NZ501874A

NZ501874A - Isolation and characterisation of receptors that are bound by members of the BT-toxin family of insecticidal proteins with respect to pesticides and pest resistance

Info

Publication number: NZ501874A
Application number: NZ501874A
Authority: NZ
Inventors: Lee A Bulla
Original assignee: Univ Wyoming
Priority date: 1997-06-20
Filing date: 1998-06-08
Publication date: 2001-10-26
Also published as: CA2292644A1; EP1005547A1; WO1998059048A1; AU7828398A; AU743234B2; WO1998059048A9; BR9810217A; ZA985372B; AR012252A1

Abstract

The cDNA encoding a glycoprotein receptor from the tobacco hornworm (Manduca sexta) that binds a Bacillus thuringiensis (BT) toxin has been obtained and sequenced. The availability of this cDNA permits the retrieval of DNAs encoding homologous receptors in other insects and organisms as well as the design of assays for the cytotoxicity and binding affinity of potential pesticides and the development of methods to manipulate natural and/or introduced homologous receptors and thus, to destroy target cells, tissues and/or organisms. The isolation and characterisation of a receptor that is bound by members of the BT-toxin family of insecticidal proteins (BT-R1 protein) and a gene encoding the BT-toxin receptor are described. Compositions and methods for use in identifying agents that bind to the BT-R1 protein as a means for identifying insecticidal agents and for identifying other members of the BT-R1 family are also described.

Description

<div class="application article clearfix" id="description"> WO 98/59048 PCT/US98/11868 - 1 - RECEPTOR FOR A BACILLUS THURINGIENSIS TOXIN Acknowledgment of Government Support Work resulting in the present invention was supported in part by Research 5 Agreement 58-319R-3-011 from the Office of International Cooperation and Development, U S D.A and by Cooperative Agreement 58-5410-1-135 from the Arthropod-Borne Animal Disease Laboratory, Agricultural Research Service, U.S.D.A. and by Grant HD-18702 from the National Institutes of Health The U.S. government has certain rights in this invention 10 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 recombmantly produced receptors that bind BT toxin and to their use in assays for 15 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) 2 o produces bactericidal proteins that are toxic to a limited range of insects, mostly m the orders Lepidoptera, Coleoptera and Diptera. Advantage has been taken of these toxins m 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 2 5 descnbed by Hofte, H. et al Microbiol Rev (1989) 53:242. It has been established that the toxms function m the brush border of the insect midgut epithelial cells as descnbed 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 Hofinann, C et al Proc Natl Acad Sci USA (1988) 85:7844; Van Rie, J et al EurJBiochem 3 0 (1989) 186:239; and Van Rie, J. et al Appl Environ Microbiol (1990) 56:1378. dc-118781 Printed from Mimosa WO 98/59048 PCT/US98/11868 -2- 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 5 by immunoprecipitating toxin-bindmg 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. 10 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 msecticidal proteins, hereinafter the BT-R, protein. The present invention is further based on the isolation 15 and characterization of a nucleic acid molecule that encodes the BT-toxin receptor, hereinafter BT-R j gene. Based on these observations, the present invention provides compositions and methods for use in identifying agents that bind to the BT-R, protein as a means for identifying insecticidal agent and for identifying other members of the BT-R, family of proteins. 20 Brief Description of the Drawings Figure 1 show the nucleotide sequence and deduced amino acid sequence of cDNA encoding the BT-R, protein from M. sexta Figure 2 (panels a and b) shows a comparison of ammo acid sequences of 2 5 cadhenn motifs (BTRcad-1 to 11) in BT-R, to those of other cadhenns Figure 3 shows a block diagram of the cadhenn-like structure of BT-R, Figure 4 shows the clone characterization of the BamHI-SacI fragment of BT-R,. LM is Hindlll cut Lambda marker; UP is the uncut plasmid clone; NP is Nsil cut plasmid; XP is Xhol cut plasmid, BSP is BamHI and SacI cut plasmid showing Printed from Mimosa WO 98/59048 PCT/US98/11868 -3- the cloned fragment from BT-R,; RM is mRNA size marker; and RT1 and RT2 are transcribed mRNAs from the cloned BT-R, fragment. Figure 5 illustrates the detection of protein expression from the plasmid containing the Bam-Sac fragment of BT-R, using 35S-methionine as a tag LCR is a 5 luciferase control mRNA to show that the rabbit reticulocyte lysates are functional; RR1 and RR2 are expression products of the Bam-Sac fragment of BT-R, produced in rabbit reticulocytes from mRNA; LCT is a luciferase control plasmid to show that the transcription/translation kit is functional; and TT1 and TT2 are expression products of the Bam-Sac fragment of BT-R, produced in a transcription/translation kit. 10 Figure 6 shows a radio-blot of the Bam-Sac fragment of BT-R, with ,25I-labeled Cryl Ab BBMV is the brush border membrane vesicles from the midgut of M sexta containing the wild-type BT-R, receptor protein; RBK is a rabbit reticulocyte blank; RR1 and RR2 are the expression products of the Bam-Sac fragment of BT-R, produced in rabbit reticulocytes from mRNA; TBK is a 15 transcription/translation kit blank; TT1 and TT2 are expression products of the Bam-Sac fragment of BT-R, produced m a transcription/translation kit The arrows point to two of the bands. Figure 7 shows the presence of a BT-R, homologue in Pink Bollworm and European Corn Borer identified using toxin binding similar to that used to identify the 2 0 original BT-R, clone. Figure 8 shows the binding of Cryl Ab to fragments of the BT-R, protein Modes of Carrying Out the Invention I. General Description 2 5 The present invention is based, in part, on the isolation and characterization of a novel protein expressed in the midgut of Manduca sexta that binds to members of the BT-toxm family of proteins, hereinafter the BT-R, protein The present invention specifically provides purified BT-R,, the amino acid sequence of BT-R,, as well as nucleotide sequences that encode BT-R,. The BT-R, protein and nucleic acid 3 0 molecules can serve as targets in identifying insecticidal agents dc-l 18781 Printed from Mimosa WO 98/59048 . PCT/US98/11868 -4- II. Specific Embodiments A. BT-R, Protein Pnor to the present invention, although members of the BT-toxin family of 5 protein were known, no one had identified the receptor that is bound by these toxin proteins. The present invention provides, in part, the amino acid sequences of a BT-toxin receptor that is expressed m the midgut of Maduca sexta. In one embodiment, the present invention provides the ability to isolate or produce a previously unknown protein by using known purification methods, the 10 cloned nucleic acid molecules herein descnbed or by synthesizing a protein having the amino acid sequence herein disclosed As used herein, BT-R, refers to a protein that has the amino acid sequence of BT-R, provided in Figure 1, as well as allelic variants of the BT-R, sequence, and conservative substitutions mutants of the BT-R, sequence that have BT-R, activity. 15 BT-R, is compnsed of a single subunit, has a molecular weight of 210 kD, and has the amino acid sequence provided in Figure 1. A prediction of the structure of BT-R, is provided m Figure 3. The BT-R, protein of the present invention includes the specifically identified and charactenzed variant herein described, as well as allelic variants, conservative 2 0 substitution variants and homologues (Figure 7) that can be isolated/generated and characterized without undue expenmentation following the methods outlined below For the sake of convenience, all BT-R, proteins will be collectively referred to as the BT-R, proteins, the BT-R, proteins of the present invention or BT-R, The term "BT-R," includes all naturally occurring allelic vanants of the 25 Manduca sexta BT-R, protein provided m Figure 1. In general, naturally occurnng allelic vanants of Manduca sexta BT-R, will share significant homology, at least 75 %, and generally at least 90%, to the BT-R, amino acid sequence provided in Seq. ID No:2. Allelic vanants, though possessing a slightly different ammo acid sequence than Seq. ID No:2, will be expressed as a transmembrane protein in the digestive tract 30 of an insect or other organism. Typically, allelic vanants of the BT-R, protein will Printed from Mimosa WO 98/59048 PCT/US98/11868 -5- contain conservative amino acid substitutions from the BT-R, sequence herein described or will contain a substitution of an amino acid from a corresponding position in a BT-R, homologue (a BT-R, protein isolated from an organism other than Manduca sexta). 5 One class of BT-R, allelic variants will be proteins that share a high degree of homology with at least a small region of the amino acid sequence provided m Seq. ID No: , but may further contain a radical departure from the sequence, such as a non- conservative substitution, truncation, insertion or frame shift Such alleles are termed mutant alleles of BT-R, and represent proteins that typically do not perform the same 10 biological functions as does the BT-R, variant of Seq. ID No:2 The BT-R, proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the BT-R, protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard 15 purification methods to obtain an isolated BT-R, protein. The nature and degree of isolation will depend on the intended use. The cloning of the BT-R, encoding nucleic acid molecule makes it possible to generate defined fragments of the BT-R, proteins of the present invention. As discussed below, fragments of BT-R, are particularly useful in generating domain 2 0 specific antibodies; identifying agents that bind to toxin binding domain on BT-R,; identifying toxin-binding structures, identifying cellular factors that bind to BT-R,; isolating homologues or other allelic forms of BT-R,; and studying the mode of action of BT-toxins Fragments of the BT-R, proteins can be generated using standard peptide 2 5 synthesis technology and the ammo acid sequence of Manduca sexta BT-R, disclosed herein Alternatively, as illustrated in Example 5, recombinant methods can be used to generate nucleic acid molecules that encode a fragment of the BT-R, protein. Fragments of the BT-R, protein subunits that contain particularly interesting structures can be identified using art-known methods such as by using an 3 0 immunogenicity plot, Chou-Fasman plot, Garnier-Robson plot, Kyte-Doolittle plot, Printed from Mimosa WO 98/59048 PCT/US98/11868 -6- Eisenberg plot, Karplus-Schultz plot or Jameson-Wolf plot of the BT-R, protein. Fragments containing such residues are particularly useful in generating domain specific anti-BT-R, antibodies or in identifying cellular factors that bind to BT-R,. One particular fragment that is preferred for use in identifying insecticidal agents is a 5 soluble fragment of BT-R, that can bind to a member of the BT family of toxins. In Example 5, a fragment of BT-R, that binds to a BT-toxin is disclosed. As described below, members of the BT-R, family of proteins can be used for, but are not limited to: 1) a target to identify agents that bind to BT-R„ 2) a target or bait to identify and isolate binding partners and cellular factors that bind to BT-R,, 10 3) an assay target to identify BT-R, and other receptor-mediated activity, and 4) a marker of cells that express a member of the BT-R, family of proteins. B. Anti-BT-R, Antibodies The present invention further provides antibodies that bind BT-R, The most 15 preferred antibodies will selectively bind to BT-R, and will not bind (or will only bind weakly) to non-BT-R, proteins Anti- BT-R, antibodies that are especially contemplated include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complement determining regions (CDRs) of these antibodies. 2 0 Antibodies are generally prepared by immunizing a suitable mammalian host using a BT-R, protein (synthetic or isolated), or fragment, in isolated or immunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). Regions of the BT-R, protein that show immunogenic structure can readily be identified using art-known methods. Other important regions and domains can readily 2 5 be identified using protein analytical and comparative methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis Fragments containing these residues are particularly suited in generating specific classes of anti-BT-R, antibodies Particularly useful fragments include, but are not limited to, the BT-toxiri binding domain of BT-R, identified in 3 0 Example 5. Printed from Mimosa WO 98/59048 PCT/US98/11868 -7- Methods for preparing a protein for use as an lmmunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are well known m the art. In some circumstances, direct conjugation with reagents such as carbodiimide may be used; in other instances linking reagents 5 like those supplied by Pierce Chemical Co., Rockford, IL, may be effective. Administration of a BT-R, immunogen is conducted generally by injection over a suitable time period in combination with a suitable adjuvant, as is generally understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation 10 Although the polyclonal antisera produced in this way may be satisfactory for some applications, for many other applications, monoclonal antibody preparations are preferred. Immortalized cell lines which secrete a desired monoclonal antibody may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The 15 immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the BT-R, protein or BT-R, fragment. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid. The desired monoclonal antibodies are then recovered from the culture 2 0 supernatant or from the ascites supernatant Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab')2 fragments is often preferable, especially m a therapeutic context, as these fragments are generally less immunogenic than the 2 5 whole immunoglobulin The antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the BT-R, protein can also be produced in the context of chimeric or CDR grafted antibodies of multiple species origin. Printed from Mimosa WO 98/59048 PCT/US98/11868 -8- As described below, anti-BT-R, antibodies are useful as modulators of BT-R, activity, are useful in in vitro and in vivo antibody based assays methods for detecting BT-R, expression/activity, in generating toxin conjugates, for purifying homologues of Manduca sexta BT-R,, in generating anti-ideotypic antibodies that mimic the 5 BT-R, protein and in identifying competitive inhibitors of BT-toxm/BT-R, interactions. C. BT-R, Encoding Nucleic Acid Molecules As described above, the present invention is based, in part, on isolating nucleic 10 acid molecules from Manduca sexta that encode BT-R,. Accordingly, the present invention further provides nucleic acid molecules that encode the BT-R, protein, as herein defined, preferably m isolated form. For convenience, all BT-R, encoding nucleic acid molecules will be referred to as BT-R, encoding nucleic acid molecules, the BT-R i genes, or BT-R]. The nucleotide sequence of the Manduca sexta nucleic 15 acid molecule that encodes one allelic form of BT-R, is provided in Figure 1. As used herein, a "nucleic acid molecule" is defined as an RNA or DNA molecule that encodes a peptide as defined above, or is complementary to a nucleic acid sequence encoding such peptides. Particularly preferred nucleic acid molecules will have a nucleotide sequence identical to or complementary to the Manduca sexta 2 0 DNA sequences herein disclosed. Specifically contemplated are genomic DNA, cDNAs, synthetically prepared DNAs, and antisense molecules, as well as nucleic acids based on an alternative backbone or including alternative bases, whether denved from natural sources or synthesized. A skilled artisan can readily obtain these classes of nucleic acid molecules using the herein descnbed BT-R j sequences However, 2 5 such nucleic acid molecules, are defined further as being novel and unobvious over any pnor art nucleic acid molecules encoding non-BT-R, proteins For example, the BT-R2 sequences of the present invention specifically excludes previously identified nucleic acid molecules that share only partial homology to BT-R j Such excluded sequences include identified members of the cadhedrin family of proteins. Printed from Mimosa WO 98/59048 PCT/US98/11868 -9- As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules that encode polypeptides other than BT-R,. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated BT-R, encoding nucleic 5 acid molecule. The present invention further provides fragments of the BT-R, encoding nucleic acid molecules of the present invention As used herein, a fragment of a BT-R, encoding nucleic acid molecule refers to a small portion of the entire BT-R j sequence. The size of the fragment will be determined by its intended use. For example, if the 10 fragment is chosen so as to encode the toxin binding domain of BT-R, identified in Example 5, then the fragment will need to be large enough to encode the toxin binding domain of the BT-R, protein. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming. Fragments of the Manduca sexta BT-R] 15 gene that are particularly useful as selective hybndization probes or PCR primers can be readily identified from the entire BT-R] sequence using art-known methods. Another class of fragments of BT-R, encoding nucleic acid molecules are the expression control sequence found upstream and downstream from the BT-R, encoding region found in genomic clones of the BT-R j gene Specifically, tissue and 2 0 developmental specific expression control elements can be identified as being 5' to the BT-R, encoding region found in genomic clones of the BT-R; gene. Such expression control sequence are useful in generatmg expression vectors for expressing genes in the digestive tract of a transgenic organism As descnbed in more detail below, a skilled artisan can readily use the BT-R j cDNA sequence herein descnbed to isolate and 2 5 identify genomic BT-R; sequences and the expression control elements found in the BT-R j gene. Fragments of the BT-R, encoding nucleic acid molecules of the present invention (i.e., synthetic oligonucleotides) that are used as probes or specific pnmers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding BT-R, 3 0 proteins, can easily be synthesized by chemical techniques, for example, the Printed from Mimosa WO 98/59048 PCT/US98/11868 - 10- phosphotriester method of Matteucci, et al., J Am Chem Soc (1981) 103-3185-3191, or using automated synthesis methods. In addition, larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the BT-R ] gene, followed by ligation of 5 oligonucleotides to build the complete modified BT-R j gene. The BT-R, encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes As described above, such probes can be used to identify nucleic acid molecules encoding other allelic variants or homologues of the BT-R, proteins and as 10 described below, such probes can be used to identify the presence of a BT-R, protein as a means for identifying cells that express a BT-R, protein A variety of such labels are known in the art and can readily be employed with the BT-R, encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides, biotm, and the like. A skilled artisan can employ any of the art-known 15 labels to obtain a labeled BT-R, encoding nucleic acid molecule D. Isolation of Other BT-R, Encoding Nucleic Acid Molecules The identification of the BT-R, protein from Manduca sexta and the corresponding encoding nucleic acid molecules, has made possible the identification of 2 0 and isolation of 1) BT-R, proteins from organisms other than Manduca sexta, hereinafter referred to collectively as BT-R, homologues, 2) other allelic and mutant forms of the Manduca sexta BT-R, protein (descnbed above), and 3) the corresponding genomic DNA that contains the BT-R; gene. The most preferred source of BT-R, homologues are insects, the most preferred being members of the Lepidopteran, 2 5 Coleopteran and Dipteran orders of insects Evidence of the existence of BT-R, homologues is provided in Figure 7. Essentially, a skilled artisan can readily use the amino acid sequence of the Manduca sexta BT-R, protein to generate antibody probes to screen expression libranes prepared from cells and organisms. Typically, polyclonal antiserum from 3 0 mammals such as rabbits immunized with the punfied protein (as descnbed above) or Printed from Mimosa WO 98/59048 PCT/US98/11868 - 11 - monoclonal antibodies can be used to probe an expression library, prepared from a target organism, to obtain the appropriate coding sequence for a BT-R, homologue. The cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructing an expression cassette using 5 control sequences appropriate to the particular host used for expression of the enzyme. Alternatively, a portion of the BT-R, encoding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the BT-R, family of proteins from organisms other than Manduca sexta, allelic variants of the Manduca sexta BT-R, protein herein descnbed, and genomic sequence containing the 10 BT-Ri gene Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 ammo acid stretch) are prepared and used to screen genomic DNA or cDNA hbranes to obtain hybridization under stringent conditions or conditions of sufficient stnngency to eliminate an undue level of false positives. Additionally, pairs of oligonucleotide primers can be prepared for use in a 15 polymerase chain reaction (PCR) to selectively amplify/clone a BT-R,-encoding nucleic acid molecule, or fragment thereof. A PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other BT-R, encoding nucleic acid molecules Regions of the Manduca sexta BT-Ri gene that are particularly well suited for use as a probe or as pnmers can be 2 0 readily identified by one skilled in the art. Non-Manduca sexta homologues of BT-R], naturally occurnng allelic variants of the Manduca sexta BT-R] gene and genomic BT-R] sequences will share a high degree of homology to the Manduca sexta BT-R] sequence herein descnbed. In general, such nucleic acid molecules will hybridize to the Manduca sexta BT-R] 25 sequence under high stnngency. Such sequences will typically contain at least 70% homology, preferably at least 80%, most preferably at least 90% homology to the Manduca sexta BT-R] sequence of Seq. ID No:l. In general, nucleic acid molecules that encode homologues of the Manduca sexta BT-R, protein will hybridize to the Manduca sexta BT-R ] sequence under 3 0 stnngent conditions "Stnngent conditions" are those that (1) employ low ionic Printed from Mimosa WO 98/59048 PCT/US98/11868 - 12- strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50°C., or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrohdone/50 mM sodium phosphate 5 buffer at pH 6 5 with 750 mM NaCl, 75 mM sodium citrate at 42°C. Another example is use of 50% formamide, 5 x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 (ig/ml), 0.1% SDS, and 10% dextran sulfate at 42°C., with washes at 42°C. in 0.2 x SSC and 0.1% SDS. A skilled artisan can 10 readily determine and vary the stringency conditions appropnately to obtain a clear and detectable hybridization signal. The presence of similar receptors in noninsect organisms as well as other insects besides those harboring BT-R, is supported by the sequence similarity of the BT-R, protein to that of the various members of the cadherin superfamily of proteins, 15 which are membrane glycoproteins believed to mediate calcium-dependent cell aggregation and sorting. See, for example, Takeichi, M. Science (1991) 251:1451; and Takeichi, M. NRev Biochem (1990) 59:237. Included m this superfamily are desmoghen, desmocollins, the Drosophila fat tumor suppressor, Manduca sexta intestinal peptide transport protein and T-cadherin. 2 0 All of these proteins share common extracellular motifs although their cytoplasmic domains differ. Goodwin, L. et al Biochem Biophys Res Commun (1990) 173:1224; Holton, J.L et al. J Cell Sci (1990) 97:239; Bestal, D.J. / Cell Biol (1992) 119:451; Mahoney, P.A. et al. Cell (1991) 853; Dantzig, A.H. et al Science (1994) 264 430; and Sano, K et al. EMBOJ{\992>) 12.2249. Inclusion of BT-R, in the cadherin 2 5 superfamily is further supported by the report that EDTA decreases the binding of CrylAb toxin of BT to the 210 kD receptor of M sexta (Martinez-Ramirez, AC et al. Biochm Biophys Res Commun (1994) 201:782). It is noted below that the amino acid sequence of BT-R, reveals that a calcium-binding motif is present. This is consistent with the possibility that cells having 3 0 receptors to bind toxin may themselves survive although they render the tissues in Printed from Mimosa WO 98/59048 PCT/US98/11868 -13- which they are included permeable to solutes and thus effect disintegration of the tissue. Such a mechanism is proposed for the death of insects that ingest the toxin via the epithelial cells m their midgut by Knowles, B.H. et al. Biochim Biophys Acta (1987) 924:509. Such a mechanism is also supported in part by the results set forth in 5 Example 4 hereinbelow which indicate that the effect of the toxin on embryonic 293 cells modified to express the receptor at their surface is reversible. E. rDNA Molecules Containing a BT-R, Encoding Nucleic Acid Molecule The present invention further provides recombinant DNA molecules (rDNAs) 10 that contain a BT-R, encoding sequences as herein described, or a fragment thereof, such as a soluble fragment of BT-R, that contains the BT-toxin binding site. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in vitro. Methods for generating rDNA molecules are well known in the art, for example,-see Sambrook et al, Molecular Cloning (1989). In the preferred rDNA 15 molecules of the present invention, a BT-R, encoding DNA sequence that encodes a BT-R, protein or a fragment of BT-R,, is operably linked to one or more expression control sequences and/or vector sequences The choice of vector and/or expression control sequences to which the BT-R, encoding sequence is operably linked depends directly, as is well known in the art, on 2 0 the functional properties desired, e.g, protein expression, and the host cell to be transformed A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the BT-R, encoding sequence included in the rDNA molecule. Expression control elements that are used for regulating the expression of an 2 5 operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, transcription terminators and other regulatory elements. Preferably, an inducible promoter that is readily controlled, such as being responsive to a nutrient in the host cell's medium, is used Further, for soluble fragments, it may be desirable to use 3 0 secretion signals to direct the secretion of the BT-R, protein, or fragment, out of the cell. Printed from Mimosa WO 98/59048 PCT/US98/11868 - 14- In one embodiment, the vector containing a BT-R, encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence havmg the ability to direct autonomous replication and maintenance of the recombinant DNA molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed 5 therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are those that confer resistance to ampicilhn or tetracycline. Vectors that include a prokaryotic replicon can further include a prokaryotic or 10 viral promoter capable of directing the expression (transcription and translation) of the BT-R, encoding sequence in a bacterial host cell, such as E. coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for 15 insertion of a DNA segment of the present invention. Typical of such vector plasmids - are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to variant rDNA molecules that 2 0 contain a BT-R, encoding sequence Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector 2 5 pCDM8 described herein, and the like eukaryotic expression vectors Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the 3 0 neomycin phosphotransferase (neo) gene. Southern et al.,JMol Anal Genet (1982) Printed from Mimosa WO 98/59048 PCT/US98/11868 -15- 1:327-341. Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by cotransfection of the host cell, and selected by culturing in the presence of the appropriate drug for the selectable marker. 5 F. Host Cells Containing an Exogenously Supplied BT-R, Encoding Nucleic Acid Molecule The present invention further provides host cells transformed with a nucleic acid molecule that encodes a BT-R, protein of the present invention, either the entire BT-R, protein or a fragment thereof. The host cell can be either prokaryotic or eukaryotic. 10 Eukaryotic cells useful for expression of a BT-R, protein are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of a BT-R/ gene. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, the most preferred being cells that do not naturally express a BT-R, protein. 15 Any prokaryotic host can be used to express a BT-R,-encoding rDNA molecule. The preferred prokaryotic host is E. coll. Transformation of appropriate cell hosts with an rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic 2 0 host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, Proc AcadSci USA (1972) 69:2110; and Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically 25 employed, see, for example, Graham et al, Virol (1973) 52 456; Wigler et al., Proc Natl Acad Sci USA (1979) 76.1373-76 Successfully transformed cells, i e, cells that contain an rDNA molecule of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to 3 0 produce single colonies. Cells from those colonies can be harvested, lysed and their Printed from Mimosa WO 98/59048 PCT/US98/11868 - 16- DNA content examined for the presence of the rDNA using a method such as that descnbed by Southern, JMolBiol (1975) 98:503, or Berent et al, Biotech (1985) 3:208 or the proteins produced from the cell assayed via an immunological method. 5 G. Production of a BT-R, Protein Using an rDNA Molecule The present invention further provides methods for producing a BT-R, protein that uses one of the BT-R, encodmg nucleic acid molecules herein described. In general terms, the production of a recombinant BT-R, protein typically involves the following steps. 10 First, a nucleic acid molecule is obtained that encodes a BT-R, protein or a fragment thereof, such as the nucleic acid molecule depicted in Figure 1. The BT-R, encoding nucleic acid molecule is then preferably placed m an operable linkage with suitable control sequences, as descnbed above, to generate an expression unit containing the BT-R, encoding sequence. The expression unit is used to transform a suitable host 15 and the transformed host is cultured under conditions that allow the production of the BT-R, protein Optionally the BT-R, protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated. Each of the foregoing steps can be done in a vanety of ways. For example, the 2 0 desired coding sequences may be obtained from genomic fragments and used directly in an appropnate host. The construction of expression vectors that are operable in a variety of hosts is accomplished using an appropnate combination of replicons and control sequences. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail 2 5 earlier Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with BT-R, encoding sequences to produce a BT-R, protein. Printed from Mimosa WO 98/59048 PCT/US98/11868 - 17- H. Identification of Agents and Cellular Constituents that Bind to a BT-R, Protein Another embodiment of the present invention provides methods for identifying agents and cellular constituents that bind to BT-R, Specifically, agents and cellular 5 constituents that bind to BT-R, can be identified by: 1) the ability of the agent/constituent to bind to BT-R„ 2) the ability to block BT-toxin binding to BT-R,, and/or 3) the ability to kill BT-R, expressing cells Activity assays for BT-R, activity and binding and competitive assays using a BT-R, protein are suitable for use in high through-put screening methods, particularly using a soluble fragment of BT-R, that 10 contains the BT-toxm binding domain, such as that disclosed m Example 5 In detail, in one embodiment, BT-R, is mixed with an agent or cellular extract. After mixing under conditions that allow association of BT-R, with the agent or component of the extract, the mixture is analyzed to determine if the agent/component bound to the BT-R,. Binding agents/components are identified as being able to bind 15 to BT-R,. Alternatively or consecutively, BT-R, activity can be directly assessed as a means for identifying agonists and antagonists of BT-R, activity Alternatively, targets that are bound by a BT-R, protein can be identified using a yeast two-hybrid system or using a binding-capture assay. In the yeast two hybrid system, an expression unit encoding a fusion protein made up of one subunit of a two 2 0 subunit transcription factor and the BT-R, protein is introduced and expressed in a yeast cell The cell is further modified to contain 1) an expression unit encoding a detectable marker whose expression requires the two subunit transcription factor for expression and 2) an expression unit that encodes a fusion protein made up of the second subunit of the transcription factor and a cloned segment of DNA. If the cloned 2 5 segment of DNA encodes a protein that binds to the BT-R, protein, the expression results m the interaction of the BT-R, and the encoded protein This bnngs the two subunits of the transcription factor into binding proximity, allowing reconstitution of the transcription factor. This results in the expression of the detectable marker. The yeast two hybrid system is particularly useful in screening a library of cDNA 3 0 encoding segments for cellular binding partners of BT-R,. Printed from Mimosa WO 98/59048 PCT/US98/11868 - 18- The BT-R, protein used in the above assays can be: an isolated and fully characterized protein, a fragment of a BT-R, protein (such as a soluble fragment contaimng the BT-toxin binding site), a cell that has been altered to express a BT-R, protein/fragment or a fraction of a cell that has been altered to express a BT-R, 5 protein/fragment. Further, the BT-R, protein can be the entire BT-R, protein or a defined fragment of the BT-R, protein. It will be apparent to one of ordinary skill in the art that so long as the BT-R, protein or fragment can be assayed for agent binding, e g., by a shift in molecular weight or activity, the present assay can be used. The method used to identify whether an agent/cellular component binds to a 10 BT-R, protein will be based primarily on the nature of the BT-R, protein used. For example, a gel retardation assay can be used to determine whether an agent binds to BT-R, or a fragment thereof. Alternatively, immunodetection and biochip technologies can be adopted for use with the BT-R, protein. A skilled artisan can readily employ numerous art-known techniques for determining whether a particular 15 agent binds to a BT-R, protein. Agents and cellular components can be further, or alternatively, tested for the ability to block the binding of a BT-toxin to a BT-R, protein/fragment Alternatively, antibodies to the BT-toxin binding site or other agents that bind to the BT-toxin binding site on the BT-R, protein can be used m place of the BT-toxm. 2 0 Agents and cellular components can be further tested for the ability to modulate the activity of a BT-R, protein using a cell-free assay system or a cellular assay system. As the activities of the BT-R, protein become more defined, functional assays based on the identified activity can be employed. As used herein, an agent is said to antagonize BT-R, activity when the agent 2 5 reduces BT-R, activity The preferred antagonist will selectively antagonize BT-R,, not affecting any other cellular proteins Further, the preferred antagonist will reduce BT-R, activity by more than 50%, more preferably by more than 90%, most preferably eliminating all BT-R, activity. As used herein, an agent is said to agonize BT-R, activity when the agent 3 0 increases BT-R, activity. The preferred agonist will selectively agonize BT-R,, not Printed from Mimosa WO 98/59048 PCT/US98/11868 - 19- affecting any other cellular proteins. Further, the preferred antagonist will increase BT-R, activity by more than 50%, more preferably by more than 90%, most preferably more than doubling BT-R, activity. Agents that are assayed in the above method can be randomly selected or 5 rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences of the BT-R, protein or BT-toxin. An example of randomly selected agents is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism or plant extract. 10 As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis that takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the BT-R, protein and BT-toxin. For example, a rationally selected peptide 15 agent can be a peptide whose amino acid sequence is identical to a fragment of a BT-R, protein or BT-toxin. The agents tested in the methods of the present invention can be, as examples, peptides, small molecules, and vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of 2 0 the agents used in the present screening method One class of agents of the present invention are peptide agents whose ammo acid sequences are chosen based on the amino acid sequence of the BT-R, protein or BT-toxin. Small peptide agents can serve as competitive inhibitors of BT-R, protein activity. Peptide agents can be prepared using standard solid phase (or solution phase) 2 5 peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included. Printed from Mimosa WO 98/59048 PCT/US98/11868 -20- Another class of agents of the present invention are antibodies immunoreactive with critical positions of the BT-R, protein. As descnbed above, antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the BT-R, protein intended to be targeted by the 5 antibodies. Critical regions particularly include the BT-toxin binding domain identified in Example 5. Such agents can be used in competitive binding studies to identify second generation BT-R, binding agents. The cellular extracts tested in the methods of the present invention can be, as examples, aqueous extracts of cells or tissues, organic extracts of cells or tissues or 10 partially purified cellular fractions A skilled artisan can readily recognize that there is no limit as to the source of the cellular extract used in the screening method of the present invention. The preferred source for isolating cellular binding partners of BT-R, are cells that express BT-R, or cells that are in close proximity to BT-R, expressmg cells. 15 An outline of one screemng method is as follows. Cells are modified by transfection, retroviral infection, electroporation or other known means, to express a BT-R, protein and then cultured under conditions wherein the receptor protein is produced and displayed. If desired, the cells are then recovered from the culture for use in the assay, or the culture itself can be used per se 20 In the assays, the modified cells are contacted with the candidate toxin and the effect on metabolism or morphology is noted in the presence and absence of the candidate. The effect may be cytotoxic - le, the cells may themselves exhibit one of the indices of cell death, such as reduced thymidine uptake, slower increase in optical density of the culture, reduced exclusion of vital dyes (e g., trypan blue), increased 2 5 release of viability markers such as chromium and rubidium, and the like The differential response between the toxin-treated cells and the cells absent the toxin is then noted. The strength of the toxin can be assessed by noting the strength of the response. Printed from Mimosa WO 98/59048 PCT/US98/11868 -21 - These assays may be conducted directly as described above or competitively with known toxins. For example, one approach might be to measure the diminution in binding of labeled BT cry toxin in the presence and absence of the toxin candidate. In addition to simply screening candidates, the screen can be used to devise 5 improved forms of toxins which are more specific or less specific to particular classes of insects as desired The ability to determine binding affinity (K_, and KJ, dissociation and association rates, and cytotoxic effects of a candidate allows quick, accurate and reproducible screening techniques for a large number of toxins and other ligands under identical conditions which was not possible heretofore. Such 10 information will facilitate the selection of the most effective toxins and ligands for any given receptor obtained from any desired host cell. Competition assays may also employ antibodies that are specifically immunoreactive with the receptor. Such antibodies can be prepared in the conventional manner by administering the purified receptor to a vertebrate animal, 15 monitoring antibody titers and recovenng the antisera or the antibody-producing cells for immortalization, to obtain immortalized cells capable of secreting antibodies of the appropnate specificity. Techniques for obtaining immortalized B cells and for screening them for secretion of the desired antibody are now conventional m the art. The resulting monoclonal antibodies may be more effective than the polyclonal 2 0 antisera as competition reagents; furthermore, the availability of the immortalized cell line secreting the desired antibody assures uniformity of production of the same reagent over time. The information and the structural characteristics of toxins and ligands tested will permit a rational approach to designing more efficient toxins and ligands. Additionally, such assays will lead to a better understanding of the function 2 5 and the structure/function relationship of both toxin/ligand and BT-R, analogs. In turn, this will allow the development of highly effective toxins/ligands Ligands include natural and modified toxins, antibodies (anti-receptor and antiidiotype antibodies which mimic a portion of a toxin that binds to a receptor, and whatever small molecules bind the receptors. Printed from Mimosa WO 98/59048 PCT/US98/11868 -22- I. Uses of Agents that Bind to a BT-R, Protein As provided in the Background section, BT-R, is the target for the msecticidal activity of BT-toxms. Agents that bind a BT-R, protein can be used: 1) to kill BT-R, expressing cells, 2) to identify agents that block the interaction of a BT-toxin with 5 BT-R, and 3) in methods for identifying cells that express BT-R,. The methods employed in using the BT-R, binding agents will be based primarily on the nature of the BT-R, binding agent and its intended use For example, a BT-R, binding agent can be used to: deliver a conjugated toxin to a BT-R, expressing cell; modulate BT-R, activity; directly kill BT-R, expressing cells; or 10 screen for and identify competitive binding agents. An agent that inhibits the activity of BT-R, can be used to directly inhibit the growth of BT-R, expressing cells Further, identified cellular factors that bind to BT-R, can, themselves, be used in binding/competitive assays to identify agonist and antagonists of BT-R,. 15 J. Methods for Identifying the Presence of a BT-R, protein or gene The present invention further provides methods for identifying cells, tissues or organisms expressing a BT-R, protein or a BT-R j gene Such methods can be used to diagnose the presence of cells or an organism that expresses a BT-R, protein in vivo or in vitro. The methods of the present invention are particularly useful in the 2 0 determining the presence of cells that are a target for BT-toxin activity or for identifying susceptibility of an organism to a BT-toxin or BT-toxm-like agent Specifically, the presence of a BT-R, protein can be identified by determining whether a BT-R, protein, or nucleic acid encoding a BT-R, protein, is expressed in a cell, tissue or organism. 25 A variety of immunological and molecular genetic techniques can be used to determine if a BT-R, protein is expressed/produced in a particular cell or sample In general, an extract containing nucleic acid molecules or an extract containing proteins is prepared. The extract is then assayed to determine whether a BT-R, protein, or a BT-R, encoding nucleic acid molecule, is produced in the cell Printed from Mimosa WO 98/59048 PCT/US98/11868 -23- For example, to perform a diagnostic test based on nucleic acid molecules, a suitable nucleic acid sample is obtained and prepared using conventional techniques. DNA can be prepared, for example, simply by boiling a sample in SDS. The extracted nucleic acid can then be subjected to amplification, for example by using the 5 polymerase chain reaction (PCR) according to standard procedures, such as a RT-PCR method, to selectively amplify a BT-R, encoding nucleic acid molecule or fragment thereof. The size or presence of a specific amplified fragment (typically following restriction endonuclease digestion) is then determined using gel electrophoresis or the nucleotide sequence of the fragment is determined (for example, see Weber and May 10 Am J Hum Genet (1989) 44:388-339; Davies, J. et al. Nature (1994) 371 130-136)). The resulting size of the fragment or sequence is then compared to the known BT-R, proteins encoding sequences, for example via hybridization probe. Using this method, the presence of a BT-R, protein can be identified. To perform a diagnostic test based on proteins, a suitable protein sample is 15 obtained and prepared using conventional techniques. Protein samples can be prepared, for example, simply by mixing a sample with SDS followed by salt precipitation of a protein fraction. The extracted protein can then be analyzed to determine the presence of a BT-R, protein using known methods For example, the presence of specific sized or charged variants of a protein can be identified using 2 0 mobility m an electnc filed Alternatively, antibodies can be used for detection purposes. A skilled artisan can readily adapt known protein analytical methods to determine if a sample contains a BT-R, protein Alternatively, BT-R, protein or gene expression can also be used in methods to identify agents that decrease the level of expression of a BT-R j gene For example, 2 5 cells or tissues expressing a BT-R, protein can be contacted with a test agent to determine the effects of the agent on BT-R, protein/gene expression Agents that activate BT-R, protem/gene expression can be used as an agonist of BT-R, activity whereas agents that decrease BT-R, protein/gene expression can be used as an antagonist of BT-R, activity. Printed from Mimosa WO 98/59048 PCT/US98/11868 -24- K. Methods to Sensitize Cells The present invention further provides methods of sensitizing cells such that they become susceptible to killing with a BT-toxin, or a BT-toxin analog. Specifically, host cells transformed to express BT-R, receptor, or a homolog of the 5 BT-R, receptor, become sensitive to the mode of action of BT-toxins. The binding of a BT-toxin to a BT-R, receptor expressed on the surface of the transformed cells results in induction of a cellular death and apoptosis of the cell expressing the BT-R, receptor. Accordingly, the BT-R, receptor is an appropriate candidate for use in transforming cells m which it is desirable to induce cell death. 10 There are numerous situations in which it is desirable to introduce the selected gene into a selected population of cells, thus bringing about cell death One such example is m the therapeutic treatment of cancer cells. In using specifically targeted vectors for delivery of BT-R,-encoding DNA molecules into a tumor cell, tumor cells within a patient can be engineered to express a BT-R, protein. Such cells then 15 become susceptible to death upon treatment with a BT-toxin Since BT-toxin is not normally toxic to mammalian cells, this method is particularly applicable to inducing cell death in particular cells in a mammalian host Other situations where it may be desirable to stimulate cell death in particular cells or cell lines are m the treatment of autoimmune disorders and in the treatment of cells harboring pathogens, such as 2 0 malaria or HIV agents The choice of the actual steps employed to introduce a BT-R,-encoding DNA molecule into a cell to render the cells susceptible to treatment with BT-toxin is based primarily on the cell type that is to be altered, the conditions under which the cell type will be altered, and the overall use envisioned. A skilled artisan can readily adapt art- 2 5 known methods for use with the BT-R,-encoding DNA molecule of the present invention. L. Animal Models and Gene Therapy The BT-R / gene and the BT-R, protein can also serve as a target for generating 3 0 transgenic organisms in which the pattern of BT-R, expression has been altered. For Printed from Mimosa WO 98/59048 PCT/US98/11868 -25- example, in one application, BT-R, deficient insects or insect cells can be generated using standard knock-out procedures to inactivate a BT-R] gene, or, if such animals are non-viable, inducible BT-R/ antisense molecules can be used to regulate BT-R j activity/expression. Alternatively, cells or an organism can be altered so as to contain 5 a Manduca sexta BT-R, encoding nucleic acid molecule or an antisense-BT-R, expression unit that directs the expression of a BT-R, protein or an antisense molecule in a tissue specific fashion In such uses, an organism or cells, for example insects or insect cells, is generated m which the expression of a BT-R j gene is altered by inactivation or activation and/or replaced by a Manduca sexta BT-R j gene This can 10 be accomplished using a variety of art-known procedures such as targeted recombination. Once generated, the BT-R j expression altered cells or organisms can be used to 1) identify biological and pathological processes mediated by the BT-R, protein, 2) identify proteins and other genes that interact with the BT-R, protein, 3) identify agents that can be exogenously supplied to overcome a BT-R, protein 15 deficiency and 4) serve as an appropriate screen for identifying mutations within the BT-R] gene that increases or decreases activity. For example, it is possible to generate transgenic insects, such as members of the dipteran order, expressing the Manduca sexta minigene encoding BT-R, in a tissue specific-fashion and test the effect of over-expression of the protein in tissues and 2 0 cells that normally do not contain the BT-R, protein M. Use of Expression Control Elements of the BT-R, Gene The present invention further provides the expression control sequences found 5' of the of the newly identified BT-R] gene in a form that can be used in generating 2 5 expression vectors Specifically, the BT-R ] expression control elements, such as the BT-R, promoter, that can readily be identified as being 5' from the ATG start codon in the BT-R; gene, can be used to direct the expression of an operably linked protein encoding DNA sequence Since BT-R, expression is mostly tissue-specific, the expression control elements are particularly useful in directing the expression of an 3 0 introduced transgene m a tissue specific fashion. A skilled artisan can readily use the Printed from Mimosa WO 98/59048 PCT/US98/11868 -26- BT-R, gene promoter and other regulatory elements to generate expression vectors using methods known in the art. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and 5 utilize the compounds of the present invention and practice the claimed methods The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. 10 Example 1 Purification and Sequence Determination of BT-R, Protein Midguts of M sexta were extracted and the BT-R, protein purified according to the method of Vadlamudi, R.K. et al. J Biol Chem (1993) 268:1233, referenced above and incorporated herein by reference. The electroeluted band was confirmed to 15 contain BT-R, protein by binding to 125I-crylAb toxin. In gel electrophoresis, the protein bound to toxin had an apparent weight of approximately 210 kD under reducing and nonreducmg conditions. The purified electroeluted BT-R, was subjected to cyanogen bromide digestion and the cyanogen bromide fragments separated on a 17% high-resolution tncme SDS-20 polyacrylamide gel as descnbed by Schagger, H et al Anal Biochem (1987) 166.368 The separated fragments were transferred to Problott membranes (Applied Biosystems) and five bands were extracted and subjected to microsequencmg using standard instrumentation. The amino acid sequences obtained were 1 (Met)-Leu-Asp-Tyr-Glu-Val-Pro-Glu-Phe-Gln-Ser-Ile-Thr-Ile-Arg-2 5 Val-Val-Ala-Thr-Asp-Asn-Asn-Asp-Thr-Arg-His-Val-Gly-Val-Ala, 2 (Met)-X-Glu-Thr-Tyr-Glu-Leu-Ile-Ile-His-Pro-Phe-Asn-Tyr-Tyr-Ala; 3. (Met)-X-X-X-His-Gln-Leu-Pro-Leu-Ala-Gin-Asp-Ile-Lys-Asn-His; 4. (Met)-Phe/Pro-Asn/Ile-V al-Arg/T yr-V al-Asp-Ile/Gly, 5. (Met)-Asn-Phe-Phe/His-Ser-V al-Asn-Arg/Asp-Glu. Printed from Mimosa WO 98/59048 PCT/US98/11868 -27- Example 2 Recovery of cDNA An M sexta cDNA library was constructed from midgut tissue in Xgtl 0 using the Superscript Choice System according to the manufacturer's instructions (Life 5 Technologies, Inc.). Degenerate oligonucleotide probes were constructed based on the peptide sequences determined in Example 1 using the methods and approach described in Zhang, S. et al. Gene (1991) 105.61. Synthetic oligonucleotides corresponding to peptides 1-3 of Example 1 were labeled with a32P using polynucleotide kinase and used as probes as described in the standard cloning manual 10 of Mamatis, T. et al Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2nd ed 1989) A clone hybridizing to all three probes identified from 40 positive clones as hybndizing to all three of the probes was plaque-purified from a screen of 4 X 105 recombinants and subcloned into pBluescnpt (Stratagene). It contained an insert of 5571 bp. 15 Double-stranded cDNA in pBluescnpt was sequenced in both directions by the dideoxy termination method with Sequanase (USB) according to the manufacturer's instructions. The sequencing showed an open reading frame of 4584 base pairs or 1528 ammo acids along with a polyadenylation signal at position 5561. The sequence obtained and the deduced amino acid sequence is shown in Figure 1. 2 0 Thus, the deduced protein has a molecular mass of 172 kD and a pi of approximately 4.5 The ammo acid sequences of the cyanogen bromide fragments of native receptor match perfectly within the deduced ammo acid sequence. The open reading frame begins with an ATG that is flanked by the consensus translation initiation sequence GAGATGG for eucaryotic mRNAs as descnbed by Kozak, M 25 Nucleic Acids Res (1987) 15.8125. As shown in Figure 1, the deduced ammo acid sequence includes a putative signal, shown underlined, preceding the mature N-terminus Asn-Glu-Arg-etc. Eleven repeats (cadi-cadi 1) are shown m the extracellular region upstream of the membrane domain, shown with the heavy underline, at positions 1406-1427. The end of the 11th j j I Printed from Mimosa I i i WO 98/59048 PCT/US98/11868 -28- repeat is shown with an arrowhead. The positions of the five CNBR fragments are also shown under the complete sequence Figure 2 compares the BT-R, sequence obtained herein with other members of the cadhenn family. Like known cadherins, the external domain of BT-R, is highly 5 repetitive and contains 11 repeats (cadl-cadl 1; see Figure 2 A). The other cadherins compared in Figure 2 B are mouse P cadhenn (mP EC1); Drosophila fat EC 18 (fat EC 18) and protocadherin (PC42 EC2), and Manduca sexta intestinal transporter (HPT-l-EC-1) The eleven repeats of the cadherin motif m BT-R, (cadl-cadl 1) are individually aligned with a single motif sequence from each of the other members of 10 the cadherin family. Conserved residues are boxed. The greatest similanty of BT-R, to the cadherins is with the extracellular repeats of the cadhenn motif of mouse P-cadherin, Drosophila fat tumor suppressor and the protocadhenns, although homologies are not high (20-40 homology and 30-60 percent similanty). The conserved repeats of BT-R, included AXDXD, DXE, DXNDXXP, one glutamic acid 15 residue and two glycine residues (Figure 2 B) Motifs A/VXDXD, DXNDN are the consensus sequences for calcium binding and two such regions are present in a typical cadhenn repeat. In all repeats of BT-R,, the sequence DXNDN is preceded by 8 to 14 hydrophobic ammo acids. Similar hydrophobic sequences also have been observed in the cadhenns. The length of the hydrophobic stretches suggests that these areas are 2 0 not transmembrane regions buy that the represent J-sheet structures commonly present in cadherin-like repeats. BT-R, contains a putative cytoplasmic domain of 101 amino acids, smaller than vertebrate cadhenn cytoplasmic domains (160 amino acids), and shows no homology to any of the cadhenn cytoplasmic domains or to cytoplasmic domains of other proteins to which it has been compared m a current sequence data 2 5 base To confirm that the sequenced clone encoded full-length BT-R, protein, total mRNA was prepared from midguts of M sexta subjected to Northern blot by hybridization with the antisense 4.8 kb SacI fragment of the BT-R, cDNA clone. The Northern blot analysis was conducted by hybndizing to the antisense probe at 42°C 3 0 and 50% formamide, 5 X Denhardt's Reagent, 5 X SSCP and 50 fig/ml salmon sperm Printed from Mimosa WO 98/59048 PCT/US98/11868 -29- DNA. The filter was then washed two times with 1 X SSC + 0.1% SDS and two times with 0.15 X SSC + 0.1% SDS at 42°C. Each wash was roughly 20 minutes. The filter was then exposed to X-ray film for 24 hours. The 4.8 kb probe hybridized to a single 5.6 kb band. 5 The BT-R, clone was translated using rabbit reticulolysate and the resultmg translated products were immunoprecipitated with antisera raised against native protein encoded by BT-R, For the in vitro translation, pBluescnpt plasmid containing BT-R, cDNA was linearized and transcnbed with T3 polymerase (Pharmacia). The translation was conducted according to manufacturer's instructions 10 with nuclease-treated rabbit reticulolysate (Life Technologies, Inc.) After one hour of incubation at 30°C, the reaction mixture was combined with an equal volume of SDS buffer or lysed with 50 mM Tns buffer containing 1% NP40 and 250 mM NaCl (pH 8.0) for immunoprecipitation. Preimmune serum was used as a control. Translation and immunoprecipitation products were electrophoresed on a 7.5% SDS-15 polyacrylamide gel fixed, treated with Enhance (Dupont NEN), dned and exposed to X-ray film for 12 hours. Two protein bands of approximately 172 kD and 150 kD as determined by SDS-PAGE were obtained; it is postulated that the 150 kD translation product was due to initiation of translation from an internal methionine at amino acid 242. This is 2 0 consistent with the observations of Kozak, M Mol Cell Biol (1989) 9:5073. Thus, both results confirm that a full-length clone was obtained Example 3 Recombinant Production and Charactenstics of the BT-R, Protein 2 5 The BT-R, cDNA clone was subcloned into the mammalian expression vector pcDNA3 (Invitrogen) and the construct transfected into COS-7 cells Membranes isolated from the COS-7 transfectants were solubihzed, electrophoresed and ligand blotted with 125I-CrylAb toxin. The cells were harvested 60 hours after transfection, washed with phosphate-buffered saline and lysed by freezing in liquid nitrogen. Cell 3 0 membranes were prepared by differential centnfugation as descnbed by Elshourbagy, Printed from Mimosa _WO 98/59048 PCT/US98/11868 -30- N.A. et al J Biol Chem (1993) 266:3873 Control cells were COS-7 cells transfected with pcDNA3. The cell membranes (10 |ig) were separated on 7.5% SDS-PAGE blotted to a nylon membrane and blocked with Tns-buffered saline containing 5% nonfat dry milk 5 powder, 5% glycerol and 1% Tween-20. The nylon membrane was then incubated with 125I-CrylAb toxin (2 X 105 cpm/ml) for two hours with blocking buffer, dried and exposed to X-ray film at -70°C. The labeled toxin bound to a 210 ± 5 kD protein; the 210 kD band was observed only in lanes containing membranes prepared from either M. sexta or COS-7 cells transfected with the BT-R, cDNA construct containing 4810 10 bp of cDNA comprising the open reading frame The discrepancy between the 210 kD protein expressed and the calculated 172 kD molecular weight is due to glycosylation of the protein; in vitro translation of the cDNA clone, as described above, which does not result in glycosylation, does produce the 172 kD protein. To verify this, the COS-7 produced protein was subjected to 15 digestion with N-glycosidase-F by first denaturing the purified protein by boiling in 1% SDS for 5 minutes followed by addition ofNP-40 to a final concentration of 1% m the presence of 0.1% SDS, and then incubating the denatured protein in sodium phosphate buffer, pH 8 5 at 37°C with N-glycosidase-F for 10 hours Controls were incubated under the same conditions without enzyme. Digestion products were 2 0 separated on a 7.5% SDS-PAGE and stained with Coomassie brilliant blue. This glycosidase treatment reduced the molecular weight ofBT-R, protein from 210 to 190 kD, this indicates N-glycosylation at some of the 16 consensus N-glycosylation sites in the protein. Treatment ofBT-R, with O-glycosidase and neuraminidase did not alter the mobility of the protein 25 In addition, embryonic 293 cells were transfected with the BT-R, cDNA clone in pcDNA3 and incubated with the labeled toxin (0.32 nM) m the presence of increasing concentrations (0 to 10"6 M) of unlabeled toxin. Nonspecific binding was measured as bound radioactivity m the presence of 1 TM unlabeled toxm. A value for the dissociation constant (K<j) of 1015 pM was determined by Scatchard analysis; this Printed from Mimosa WO 98/59048 PCT/US98/11868 -31 - is approximately the same value that was obtained for the natural receptor as descnbed by Vadlamudi, R.K. et al. J Biol Chem (1993) {supra). Example 4 5 Physiological Effect of BT Toxin on Modified Embryonic 293 Cells Both unmodified embiyonic 293 cells, and 293 cells which have been modified to produce the BT-R, receptor as descnbed in Example 3, when cultured in vitro form adherent star-shaped clusters. When BT toxin (200 nM) is added to serum-free medium, the clusters round up and release from the plastic surfaces of the culture 10 dish. This effect is also observed under known conditions of cytotoxicity for 293 cells. The foregoing effect is observed only when the cells are cultured in serum-free medium since the toxin binds to serum and would thus be ineffective under conditions where serum is present. However, m the presence of anti-receptor antisera, this effect of BT toxin is 15 blocked. Also, when serum is added back to a culture of modified E293 cells which has been treated m serum-free conditions with the toxin, the cells revert to their normal star-shaped adherent cluster shapes. This indicates that the effect of the toxin is reversible. 20 Example 5 Identification Of A Fragment OfBT-R, That Binds To A BT Toxm To understand some of the properties ofBT-R,, research has been undertaken to define the location of the BT-R,/Cryl Ab protem-protein interaction The full-length wild-type ammo acid sequence ofBT-R, is provided in Fig. 1 with a block 2 5 diagram of a possible cadhenn-like structure for BT-R, shown in Fig 3 In both figures, restriction digest sites from the cDNA are provided relative to the positions at which they would disrupt the ammo acid coding sequence. A small fragment lying between the BamHI and SacI restnction sites of wild-type BT-R, was cloned into the vector pCITE (Novagen) This vector contains 3 0 transcnption/translation sequences designed for use in a rabbit reticulocyte lysate Printed from Mimosa WO 98/59048 PCT/US98/11868 -32- (RRL) system. The clone has been analyzed by restriction mapping and mRNA expression (Fig. 4). Lane UP shows the uncut plasmid and lanes NP and XP show restnction digests using Nsil and Xhol, respectively. Nsil is used because it has only one restriction site lying within the Bam-Sac fragment and does not cut anywhere 5 within the pCITE vector. The BSP lane shows the restriction digest of the clone using BamHI and Sacl. The digest releases the cloned fragment which separates at about 700 base pairs. The RT1 and RT2 lanes show mRNA transcription from the clone after linearization with Xhol The mRNA separates at the expected 1350 base pairs. 10 Protein for analysis has been prepared from this clone in two ways. First, an RRL translation kit was employed to produce protein from the mRNA transcnption reaction described above. Second, the plasmid was added directly to an RRL based transcription and translation (TNT) coupled kit. Protein production was detected usmg 35S-methiomne as a tag (Fig. 5). The LCR lane shows production of luciferase 15 protein from mRNA in an RRL kit and the LCT lane is luciferase protein from a plasmid containing the luciferase coding sequence translated in the TNT kit. Both are positive controls to demonstrate that the two translation kits are operational. The major bands for luciferase translation are observed at 66 kDa. The lanes labeled as RR, and RR2 show expression of the polypeptide sequence of the Bam-Sac fragment 2 0 ofBT-R, translated from mRNA in the RRL kit. The lanes TT1 and TT2 are translations from the pCITE plasmid containing the Bam-Sac fragment from the TNT kit. All four lanes possess a major band at 30 kDa which is the expected size of the Bam-Sac fragment with the addition of a coded antibody tag called S-tag. S-tag is part of the multicloning site of pCITE. 2 5 The clone was then tested for its ability to bind the insecticidal toxin Cry 1 Ab. Polypeptide translation of the Bam-Sac fragment ofBT-R, was earned out in duplicate as described above. The only change is that the 35S-methionme tag was left out of the reaction mixtures to produce non-radiolabeled proteins The proteins were separated by SDS-PAGE, blotted to nitrocellulose and hybridized with 125I-labeled 30 CrylAb(Fig 6). BBMV is wild-type BT-R, prepared from the midgut brush border Printed from Mimosa WO 98/59048 PCT/US98/11868 -33- membrane vesicles (BBMV) of M. sexta, and, is used as a positive control. RBK and TBK are RRL and TNT control reactions prepared without mRNA or plasmid present to determine whether proteins endogenous to either kit bind Cryl Ab. R, and RR2 are translations from the RRL kit and TT1 and TT2 are from the TNT kit. A single 30-5 kDa band appears in each of these lanes. Two are marked by arrows. These bands demonstrate that the Bam-Sac fragment ofBT-R, is capable of binding CiylAb insecticidal toxin. To further understand the nature of this binding site, a set of truncation mutants of BT-R, was prepared through the use of restnction digests. The cDNA was 10 digested at specific sites to remove increasingly larger portions of the C-terminus. The restnction enzymes used were Nsil, BamHI, Nrul, Clal, Xhol and StuI (Figs. 1 and 3). The procedure involved lineanzing the plasmid at each one of these sites and transcribing up to the truncation. The shortened mRNAs then were translated in an RRL kit blotted to nitrocellulose and hybndized with ,25I-labeled Cryl Ab. 15 Translation of the wild-type BT-R, from the cDNA showed binding to a 172-kDa protein band, the expected size of wild-type BT-R,. It also shows smaller bands that bind Cryl Ab although the nature of these bands has not been determined. A blank made by preparing an RRL reaction mixture without any mRNA gaves several bands below 66 kDa that show some type of binding of Cryl Ab to the reticulocytes. The 2 0 specificity of this binding has not been determined. The truncation mutants created by Nsil, BamHI, Nrul, Clal, Xhol and StuI restriction digests did not show any binding to CrylAb except in the region where the reticulocytes bind Cryl Ab. This data demonstrates that the removal of the last 100 ammo acids from wild type BT-R, by Nsil restnction results in the loss of the ability ofBT-R, to bind CrylAb. This 2 5 localizes the toxin binding site on the BT-R, clone and provides a soluble fragment of the receptor that can be used in toxin and other binding studies. A clone of a fragment of BT-R„ called the Bam-Sac fragment, has been prepared. It was prepared using BamHI and SacI restriction digests (Fig l)and cloning of the resulting fragment into a vector called pCITE The polypeptide 3 0 sequence was translated and tested for binding to the insecticidal toxin CrylAb Printed from Mimosa WO 98/59048 PCT/US98/11868 -34- (Figure 8). The Bam-Sac fragment binds to CrylAb, providing first insight into the location of the CrylAb binding site within the BT-R, sequence. It lies m the last 234 C-terminal amino acids. This evidence is further supported by a set of truncation mutants that has been prepared Removal of the 100 most C-terminal amino acids 5 from wild type BT-R, results in the loss of CrylAb binding. The C-terminal end of BT-R, is the location of the CrylAb binding site. Example 6 Identification Of Homologue ofBT-R, That Binds To A BT Toxin 1 o Western blots of tissue extracts prepared from Pink bollworm and European corn borer were prepare and probed with labeled Cry la (Figure 7). The results show that homologues ofBT-R, are present in these two insects and can be readily isolated using the methods described herein. Printed from Mimosa </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO 98/59048 PCT/US98/11868 -35-Claims

1. A method to identify agents that bind to a BT-toxin receptor, said method comprising the steps of: 5 i) contacting an agent with a BT-toxin binding receptor selected from the group consisting of a) a cell that has been altered to contain a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2, b) a cell that has been altered to contain a nucleic acid molecule that encodes a fragment of the amino acid sequence of SEQ ID No:2 that binds to a BT toxm, c) a cell that has been altered to contain a 10 nucleic acid molecule encoding a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID No l under high stringency, d) a cell that has been altered to contain a nucleic acid molecule that encodes a fragment of a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID No.l under high stringency and that binds to a BT toxin, e) an isolated protein with an amino acid sequence of SEQ 15 ID No.2, f) an isolated fragment of a protein with an amino acid sequence of SEQ ID No.2, said fragment containing a BT-toxin binding domain, g) an isolated BT-toxin receptor that is encoded by a nucleic acid molecule that hybndizes to the nucleic acid sequence of SEQ ID No 1 under high stnngency, and h) an isolated fragment of a BT-toxin receptor that is encoded by a nucleic acid molecule that hybridizes to the 2 0 nucleic acid sequence of SEQ ID No.l under high stnngency, and li) determining whether said agent binds to said BT-toxm receptor.

2. The method of claim 1, wherein said method further compnses the step of determining whether said agent blocks the binding of a BT-toxm to said BT-toxm 2 5 receptor.

3. The method of claim 1, wherein said cell that has been altered is a eukaryotic cell. 30

4. The method of claim 3, wherein eukaryotic cell is an insect cell. Printed from Mimosa WO 98/59048 PCT/US98/11868 -36- 5 A method to identify agents that block the binding of a BT-toxin to a BT-toxin receptor, said method comprising the steps of: i) contacting an agent, m the presence and absence of a BT-toxin, with a

5 BT-toxm binding receptor selected from the group consisting of a) a cell that has been altered to contain a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No:2, b) a cell that has been altered to contain a nucleic acid molecule that encodes a fragment of the amino acid sequence of SEQ ID No:2 that binds to a BT toxin, c) a cell that has been altered to contain a nucleic acid molecule encoding a 10 BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID No:l under high stnngency, d) a cell that has been altered to contain a nucleic acid molecule that encodes a fragment of a BT-toxin receptor that hybndizes to the nucleic acid sequence of SEQ ID No:l under high stnngency and that binds to a BT toxin, e) an isolated protein with an amino acid sequence of SEQ ID No:2, f) an isolated fragment of a 15 protein with an ammo acid sequence of SEQ ID No.2, said fragment containing a BT-toxin binding domain, g) an isolated BT-toxin receptor that is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID No:l under high stnngency, and h) an isolated fragment of a BT-toxin receptor that is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of 2 0 SEQ ID No • 1 under high stringency, and ii) determining whether said agent blocks the binding of said BT-toxin to said BT-toxin receptor.

6. The method of claim 5, wherein said BT-toxin is a member of the BT- 2 5 cry( 1) family of toxins

7. The method of claim 5, wherein said cell that has been altered is a eukaryotic cell 3 0

8. The method of claim 7, wherein eukaryotic cell is an insect cell. Printed from Mimosa WO 98/59048 PCT/US98/11868 501874 "A

9. Ail isolated antibody, wherein said antibody binds to a protein selected from the group consisting of a) a BT-toxin receptor protein with an amino acid sequence of SEQ ID No:2, and b) a BT-toxin receptor protein that is encoded by a 5 nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID No: 1 under high stringency, or a fragment of said antibody, wherein said antibody fragment binds to said BT-toxin.

10. The antibody of claim 9, wherein said antibody binds to said BT-toxin 10 receptor and blocks the binding of a BT-toxin to said receptor.

11. The antibody of claim 10, wherein said antibody binds to an epitope located within the 232 c-terminal amino acids of the BT-toxin receptor depicted in SEQ ID No:2. 15

12. An unglycosylated BT-toxin receptor protein fragment which binds CrylA(h) toxin selected from the group consisting of a) a BT-toxin receptor protein fragment which binds CrylA(b) toxin and has an amino acid sequence found within the amino acid sequence of SEQ ID 20 NO:2, and b) a BT-toxin receptor protein fragment which binds CrylA(b) toxin and is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under stringent conditions 25 -3-0- INTELLECTUAL PROPERTY OFFICE OF NZ. 2 8 AUG 2001

13. A method to produce BT-toxin receptor protein, or a fragment thereof, said method comprising the steps of: i) culturing a cell that has been altered to contain a nucleic acid molecule that encodes a BT-toxin receptor protein,or BT-toxin binding fragment thereof, . wherein said cell has been altered to contain a nucleic acid molecule selected from the El VED WO 98/59048 PCT/US98/11868 -38- 501874 group consisting of a) a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No:2, b) a nucleic acid molecule that encodes a fragment of the amino acid sequence of SEQ ED No:2 that binds to a BT toxin, c) a nucleic acid molecule encoding a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID of a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID No: 1 under high stringency and that binds to a BT toxin, under condition in which said nucleic acid molecule is expressed and ii) isolating said BT-toxin receptor protein or fragment.

14. The method of claim 13, wherein said cell that has been altered is a eukaryotic cell.

15. The method of claim 14, wherein eukaryotic cell is an insect cell.

16. The BT-toxin receptor protein fragment of claim 12, wherein the fragment is a Bam-Sac fragment.

17. A BT-loxin leceptor protein fragment which binds CrylA(b) toxin selected from the group consisting of a) a BT-toxin receptor protein fragment which binds CrytA(b) toxin and has an amino acid sequence encoded by the nucleic acid fragment lying between the BamHI and Sacl restriction sides in SEQ ID NO-1, and b) a BT-toxin receptor protein fragment which binds CrylA(b) toxin and is encoded by a nucleic acid molecule that hybridizes to the nucleic acid fragment lying between the BamHI and Sacl restriction sites in SEQ ID NO:1 under stringent conditions.

18. The BT-toxin receptor protein fragment of claim 17, which is unglycosylated. 5 No: 1 under high stringency, and d) a nucleic acid molecule that encodes a fragment 10 INTELLECTUAL PROPERTY OFFICE OF N Z 2 8 AUG 2001 RECEIVES SEQUENCE LISTING 74 <110> BULLA, Lee A. <120> RECEPTOR FOR A BACILLUS THURINGIENSIS TOXIN <130> 27112-20037.11 <140> 09/178,176 <141^ 1998-12-22 <150.- US 08/982,129 <153 > 1997-12-01 <150> US 08/326,117 <153 ^ 1994-10-li< <160.> 11 <170;• FastSEQ fc.iT Windows Version 4.0 <210> 1 <r211"> 5582, <212> DNA <213> M. sexta <220> <221> CDS <222> (197)...(5348) <400> 1 gaccaatcgg agtgtggtga atttttggaa aatattttgt gcggttcctt tagttgtgta 60 atafagtact ttagttacaa atttggaata atttggcagc aaaaccatct gcagcaacaa 120 aatcatctgc agctucgaaa tcatctgcag cagcaaaagc atcttcagga gcgagaaaag 18 0 ccce ^aataa tgtq.^g atg gca gtt gac gtc cga ate get gcc ttc ctg ctg 232 Met Ala Va'i Asp Val Arg He Ala Ala Phe Leu Leu 1 5 10 gtg ttt ata gcg cct gca gtt tta get caa gag aga tgt ggg tat atg 280 Val Phe lie Ala Pro Ala Val Leu Ala Gin Glu Arg Cys Gly Tyr Met 15 20 25 acc gcc ate ccd agg eta cca rga ccg gat aat ttg cca gta eta aat 328 Thr Ala lie Pro Arg Leu Pro Arg Pro Asp Asn Leu Pro Val Leu Asn 30 35 40 ttt gaa ggc cag aca tgg agt cag agg ccc ctg ctc ccc gcc ccg gag 376 Phe Glu Gly Gli) Thr Trp Ser Gin Arg Pro Leu Leu Pro Ala Pro Glu 45 50 55 60 egg gat gac ctg tgc atg gac gcc tac cac gtg ata aca gcc aac ctc 424 Arg Asp Asp Leu Cys Met Asp Ma Tyr His Val lie Thr Ala Asn Leu 65 70 75 ggc acg cag gtc ate tac atg gat gaa gag ata gaa gac gaa ate acc 472 Gly Thr Gin Val lie Tyr Met Asp Glu Glu lie Glu Asp Glu lie Thr 80 85 90 ate gcc ata ctt aat tat aac gga cca tea act ccg ttc att gaa ctg 520 lie Ala lie Leu Asn Tyr Asn Gly Pro Ser Thr Pro Phe lie Glu Leu 95 100 . 105 cca ttt tta tec ggt teg tac aat ctg ctg atg ccg gtc ate agg aga 568 Pro Phe Leu Ser Gly Ser Tyr Asn Leu Leu Met Pro Val lie Arg Arg 110 115 120 gtt gac aac ggg gag tgg cat ctc ate ate acg caa aga cag cat tac 616 Val Asp Asn Gly Glu Trp His Leu lie lie Thr Gin Arg Gin His Tyr 125 130 135 140 gag ttg ccc ggc atg cag cag tac atg ttc aat gtg cgc gtg gac ggc 664 Glu Leu Pro Gly Met Gin Gin Tyr Met Phe Asn Val Arg Val Asp Gly 145 150 155 cag teg ctg gtg gca ggc gtg tct ctc get ate gtc aac ata gat gac 712 Gin Ser Leu Val Ala Gly Val Sei Leu Ala lie Val Asn lie Asp Asp 160 165 170 aac gcg ccc ate ata caa aac ttc gag cct tgc egg gtt cct gaa ctg 760 Asn Ala Pro lie lie Gin Asn Phe Glu Pro Cys Arg Val Pro Glu Leu 175 180 185 ggc gag cca ggg ttg aca gaa tgc aca tac caa gta teg gac gcg gac 80 8 Gly Glu Pro Gly Leu Thr Glu Cys Thr Tyr Gin Val Ser Asp Ala Asp 190 195 200 gga egg ate age aca gag ttc atg acg ttc agg ate gac age gtt cgt 856 Gly Arg lie Ser Thr Glu Phe Met Thr Phe Arg lie Asp Ser Val Arg 205 210 215 220 ggc gac gag gag acc ttc tac ate gaa egg acg aat ate ccc aac caa 304 Gly Asp Glu Glu Thr Phe Tyr lie, Glu Arg Thr Asn lie Pro Asn Gin 225 - 230 235 tgg atg tgg eta aat atg Trp Met Trp Leu Asn Met 240 gtc acc agt ccg ctg cat Val Thr Ser Pro Leu His 255 ccg aac acc cac acg gtg Pro Asn Thr His Thr Val 270 age cgt ccg ccg cgc tgg Ser Arg Pro Pro Arg Trp 285 290 gag aaa tct tac caa aac Glu Lys Ser Tyr Gin Asn acc ata ggc gtt aat acc Thr lie Gly Val Asn Thr 245 ata ttc. .age gtg aca gcc lie Phe Ser Val Thr Ala 260 act atg atg gtg caa gtg Thr Met Met Val Gin Val 275 280 ctg gag ate ttc get gtc Leu Glu lie Phe Ala Val 295 ttc aca gtg agg gcg ate Phe Thr Val Arg Ala lie teg ctc aac ttc 952 Ser Leu Asn Phe 250 ctg gac teg ctc 1000 Leu Asp Ser Leu 265 gcg aat gtg aac 104 8 Ala Asn Val Asn caa cag ttt gaa 1096 Gin Gin Phe Glu 300 gac gga gac act 1144 Asp Gly Asp Thr 2 305 310 315 gag ate aat atg cct ate aac tac agg ctg ate aca aat gag gaa gac 1192 Glu lie Asn Met Pro lie Asn Tyr Arg Leu lie Thr Asn Glu Glu Asp 320 325 330 aca ttc ttc age att gag gcc ctg cct ggt gga aaa age ggg get gta 1240 Thr Phe Phe Ser lie Glu Ala Leu Pro Gly Gly Lys Ser Gly Ala Val 335 340 345 ttc ctc gtg teg cca att gac cgc gac aca ctg caa cga gag gtg ttt 1288 Phe Leu Val Ser Pro lie Asp Arg Asp Thr Leu Gin Arg Glu Val Phe 350 355 360 cca ctt acg ate gtc get tac aaa tat gat gag gag gcc ttc tee aca 133S Pro Leu Thr lie Val Ala Tyr Lys Tyr Asp Glu Glu Ala Phe Ser Thr 365 370 375 380 tea aca aac gtg gtc ate att gtg aca gac ate aac gac caa aga cct 1384 Ser Thr Asn Val Val lie lie Val Thr Asp lie Asn Asp Gin Arg Pro 385 390 395 gaa ret ata cac aag gaa tat cga ctg gca ate atg gag gag acg ccc 1432 Glu Pro lie His Lys Glu Tyr Arg Leu Ala lie Met Glu Glu Thr Pro 400 405 410 ctg acc ctc aac ttc gat aaa gaa ttc gga ttt cat gat aag gat tta 14 80 Leu Thr Leu Asn Phe Asp Lys Glu Phe Gly Phe His Asp Lys Asp Leu 415 420 425 ggt caa aac get cag tac acg gtg cgt eta gag age gtg gac cct cca 1528 Gly Gin Asn Ala Gin Tyr Thr Val Arg Leu Glu Ser Val Asp Pro Pro 430 435 440 ggc get get gag gca ttc tac ata gcg cct gaa gtc ggc tac cag cga 1576 Gly Ala Ala Glu Ala Phe Tyr lie Ala Pro Glu Val Gly Tyr Gin Arg 445 450 455 460 cag acc ttc ate atg ggc acc ctc aat cac tee atg ctg gat tac gaa 1624 Gin Thr Phe lie Met Gly Thr Leu Asn His Ser Met Leu Asp Tyr Glu 465 470 475 gtg cca gag ttt cag agt att acg att egg gtg gta gcg acc gac aac 1672 Val Pro Glu Phe Gin Ser lie Thr .lie Arg Val Val Ala Thr Asp Asn 480 485 490 aac gac acg agg cac gtg ggc gtc gcg ttg gtt cac att gac ctc ate 1720 Asn Asp Thr Arg His Val Gly Val Ala Leu Val His lie Asp Leu lie 495 500 505 aat tgg aac gat gag cag ccg ate ttc gaa cac gcc gtg cag acc gtc 1768 Asn Trp Asn Asp Glu Gin Pro lie Phe Glu His Ala Val Gin Thr Val 510 515 520 acc ttc gac gag act gaa ggc gag ggg ttc ttc gtc gcc aag gcg gtt 1816 Thr Phe Asp Glu Thr Glu Gly Glu Gly Phe Phe Val Ala Lys Ala Val 525 530 535 540 3 gca cac gac aga gac ate ggg gat gtc gtc gag cat act tta ttg ggt Ala His Asp Arg Asp lie Gly Asp Val Val Glu His Thr Leu Leu Gly 545 550 555 aac get gtt aac ttc ctg acc ate gac aaa ctc acc ggc gac ate cgc Asn Ala Val Asn Phe Leu Thr lie Asp Lys Leu Thr Gly Asp lie Arg 560 565 570 gtc tea get aac gac tcc ttc aac tac cat cga gaa agt gaa tta ttt Val Ser Ala Asn Asp Ser Phe Asn Tyr His Arg Glu Ser Glu Leu Phe 575 580 585 gtg cag gtg cga get aca gac acg ctg ggc gaa ccc ttc cac acg gcg Val Gin Val Arg Ala Thr Asp Thr Leu Gly Glu Pro Phe His Thr Ala 590 595 600 acg tea cag ctg gtc ata cga eta aat gac ate aac aac acg cca ccc Thr Ser Gin Leu Val lie Arg Leu Asn Asp lie Asn Asn Thr Pro Pro 605 610 615 620 acc tta egg ctg cct cga ggc agt ccc caa gtg gag gag aac gtg cct Thr Leu Arg Leu Pro Arg Gly Ser Pro Gin Val Glu Glu Asn Val Pro 625 630 635 gat ggc cac gtc ate acc cag gag tta cgc gcc acc gac ccc gac acc Asp Gly His Val lie Thr Gin Glu Leu Arg Ala Thr Asp Pro Asp Thr 640 645 650 acg gcc gat ctg cgc ttc gag ata aac tgg gac acc tct ttc gcc acc Thr Ala Asp Leu Arg Phe Glu lie Asn Trp Asp Thr Ser Phe Ala Thr 655 660 665 aag caa ggc cgc cag get aac ccc gac gag ttt agg aat tgc gtg gaa Lys Gin Gly Arg G3n Ala Asn Pro Asp Glu Phe Arg Asn Cys Val Glu 670 675 680 ate gag acc ate ttc ccc gag att aac aac egg gga ctg get acc ggc lie Glu Thr lie Phe Pro Glu lie Asn Asn Arg Gly Leu Ala He Gly 685 690 695 700 cgc gtt gta gcg cgc gaa ate aga cac aac gtg acc ata gac tac gag Arg Val Val Ala Arg Glu lie Arg His Asn Val Thr lie Asp Tyr Glu . 705 - . 710 715 gag ttt gag gtc ctc tcc ctc aca gtg agg gtg cgt gac ctt aac acc Glu Phe Glu Val Leu Ser Leu Thr Val Arg Val Arg Asp Leu Asn Thr 720 725 730 gtc tac gga gac gac tac gac gaa teg atg ctc aca ata act ata ate Val Tyr Gly Asp Asp Tyr Asp Glu Ser Met Leu Thr lie Thr lie lie 735 740 745 gat atg aac gac aac gcg ccg gtg tgg gtg gag ggg act ctg gag cag Asp Met Asn Asp Asn Ala Pro Val Trp Val Glu Gly Thr Leu Glu Gin 750 755 760 4 t aac ttc cga gtc cgc gag atg teg gcg ggc ggg ctc gtg gtg ggc tcc 2536 Asn Phe Arg Val Arg Glu Met Ser Ala Gly Gly Leu Val Val Gly Ser 765 770 775 780 gtg cgc gcg gac gac ate gac gga ccg ctc tac aac caa gtg cga tac 2584 Val Arg Ala Asp Asp lie Asp Gly Pro Leu Tyr Asn Gin Val Arg Tyr 735 790 795 acc att ttc cct cgt gaa gac aca gat aag gac ctg ata atg ate gac Thr lie Phe Pro Arg Glu Asp Thr Asp Lys Asp Leu lie Met lie Asp 800 805 810 2632 ttc ctc acg ggt caa att tcc gtg aac aca age ggc gcc ate gac gcg 2680 Phe Leu Thr Gly Gin lie Ser Val Asn Thr Ser Gly Ala lie Asp Ala 815 820 825 gat act cct cca cgc ttc cac ctc tac tat aca gtg gtc get agt gac 2728 Asp Thr Pro Pro Arg Phe His Leu Tyr Tyr Thr Val Val Ala Ser Asp 830 835 840 cga tgc teg aca gaa gat cct gca gat tgc ccc cct gac ccg act tat 27 76 Arg Cys Ser Thr Glu Asp Pro Ala Asp Cys Pro Pro Asp Pro Thr Tyr 845 850 855 860 tgg gaa acc gaa gga aat ate aca ate cac ate acc gac acg aac aac 2824 Trp Glu Thr Glu Gly Asn lie Thr lie His lie Thr Asp Thr Asn Asn 865 870 875 aag gtc ccg cag gcg gaa acg act aag ttc gat acc gtc gtg tat att 2 872 Lys Val Pro Gin Ala Glu Thr Thr Lys Phe Asp Thr Val Val Tyr lie 880 885 890 tac gag aac gca acc cac tta gac gag gtg gtc act ctg ata gcc agt 2920 Tyr Glu Asn Ala Thr His Leu Asp Glu Val Val Thr Leu lie Ala Ser 895 900 905 gat ctt gac aga gac gaa ata cac cac acg gtg age tac gtc acc aat 2968 Asp Leu Asp Arg Asp Glu He Tyr His Thr Val Ser Tyr Val lie Asn 910 915 920 tat gca gtg aac cct cga ctg atg aac ttc ttc tcc gtg aac cga gag 3016 Tyr Ala Val Asn Pro Arg Leu Met Asn Phe Phe Ser Val Asn Arg Glu 925 930 935 940 acc ggc ctg gtg tac gtg gac tat gag acc cag ggt agt ggc gag gtg 3064 Thr Gly Leu Val Tyr Val Asp Tyr Glu Thr Gin Gly Ser Gly Glu Val 945 950 955 ctg gac cgt gat ggt gat gaa cca acg cac cgt ate ttc ttc aac ctc 3112 Leu Asp Arg Asp Gly Asp Glu Pro Thr His Arg lie Phe Phe Asn Leu 960 965 970 ate gac aac ttc atg ggg gaa gga gaa ggt aac aga aat cag aac gac 3160 lie Asp Asn Phe Met Gly Glu Gly Glu Gly Asn Arg Asn Gin Asn Asp 975 980 985 aca gaa gtt ctc gtt ate ttg ttg gat gtg aat gac aat get cct gaa 3208 5 Thr Glu Val Leu Val lie Leu Leu Asp Val Asn Asp Asn Ala Pro Glu 990 995 1000 ttg cca ccg ccg age gaa ctc tct tgg act ata tct gag aac ctt aag Leu Pro Pro Pro Ser Glu Leu Ser Trp Thr lie Ser Glu Asn Leu Lys 1005 1010 1015 1020 3256 cag ggc gtc cgt ctt gaa cca cat ate ttc gcc ccg gac cgc gac gag Gin Gly Val Arg Leu Glu Pro His lie Phe Ala Pro Asp Arg Asp Glu 1025 1030 1035 3304 ccc gac aca gac aac tcc agg gtc ggc tac gag ate ctg aac ctc age Pro Asp Thr Asp Asn Ser Arg Val Gly Tyr Glu lie Leu Asn Leu Ser 1040 1045 1050 3352 acg gag egg gac ate gaa gtg ccg gag ctg ttt gtg atg ata cag ate Thr Glu Arg Asp lie Glu Val Pro Glu Leu Phe Val Met lie Gin lie 1055 1060 1065 3400 gcg aac gtc acg gga gag ctg gag acc gcc atg gac ctc aag gga tat Ala Asn Val Thr Gly Glu Leu Glu Thr Ala Met Asp Leu Lys Gly Tyr 1070 1075 1080 3448 tgg ggg acg tac get ata cat ata egg gca ttc gac cac ggc att ccg Trp Gly Thr Tyr Ala lie His lie Arg Ala Phe Asp His Gly lie Pro 1085 1090 1095 1100 3496 caa atg tcc atg aac gag aca tat gag ccg ate ate cat ccg ttc aac Gin Met Ser Met Asn Glu Thr Tyr Glu Leu lie lie His Pro Phe Asn 1105 1110 1115 3544 tac tac gcg cct gag ttc gtc ttc ccg acc aac gat gcc gtc ata cga Tyr Tyr Ala Pro Glu Phe Val Phe Pro Thr Asn Asp Ala Val lie Arg 1120 1125 1130 3592 ctt gcg agg gaa cga get gta ate aat gga gtt eta gcg aca gtg aac Leu Ala Arg Glu Arg Ala Val He Asn Gly Val Leu Ala Thr Val Asn 1135 1140 1145 3640 gga gag ttc ttg gag egg ata teg gcg act gat ccg gac gga ctc cac Gly Glu Phe Leu Glu Arg lie Ser Ala Thr Asp Pro Asp Gly Leu His 1150 1155 1160 3688 gcg ggc gtc gtc acc ttc caa gtg gta ggc gat gag gaa tea caa egg Ala Gly Val Val Thr Phe Gin Val Val Gly Asp Glu Glu Ser Gin Arg 1165 1170 1175 1180 3736 tac ttt caa gta gtt aac gat ggc gag aac ctc ggc teg ttg agg tta Tyr Phe Gin Val Val Asn Asp Gly Glu Asn Leu Gly Ser Leu Arg Leu 1185 1190 1195 3784 ctg caa gcc gtt cca gag gag ate agg gag ttc egg ata acg att cgc Leu Gin Ala Val Pro Glu Glu lie Arg Glu Phe Arg lie Thr lie Arg 1200 1205 1210 3832 get aca gac cag gga acg gac cca gga ccg ctg tcc acg gac atg acg Ala Thr Asp Gin Gly Thr Asp Pro Gly Pro Leu Ser Thr Asp Met Thr 3880 6 1215 1220 1225 ttc aga gtt gtt ttt gtg ccc acg caa gga gaa cct aga ttc gcg tcc 3928 Phe Arg Val Val Phe Val Pro Thr Gin Gly Glu Pro Arg Phe Ala Ser 1230 1235 1240 tea gaa cat get gtc get ttc ata gaa aag agt gcc ggc atg gaa gag 3976 Ser Glu His Ala Val Ala Phe lie Glu Lys Ser Ala Gly Met Glu Glu 1245 1250 1255 1260 tct cac caa ctt cct eta gca caa gac ate aag aac cat ctc tgt gaa 4024 Ser His Gin Leu Pro Leu Ala Gin Asp lie Lys Asn His Leu Cys Glu • 1265 1270 1275 gac gac tgt cac age att tac tat cgt att ate gat ggc aac age gaa 4072 Asp Asp Cys His Ser lie Tyr Tyr Arg lie lie Asp Gly Asn Ser Glu 1280 1285 1290 gt,,t cat ttc ggc ctg gat cct gtt cgc aac agg ttg ttc ctg aag aaa 4120 G?y His Phe Gly Leu Asp Pro Val Arg Asn Arg Leu Phe Leu Lys Lys 1295 1300 1305 grtg ctg ata agg gaa caa agt gcc tcc cac act ctg caa gtg gcg get _ 4168 Giu Leu lie Arg Glu Gin Ser Ala Ser His Thr Leu Gin Val Ala Ala 1310 1315 1320 agt aac teg ccc gat ggt ggc att cca ctt cct get tcc ate ctt act 4216 Ser Asn Ser Pro Asp Gly Gly lie Pro Leu Pro Ala Ser lie Leu Thr 1325 1330 1335 1340 gtc act gtt acc gtg agg gag gca gac cct cgt cca gtg ttt gtg agg 42 64 Val Thr Val Thr Val Arg Glu Ala Asp Pro Arg Pro Val Phe Val Arg 1345 1350 1355 gaa ttg tac acc gca ggg ata tcc aca gcg gac tcc ate ggc aga gag 4312 C'JLu Leu Tyr Thr Ala Gly lie Ser Thr Ala Asp Ser lie Gly Arg Glu 1360 1365 1370 ctg ctc aga tta cat gcg acc cag tct gaa ggc teg gcc att act tat 4360 Leu Leu Arg Leu His Ala Thr Gin Ser Glu Gly Ser Ala He Thr Tyr 1375 1380 1385 get ata gac tac gat aca atg gta gtg gac ccc age ctg gag gca gtg 4408 Ala He Asp Tyr Asp Thr Met Val -Val Asp Pro Ser Leu Glu Ala Val 1390 1395 1400 aga cag teg get ttc gta ctg aac get caa acc gga gtg ctg acg ctt 4456 Arg Gin Ser Ala Phe Val Leu Asn Ala Gin Thr Gly Val Leu Thr Leu 3405 1410 1415 1420 aat ate cag ccc acg gcc acg atg cat gga ctg ttc aaa ttc gaa gtc 4504 Asn lie Gin Pro Thr Ala Thr Met His Gly Leu Phe Lys Phe Glu Val 1425 1430 1435 aca get act gac acg gcc ggc get cag gac cgc acc gac gtc acc gtg 4552 Thr Ala Thr Asp Thr Ala Gly Ala Gin Asp Arg Thr Asp Val Thr Val 1440 1445 1450 7 tac gtg gta tcc teg cag aac cgc gtc tac ttc gtg ttc gtc aac acg Tyr Val Val Ser Ser Gin Asn Arg Val Tyr Phe Val Phe Val Asn Thr 1455 1460 1465 4600 ctg caa cag gtc gaa gac aac aga gac ttt ate gcg gac acc ttc age Leu Gin Gin Val Glu Asp Asn Arg Asp Phe lie Ala Asp Thr Phe Ser 1470 1475 1480 4648 get ggg ttc aac atg acc tgc aac ate gac caa gtg gtg ccc get aac Ala Gly Phe Asn Met Thr Cys Asn lie Asp Gin Val Val Pro Ala Asn 1485 1490 1495 1500 4696 gac ccc gtc acc ggc gtg gcg ctg gag cac age acg cag atg cgc ggc Asp Pro Val Thr Gly Val Ala Leu Glu His Ser Thr Gin Met Arg Gly 1505 1510 1515 4744 cac ttc ata egg gac aao gta ccc gta ctc get gat gag ata gaa cag His Phe lie Arg Asp Asn Val Pro Val Leu Ala Asp Glu lie Glu Gin 1520 1525 1530 4792 ate cgt agt gac eta gtc ctc ctg age teg ata caa aca acg ctg gcg lie Arg Ser Asp Leu Vp'i Leu Leu Ser Ser lie Gin Thr Thr Leu Ala 1535 1540 1545 4840 gcg cga teg ctg gtg ttg cag gac ttg ttg acc aac tcc age ccg gac Ala Arg Ser Leu Val Leu Gin Asp Leu Leu Thr Asn Ser Ser Pro Asp 1550 1555 1560 4888 teg gcg cct gac teg age ctc acg gtg tac gtg ctg gcc tea ctg tct Ser Ala Pro Asp Ser Ser Leu Thr Val Tyr Val Leu Ala Ser Leu Ser 1565 1570 1575 1580 4936 get gtg ctc ggt ttc atg tgc ctt gtg eta ctg ctt acc ttc ate ate Ala Val Leu Gly Phe Mf-t Cys Leu Val Leu Leu Leu Thr Phe lie lie 1585 15 90 1595 4984 agg act aga gcg eta aac cga egg ttg gaa gcc ctg teg atg acg aag Arg Thr Arg Ala Leu Asn Arg Arg Leu Glu Ala Leu Ser Met Thr Lys 1600 1605 1610 5032 tac ggc tea ctg gac tct gga ttg aac cgc gcc ggc ate gcc gcc ccc Tyr Gly Ser Leu Asp Ser Gly Leu Asn Arg Ala Gly He Ala Ala Pro 1615 1620-- 1625 5080 ggc acc aac aaa cac act gtg gaa ggc tcc aac cct ate ttc aat gaa Gly Thr Asn Lys His Thr Val Glu Gly Ser Asn Pro lie Phe Asn Glu 1630 1635 1640 5128 gca ata aag acg cca gat tta gat gcc att age gag ggt tcc aac gac Ala lie Lys Thr Pro Asp Leu Asp Ala lie Ser Glu Gly Ser Asn Asp 1645 1650 1655 1660 5176 tct gat ctg ate ggc ate gaa gat ctt ccg cac ttt ggc aac gtc ttc Ser Asp Leu lie Gly lie Glu Asp Leu Pro His Phe Gly Asn Val Phe 1665 1670 1675 5224 8 i 874 atg gat cct gag gtg aac gaa aag gca aat ggt tat ccc gaa gtc gca 5272 Met Asp Pro Glu Val Asn Glu Lys Ala Asn Gly Tyr Pro Glu Val Ala 1680 1685 1690 aac cac aac aac aac ttc get ttc aac ccg act ccc ttc teg cct gag 5320 Asn His Asn Asn Asn Phe Ala Phe Asn Pro Thr Pro Phe Ser Pro Glu 1695 1700 1705 ttc gtt aac gga cag ttc aga aag ate t agaagataac aacactagtt 5368 Phe Val Asn Gly Gin Phe Arg Lys lie 1710 1715 aagatcatta attttggagt ttggaattaa gatttttgaa aggatagttg tgataagcct 5428 gtgattttta aaactgtaat tgaaaaaaaa aattgagacc tccatttaag ctcttgctct 5488 catctcatca aattttataa aatgccatta gtcattaaga tactcgattt aatttaagat 554 8 tatttaagat attatgtaaa ataaatatat tgtc 5582 <210> 2 <211> 1717 <212> PRT <213> M. sexta <400> 2 Met Ala Val Asp Val Arg lie Ala Ala Phe Leu Leu Val Phe lie Ala 15 10 15 Pro Ala Val Leu Ala Gin Glu Arg Cys Gly Tyr Met Thr Ala lie Pro 20 25 30 Arg Leu Pro Arg Pro Asp Asn Leu Pro Val Leu Asn Phe Glu Gly Gin 35 40 45 Thr Trp Ser Gin Arg Pro Leu Leu Pro Ala Pro Glu Arg Asp Asp Leu 50 55 60 Cys Met Asp Ala Tyr His Val lie Thr Ala Asn Leu Gly Thr Gin Val 65 70 75 80 lie Tyr Met Asp Glu Glu lie Glu Asp Glu lie Thr lie Ala lie Leu 85 90 95 Asn Tyr Asn Gly pre Sex Thr Pro Phe lie Glu Leu Pro Phe Leu Ser 100 105 110 Gly Ser Tyr Asn Leu Leu Met Pro Val lie Arg Arg Val Asp Asn Gly 115 120 125 Glu Trp His Leu lie lie Thr Gin Arg Gin His Tyr Glu Leu Pro Gly 130 -135 140 Met Gin Gin Tyr Met Phe Asn Val Arg Val Asp Gly Gin Ser Leu Val 145 150 155 160 Ala Gly Val Ser Leu Ala lie Val Asn lie Asp Asp Asn Ala Pro lie 165 170 175 lie Gin Asn Phe Glu Pro Cys Arg Val Pro Glu Leu Gly Glu Pro Gly 180 185 190 Leu Thr Glu Cys Thr Tyr Gin Val Ser Asp Ala Asp Gly Arg lie Ser 195 200 205 Thr Glu Phe Met Thr Phe Arg lie Asp Ser Val Arg Gly Asp Glu Glu 210 215 220 Thr Phe Tyr lie Glu Arg Thr Asn lie Pro Asn Gin Trp Met Trp Leu 225 230 235 240 Asn Met Thr lie Gly Val Asn Thr Ser Leu Asn Phe Val Thr Ser Pro 245 250 255 Leu His lie Phe Ser Val Thr Ala Leu Asp Ser Leu Pro Asn Thr His 260 265 270 9 50 Thr Val Thr Met Met Val Gin Val Ala Asn Val Asn Ser Arg Pro Pro 275 280 285 Arg Trp Leu Glu lie Phe Ala Val Gin Gin Phe Glu Glu Lys Ser Tyr 290 295 300 Gin Asn Phe Thr Val Arg Ala lie Asp Gly Asp Thr Glu lie Asn Met 305 310 315 320 Pro lie Asn Tyr Arg Leu lie Thr Asn Glu Glu Asp Thr Phe Phe Ser 325 330 335 lie Glu Ala Leu Pro Gly Gly Lys Ser Gly Ala Val Phe Leu Val Ser 340 345 350 Pro lie Asp Arg Asp Thr Leu Gin Arg Glu Val Phe Pro Leu Thr lie 355 360 365 Val Ala Tyr Lys Tyr Asp Glu Glu Ala Phe Ser Thr Ser Thr Asn Val 370 375 380 Val lie lie Val Thr Asp lie Asn Asp Gin Arg Pro Glu Pro lie His 385 390 395 400 Lys Glu Tyr Arg Leu Ala lie Met Glu Glu Thr Pro Leu Thr Leu Asn 405 410 415 Phe Asp Lys Glu Phe Gly Phe His Asp Lys Asp Leu Gly Gin Asn Ala 420 425 430 Gin Tyr Thr Val A> g Leu Glu Ser Val Asp Pre- Pro Gly Ala AJa Glu 435 440 445 Ala Phe Tyr He A^ a Pro Glu Val Gly Tyr Gin Arg Gin Thr Phe lie 450 455 460 Met Gly Thr Leu Asn His Ser Met Leu Asp Tyi Glu Val Pro Glu Phe 465 470 475 480 Gin Ser lie Thr lie Arg Val Val Ala Thr Asp Asn Asn Asp Thr Arg 485 490 495 His Val Gly Val Ala Leu Val His lie Asp Leu lie Asn Trp Asn Asp 500 505 510 Glu Gin Pro lie Phe Glu His Ala Val Gin Thr Val Thr Phe Asp Glu 515 520 525 Thr Glu Gly Glu Gly Phe Phe Val Ala Lys Ala Val Ala His Asp Arg 530 535 540 Asp He Gly Asp Val Val Glu His Thr Leu Leu Gly Asn Ala Val Asn 545 550 55 i> 560 Phe Leu Thr He Asp Lys Leu Thr Gly Asp He Arg Val Ser A]a Asn 565 570 575 Asp Ser Phe Asn Tyr His Arg Glu Ser Glu Leu Phe Val Gin Val Arg 580 585 590 Ala Thr Asp Thr Leu Gly Glu Pro Phe His Thr Ala Thr Ser Gin Leu 595 600 605 Val lie Arg Leu Asn Asp lie Asn Asn Thr Pro Pro Thr Leu Arg Leu 610 615 620 Pro Arg Gly Ser Pro Gin Val Glu Glu Asn Val Pro Asp Gly His Val 625 63 0 635 640 lie Thr Gin Glu Leu Arg Ala Thr Asp Pro Asp Thr Thr Ala Asp Leu 645 650 655 Arg Phe Glu lie Asn Trp Asp Thr Ser Phe Ala Thr Lys Gin Gly Arg 660 665 670 Gin Ala Asn Pro Asp Glu Phe Arg Asn Cys Val Glu lie Glu Thr lie 675 680 685 Phe Pro Glu lie Asn Asn Arg Gly Leu Ala lie Gly Arg Val Val Ala 690 695 700 Arg Glu lie Arg His Asn Val Thr lie Asp Tyr Glu Glu Phe Glu Val 705 710 715 720 Leu Ser Leu Thr Val Arg Val Arg Asp Leu Asn Thr Val Tyr Gly Asp 10 501 725 730 735 Asp Tyr Asp Glu Ser Met Leu Thr lie Thr lie lie Asp Met Asn Asp 740 745 750 Asn Ala Pro Val Trp Val Glu Gly Thr Leu Glu Gin Asn Phe Arg Val 755 760 765 Arg Glu Met Ser Ala Gly Gly Leu Val Val Gly Ser Val Arg Ala Asp 770 775 780 Asp lie Asp Gly Pro Leu Tyr Asn Gin Val Arg Tyr Thr lie Phe Pro 785 790 795 800 Arg Glu Asp Thr Asp 805 Lys Asp Leu lie Met 810 lie Asp Phe Leu Thr 815 Gly Gin lie Ser Val 820 Asn Thr Ser Gly Ala 825 lie Asp Ala Asp Thr 830 Pro Pro Arg Phe His 835 Leu Tyr Tyr Thr Val 840 Val Ala Ser Asp Arg 845 Cys Ser Thr Glu Asp 850 Pro Ala Asp Cys Pro 855 Pro Asp Pro Thr Tyr 860 Trp Glu Thr Glu Gly Asn lie Thr lie His lie Thr Asp Thr Asn Asn Lys Val Pro Gin 865 870 875 880 Ala Glu Thr Thr Lys Phe Asp Thr Val Val Tyr He Tyr Glu Asn Ala 885 890 895 Thr His Leu Asp 900 Glu Val Val Thr Leu 905 lie Ala Ser Asp Leu Asp 910 Arg Asp Glu lie Tyr His Thr Val Ser Tyr Val lie Asn Tyr Ala Val Asn 915 920 925 Pro Arg 930 Leu Met Asn Phe Phe 935 Ser Val Asn Arg Glu 940 Thr Gly Leu Val Tyr Val Asp Tyr Glu Thr Gin Gly Ser Gly Glu Val Leu Asp Arg Asp 945 950 955 960 Gly Asp Glu Pro Thr 965 His Arg lie Phe Phe 970 Asn Leu lie Asp Asn 975 Phe Met Gly Glu Gly 980 Glu Gly Asn Arg Asn 985 Gin Asn Asp Thr Glu Val 990 Leu Val lie Leti Leu Asp Val Asn Asp Asn Ala Pro Glu Leu Pro Pro Pro 99f> 1000 1005 Ser Glu Leu Ser Trp Thr He Ser Glu Asn Leu Lys Gin Gly Val Arg 1010 1015 1020 Leu Glu Pro His lie Phe Ala Pro Asp Arg Asp Glu Pro Asp Thr Asp 1025 1030 1035 . 1040 Asn Ser Arg Val Gly Tyr Glu lie Leu Asn Leu Ser Thr Glu Arg Asp 1045 1050 1055 lie Glu Val Pro Glu Leu Phe Val Met lie Gin lie Ala Asn Val Thr 1060 1065 1070 Gly Glu Leu Glu Thr Ala Met Asp Leu Lys Gly Tyr Trp Gly Thr Tyr 1075 1 1080 1085 Ala lie His lie Arg Ala Phe Asp His Gly lie Pro Gin Met Ser Met 1090 1095 1100 Asn Glu Thr Tyr Glu Leu lie lie His Pro Phe Asn Tyr Tyr Ala Pro 1105 1110 1115 1120 Glu Phe Val Phe Pro Thr Asn Asp Ala Val lie Arg Leu Ala Arg Glu 1125 1130 1135 Arg Ala Val lie Asn Gly Val Leu Ala Thr Val Asn Gly Glu Phe Leu 1140 1145 1150 Glu Arg lie Ser Ala Thr Asp Pro Asp Gly Leu His Ala Gly Val Val 1155 1160 1165 Thr Phe Gin Val Val Gly Asp Glu Glu Ser Gin Arg Tyr Phe Gin Val 1170 1175 1180 11 501 Val Asn Asp Gly Glu Asn Leu Gly Ser Leu Arg Leu Leu Gin Ala Val 1185 1190 1195 1200 Pro Glu Glu lie Arg Glu Phe Arg lie Thr lie Arg Ala Thr Asp Gin 1205 1210 1215 Gly Thr Asp Pro Gly Pro Leu Ser Thr Asp Met Thr Phe Arg Val Val 1220 1225 1230 Phe Val Pro Thr Gin Gly Glu Pro Arg Phe Ala Ser Ser Glu His Ala 1235 1240 1245 Val Ala Phe lie Glu Lys Ser Ala Gly Met Glu Glu Ser His Gin Leu 1250 1255 1260 Pro Leu Ala Gin Asp lie Lys Asn His Leu Cys Glu Asp Asp Cys His 1265 L270 1275 1280 Ser lie Tyr Tyr Arg tie lie Asp Gly Asn Ser Glu Gly His Phe Gly 1285 1290 1295 Leu Asp Pro Val Arg Asn Arg Leu Phe Leu Lys Lys Glu Leu lie Arg 1300 1305 1310 Glu Gin Ser Ala Ser His Thr Leu Gin Val Ala Ala Ser Asn Ser Pro 1315 1320 1325 Asp "-ly Gly lie Pro Leu Pro Ala Ser lie Leu Thr Val Thr Val Thr :330 1335 1340 Val rrg Olu Ala Asp Pro Arg Pro Val Phe Val Arg Glu Leu Tyr Thr 1345 1350 1355 1360 Ala -ly tie Ser Thr ^la Asp Ser lie Gly Arg Glu Leu Leu Arg Leu 136'j- 1370 1375 His Ala Thr Gin Ser Glu Gly Ser Ala lie Thr Tyr Ala lie Asp Tyr 1380 1385 1390 Asp Thr Met Val Val Asp Pro Ser Leu Glu Ala Val Arg Gin Ser Ala 1395 1400 1405 Phe Val Leu Asn Ala Gin Thr Gly Val Leu Thr Leu Asn lie Gin Pro 1410 1415 1420 Thr Ala Thr Met His Gly Leu Phe Lys Phe Glu Val Thr Ala Thr Asp 1425 1430 1435 1440 Thr Ala Gly Ala Gin Asp Arg Thr Asp Val Thr Val Tyr Val Val Ser 1445 1450 1455 Ser Gin Asn Arg VaJ Tyr Phe Val Phe Val Asn Thr Leu Gin Gin Val 1460 1465 1470 Glu Asp Asn Arg Asp Phe lie Ala Asp Thr Phe Ser Ala Gly Phe Asn 1475 1480 1485 Met Thr Cys Asn lie Asp Gin Val Val Pro Ala Asn Asp Pro Val Thr 1490 1495 1500 Gly Val Ala Leu Glu His Ser Thr Gin Met Arg Gly His Phe lie Arg 1505 1510 1515 1520 Asp Asn Val Pro Va3 Leu Ala Asp Glu lie Glu Gin lie Arg Ser Asp 152 ri . 1530 1535 Leu Val Leu Leu Ser Ser lie Gin Thr Thr Leu Ala Ala Arg Ser Leu 1540 1545 1550 Val Leu Gin Asp Leu Leu Thr Asn Ser Ser Pro Asp Ser Ala Pro Asp 1555 1560 1565 Ser Ser Leu Thr Val Tyr Val Leu Ala Ser Leu Ser Ala Val Leu Gly 1570 1575 1580 Phe Met Cys Leu Val Leu Leu Leu Thr Phe lie lie Arg Thr Arg Ala 1585 1590 1595 1600 Leu Asn Arg Arg Leu Glu Ala Leu Ser Met Thr Lys Tyr Gly Ser Leu 1605 1610 1615 Asp Ser Gly Leu Asn Arg Ala Gly lie Ala Ala Pro Gly Thr Asn Lys 1620 1625 1630 His Thr Val Glu Gly Ser Asn Pro lie Phe Asn Glu Ala lie Lys Thr 12 i 1635 1640 1645 Pro Asp Leu Asp Ala lie Ser Glu Gly Ser Asn Asp Ser Asp Leu lie 1650 1655 1660 Gly He Glu Asp Leu Pro His Phe Gly Asn Val Phe Met Asp Pro Glu 1665 1670 1675 1680 Val Asn Glu Lys Ala Asn Gly Tyr Pro Glu Val Ala Asn His Asn Asn 1685 1690 1695 Asn Phe Ala Phe Asn Pro Thr Pro Phe Ser Pro Glu Phe Val Asn Gly 1700 1705 1710 Gin Phe Arg Lys lie 1715 <210> 3 «,211> 30 •. 212> PRT <213> M. sexta <400> 3 Met Leu Asp Tyr Glu Val Pro Glu Phe Gin Ser lie Thr lie Arg Val 15 10 15 ^al Ala Thr Asp Asn Asn Asp Thr Arg His Val Gly Val Ala 20 25 30 <■ 210> 4 <211> 16 <212> PRT <213> M. sexta <220> <221> VARIANT <222> (1)...(16) <223> Xaa = Any Amino Acid <400> 4 Met Xaa Glu Thr Tyr Glu Leu lie He His Pro Phe Asr. Tyr Tyr Ala 15 10 15 <210> 5 <211> 16 <212> PRT <213> M. sexta ■z220> <221> VARIANT <222> (1)..-(16) <223 > Xaa - Any Amino Acid <400> 5 Met Xaa Xaa Xaa His Gin Leu Pro Leu Ala Gin Asp lie Lys Asn His 15 10 15 <210> 6 <211> 8 <212> PRT <213> M. sexta <220> 13 <221> VARIANT <222> (1)..•(8) <223> Xaa = Any Amino Acid <4Q0> 6 Met Xaa Xaa Val Xaa Val Asp Xaa 1 S <210> 7 <211> 9 <212 > PRT <213> M. sexta <220> <221> VARIANT <222> (1).-.(9) <223> Xaa == Any Amino Acid <4Q0> 7 Met Asn Phe Xaa Ser Val Asn Xaa Glu X 5 <210> 8 <211> 109 <212> PRT <213> M. sexta <400> 8 Glu Trp Val Met Pro Pro lie Phe Val Pro 1 5 10 Phe Pro Gin Arg Leu Asn Gin Leu Lys Ser 20 25 Lys He Phe Tyr Tyr Ser lie Thr Gly Pro 35 40 Glu Gly Val Phe Thr He Glu Lys Glu Ser 50 55 Met Pro Leu Asp Arg Glu Lys He Val Lys 65 70 Ala Val Ser Glu Asn Gly Ala Ser Val Glu 85 90 lie lie Val Thr Asp Gin Asn Asp Asn Lys 100 105 <210> 9 <211> 105 <212> PRT <213> M. sexta <400> 9 Glu Asp Thr Val Tyr 1 5 Tyr Gin Val Gly Gin 20 Ala Gin Leu Ser Tyr 35 Ser Leu Asn Pro Gin 50 Ser Phe Asp lie Asp 10 lie Val Ala Arg Asp 25 Gly Val Val Ser Asp 40 Thr Gly Met Leu Thr / J Glu Asn Gly Lys Gly Pro 15 Asn Lys Asp Arg Gly Thr 30 Gly Ala Asp Ser Pro Pro 45 Gly Trp Leu Leu Leu His 60 Tyr Glu Leu Tyr Gly His 75 SO Glu Pro Met Asn He Ser 95 Pro Lys Phe Glu Asn Ala Gin Arg Gly 15 Ala Asp Leu Gly Gin Asn 30 Trp Ala Asn Asp Val Phe 45 Leu Thr Ala Arg Leu Asp 60 8 14 Tyr Glu Glu Val Gin His Tyr lie Leu lie Val Gin Ala Gin Asp Asn 65 70 75 80 Gly Gin Pro Ser Leu Ser Thr Thr lie Thr Val Tyr Cys Asn Val Leu 85 90 95 Asp Leu Asn Asp Asn Ala Pro lie Phe 100 105 <210> 10 <211> 92 <212> PRT <213> M. sexta <400> 10 Ala Ser Pro Val lie Thr Leu Ala 1 5 Leu Phe Pro lie Pro Leu Ala Ser 20 Val Ala Glu Asp Gin Glu Glu Lys 35 40 Asn Leu Asp Arg Glu Arg Trp Asp 50 55 Gin Asp Gly Gly Ser Pro Pro Arg 65 70 Thr Val Leu Asp Thr Asn Asp Asn 85 lie Pro Glu Asn Thr Asn Gly Ser 10 15 Asp Arg Asp Ala Asn Glu Leu Gin 25 30 Gin Pro Gin Leu lie Val Met Gly 45 Ser Tyr Asp Leu Thr lie Lys Val 60 Ala Thr Ser Ala Leu Leu Arg Val 75 80 Ala Pro Lys Phe 90 <210> 11 <211> 106 <212> PRT <213> M. sexta <400> 11 lie Val Thr Glu Asn lie Trp Lys Ala Pro Lys Pro Val Glu Met Val 15 10 15 Glu Asn Ser Thr Pio His Pro He Lys lie Thr Gin Val Arg Trp Asn 20 25 30 Asp Pro Gly Ala Gin Tyr Ser Leu Val Asp Lys Glu Lys Leu Pro Arg 35 40 45 Phe Pro Phe Ser lie Asp Gin Glu Gly Asp lie Tyr Val Thr Gin Pro 50 55 60 lie Asp Arg Glu Glu Lys Asp Ala Tyr Val Phe Tyr Ala Val Ala Lys 65 70 75 80 Asp Glu Tyr Gly Lys Pro Leu Ser Tyr Pro Leu Glu lie His Val Lys 85 .. 90 95 Val Lys Asp Asn Asp Asn Pro Pro Thr Cys 100 105 15 </div>