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United States Patent (19) 11 Patent Number: 5,756,459 Jackson Et Al IIIUS005756459A United States Patent (19) 11 Patent Number: 5,756,459 Jackson et al. 45 Date of Patent: May 26, 1998 54 INSECTICDALLY EFFECTIVE PEPTIDES Dunwiddie, T.V., 'The Use of In Vitro Brain Slices in SOLATABLE FROM PHDPPUS SPIDER Neuropharmacology". Electrophysiological Techniques in VENOM Pharmacology, H.M. Geller, 25ed. Alan R. Liss, Inc., New York, pp. 65-90 (1986). 75) Inventors: John Randolph Hunter Jackson; Eric Fuqua, et al., "A simple PCR Method for detection and DelMar; Janice Helen Johnson; cloning low abundant transcript". Biotechniques, 9:206-211 Robert Marden Kral, Jr., all of Salt (1990). Lake City, Utah Hink, et al., "Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines". Biotechnol. 73) Assignees: FMC Corporation, Philadelphia, Pa.; Prog, 7:9-14 (1991). 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Wax Toxins from the Venom of The Funnel Web Spider, Agele Assistant Examiner-Kawai Lau nopsis aperta", in Insect Neurochemistry and Neurophysi Attorney, Agent, or Firm-Woodcock Washburn Kurtz ology 1986, Borkevec and Gelman, eds. Humana Press, Mackiewicz & Norris LLP New Jersey, 1986. Carbonell, et al. "Synthesis of a gene coding for an 57 ABSTRACT insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors", Gene, 73:409-418 (1988). This invention provides a family of insecticidally effective Chomczynski, et al., "Single-Step Method of RNAIsolation proteins and particular members of that family which may by Acid Guanidinium. Thiocyanate-Phenol-Chloroform be isolated from the venom of the spider Phidippus audax, Extraction". Analytical Biochemistry, 162:156-159 (1987). DNA encoding such proteins, insecticidal compositions of Cutler, et al., "Electroporation: Being Developed to Trans these proteins or the DNA encoding them, and methods for form Crops, (success with model crop confirmed)", AG controlling invertebrate pests. Recombinant expression vec Biotech. News, (5):3 & 17 (1990). tors and host cells and methods for producing insecticidally Davies, et al., "Recombinant baculovirus vectors expressing effective peptides are also provided. gluthathione-S-transferase fusion proteins", Biotech, 11:933-936 (1993). 3 Claims, 5 Drawing Sheets 5,756,459 Page 2 OTHER PUBLICATIONS Raymond, "Presentation d'un programme Basic d'analyse log-probit pour micro-ordinateur", Set. Ent. med et Para Vialard, et al. "Synthesis of the Membrane Fusion and sitol., 23:117-121 1985. Hemagglutinin Proteins of Measles Virus Using a Novel Baculovirus Vector Containing the B-Galactosidase Gene". Luria, et al. In General Virology, John Wiley and Sons: New J. 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Biotechnology, 9:848-851, 1991. insect larvau of antigenic proteins of hepatitis B virus and Bentzien, "Biology of the Spider Diguetia imperiosa", The influenza virus", Proc. Natl. Acad. Sci., USA, 86:1453-1456, Pan-Pacific Entomologist, 49:110-123, 1973. 1989, Quicke, et al., "Extended summaries Pesticides Group and Bers, et al., "Protein and Nucleic Acid Blotting and Immu Physicochemical and Biophysical Panel Symposium Novel nobiochemical Detection". BioTechniques, 3:276-288, Approaches in Agrochemical Research". Pestic. Sci., 1985. 20:315-317, 1987. Quistad, et al., “Insecticidal Activity of Spider (Araneae), Bronstein, et al. "Rapid and Sensitive Detection of DNA in Centipede (Chilopoda), Scorpion (Scorpionida), and Snake Southern with Chemiluminescence". BioTechniques, (Serpentes) Venoms". J. Econ. Entom, 85:33-39. 8:310-314, 1990. Nentwig, et al. "Comparative Investigations on the Effect of Quistad, et al., "Paralytic and Insecticidal Toxins from the the Venoms of 18 Spider Species onto the Cockroach Blatta Funnel Web Spider, Hololena curta”, Toxicon, 29:329-336, orientalis(Blattodea)", Zool. Jb. Physiol. 96:279-290 1991. (1992). Friedel, et al., "Immobilizing and Lethal Effects of Spider Bowers, et al., "Identification and purification of an irre Venoms on the Cockroach and the Common Mealbeetle". versible presynaptic neurotoxin from the venom of spider Todicon, 27:305-316, 1989. Hololena curta', Proc. Natl. Acad. Sci. USA, Roth, "The Spider Genus Tegenaria in the Western Hemi 84:3506-3510, 1987. sphere (Agelenidae)". Amer: Museum Novitates, 2323:1-33, Geren, "Neurotoxins and Necrotoxins of Spider Venoms". J. 1968, Toxicol. -Toxin Reviews, 5:161-170, 1986. West, "Necrotic Arachnidism in the Northwest United States Grishin, "Toxic components from Buthus eupeus and Lycosa and Its Probably Relationship to Tegenaria agrestis (Wal singoriensis venoms". Front. Bioorg. Chem. Mol. Biol. ckenaer) Spiders". Toxicon, 25:175-184, 1987. Proc. Int. Symp. (1980). Ed: Ananchenko. Pergamon: Creighton, "Modification of the Amino and Carboxyl Ter Oxford, England, pp. 93-96. minal Groups". Proteins. Structure and Molecular Proper ties, W.H. Freeman and Company: New York, pp. 75-76, Hermann and Frischauf, "Isolation of Genomic DNA", 1983. Methods in Enzymology, 152:180-183, 1987. Blobel, "Intracellular Protein Topogenesis". Proc. Natl. Kaneda, et al., "Scorpion toxin prolongs an inactivation Acad. Sci. USA, 77:1496-1500, 1980. phase of the voltage dependent sodium current in rat isolated Raineri, et al., "Agrobacterium-Mediated Transformation of single hippocampal neurons", Brain Res., 487: 192-195, Rice (Oryza satiya L.)". Biotechnology, 8:33-38, 1990. 1989. Fromm, et al. "Inheritance and Expression of Chimeric Genes in the Progeny of Transgenic Maize Plants", Biotech McKnight, et al., "Transcriptional Control Signals of an nology, 8:833-839, 1990. Eucaryotic Protein-Coding Gene". Science, 217, 316-324, Smith et al. "M, 26,000 antigen of Schistosoma japonicum 1982. recognized by resistant WEHI 129/J mice is a parasite Barrett, S.M.et al. (1993) "Passive hemagglutination inhi gluthathione S-transferase". Proc. Natl. Acad. Sci. USA, bition test for diagnosis of brown recluse spider bite enveno 83:8703-8707, 1986. mation" Clin. Chem. 39(10):2104-2107, Oct. 1993. U.S. Patent May 26, 1998 Sheet 1 of 5 5,756,459 O : 3OO O.OO OO 2OO 3OO 4OO Time (min) F. G. U.S. Patent May 26, 1998 Sheet 2 of 5 5,756,459 len 9, . U.S. Patent May 26, 1998 Sheet 3 of 5 5,756,459 CN N is dN N leyn 8 U.S. Patent May 26, 1998 Sheet 4 of 5 5,756,459 o O C. MO C. 8RE VN. > CO L O 9 C O C O O do S3 S. S. & O leyn 99% U.S. Patent May 26, 1998 Sheet 5 of 5 5,756,459 Jen 9 8. A. 5,756,459 1 2 NSECTICDALLY EFFECTIVE PEPTDES quinquestriatus were revealed in Zlotkin, et al. Arch Bio SOLATABLE FROM PHDIPPUS SPIDER chem and Biophysics, 1985,240, 877-87.
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