CANNON RAYMOND J C (GB)
BAGLEY ANGELA L (GB)
US4849217A | 1989-07-18 | |||
EP0303426A2 | 1989-02-15 |
CHEMICAL ABSTRACTS, vol. 101, no. 23, 3 December 1984, Columbus, Ohio, US; abstract no. 206066P, R.A.LI ET. AL.: 'Biology of ectoparasites of birds and acaricides to control them.' page 222 ;
JOURNAL OF INVERTEBRATE PATHOLOGY vol. 56, no. 2, September 1990, pages 258 - 266; S.C.MACINTOSH ET. AL.: 'Specificity and Efficacity of Purified Bacillus thuringiensis Proteins against Agronomically Important Insects'
Perry, Robert (53-64 Chancery Lane, London WC2A 1HN ())
1. | A method for controlling acarid pests wherein said method comprises contacting said pests with an acaridcontrolling effective amount of a B.t endotorin. |
2. | The method, according to claim 1, wherein said toxin is obtainable from a B. t isolate selected from the group consisting of Rt PS50C, Rt. PS86A1, Rt. PS69D1, Rt. PS72L1, Rt PS75J1, Rt PS83E5, Rt PS45B1, Rt PS24J, R PS94R3, Rt PS17, Rt PS62B1 and Rt PS74G1, and mutants thereof. |
3. | The method, according to claim 2, wherein said isolate is PS50C. |
4. | The method, according to claim 2, wherein said isolate is PS86A1. |
5. | The method, according to claim 2, wherein said isolate is PS69D1. |
6. | The method, according to claim 2, wherein said isolate is PS7 I2. |
7. | The method, according to claim 2, wherein said isolate is PS75J2. |
8. | The method, according to claim 2, wherein said isolate is PS83E5. |
9. | The method, according to claim 2, wherein said microbe is PS45B1. |
10. | The method, according to claim 2, wherein said isolate is PS24J. |
11. | '. |
12. | The method, according to claim 2, wherein said isolate is PS94R3. |
13. | The method, according to claim 2, wherein said isolate is PS17. |
14. | The method, according to claim 2, wherein said isolate is PS62B1. |
15. | The method, according to claim 2, wherein said isolate is PS74G1. |
16. | The method, according to claim 3, wherein said toxin has the amino acid sequence of SEQ ID NO.28. |
17. | The method, according to claim 4, wherein said toxin has the amino acid sequence of SEQ ID NO. 30. |
18. | The method, according to claim 5, wherein said toxin has the amino acid sequence of SEQ ID NO. 10. |
19. | The method, according to claim 12, wherein said toxin has the amino acid sequence of SEQ ID NO. 2. |
20. | The method, according to claim 12, wherein said toxin has the amino acid sequence of SEQ ID NO. 4. |
21. | The method, according to claim 1, wherein said acarid pest is a mite. |
22. | The method, according to claim 20, wherein said mite is the Two Spotted Spider Mite. |
23. | A composition of matter comprising a Bacillus thurinpiensis isolate selected from the group consisting of Rt PS72L1, Rt PS75J1, Rt PS83E5, Rt PS45B1, Rt PS24J, Rt PS94R3, Rt PS62B1 and Rt PS74G1, and mutants thereof, or proteins, toxic crystals, or spores of said isolates, in association with an inert carrier. |
24. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS24J. |
25. | *. |
26. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS94R3/. |
27. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS45B1. |
28. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS62B1. |
29. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS74G1. |
30. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS72L1. |
31. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS75J1. |
32. | The composition of matter, according to claim 22, comprising Bacillus thuringiensis PS83E5. |
33. | A composition for controlling an acaride pest wherein said composition comprises substantially intact, treated cells having pestiddal activity and prolonged persistence in the feeding zone of said pests when applied to the environment of acaride pests, wherein said pestidde is a polypeptide toxic to acaride pests, is intracellular, and is produced by a Bacillus thuringiensis isolate selected from the group consisting of Rt PS50C, Rt PS86A1, Rt PS69D1, Rt PS72L1, Rt PS75J1, R PS83E5, Rt PS45B1, Rt PS24J, Rt PS94R3, Rt PS17, Rt PS62B1 and Rt PS74G1, and mutants thereof. |
34. | The pestiddal composition, according to claim 18, wherein said treated cells are treated by chemical or physical means to prolong the pestiddal activity in the environment. |
35. | A gene encoding a toxin which is active against acarides wherein said gene is obtainable from a Bacillus thuringiensis isolate seleded from the group consisting of Rt PS72L1, Rt PS75J1, Rt PS83E5, Rt PS45B1, Rt PS24J, Rt PS94R3, Rt PS62B1 and Rt PS74G1, and mutants thereof or is equivalent to one of said genes. |
36. | A toxin encoded by a gene obtainable from a Bacillus thuringiensis isolate seleded from the group consisting of Rt PS72L1, Rt PS75J1, Rt PS83E5, Rt PS45B1, Rt PS24J, Rt PS94R3, Rt PS62B1 and Rt PS74G1, and mutants thereof, wherein said toxin is active against acaride pests. |
37. | A transformed host seleded from the group consisting of a plant, a microbe, and a baculovirus transformed by a gene obtainable from a Bacillus thuringiensis isolate seleded from the group consisting of Rt PS72L1, R PS75J1, Rt PS83E5, Rt PS45B1, Rt PS24J, R PS94R3, Rt PS62B1 and Rt PS74G1, and mutants thereof:. |
38. | A biologically pure culture of a Bacillus thuringiensis seleded from the group consisting of Rt PS72L1, Rt PS75J1, Rt PS83E5, Rt PS45B1, Rt PS24J, Rt PS94R3, Rt PS62B1 and Rt PS74G1, and mutants thereof. |
NOVEL BACILLUS THURINGIENSIS ISOLATES FOR CONTROLLING ACARIDES
Cross-Reference to a Related Application This is a continuation-in-part of co-pending application Serial No.07/693,210, filed on April 30, 1991. This is also a continuation-in-part of application Serial No. 07 768,141, filed on September 30, 1991 which is a continuation-in-part of application Serial No. 07/759,248, filed on September 13, 1991.
Background of the Invention The spore-forming microorganism Bacillus thuringiensis (B.t) produces the best- known insect toxin. The toxin is a protein, designated as ό-endotoxin. It is synthesized by the B . sporulating cell. The toxin, upon being ingested in its crystalline form by susceptible insect larvae, is transformed into biologically active moieties by the insect gut juice proteases. The primary target is inse * -t cells of the gut epithelium, which are rapidly destroyed. Experience has shown that the activity of the BΛ. toxin is so high that only nanogram amounts are required to kill susceptible insects. The reported activity spectrum of BΛ. covers insect species within the order
Lepidoptera, which is a major insect problem in agriculture and forestry. The activity spectrum also includes the insect order Diptera, wherein reside mosquitoes and blackflies. See Couch, T.L., (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis." Developments in Industrial Microbiology, 22:61-67; Beegle, CC, (1978) "Use of Entomogeneous Bacteria in Agroecosystems," Developments in Industrial Microbiology, 20:97-
104. i U.S. Patent 4,771,131 discloses a toxin gene isolated from a strain of Bacillus thuringiensis. This gene encodes a toxin which is active against beetles of the order Coleoptera. There have been published reports concerning the use of Bacillus thuringiensis preparations for the control of mites. These publications are as follow:
Royalty, R.N., Hall, F.R. and Taylor, R.A.J. 1990. Effects of thuringiensin on Tetranychus urticae (Acari: Tetranychidae) mortality, fecundity, and feeding. J. Econ. Entomol. 83:792-798.
Neal, J.W., L dquist, R.K., Gott, KM. and Casey, M.L. 1987. Activity of the themostable beta-exotoxin of Bacillus thuringiensis Berliner on Tetranychus urticae and Tetranychus cinnabarinus. J. Agric. Entomol. 4:33-40.
Y-ayen, P., Impe, G. and Van Semaille, R. 1978. Effect of a commercial preparation of Bacillus thuringiensis on the spider mite Tetranychus urticae Koch. (Acari: Tetranychidae). Mededelingen 43:471-479.
In the above published studies, the active ingredient in the B.t. preparations was beta-exoto-rin (also called thuringiensin).
U.S. Patent No. 4,695,455 concerns methods and compositions for preparing and using biological pesticides, where the pesticides are encapsulated in non-proliferating cells.
U.S. Patent No. 4,849,217 concerns BΛ. isolates active against the alfalfa weevil.
Brief Summary of the Invention
The subject invention concerns Bacillus thuringiensis isolates and toxins which have acaricidal properties. Unlike published reports of the use of B.t β-exotøxins to control mites, the subject invention isolates express <3-endotoxins which control mites. The use of δ- endotoxins is highly advantageous in view of the known general toxici-y of β-exotoxins to humans and animals.
More specifically, the subject invention concerns Bacillus thuringiensis isolates designated BΛ. PS50C, BΛ. PS86A1, BΛ. PS69D1, BΛ. PS72L1, BΛ. PS75J1, BΛ. PS83E5, BΛ.
PS45B1, Rt PS24J, BΛ. PS94R3, BΛ. PS17, Rt PS62B1 and BΛ. PS74G1. The B.t isolates of the subject invention are toxic to the Two Spotted Spider Mite,
Tetranychus urticae. Thus, these isolates can be used to control this mite. Further, the δ- endotoxins from these BΛ. isolates can be isolated by standard procedures, e.g. ion exchange, and formulated by standard procedures to control the Two Spotted Spider Mite. These B.t. isolates can also be used against non-phytophagus mites such as acarid pests of livestock, fowl and stored products. Still further, the gene(s) from the BΛ. isolates of the invention which encode the acaricidal toxin can be cloned from the isolates and then used to transform other
'hosts, eg., prokaiyotic, eukaiyotic or plants, which transformed host can be used to control mites, or, in the case of transgenic plants, be resistant to mites.
Brief Description of the Drawings
FIGURES 1, 2A and 2B are photographs of 12% SDS polyacrylamide gels showing alkali-soluble proteins of the isolates of the invention.
Brief Description of the Sequences SEQ ID NO. 1 discloses the DNA of 17a.
SEQ ID NO. 2 discloses the amino acid sequence of the toxin encoded by 17a. SEQ ID NO.3 discloses the DNA of 17b.
SEQ ID NO. 4 discloses the amino acid sequence of the toxin encoded by 17b. SEQ ID NO. 5 is the nucleotide sequence of gene 33F2.
SEQ ID NO. 6 is the nucleotide sequence of a gene from 52A1.
SEQ ID NO. 7 is the amino add sequence of the protein expressed by the gene from 52A1.
SEQ ID NO. 8 is the nucleotide sequence of a gene from 69D1. SEQ ID NO. 9 is the amino add sequence of the protein expressed by the gene from 69D1.
SEQ ID NO. 10 is the DNA coding for the amino add sequence of SEQ ID NO. 13.
SEQ ID NO. 11 is the amino add sequence of a probe which can be used according to the subject invention.
SEQ ID NO. 12 is the N-terminal amino add sequence of 17a.
SEQ ID NO. 13 is the N-terminal amino add sequence of 17b.
SEQ ID NO. 14 is the N-terminal amino add sequence of 52A1.
SEQ ID NO. 15 is the N-terminal amino add sequence of 69D1. SEQ ID NO. 16 is a synthetic oligonucleotide derived from 17.
SEQ ID NO. 17 is an oligonucleotide probe designed from the N-terminal amino add sequence of 52A1.
SEQ ID NO. 18 is the synthetic oligonucleotide probe designated as 69D1-D.
SEQ ID NO. 19 is the forward oligonucleotide primer from 63B. SEQ ID NO. 20 is the reverse complement primer to SEQ ID NO. 29, used according to the subject invention.
SEQ ID NO. 21 is the DNA coding for the primer of SEQ ID NO. 31.
SEQ ID NO. 22 is a forward primer according to the subject invention.
SEQ ID NO. 23 is a probe according to the subject invention. SEQ ID NO. 24 is a probe according to the subject invention.
SEQ ID NO. 25 is a probe according to the subject invention.
- SEQ ID NO. 26 is a forward primer according to the subject invention.
SEQ ID NO. 27 is the nucleotide sequence of a gene from PS50C
SEQ ID NO. 28 is the amino add sequence of the protein expressed by the gene from PS50C.
SEQ ID NO. 29 is the nucleotide sequence of a gene from PS86A1.
SEQ ID NO. 30 is the amino add sequence of the protein expressed by the gene from PS86A1.
Detailed Disclosure of the Invention
The subject invention concerns B.t <5-endotoxins having acariddal activity. In addition to having acariddal activity, the toxins of the subject invention may have one or more of the following characteristics:
1. A high degree of amino add homology with specific toxins disclosed herein,
2. A DNA sequence encoding the toxin which hybridizes with probes or genes disclosed herein.
3. A nucleotide sequence which can be amplified using primers disclosed herein,
4. --πununoreact-vity to an antibody raised to a specific toxin disclosed herein.
Acaride-active toxins according to the subject invention are specifically exemplified herein by the toxins encoded by the genes designated 17a, 17b, and 69D1. Since these toxins are merely exemplary of the toxins presented herein, it should be readily apparent that the subject invention further comprises toxins from the other disclosed isolates as well as equivalent toxins (and ήudeotide sequences coding for equivalent toxins) having the same or similar biological activity of the specific toxins disclosed or claimed herein. These equivalent toxins will have amino add homology with the toxins disclosed and claimed herein. This amino add homology will typically be greater than 50%, preferably be greater than 75%, and most preferably be greater than 90%. The amino add 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 add substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino add substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino adds may be placed in the following classes: non-polar, uncharged polar, basic, and addic Conservative substitutions whereby an amino add of one class is replaced with another amino add of the same type -all within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 1 provides a listing of examples of amino adds belonging to each class.
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 information presented in the generic formulae of the subjed invention provides clear guidance to the person skilled in this art in making various amino add substitutions. The BΛ. isolates of the invention have the following chararteristics:
Strain
B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis
Additionally, the isolates have the following common characteristics: Colony morphology — large colony, dull surface, typical BΛ.
Vegetative cell morphology - typical BΛ.
The toxins of the subject invention can be accurately characterized in terms of the shape and location of crystal toxin inclusions. Specifically, acaride-active inclusions typically remain attached to the spore after cell lysis. These inclusions are not inside the exosporium, as in previous descriptions of attached inclusions, but are held within the spore by another mechanism. Inclusions of the acaride-adive isolates are typically amorphic, generally long and/or multiple. These inclusions are distinguishable from the larger round/amorphic inclusions that remain attached to the spore. No B.t strains that fit this description have been found to have activity against the conventional target — Lepidoptera, Diptera, or Colorado Potato Beetle. We have found a very high correlation between this crystal structure and acaride activity.
The genes and toxins according to the subjed invention include not only the full length sequences disclosed herein but also fragments of these sequences, or fusion proteins, which retain the characteristic acariddal activity of the sequences specifically exemplified herein.
It should be apparent to a person skilled in this art that genes coding for acaride- active toxins can be identified and obtained through several means. The specific genes may be obtained from a culture depository as described below. These genes, or portions thereof, may be construded synthetically, for example, by use of a gene machine. Variations of these genes may be readily construded 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 acaride-active toxins of the instant invention which occur in nature. For example, antibodies to the acaride-active toxins disclosed and claimed herein can be used to identity and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the acaride-active toxins using procedures which are well known in the art These antibodies can then be used to specifically identity equivalent toxins with the characteristic acariddal activityby immunopredpitation, enzyme linked immunoassay (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 subjed invention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a detectable label. As is well known in the art, if the probe molecule and nudeic add sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying nematiάdal endo toxin genes of the subjed 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 the 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 -^P, 12S Ϊ, 35 S, or the like. A probe labeled with a radioactive isotope can be construded 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. 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 chemttuminescers such as ludferin, 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, and, as noted above, a certain degree of mismatch can be tolerated.
Therefore, the probes of the subjed 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, insertion-.!, and deletion--! variants generated from a given probe may be more or less effident than the original probe. Notwithstanding such differences in effidency, these variants are within the scope of the present invention.
Thus, mutational, insertional, and deletional variants of the disclosed test 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 suffirient to enable the variant to function in the same capadty 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 capadty 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 subjed invention, in the rapid identification of acaride-adive genes can be prepared utilizing the sequence information provided herein.
The potential variations in the probes listed is due, in part, to the redundancy of the genetic code. 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 adds used to make proteins. Therefore different nucleotide sequences can code for a particular amino add. Thus, the amino add sequences of the B.t toxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino add sequence of the protein or peptide. Accordingly, the subjed invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an asped of the subjed invention and can be readily used by a person skilled in this art In addition it has been shown that proteins of identified strudure and function
may be construded by changing the amino add sequence if such changes do not alter the protein secondary structure (Kaiser, E.T. and Kezdy, FJ. [1984] Sdence 223:249-255). Thus, the subject invention includes mutants of the amino add sequence depided herein which do not alter the protein secondary strudure, or if the strudure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a toxin encoding a gene of the invention. Such microbial mutants can be made by techniques well known to persons skilled in the art For example, UV irradiation can be used to prepare mutants of host organisms. likewise, such mutants may include asporogenous host cells which also can be prepared by procedures well known in the art
The BΛ. isolates of the invention, and mutants thereof can be cultured using standard known media and fermentation techniques. Upon completion of the fermentation cycle, the baderia can be harvested by first separating the BΛ. spores and crystals from the fermentation broth by means well known in the art The recovered BΛ. spores and crystals can be formulated into a wettable powder, a liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers and other components to facilitate handling and application for particular target pests. The formulation and application procedures are all well known in the art and are used with commercial strains. The novel B.t isolates, and mutants thereof; can be used to control target pests.
The cultures of the subjed invention were deposited in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois, 61604 USA.
Culture Accession No. Deposit Date
BΛ. PS50C NRRL B-18746 January 9, 1991
BΛ. PS86A1 NRRL B-18400 August 16, 1988
B.t PS69D1 NRRL B-18247 July 28, 1987
B.t PS72L1 NRRL B-18780 March 7, 1991
B.t PS75J1 NRRL B-18781 March 7, 1991
B.t PS83E5 NRRL B-18782 March 7, 1991
BΛ. PS45B1 NRR B-18396 August 16, 1988
BΛ. PS24J NRRL B-18881 August 30, 1991
B.t PS94R3 NRRL B-18882 August 30, 1991
B.t PS17 NRRL B-18243 July 28, 1987
B.t PS62B1 NRRL B-18398 August 16, 1988
BΛ. PS74G1 NRRL B-18397 August 16, 1988
R coU NM522(pMYC 2321) NRRL B-18770 February 14, 1991 R coli NM522(pMYC 2317) NRRL B-18816 April 24, 1991 R coli NM522(pMYC 1627) NRRL B-18651 May 11, 1990 R coli NM522(pMYC 1628) NRRL B-18652 May 11, 1990 R coli NM522(pMYC 1638) NRRL B-18751 January 11, 1991
E. coli NM522(pMYC 1638) NRRL B-18769 February 14, 1991
The subjed cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37
CFR 1.14 and 35 U.S.C. 122. These deposits are available as required by foreign patent laws in countries wherein counterparts of the subjed application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to pradice the subjed invention in derogation of patent rights granted by governmental adion. Further, the subjed 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 a deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing a culture. The depositor acknowledges the duty to replace a deposit should the depository be unable to furnish a sample when requested, due to the condition of a deposit All restrictions on the availability to the public of the subjed culture deposits will be irrevocably removed upon the granting of a patent disclosing them. Upon applying an acariddal-effective amount of a microbe, or toxin, as disclosed herein, in a suitable acariddal formulation to the environment of the target pest, there is obtained effective control of these pests. An acariddal-effective amount can vary from about 1 to about 121/ha, depending upon the nature and quantity of the pests to be controlled, the time of year, temperature, humidity, and other known factors which may affect a bioinsectidde. It is well within the skill of those trained in this art to determine the quantity of bioinsectidde to apply in order to obtain effective control of target pests. The intracellular ό-endotox-n protein can be combined with other insediddal proteins (including those obtained from sources other than Bacillus thuringiensis'. to increase the spectrum of adivity to give complete control of target pests. The BΛ. cells may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pestiddal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.
The pestiddal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used diredly. The
pestidde 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 pestidde 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 2 to about 10 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 target pest(s), e.g., plants, livestock, fowl, soil or water, by spraying, dusting, sprinkling, or the like.
The toxin genes harbored by the novel isolates of the subjed invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pestidde. With suitable hosts, e.g., Pseudomonas. the microbes can be applied to the situs of mites where they will proliferate and be ingested by the mites. The result is a control of the mites. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of the target pest The resulting product retains the toxidty of the Rt toxin.
Where the BΛ. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are seleded which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and or rhizoplane) of one or more crops of interest These microorganisms are seleded so as to be capable of successfully competing in the particular environment (crop and other insed habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pestidde, and, desirably, provide for improved protection of the pestidde from environmental degradation and inactivation. A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots). These -hicroorganisms include baderia, algae, and fungi Of particular interest are π-icroorganisn-s, such as baderia, e.g-, genera Bacillus. Pseudomonas. Erwinia. Serratia, Klebsiella. Xanthomonas.Streptomyces.R----∞bium.-^odopseudomonas.Methy loph--Uus.Atπ , obaderium. Acetobader, Ladobadllus. Arthrobader. Azotobader, Leuconostoc Alcaligenes and
Clostridium; fungi, particularly yeast, e.g., genera Saccharomvces. Cryptococcus. Kluweromvces. Sporobolomvces. Rhodotorula. and Aureobasidium: microalgae, e.g., families Cvanophvceae. Prochlorophvceae. Rhodophyceae. Dinophvceae. Chrvsophyceae. Prvmnesiophvceae. Xanthophvceae. Raphidophvceae. Bacillariophvceae. Eustigmatophvceae. Crvptophvceae. Euglenophvceae. Prasinophvceae. and Chlorophvceae. Of particular interest are such phytosphere baderial spedes as Pseudomonas syringae. Pseudomonas fluorescens. Serratia marcescens.A∞tobaderxy---Qum.Agrobaderiumtnmefadens.Rhodop seudomonasspheroides. Xanthomonas campestris. Rhizobiu melioti. Alcaligenes entrophus. and Azotobader vinlandii; and phytosphere yeast spedes such as Rhodotorula rubra. R. glutinis. R. marina. R.
aurantiaca. Crvptococcus albidus. C. diff-uens, C. laurentϋ, Saccharomvces rosei. S. pretoriensis. S. cerevisiae. Sporobolomvces roseus. S. odorus, Kluweromvces veronae. and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A wide variety of ways are available for introducing a Rt gene expressing a toxin 5 into the microorganism host under conditions which allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the toxin gene, the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host,
10 whereby integration or stable maintenance will occur.
The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the toxin, where expression of the toxin will only occur after release into the environment. This can be achieved with operators or a region binding to an adivator or enhancers, which
15 are capable of induction upon a change in the physical or chemical environment of the microorganisms. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a toxin, but upon release into the environment, expression would begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where
20 the nutriei-- medium in the environment would allow for expression of the toxin. For translational initiation, a ribosomal binding site and an initiation codon will be present
Various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA. The transcriptional and translational
25 termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. A hydrophobic "leader" sequence may be employed at the amino 1 terminus of the translated polypeptide sequence in order to promote secretion of the protein across the inner membrane.
In the direction of transcription, namely in the 5' to 3' direction of the coding
-... or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the strudural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for
35 transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construd during introduction of the DNA into the host
By a marker is intended a strudural gene which provides for selection of those hosts which have been modified or transformed. The marker will normally provide for
selective advantage, for example, providing for biodde resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Preferably, complementation is employed, so that the modified host may not only be seleded, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as for modifying the host The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, e.g., siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment
Where no functional replication system is present, the construd will also include a sequence of at least 50 basepairs ( p), preferably at least about 100 bp, and usually not more than about 5000 bp of a sequence homologous with a sequence in the host In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intad construct
A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as baderia, baderiophage, cyanobaderia, algae, fungi, and the like. Various transcriptional regulatory regions include the regions associated with the tig gene, lac gene, gal gene, the lambda left and right promoters, the tac promoter, the naturally- occurring promoters associated with the toxin gene, where functional in the host See for example, U.S. Patent Nos.4,332,898, 4342,832 and 4,356,270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host
Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host A large number of plasmids are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the like. See for example, Olson et al., (1982) J. Baderio 150:6069, and Bagdasarian et aL, (1981) Gene 16:237, and U.S. Patent Nos. 4,356,270, 4,362,817, and 4-371,625.
The Rt gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under
the regulatory control of the initiation region. This construd will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.
The transformants can be isolated in accordance with conventional ways, usually employing a seledion technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present The transformants then can be tested for pestiddal adivity.
Suitable host cells, where the pestidde-cont-uning cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application suffitiently low as to avoid any possibility of toxidty to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi, as disclosed previously.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the Rt gene into the host, availability of expression systems, effidency of expression, stability of the pestidde in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pestidde microcapsule include protedive qualities for the pestidde, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxidty; attradiveness 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.
• ■ 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 Rt 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 affed the properties of the toxin, nor diminish the cellular capability in 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 suffldent time to achieve i e desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti- infedives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, and Kelly'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 animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. The cells generally will have enhanced strudural stability which will enhance resistance to environmental conditions. Where the pestidde is in a profoπn, the method of inactivation should be seleded so as not to inhibit processing of the profoπn to the mature form of the pestidde by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pestidde. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin.
The cellular host containing the Rt insediddal 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 Rt gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The Rt 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 Rt spores and crystals from the fermentation broth by means well known in the art The recovered Rt 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 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 Rt isolates disclosed herein, can be applied to the soil or in the vicinity of stored products. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. 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 proteded, encapsulated toxin protein.
To prepare a phage resistant variant of said asporogenous mutant, an aliquot of the phage lysate is spread onto nutrient agar and allowed to dry. An aliquot of the phage sensitive bacterial strain is then plated directly over the dried lysate and allowed to dry. The plates are incubated at 30 °C. The plates 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 °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°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
A subculture of the Rt isolates, or mutants thereof, can be used to inoculate the following medium, a peptone, glucose, salts medium.
Bacto Peptone 7.5 gΛ
Glucose 1.0 gΛ
KH 2 PO 4 3.4 gΛ - 2 HPO 4 4.35 g/1
Salt Solution 5.0 ml/1
CaCl 2 Solution 5.0 ml/1 pH 7.2
Salts Solution (100 ml) MgSO 4 .7H 2 O 2.46 g
MnSO 4 .H 2 O 0.04 g ZnSO 4 .7H 2 O 0.28 g FeS0 4 .7H 2 O 0.40 g
CaCl 2 Solution (100 ml)
CaCl 2 .2H 2 O 3.66 g
The salts solution and CaCl 2 solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30° C on a rotary shaker at 200 rpm for 64 hr.
The above procedure can be readily scaled up to large feπnentors by procedures well known in the art
The Rt 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 subjed the harvested fermentation broth to separation techniques, e.g., centrifugation.
Example 2 — Purification of Protein and Amino Add Sequencing
The B.t isolates PS17, PS52A1 and PS69D1 were cultured as described in Example 1. The parasporal inclusion bodies were partially purified by sodium bromide (28-38%) isopycnic gradient centrifugation (Pfannenstiel, M-A., EJ. Ross, V.C Kramer, and K.W. Nickerson [1984] EEMS MicrobioL Lett 2139). The proteins were bound to PVDF membranes (Millipore, Bedford, MA) by western blotting techniques (Towbin, H., T.
Staehlelin, and K. Gordon [1979] Proα Natl. Acad. Sci. USA 76:4350) and the N-tem-inal amino add sequences were determined by the standard Edman reaction with an automated gas- phase sequenator (Hunkapiller, M.W., R.M. Hewick, WX. Dreyer, and L.E. Hood [1983] Meth. EnzymoL 91399). The sequences obtained were: PS17a: AILNEL YP S VP YN V(SEQ ID NO.12)
PS17b: AILNELYPSVPYNV(SEQIDNO.13) PS52A1: M IID S KTTLP RHS LINT (SEQ ID NO.14) PS69D1: MILGNGKTLPKHIRLAHIFATQNS (SEQ ID NO.15)
Example 3 — Cloning of Novel Toxin Genes and Transformation into Escherichia coli
Total cellular DNA was prepared by growing the cells Rt PS17 to a low optical
' density (OD^ = 1.0) and recovering the cells by centrifugation. The cells were protoplasted in TES buffer (30 mM Tϊis-Cl, 10 mM EDTA, 50 mM NaCl, pH = 8.0) containing 20 % sucrose and 50 mgml lysozyme. The protoplasts were lysed by addition of SDS to a final concentration of 4%. The cellular material was predpitated overnight at 4°C in 100 mM
(final concentration) neutral potassium chloride. The supernate was extraded twice with phenol/chloroform (1:1). The DNA was predpitated with ethanol and purified by isopycnic banding on a cesium chloride-ethidium bromide gradient
Total cellular DNA from PS17 was digested with EcoRI and separated by electrophoresis on a 0.8% (wv) 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 a [ 32 P] - radiolabeled oligonucleotide probe derived from the N-terminal amino add sequence of purified 130 kDa protein from PS17. The sequence of the oligonucleotide synthesized is (GCAATTTTAAATGAATTATATCC) (SEQ ID NO. 16). Results showed that the
hybridizing EcoRI fragments of PS17 are 5.0 kb, 4.5 kb, 2.7 kb and 1.8 kb in size, presumptively identifying at least four new acaride-active toxin genes, PS17d, PS17b, PS17a and PS17e, respectively.
A library was construded from PS17 total cellular DNA partially digested with Sau3A and size fradionated by electrophoresis. The 9 to 23 kb region of the gel was excised and the DNA was electroeluted and then concentrated using an Elutip™ ion exchange column (Schleicher and Schuel, Keene NH). The isolated Sau3A fragments were ligated into LambdaGEM-11™ (PROMEGA). The packaged phage were plated on KW251 E. coli cells (PROMEGA) at a high liter and screened using the above radiolabeled synthetic oligonucleotide as a nucleic add hybridization probe. Hybridizing plaques were purified and rescreened at a lower plaque density. Single isolated purified plaques that hybridized with the probe were used to infed KW251 E. coli cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures.
Recovered recombinant phage DNA was digested with EcoRI and separated by eledrophoresis on a 0.8% agarose-TAE gel. The gel was Southern blotted and hybridized with the oligonucleotide probe to characterize the toxin genes isolated from the lambda library. Two pε.-erns were present, clones containing the 4.5 kb (PS17b) or the 2.7 kb (PS17a) EcoRI fragments. Preparative amounts of phage DNA were digested with Sail (to release the inserted DNA from lambda arms) and separated by electrophoresis on a 0.6% agarose-TAE geL The large fragments, electroeluted and concentrated as described above, were ligated to Sall- digested and dephosphorylated pBClac, an E. coli/B.t shuttle vedor comprised of replication origins from pBC16 and pUC19 . The ligation mix was introduced by transformation into NM522 competent E. coli cells and plated on LB agar containing ampicillin, isopropyl-(Beta)- D-thiogaladoside (IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galadoside (XGAL). White colonies, with putative insertions in the (Beta)-galadosidase gene of pBClac, were subjected to standard rapid plasmid purification procedures to isolate the desired plasmids. The seleded plasmid containing the 2.7 kb EcoRI fragment was named pMYC1627 and the plasmid containing the 4:5 kb EcoRI fragment was called pMYC1628.
The toxin genes were sequenced by the standard Sanger dideoxy chain termination method using the synthetic oligonucleotide probe, disclosed above, and by "walking' 1 with primers made to the sequence of the new toxin genes.
The PS17 toxin genes were subcloned into the shuttle vedor pHT3101 (Lereclus, D. et al. [1989] FEMS Microbiol. Lett. 60:211-218) using standard methods for expression in B.t Briefly, Sail fragments containing the 17a and 17b toxin genes were isolated from pMYC1629 and pMYC1627, respedively, by preparative agarose gel electrophoresis, electroelution, and concentrated, as described above. These concentrated fragments were ligated into Sail-cleaved and dephosphorylated pHT3101. The ligation mixtures were used separately to transform frozen, competent E. coli NM522. Plasmids from each respedive recombinant E. coli strain were prepared by alkaline lysis and analyzed by agarose gel
electrophoresis. The resulting subclones, pMYC2311 and pMYC2309, harbored the 17a and 17b toxin genes, respectively. These plasmids were transformed into the aciystalliferous B.t strain, HD-1 cryB (Aronson, A, Purdue University, West Lafayette, IN), by standard electroporation techniques (Instruction Manual, Biorad, Richmond, CA). Recombinant B.t strains HD-1 αyB [pMYC2311] and [pMYC2309] were grown to sporulation and the proteins purified by NaBr gradient centrifugation as described above for the wild-type B.t proteins.
Example 4 — Molecular Cloning of Gene Encoding a Novel Toxin From Bacillus thuringiensis strain PS52A1
Total cellular DNA was prepared from Bacillus thuringiensis PS52A1 (B.t PS52A1) as disdosed in Example 3.
RFLP analyses were performed by standard hybridization of Southern blots of PS52A1 DNA with a 32 P-labeled oligonucleotide probe designed from the N-terminal amino add sequence disclosed in Example 2. The sequence of this probe is:
5' ATG ATT ATT GAT TCT AAA ACA ACA TTA CCA AGA CAT TCA T TTA ATA/T AAT ACA/T ATA/T AA 3' (SEQ ID NO. 17) This probe was designated 52A1-C Hybridizing bands included an approximately 3.6 kbp Hy-diπ fragment and an approximately 8.6 kbp -EcoRV fragment A gene library was constructed from PS52A1 DNA partially digested with SmiSAx. Partial restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 6.6 to 23 kbp in size were excised from the gel, electroeluted from the gel slice, and recovered by ethanol predpitation after purification on an Elutip-D ion exchange column. The Sau3A inserts were ligated into -5-z ΗI-digested LambdaGem-11 (Promega). Recombinant phage were packaged and plated on £ coli KW251 cells (Promega). Plaques were screened by hybridization with the radiolabeled 52A1-C oligonucleotide probe disclosed above. Hybridizing phage were plaque- purified and used to infed liquid cultures of R coli KW251 cells for isolation of phage DNA by standard procedures (Maniatis et al.). For subcloning, preparative amounts of DNA were digested with -EcoRI and Sail, and electrophoresed on an agarose gel. The approximately 3.1 kbp band containing the toxin gene was excised from the gel, electroeluted from the gel slice, and purified by ion exchange chromatography as above. The purified DNA insert was ligated into -EfcoRI + 5-7/I-digested pHTBluell (an R coli/B. thuringiensis shuttle vedor comprised of pBIuescript S/K [Stratagene] and the replication origin from a resident B.t plasmid p. Lereclus et aL 1989. FEMS Microbiology Letters 60:211-218]). The ligation mix was used to transform frozen, competent R coli NM522 cells (ATCC 47000). Transformants were plated on LB agar containing ampicillin, isopropyl-(Beta)-D-thiogaladoside (IPTG), and 5-Bromo-4- Chloro-3-indoIyl-(Beta)-D-galadoside (XGAL). Plasmids were purified from putative recombinants by alkaline lysis (Maniatis et aL) and analyzed by electrophoresis of E oRI and
Sail digests on agarose gels. The desired plasmid construd, pMYC2321 contains a toxin gene that is novel compared to the maps of other toxin genes encoding acariddal proteins.
Plasmid pMYC2321 was introduced into an aciystalliferous (Cry- ) B.t host by eledroporation. Expression of an approximately 55-60 kDa crystal protein was verified by SDS-PAGE analysis.
Example 5 — Molecular Cloning of Gene Encoding a Novel Toxin From Bacillus Thurinsiensis strain PS69D1
Total cellular DNA was prepared from PS69D1 {B.t PS69D1) as disclosed in Example 3. RFLP analyses were performed by standard hybridization of Southern blots of
PS69D1 DNA with a 32P-labeled oligonucleotide probe designated as 69D1-D. The sequence of the 69D1-D probe was:
5* AAA CAT ATT AGA TTA GCA CAT ATT TTT GCA ACA CAA AA 3' (SEQ ID NO. 18) Hybridizing bands included an approximately 2.0 kbp Hiwdlll fragment
A gene library was construded from PS69D1 DNA partially digested with Sau3A. Partial restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 6.6 to 23 kbp in size were excised from the gel, electroeluted from the gel slice, and recovered by ethanol predpitation after purification on an Elutip-D ion exchange column. The Sau3A inserts were ligated into -B-wiΗI-digested LambdaGem-11 (Promega, Madison, WI).
Recombinant phage were packaged and plated on E. coli KW251 cells (Promega, Madison, WI). Plaques were screened by hybridization with the radiolabeled 69D1-D oligonucleotide probe. Hybridizing phage were plaque-purified and used to infed liquid cultures of E. coli KW251 cells for isolation of phage DNA by standard procedures (Maniatis et aL [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY). For subcloning, preparative amounts of DNA were digested with Hwidlll and electrophoresed on an agarose gel. The approximately, 2.0 kbp band containing the toxin gene was excised from the gel, electroeluted from the gel slice, and purified by ion exchange chromatography as above. The purified DNA insert was ligated into H-wdlH-digestedpHTBluell (and-E. colilB shuttle vedor comprised of pBluescript S/K (Stratagene, San Diego, CA) 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). Transformants were plated on LB agar containing 5-bromo-4-chloro-3-indolyl-(Beta)-D- galadoside (XGAL). Plasmids were purified from putative recombinants by alkaline lysis (Maniatis et al., supra) and analyzed by electrophoresis of Hindlll digests on agarose gels. The desired plasmid construd, pMYC2317, contains a toxin gene that is novel compared to the maps of other toxin genes encoding insediddal proteins.
Example 6 — Activity of B.t Isolates Against Mites
B. thuringiensis isolates of the invention were tested as spray-dried powders of fermentation broths which were concentrated by centrifugation. Pellets, which consist of water and biomass (spores, crystalline delta-endotoxins, cellular debris and growth media) were mixed with a standard carrier, preservative and surfactant Powders, which consisted of 25% biomass, were made using a Yamato spray drier. (Sold by Yamato Sdentific Co., Ltd. Tokoyo, Japan)
All broths were tested for the presence of beta-exotoxin by a larval house fly bioassay (Campbell, D.P., Dieball, D.E. and Brackett, J.M., 1987, Rapid HPLC assay for the β-exotoxin of Bacillus thuringiensis. J. Agric Food Chem, 35:156-158). Only isolates which tested free of B-exoto-dn were used in the assays against mites.
B. thuringiensis isolates were tested using an artificial feeding assay. Spray-dried powders were prepared for testing by mixing 25mg of powder in 5 ml of a 10% sucrose solution. This mixture was then sonicated for 8 min to produce a suspension.
Two ml of suspension was placed in a reservoir consisting of a metal ring with a Parafilm ™ M film bottom, A petri dish containing approximately 30 female Two-spotted spider mites (Tetranychus urticae') was placed on the underside of the film. Mites were allowed to feed on the sucrose solution for 24 hrs and then transfered to 2 cm French bean leaf discs (20 mites per disc). Mortality was determined after 7 days (Table 2). Each assay was done in triplicate.
TABLE 2. Toxidty of Bacillus thuringiensis isolates to the two spotted spider mite, Tetranychus urticae. Mortality was deteπnined after 7 days of treatment
Example 7 — Cloning of Novel Acaride-Active Genes Using Generic Oligonucleotide Primers The acariddal gene of a new acariddal B.t isolate can be obtained from DNA of the strain by performing the standard polymerase chain reaction using the oligonucleotides of SEQ ID NO. 21 or SEQ ID NO.20 as reverse primers and SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 16, Probe B of SEQ ED NO. 5 (AAT GAA GTA/T TAT CCA/T GTA/T AAT), or SEQ ID NO. 19 as forward primers. The expeded PCR fragments would be approximately 330 to 600 bp (with either reverse primer and SEQ ID NO. 10), 1000 to 1400 bp (with either reverse primer and SEQ ID NO. 11), and 1800 to 2100 bp (with either reverse primer and any of the three N-terminal primers, SEQ ID NO.5 (Probe B), SEQ ID NO. 16, and SEQ ID NO. 19). Alternatively, a complement from the primer family described by SEQ ID NO. 10 can be used as reverse primer with SEQ ID NO. 11, SEQ ID NO. 16, SEQ ID NO. 5 (Probe B), or SEQ ID NO. 19 as forward primers. The expeded PCR fragments would be approximately 650 to 1000 bp with SEQ ID NO. 11, and 1400 to 1800 bp (for the three N-terminal primers, SEQ ID NO.5 (Probe B), SEQ ID NO. 16, and SEQ ID NO. 19). Amplified DNA fragments of the indicated sizes can be radiolabeled and used as probes to clone the entire gene.
Example 8 - Further Clrming of Novel Acaride-Adive Genes Using Generic Oligonucleotide Primers
A gene coding for a acariddal toxin of an acariddal B.t isolate can also be obtained from DNA of the strain by performing the standard polymerase chain reaction using oligonucleotides derived from the PS52A1 and PS69D1 gene sequences as follows:
1. Forward primer "TGATTTT(T or A)(C or A)TCAATTATAT(A or G)A(G or T)GTTTAT" (SEQ ID NO. 22) can be used with primers complementary to probe "AAGAGTTA(C or T)TA(A or G)A(G or A)AAAGTA" (SEQ ID NO. 23), probe "TTAGGACCATT(A or G)(C or T)T(T or A)GGATTTGTTGT(A or T)TATGAAAT" (SEQ
ID NO. 24), and probe "GA(C or T)AGAGATGT(A or T)AAAAT(C or T)(T or A)TAGGAATG" (SEQ ID NO.25) to produce amplified fragments of approximately 440, 540, and 650 bp, respedively.
2. Forward primer TT(A or C)TTAAA(A or T)C(A or T)GCTAATGATATT" (SEQ ID NO. 26) can be used with primers complementary to SEQ ID NO. 23, SEQ ID NO.
24, and SEQ ID NO. 25 to produce amplified fragments of approximately 360, 460, and 570 bp, respedively.
3. Forward primer SEQ ID NO. 23 can be used with primers complementary to SEQ ID NO. 24 and SEQ ID NO. 25 to produce amplified fragments of approx-mately 100 and 215 bp, respedively.
Amplified DNA fragments of the indicated sizes can be radiolabeled and used as probes to clone the entire gene.
Example 9 — Insertion of Toxin Genes Into Plants
One asped of the subjed invention is the transformation of plants with genes coding for a acariddal toxin. The transformed plants are resistant to attack by acarides.
Genes coding for acariddal 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 coding for 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 -ysed. 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. f, 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 f-anking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and suffidently described in EP 120 516; Hoekema (1985) In: The Binary Plant Vedor System, Of-set-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et aL, Crit Rev. Plant Sd. 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 biodde or an antibiotic, such as k-u-amycin, 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 Agrobaderium tumefadens or Agrobaderium rhizogenes as transformation agent, fusion, injection, or electroporation as well as other possible methods. If agrobaderia are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vedor or into a binary vedor. The intermediate vedors 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 necessaiy for the transfer of the
T-DNA Intermediate vedors cannot replicate themselves in agrobaderia. The intermediate vedor can be transferred into Agrobaderium tumefadens by means of a helper plasmid (conjugation). Binary vedors can replicate themselves both in E. coli and in agrobaderia. 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 agrobaderia
(Holsters et al. [1978] Mol. Gen. Genet 163:181-187). The agrobaderium 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 baderium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobaderium tumefadens or Agrobaderium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infeded 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 bioddes 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 fadors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
Example 10 — Cloning of Bacillus thuringiensis Genes Into Baculoviruses
The genes coding for the insectiddal toxins, as disclosed herein, can be cloned into baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids can be construded that contain the AcNPV genome cloned into a commercial cloning vector such as pUCδ. The AcNPV genome is modified so that the coding region of the polyhedrin gene is removed and a unique cloning site for a passenger gene is placed directly behind the polyhedrin promoter. Examples of such vedors are pGP-B6874, described by Pennock et al. (Pennock, G.D., Shoemaker, C. and Miller, L.K. [1984] Mol. Cell. Biol. 4399-406), and ρAC380, described by Smith et al. (Smith, G.E., Summers, M.D. and Fraser, MJ. [1983] Mol CelL Biol. 3:2156-2165). The genes coding for the protein toxins of the invention can be modified with BamHI linkers at appropriate regions both upstream and downstream from the coding region and inserted into the passenger site of one of the AcNPV vedors.
It should be imderstood 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:
(i) APPLICANT: Payne, Jewel M.
Cannon, Raymond J.C. Bagley, Angela L.
(ii) TITLE OF INVENTION: Novel Bacillus thuringiensis Isolates for Controlling Acarides
(iii) NUMBER OF SEQUENCES: 30
(iv) CORRESPONDENCE ADDRESS:
A) ADDRESSEE: David R. Saliwanchik
B) STREET: 2421 N.W. 41st Street, Suite A-l
C) CITY: Gainesville ) STATE: FL
E) COUNTRY: USA
F) ZIP: 32606
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Saliwanchik, David R.
(B) REGISTRATION NUMBER: 31,794
(C) REFERENCE/DOCKET NUMBER: M/S 104
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 904-375-8100
(B) TELEFAX: 904-372-5800
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 4155 base pairs 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:
! (AA)) ORGANISM: Bacillus thuringiensis B 81) STRAIN: PS17
INDIVIDUAL ISOLATE: PS17a
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 1627) NRRL B-18651
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGGCAATTT TAAATGAATT ATATCCATCT GTACCTTATA ATGTATTGGC GTATACGCCA 60
CCCTCTTTTT TACCTGATGC GGGTACACAA GCTACACCTG CTGACTTAAC AGCTTATGAA 120
CAATTGTTGA AAAATTTAGA AAAAGGGATA AATGCTGGAA CTTATTCGAA AGCAATAGCT 180
GATGTACTTA AAGGTATTTT TATAGATGAT ACAATAAATT ATCAAACATA TGTAAATATT 240
GGTTTAAGTT TAATTACATT AGCTGTACCG GAAATTGGTA TTTTTACACC TTTCATCGGT 300
TTGTTTTTTG CTGCATTGAA TAAACATGAT GCTCCACCTC CTCCTAATGC AAAAGATATA 360
TTTGAGGCTA TGAAACCAGC GATTCAAGAG ATGATTGATA GAACTTTAAC TGCGGATGAG 420
CAAACATTTT TAAATGGGGA AATAAGTGGT TTACAAAATT TAGCAGCAAG ATACCAGTCT 480
ACAATGGATG ATATTCAAAG CCATGGAGGA TTTAATAAGG TAGATTCTGG ATTAATTAAA 540
AAGTTTACAG ATGAGGTACT ATCTTTAAAT AGTTTTTATA CAGATCGTTT ACCTGTATTT 600
ATTACAGATA ATACAGCGGA TCGAACTTTG TTAGGTCTTC CTTATTATGC TATACTTGCG 660 AGCATGCATC TTATGTTATT AAGAGATATC ATTACTAAGG GTCCGACATG GGATTCTAAA 720 ATTAATTTCA CACCAGATGC AATTGATTCC TTTAAAACCG ATATTAAAAA TAATATAAAG 780 CTTTACTCTA AAACTATTTA TGACGTATTT CAGAAGGGAC TTGCTTCATA CGGAACGCCT 840 TCTGATTTAG AGTCCTTTGC AAAAAAACAA AAATATATTG AAATTATGAC AACACATTGT 900 TTAGATTTTG CAAGATTGTT TCCTACTTTT GATCCAGATC TTTATCCAAC AGGATCAGGT 960 GATATAAGTT TACAAAAAAC ACGTAGAATT CTTTCTCCTT TTATCCCTAT ACGTACTGCA 1020 GATGGGTTAA CATTAAATAA TACTTCAATT GATACTTCAA ATTGGCCTAA TTATGAAAAT 1080 GGGAATGGCG CGTTTCCAAA CCCAAAAGAA AGAATATTAA AACAATTCAA ACTGTATCCT 1140 AGTTGGAGAG CGGGACAGTA CGGTGGGCTT TTACAACCTT ATTTATGGGC AATAGAAGTC 1200 CAAGATTCTG TAGAGACTCG TTTGTATGGG CAGCTTCCAG CTGTAGATCC ACAGGCAGGG 1260 CCTAATTATG TTTCCATAGA TTCTTCTAAT CCAATCATAC AAATAAATAT GGATACTTGG 1320 AAAACACCAC CACAAGGTGC GAGTGGGTGG AATACAAATT TAATGAGAGG AAGTGTAAGC 1380 GGGTTAAGTT TTTTACAACG AGATGGTACG AGACTTAGTG CTGGTATGGG TGGTGGTTTT 1440 GCTGATACAA TATATAGTCT CCCTGCAACT CATTATCTTT CTTATCTCTA TGGAACTCCT 1500 TATCAAACTT CTGATAACTA TTCTGGTCAC GTTGGTGCAT TGGTAGGTGT GAGTACGCCT 1560 CAAGAGGCTA CTCTTCCTAA TATTATAGGT CAACCAGATG AACAGGGAAA TGTATCTACA 1620 ATGGGATTTC CGTTTGAAAA AGCTTCTTAT GGAGGTACAG TTGTTAAAGA ATGGTTAAAT 1680 GGTGCGAATG CGATGAAGCT TTCTCCTGGG CAATCTATAG GTATTCCTAT TACAAATGTA 1740 ACAAGTGGAG AATATCAAAT TCGTTGTCGT TATGCAAGTA ATGATAATAC TAACGTTTTC 1800 TTTAATGTAG ATACTGGTGG AGCAAATCCA ATTTTCCAAC AGATAAACTT TGCATCTACT 1860 GTAGATAATA ATACGGGAGT ACAAGGAGCA AATGGTGTCT ATGTAGTCAA ATCTATTGCT 1920
ACAACTGATA ATTCTTTTAC AGAAATTCCT GCGAAGACGA TTAATGTTCA TTTAACCAAC 1980
CAAGGTTCTT CTGATGTCTT TTTAGACCGT ATTGAATTTA TACCTTTTTC TCTACCTCTT 2040
ATATATCATG GAAGTTATAA TACTTCATCA GGTGCAGATG ATGTTTTATG GTCTTCTTCA 2100
AATATGAATT ACTACGATAT AATAGTAAAT GGTCAGGCCA ATAGTAGTAG TATCGCTAGT 2160
TCTATGCATT TGCTTAATAA AGGAAAAGTG ATAAAAACAA TTGATATTCC AGGGCATTCG 2220
GAAACCTTCT TTGCTACGTT CCCAGTTCCA GAAGGATTTA ATGAAGTTAG AATTCTTGCT 2280
GGCCTTCCAG AAGTTAGTGG AAATATTACC GTACAATCTA ATAATCCGCC TCAACCTAGT 2340'
AATAATGGTG GTGGTGATGG TGGTGGTAAT GGTGGTGGTG ATGGTGGTCA ATACAATTTT 2400
TCTTTAAGCG GATCTGATCA TACGACTATT TATCATGGAA AACTTGAAAC TGGGATTCAT 2460
GTACAAGGTA ATTATACCTA TACAGGTACT CCCGTATTAA TACTGAATGC TTACAGAAAT 2520
AATACTGTAG TATCAAGCAT TCCAGTATAT TCTCCTTTTG ATATAACTAT ACAGACAGAA 2580
GCTGATAGCC TTGAGCTTGA ACTACAACCT AGATATGGTT TTGCCACAGT GAATGGTACT 2640
GCAACAGTAA AAAGTCCTAA TGTAAATTAC GATAGATCAT TTAAACTCCC AATAGACTTA 2700
CAAAATATCA CAACACAAGT AAATGCATTA TTCGCATCTG GAACACAAAA TATGCTTGCT 2760
CATAATGTAA GTGATCATGA TATTGAAGAA GTTGTATTAA AAGTGGATGC CTTATCAGAT 2820
GAAGTATTTG GAGATGAGAA GAAGGCTTTA CGTAAATTGG TGAATCAAGC AAAACGTTTG 2880
AGTAGAGCAA GAAATCTTCT GATAGGTGGG AGTTTTGAAA ATTGGGATGC ATGGTATAAA 2940
GGAAGAAATG TAGTAACTGT ATCTGATCAT GAACTATTTA AGAGTGATCA TGTATTATTA 3000
CCACCACCAG GATTGTCTCC ATCTTATATT TTCCAAAAAG TGGAGGAATC TAAATTAAAA 3060
CCAAATACAC GTTATATTGT TTCTGGATTC ATCGCACATG GAAAAGACCT AGAAATTGTT 3120
GTTTCACGTT ATGGGCAAGA AGTGCAAAAG GTCGTGCAAG TTCCTTATGG AGAAGCATTC 3180
CCGTTAACAT CAAATGGACC AGTTTGTTGT CCCCCACGTT CTACAAGTAA TGGAACCTTA 3240
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
LENGTH: 1385 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS17
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 1627) NRRL B-18651
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala lie Leu Asn Glu Leu Tyr Pro Ser Val Pro 1 5 10
Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp Ala Gly -20 * 25
Pro Ala Asp Leu Thr Ala Tyr Glu Gin Leu Leu Lys 35 40
Gly lie Asn Ala Gly Thr Tyr Ser Lys Ala lie Ala 50 55 60
Gly lie Phe lie Asp Asp Thr lie Asn Tyr Gin Thr 65 70 75
Gly Leu Ser Leu lie Thr Leu Ala Val Pro Glu lie 85 90
Pro Phe lie Gly Leu Phe Phe Ala Ala Leu Asn Lys
10lo " 105
Pro Pro Pro Asn Ala Lys Asp lie Phe Glu Ala Met 115 120
Gin Glu Met lie Asp Arg Thr Leu Thr Ala Asp Glu 130 135 140
Asn Gly Glu lie Ser Gly Leu Gin Asn Leu Ala Ala 145 150 155
Thr Met Asp Asp He Gin Ser His Gly Gly Phe Asn Lys Val Asp Ser 165 170 175
Gly Leu He Lys Lys Phe Thr Asp Glu Val Leu Ser Leu Asn Ser Phe 180 185 190
Tyr Thr Asp Arg Leu Pro Val Phe He Thr Asp Asn Thr Ala Asp Arg 19§ 200 205
Thr Leu Leu Gly Leu Pro Tyr Tyr Ala He Leu Ala Ser Met His Leu 210 215 220
Met Leu Leu Arg Asp He He Thr Lys Gly Pro Thr Trp Asp Ser Lys 225 230 235 240
He Asn Phe Thr Pro Asp Ala He Asp Ser Phe Lys Thr Asp He Lys 245 250 255
Asn Asn He Lys Leu Tyr Ser Lys Thr He Tyr Asp Val Phe Gin Lys 260 265 270
Gly Leu Ala Ser Tyr Gly Thr Pro Ser Asp Leu Glu Ser Phe Ala Lys 275 280 285
Lys Gin Lys Tyr He Glu He Met Thr Thr His Cys Leu Asp Phe Ala 290 295 300
Arg Leu Phe Pro Thr Phe Asp Pro Asp Leu Tyr Pro Thr Gly Ser Gly 305 310 315 320
Asp He Ser Leu Gin Lys Thr Arg Arg He Leu Ser Pro Phe He Pro 325 330 335
He Arg Thr Ala Asp Gly Leu Thr Leu Asn Asn Thr Ser He Asp Thr 340 345 350
Ser Asn Trp Pro Asn Tyr Glu Asn Gly Asn Gly Ala Phe Pro Asn Pro 355 360 365
Lys Glu Arg He Leu Lys Gin Phe Lys Leu Tyr Pro Ser Trp Arg Ala 370 375 380
Gly Gin Tyr Gly Gly Leu Leu Gin Pro Tyr Leu Trp Ala He Glu Val
385 390 395 400
Gin Asp Ser Val GGlluu TThhrr AArrgg LLeeuu TTyyrr GGllyy GGiinn LLeeuu PPrroo AAllaa VVaall Asp 405 410 415
Pro Gin Ala Gly PPrroo AAssnn TTyyrr VVaall SSeerr HHee AAsspp SSeerr SSeerr AAssnn Pro He 4"2200 425 4"3"0"
He Gin lie AAssnn MMeett AAsspp TThhrr TTrrpp LLyyss TThhrr PPrroo PPrroo GGiinn Gly Ala Ser 435 440 445
Gly Trp Asn Thr Asn Leu Met Arg Gly Ser Val Ser Gly Leu Ser Phe 450 455 460
Leu Gin Arg Asp Gly Thr Arg Leu Ser Ala Gly Met Gly Gly Gly Phe 465 470 475 480
Ala Asp Thr He Tyr Ser Leu Pro Ala Thr His Tyr Leu Ser Tyr Leu 485 490 495
Tyr Gly Thr Pro Tyr Gin Thr Ser Asp Asn Tyr Ser Gly His Val Gly 500 505 510
Ala Leu Val Gly Val Ser Thr Pro Gin Glu Ala Thr Leu Pro Asn He 515 520 525
He Gly Gin Pro Asp Glu Gin Gly Asn Val Ser Thr Met Gly Phe Pro 530 535 540
Phe Glu Lys Ala Ser Tyr Gly Gly Thr Val Val Lys Glu Trp Leu Asn 545 550 555 560
Gly Ala Asn Ala Met Lys Leu Ser Pro Gly Gin Ser He Gly He Pro 565 570 575
He Thr Asn Val Thr Ser Gly Glu Tyr Gin He Arg Cys Arg Tyr Ala 580 585 590
Ser Asn Asp Asn Thr Asn Val Phe Phe Asn Val Asp Thr Gly Gly Ala 595 600 605
Asn Pro He Phe Gin Gin He Asn Phe Ala Ser Thr Val Asp Asn Asn 610 615 620
Thr Gly Val Gin Gly Ala Asn Gly Val Tyr Val Val Lys Ser He Ala 625 630 635 640
Thr Thr Asp Asn Ser Phe Thr Glu He Pro Ala Lys Thr He Asn Val 645 650 655
His Leu Thr Asn Gin Gly Ser Ser Asp Val Phe Leu Asp Arg He Glu 660 665 670
Phe He Pro Phe Ser Leu Pro Leu He Tyr His Gly Ser Tyr Asn Thr 675 680 685
Ser Ser Gly Ala Asp Asp Val Leu Trp Ser Ser Ser Asn Met Asn Tyr 690 695 700
Tyr Asp He He Val Asn Gly Gin Ala Asn Ser Ser Ser He Ala Ser 705 710 715 720
Ser Met His Leu Leu Asn Lys Gly Lys Val He Lys Thr He Asp He 725 730 735
Pro Gly His Ser Glu Thr Phe Phe Ala Thr Phe Pro Val Pro Glu Gly 740 745 750
Phe Asn Glu Val Arg He Leu Ala Gly Leu Pro Glu Val Ser Gly Asn 755 760 765
He Thr Val Gin Ser Asn Asn Pro Pro Gin Pro Ser Asn Asn Gly Gly 770 775 780
Gly Asp Gly Gly Gly Asn Gly Gly Gly Asp Gly Gly Gin Tyr Asn Phe 785 790 795 800
Ser Leu Ser Gly Ser Asp His Thr Thr He Tyr His Gly Lys Leu Glu 805 810 815
Thr Gly He His Val Gin Gly Asn Tyr Thr Tyr Thr Gly Thr Pro Val 820 825 830
Leu He Leu Asn Ala Tyr Arg Asn Asn Thr Val Val Ser Ser He Pro 835 840 845
Val Tyr Ser Pro Phe Asp He Thr He Gin Thr Glu Ala Asp Ser Leu 850 855 860
Glu Leu Glu Leu Gin Pro Arg Tyr Gly Phe Ala Thr Val Asn Gly Thr 865 870 875 880
Ala Thr Val Lys Ser Pro Asn Val Asn Tyr Asp Arg Ser Phe Lys Leu 885 890 895
Pro He Asp Leu Gin Asn He Thr Thr Gin Val Asn Ala Leu Phe Ala 900 905 910
Ser Gly Thr Gin Asn Met Leu Ala His Asn Val Ser Asp His Asp He 915 920 925
Glu Glu Val Val Leu Lys Val Asp Ala Leu Ser Asp Glu Val Phe Gly ' 930 935 940
Asp Glu Lys Lys Ala Leu Arg Lys Leu Val Asn Gin Ala Lys Arg Leu 945 * 950 955 960
Ser Arg Ala Arg Asn Leu Leu He Gly Gly Ser Phe Glu Asn Trp Asp 965 970 975
Ala Trp Tyr Lys Gly Arg Asn Val Val Thr Val Ser Asp His Glu Leu 980 985 990
Phe Lys Ser Asp His Val Leu Leu Pro Pro Pro Gly Leu Ser Pro Ser 995 1000 1005
Tyr He Phe Gin Lys Val Glu Glu Ser Lys Leu Lys Pro Asn Thr Arg 1010 1015 1020
Sr He Val Ser Gly Phe He Ala His Gly Lys Asp Leu Glu He Val 25 1030 1035 1040
Val Ser Arg Tyr Gly Gin Glu Val Gin Lys Val Val Gin Val Pro Tyr 1045 1050 1055
Gly Glu Ala Phe Pro Leu Thr Ser Asn Gly Pro Val Cys Cys Pro Pro 1060 1065 1070
Arg Ser Thr Ser Asn Gly Thr Leu Gly Asp Pro His Phe Phe Ser Tyr 1075 1080 1085
Ser He Asp Val Gly Ala Leu Asp Leu Gin Ala Asn Pro Gly He Glu 1090 1095 1100
Phe Gly Leu Arg He Val Asn Pro Thr Gly Met Ala Arg Val Ser Asn 1105 1110 1115 1120
Leu Glu He Arg Glu Asp Arg Pro Leu Ala Ala Asn Glu He Arg Gin 1125 1130 1135
Val Gin Arg Val Ala Arg Asn Trp Arg Thr Glu Tyr Glu Lys Glu Arg 1140 1145 1150
Ala Glu Val Thr Ser Leu He Gin Pro Val He Asn Arg He Asn Gly 1155 1160 1165
Leu Tyr Glu Asn Gly Asn Trp Asn Gly Ser He Arg Ser Asp He Ser 1170 1175 1180
2r Gin Asn He Asp Ala He Val Leu Pro Thr Leu Pro Lys Leu Arg 85 1190 1195 1200
His Trp Phe Met Ser Asp Arg Phe Ser Glu Gin Gly Asp He Met Ala 1205 1210 1215
Lys Phe Gin Gly Ala Leu Asn Arg Ala Tyr Ala Gin Leu Glu Gin Ser 1220 1225 1230
Thr Leu Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr 1235 1240 1245
He Glu Gly Asp Ala His Gin He Thr Leu Glu Asp Gly Arg Arg Val 1250 1255 1260
Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gin Met He Glu He 1265 1270 1275 1280
Glu Asn Phe Asn Pro Asp Lys Glu Tyr Asn Leu Val Phe His Gly Gin 1285 1290 1295
Gly Glu Gly Thr Val Thr Leu Glu His Gly Glu Glu Thr Lys Tyr He 1300 1305 1310
Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Ser Gin Arg Gin 1315 1320 1325
Gly Leu Thr Phe Glu Ser Asn Lys Val Thr Val Thr He Ser Ser Glu 1330 1335 1340
Asp Gly Glu Phe Leu Val Asp Asn He Ala Leu Val Glu Ala Pro Leu 1345 1350 1355 1360
Pro Thr Asp Asp Gin Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn 1365 1370 1375
Ser Asp Thr Ser Met Asn Asn Asn Gin 1380 1385
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: ,
LENGTH: 3867 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(B) STRAIN: PS17
(C) INDIVIDUAL ISOLATE: PSl7b
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 1628) NRRL B-18652
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGGCAATTT TAAATGAATT ATATCCATCT GTACCTTATA ATGTATTGGC GTATACGCCA 60 CCCTCTTTTT TACCTGATGC GGGTACACAA GCTACACCTG CTGACTTAAC AGCTTATGAA 120
CAATTGTTGA AAAATTTAGA AAAAGGGATA AATGCTGGAA CTTATTCGAA AGCAATAGCT 180
GATGTACTTA AAGGTATTTT TATAGATGAT ACAATAAATT ATCAAACATA TGTAAATATT 240
GGTTTAAGTT TAATTACATT AGCTGTACCG GAAATTGGTA TTTTTACACC TTTCATCGGT 300
TTGTTTTTTG CTGCATTGAA TAAACATGAT GCTCCACCTC CTCCTAATGC AAAAGATATA 360
TTTGAGGCTA TGAAACCAGC GATTCAAGAG ATGATTGATA GAACTTTAAC TGCGGATGAG 420
CAAACATTTT TAAATGGGGA AATAAGTGGT TTACAAAATT TAGCAGCAAG ATACCAGTCT 480
ACAATGGATG ATATTCAAAG CCATGGAGGA TTTAATAAGG TAGATTCTGG ATTAATTAAA 540
AAGTTTACAG ATGAGGTACT ATCTTTAAAT AGTTTTTATA CAGATCGTTT ACCTGTATTT 600
ATTACAGATA ATACAGCGGA TCGAACTTTG TTAGGTCTTC CTTATTATGC TATACTTGCG 660
AGCATGCATC TTATGTTATT AAGAGATATC ATTACTAAGG GTCCGACATG GGATTCTAAA 720
ATTAATTTCA CACCAGATGC AATTGATTCC TTTAAAACCG ATATTAAAAA TAATATAAAG 780
CTTTACTCTA AAACTATTTA TGACGTATTT CAGAAGGGAC TTGCTTCATA CGGAACGCCT 840
TCTGATTTAG AGTCCTTTGC AAAAAAACAA AAATATATTG AAATTATGAC AACACATTGT 900
TTAGATTTTG CAAGATTGTT TCCTACTTTT GATCCAGATC TTTATCCAAC AGGATCAGGT 960
GATATAAGTT TACAAAAAAC ACGTAGAATT CTTTCTCCTT TTATCCCTAT ACGTACTGCA 1020
GATGGGTTAA CATTAAATAA TACTTCAATT GATACTTCAA ATTGGCCTAA TTATGAAAAT 1080
GGGAATGGCG CGTTTCCAAA CCCAAAAGAA AGAATATTAA AACAATTCAA ACTGTATCCT 1140
AGTTGGAGAG CGGCACAGTA CGGTGGGCTT TTACAACCTT ATTTATGGGC AATAGAAGTC 1200
CAAGATTCTG TAGAGACTCG TTTGTATGGG CAGCTTCCAG CTGTAGATCC ACAGGCAGGG 1260
CCTAATTATG TTTCCATAGA TTCTTCTAAT CCAATCATAC AAATAAATAT GGATACTTGG 1320
AAAACACCAC CACAAGGTGC GAGTGGGTGG AATACAAATT TAATGAGAGG AAGTGTAAGC 1380
GGGTTAAGTT TTTTACAACG AGATGGTACG AGACTTAGTG CTGGTATGGG TGGTGGTTTT 1440
GCTGATACAA TATATAGTCT CCCTGCAACT CATTATCTTT CTTATCTCTA TGGAACTCCT 1500
TATCAAACTT CTGATAACTA TTCTGGTCAC GTTGGTGCAT TGGTAGGTGT GAGTACGCCT 1560
CAAGAGGCTA CTCTTCCTAA TATTATAGGT CAACCAGATG AACAGGGAAA TGTATCTACA 1620
ATGGGATTTC CGTTTGAAAA AGCTTCTTAT GGAGGTACAG TTGTTAAAGA ATGGTTAAAT 1680
GGTGCGAATG CGATGAAGCT TTCTCCTGGG CAATCTATAG GTATTCCTAT TACAAATGTA 1740
ACAAGTGGAG AATATCAAAT TCGTTGTCGT TATGCAAGTA ATGATAATAC TAACGTTTTC 1800
TTTAATGTAG ATACTGGTGG AGCAAATCCA ATTTTCCAAC AGATAAACTT TGCATCTACT I860
GTAGATAATA ATACGGGAGT ACAAGGAGCA AATGGTGTCT ATGTAGTCAA ATCTATTGCT 1920
ACAACTGATA ATTCTTTTAC AGTAAAAATT CCTGCGAAGA CGATTAATGT TCATTTAACC 1980
AACCAAGGTT CTTCTGATGT CTTTTTAGAT CGTATTGAGT TTGTTCCAAT TCTAGAATCA 2040
AATACTGTAA CTATATTCAA CAATTCATAT ACTACAGGTT CAGCAAATCT TATACCAGCA 2100
ATAGCTCCTC TTTGGAGTAC TAGTTCAGAT AAAGCCCTTA CAGGTTCTAT GTCAATAACA 2160
GGTCGAACTA CCCCTAACAG TGATGATGCT TTGCTTCGAT TTTTTAAAAC TAATTATGAT 2220
ACACAAACCA TTCCTATTCC GGGTTCCGGA AAAGATTTTA CAAATACTCT AGAAATACAA 2280
GACATAGTTT CTATTGATAT TTTTGTCGGA TCTGGTCTAC ATGGATCCGA TGGATCTATA 2340
AAATTAGATT TTACCAATAA TAATAGTGGT AGTGGTGGCT CTCCAAAGAG TTTCACCGAG 2400
CAl'-A TGATT TAGAGAATAT CACAACACAA GTGAATGCTC TATTCACATC TAATACACAA 2460
GATGCACTTG CAACAGATGT GAGTGATCAT GATATTGAAG AAGTGGTTCT AAAAGTAGAT 2520
GCATTATCTG ATGAAGTGTT TGGAAAAGAG AAAAAAACAT TGCGTAAATT TGTAAATCAA 2580
GCGAAGCGCT TAAGCAAGGC GCGTAATCTC CTGGTAGGAG GCAATTTTGA TAACTTGGAT 2640
GCTTGGTATA GAGGAAGAAA TGTAGTAAAC GTATCTAATC ACGAACTGTT GAAGAGTGAT 2700
CATGTATTAT TACCACCACC AGGATTGTCT CCATCTTATA TTTTCCAAAA AGTGGAGGAA 2760
TCTAAATTAA AACGAAATAC ACGTTATACG GTTTCTGGAT TTATTGCGCA TGCAACAGAT 2820
TTAGAAATTG TGGTTTCTCG TTATGGGCAA GAAATAAAGA AAGTGGTGCA AGTTCCTTAT 2880
GGAGAAGCAT TCCCATTAAC ATCAAGTGGA CCAGTTTGTT GTATCCCACA TTCTACAAGT 2940
AATGGAACTT TAGGCAATCC ACATTTCTTT AGTTACAGTA TTGATGTAGG TGCATTAGAT 3000
GTAGACACAA ACCCTGGTAT TGAATTCGGT CTTCGTATTG TAAATCCAAC TGGAATGGCA 3060
CGCGTAAGCA ATTTGGAAAT TCGTGAAGAT CGTCCATTAG CAGCAAATGA AATACGACAA 3120
GTACAACGTG TCGCAAGAAA TTGGAGAACC GAGTATGAGA AAGAACGTGC GGAAGTAACA 3180
AGTTTAATTC AACCTGTTAT CAATCGAATC AATGGATTGT ATGACAATGG AAATTGGAAC 3240
GGTTCTATTC GTTCAGATAT TTCGTATCAG AATATAGACG CGATTGTATT ACCAACGTTA 3300
CCAAAGTTAC GCCATTGGTT TATGTCAGAT AGATTTAGTG AACAAGGAGA TATCATGGCT 3360
AAATTCCAAG GTGCATTAAA TCGTGCGTAT GCACAACTGG AACAAAATAC GCTTCTGCAT 3420
AATGGTCATT TTACAAAAGA TGCAGCCAAT TGGACGGTAG AAGGCGATGC ACATCAGGTA 3480
GTATTAGAAG ATGGTAAACG TGTATTACGA TTGCCAGATT GGTCTTCGAG TGTGTCTCAA 3540
ACGATTGAAA TCGAGAATTT TGATCCAGAT AAAGAATATC AATTAGTATT TCATGGGCAA 3600
GGAGAAGGAA CGGTTACGTT GGAGCATGGA GAAGAAACAA AATATATAGA AACGCATACA 3660
CATCATTTTG CGAATTTTAC AACTTCTCAA CGTCAAGGAC TCACGTTTGA ATCAAATAAA 3720
GTGACAGTGA CCATTTCTTC AGAAGATGGA GAATTCTTAG TGGATAATAT TGCGCTTGTG 3780
GAAGCTCCTC TTCCTACAGA TGACCAAAAT TCTGAGGGAA ATACGGCTTC CAGTACGAAT 3840
AGCGATACAA GTATGAACAA CAATCAA 3867
(2) INFORMATION FOR SEQ ID NO: :
(i) SEQUENCE CHARACTERISTICS:
LENGTH: 1289 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(j"A C ) ) ■ OORRGGAANNIISSMM:: BBACILLUS THURINGIENSIS
INDIVIDUAL ISOLATE: PS17
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 1628) NRRL B-18652
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ala He Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu 1 5 10 15
Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp Ala Gly Thr Gin Ala Thr 20 25 30
Pro Ala Asp Leu Thr Ala Tyr Glu Gin Leu Leu Lys Asn Leu Glu Lys 35 40 45
Gly He Asn Ala Gly Thr Tyr Ser Lys Ala He Ala Asp Val Leu Lys 50 55 60
Gly He Phe He Asp Asp Thr He Asn Tyr Gin Thr Tyr Val Asn He 65 70 75 80
Gly Leu Ser Leu He Thr Leu Ala Val Pro Glu He Gly He Phe Thr 85 90 95
Pro Phe He Gly Leu Phe Phe Ala Ala Leu Asn Lys His Asp Ala Pro 100 105 110
Pro Pro Pro Asn Ala Lys Asp He Phe Glu Ala Met Lvs Pro Ala He
115 120 1 £25
Gin Glu Met He Asp Arg Thr Leu Thr Ala Asp Glu Gin Thr Phe Leu 130 135 140
Asn Gly Glu He Ser Gly Leu Gin Asn Leu Ala Ala Arg Tyr Gin Ser
145 150 155 160
Thr Met Asp Asp Hliee GGiinn SSeerr HHiiss GGllyy GGllyy PPhhee AAssnn LLyyss VVaall AAsstp Ser 165 170 17ϊ
Gly Leu He Lys Lys Phe Thr Asp Glu Val Leu Ser Leu Asn Ser Phe 180 185 190
Tyr Thr Asp Arg Leu Pro Val Phe He Thr Asp Asn Thr Ala Asp Arg 195 200 205
Thr Leu Leu Gly Leu Pro Tyr Tyr Ala He Leu Ala Ser Met His Leu 210 215 220
Met Leu Leu Arg Asp He He Thr Lys Gly Pro Thr Trp Asp Ser Lys 225 230 235 240
He Asn Phe Thr Pro Asp Ala He Asp Ser Phe Lys Thr Asp He Lys 245 250 255
Asn Asn He Lys Leu Tyr Ser Lys Thr He Tyr Asp Val Phe Gin Lys 260 265 270
Gly Leu Ala Ser Tyr Gly Thr Pro Ser Asp Leu Glu Ser Phe Ala Lys 275 280 285
Lys Gin Lys Tyr He Glu He Met Thr Thr His Cys Leu Asp Phe Ala 290 295 300
Arg Leu Phe Pro Thr Phe Asp Pro Asp Leu Tyr Pro Thr Gly Ser Gly 305 310 315 320
Asp He Ser Leu G32in5 Lys Thr Arg * - » Arg * => 3 H 3 e 0 Leu Ser Pro Phe 3 H 3 e 5 Pro
He Arg Thr Ala Asp Gly Leu Thr Leu Asn Asn Thr Ser He Asp Thr 340 345 350
Ser Asn Trp Pro Asn Tyr Glu Asn Gly Asn Gly Ala Phe Pro Asn Pro 355 360 365
Lys Glu Arg He Leu Lys Gin Phe Lys Leu Tyr Pro Ser Trp Arg Ala ' 370 375 380
Ala Gin Tyr Gly Gly Leu Leu Gin Pro Tyr Leu Trp Ala He Glu Val 385 390 395 400
Gin Asp Ser Val Glu Thr Arg Leu Tyr Gly Gin Leu Pro Ala Val Asp 405 410 415
Pro Gin Ala Gly Pro Asn Tyr Val Ser He Asp Ser Ser Asn Pro He 420 425 430
He Gin He Asn Met Asp Thr Trp Lys Thr Pro Pro Gin Gly Ala Ser , 435 440 445
Gly Trp Asn Thr Asn Leu Met Arg Gly Ser Val Ser Gly Leu Ser Phe 450 455 460
Leu Gin Arg Asp Gly Thr Arg Leu Ser Ala Gly Met Gly Gly Gly Phe 465 470 475 480
Ala Asp Thr He Tyr Ser Leu Pro Ala Thr His Tyr Leu Ser Tyr Leu 485 490 495
Tyr Gly Thr Pro Tyr Gin Thr Ser Asp Asn Tyr Ser Gly His Val Gly 500 505 510
Ala Leu Val Gly Val Ser Thr Pro Gin Glu Ala Thr Leu Pro Asn He 515 520 525
He Gly Gin Pro Asp Glu Gin Gly Asn Val Ser Thr Met Gly Phe Pro 530 535 540
Phe Glu Lys Ala Ser Tyr Gly Gly Thr Val Val Lys Glu Trp Leu Asn 545 550 555 560
Gly Ala Asn Ala Met Lys Leu Ser Pro Gly Gin Ser He Gly He Pro 565 570 575
He Thr Asn Val Thr Ser Gly Glu Tyr Gin He Arg Cys Arg Tyr Ala 580 585 590
Ser Asn Asp Asn Thr Asn Val Phe Phe Asn Val Asp Thr Gly Gly Ala 595 600 605
Asn Pro He Phe Gin Gin He Asn Phe Ala Ser Thr Val Asp Asn Asn 610 615 620
Thr Gly Val Gin Gly Ala Asn Gly Val Tyr Val Val Lys Ser He Ala 625 630 635 640
Thr Thr Asp Asn Ser Phe Thr Val Lys He Pro Ala Lys Thr He Asn 645 650 655
Val His Leu Thr Asn Gin Gly Ser Ser Asp Val Phe Leu Asp Arg He 660 665 670
Glu Phe Val Pro He Leu Glu Ser Asn Thr Val Thr He Phe Asn Asn 675 680 685
Ser Tyr Thr Thr Gly Ser Ala Asn Leu He Pro Ala He Ala Pro Leu 690 695 700
Trp Ser Thr Ser Ser Asp Lys Ala Leu Thr Gly Ser Met Ser He Thr 705 710 715 720
Gly Arg Thr Thr Pro Asn Ser Asp Asp Ala Leu Leu Arg Phe Phe Lys 725 730 735
Thr Asn Tyr Asp Thr Gin Thr He Pro He Pro Gly Ser Gly Lys Asp 740 745 750
Phe Thr Asn Thr Leu Glu He Gin Asp He Val Ser He Asp He Phe 755 760 765
Val Gly Ser Gly Leu His Gly Ser Asp Gly Ser He Lys Leu Asp Phe 770 775 780
Thr Asn Asn Asn Ser Gly Ser Gly Gly Ser Pro Lys Ser Phe Thr Glu 785 790 795 800
Gin Asn Asp Leu Glu Asn He Thr Thr Gin Val Asn Ala Leu Phe Thr 805 810 815
Ser Asn Thr Gin Asp Ala Leu Ala Thr Asp Val Ser Asp His Asp He 820 825 830
Glu Glu Val Val Leu Lys Val Asp Ala Leu Ser Asp Glu Val Phe Gly 835 840 845
Lys Glu Lys Lys Thr Leu Arg Lys Phe Val Asn Gin Ala Lys Arg Leu 850 855 860
Ser Lys Ala Arg Asn Leu Leu Val Gly Gly Asn Phe Asp Asn Leu Asp
865 8 ■ "7"0 *■ 8 * --75- 8"8",o
Ala Trp Tyr Arg GGllyy AArrgg AAssnn VVaall VVaall AAssnn VVaall SSeerr AAssnn HHiiss GGlluu Leu 885 890 895
Leu Lys Ser Asp His Val Leu Leu Pro Pro Pro Gly Leu Ser Pro Ser 900 905 910
Tyr He Phe Gin Lys Val Glu Glu Ser Lys Leu Lys Arg Asn Thr Arg 915- • 920 925
Tyr Thr Val Ser Gly Phe He Ala His Ala Thr Asp Leu Glu He Val 930 935 940
Val Ser Arg Tyr Gly Gin Glu He Lys Lys Val Val Gin Val Pro Tyr 945 950 955 950
Gly Glu Ala Phe Pro Leu Thr Ser Ser Gly Pro Val Cys Cys He Pro 965 970 975
His Ser Thr Ser Asn Gly Thr Leu Gly Asn Pro His Phe Phe Ser Tyr 980 985 990
Ser He Asp Val Gly Ala Leu Asp Val Asp Thr Asn Pro Gly He Glu
995 1000 1005
Phe Gly Leu Arg He Val Asn Pro Thr Gly Met Ala Arg Val Ser Asn 1010 1015 1020
Leu Glu He Arg Glu Asp Arg Pro Leu Ala Ala Asn Glu He Arg Gin 1025 1030 1035 1040
Val Gin Arg Val Ala Arg Asn Trp Arg Thr Glu Tyr Glu Lys Glu Arσ 1045 1050 1055
Ala Glu Val Thr Ser Leu He Gin Pro Val He Asn Arg He Asn Gly 1060 1065 1070
Leu Tyr Asp Asn Gly Asn Trp Asn Gly Ser He Arg Ser Asp He Ser 1075 1080 1085
Tyr Gin Asn He Asp Ala He Val Leu Pro Thr Leu Pro Lys Leu Arg 1090 1095 1100
His Trp Phe Met Ser Asp Arg Phe Ser Glu Gin Gly Asp He Met Ala 1105 1110 1115 1120
Lys Phe Gin Gly Ala Leu Asn Arg Ala Tyr Ala Gin Leu Glu Gin Asn 1125 1130 1135
Thr Leu Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr 1140 1145 1150
Val Glu Gly Asp Ala His Gin Val Val Leu Glu Asp Gly Lys Arg Val 1155 1160 1165
Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gin Thr He Glu He 1170 1175 1180
Glu Asn Phe Asp Pro Asp Lys Glu Tyr Gin Leu Val Phe His Gly Gin 1185 1190 1195 1200
Gly Glu Gly Thr Val Thr Leu Glu His Gly Glu Glu Thr Lys Tyr He 1205 1210 1215
Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Ser Gin Arg Gin 1220 1225 1230
Gly Leu Thr Phe Glu Ser Asn Lys Val Thr Val Thr He Ser Ser Glu 1235 1240 1245
Asp Gly Glu Phe Leu Val Asp Asn lie Ala Leu Val Glu Ala Pro Leu 1250 1255 1260
Pro Thr Asp Asp Gin Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn 1265 1270 1275 1280
Ser Asp Thr Ser Met Asn Asn Asn Gin 1285
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3771 base pairs
(B) TYPE: nucleic acid
(C) STRANDΞDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
A- ' ) OORRGGAANNIISSMM:: BBacillus thuringiensis
IS! INDIVIDUAL ISOLATE: 33f2
( ii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 2316) B-18785
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 4..27
(D) OTHER INFORMATION: /function= "oligonucleotide hybridization probe"
/produc = "GCA/T ACA/T TTA AAT GAA GTA/T TAT" /standard name= "probe a" /note= "Probe A"
(ix)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATGGCTACAC TTAATGAAGT ATATCCTGTG AATTATAATG TATTATCTTC TGATGCTTTT 60
CAACAATTAG ATACAACAGG TTTTAAAAGT AAATATGATG AAATGATAAA AGCATTCGAA 120
AAAAAATGGA AAAAAGGGGC AAAAGGAAAA GACCTTTTAG ATGTTGCATG GACTTATATA 180
ACTACAGGAG AAATTGACCC TTTAAATGTA ATTAAAGGTG TTTTATCTGTATTAACTTTA 240
ATTCCTGAAG TTGGTACTGT GGCCTCTGCA GCAAGTACTA TTGTAAGTTT TATTTGGCCT 300
AAAATATTTG GAGATAAACC AAATGCAAAA AATATATTTG AAGAGCTCAA GCCTCAAATT 360
GAAGCATTAA TTCAACAAGA TATAACAAAC TATCAAGATG CAATTAATCA AAAAAAATTT 420
GACAGTCTTC AGAAAACAAT TAATCTATAT ACAGTAGCTA TAGATAACAA TGATTACGTA 480
ACAGCAAAAA CGCAACTCGA AAATCTAAAT TCTATACTTA CCTCAGATAT CTCCATATTT 540
ATTCCAGAAG GATATGAAAC TGGAGGTTTA CCTTATTATG CTATGGTTGC TAATGCTCAT 600
ATATTATTGT TAAGAGACGC TATAGTTAAT GCAGAGAAAT TAGGCTTTAG TGATAAAGAA 660
GTAGACACAC ATAAAAAATA TATCAAAATG ACAATACACA ATCATACTGA AGCAGTAATA 720
AAAGCATTCT TAAATGGACT TGACAAATTT AAGAGTTTAG ATGTAAATAG CTATAATAAA 780
AAAGCAAATT ATATTAAAGG TATGACAGAA ATGGTTCTTG ATCTAGTTGC TCTATGGCCA 840
ACTTTCGATC CAGATCATTA TCAAAAAGAA GTAGAAATTG AATTTACAAG AACTATTTCT 900
TCTCCAATTT ACCAACCTGT ACCTAAAAAC ATGCAAAATA CCTCTAGCTC TATTGTACCT 960
AGCGATCTAT TTCACTATCA AGGAGATCTT GTAAAATTAG AATTTTCTAC AAGAACGGAC 1020
AACGATGGTC TTGCAAAAAT TTTTACTGGT ATTCGAAACA CATTCTACAA ATCGCCTAAT 1080
ACTCATGAAA CATACCATGT AGATTTTAGT TATAATACCC AATCTAGTGG TAATATTTCA 1140
AGAGGCTCTT CAAATCCGAT TCCAATTGAT CTTAATAATC CCATTATTTC AACTTGTATT 1200
AGAAATTCAT TTTATAAGGC AATAGCGGGA TCTTCTGTTT TAGTTAATTT TAAAGATGGC 1260
ACTCAAGGGT ATGCATTTGC CCAAGCACCA ACAGGAGGTG CCTGGGACCA TTCTTTTATT 1320
GAATCTGATG GTGCCCCAGA AGGGCATAAA TTAAACTATA TTTATACTTC TCCAGGTGAT 1380
ACATTAAGAG ATTTCATCAA TGTATATACT CTTATAAGTA CTCCAACTAT AAATGAACTA 1440
TCAACAGAAA AAATCAAAGG CTTTCCTGCG GAAAAAGGAT ATATCAAAAA TCAAGGGATC 1500
ATGAAATATT ACGGTAAACC AGAATATATT AATGGAGCTC AACCAGTTAA TCTGGAAAAC 1560
CAGCAAACAT TAATATTCGA ATTTCATGCT TCAAAAACAG CTCAATATAC CATTCGTATA 1620
CGTTATGCCA GTACCCAAGG AACAAAAGGT TATTTTCGTT TAGATAATCA GGAACTGCAA 1680
ACGCTTAATA TACCTACTTC ACACAACGGT TATGTAACCG GTAATATTGG TGAAAATTAT 1740
GATTTATATA CAATAGGTTC ATATACAATT ACAGAAGGTA ACCATACTCT TCAAATCCAA 1800
CATAATGATA AAAATGGAAT GGTTTTAGAT CGTATTGAAT TTGTTCCTAA AGATTCACTT 1860
CAAGATTCAC CTCAAGATTC ACCTCCAGAA GTTCACGAAT CAACAATTAT TTTTGATAAA 1920
TCATCTCCAA CTATATGGTC TTCTAACAAA CACTCATATA GCCATATACA TTTAGAAGGA 1980
TCATATACAA GTCAGGGAAG TTATCCACAC AATTTATTAA TTAATTTATT TCATCCTACA 2040
GACCCTAACA GAAATCATAC TATTCATGTT AACAATGGTG ATATGAATGT TGATTATGGA 2100
AAAGATTCTG TAGCCGATGG GTTAAATTTT AATAAAATAA CTGCTACGAT ACCAAGTGAT 2160
GCTTGGTATA GCGGTACTAT TACTTCTATG CACTTATTTA ATGATAATAA TTTTAAAACA 2220
ATAACTCCTA AATTTGAACT TTCTAATGAA TTAGAAAACA TCACAACTCA AGTAAATGCT 2280
TTATTCGCAT CTAGTGCACA AGATACTCTC GCAAGTAATG TAAGTGATTA CTGGATTGAA 2340
CAGGTCGTTA TGAAAGTCGA TGCCTTATCA GATGAAGTAT TTGGAAAAGA GAAAAAAGCA 2400
TTACGTAAAT TGGTAAATCA AGCAAAACGT CTCAGTAAAA TACGAAATCT TCTCATAGGT 2460
GGTAATTTTG ACAATTTAGT CGCTTGGTAT ATGGGAAAAG ATGTAGTAAA AGAATCGGAT 2520
CATGAATTAT TTAAAAGTGA TCATGTCTTA CTACCTCCCC CAACATTCCA TCCTTCTTAT 2580
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1425 base pairs iB) TYPE: nucleic acid |C) STRANDEDNESS: double D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
( i) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS52A1
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 2321) B-18770
(ix) FEATURE:
((AA)) NNAAMME/KEY: mat peptide B) LLOOCCAATTIIOONN:: 1..T425
D) OTHER INFORMATION: /product= "OPEN READING FRAME OF MATURE PROTEIN"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGATTATTG ATAGTAAAAC GACTTTACCT AGACATTCAC AATTCTAATA AGAAATATGG TCCTGGTGAT ATGACTAATG AAACAAGAAT GGGCTACGAT TGGAGCATAT ATTCAGACTG GAACAACAAT TAAGAACACA TGTTAATTTA AGTCAGGATA TCTCAATTAT ATGATGTTTA TTGTTCTGAT AAAACTTCAG TTATATCCTT TAATTATTAA ATCTGCTAAT GATATTGCTT GGTGATCCTT CTATTAAGAA AGATGGATAT TTTAAAAAAT ATTGTTGATA ATAATTCCGA TGATGATGCA ATAGCTAAAG CGATGTGGTA TTTTAATTAA AGAAGCTAAA CAATATGAAG
ACATCTTTAG ATCAATTTTT ACATGGTGAT CAGAAAAAAT TAGAAGGTGT TATCAATATT 600
CAAAAACGTT TAAAAGAAGT TCAAACAGCT CTTAATCAAG CCCATGGGGA AAGTAGTCCA 660
GCTCATAAAG AGTTATTAGA AAAAGTAAAA AATTTAAAAA CAACATTAGA AAGGACTATT 720
AAAGCTGAAC AAGATTTAGA GAAAAAAGTA GAATATAGTT TTCTATTAGG ACCATTGTTA 780
GGATTTGTTG TTTATGAAAT TCTTGAAAAT ACTGCTGTTC AGCATATAAA AAATCAAATT 840
GATGAGATAA AGAAACAATT AGATTCTGCT CAGCATGATT TGGATAGAGA TGTTAAAATT 900
ATAGGAATGT TAAATAGTAT TAATACAGAT ATTGATAATT TATATAGTCA AGGACAAGAA 960
GCAATTAAAG TTTTCCAAAA GTTACAAGGT ATTTGGGCTA CTATTGGAGC TCAAATAGAA 1020
AATCTTAGAA CAACGTCGTT ACAAGAAGTT CAAGATTCTG ATGATGCTGA TGAGATACAA 1080
ATTGAACTTG AGGACGCTTC TGATGCTTGG TTAGTTGTGG CTCAAGAAGC TCGTGATTTT 1140
ACACTAAATG CTTATTCAAC TAATAGTAGA CAAAATTTAC CGATTAATGT TATATCAGAT 1200
TCATGTAATT GTTCAACAAC AAATATGACA TCAAATCAAT ACAGTAATCC AACAACAAAT 1260
ATGACATCAA ATCAATATAT GATTTCACAT GAATATACAA GTTTACCAAA TAATTTTATG 1320
TTATCAAGAA ATAGTAATTT AGAATATAAA TGTCCTGAAA ATAATTTTAT GATATATTGG 1380
TATAATAATT CGGATTGGTA TAATAATTCG GATTGGTATA ATAAT 1425
(2) INFORMATION FOR SEQ ID NO:7 (PS52A1) :
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 475 amino acids B) TYPE: amino acid C) STRANDΞDNESS: single D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS52A1
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC 2321) B-18770
(ix) FEATURE: ,
(A) NAME/KEY: Protein
(B) LOCATION: 1..475
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met He He Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu He His 1 5 10 15
Thr He Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30
Asn Gly Asn Gin Phe He He Ser Lys Gin Glu Trp Ala Thr He Gly 35 40 45
Ala Tyr He Gin Thr Gly Leu Gly Leu Pro Val Asn Glu Gin Gin Leu 50 55 60
Arg Thr His Val Asn Leu Ser Gin Asp He Ser He Pro Ser Asp Phe 65 70 75 80
Ser Gin Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95
Trp Asn Lys Asn Leu Tyr Pro Leu He He Lys Ser Ala Asn Asp He 100 105 110
Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser He Lys Lys Asp 115 120 125
Gly Tyr Phe Lys Lys Leu Gin Asp Glu Leu Asp Asn He Val Asp Asn 130 135 140
Asn Ser Asp Asp Asp Ala He Ala Lys Ala He Lys Asp Phe Lys Ala 145 150 155 160
Arg Cys Gly He Leu He Lys Glu Ala Lys Gin Tyr Glu Glu Ala Ala 165 170 175
Lys Asn He Val Thr Ser Leu Asp Gin Phe Leu His Gly Asp Gin Lys 180 185 190
Lys Leu Glu Gly Val He Asn He Gin Lys Arg Leu Lys Glu Val Gin 195 200 205
Thr Ala Leu Asn Gin Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220
Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr He 225 230 235 240
Lys Ala Glu Gin Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255
Gly Pro Leu Leu Gly Phe Val Val Tyr Glu He Leu Glu Asn Thr Ala 260 265 270
Val Gin His He Lys Asn Gin He Asp Glu He Lys Lys Gin Leu Asp 275 280 285
Ser Ala Gin His Asp Leu Asp Arg Asp Val Lys He He Gly Met Leu 290 295 300
Asn Ser He Asn Thr Asp He Asp Asn Leu Tyr Ser Gin Gly Gin Glu 305 310 315 320
Ala He Lys Val Phe Gin Lys Leu Gin Gly He Trp Ala Thr He Gly 325 330 335
Ala Gin He Glu Asn Leu Arg Thr Thr Ser Leu Gin Glu Val Gin Asp 340 345 350
Ser Asp Asp Ala Asp Glu He Gin He Glu Leu Glu Asp Ala Ser Asp 355 360 365
Ala Trp Leu Val Val Ala Gin Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 r Ser Thr Asn Ser Arg 8 GGiinn AAssnn LLeeuu PPrroo HHee AAssnn VVaall HHee SSeerr AAssp 395 400
Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gin Tyr Ser Asn 405 410 415
Pro Thr Thr Asn Met Thr Ser Asn Gin Tyr Met He Ser His Glu Tyr 420 425 430
Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445
Tyr Lys Cys Pro Glu Asn Asn Phe Met He Tyr Trp Tyr Asn Asn Ser 450 455 460
Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1185 base pairs
(B) TYPE: nucleic acid
(C) STRANDΞDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS69D1
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2317) NRRL B-18816
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 1..T185
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATGATTTTAG GGAATGGAAA GACTTTACCA AAGCATATAA GATTAGCTCA TATTTTTGCA 60
ACACAGAATT CTTCAGCTAA GAAAGACAAT CCTCTTGGAC CAGAGGGGAT GGTTACTAAA 120
GACGGTTTTA TAATCTCTAA GGAAGAATGG GCATTTGTGC AGGCCTATGT GACTACAGGC 180
ACTGGTTTAC CTATCAATGA CGATGAGATG CGTAGACATG TTGGGTTACC ATCACGCATT 240
CAAATTCCTG ATGATTTTAA TCAATTATAT AAGGTTTATA ATGAAGATAA ACATTTATGC 300
AGTTGGTGGA ATGGTTTCTT GTTTCCATTA GTTCTTAAAA CAGCTAATGA TATTTCCGCT 360
TACGGATTTA AATGTGCTGG AAAGGGTGCC ACTAAAGGAT ATTATGAGGT CATGCAAGAC 420
GATGTAGAAA ATATTTCAGA TAATGGTTAT GATAAAGTTG CACAAGAAAA AGCACATAAG 480
GATCTGCAGG CGCGTTGTAA AATCCTTATT AAGGAGGCTG ATCAATATAA AGCTGCAGCG 540
GATGATGTTT CAAAACATTT AAACACATTT CTTAAAGGCG GTCAAGATTC AGATGGCAAT 600
GATGTTATTG GCGTAGAGGC TGTTCAAGTA CAACTAGCAC AAGTAAAAGA TAATCTTGAT 660
GGCCTATATG GCGACAAAAG CCCAAGACAT GAAGAGTTAC TAAAGAAAGT AGACGACCTG 720
AAAAAAGAGT TGGAAGCTGC TATTAAAGCA GAGAATGAAT TAGAAAAGAA AGTGAAAATG 780
AGTTTTGCTT TAGGACCATT ACTTGGATTT GTTGTATATG AAATCTTAGA GCTAACTGCG 840
GTCAAAAGTA TACACAAGAA AGTTGAGGCA CTACAAGCCG AGCTTGACAC TGCTAATGAT 900
GAACTCGACA GAGATGTAAA AATCTTAGGA ATGATGAATA GCATTGACAC TGATATTGAC 960
AACATGTTAG AGCAAGGTGA GCAAGCTCTT GTTGTATTTA GAAAAATTGC AGGCATTTGG 1020
AGTGTTATAA GTCTTAATAT CGGCAATCTT CGAGAAACAT CTTTAAAAGA GATAGAAGAA 1080
GAAAATGATG ACGATGCACT GTATATTGAG CTTGGTGATG CCGCTGGTCA ATGGAAAGAG 1140
ATAGCCGAGG AGGCACAATC CTTTGTACTA AATGCTTATA CTCCT 1185
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 395 amino acids B'ι TYPE: amino acid Ci STRANDEDNESS: single D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS ' (C) INDIVIDUAL ISOLATE: PS69D1
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2317) NRRL B-18816
(ix) FEATURE: ,
(A) NAME/KEY: Protein
(B) LOCATION: 1..395
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met He Leu Gly Asn Gly Lys Thr Leu Pro Lys His He Arg Leu Ala 1 5 10 15
His He Phe Ala Thr Gin Asn Ser Ser Ala Lys Lys Asp Asn Pro Leu 20 25 30
Gly Pro Glu Gly Met Val Thr Lys Asp Gly Phe He He Ser Lys Glu 35 40 45
Glu Trp Ala Phe Val Gin Ala Tyr Val Thr Thr Gly Thr Gly Leu Pro 50 55 60
He Asn Asp Asp Glu Met Arg Arg His Val Gly Leu Pro Ser Arg He 65 70 75 80
Gin He Pro Asp Asp Phe Asn Gin Leu Tyr Lys Val Tyr Asn Glu Asp 85 90 95
Lys His Leu Cys Ser Trp Trp Asn Gly Phe Leu Phe Pro Leu Val Leu 100 105 110
Lys Thr Ala Asn Asp He Ser Ala Tyr Gly Phe Lys Cys Ala Gly Lys 115 120 125
Gly Ala Thr Lys Gly Tyr Tyr Glu Val Met Gin Asp Asp Val Glu Asn 130 135 140
He Ser Asp Asn Gly Tyr Asp Lys Val Ala Gin Glu Lys Ala His Lys 145 150 155 160
Asp Leu Gin Ala Arg Cys Lys He Leu He Lys Glu Ala Asp Gin Tyr 165 170 175
Lys Ala Ala Ala Asp Asp Val Ser Lys His Leu Asn Thr Phe Leu Lys 180 185 190
Gly Gly Gin Asp Ser Asp Gly Asn Asp Val He Gly Val Glu Ala Val 195 200 205
Gin Val Gin Leu Ala Gin Val Lys Asp Asn Leu Asp Gly Leu Tyr Gly 210 215 220
Asp Lys Ser Pro Arg His Glu Glu Leu Leu Lys Lys Val Asp Asp Leu 225 230 235 240
Lys Lys Glu Leu Glu Ala Ala He Lys Ala Glu Asn Glu Leu Glu Lys 245 250 255
Lys Val Lys Met Ser Phe Ala Leu Gly Pro Leu Leu Gly Phe Val Val 260 265 270
Tyr Glu He Leu Glu Leu Thr Ala Val Lys Ser He His Lys Lys Val 275 280 285
Glu Ala Leu Gin Ala Glu Leu Asp Thr Ala Asn Asp Glu Leu Asp Arg 290 295 300
Asp Val Lys He Leu Gly Met Met Asn Ser He Asp Thr Asp He Asp 305 310 315 320
Asn Met Leu Glu Gin Gly Glu Gin Ala Leu Val Val Phe Arg Lys He 325 330 335
Ala Gly He Trp Ser Val He Ser Leu Asn He Gly Asn Leu Arg Glu 340 345 350
Thr Ser Leu Lys Glu He Glu Glu Glu Asn Asp Asp Asp Ala Leu Tyr 355 360 365
He G3l7u0 Leu Gly J Asp t Ala A3l7a5 Gly J Gin Trp*" Lys G 38 lu 0 He Ala Glu Glu
Ala Gin Ser Phe Val Leu Asn Ala Tyr Thr Pro
385 390 395 ,
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AGARTRKWTW AATGGWGCKM AW 22
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid ' (C STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Pro Thr Phe Asp Pro Asp Leu Tyr 1 5
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ala He Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val 1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 14 amino acids B) TYPE: amino acid C) STRANDEDNESS: single D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ala He Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val 1 5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 17 amino acids B'ι TYPE: amino acid Ci STRANDEDNESS: single D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met He He Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu He Asn 1 5 10 15
Thr
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met He Leu Gly Asn Gly Lys Thr Leu Pro Lys His He Arg Leu Ala 1 5 10 15
His He Phe Ala Thr Gin Asn Ser 20
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 bases i B) TYPE: nucleic acid iC STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GCAATTTTAA ATGAATTATA TCC 23
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 bases
(B) TYPE: nucleic acid
(C) STRMIDEDNESS^ single
TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATGATTATTG ATTCTAAAAC AACATTACCA AGACATTCWT TAATWAATAC WATWAA 56
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 bases
(B) TYPE: nucleic acid
C STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AAACATATTA GATTAGCACA TATTTTTGCA ACACAAAA 38
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CAAYTACAAG CWCAACC 17
(2) INFORMATION FOR SEQ ID NO:20:
(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:20: AGGAACAAAY TCAAKWCGRT CTA 23
I
(2) INFORMATION FOR SEQ ID NO:21:
(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:21: TGGAATAAAT TCAATTYKRT CWA 23
(2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS:
LENGTH: 28 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TGATTTTWMT CAATTATATR AKGTTTAT 28
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AAGAGTTAYT ARARAAAGTA 20
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TTAGGACCAT TRYTWGGATT TGTTGTWTAT GAAAT 35
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GAYAGAGATG TWAAAATYWT AGGAATG 27
(2) INFORMATION FOR SEQ ID NO:26:
(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:26: TTMTTAAAWC WGCTAATGAT ATT 23
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 1425 base pairs B) TYPE: nucleic acid Ci STRANDEDNESS: double D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS86A1
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC1638) NRRL B-18751
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 1..1425
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27
ATGATTATTG ATAGTAAAAC GACTTTACCT AGACATTCAC
AATTCTAATA AGAAATATGG TCCTGGTGAT ATGACTAATG
AAACAAGAAT GGGCTACGAT TGGAGCATAT ATTCAGACTG
GAACAACAAT TAAGAACACA TGTTAATTTA AGTCAGGATA
TCTCAATTAT ATGATGTTTA TTGTTCTGAT AAAACTTCAG TTATATCCTT TAATTATTAA ATCTGCTAAT GATATTGCTT
GGTGATCCTT CTATTAAGAA AGATGGATAT TTTAAAAAAT
ATTGTTGATA ATAATTCCGA TGATGATGCA ATAGCTAAAG
CGATGTGGTA TTTTAATTAA AGAAGCTAAA CAATATGAAG
ACATCTTTAG ATCAATTTTT ACATGGTGAT CAGAAAAAAT
CAAAAACGTT TAAAAGAAGT TCAAACAGCT CTTAATCAAG
GCTCATAAAG AGTTATTAGA AAAAGTAAAA AATTTAAAAA
AAAGCTGAAC AAGATTTAGA GAAAAAAGTA GAATATAGTT
GGATTTGTTG TTTATGAAAT TCTTGAAAAT ACTGCTGTTC
GATGAGATAA AGAAACAATT AGATTCTGCT CAGCATGATT
ATAGGAATGT TAAATAGTAT TAATACAGAT ATTGATAATT
GCAATTAAAG TTTTCCAAAA GTTACAAGGT ATTTGGGCTA
AATCTTAGAA CAACGTCGTT ACAAGAAGTT CAAGATTCTG
ATTGAACTTG AGGACGCTTC TGATGCTTGG TTAGTTGTGG
ACACTAAATG CTTATTCAAC TAATAGTAGA CAAAATTTAC
TCATGTAATT GTTCAACAAC AAATATGACA TCAAATCAAT
ATGACATCAA ATCAATATAT GATTTCACAT GAATATACAA
TTATCAAGAA ATAGTAATTT AGAATATAAA TGTCCTGAAA
TATAATAATT CGGATTGGTA TAATAATTCG GATTGGTATA
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
!A) LENGTH: 475 amino acids B) TYPE: amino acid C) STRANDEDNESS: single D) TOPOLOGY: linear
(ii)' MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS (C) INDIVIDUAL ISOLATE: PS86A1
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC1638) NRRL B-18751
(ix) FEATURE: ,
(A) NAME/KEY: Protein
(B) LOCATION: 1..475
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Met He He Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu He His 1 5 10 15
Thr He Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30
Asn Gly Asn Gin Phe He He Ser Lys Gin Glu Trp Ala Thr He Gly 35 40 45
Ala Tyr He Gin Thr Gly Leu Gly Leu Pro Val Asn Glu Gin Gin Leu 50 55 60
Arg Thr His Val Asn Leu Ser Gin Asp He Ser He Pro Ser Asp Phe 65 70 75 80
Ser Gin Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95
Trp Asn Lys Asn Leu Tyr Pro Leu He He Lys Ser Ala Asn Asp He 100 105 110
Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser He Lys Lys Asp 115 120 125
Gly Tyr Phe Lys Lys Leu Gin Asp Glu Leu Asp Asn He Val Asp Asn 1 130 135 140
Asn Ser Asp Asp Asp Ala He Ala Lys Ala He Lys Asp Phe Lys Ala 145 150 155 160
Arg Cys Gly He Leu He Lys Glu Ala Lys Gin Tyr Glu Glu Ala Ala 165 170 175
Lys Asn He Val Thr Ser Leu Asp Gin Phe Leu His Gly Asp Gin Lys 180 185 190
Lys Leu Glu Gly Val He Asn He Gin Lys Arg Leu Lys Glu Val Gin 195 200 205
Thr Ala Leu Asn Gin Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220
Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr He 225 230 235 240
Lys Ala Glu Gin Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255
Gly Pro Leu Leu Gly Phe Val Val Tyr Glu He Leu Glu Asn Thr Ala 260 265 270
Val Gin His He Lys Asn Gin He Asp Glu He Lys Lys Gin Leu Asp 275 280 285
Ser Ala Gin His Asp Leu Asp Arg Asp Val Lys He He Gly Met Leu 290 295 300
Asn Ser He Asn Thr Asp He Asp Asn Leu Tyr Ser Gin Gly Gin Glu 305 310 315 320
Ala He Lys Val Phe Gin Lys Leu Gin Gly He Trp Ala Thr He Gly 325 330 335
Ala Gin He Glu Asn Leu Arg Thr Thr Ser Leu Gin Glu Val Gin Asp 340 345 350
Ser Asp Asp Ala Asp Glu He Gin He Glu Leu Glu Asp Ala Ser Asp 355 360 365
Ala Trp Leu Val Val Ala Gin Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 ' 375 380 rr Ser Thr Asn Ser Arg G Giinn AAssnn LLeeuu PPrroo Hliee AAssnn Vvaall Hliee Sseerr AAssp
385 39o " 395 400
Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gin Tyr Ser Asn 405 410 415
Pro Thr Thr Asn Met Thr Ser Asn Gin Tyr Met He Ser His Glu Tyr 420 425 430
Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445
Tyr Lys Cys Pro Glu Asn Asn Phe Met He Tyr Trp Tyr Asn Asn Ser 450 455 460
Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3471 base pairs
(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:
(A) ORGANISM: Bacillus thuringiensis
(B) STRAIN: kumamotoensis
(C) INDIVIDUAL ISOLATE: PS50C
( ii) IMMEDIATE SOURCE:
(B) CLONE: Ξ. coli NM522(pMYC2320) NRRL B-18769
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
ATGAGTCCAA ATAATCAAAA TGAATATGAA ATTATAGATG CGACACCTTC TACATCTGTA 60
TCCAGTGATT CTAACAGATA CCCTTTTGCG AATGAGCCAA CAGATGCGTT ACAAAATATG 120
AATTATAAAG ATTATCTGAA AATGTCTGGG GGAGAGAATC CTGAATTATT TGGAAATCCG 180
GAGACGTTTA TTAGTTCATC CACGATTCAA ACTGGAATTG GCATTGTTGG TCGAATACTA 240
GGAGCTTTAG GGGTTCCATT TGCTAGTCAG ATAGCTAGTT TCTATAGTTT CATTGTTGGT 300
CAATTATGGC CGTCAAAGAG CGTAGATATA TGGGGAGAAA TTATGGAACG AGTGGAAGAA 360
CTCGTTGATC AAAAAATAGA AAAATATGTA AAAGATAAGG CTCTTGCTGA ATTAAAAGGG 420
CTAGGAAATG CTTTGGATGT ATATCAGCAG TCACTTGAAG ATTGGCTGGA AAATCGCAAT 480
GATGCAAGAA CTAGAAGTGT TGTTTCTAAT CAATTTATAG CTTTAGATCT TAACTTTGTT 540
AGTTCAATTC CATCTTTTGC AGTATCCGGA CACGAAGTAC TATTATTAGC AGTATATGCA 600
CAGGCTGTGA ACCTACATTT ATTGTTATTA AGAGATGCTT CTATTTTTGG AGAAGAGTGG 660
GGATTTACAC CAGGTGAAAT TTCTAGATTT TATAATCGTC AAGTGCAACT TACCGCTGAA 720
TATTCAGACT ATTGTGTAAA GTGGTATAAA ATCGGCTTAG ATAAATTGAA AGGTACCACT 780
TCTAAAAGTT GGCTGAATTA TCATCAGTTC CGTAGAGAGA TGACATTACT GGTATTAGAT 840
TTGGTGGCGT TATTTCCAAA CTATGACACA CATATGTATC CAATCGAAAC AACAGCTCAA 900
CTTACACGGG ATGTGTATAC AGATCCGATA GCATTTAACA TAGTGACAAG TACTGGATTC 960
TGCAACCCTT GGTCAACCCA CAGTGGTATT CTTTTTTATG AAGTTGAAAA CAACGTAATT 1020
CGTCCGCCAC ACTTGTTTGA TATACTCAGC TCAGTAGAAA TTAATACAAG TAGAGGGGGT 1080
ATTACGTTAA ATAATGATGC ATATATAAAC TACTGGTCAG GACATACCCT AAAATATCGT 1140,
AGAACAGCTG ATTCGACCGT AACATACACA GCTAATTACG GTCGAATCAC TTCAGAAAAG 1200
AATTCATTTG CACTTGAGGA TAGGGATATT TTTGAAATTA ATTCAACTGT GGCAAACCTA 1260
GCTAATTACT ACCAAAAGGC ATATGGTGTG CCGGGATCTT GGTTCCATAT GGTAAAAAGG 1320
GGAACCTCAT CAACAACAGC GTATTTATAT TCAAAAACAC ATACAGCTCT CCAAGGGTGT 1380
ACACAGGTTT ATGAATCAAG TGATGAAATA CCTCTAGATA GAACTGTACC GGTAGCTGAA 1440
AGCTATAGTC ATAGATTATC TCATATTACC TCCCATTCTT TCTCTAAAAA TGGGAGTGCA 1500
TACTATGGGA GTTTCCCTGT ATTTGTTTGG ACACATACTA GTGCGGATTT AAATAATACA 1560
ATATATTCAG ATAAAATCAC TCAAATTCCA GCGGTAAAGG GAGACATGTT ATATCTAGGG 1620
GGTTCCGTAG TACAGGGTCC TGGATTTACA GGAGGAGATA TATTAAAAAG AACCAATCCT 1680
AGCATATTAG GGACCTTTGC GGTTACAGTA AATGGGTCGT TATCACAAAG ATATCGTGTA 1740
AGAATTCGCT ATGCCTCTAC AACAGATTTT GAATTTACTC TATACCTTGG CGACACAATA 1800
GAAAAAAATA GATTTAACAA AACTATGGAT AATGGGGCAT CTTTAACGTA TGAAACATTT 1860
AAATTCGCAA GTTTCATTAC TGATTTCCAA TTCAGAGAAA CACAAGATAA AATACTCCTA 1920
TCCATGGGTG ATTTTAGCTC CGGTCAAGAA GTTTATATAG ACCGAATCGA ATTCATCCCA 1980
GTAGATGAGA CATATGAGGC GGAACAAGAT TTAGAAGCGG CGAAGAAAGC AGTGAATGCC 2040
TTGTTTACGA ATACAAAAGA TGGCTTACGA CCAGGTGTAA CGGATTATGA AGTAAATCAA 2100
GCGGCAAACT TAGTGGAATG CCTATCGGAT GATTTATATC CAAATGAAAA ACGATTGTTA 2160 TTTGATGCGG TGAGAGAGGC AAAACGCCTC AGTGGGGCAC GTAACTTACT ACAAGATCCA 2220 GATTTCCAAG AGATAAACGG AGAAAATGGA TGGGCGGCAA GTACGGGAAT TGAGATTGTA 2280 GAAGGGGATG CTGTATTTAA AGGACGTTAT CTACGCCTAC CAGGTGCACG AGAAATTGAT 2340 ACGGAAACGT ATCCAACGTA TCTGTATCAA AAAGTAGAGG AAGGTGTATT AAAACCATAC 2400
ACAAGATATA GACTGAGAGG GTTTGTGGGA AGTAGTCAAG GATTAGAAAT TTATACGATA 2460
CGTCACCAAA CGAATCGAAT TGTAAAGAAT GTACCAGATG ATTTATTGCC AGATGTATCT 2520
CCTGTAAACT CTGATGGCAG TATCAATCGA TGCAGCGAAC AAAAGTATGT GAATAGCCGT 2580
TTAGAAGGAG AAAACCGTTC TGGTGATGCA CATGAGTTCT CGCTCCCTAT CGATATAGGA 2640
GAGCTGGATT ACAATGAAAA TGCAGGAATA TGGGTTGGAT TTAAGATTAC GGACCCAGAG 2700
GGATACGCAA CACTTGGAAA TCTTGAATTA GTCGAAGAGG GACCTTTGTC AGGAGACGCA 2760
TTAGAGCGCT TGCAAAGAGA AGAACAACAG TGGAAGATTC AAATGACAAG AAGACGTGAA 2820
GAGACAGATA GAAGATACAT GGCATCGAAA CAAGCGGTAG ATCGTTTATA TGCCGATTAT 2880
CAGGATCAAC AACTGAATCC TGATGTAGAG ATTACAGATC TTACTGCGGC TCAAGATCTG 2940
ATACAGTCCA TTCCTTACGT ATATAACGAA ATGTTCCCAG AAATACCAGG GATGAACTAT 3000
ACGAAGTTTA CAGAATTAAC AGATCGACTC CAACAAGCGT GGAATTTGTA TGATCAGCGA 3060
AATGCCATAC CAAATGGTGA TTTTCGAAAT GGGTTAAGTA ATTGGAATGC AACGCCTGGC 3120
GTAGAAGTAC AACAAATCAA TCATACATCT GTCCTTGTGA TTCCAAACTG GGATGAACAA 3180
GTTTCACAAC AGTTTACAGT TCAACCGAAT CAAAGATATG TATTACGAGT TACTGCAAGA 3240
AAAGAAGGGG TAGGAAATGG ATATGTAAGT ATTCGTGATG GTGGAAATCA ATCAGAAACG 3300
CTTACTTTTA GTGCAAGCGA TTATGATACA AATGGTGTGT ATAATGACCA AACCGGCTAT 3360
ATCACAAAAA CAGTGACATT CATCCCGTAT ACAGATCAAA TGTGGATTGA AATAAGTGAA 3420
ACAGAAGGTA CGTTCTATAT AGAAAGTGTA GAATTGATTG TAGACGTAGA G 3471
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1157 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO
( i) ORIGINAL SOURCE: '
(A) ORGANISM: Bacillus thuringiensis
(B) STRAIN: kumamotoensis
(C) INDIVIDUAL ISOLATE: PS50C
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2320) NRRL B-18769
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: Met Ser Pro Asn Asn Gin Asn Glu Tyr Glu 1 5 10
Ser Thr Ser Val Ser Ser Asp Ser Asn Arg 20 25
Pro Thr Asp Ala Leu Gin Asn Met Asn Tyr 35 40
Ser Gly Gly Glu Asn Pro Glu Leu Phe Gly 50 55
Ser Ser Ser Thr He Gin Thr Gly He Gly 65 70 x
Gly Ala Leu Gly Val Pro Phe Ala Ser Gin He Ala Ser Phe 85 90 IF Ser
Phe He Val Gly Gin Leu Trp Pro Ser Lys Ser Val Asp He Trp Gly 100 105 110
Glu He Met Glu Arg Val Glu Glu Leu Val Asp Gin Lys He Glu Lys 115 120 125
Tyr Val Lys Asp Lys Ala Leu Ala Glu Leu Lys Gly Leu Gly Asn Ala * 130 * 135 * 140
Leu Asp Val Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asn 145 150 155 160
Asp Ala Arg Thr Arg Ser Val Val Ser Asn Gin Phe He Ala Leu Asp 165 170 175
Leu Asn Phe Val Ser Ser He Pro Ser Phe Ala Val Ser Gly His Glu 180 185 190
Val Leu Leu Leu Ala Val Tyr Ala Gin Ala Val Asn Leu His Leu Leu 195 200 205
Leu Leu Arg Asp Ala Ser He Phe Gly Glu Glu Trp Gly Phe Thr Pro 210 215 220
Gly Glu He Ser Arg Phe Tyr Asn Arg Gin Val Gin Leu Thr Ala Glu 225 230 235 240
Tyr Ser Asp Tyr Cys Val Lys Trp Tyr Lys He Gly Leu Asp Lys Leu 2 5 250 255
Lys Gly Thr Thr Ser Lys Ser Trp Leu Asn Tyr His Gin Phe Arg Arg 260 265 270
Glu Met Thr Leu Leu Val Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr 275 280 285
Asp Thr His Met Tyr Pro He Glu Thr Thr Ala Gin Leu Thr Arg Asp 290 295 300
Val Tyr Thr Asp Pro He Ala Phe Asn He Val Thr Ser Thr Gly Phe 305 310 315 320
Cys Asn Pro Trp Ser Thr His Ser Gly He Leu Phe Tyr Glu Val Glu 325 330 335
Asn Asn Val He Arg Pro Pro His Leu Phe Asp He Leu Ser Ser Val 340 345 350
Glu He Asn Thr Ser Arg Gly Gly He Thr Leu Asn Asn Asp Ala Tyr 355 360 365
He Asn Tyr Trp Ser Gly His Thr Leu Lys Tyr Arg Arg Thr Ala Asp 370 375 380
Ser Thr Val Thr Tyr Thr Ala Asn Tyr Gly Arg He Thr Ser Glu Lys 385 390 395 400
Asn Ser Phe Ala Leu Glu Asp Arg Asp He Phe Glu He Asn Ser Thr * 405 410 415
Val Ala Asn Leu Ala Asn Tyr Tyr Gin Lys Ala Tyr Gly Val Pro Gly - 420 425 430
Ser Trp Phe His Met Val Lys Arg Gly Thr Ser Ser Thr Thr Ala Tyr 435 440 445
Leu Tyr Ser Lys Thr His Thr Ala Leu Gin Gly Cys Thr Gin Val Tyr 450 455 450
Glu Ser Ser Asp Glu He Pro Leu Asp Arg Thr Val Pro Val Ala Glu 465 470 475 480
Ser Tyr Ser His Arg Leu Ser His He Thr Ser His Ser Phe Ser Lys 485 490 495
Asn Gly Ser Ala Tyr Tyr Gly Ser Phe Pro Val Phe Val Trp Thr His 500 505 510
Thr Ser Ala Asp Leu Asn Asn Thr He Tyr Ser Asp Lys He Thr Gin 515 520 525
He Pro Ala Val Lys Gly Asp Met Leu Tyr Leu Gly Gly Ser Val Val 530 535 540
Gin Gly Pro Gly Phe Thr Gly Gly Asp He Leu Lys Arg Thr Asn Pro 545 550 555 560
Ser He Leu Gly Thr Phe Thr Val Asn Gly Ser Leu Ser Gin 565 570 575 Arg Tyr Arg Val Arg He Ala Ser Thr Thr Asp Phe Glu Phe 580 585 590 Thr Leu r Leu Gly Asp Glu Lys Asn Arg Phe Asn Lys Thr 5 605
Met As Έp Asn Gly Ala Ser Tyr Glu Thr Phe Lys Phe Ala Ser
61 620
Phe He Thr Asp Phe Gin Glu Thr Gin Asp Lys He Leu Leu 625 630 635 640
Ser Met Gly Asp Phe Ser Gin Glu Val Tyr He Asp Arg He 645 650 655
Glu Phe He Leu Glu
Ala Ala Ly ^ s LLyyss Ala Val Asn Ala Leu Phe Thr Asn Thr Lys Asp Gly 6755 6'80 685
Leu Arg Pro Gly Val Thr Asp Tyr Glu Val Asn Gin Ala Ala Asn Leu 690 695 700
Val Glu Cys Leu Ser Asp Asp Leu Tyr Pro Asn Glu Lys Arg Leu Leu 705 710 715 720
Phe Asp Ala Val Arg Glu Ala Lys Arg Leu Ser Gly Ala Arg Asn Leu 725 730 735
Leu Gin Asp Pro Asp Phe Gin Glu He Asn Gly Glu Asn Gly Trp Ala 740 745 750
Ala Ser Thr Gly He Glu He Val Glu Gly Asp Ala Val Phe Lys Gly 755 760 765
Arg Tyr Leu Arg Leu Pro Gly Ala Arg Glu He Asp Thr Glu Thr Tyr 770 775 780
Pro Thr Tyr Leu Tyr Gin Lys Val Glu Glu Gly Val Leu Lys Pro Tyr 785 790 795 800
Thr Arg Tyr Arg Leu Arg Gly Phe Val Gly Ser Ser Gin Gly Leu Glu 805 810 815
He Tyr Thr He Arg His Gin Thr Asn Arg He Val Lys Asn Val Pro 820 825 830
Asp Asp Leu Leu Pro Asp Val Ser Pro Val Asn Ser Asp Gly Ser He 835 840 845
Asn Arg Cys Ser Glu Gin Lys Tyr Val Asn Ser Arg Leu Glu Gly Glu 850 855 860
Asn Arg Ser Gly Asp Ala His Glu Phe Ser Leu Pro He Asp He Gly 865 870 875 880
Glu Leu Asp Tyr Asn Glu Asn Ala Gly He Trp Val Gly Phe Lys He 885 890 895
Thr Asp Pro Glu Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu Val Glu 900 905 910
Glu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg Leu Gin Arg Glu Glu 915 920 925
Gin Gin Trp Lys He Gin Met Thr Arg Arg Arg Glu Glu Thr Asp Arg 930 935 940
Arg Tyr Met Ala Ser Lys Gin Ala Val Asp Arg Leu Tyr Ala Asp Tyr 945 * 950 * 955 * 950
Gin Asp Gin Gin Leu Asn Pro Asp Val Glu He Thr Asp Leu Thr Ala 965 970 975
Ala Gin Asp Leu He Gin Ser He Pro Tyr Val Tyr Asn Glu Met Phe 980 985 990
Pro Glu He Pro Gly Met Asn Tyr Thr Lys Phe Thr Glu Leu Thr Asp 995 1000 1005
Arg LLeeuu GGiinn GGiinn AAllaa TTrrpp AAssnn LLeeuu TTyyrr AAsspp GGiinn AArrcg Asn Ala He Pro l "o"io" 1 ■ "0 , 1"5 ι-o--.:--o
Asn Gly Asp Phe Arg Asn Gly Leu Ser Asn Trp Asn Ala Thr Pro Gly 1025 1030 1035 1040
Val Glu Val Gin Gin He Asn His Thr Ser Val Leu Val He Pro Asn 1045 1050 1055
Trp Asp Glu Gin Val Ser Gin Gin Phe Thr Val Gin Pro Asn Gin Arg 1060 1065 1070
Tyr Val Leu Arg Val Thr Ala Arg Lys Glu Gly Val Gly Asn Gly Tyr 1075 1080 1085
Val Ser He Arg Asp Gly Gly Asn Gin Ser Glu Thr Leu Thr Phe Ser 1090 1095 1100
Ala Ser Asp Tyr Asp Thr Asn Gly Val Tyr Asn Asp Gin Thr Gly Tyr 1105 1110 1115 1120
He Thr Lys Thr Val Thr Phe He Pro Tyr Thr Asp Gin Met Trp He 1125 1130 1135
Glu He Ser Glu Thr Glu Gly Thr Phe Tyr He Glu Ser Val Glu Leu 1140 1145 1150
He Val Asp Val Glu 1155