Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-12-10
2003-09-02
Spector, Lorraine (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387900, C530S389100
Reexamination Certificate
active
06613886
ABSTRACT:
TECHNICAL FIELD
The invention relates to receptors that bind toxins from
Bacillus thuringiensis
and thus to pesticides and pest resistance. More particularly, the invention concerns recombinantly produced receptors that bind BT toxin and to their use in assays for improved pesticides, as well as in mediation of cell and tissue destruction, dissociation, dispersion, cell-to-cell association, and changes in morphology.
BACKGROUND ART
It has long been recognized that the bacterium
Bacillus thuringiensis
(BT) produces bactericidal proteins that are toxic to a limited range of insects, mostly in the orders Lepidoptera, Coleoptera and Diptera. Advantage has been taken of these toxins in controlling pests, mostly by applying bacteria to plants or transforming plants themselves so that they generate the toxins by virtue of their transgenic character. The toxins themselves are glycoprotein products of the cry gene as described by Höfte, H. et al.
Microbiol Rev
(1989) 53:242. It has been established that the toxins function in the brush border of the insect midgut epithelial cells as described by Gill, S. S. et al.
Annu Rev Entomol
(1992) 37:615. Specific binding of BT toxins to midgut brush border membrane vesicles has been reported by Hofmann, C. et al.
Proc Natl Acad Sci USA
(1988) 85:7844; Van Rie, J. et al.
Eur J Biochem
(1989) 186:239; and Van Rie, J. et al.
Appl Environ Microbiol
(1990) 56:1378.
Presumably, the toxins generated by BT exert their effects by some kind of interaction with receptors in the midgut. The purification of a particular receptor from
Manduca sexta
was reported by the present inventors in an article by Vadlamudi, R. K. et al.
J Biol Chem
(1993) 268:12334. In this report, the receptor protein was isolated by immunoprecipitating toxin-binding protein complexes with toxin-specific antisera and separating the complexes by SDS-PAGE followed by electroelution. However, to date, there has been no structural information concerning any insect receptor which binds BT toxin, nor have, to applicants' knowledge, any genes encoding these receptors been recovered.
DISCLOSURE OF THE INVENTION
The present invention is based, in part, on the isolation and characterization of a receptor that is bound by members of the BT-toxin family of insecticidal proteins, hereinafter the BT-R
1
protein. The present invention is further based on the isolation and characterization of a nucleic acid molecule that encodes the BT-toxin receptor, hereinafter BT-RBT-R
1
gene. Based on these observations, the present invention provides compositions and methods for use in identifying agents that bind to the BT-RBT-R
1
protein as a means for identifying insecticidal agent and for identifying other members of the BT-R
1
family of proteins.
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patent: 5665595 (1997-09-01), Petell et al.
patent: 5804393 (1998-09-01), Geiser et al.
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patent: 0 526 397 (1996-01-01), None
patent: WO 96 12964 (1996-05-01), None
Oddou et al., Immunologically unrelated Heliothis sp. and Spodoptera sp. midgut membrane-proteins bind Bacillus thuringiensis CrylA(b) delta-endotoxin, Eur. J. Biochem. 212: 145-150 (1993).*
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Hoffman et al., Specificity of Bacillus Thuringiensis &ggr;-endotoxins is Correlated with the Presence of High-affinity Binding Sites in the Brush Border Membrane of Target Insect Midguts; Proc. Natl. Acad. Sci., USA (1988) 85:7844-7848.
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Knight et al., The eceptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrance of the lepidopteran Manduca sexta is aminopeptidase N, Molecular Microbiology (1994) 11(3):429-436 (1994).
Knowles et al., The crystal &ggr;-endotoxins of Bacillus thuringiensis: Models for their mechanism of action on the insect gut, BioEssays 15(7):469-476 (1993).
Lee et al., Location of a Bombyx mori Receptor Binding Region on a Bacillus thuringiensis &ggr;-Endotoxin, J. Biol. Chem., vol. 167, No. 5, pp. 3115-3121 (1992).
Sanchis et al., Identification and partial purification of a Bacillus thuringiensis CryIC &ggr;-endotoxin binding protein from Spodoptera littoralis gut membrances, FEBS 316(3):264-268 (1993).
Sangadala et al., A Mixture of Manduca Sectra Aminopeptidase and Phosphatase Enhances Bacillus Thuringiensis Insecticidal CrylA(c) Toxin Binding and Rb+-K+Efflux in vitro, vol. 269, No. 13, pp. 10088-10092 (1994).
Vadlamudi et al., Cloning and Expression of a Receptor for an nsecticidal Toxin of Bacillus thuringiensis, J. Biol. Chem. 270(10):5490-5494 (1995).
Vadlamudi et al., A Specific Binding Protein from Manduca Sexta for the Insecticidal Toxin of Bacillus Thuringiensis Subsp., Berliner; J. Biol. Chem. 268(17):12334-12340 (1993).
Van Rie et al., Specificity of Bacillus Thuringiensis &ggr;-endotoxins; Eur. J. Biochem. (1989) 186:239-247.
Van Rie et al., Receptors on the Brush Border Membrane of the Insect Midgut as Determinants of the Specificity of Bacillus Thuringiensis Belta-Endotoxins (1990) 56(5):1378-1385.
Morrison & Foerster / LLP
Spector Lorraine
The University of Wyoming
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