Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-23
2002-07-23
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S325000, C435S320100, C435S069100, C435S252300, C435S254110, C530S300000, C530S350000, C536S023100, C536S023500, C536S023710
Reexamination Certificate
active
06423502
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 bacteriocidal 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 Michrobiol
(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 invention provides recombinant materials for the production of BT toxin-binding receptors as well as methods to employ these materials to generate receptors for use in screening assays for candidate pesticides. Since the native cDNA sequence encoding this receptor, designated BT-R
1
, has been retrieved-from the tobacco hornworm, encoding DNA for receptors in other species of insects, as well as in other organisms, which have homology to hornworm receptor can be obtained.
Thus, in one aspect, the invention is directed to a polynucleotide in purified and isolated form which comprises a nucleotide sequence encoding a receptor that binds a BT toxin and other ligands and which has the requisite homology to the BT-R
1
protein.
In other aspects, the invention is directed to expression systems for nucleotide sequences encoding the receptor, to methods of producing the receptor recombinantly, to the receptor as thus produced, to antibodies specifically immunoreactive with the receptor, to assay methods useful for screening candidate pesticides, to antisense polynucleotides corresponding to the coding sequence, to methods of targeting tissues and/or cells using the binding characteristics of the receptor, and to methods of manipulating tissues and/or cells using the function of the receptor.
REFERENCES:
patent: 4675285 (1987-06-01), Clark et al.
patent: 5071654 (1991-12-01), English
patent: WO 96 12964 (1996-05-01), None
Oddou et al., Immunologically unrelated Heliothis sp. and Spodoptera sp. midgut membrane-proteins bindBacillus thuringiensisCrylA(b) delta-endotoxin, Eur. J. Biochem., 212: 145-150, 1993.*
Almond et al., Suppression of Protein Structure Destabilizing Mutations inBacillus thuringiensis&dgr;-endotoxin by Second Site Mutations; Biochemistry 32:1040-1046 (1993).
Chen et al., Site-directed mutations in a highly conserved region ofBacillus thuringiensis&dgr;-endotoxin affect inhibition of short circuit current across Bombyx mori midguts, Proc. Natl. Acad. Sci. USA 90:9041-9045 (1993).
Sorsch, J. A. et al., “Determination of the specific region of BT-R1 to which the CryaAb toxin ofBacillus thuringiensissusp. Berliner binds”,FASEB Journal(Abstracts), vol. 11, No. 9, Jul. 31, 1997, p. A1050.
English, L., Mode of action of delta-endotoxins fromBacillus Thuringiensis: A comparison with other bacterial toxins, Insect Biochem. Molec. Biol. 22(1):1-7.
Gill et al., The Mode of Action ofBacillusThuringiensisEndotoxins; Ammun. Rev. Entomol. (1992) 37:615-36.
Hoffman et al., Specificity ofBacillus Thuringiensis&dgr;-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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Ishihara et al., Molecular cloning and expression of a cDNA encoding the secretin receptor, EMOB J., vol. 10, No. 7, 1635-1641.
Knight et al., The eceptor forBacillus thuringiensisCrylA(c) delta-endotoxin in the brush border membrance of the lepidopteranManduca sextais aminopeptidase N, Molecular Microbiology (1994) 11(3):429-436 (1994).
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Lee et al., Location of aBombyx moriReceptor Binding Region on aBacillus thuringiensis&dgr;-Endotoxin, J. Biol. Chem., vol. 167, No. 5, pp. 3115-3121 (1992).
Sanchis et al., Identification and partial purification of aBacillus thuringiensisCryIC &dgr;-endotoxin binding protein fromSpodoptera littoralisgut membrances, FEBS 316(3):264-268 (1993).
Sangadala et al., A Mixture ofManduca SectraAminopeptidase and Phosphatase EnhancesBacillus ThuringiensisInsecticidal CrylA(c) Toxin Biding 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 insecticidal Toxin ofBacillus thuringiensis, J. Biol. Chem. 270(10):5490-5494 (1995).
Vadlamudi et al., A Specific Binding Protein fromManduca Sextafor the Insecticidal Toxin ofBacillus ThuringiensisSubsp., Berliner, J. Biol. Chem. 268(17):12334-12340 (1993).
Van Rie et al., Specificity ofBacillus Thuringiensis&dgr;-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 ofBacillus ThuringiensisBelta-Endotoxins (1990) 56(5):1378-1385.
Kaufman Claire M.
Spector Lorraine
University of Wyoming
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