAGAGCGG AAAAAAAATG GA - #GAGACAAA2640- CGGGAAAAAC TACAATTGGA AACAAAACGA GTATATACAG AGGCAAAAGA AG - #CTGTGGAT2700- GCTTTATTCG TAGATTCTCA ATATGATAGA TTACAAGCAG ATACAAACAT TG - #GTATGATT2760- CATGCGGCAG ATAGACTTGT TCATCAGATC CACGAGGCTT ATCTTCCAGA AC - #TACCTTTC2820- ATTCCAGGAA TAAATGTGGT GATTTTTGAA GAATTAGAAA ACCGTATTTC TA - #CTGCATTA2880- TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGCGATT TCAATAATGG CT - #TATCATGC2940- TGGAACGTGA AAGGGCATGT AGATGTAGTA GAACAAAACA ACCACCGTTC GG - #TCCTTGTT3000- GTCCCGGAAT GGGAAGCAGA AGTGTCACAA ACAATTCGTG TCTGTCCGGG GC - #GTGGCTAT3060- ATCCTCCGTG TTACAGCGTA CAAAGAGGGA TATGGAGAAG GTTGCGTAAC CA - #TCCATGAG3120- ATCGAGAACA ATACAGACGA ACTAAAATTT AAAAACTGTG AAGAAGAGGA AG - #TGTATCCA3180- ACGGATACAG GAACGTGTAA TGATTATACT GCACACCAAG GTACAGCAGG AT - #CCACAGAT3240- TCATGTAATT CCCGTAATAT CAGATATGAG GATGCATATG AAATGAATAC TA - #CAGCATCT3300- GTTAATTACA AACCGACTTA CGAAGAAGAA AGGTATACAG ATGTACAAGG AG - #ATAATCAT3360- TGTGAATATG ACAGAGGGTA TGTGAATTAT CGACCAGTAC CAGCTGGTTA TG - #TGACAAAA3420- GAATTAGAGT ACTTCCCAGA AACCGATAAG GTATGGATTG AGATCGGAGA AA - #CGGAAGGG3480#3522 ATGT CGAATTACTC CTTATGGAGG AA- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1174 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (iii) HYPOTHETICAL: YES- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE:#thuringiensisORGANISM: Bacillus (B) STRAIN: aizawai#PS81A2 (C) INDIVIDUAL ISOLATE:- (vii) IMMEDIATE SOURCE:#- 11 (tm) Library of August Sick (B) CLONE: 81A2- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Glu Asn Asn Ile Glu Asn Gln Cys Ile Pr - #o Tyr Asn Cys Leu Asn# 15- Asn Pro Glu Val Glu Ile Leu Gly Ile Glu Ar - #g Ser Asn Ser Asn Val# 30- Ala Ala Glu Ile Gly Leu Gly Leu Ser Arg Le - #u Leu Val Ser Arg Ile# 45- Pro Leu Gly Asp Phe Ile Leu Gly Leu Phe As - #p Val Ile Trp Gly Ala# 60- Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Gl - #u Gln Ile Glu Leu Leu#80- Ile Gly Gln Arg Ile Glu Glu Phe Ala Arg As - #n Gln Ala Ile Ser Arg# 95- Leu Gln Gly Leu Ser Asn Leu Tyr Arg Ile Ty - #r Thr Asn Ala Phe Lys# 110- Asn Trp Glu Val Asp Pro Thr Asn Pro Ala Le - #u Arg Glu Glu Met Arg# 125- Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Th - #r Thr Ala Ile Pro Leu# 140- Phe Ser Val Gln Gly Tyr Glu Ile Pro Leu Le - #u Ser Val Tyr Val Gln145 1 - #50 1 - #55 1 -#60- Ala Ala Asn Leu His Leu Ser Val Leu Arg As - #p Val Ser Val Phe Gly# 175- Gln Arg Trp Gly Phe Asp Val Ala Thr Ile As - #n Ser Arg Tyr Asn Asp# 190- Leu Thr Arg Leu Ile Gly Glu Tyr Thr Asp Ty - #r Ala Val Arg Trp Tyr# 205- Asn Thr Gly Leu Asn Arg Leu Pro Arg Asn Gl - #u Gly Val Arg Gly Trp# 220- Ala Arg Phe Asn Arg Phe Arg Arg Glu Leu Th - #r Ile Ser Val Leu Asp225 2 - #30 2 - #35 2 -#40- Ile Ile Ser Phe Phe Gln Asn Tyr Asp Ser Ar - #g Leu Tyr Pro Ile Pro# 255- Thr Ile Tyr Gln Leu Thr Arg Glu Val Tyr Th - #r Asp Pro Val Ile Asn# 270- Ile Thr Asp Tyr Arg Val Thr Pro Ser Phe Gl - #u Ser Ile Glu Asn Ser# 285- Ala Ile Arg Ser Pro His Leu Met Asp Phe Le - #u Asn Asn Ile Ile Ile# 300- Asp Thr Asp Leu Ile Arg Gly Val His Tyr Tr - #p Ala Gly His Arg Val305 3 - #10 3 - #15 3 -#20- Thr Ser His Phe Thr Gly Ser Ser Gln Val Il - #e Ser Ser Pro Gln Tyr# 335- Gly Ile Thr Ala Asn Ala Glu Pro Ser Arg Th - #r Ile Ala Pro Ser Thr# 350- Phe Pro Gly Leu Asn Leu Phe Tyr Arg Thr Le - #u Ser Asp Pro Phe Phe# 365- Arg Arg Ser Asp Asn Ile Met Pro Thr Leu Gl - #y Ile Asn Val Val Gln# 380- Gly Val Gly Phe Ile Gln Pro Asn Asn Gly Gl - #u Val Leu Tyr Arg Arg385 3 - #90 3 - #95 4 -#00- Arg Gly Thr Val Asp Ser Leu Asp Glu Leu Pr - #o Ile Asp Gly Glu Asn# 415- Ser Leu Val Gly Tyr Ser His Arg Leu Ser Hi - #s Val Thr Leu Thr Arg# 430- Ser Leu Tyr Asn Thr Asn Ile Thr Ser Leu Pr - #o Thr Phe Val Trp Thr# 445- His His Ser Ala Thr Asp Arg Asn Ile Ile Ty - #r Pro Asp Val Ile Thr# 460- Gln Ile Pro Leu Val Lys Ser Phe Ser Leu Th - #r Ser Gly Thr Ser Val465 4 - #70 4 - #75 4 -#80- Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Il - #e Ile Arg Thr Asn Val# 495- Asn Gly Asn Val Leu Ser Met Ser Leu Asn Ph - #e Ser Asn Thr Ser Leu# 510- Gln Arg Tyr Arg Val Arg Val Arg Tyr Ala Al - #a Ser Gln Thr Met Val# 525- Met Arg Val Asn Val Gly Gly Ser Thr Thr Ph - #e Asp Gln Gly Phe Pro# 540- Ser Thr Met Ser Ala Asn Gly Ser Leu Thr Se - #r Gln Ser Phe Arg Phe545 5 - #50 5 - #55 5 -#60- Ala Glu Phe Pro Val Gly Ile Ser Thr Ser Gl - #y Ser Gln Thr Ala Gly# 575- Ile Ser Ile Ser Asn Asn Pro Gly Arg Gln Th - #r Phe His Leu Asp Arg# 590- Ile Glu Phe Ile Pro Val Asp Ala Thr Phe Gl - #u Ala Glu Tyr Asp Leu# 605- Glu Arg Ala Gln Lys Ala Val Asn Ser Leu Ph - #e Thr Ser Ser Asn Gln# 620- Ile Glu Leu Lys Thr Asp Val Thr Asp Tyr Hi - #s Ile Asp Gln Val Ser625 6 - #30 6 - #35 6 -#40- Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cy - #s Leu Asp Glu Lys Arg# 655- Glu Leu Ser Glu Lys Val Lys His Ala Lys Ar - #g Leu Ser Asp Glu Arg# 670- Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Il - #e Asn Arg Gln Pro Asp# 685- Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Il - #e Gln Gly Gly Asp Asp# 700- Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gl - #y Thr Phe Asp Glu Cys705 7 - #10 7 - #15 7 -#20- Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Gl - #u Ser Lys Leu Lys Ala# 735- Tyr Asn Arg Tyr Gln Leu Arg Gly Tyr Ile Gl - #u Asp Ser Gln Asp Leu# 750- Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys Hi - #s Glu Thr Val Asn Val# 765- Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Va - #l Glu Ser Pro Ile Gly# 780- Arg Cys Gly Glu Pro Asn Arg Cys Val Pro Hi - #s Leu Glu Trp Asn Pro785 7 - #90 7 - #95 8 -#00- Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Ly - #s Cys Ala His His Ser# 815- His His Phe Ser Leu Asp Ile Asp Val Gly Cy - #s Thr Asp Leu Gln Glu# 830- Asp Leu Gly Val Trp Val Val Phe Lys Ile Ly - #s Thr Gln Glu Gly Tyr# 845- Ala Arg Leu Gly Asn Leu Glu Phe Ile Glu Gl - #u Lys Pro Leu Ile Gly# 860- Glu Ala Leu Ser Arg Val Lys Arg Ala Glu Ly - #s Lys Trp Arg Asp Lys865 8 - #70 8 - #75 8 -#80- Arg Glu Lys Leu Gln Leu Glu Thr Lys Arg Va - #l Tyr Thr Glu Ala Lys# 895- Glu Ala Val Asp Ala Leu Phe Val Asp Ser Gl - #n Tyr Asp Arg Leu Gln# 910- Ala Asp Thr Asn Ile Gly Met Ile His Ala Al - #a Asp Arg Leu Val His# 925- Gln Ile His Glu Ala Tyr Leu Pro Glu Leu Pr - #o Phe Ile Pro Gly Ile# 940- Asn Val Val Ile Phe Glu Glu Leu Glu Asn Ar - #g Ile Ser Thr Ala Leu945 9 - #50 9 - #55 9 -#60- Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys As - #n Gly Asp Phe Asn Asn# 975- Gly Leu Ser Cys Trp Asn Val Lys Gly His Va - #l Asp Val Val Glu Gln# 990- Asn Asn His Arg Ser Val Leu Val Val Pro Gl - #u Trp Glu Ala Glu Val# 10050- Ser Gln Thr Ile Arg Val Cys Pro Gly Arg Gl - #y Tyr Ile Leu Arg Val# 10205- Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cy - #s Val Thr Ile His Glu# 10401030 - # 1035- Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ly - #s Asn Cys Glu Glu Glu# 10550- Glu Val Tyr Pro Thr Asp Thr Gly Thr Cys As - #n Asp Tyr Thr Ala His# 10705- Gln Gly Thr Ala Gly Ser Thr Asp Ser Cys As - #n Ser Arg Asn Ile Arg# 10850- Tyr Glu Asp Ala Tyr Glu Met Asn Thr Thr Al - #a Ser Val Asn Tyr Lys# 11005- Pro Thr Tyr Glu Glu Glu Arg Tyr Thr Asp Va - #l Gln Gly Asp Asn His# 11201110 - # 1115- Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Ar - #g Pro Val Pro Ala Gly# 11350- Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Gl - #u Thr Asp Lys Val Trp# 11505- Ile Glu Ile Gly Glu Thr Glu Gly Lys Phe Il - #e Val Asp Asn Val Glu# 11650- Leu Leu Leu Met Glu Glu 1170- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 3504 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE:#thuringiensisORGANISM: Bacillus (B) STRAIN: Morrissoni#PS91C2 (C) INDIVIDUAL ISOLATE:- (vii) IMMEDIATE SOURCE: 11 Library of Teresa: LambdaGem Thompson (B) CLONE: 91C2- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- ATGAAGAATA ACATTCAAAA TCAATGCGTA CCTTACAATT GTTTAAGTAA TC - #CTGAAGTA 60- GAAATATTAA GTGAAGAAAG AAGTACTGGC AGATTACCGT TAGATATATC CT - #TGTCGCTT 120- ACACGTTTCC TTTTGAGTGA ATTTGTTCCA GGTGTGGGAG TTGCGTTTGG AT - #TATTTGAT 180- TTAATATGGG GTTTTATAAC TCCTTCTGAA TGGAGTTTAT TTCTTTTACA GA - #TTGAACAA 240- CTGATTGAAC AAAGAATTGA AACATTGGAA AGGAACCGGG CAATTACTAC AT - #TACGAGGG 300- TTAGCGGATA GCTATGAAGT TTACCTTGAG GCACTAAGAG AGTGGGAAGA AA - #ATCCTAAT 360- AATGCACAAT TAAGGGAAGA TGTGCGTATT CGATTTGCTA ATACAGACGA CG - #CTTTAATA 420- ACAGCAATAA ATAATTTTAC ACTTACAAGT TTTGAAATCC CTCTTTTATC GG - #TCTATGTT 480- CAAGCGGCGA ATCTACATTT ATCACTATTA AGAGATGCTG TATCGTTTGG GC - #AGGGTTGG 540- GGGCTGGATA TAGCTACTGT TAATAATCAT TATAATAGAT TAATAAATCT TA - #TTCATAGA 600- TATACGGAAC ATTGTTTGGA CACATACAAT CAAGGATTAG AAAACTTAAG AG - #GTACTAAT 660- ACTCGACAAT GGTCAAGATT CAATCAGTTT AGGAGAGAGT TAACATTGAC TG - #TATTAGAT 720- ATCGTTGCTC TTTTTCCGAA CTACGATGCT AGAGCATATC CAATTCAAAC GT - #CATCCCAA 780- TTAACAAGGG AAATTTATAC AAGTTCAGTA ATTGAAGATT CTCCAGTTTC TG - #CTAATATA 840- CCTAATGGTT TTAATAGAGC GGAATTTGGA GTTAGACCGC CCCATCTTAT GG - #ACTTTATG 900- AATTCTTTGT TTGTAACTGC AGAGACTGTT AGAAGTCAAA CTGTGTGGGG AG - #GACACTTA 960- GTTAGTTCAC GAAATACGGC TGGTAACCCT ATAAATTTCC CTATTTATGG GG - #TCTTCAAT1020- CCTGGTGGCG CCATTTGGAT TGCAGATGAG GATCCACGTC CTTTTTATCG GA - #CATTATCA1080- GATCCTGTTT TTGTCCGAGG AGGATTTGGG GATCCTCATT ATGTACTTGG GC - #TTAGGGGA1140- GTAGGATTTC AACAAACTGG TACGAACCAC ACCCGAACAT TTAGAAATAG TG - #GGACCATA1200- GATTCTCTAG ATGAAATCCC ACCTCAGGAT AATAGTGGGG CACCTTGGAA TG - #ATTATAGT1260- CATGTATTAA ATCATGTTAC ATTTGTAAGG TGGCCTGGTG AGATTGCAGG AA - #GTGATTCA1320- TGGAGAGCGC CAATGTTTTC TTGGACACAC CGTAGTGCAG ATCGTACAAA TA - #TCATTAAT1380- CCAAATATAA TTACACAAAT ACCTGCTGTA AAAGCACACA ATCTTCATTC GG - #GTTCTACG1440- GTTGTTAGAG GACCCGGGTT TACAGGTGGT GATCTCTTAC GAAGAACGAA TA - #CTGGTACA1500- TTTGCAGATA TAAGAGTAAA TATTACTGGG CCATTATCTC AAAGATATCG TG - #TAAGAATT1560- CGCTATGCTT CTACGACAGA TTTACAATTT TTCACGAGAA TCAATGGAAC TT - #CTGTAAAT1620- CAAGGTAATT TCCAAAGAAC TATGAATAGA GGGGATAATT TAGAATCTGG AA - #ACTTTAGG1680- ACTGCAGGAT TTAGTACGCC TTTTAGTTTT TCAAATGCGC AAAGTACATT CA - #CATTGGGT1740- ACTCAGGCTT TTTCAAATCA GGAAGTTTAT ATAGATCGAA TTGAATTTGT CC - #CGGCAGAA1800- GTAACATTCG AGGCAGAATC TGATTTAGAA AGAGCGCAAA AGGCGGTGAA TG - #CCCTGTTT1860- ACTTCTACAA GCCAACTAGG GCTAAAAACA AATGTAACGG GTTACCATAT TG - #ATCAAGTG1920- TCCAATTTAG TTGCGTGTTT ATCGGATGAA TTTTGTCTGG ATGAAAAGAG AG - #AATTGTCC1980- GAGAAAGTTA AACATGCGAA GCGACTCAGT GATAAGCGGA ATTTACTTCA AG - #ATCCAAAC2040- TTCAGAGGGA TCAATAGGCA ACCAGACCAT GGCTGGAGAG GAAGTACGGA TA - #TTACTATC2100- CAAGGAGGAG ATGACGTATT CAAAGAGAAT TACGTTACGC TACCGGGTAC TT - #TTGATGAG2160- TGCTATCCAA CGTATTTATA TCAAAAAATA GATGAGTCGA AATTAAAAGC CT - #ATACCCGT2220- TATCAATTAA GAGGGTATAT CGAAGATAGT CAAGACTTAG AAATCTATTT AA - #TTCGTTAC2280- AATTCAAAAC ACGAAATAGT AAATGTACCA GGTACAGGGA GTTTATGGCC TC - #TTTCTGTA2340- GAAAATCAAA TTGGACCTTG TGGAGAACCG AATCGATGCG CGCCACACCT TG - #AATGGAAT2400- CCTGATTTAC ACTGTTCCTG CAGAGACGGG GAAAAATGTG TGCATCATTC TC - #ATCATTTC2460- TCTTTGGACA TTGATGTCGG ATGTACAGAT TTAAATGAGG ACCTAGGTGT AT - #GGTTGATA2520- TTCAAGATTA AGACGCAAGA TGGCCACGCA AGACTAGGGA ATCTAGAGTT TC - #TCGAAGAG2580- GAACCGTTAT TAGGCGAAGC GTTAGGACGT GTGAAGAGAG CGGAGAAGAA GT - #GGAGAGAC2640- AAACGCGAGA AACTGCAGTT GGAAACAAAT ATTGTCTATA AAGAGGCAAA AG - #AATCTGTA2700- GATGCTTTAT TTGTAAACTC TCAATATGAT AGATTACAAG CGGATACGAA CA - #TCGCGATG2760- ATTCATGCGG CAGATAAACG CGTTCATAGA ATCCGGGAAG CGTATCTGCC AG - #AGTTGTCT2820- GTGATTCCAG GTGTCAATGC GGCCATTTTC GAAGAATTAG AGGGACGTAT TT - #TTACAGCG2880- TATTCCTTAT ATGATGCGAG AAATGTTATT AAAAATGGCA ATTTCAATAA TG - #GCTTATTA2940- TGCTGGAACG TGAAAGGGCA TGTAGATGTA GAAGAGCAAA ACAACCACCG TT - #CGGTCCTT3000- GTTGTTCCGG AATGGGAAGC AGAAGTGTCA CAAGAAGTTC GTGTCTGTCC GG - #GTCGTGGC3060- TATATCCTTC GTGTCACAGC GTACAAAGAG GGATATGGAG AAGGCTGCGT AA - #CTATTCAT3120- GAAGTCGATA ATAATACAGA CGAATTGAAG TTTAGCAACT GTGAGAAAGA AC - #AAGTATAT3180- CCAGGTAATA CGGTAGCATG TAATGATTAT AATAAGAATC ACGGTGCGAA TG - #CATGTAGT3240- TCTCGTAATC GTGGATATGA CGAATCTTAT GAAAGTAATT CTTCCATACC AG - #CTGATTAT3300- GCACCGGTTT ATGAAGAAGA AGCGTATACA GATGGACAAA GAGGGAATCC TT - #GTGAATTT3360- AACAGAGGGC ATACACCATT ACCAGCTGGT TATGTGACAG CAGAGTTAGA GT - #ACTTCCCA3420- GAAACGGATA CAGTATGGGT TGAGATTGGA GAAACGGAAG GAACATTTAT CG - #TGGACAGT3480# 3504TGGA GGAA- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1168 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (iii) HYPOTHETICAL: YES- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE:#THURINGIENSISORGANISM: BACILLUS (B) STRAIN: Morrissoni#PS91C2 (C) INDIVIDUAL ISOLATE:- (vii) IMMEDIATE SOURCE: 11 LIBRARY OF TERESA: LAMBDAGEM THOMPSON (B) CLONE: 91C2- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Met Lys Asn Asn Ile Gln Asn Gln Cys Val Pr - #o Tyr Asn Cys Leu Ser# 15- Asn Pro Glu Val Glu Ile Leu Ser Glu Glu Ar - #g Ser Thr Gly Arg Leu# 30- Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Ph - #e Leu Leu Ser Glu Phe# 45- Val Pro Gly Val Gly Val Ala Phe Gly Leu Ph - #e Asp Leu Ile Trp Gly# 60- Phe Ile Thr Pro Ser Glu Trp Ser Leu Phe Le - #u Leu Gln Ile Glu Gln#80- Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Ar - #g Asn Arg Ala Ile Thr# 95- Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Va - #l Tyr Leu Glu Ala Leu# 110- Arg Glu Trp Glu Glu Asn Pro Asn Asn Ala Gl - #n Leu Arg Glu Asp Val# 125- Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Le - #u Ile Thr Ala Ile Asn# 140- Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Le - #u Leu Ser Val Tyr Val145 1 - #50 1 - #55 1 -#60- Gln Ala Ala Asn Leu His Leu Ser Leu Leu Ar - #g Asp Ala Val Ser Phe# 175- Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Va - #l Asn Asn His Tyr Asn# 190- Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Gl - #u His Cys Leu Asp Thr# 205- Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Th - #r Asn Thr Arg Gln Trp# 220- Ser Arg Phe Asn Gln Phe Arg Arg Glu Leu Th - #r Leu Thr Val Leu Asp225 2 - #30 2 - #35 2 -#40- Ile Val Ala Leu Phe Pro Asn Tyr Asp Ala Ar - #g Ala Tyr Pro Ile Gln# 255- Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Th - #r Ser Ser Val Ile Glu# 270- Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gl - #y Phe Asn Arg Ala Glu# 285- Phe Gly Val Arg Pro Pro His Leu Met Asp Ph - #e Met Asn Ser Leu Phe# 300- Val Thr Ala Glu Thr Val Arg Ser Gln Thr Va - #l Trp Gly Gly His Leu305 3 - #10 3 - #15 3 -#20- Val Ser Ser Arg Asn Thr Ala Gly Asn Pro Il - #e Asn Phe Pro Ile Tyr# 335- Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Il - #e Ala Asp Glu Asp Pro# 350- Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Va - #l Phe Val Arg Gly Gly# 365- Phe Gly Asp Pro His Tyr Val Leu Gly Leu Ar - #g Gly Val Gly Phe Gln# 380- Gln Thr Gly Thr Asn His Thr Arg Thr Phe Ar - #g Asn Ser Gly Thr Ile385 3 - #90 3 - #95 4 -#00- Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp As - #n Ser Gly Ala Pro Trp# 415- Asn Asp Tyr Ser His Val Leu Asn His Val Th - #r Phe Val Arg Trp Pro# 430- Gly Glu Ile Ala Gly Ser Asp Ser Trp Arg Al - #a Pro Met Phe Ser Trp# 445- Thr His Arg Ser Ala Asp Arg Thr Asn Ile Il - #e Asn Pro Asn Ile Ile# 460- Thr Gln Ile Pro Ala Val Lys Ala His Asn Le - #u His Ser Gly Ser Thr465 4 - #70 4 - #75 4 -#80- Val Val Arg Gly Pro Gly Phe Thr Gly Gly As - #p Leu Leu Arg Arg Thr# 495- Asn Thr Gly Thr Phe Ala Asp Ile Arg Val As - #n Ile Thr Gly Pro Leu# 510- Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr Al - #a Ser Thr Thr Asp Leu# 525- Gln Phe Phe Thr Arg Ile Asn Gly Thr Ser Va - #l Asn Gln Gly Asn Phe# 540- Gln Arg Thr Met Asn Arg Gly Asp Asn Leu Gl - #u Ser Gly Asn Phe Arg545 5 - #50 5 - #55 5 -#60- Thr Ala Gly Phe Ser Thr Pro Phe Ser Phe Se - #r Asn Ala Gln Ser Thr# 575- Phe Thr Leu Gly Thr Gln Ala Phe Ser Asn Gl - #n Glu Val Tyr Ile Asp# 590- Arg Ile Glu Phe Val Pro Ala Glu Val Thr Ph - #e Glu Ala Glu Ser Asp# 605- Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Le - #u Phe Thr Ser Thr Ser# 620- Gln Leu Gly Leu Lys Thr Asn Val Thr Gly Ty - #r His Ile Asp Gln Val625 6 - #30 6 - #35 6 -#40- Ser Asn Leu Val Ala Cys Leu Ser Asp Glu Ph - #e Cys Leu Asp Glu Lys# 655- Arg Glu Leu Ser Glu Lys Val Lys His Ala Ly - #s Arg Leu Ser Asp Lys# 670- Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gl - #y Ile Asn Arg Gln Pro# 685- Asp His Gly Trp Arg Gly Ser Thr Asp Ile Th - #r Ile Gln Gly Gly Asp# 700- Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pr - #o Gly Thr Phe Asp Glu705 7 - #10 7 - #15 7 -#20- Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile As - #p Glu Ser Lys Leu Lys# 735- Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Il - #e Glu Asp Ser Gln Asp# 750- Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ser Ly - #s His Glu Ile Val Asn# 765- Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Se - #r Val Glu Asn Gln Ile# 780- Gly Pro Cys Gly Glu Pro Asn Arg Cys Ala Pr - #o His Leu Glu Trp Asn785 7 - #90 7 - #95 8 -#00- Pro Asp Leu His Cys Ser Cys Arg Asp Gly Gl - #u Lys Cys Val His His# 815- Ser His His Phe Ser Leu Asp Ile Asp Val Gl - #y Cys Thr Asp Leu Asn# 830- Glu Asp Leu Gly Val Trp Leu Ile Phe Lys Il - #e Lys Thr Gln Asp Gly# 845- His Ala Arg Leu Gly Asn Leu Glu Phe Leu Gl - #u Glu Glu Pro Leu Leu# 860- Gly Glu Ala Leu Gly Arg Val Lys Arg Ala Gl - #u Lys Lys Trp Arg Asp865 8 - #70 8 - #75 8 -#80- Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Il - #e Val Tyr Lys Glu Ala# 895- Lys Glu Ser Val Asp Ala Leu Phe Val Asn Se - #r Gln Tyr Asp Arg Leu# 910- Gln Ala Asp Thr Asn Ile Ala Met Ile His Al - #a Ala Asp Lys Arg Val# 925- His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Le - #u Ser Val Ile Pro Gly# 940- Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gl - #y Arg Ile Phe Thr Ala945 9 - #50 9 - #55 9 -#60- Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Ly - #s Asn Gly Asn Phe Asn# 975- Asn Gly Leu Leu Cys Trp Asn Val Lys Gly Hi - #s Val Asp Val Glu Glu# 990- Gln Asn Asn His Arg Ser Val Leu Val Val Pr - #o Glu Trp Glu Ala Glu# 10050- Val Ser Gln Glu Val Arg Val Cys Pro Gly Ar - #g Gly Tyr Ile Leu Arg# 10205- Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gl - #y Cys Val Thr Ile His# 10401030 - # 1035- Glu Val Asp Asn Asn Thr Asp Glu Leu Lys Ph - #e Ser Asn Cys Glu Lys# 10550- Glu Gln Val Tyr Pro Gly Asn Thr Val Ala Cy - #s Asn Asp Tyr Asn Lys# 10705- Asn His Gly Ala Asn Ala Cys Ser Ser Arg As - #n Arg Gly Tyr Asp Glu# 10850- Ser Tyr Glu Ser Asn Ser Ser Ile Pro Ala As - #p Tyr Ala Pro Val Tyr# 11005- Glu Glu Glu Ala Tyr Thr Asp Gly Gln Arg Gl - #y Asn Pro Cys Glu Phe# 11201110 - # 1115- Asn Arg Gly His Thr Pro Leu Pro Ala Gly Ty - #r Val Thr Ala Glu Leu# 11350- Glu Tyr Phe Pro Glu Thr Asp Thr Val Trp Va - #l Glu Ile Gly Glu Thr# 11505- Glu Gly Thr Phe Ile Val Asp Ser Val Glu Le - #u Leu Leu Met Glu Glu# 11650- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:# 19 ATA- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:# 36 TTAA TAATGCACAA TTAAGG- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:# 25 ATAR TCCGT- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:# 23TTTG CGC- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Ser Thr Gly Arg Leu Pro Leu Asp#5- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:# 24CRTT RGAY- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Glu Asp Ser Pro Val Ser Ala Asn#5- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:# 24CWGC WAAT- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 10 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:- Asn Gly Phe Asn Arg Ala Glu Phe Gly Val#10- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:# 31 CTGA ATTTGGGAGT W- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 10 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:- Val Thr Ala Glu Thr Val Arg Ser Gln Thr#10- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:# 30 TWAG WAGTCAAACW- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 13 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:- Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Al - #a Asp Glu#10- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:# 42 GMGC MATWTGGATW GCWGATGARG AT- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 6 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- Val Arg Gly Gly Phe Gly#5- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:# 18 GR- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 7 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:- Gly Thr Asn His Thr Arg Thr#5- (2) INFORMATION FOR SEQ ID NO:22:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:#21 GAAC W- (2) INFORMATION FOR SEQ ID NO:23:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 7 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:- Val Arg Trp Pro Gly Glu Ile#5- (2) INFORMATION FOR SEQ ID NO:24:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:#21 ARAT W- (2) INFORMATION FOR SEQ ID NO:25:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 6 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:- Ser Asp Ser Trp Arg Ala#5- (2) INFORMATION FOR SEQ ID NO:26:- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (synthetic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:# 18 CW- (2) INFORMATION FOR SEQ ID NO:27:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 1174 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:- Met Xaa Asn Asn Ile Gln Asn Gln Cys Val Pr - #o Tyr Asn Cys Leu Xaa# 15- Asn Pro Glu Val Glu Ile Leu Xaa Glu Glu Ar - #g Ser Thr Gly Arg Leu# 30- Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Ph - #e Leu Leu Ser Glu Phe# 45- Val Pro Gly Val Gly Val Ala Phe Gly Leu Ph - #e Asp Leu Ile Trp Gly# 60- Phe Ile Thr Pro Ser Xaa Trp Ser Leu Phe Le - #u Leu Gln Ile Glu Gln#80- Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Ar - #g Asn Arg Ala Ile Thr# 95- Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Xa - #a Tyr Xaa Glu Ala Leu# 110- Arg Glu Trp Glu Xaa Asn Pro Asn Asn Ala Gl - #n Leu Arg Glu Asp Val# 125- Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Le - #u Ile Thr Ala Ile Asn# 140- Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Le - #u Leu Ser Val Tyr Val145 1 - #50 1 - #55 1 -#60- Gln Ala Ala Asn Leu His Leu Ser Leu Leu Ar - #g Asp Ala Val Ser Phe# 175- Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Va - #l Asn Asn His Tyr Asn# 190- Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Xa - #a His Cys Leu Asp Thr# 205- Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Th - #r Asn Thr Arg Gln Trp# 220- Xaa Arg Phe Asn Gln Phe Arg Arg Xaa Leu Th - #r Leu Thr Val Leu Asp225 2 - #30 2 - #35 2 -#40- Ile Val Ala Leu Phe Pro Asn Tyr Asp Xaa Ar - #g Xaa Tyr Pro Ile Gln# 255- Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Th - #r Ser Ser Val Ile Glu# 270- Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gl - #y Phe Asn Arg Ala Glu# 285- Phe Gly Val Arg Pro Pro His Leu Met Asp Ph - #e Met Asn Ser Leu Phe# 300- Val Thr Ala Glu Thr Val Arg Ser Gln Thr Va - #l Trp Gly Gly His Leu305 3 - #10 3 - #15 3 -#20- Val Ser Ser Arg Asn Thr Ala Gly Asn Xaa Il - #e Asn Phe Pro Xaa Tyr# 335- Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Il - #e Ala Asp Glu Asp Pro# 350- Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Va - #l Phe Val Arg Gly Gly# 365- Phe Gly Xaa Pro His Tyr Val Leu Gly Leu Ar - #g Gly Val Xaa Phe Gln# 380- Gln Thr Gly Thr Asn His Thr Arg Thr Phe Ar - #g Asn Ser Gly Thr Ile385 3 - #90 3 - #95 4 -#00- Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp As - #n Ser Gly Ala Pro Trp# 415- Asn Asp Tyr Ser His Val Leu Asn His Val Th - #r Phe Val Arg Trp Pro# 430- Gly Glu Ile Xaa Gly Ser Asp Ser Trp Arg Al - #a Pro Met Phe Ser Trp# 445- Thr His Arg Ser Ala Xaa Xaa Thr Asn Xaa Il - #e Xaa Pro Xaa Xaa Ile# 460- Thr Gln Ile Pro Xaa Val Xaa Ala His Xaa Le - #u Xaa Ser Gly Xaa Thr465 4 - #70 4 - #75 4 -#80- Val Val Arg Gly Pro Gly Phe Thr Gly Gly As - #p Xaa Leu Arg Arg Thr# 495- Xaa Xaa Gly Xaa Phe Ala Xaa Xaa Xaa Val As - #n Ile Xaa Gly Xaa Leu# 510- Xaa Gln Arg Tyr Arg Xaa Arg Ile Arg Tyr Al - #a Ser Thr Thr Xaa Leu# 525- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xa - #a Xaa Xaa Gly Xaa Phe# 540- Xaa Xaa Thr Met Xaa Xaa Gly Asp Xaa Leu Xa - #a Xaa Xaa Xaa Phe Xaa545 5 - #50 5 - #55 5 -#60- Xaa Ala Xaa Xaa Xaa Thr Xaa Phe Xaa Phe Xa - #a Xaa Xaa Gln Ser Xaa# 575- Phe Thr Xaa Gly Xaa Xaa Xaa Phe Xaa Ser Xa - #a Xaa Glu Val Tyr Ile# 590- Asp Xaa Xaa Glu Xaa Xaa Pro Xaa Xaa Xaa Th - #r Phe Glu Ala Glu Xaa# 605- Asp Xaa Glu Arg Ala Gln Xaa Ala Val Asn Al - #a Leu Phe Thr Ser Xaa# 620- Xaa Gln Xaa Gly Xaa Xaa Thr Xaa Val Thr Xa - #a Tyr His Ile Asp Gln625 6 - #30 6 - #35 6 -#40- Val Ser Asn Leu Val Xaa Cys Leu Ser Asp Gl - #u Phe Cys Leu Asp Glu# 655- Xaa Arg Glu Leu Ser Glu Xaa Val His Xaa Al - #a Xaa Arg Leu Ser Asp# 670- Xaa Arg Asn Leu Leu Gln Asp Pro Asn Phe Xa - #a Gly Ile Asn Arg Gln# 685- Xaa Asp Xaa Gly Trp Arg Gly Ser Thr Asp Il - #e Thr Ile Gln Xaa Gly# 700- Asp Asp Val Phe Xaa Glu Asn Tyr Val Thr Le - #u Pro Gly Thr Phe Asp705 7 - #10 7 - #15 7 -#20- Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Xaa Il - #e Asp Glu Ser Xaa Leu# 735- Xaa Xaa Tyr Thr Arg Tyr Gln Leu Arg Gly Ty - #r Ile Glu Asp Ser Gln# 750- Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Xa - #a Xaa His Glu Pro Val# 765- Asn Val Xaa Gly Thr Gly Ser Leu Trp Pro Le - #u Ser Val Xaa Xaa Xaa# 780- Ile Xaa Xaa Cys Gly Glu Pro Asn Arg Cys Al - #a Pro His Leu Glu Trp785 7 - #90 7 - #95 8 -#00- Asn Pro Asp Leu Xaa Cys Ser Cys Arg Asp Gl - #y Glu Xaa Cys Xaa His# 815- His Ser His His Phe Ser Leu Asp Ile Asp Va - #l Gly Cys Thr Asp Leu# 830- Asn Glu Asp Leu Xaa Val Trp Xaa Ile Phe Xa - #a Ile Xaa Thr Gln Asp# 845- Gly His Ala Arg Leu Gly Asn Leu Glu Phe Le - #u Glu Glu Xaa Pro Leu# 860- Xaa Gly Glu Ala Leu Xaa Arg Val Xaa Arg Al - #a Glu Xaa Xaa Trp Arg865 8 - #70 8 - #75 8 -#80- Asp Xaa Arg Glu Xaa Leu Xaa Leu Glu Thr As - #n Ile Val Tyr Xaa Glu# 895- Ala Xaa Glu Ser Val Asp Ala Leu Phe Val As - #n Ser Gln Tyr Asp Xaa# 910- Leu Gln Ala Asp Thr Asn Ile Ala Met Ile Hi - #s Ala Ala Asp Xaa Arg# 925- Val His Arg Ile Arg Glu Ala Tyr Leu Pro Gl - #u Leu Ser Val Ile Pro# 940- Gly Val Asn Xaa Xaa Ile Phe Glu Glu Leu Xa - #a Gly Arg Ile Phe Thr945 9 - #50 9 - #55 9 -#60- Ala Xaa Xaa Leu Tyr Asp Ala Arg Asn Val Il - #e Xaa Asn Gly Xaa Phe# 975- Asn Asn Gly Leu Xaa Cys Trp Asn Val Xaa Gl - #y His Val Asp Val Glu# 990- Glu Gln Asn Asn His Arg Ser Val Leu Val Va - #l Pro Glu Trp Glu Ala# 10050- Glu Val Ser Gln Glu Val Arg Val Cys Pro Gl - #y Arg Gly Tyr Ile Leu# 10205- Arg Val Thr Ala Tyr Xaa Glu Gly Tyr Gly Gl - #u Gly Cys Val Thr Ile# 10401030 - # 1035- His Glu Xaa Xaa Asn Asn Thr Asp Glu Leu Xa - #a Phe Ser Asn Cys Xaa# 10550- Xaa Glu Xaa Val Tyr Pro Xaa Asn Thr Val Xa - #a Cys Asn Asp Tyr Xaa# 10705- Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xa - #a Ser Arg Asn Arg Gly# 10850- Tyr Asp Glu Xaa Tyr Xaa Ser Asn Ser Ser Xa - #a Pro Ala Asp Tyr Ala# 11005- Xaa Val Tyr Glu Glu Xaa Xaa Tyr Thr Asp Gl - #y Xaa Arg Xaa Asn Pro# 11201110 - # 1115- Cys Glu Xaa Asn Arg Gly Xaa Xaa Xaa Xaa Th - #r Pro Leu Pro Ala Gly# 11350- Tyr Val Thr Xaa Glu Leu Glu Tyr Phe Pro Gl - #u Thr Asp Xaa Val Trp# 11505- Xaa Glu Ile Gly Glu Thr Glu Gly Thr Phe Il - #e Val Asp Ser Val Glu# 11650- Leu Leu Leu Met Glu Glu 1170__________________________________________________________________________

Number	Name	Date
4448885	Schnepf et al.	May 1984
4467036	Schnepf et al.	Aug 1984
4797276	Herrstadt et al.	Jan 1989
4853331	Herrstadt et al.	Aug 1989
5151363	Payne	Sep 1992
5164180	Payne et al.	Nov 1992

Number	Date	Country
0 405 810	Jan 1991	EPX
9116434	Oct 1991	WOX
9314641	Aug 1993	WOX
9502694	Jan 1995	WOX

	Number	Date	Country
Parent	291368	Aug 1994
Parent	597607	Oct 1990

Protein toxins active against lepidopteran pests

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