AU743234B2 - Receptor for a bacillus thuringiensis toxin - Google Patents

Description

RECEPTOR FOR A BACILLUS THURINGIENSIS TOXIN Technical Field The invention relates to receptors that bind toxins from Bacillus thuringiensis and thus to pesticides and pest resistance. More particularly, the invention concerns recombinantly produced receptors that bind BT toxin and to these use in assays for improved pesticides, as well as in mediation of cell and tissue destruction, dissociation, dispersion, cell-to-cell association, and changes in morphology.

Background Art It has long been recognized that the bacterium Bacillus thuringiensis (BT) produces bactericidal proteins that are toxic to a limited range of insects, 15 mostly in the orders Lepidoptera, Coleoptera and Diptera. Advantage has been taken of these toxins in controlling pests, mostly by applying bacteria to plants or transforming plants themselves so that they generate the toxins by virtue of their transgenic character. The toxins themselves are glycoprotein products of the cry gene as described by Hofte, H. et al. Microbiol Rev (1989) 53:242. It has been established that the toxins function in the brush border of the insect midgut epithelial cells as described by Gill, S.S. et al. Annu Rev Entomol (1992) 37:615. Specific binding of BT toxins to midgut brush border membrane vesicles has been reported by Hofmann, C. et al. Proc NatlAcad Sci USA (1988) 85:7844; Van Rie, J. et al. Eur JBiochem (1989) 186:239; and Van Rie, J. et al. Appl Environ Microbiol (1990) 56:1378.

Presumably, the toxins generated by BT exert their effects by some kind of interaction with receptors in the midgut. The purification of a particular receptor form Manduca sexta was reported by the present inventors in an article by Vadlamudi, R.K. et al. J Biol Chem (1993) 268:12334. In this report, the receptor protein was isolated by immunoprecipitating toxin-binding protein complexes with toxin-specific antisera and separating the complexes by SDS-PAGE followed by electroelution. However, to date, there has been no structural information concerning any insect receptor which binds BT toxin, nor have, to applicants' knowledge, any genes encoding these receptors been recovered.

Disclosure of the Invention The present invention is based, in part, on the isolation and characterization of a receptor that is bound by members of the BT-toxin family of insecticidal proteins, hereinafter the BT-R, protein. The present invention is further based on the isolation and characterization of a nucleic acid molecule that encodes the BT-toxin receptor, hereinafter BT-R, gene.

Based on these observations, the present invention provides compositions and methods for use in identifying agents that bind to the BT-R 1 protein as a means for identifying insecticidal agent and for identifying other members of the BT-R, family of proteins.

In one aspect, the present invention provides a method to identify an agent that binds to a BT-toxin receptor, said method comprising the steps of: i) contacting an agent with a BT-toxin receptor fragment selected from the group consisting of 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, 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:1 under high stringency and that binds to a BT-toxin, an isolated BT-toxin receptor fragment which binds to a BT-toxin and is encoded by the Bam-Sac nucleic acid fragment, an isolated BT-toxin receptor fragment which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the Bam-Sac nucleic acid fragment under high stringency, an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin having an amino acid sequence found within SEQ ID NO:2, and an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high stringency, and ii) determining whether said agent binds to said BT-toxin receptor fragment.

In another aspect, the present invention provides 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, in the presence and absence of a BT-toxin, with a BT-toxin binding receptor fragment selected from the group consisting of a cell that has been altered to contain a nucleic acid molecule that -2Aencodes a fragment of the amino acid sequence of SEQ ID NO:2 that binds to a BT-toxin, 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:1 under high stringency and that binds to a BTtoxin, an isolated BT-toxin receptor fragment which binds to a BT-toxin and is encoded by the Bam-Sac nucleic acid fragment, an isolated BTtoxin receptor fragment which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the Bam-Sac nucleic acid fragment under high stringency, an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin having an amino acid sequence found within SEQ ID NO:2, and (f) .i an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high stringency, and ii) determining whether said agent blocks the binding of said BTtoxin to said BT-toxin receptor fragment.

In another aspect, the present invention provides an isolated antibody, wherein said antibody selectively binds to a BT-toxin receptor fragment selected from the group consisting of a) a BT-toxin receptor fragment which binds a BT-toxin encoded by the Bam-Sac fragment, b) a BT-toxin receptor 20 protein fragment which binds a BT-toxin encoded by a nucleic acid molecule Sthat hybridizes to the Bam-Sac nucleic acid sequence under high stringency, c) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin having an amino acid sequence found within SEQ ID NO:2, and d) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high stringency.

In another aspect, the present invention provides an isolated BT-toxin receptor protein selected from the group consisting of a) a BT-toxin receptor fragment which binds a BT-toxin encoded by the Bam-Sac fragment, b) a BTtoxin receptor protein fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes to the Bam-Sac nucleic acid sequence under high stringency, c) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin having an amino acid sequence found within SEQ ID NO:2, and d) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high stringency.

2B In another aspect, the present invention provides a method to produce BT-toxin receptor protein fragment, 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 fragment, wherein said cell has been altered to contain a nucleic acid molecule selected from the group consisting of a) the Bam-Sac nucleic acid, b) a nucleic acid molecule which encodes a BT-toxin receptor fragment which binds a BT-toxin having an amino acid sequence found within SEQ ID NO:2, c) a nucleic acid molecule encoding a BT-toxin receptor fragment which binds a BT-toxin receptor that hybridizes to the Bam-Sac nucleic acid under high stringency, and d) a I.ii nucleic acid molecule which encodes a BT-toxin receptor fragment which binds a BT-toxin that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high stringency, under conditions in which said nucleic acid molecule is expressed and 15 ii) isolating said BT-toxin receptor protein fragment.

Any discussion of documents, acts, materials, devices, articles or the :like which has been included in the present specification is solely for the *purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present S" invention as it existed, particularly in Australia, before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Brief Description of the Drawings Figure 1 (SEQ ID NOS:1 and 2) show the nucleotide sequence and deduced amino acid sequence of cDNA encoding the BT-R 1 protein from M.

sexta.

Figure 2 (panels a and b) (SEQ ID NO:2) shows a comparison of amino acid sequences of cadherin motifs (BTRcad-1 to 11) in BT-R 1 to those of other cadherins.

2C- Figure 3 (SEQ ID NOS:3 to 17) shows a block diagram of the cadherinlike structure of BT-R 1 Figure 4 shows the clone characterization of the BamHI-SacI fragment of BT-R 1 LM is HindIII cut Lambda marker; UP is the uncut plasmid clone; NP is NsiI cut plasmid; XP is XhoI cut plasmid; BSP is BamHI and SacI cut plasmid showing e *a WO 98/59048 PCT/US98/11868 -3the 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 35 S-methionine as a tag. LCR is a 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-Ri produced in a transcription/translation kit.

Figure 6 shows a radio-blot of the Bar-Sac fragment of BT-R, with 1 25 I-labeled CrylAb. BBMV is the brush border membrane vesicles from the midgut ofM. 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 transcription/translation kit blank; TT 1 and TT2 are expression products of the Bam- Sac fragment of BT-R, produced in 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 original BT-R, clone.

Figure 8 shows the binding of CrylAb to fragments of the BT-R, protein.

Modes of Carrying Out the Invention I. General Description The present invention is based, in part, on the isolation and characterization of a novel protein expressed in the midgut ofManduca sexta that binds to members of the BT-toxin family of proteins, hereinafter the BT-R, protein. The present invention specifically provides purified BT-R,, the amino acid sequence ofBT-R,, as well as nucleotide sequences that encode BT-R,. The BT-R, protein and nucleic acid molecules can serve as targets in identifying insecticidal agents.

dc-118781 WO 98/59048 PCT/US98/11868 -4- II. Specific Embodiments A. BT-R, Protein Prior to the present invention, although members of the BT-toxin family of 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 in the midgut ofMaduca sexta.

In one embodiment, the present invention provides the ability to isolate or produce a previously unknown protein by using known purification methods, the cloned nucleic acid molecules herein described 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.

BT-R, is comprised 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 in Figure 3.

The BT-R, protein of the present invention includes the specifically identified and characterized variant herein described, as well as allelic variants, conservative substitution variants and homologues (Figure 7) that can be isolated/generated and characterized without undue experimentation 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 includes all naturally occurring allelic variants of the Manduca sexta BT-R, protein provided in Figure 1. In general, naturally occurring allelic variants ofManduca sexta BT-R) will share significant homology, at least and generally at least 90%, to the BT-R, amino acid sequence provided in Seq. ID No:2. Allelic variants, though possessing a slightly different amino acid sequence than Seq. ID No:2, will be expressed as a transmembrane protein in the digestive tract of an insect or other organism. Typically, allelic variants of the BT-R, protein will 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).

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 in SEQ ID NO:1, but may further contain a radical departure from the sequence, such as a nonconservative 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 biological functions as does the BT-R, variant of SEQ ID NO:2.

S* 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 1 5 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 s ee. discussed below, fragments of BT-R, are particularly useful in: generating domain 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 synthesis technology and the amino 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 R6 immunogenicity plot, Chou-Fasman plot, Gamier-Robson plot, Kyte-Doolittle plot, dc-118781 q2 1 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 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,, 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 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.

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 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-toxin binding domain of BT-R, identified in Example WO 98/59048 PCT/US98/11868 -7- Methods for preparing a protein for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation with reagents such as carbodiimide may be used; in other instances linking reagents like those supplied by Pierce Chemical Co., Rockford, IL, may be effective.

Administration of a BT-R 1 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.

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 immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigenis the BT-R, protein or BT-R 1 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 supernatant or from the ascites supemrnatant. 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 in a therapeutic context, as these fragments are generally less immunogenic than the 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.

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 BT-R, protein and in identifying competitive inhibitors of BT-toxin/BT-R, interactions.

C. BT-R, Encoding Nucleic Acid Molecules As described above, the present invention is based, in part, on isolating nucleic 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 in isolated form. For convenience, all BT-R, encoding nucleic acid molecules will be referred to as BT-R, encoding nucleic acid molecules, the BT-RI genes, or BT-R1. The nucleotide sequence of the Manduca sexta nucleic 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 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 derived from natural sources or synthesized. A skilled artisan can readily obtain these classes of nucleic acid molecules using the herein described BT-R 1 sequences. However, such nucleic acid molecules, are defined further as being novel and unobvious over any prior art nucleic acid molecules encoding non-BT-R, proteins. For example, the BT-R1 sequences of the present invention specifically excludes previously identified nucleic acid molecules that share only partial homology to BT-R]. Such excluded sequences include identified members of the cadhedrin family of proteins.

WO 98/59048 PCT/US98/ 1868 -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 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-R1 sequence.

The size of the fragment will be determined by its intended use. For example, if the 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-Rj gene that are particularly useful as selective hybridization probes or PCR primers can be readily identified from the entire BT-Rj 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 1 gene. Specifically, tissue and developmental specific expression control elements can be identified as being 5' to the BT-R, encoding region found in genomic clones of the BT-Rj gene. Such expression control sequence are useful in generating expression vectors for expressing genes in the digestive tract of a transgenic organism. As described in more detail below, a skilled artisan can readily use the BT-R 1 cDNA sequence herein described to isolate and identify genomic BT-Rj sequences and the expression control elements found in the

BT-R

1 gene.

Fragments of the BT-R, encoding nucleic acid molecules of the present invention synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding BT-R, proteins, can easily be synthesized by chemical techniques, for example, the WO 98/59048 PCT/US98/11868 phosphotriester method of Matteucci, et al., JAm 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-Rj gene, followed by ligation of oligonucleotides to build the complete modified BT-Rj 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 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, biotin, and the like. A skilled artisan can employ any of the art-known 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 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 (described above), and 3) the corresponding genomic DNA that contains the BT-R 1 gene. The most preferred source of BT-R, homologues are insects, the most preferred being members of the Lepidopteran, 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 libraries prepared from cells and organisms. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described above) or 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 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 described, and genomic sequence containing the BT-Rj gene. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.

Additionally, pairs of oligonucleotide primers can be prepared for use in a 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-R

1 gene that are particularly well suited for use as a probe or as primers can be readily identified by one skilled in the art.

Non-Manduca sexta homologues of BT-R1, naturally occurring allelic variants of the Manduca sexta BT-Rj gene and genomic BT-Rj sequences will share a high degree of homology to the Manduca sexta BT-R1 sequence herein described. In general, such nucleic acid molecules will hybridize to the Manduca sexta BT-Rj sequence under high stringency. Such sequences will typically contain at least homology, preferably at least 80%, most preferably at least 90% homology to the Manduca sexta BT-R 1 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-Rj sequence under stringent conditions. "Stringent conditions" are those that employ low ionic WO 98/59048 PCT/US98/11868 -12strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50 0 or 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% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaC1, 75 mM sodium citrate at 42 0 C. Another example is use of 50% formamide, 5 x SSC (0.75M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42 0 with washes at 42 0 C. in 0.2 x SSC and 0.1% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately 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, 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 in this superfamily are desmoglien, desmocollins, the Drosophila fat tumor suppressor, Manduca sexta intestinal peptide transport protein and T-cadherin.

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. 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(1993) 12:2249. Inclusion of BT-R, in the cadherin 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, A.C. et al.

Biochm Biophys Res Commun (1994) 201:782).

It is noted below that the amino acid sequence of BT-R, reveals that a calciumbinding motif is present. This is consistent with the possibility that cells having receptors to bind toxin may themselves survive although they render the tissues in WO 98/59048 PCT/US98/11868 -13which 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 in 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 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) 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 molecules of the present invention, a BT-R, encoding DNA sequence that encodes a BT-R, protein or a fragment of BT-R 1 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 the functional properties desired, 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 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 secretion signals to direct the secretion of the BT-R, protein, or fragment, out of the cell.

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, a DNA sequence having 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 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 ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) of the BT-RI 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 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 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- 1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the vector 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, the neomycin phosphotransferase (neo) gene. Southern et al., JMolAnal Genet (1982) WO 98/59048 PCT/US98/11868 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.

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.

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-Rj 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.

Any prokaryotic host can be used to express a BT-R,-encoding rDNA molecule.

The preferred prokaryotic host is E. coli.

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 host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al., Proc Acad Sci 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 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, 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 produce single colonies. Cells from those colonies can be harvested, lysed and their WO 98/59048 PCT/US98/11868 -16- DNA content examined for the presence of the rDNA using a method such as that described by Southern, JMol Biol (1975) 98:503, or Berent et al., Biotech (1985) 3:208 or the proteins produced from the cell assayed via an immunological method.

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, encoding nucleic acid molecules herein described. In general terms, the production of a recombinant BT-R, protein typically involves the following steps.

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 in an operable linkage with suitable control sequences, as described above, to generate an expression unit containing the BT-R, encoding sequence. The expression unit is used to transform a suitable host 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 variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in an appropriate host. The construction of expression vectors that are operable in a variety of hosts is accomplished using an appropriate combination ofreplicons 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 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.

WO 98/59048 PCT/US98/11868 -17- H. Identification of Agents and Cellular Constituents that Bind to a

BT-R

1 Protein Another embodiment of the present invention provides methods for identifying agents and cellular constituents that bind to BT-R,. Specifically, agents and cellular constituents that bind to BT-R 1 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 contains the BT-toxin binding domain, such as that disclosed in Example 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 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 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 segment of DNA encodes a protein that binds to the BT-R, protein, the expression results in the interaction of the BT-R, and the encoded protein. This brings 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 encoding segments for cellular binding partners of BT-R,.

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 containing 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, 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, 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 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 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 in place of the BT-toxin.

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 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 increases BT-R, activity. The preferred agonist will selectively agonize BT-R), not WO 98/59048 PCT/US98/11868 -19affecting 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 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.

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 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 the agents used in the present screening method. One class of agents of the present invention are peptide agents whose amino 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) 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.

IYO 98/59048 PCT/US98/1 1868 Another class of agents of the present invention are antibodies immunoreactive with critical positions of the BT-R, protein. As described above, antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the BT-R 1 protein intended to be targeted by the 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 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, expressing cells.

An outline of one screening method is as follows. Cells are modified by transfection, retroviral infection, electroporation or other known means, to express a

BT-R

1 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.

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 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 trypan blue), increased 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.

WO 98/59048 PCT/US98/ 1868 -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 improved forms of toxins which are more specific or less specific to particular classes of insects as desired. The ability to determine binding affinity (Ka and Kd), 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 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, monitoring antibody titers and recovering the antisera or the antibody-producing cells for immortalization, to obtain immortalized cells capable of secreting antibodies of the appropriate specificity. Techniques for obtaining immortalized B cells and for screening them for secretion of the desired antibody are now conventional in the art.

The resulting monoclonal antibodies may be more effective than the polyclonal 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 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 antiidiotypic antibodies which mimic a portion of a toxin that binds to a receptor, and whatever small molecules bind the receptors.

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 insecticidal activity of BT-toxins. 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 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 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,.

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-R1 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 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-toxin-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.

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.

Wo 98/59048 PCTIUS98/11868 -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 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 ofa 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 Am JHum 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 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 mobility in an electric 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-R1 gene. For example, 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, protein/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.

WO 98/59048 PCTUS98/11868 -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 BT-R, receptor, become sensitive to the mode of action ofBT-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 in which it is desirable to induce cell death.

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 in 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 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 in the treatment of autoimmune disorders and in the treatment of cells harboring pathogens, such as 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 artknown methods for use with the BT-R,-encoding DNA molecule of the present invention.

L. Animal Models and Gene Therapy The BT-R1 gene and the BT-R, protein can also serve as a target for generating transgenic organisms in which the pattern of BT-R, expression has been altered. For WO 98/59048 PCT/US98/11868 example, in one application, BT-R, deficient insects or insect cells can be generated using standard knock-out procedures to inactivate a BT-Rj gene, or, if such animals are non-viable, inducible BT-R 1 antisense molecules can be used to regulate BT-Rj activity/expression. Alternatively, cells or an organism can be altered so as to contain 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 in which the expression of a BT-R 1 gene is altered by inactivation or activation and/or replaced by a Manduca sexta BT-Rj gene. This can be accomplished using a variety of art-known procedures such as targeted recombination. Once generated, the BT-Rj 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 deficiency and 4) serve as an appropriate screen for identifying mutations within the BT-Rj 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 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 of the of the newly identified BT-Rj gene in a form that can be used in generating expression vectors. Specifically, the BT-R1 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 introduced transgene in a tissue specific fashion. A skilled artisan can readily use the 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 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.

1 0 Example 1 Purification and Sequence Determination of BT-R, Protein Midguts ofM. sexta were extracted and the BT-R, protein purified according to the method ofVadlamudi, R.K. et al. JBiol Chem (1993) 268:1233, referenced above and incorporated herein by reference. The electroeluted band was confirmed to 1.5 contain BT-R, protein by binding to 25 I-crylAb toxin. In gel electrophoresis, the protein bound to toxin had an apparent weight of approximately 210 kD under reducing and nonreducing conditions.

The purified electroeluted BT-R, was subjected to cyanogen bromide digestion and the cyanogen bromide fragments separated on a 17% high-resolution tricine SDSpolyacrylamide gel as described 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 microsequencing using standard instrumentation. The amino acid sequences obtained were (SEQ ID NOS:18-22): 1. (Met)-Leu-Asp-Tyr-Glu-Val-Pro-Glu-Phe-Gln-Ser-Ile-Thr-Ile-Arg- Val-Val-Ala-Thr-Asp-Asn-Asn-Asp-Thr-Arg-His-Val-Gly-Val-Ala; 2. (Met)-X-Glu-Thr-Tyr-Glu-Leu-Ile-le-IHis-Pro-Phe-Asn-Tyr-Tyr-Ala; 3. (Met)-X-X-X-His-Gln-Leu-Pro-Leu-Ala-Gln-Asp-Ile-Lys-Asn-His; 4. (Met)-Phe/Pro-Asn/Ile-Val-Arg/Tyr-Val-Asp-Ile/Gly; 5. (Met)-Asn-Phe-Phe/His-Ser-Val-Asn-Arg/Asp-Glu.

K dc-1 18781I WO 98/59048 PCT/US98/11868 -27- Example 2 Recovery of cDNA An M. sexta cDNA library was constructed from midgut tissue in Xgtl0 using the Superscript Choice System according to the manufacturer's instructions (Life 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 at 2 P using polynucleotide kinase and used as probes as described in the standard cloning manual ofManiatis, 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 hybridizing to all three of the probes was plaque-purified from a screen of 4 X 105 recombinants and subcloned into pBluescript (Stratagene). It contained an insert of 5571 bp.

Double-stranded cDNA in pBluescript 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 amino acids along with a polyadenylation signal at position 5561. The sequence obtained and the deduced amino acid sequence is shown in Figure 1.

Thus, the deduced protein has a molecular mass of 172 kD and a pi of approximately 4.5. The amino acid sequences of the cyanogen bromide fragments of native receptor match perfectly within the deduced amino acid sequence. The open reading frame begins with an ATG that is flanked by the consensus translation initiation sequence GAGATGG for eucaryotic mRNAs as described by Kozak, M.

Nucleic Acids Res (1987) 15:8125.

As shown in Figure 1, the deduced amino acid sequence includes a putative signal, shown underlined, preceding the mature N-terminus Asn-Glu-Arg-etc. Eleven repeats (cadl-cadl 1) are shown in the extracellular region upstream of the membrane domain, shown with the heavy underline, at positions 1406-1427. The end of the 11th -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 cadherin family. Like known cadherins, the external domain of BT-R, is highly repetitive and contains 11 repeats (cadl-cadl 1; see Figure 2 The other cadherins compared in Figure 2 B are mouse P cadherin (mP EC1); Drosophilafat EC18 (fat EC 18) and protocadherin (PC42 EC2), and Manduca sexta intestinal transporter (HPT- EC-1). The eleven repeats of the cadherin motif in BT-R, (cadi-cadl 1) are individually aligned with a single motif sequence from each of the other members of the cadherin family. Conserved residues are boxed. The greatest similarity of BT-R, to the cadherins S* is with the extracellular repeats of the cadherin motif of mouse P-cadherin, Drosophila fat tumor suppressor and the protocadherins, although homologies are not high (20-40 homology and 30-60 percent similarity). The conserved repeats of BT-R, included AXDXD (SEQ ID NO:23), DXE, DXNDXXP (SEQ ID NO:24), one glutamic acid residue and two glycine residues (Figure 2 Motifs A/VXDXD (SEQ ID DXNDN (SEQ ID NO:26) are the consensus sequences for calcium binding and two such regions are present in a typical cadherin repeat. In all repeats of BT-R,, the sequence DXNDN is preceded by 8 to 14 hydrophobic amino acids. Similar hydrophobic sequences also have been observed in the cadherins. The length of the hydrophobic stretches suggests that these areas are not transmembrane regions buy that *00 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 cadherin cytoplasmic domains (160 amino acids), and shows no homology to any of the cadherin cytoplasmic domains or to cytoplasmic domains of other proteins to which it has been compared in a current sequence data 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 hybridizing to the antisense probe at 42 0 C and SR 50% formamide, 5 X Denhardt's Reagent, 5 X SSCP and 50 tg/ml salmon sperm dc-1 18781 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 0 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.

The BT-R, clone was translated using rabbit reticulolysate and the resulting translated products were immunoprecipitated with antisera raised against native protein encoded by BT-R,. For the in vitro translation, pBluescript plasmid containing BT-R, cDNA was linearized and transcribed with T 3 polymerase (Pharmacia). The translation was conducted according to manufacturer's instructions with nuclease-treated rabbit reticulolysate (Life Technologies, Inc.). After one hour of incubation at 30 0 C, the reaction mixture was combined with an equal volume of SDS buffer or lysed with 50 mM Tris 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% SDSpolyacrylamide gel fixed, treated with Enhance (Dupont NEN), dried 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 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 Characteristics of the BT-R, Protein 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 solubilized, electrophoresed and ligand blotted with 2 I-CrylAb toxin. The cells were harvested 60 hours after transfection, washed with phosphate-buffered saline and lysed by freezing in liquid nitrogen. Cell membranes were prepared by differential centrifugation as described by Elshourbagy, NO~ 98/59048 PCT/US98/11868 N.A. et al. JBiol Chem (1993) 266:3873. Control cells were COS-7 cells transfected with pcDNA3.

The cell membranes (10 jg) were separated on 7.5% SDS-PAGE blotted to a nylon membrane and blocked with Tris-buffered saline containing 5% nonfat dry milk powder, 5% glycerol and 1% Tween-20. The nylon membrane was then incubated with 25 I-CrylAb toxin (2 X 105 cpm/ml) for two hours with blocking buffer, dried and exposed to X-ray film at -70 0 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 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 digestion with N-glycosidase-F by first denaturing the purified protein by boiling in 1% SDS for 5 minutes followed by addition of NP-40 to a final concentration of 1% in the presence of 0.1% SDS, and then incubating the denatured protein in sodium phosphate buffer, pH 8.5 at 37 0 C with N-glycosidase-F for 10 hours. Controls were incubated under the same conditions without enzyme. Digestion products were separated on a 7.5% SDS-PAGE and stained with Coomassie brilliant blue. This glycosidase treatment reduced the molecular weight of BT-R, protein from 210 to 190 kD; this indicates N-glycosylation at some of the 16 consensus N-glycosylation sites in the protein. Treatment of BT-R, with O-glycosidase and neuraminidase did not alter the mobility of the protein.

In addition, embryonic 293 cells were transfected with the BT-R, cDNA clone in pcDNA3 and incubated with the labeled toxin (0.32 nM) in the presence of increasing concentrations (0 to 10 6 M) of unlabeled toxin. Nonspecific binding was measured as bound radioactivity in the presence of 1 TM unlabeled toxin. A value for the dissociation constant (Kd) of 1015 pM was determined by Scatchard analysis; this WO 98/59048 PCT/US98/11868 -31 is approximately the same value that was obtained for the natural receptor as described by Vadlamudi, R.K. et al. JBiol Chem (1993) (supra).

Example 4 Physiological Effect of BT Toxin on Modified Embryonic 293 Cells Both unmodified embryonic 293 cells, and 293 cells which have been modified to produce the BT-R, receptor as described in Example 3, when cultured in vitro form adherent star-shaped clusters. When BT toxin (200 nM) is added to serumfree medium, the clusters round up and release from the plastic surfaces of the culture 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, in the presence of anti-receptor antisera, this effect of BT toxin is blocked. Also, when serum is added back to a culture of modified E293 cells which has been treated in 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.

Example Identification Of A Fragment Of BT-R, That Binds To A BT Toxin To understand some of the properties of BT-R,, research has been undertaken to define the location of the BT-R,/CrylAb protein-protein interaction. The fulllength wild-type amino acid sequence of BT-R, is provided in Fig. 1 with a block diagram of a possible cadherin-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 amino acid coding sequence.

A small fragment lying between the BamHI and SacI restriction sites of wildtype BT-R, was cloned into the vector pCITE (Novagen). This vector contains transcription/translation sequences designed for use in a rabbit reticulocyte lysate WO 98/59048 PCT/US98/11868 -32- (RRL) system. The clone has been analyzed by restriction mapping and mRNA expression (Fig. Lane UP shows the uncut plasmid and lanes NP and XP show restriction digests using NsiI and XhoI, respectively. NsiI is used because it has only one restriction site lying within the Bar-Sac fragment and does not cut anywhere within the pCITE vector. The BSP lane shows the restriction digest of the clone using BamHI and SacI. 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 XhoI. The mRNA separates at the expected 1350 base pairs.

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 transcription reaction described above. Second, the plasmid was added directly to an RRL based transcription and translation (TNT) coupled kit. Protein production was detected using 3 5 S-methionine as a tag (Fig. The LCR lane shows production of luciferase 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 of BT-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.

The clone was then tested for its ability to bind the insecticidal toxin CrylAb.

Polypeptide translation of the Bam-Sac fragment of BT-R, was carried out in duplicate as described above. The only change is that the "S-methionine 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 2 I-labeled CrylAb (Fig. BBMV is wild-type BT-R, prepared from the midgut brush border WO 98/59048 PCT/US98/11868 -33membrane vesicles (BBMV) ofM. 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 CrylAb. R, and RR2 are translations from the RRL kit and TTI and TT2 are from the TNT kit. A single kDa band appears in each of these lanes. Two are marked by arrows. These bands demonstrate that the Bam-Sac fragment of BT-R, is capable of binding CrylAb 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 restriction digests. The cDNA was digested at specific sites to remove increasingly larger portions of the C-terminus.

The restriction enzymes used were NsiI, BamHI, NruI, Clal, XhoI and StuI (Figs. 1 and The procedure involved linearizing 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 hybridized with '"I-labeled CrylAb.

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 CrylAb 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 CrylAb to the reticulocytes. The specificity of this binding has not been determined. The truncation mutants created by NsiI, BamHI, NruI, Clal, XhoI and StuI restriction digests did not show any binding to CrylAb except in the region where the reticulocytes bind CrylAb. This data demonstrates that the removal of the last 100 amino acids from wild type BT-R, by NsiI restriction results in the loss of the ability of BT-R, to bind CrylAb. This 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. 1) and cloning of the resulting fragment into a vector called pCITE. The polypeptide sequence was translated and tested for binding to the insecticidal toxin CrylAb WO 98/59048 PCT/US98/11868 -34- (Figure 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 in 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 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 OfHomologue of BT-R, That Binds To A BT Toxin Western blots of tissue extracts prepared from Pink bollworm and European corn borer were prepare and probed with labeled Cryl a (Figure The results show that homologues of BT-R, are present in these two insects and can be readily isolated using the methods described herein.

SEQUENCE LISTING GENERAL INFOR-MATION: APPLICANT: UNIVERSITY OF WYOMING (ii) TITLE OF INVENTION: RECEPTOR FOR A BACILLUS THURINGIENSIS

TOXIN

(ii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: MORRISON FOERSTER LLP STREET: 2000 Pennsylvania Ave. N.W.

CITY: Washington STATE: DC COUNTRY: USA ZIP: 20006-1812 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US98/11868 FILING DATE: 08-JUN-1998

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/982,129 *So FILING DATE: 01-DEC-1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/880,042 FILING DATE: 20-JUN-1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PCT/US95/13256 FILING DATE: 10-OCT-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/326,117 FILING DATE: 19-OCT-1994

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: LIVNAT, SHMUEL REGISTRATION NUMBER: 33,949 REFERENCE/DOCKET NUMBER: 271122003750 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (202) 887-1500 TELEFAX: (202) 887-0763 TELEX: 90-4030 I- POR ,I-7TON SEQ ID NO1:: SEQUENCE CHARCTE R T 7r LENGTH: 5577 base cairs TYPE: nucle-ic acidi STRANDEDNESS: -Jouble CD) TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (ix) FEATURE: NAaME/KEY: CDS LOCATION: 197. .4780 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GACAATCGG AGTGTGGTGA ATTTTTGGAA AATATTTTGT GCSGTTCCTT TAGTTC-TGT1A ATATACTACT TTAGTTACAA ATT'Tc-GAATA ATTTGGCAGC AAAACCATCT GCAGCAACAA AATCATCTGC AGCTGCGAAA TCATCTGCAG CAGCAAAAGC ATCTTCAGGA GCGAC-AXAAG so **CCCCAAATAA TGTOAG ATG GCA GTT GAC GC CGA ATC GCT GCC TTC CTG 120 180 229

S.

0 0 00 S 0

S.

00 0

S.

@5 00 a S

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OS

@0

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CTG

Leu

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Myet

AAT

Asn

GAG

Giu 60 C7C Leu

ACC

Arg GrnG TTT Val Phie ACC GC C Thr Al a TTT GAA Phe Glu 45 OGG GAT Arq Aso GGC ACG Gly Thr ATC GCC 71 e Ala CCA TTT Pro P, e 110 GTT GAC Val A sp 12-5

ATA

Ile 1z

ATO

GGO

Gly

GAC

Asp

CAG

Gln

ATA

le T7A L e 11AC GCG CCT GCA GTT TTA GCT CAA GAG AGA TGT GCCG TAT Al a

CCA

Pro

CAG

Gin

CTG

Leui

GTC

ValI 80

OTT

Le u

TOO

Ser

GGG

G iv Pro Ala AGG OTA Arg Leui ACA TGG Thr Tro s0 TGC ATG Cys Met 65 ATO TAO Ile Tyr AAT TAT Asn Tyr GGT TOO Gi1v Ser ACT GOA S e A ~-a ValI

OCA

Pro 35

AGCT

Ser

GAC

Asp

ATG

Met

AAO

As n

TAC

TOT

Ser Leu 20 OAg

CAG

Glin

GC

Ala

GAT

Aso

GGA

GlIy 100

AAT

OAT.s

COG

Pro Ar g

TAO

Tyr G A G 1u 3 5

OCA

Pro LC 7 1 Gin

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Aso

COO,

CAC

H i s 70

GAG

Giu

TCA

Ser

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L e C-A C Glu

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GC

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Glu AT T ile

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T yr

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Ty r 469 13 0 T TG COO'- GCC ATO- CAG CAC TAO' ATG T T A OOCOGT A G7G CGC GTG GAC GGC

I.

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GC

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GA

Gly

GC

Giy 220

TG

T rp

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Val1

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AGC

Ser

GAG

Giu 300

GAG

Giu

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Thr

TTC

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CCA

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GC(

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CGG

Arg 205

GAC

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ATG

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AAC

As n

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AA

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LeuL 365S Pro Pro 190

ATC

Ile

GAO

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Ser

ACC

T h r 270

CCC

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Ser

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As n

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175

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Oiu

CTA

Leu

CCG

Pro 255

CAC

FHi s

CCC

Pro

TAC

T yr

ATC

Met

AOC

Ser 33 5 T CG Ser

ATC

Ile

GCA

Al a 16 0 A TA 7 e

TTG

Leua

ACA

Thr

ACC

Thr

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As n 240

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Leu

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As n 305

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Giu kla

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TAC

T yr

ACC

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ACT

Thr

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TC

C ys 195

ATO

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ATC

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GAG

Ciu

ACA

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Leua C GC Ara 355

A

Lys

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Clii

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Thr

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Thr

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Glu

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Ser 260 AT C Mel: A T C Ile

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Arg

CCT

Pro 340

CAC

As o Tyr Aia 165

CT

Pro

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Val1 245

G

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Leu 3 25

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Gly AClA Thb.r GA2O As o

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PAT

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GCC

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OTT

Val1

TCG

Ser 200

CAC

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CTC

Leu

CC

Al a 280 CPaA Gin

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PAT

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Ser

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GA

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As n

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As n 250

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OCT

Al a

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CTC

LeuL

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Arg

CPA

Gin 235

TTC

Phe

CTC

Leua

PAC

As n

CPA

Gilu

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Thr 315

CAC

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TT

Phe

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T hr 709 757 805 853 901 949 997 1045 1093 1141 1199 1237 1285 1333 1381 7CA ACa AC GTG GOC ATC AT7 COG ACA GAC ATO AC,- GAO CP A AGA COO Ser Thr A'sni Val Val Ie- ile Val Ohr Aso Tle Asn Aso Gi-n Arq Pro 37 380330 395 GA cc: aTa CAC *A-G G~AA C: GCA ATC AG cAG GAG ACG C CC 142 9 c-lu Pro !Ie- His Lys Glu Tyr Arg Leu Ala lie Met c-lu c-lu 71-1r Pro 400 405 4 CTG ACC CTC AAC TTC GAT AA G-kA TTC c-GA TTT CAT C AT At" C AT TTA 1477 Leu Thr Leu Asn Phe Asp Lys c-lu Pbhe Gly Phe H is Asp Lys Aso Leu 415 420 42-5 GGT CXAA. AC GCT CAG TAC ACO GTG Cc-T CTA G-AG AGC GTG c-AC CCT CCA 1525 Gly c-in Asn Ala c-in Tyr Thr Val Arg Leu Giu Ser Val Asp Pro Pro 430 435 440 c--C c-CT c-CT GAG c-CA TTC TAC ATA GCG CCT GAA GTC GGC TAC CAG Cc-A 1573 Gly Ala Ala c-lu Ala Phe Tyr le Ala Pro c-lu Val C-ly Tyr G In Arc- 445 450 455 CAG ACC TTC ATC ATG Cc-C ACC CTC AAT CAC TCC ATG C TG GAT TAC GAA 1621 Gln Thr Phe Ile Met Gly Thr Leu Asn HiJs Ser Met Leu Asp Tyr c-lu 460 465 470 475 GTG CCA c-Ac- TTT CAG AGT ATT ACG ATT CGc- GTG c-TA c-Cc- ACC c-AC AAC 1669 V9600 Val Pro c-lu Phe Giln Ser ile Thr Ile Arc- Val Val Ala Thr Asp Asn 0 0 480 485 490 oAAC c-AC ACG Ac-c CAC GTG Gc-c GTC ccc TTG GTT CAPC ATT c-AC CTC ATC 1717 *.*Asn Asp Thr Arc- His Val Gly Val Ala Leu Val His Ile Asp Leu le 00e 495 500 AA T TGG AAC c-AT GAG CAG CCc- ATC TTC GA A CAC c-CC GTG CAG ACC GTC 1765 Asn Trp Asn Aso c-lu c-in Pro Ile Phe c-lu His Ala Val Giln Th r Val I-510 515 520 ACC TTC c-AC c-Ac- ACT GAA c-cC GAG c-c-c T TC TT C GTC c-CC AA- c-Cc- GTT 1813 Thr Ph-e Asp c-lu Thr c-lu Gly c-lu Gly P he Phe Val Ala Lys AlIa Val 525 50535 0*0' c-CA CAC c-AC Ac-A c-AC ATC c-c-c c-AT GTC GTC c-Ac- CAT ACT TTA TTG c-CT 1861 dOSS. AlIa His A sp Arc- Asp Ile Cly Asp Val Val c-lu HiJs Thr Leu Leu Gly 009 540 545 550 555 c-CT GTT AAC TTC CTG ACC ATC c-AC AAA CTC ACC Gc-c c-AC ATC Cc-C 1909 Asn Ala Val Asn Phe Leu Thr TIe Asp Lys Leu Thr Gly Asp Ile Arc- 560 565 570 CTC TCA c-CT AAC c-AC TCC TTC JAC TAC CAT Cc-A GA.A ACT CA.A TTA TTT 1957 ***Val Ser Ala Asn Asp Ser Phe Asn Tyr His Arc- c-lu Ser c-lu Leu Phe 575 580 585 590 5o 600 A C G TCA CAG CTc- CTC ATA Cc-A CTA AAT c-AC ATC AAC A.AC ACG CCA ccc 2053 Thr Ser c-I n Leu Val TIe Arg Leu Asn Aso 7ie Asn Asn Thr Pro Pro 605 610 615 ACC T TA CCC CTG CCT Cc-A c-cC AGT CCC C AA c-TC GAG c-AG AAZC CTC CCT 2101 T~n r eu Ar; Leu Pro Ara Clv Ser Pro Gin Val c-lIu c-lu Asn Val Pro S620 625 630 635 k. GAT CCC- CAC CT.C ATC ACC CAG GAS TTA CGC CCC ACC GAC CCC GAC ACC 2 14 9 Aso Cly His Val Ile Thr Giln Leu Arg Ala Th-r AsD Pro Aso Thr a. 6* 0e 6

C

00 0 a.

60 6 6* *6 a.

a 0 c *0 6 eq.,.

60 a 6*a* 4.J~ I. lee.

a a *6 qq* 0 a

S

a a. ma.

S

ACG

Thr

AAG

Lys

ATC

Ile

CCC

Arg 700

GAG

Glu

GTC

Val1

GAT

Asp

,AAC

GTG

ValI 780

ACC

Thr

TTC

Phe

CA

Arg A I

CA;

G ir

GAC'.

Gil 685

GTT

Val1

TTT

Phe T AC T yr AT G Met

TTC

Phe 765

CGC

Arg

ATT

Ile

CTC

Leu

ACT

rhr ys 3 45-

GAT

Aso

GGC

Gly 670

ACC

Thr G TA Val1

GAG

Giu

GGA

Gly

AAC

Asn 750

COA

Arg

GCG

Al a

TTC

Sh e

ACG

Thr

CCT

Pro Q83 0 Ser 655

CC

Ar g

ATC

Ile

GCG

Al a

GTC

Val1

GAC

Asp 735

GAC

Asp

GTC

ValI

GAC

Asp

CCT

Pro

GGT

Cly 815

~CCA

Pro AC A 642 Arc

CAG

Ginr

TTC

Phe

CGC

Ar g

CTC

Leui 720

CAC

Asp

.AAC

As n

CC

Ar q

GAC

Asp

CGT

Arg 800

CAA

CGC

A.r

GAA

T.C

Phe

GCT

Al a

*CCC

Pr o

GAA

Glu 705

TCC

Ser

TAC

T yr G CG Al a

GAG

Glu

ATC

Ile 785

GAA

Glu

ATT

Ile

T.TC

Phe

GAT

As o

GAG

Giu

AAC

Asr.

GAG

Glu 690

ATC

le

CTC

Le u

GAC

Asp

CCC

Pro

AT-

Met 770 Asp

GAC

Asp

TCC

Ser

CAC

Pro 850 Ile

CCC

Pro 675 AT T Ile

AGA

Arg ACA:k Tnr

GA.A

Glu Val 755

TCG

Ser

GGA

Gly

ACA

Thr

GTG

ValI

CTC

Leu 8 35 GCA2 Ala 660

GAC

Asp

AAC

As n

CAC

H i S

GTG

Val

TCG

Ser 740 T ro Al a

CCG

Pro

GAT

Aso A.Ac As n 820 T y r Asp Trp

GAG

Glu

AAC

Asn

AAC

As n

AGG

Arg 725

ATG

Met

GTG

ValI

GGC

Gly

CTC,

Le u

AAG

L ys 805

ACA

Thr T AT T yr

TC

C ys

CAC

GAC

Asp

TTT

Phe

CGG

Arg

GTG

ValI 710

GTG

ValI

CTC

Le u

GAG

Glu

GGG

Giy

TAC

Tyr 790

GAC

Aso

AGC

Ser

ACA:

Th r

CCC

Pro AT C Ile 870

ACC

Thr

AGG

Arqg

GGA

Gly 695

ACC

Thr

CCT

Arg

ACA

*Thr

GGG

Gly

CTC

Le u 775

AAC

Asn

CTG

Leu

GGC

Cl y

CTC

ValI

CCT

Pro 855

AP-C

Thr T C T *Ser

AAT

Asnr 680

CTG

Leu

ATA

Ile

GAC

Asp

ATA

Ile

ACT

Thb.r 760

GTG

Val1

CAA

Gin

ATA

Ile

GCC

Ala

GTC

Val1 840

CAC

Asp

GAC

Asp

TTC

Phe 665

TGC

C ys

GCT

Al a

GAC

Asp

CTT

Leu

ACT

Thr 745

CTG

Leu

GTG

Val1

GTG

Val1

ATG

Met

ATC

Ile 825 GC T Ala

CCG

Pro

ACG

Tnhr

GCC

Ala

GTG

ValI AT C Ile

TAC

T yr

AAC

Asn 730

ATA

Ile

GAG

Giu

GGC

CGA

Arg

ATC

le 810 GA'tC Aso

ACT

Ser

ACT

Tnhr

AAC

As n

ACC

Th-r

GAA

Giu

GGC

Gly

GAG

Giu 715

ACC

Thr

ATC

Ile

CAG

Gin

TCC

Ser

TAC

T yr 795

GAC

Aso

GCG

Al a

GAC

Aso

TAT

T yr

AAC

As n 875 2197 2245 2293 2341 2389 2437 2485 2533 258 1 2629 2677 2725 2773 2921 650 7CC GAA' ACC GA; GA. AraT ATC ACA ATC Tro C hi Thr C-lu Cly Asn Ile Thr Tie 865 AG GTC C:.G GCG GA.A ACG AAG GAT ACC GTC G7G 7aT 1-T LYS Val Pro Gin Ala Giu 880 eke 00 6*64 9400 890008 4. a 00 0 TAC GAG yr Glu Asn GAT CTT GAC -asp Leu aso 910 TAT GCA GTG Tyr Ala Val 925 ACC GGC CTG Thr Gly Leu 940 CTG GAC CGT Leu Asp Arg ATC GAC AAC Ile Asp Asn A C A GAA GTT Thr Glu Val 990 TTG CCA CCG Leu Pro Pro 1005 CAG GGC GTC Gin Gly Val 1020 CCC GAC ACA Pro Aso Thr ACG GAG CGG Tlm. r Glu Arg GCG a-AC G7C Ala Asn Val 1070 TGG GGG ACGG Giy Thr j loss CA.A ATG TCC G-In Met Ser iiOO

GC

A!

89

AG

Ar

A_:

As

GT

Va

GAI

AsE

TTC

Phe 975

CTC

Leu

CCG

Pro

CGT

Arg

GAC

Aso

GAC

Aso 105 kCG Thr

AC

Yr 7G e Z: Pk ACC a 7hr 5 A G A C g Asp C CCT n Pro 3 TAC L Tyr

GGT

Gly 960

ATG

Met

GTT

Val

AGC

Ser

CTT

Leu

AAC

Asn 1040

ATC

Ile 5

GGA

Gly GC7 Al a

AAC

Asn C Ac His

GAA

Glu

CGA

Arg

GTG

Val 945

GAT

Aso

GGG

Gly'0

ATC_

Ile

GAA

Glu

GAA

Glu 102E

TCC

Ser

GAA

Glu

GAG

Glu

ATA

Ile 3 A G Ilu 1105 nr li.r Lvs Phe Aso Thr Vail Val Tyr Ile 890 T-P. GAC GAG GTG GTC ACT C7G AT GCC AGT Leu Aso Glu Val Val- TI-ir L 7 e_ Ala Ser 900 905 ATA TAC CAC ACG GTG AGC TAC GTC A7C AA7 Ile Tyr His Thr Val Ser Tyr Val iie Asn 915 920 CTG ATG A-zkC TTC TTC TCC GTG AAC CGA GAG Leu Met Asn Phe Phe Ser Val Asn Arg Glu 930 935 GAC TAT GAG ACC CAG GGT AGT GGC GAG GTG Aso Tyr Glu Thr Gin Gly Ser Gly Glu Val 950 955 G.AA CCA ACG CAC CGT ATC TTC TTC AAC CTC Glu Pro Thr His Arg Ile Phe Phe Asn Leu 963 970 GAA GGA GAA GGT AAC AGA AAT CAG AAC GAC Glu Gly Glu Gly Asn Arg Asn Gin Asn Asp 980 985 TTG TTG GAT GTG AAT GAC AAT GCT CCT GA-A Leu Leu Asp Val Asn Aso Asn Ala Pro Glu 995 iOOO CTC TCT TGG ACT ATA TCT GAG AAC CTTI AAG Leu Ser Tro Thr Ile Ser Glu Asn Leu Lvs 1010 1015 CCA CAT ATC TTC GCC CCG GAC CGC GAC GAGG Pro His Ile Phe Ala Pro Asp Arg Asp Glu 1030 1035 AGG GTC_ GGC TAC GAG ATC CTG AAC CTC AGC Arg Val Gly Tyr Glu Ile Leu Asn Leu Ser 1045 1050 GTG CCG GAG CTG TTT GTG ATG ATA CAG ATC Val Pro Glu Leu Phe Val Met ile Gin Ile 1060 1063 CTG GAG ACC GCC ATG GA'C_ CTC AAG GGA 7AT Leu Glu Thr Ala Met Aso Leu Lys Gly Tyr 1075 1080 CAT A7.a. CGG GCA T7C GAC cC GG-C ATT C C G His Ile Arg Ala Phe Aso His G_1v ile Pro 1090 1095 CAT CCG TTC AAC 'Ca TAT GAG CTG 'TC ATC Thr Tvr Glu Leu ile ile His P-ro Phe Asn 1115 2S69 2917 2965 3013 3061 3109 3157 3205 3253 3301 3349 3397 3445 3493 3541 3589 X, C TAC GCO CCT GAG TTC GTC TTC CCG ACC AAC GAT GCC GTC ATA CGA TVr Tyr Ala Pro s-lu Phe Val P- -e Pro Thr Asn ASO Al1a Val Tle Arg 112 0 112 3 110 CT? GC AGG GAA CGA GCT GA aTC GGA CTA GC 'CA fG7- A,.C 33 eu Ala Ar= G Vllu Arg Ala Va e Asn Gly Val L eu Al-!a ?hr Val Asn 1135 1140 1145 GGA GAG TC TTG GAG CGC ATA TCC GCG ACT GA? CCG GAC GGA CC CAC 3685 Gly Glu Phe Leu Glu Arg Tie Ser Ala ?.hr Aso Pro Aso Gly Leu His 1130 1155 1160 GCIG CCC GTC GTC ACC TTC CAA CTG GTA CCC CAT GAG CAA TCA CAA CGG 3733 Ala Gly Val Val ?hr Phe Gin Val Val Gly Aso Clu Clu Ser Gin Arg 1165 1170 1175 TAC TTT CAA CTA CT? AC GAT CCC GAG ?-AC CTC CGC TCG TTC AGG TTA 3781 Tyr Phe Cln Val Val Asn Asp Cly Glu Asn Leu Cly Ser Leu Arg Lau 1180 1185 1190 1195 CC CAA CCC CCA GAG GAG AC AGC GAG TTC CCC AA ACG CCC 3829 Leu GIn Ala Val Pro Clu Clu Ile Arg Clu Phe Arg Tle ?hr Ile Arq 1200 1205 1210 GC ACA CAC CAG CCA ACC CAC CCA CCA CCC CTG TCC ACG CAC ATG ACG 3877 Ala ?hr Asp Cln Cly ?hr Asp Pro Gly Pro Leu Ser Thr Asp Met Thr 1215 1220 1225 AGA CT? ?TT CC CCC ACC CAA GGA GPA. CC? AGA TTC CC TCC 3925 *Phe Arg Val Val Phe Val Pro ?hr Gin CGlv C u Pro Arg Phe Ala Ser 00* 1230 1235 1240 CA GAA CAT GC CC C? AA GAA AAG ACT CCC CCC AC GAA GAG 3973 00* Ser Glu His Ala Val Ala Phe Ilie Glu Ly's Ser Ala Cly Met Glu Glu 0. 1245 1250 1255 0., 0 Te C? CAC CAA CC? CA GCA C.AA GAC A7C .AC CA? CC ?GT GAA 4021 Ser His Gin Leu Pro Leu Ala Gin Aso Ile Ly's Asn His Leu Cys Glu 1260 1265 1270 1275 GAC CAC CAC ACC TAC TA? CC? AC CA? CCC AAC ACC GA.A 4069 *00 Asp Asp Cys His Ser Ile Tyr Tyr Ar Ile Ile Aso Gly Asn Ser Clu 6001280 1285 1290 CC? CA? CCC CC CAT CC? CCC AC. AGC TC CC .AAG AT-A 4117 Cly His Phe Gly Leu Asp Pro Val Arg Asn Arg Leu Phe Leu Lys Lys e~..1295 1300 1305 GAG CG A?.A AGG GAA CAA AC? CCC 7CC CAPC ACT CTC CAA GC CC C? 4165 Clu LeU lie Arg Clu Gin Ser Ala Ser His ?h1-r Leu Gin Val Ala Ala 1310 1315 1320 AC? AAC ?CC CCC GA? CC? CCC AT? CCA CCT C? TCC AC CT? AC? 4213 Asn Ser Pro Asp Gly Cly Tle 1Pro Leu Pro Ala Ser Tie Le Thr 1325 1330 1335 CC AC? ACC CC ACG GAG GC' CAC CC? CC? C CA GCT GTT CTC ACC 4261 Val ?hr Val ?hr Val Arg Clu Ala ASc Pro Arg Pro Val Phe Val!-1 Arg 134110 1345 13 50 1353 T7C ?AC ACC GCA CCC ATA ?CC A CC CAC :CC AC CCC AGA GAG 4309 R- PuLeu ?vir ?hr Ala ClV Tle Ser T, r Ala Aso Ser lie Gly t'.rgu Cli 13-65 7

C)

CTG CTC Ac-A TT' CAT GC ACC CAC TCT GLA 7eu Leu Arc- Leu His Thr -In Se-r c,-lu 1373 1380 c-CT ATA G-A C TAC CGA: ACA ATG GTA GTG GAC Ala 7ie Aso Tyr Aso Th-r Met Val Val Aso 1390 19 Ac-A CAc- TCG GCT TTC- c-A CTG AAC GCT CAA Arc- Gin Ser Ala 2he Val Leu Asn Ala c-in 1405 1410 Gly Ser Ala CCC AGC C7' Pro Ser Leu 14An00 "7T ACT TAT 71 mr Tyr 1385 c-Ac- GCA GTG c-lu Ala Val 4357 4405 4453 ACC c-cA GTG CTG ACG CTT Thr c-ly Val Leu Thr Leu 1415 A.AT ATC Asn Ile 1420 ACA c-CT Thmr Ala TAC c-T- Tyr Val CAc- CCC ACG c-in Pro Thr c-CC ACG AlIa Thr 1425 ATc- CAT c-cA Met His c-1y ACT c-AC ACc- c-CC Thr Aso Thr Al1a 1440 c-cC c-iy c-CT CAG Ala Gin CTG TTC Leu Phe 1430 Cc-C ACC Arc- Thr AAA TT C Lys Phe c-AC c-TC Asp Val GA A GTC Glu Val 1435 ACC c-T- Tnr Val 1450 c-AC As o 1445 c-TA ValI 0* *e S S 0 es

S

00 0S 0 S S

S.

S.

S S 0O

S..

S

CT c CAA CAGI Leu c-in c-in 1470 c-CT c-c-c TTC Ala c-iy 2Phe 1485 c-AC CCC c-TC Aso Pro Val TCC TCG Ser Ser 145 GTC c-A Val c-lu AAC ATG Asn Met kCC c-cC Thr Gly CAc- G in AAC Cc-C GTC TAC Asn Arc- Val Tyr 1460 c-AC AAC Ac-A Asp Asn Ara 1475 ACC Tc-C A.AC Thr Cys Asn 1490 c-Cc- CTC Val Ala Leu c-AC TTT Aso Phe ATC c-AC I le Asp c-Ac- CAC c-lu H i s TAC TCc- TTC GTG Phe Val ATC c-Cc- Ile Ala CAAZ c-:c c-in Val 14 9~ Ac-C ACc- Ser Thr TTC GTC AAC ACc- Phe Val Asn Thr 1465 c-AC ACC TTC Ac-C Aso Thr Phe Ser 1480 c-Tc CCC c-CT AAC- Val Pro Ala Asn CAc- Ac- ccc c-CC c-in Met Ala Ala 4501 4549 4597 4645 4693 4741 1500 ACT TCA TAC c-c-c 1505 ACA ACc- TAC CCG 1510 CTc- 1515s ATc- Ac-A TAc-ACAc-ATC Thr Ser Tyr c-ly Thr Thr Tyr Pro Tyr Ser Leu Met Arc- 1520 1525 Cc-TAc-Tc-ACC Tc-CAc-cACTT cTACc-TCT-c

TTCATCATCA

GG ccCTCACT-c

AC:TGGAAC

AT TAC c-c-c AAC73TCTTCA- C AC AAC AAC A 77C Ac G A -A

TAGTCCTCCT

c-TTc-ACCAAC CCTCACTc-TC c-cACTA-Ac-C ccT CCAACCC u'TTCCA ACGA T c-c-TC-a .,CTTCc-TT CTAc-AGAT c-AGCTCGATA TCCA-CCC-c Tc-CTc-Tc-CTC c-CTAA-zCCc-A c.-AkCcGcccCC TATCT T CAAT CTCTcG-TCT- CAACCcAC: AA.CAtACAC7TA.

CAA-'ACAA-GC

ACT TCc-cCc-C c--TTTCATc-T Cc--T~-cAA- .a.Cc-cCATC- GC T- C-C:TrccC c-:TA 'C-AT7CA CTGcACTCc-Ac-

GCCTTGTGCT

CCCTc-TCGcAT CC CC Ccc-CAC Ac-CCCaGA A=Ac-AT CTTGC cT TAT C TC c-A AT Cc-TGcc-TGcT CCTc-AC-cT ACTc-CTTACC c-ACc-AAcTAC

CAACA.A.ACAC

TTTAc-ATc-CC c-CACTTTGc- Ac-TCGCP-CC TA.ACGGcACA- Ac-TTTc-cAAT 4790 4850 4910 4970 5030 5090 5 15 0 5 210 5270 5330 5390 TAAGA:::r GA.AGGATAG TTrGTGATAC- CCTGCATTT T7T12a-aACT AA:: GAA: A-AATGAG ACCTCCATTT AAGCTCTTOC TCTCATCTCA TCAAATT 7 T- ATGCC ::?.o7CATTA AGATACTOGA TTTAATT:AA G-ATTATTTA ATAATaTGT ATA A,-

TATTGTC

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHAPACTERISTICS: LENGTH: 1528 amino acids TYPE: amino acid TOPOLOGY: linear 3450 5510 5570 5577 SS Se 0 S S 0 00 0 0 0* S.

0 S 0 0 0 0 5

S

@000 0 0

S

S

Met 1 Pro Arg T.hr s 65 As n Gly Ser GIn 145 Gly G In, (ii) (xi) Ala Val Ala Val Leu Pro Tro Ser Met Aso Tyr Met Tyr Asn Ser Tyr 115 Ala Ser 130 Gin Tyr Val Ser Asn he Glu Cvs 195

SEQUENCE

Aso Val 5 Leu Ala 20 Arg Pro Gln Arg Ala Tvr Asp Giu 85 Gly Pro 100 Asn Leu His His Met Phe reu Ala 165 Glu Pro 130 Thr Tyr MOLECULE TYPE: protein Arg Ile Ala Ala Phe Gin Asp Pro His 70 Glu Ser Leu His Asn 150 Ile Cys GLn Glu Asn Leu 55 Va1 Ile Thr Met Ala 135 Va1 Val Arg Val Arg Cys 25 Leu Pro 40 Leu Pro Ile Thr G Asp Pro Phe 105 Pro Val 120 Arg Gin Arg Val Asn lie Val Pro 1 9: Ser Asp 200 10 Gly Va1 Ala Ala Glu 90 Ile lie His Aso Aso 170 Glu Leu Tyr Leu Pro Asn 75 lie Glu Arg Tyr Gly 155 Aso Leu As Leu Met Asn Glu Leu Thr Leu Arg Glu 140 Gin Asn Glv Giy Val Thr Phe Arg Gly Ile Pro Val 125 Leu Ser Ala Glu Arg 205 Phe Ala Glu Asp Thr Ala Phe 110 Asp Pro Leu Pro Pro 190 Ile Ile Ile Gly Asp Gin Ile Leu Asn Gly VaI Ile Gl v rer Ala Pro Gin Leu Val Leu Ser Gly Met Al a 160 Ile Leu Thr DESCRIPTION: SEQ ID NO:2: cj, Ri;~ L aI, j t ~7 O~: Phe Me- Thr Phe Arq Ile Asp Ser Val Arg Gly Aso Glu Glu Thr I I 210 The Tvr Iie Glu Arg Th: 0@: 0 0 0 0 0 0 0 00 @05e 5055

C

0 0

C

Me Hi Va Trr Asr 305 lIe Glu Ile Ala Ile 3935 Glu AsD Tyr Phe Gly 465 Ser Val Gln G STr S Ile L Thr Leu 290 Phe Asn Ala Asp Tyr 370 Ile Tyr Lys Tbr Tyr 450 Thr Ile Glv C 530 PhE Met 275 Glu Thr Tyr Leu Arg 355 Lys Val Arg Val 435 Ile .eu /ai 310 Gi/ Ser 260 Met Ile Val Arg Pro 340 Asp Tyr Thr Leu Phe 420 Arg Ala Asn Ile Slo 500 Phe G 1 Val 245 Val Va1 Phe Arg Leu 325 Gly Thr Aso Asp Al a 405 Gly Leu Pro His Arg 485 Leu Phe Asr Thr Gin Ala Ala 310 Ile Gly Leu Glu iIe 390 Ile Phe Glu Glu Ser 470 Val VaI Hi5 The 2 1 Ala Val Va1 295 Ile Thr Lys Gin Glu 375 Asp.

Met His Ser Val 455 Met Val '-s Ala Val 535 Ser Leu Ala 280 Gin Asp Asn Ser Arg 360 Ala Asp Glu As p Va1 440 Gly Leu Sla Val 520 Ala Leu Asp 265 Asn Gin Gly Glu Gly 345 Glu The Gin Glu Lys 425 Asc Tyr As o Tn r 505 GIn Lys Asn 250 Ser Val Phe Asp Glu 330 Ala Val Ser Arg Thr 410 Aso Pro Gin Tyr 490 Ala Al 7ie Pro sn Gin Phe Leu Asn Glu Thr 315 Asp Va1 Phe Thr Pro 395 Pro Leu Pro Arg Glu 475 Asn Val Val 220 Tr: Val Pro Ser Glu 300 Glu Thr The .Pro Ser 380 Glu Leu Gly Gly Gin 460 VaI Asn Asn Thr Ala 540 Thr Asn Arg 285 Lys Ile Phe Leu Leu 365 Thr Pro Thr Gin Ala 445 Thr Pro Aso Trp Pbe 525 His Met Tro Leu Asn Ser Thr 270 Pro Ser Asn Phe Val 350 Thr Asn Leu Asn 430 Ala The Glu Thr As n 510 Asp Asp Pro 255 His Pro Tyr Met Ser 335 Ser Ile Val His Asn 415 Al a Glu Ile The Arg 495 Aso Glu Arg 240 Leu Thr Arg Gin Pro 320 Tie Pro VaI Va1 Lys 400 Phe Gin Ala Met Gin 480 His G1, Thr Aso i Ile Gly Aso Val Val Glu His Thr Leu Leu Gly Asn Ala Val Asn Phe 545 0 0 0

S

*00.

0@ 0 *4 00 0 0 0 90

S,

0 0

S

S

Leu Ser Thr Ile Arg 625 Thr Phe Ala Pro Glu 705 Ser Tyr Ala Glu Ile 2 785 C-lu Ile Phe H Th: Aso Arg 610 Gly Gin Glu Asn Glu 690 Ile Leu Aso Pro Met 770 kso ksp er i s As: Thi 595 Lei Ser Glu Ile Pro 675 Ile Arg Thr Glu Val 755 Ser Gly Thr Val Leu 835 Aso i Tvr 580 Leu i Asn Pro Leu Asn 660 Aso Asn His Val Ser 740 Tro Ala Pro Asp I A-sn 'I 820 Tyr T Lys 565 His Gly Asp Gin Arq 645 Trp Glu Asn Asn Arg 725 Met Val ly Leu -vs 305 hr yr Leu Arg Giu Ile Val 630 Ala Aso Phe Arg Va1 710 VaI Leu -lu Gly Tyr 790 Asp Ser Thr Thr Glu Pro Asn 615 Glu Thr Thr Arg Gly 695 Thr Arg Thr Gly Leu 775 Asn Leu 1 y /a! C-ly Ser Phe 600 Asn Glu Asp Ser Asn 680 Leu Ile Asp Ile Thr 760 Val Gin Ile Ala Val 840 As o Glu 585 His Thr Asn Pro Phe 665 Cys Ala Asp Leu Thr 745 Leu Va1 Va1 Met i e 825 Ala 570 Leu Thr Pro Va1 Asp 650 Ala Va1 Ile Tyr Asn 730 Ile Glu Gly Arg 810 Asp Ser ,Arc Phe Ala Pro Pro 635 Thr Thr Glu Cly Glu 715 Thr Ile Gin Ser Tyr 795 Asp Ala Aso Val Val Thr Thr 620 Asp Thr Lys Ile Arg 700 Glu Va1 Asp Asn Val 780 Thr Phe As o Arc Ser GIn Ser 605 Leu Gly Ala Gin Glu 685 Va1 Phe Tyr Met Phe 765 Arg Ile Leu Thr Cys 845 Ala Val 590 Gin Arg His Asp Gly 670 Thr Val Glu Gly Asn 750 Arg Ala Phe Thr Pro 830 Ser Asn 575 Arg Leu Leu Va1 Leu 655 Arg Ile Ala Val As p 735 Aso Val Asp Pro Gly Pro Thr Asp Ala Va1 Pro Ile 640 Arg Gin Phe Arg Leu 720 Asp Asn Arg Asp Ara 800 Gin Arg Glu Asp Pro Ala Asp Cys Pro Pro Aso Pro Thr Tyr 850 355 Trp Glu Thr Glu Gly 860

I.

Asn !lI Thr 7le His 7ie Tr Asc Thr Asn Asn Lys Val Pro GIn Ala 965 870 875 380 Glu Thr Thr Lys ?he Asp Thr Val Val 7Vr 7le Tyr G7u Asn Ala Thr 835 890 395 His Leu Aso Giu Val Val Thr Leu Ile Ala Ser Asp Leu Asp Arg Asp 900 905 910 Glu Ile Tyr His Thr Val Ser Tyr Val Ile Asn Tyr Ala Val Asn Pro 915 920 925 Arg Leu Met Asn Phe Phe Ser Val Asn Ar; Glu Thr Gly Leu Val Tyr 930 935 940 Val Asp Tyr Giu Thr Gin Gly Ser Gly Giu Val Leu Asp Ar; Asp Gly 945 950 955 960 Asp Glu Pro Thr His Arg Ile Phe Phe Asn Leu lie Asp Asn Phe Met 965 970 975 Gly Glu Gly Giu Gly Asn Arg Asn Gin Asn Asp Thr 0lu Val Leu Val 980 985 990 Ile Leu Leu Asp Val Asn Asp Asn Ala Pro Glu Leu Pro Pro Pro Ser 995 1000 1005 f Glu Leu Ser Tro Thr Ile Ser Glu Asn Leu Lys Gin Gly Val Arg Leu 1010 1015 1020 u Pro His Ile Phe Ala Pro Asp Arg Asp Glu Pro Asp Thr Asp Asn 1025 1030 1035 1040 Ser Ar; Val Gly Tyr Glu Ile Leu Asn Leu Ser Thr Glu Ar; Asp Tie 1045 1050 1055 Glu Val Pro Giu Leu Phe Val Met Ile Gin Ile Ala Asn Val Thr Gly 1060 1065 1070 Glu Leu Giu Thr Ala Met Asp Leu Lys Gly Tyr Trp Gly Thr Tyr Ala eec. *1075 1030 1085 0000 Ile His Ile Ar; Ala Phe Asp His Gly Ile Pro Gin Met Ser Met Asn 1090 1095 1100 Glu Thr Tyr Giu Leu Ile Ile His Pro Phe Asn Tyr Tyr Ala Pro Glu 1105 1110 1115 1120 Phe Val Phe Pro Thr Asn Asp Ala Val Ile Ar; Leu Ala Arg Giu Ar; 1125 1130 1135 Ala Val Tle Asn Gly Val Leu Ala Thr Val Asn Gly Glu she Leu Glu *:fee: 1140 1145 1150 Ar Ile Ser Ala Thr Asp Pro Asp Gly Leu His Ala Gly Val Val Thr 1155 1160 1165 P-e Gin Val Val Gly Asp Glu Glu Ser GIn Ar; Tyr Phe -:in Val Val 1170 1175 1130 Asn Asp Gly Gl Asn Leu Gly Ser Leu Ar; Leu Leu Gin Ala Val Pro 46

I.

1185 1190 1195 1200 Glu Glu Ile Arg Glu Phe Arg Ile Thr Ile Arg Ala Thr Aso Gin Gly 1205 1210 1215 Thr Asp Pro Gly Pro Leu Ser Thr Asp Met Thr Phe Arg Val Val Phe 1220 1225 1230 Val Pro Thr Gin Gly Glu Pro Arg Phe Ala Ser Ser Glu His Ala Val 1235 1240 1245 Ala Phe Ile Glu Lys Ser Ala Gly Met Glu Glu Ser His Gin Leu Pro 1250 1255 1260 Leu Ala Gin Asp Ile Lys Asn His Leu Cys Glu Asp Asp Cys His Ser 1265 1270 1275 1280 Ile Tyr Tyr Arg Ile Ile Asp Gly Asn Ser Glu Gly His Phe Gly Leu 1285 1290 1295 Asp Pro Val Arg Asn Arg Leu Phe Leu Lys Lys Glu Leu Ile Arg Glu 1300 1305 1310 Gin Ser Ala Ser His Thr Leu Gin Val Ala Ala Ser Asn Ser Pro Asp 1315 1320 1325 0O Gly Gly Ile Pro Leu Pro Ala Ser Ile Leu Thr Val Thr Val Thr Val 1330 1335 1340 Arg Glu Ala Asp Pro Arg Pro Val Phe Val Arg Glu Leu Tyr Thr Ala 1345 1350 1355 1360 *e Gly Ile Ser Thr Ala Asp Ser Ile Gly Arg Glu Leu Leu Arg Leu His 1365 1370 1375 Ala Thr Gin Ser Glu Gly Ser Ala Ile Thr Tyr Ala Ile Asp Tyr Asp 1380 1385 1390 Thr Met Val Val Asp Pro Ser Leu Glu Ala Val Arg Gin Ser Ala Phe 1395 1400 1405 Val Leu Asn Ala Gin Thr Gly Val Leu Thr Leu Asn Ile Gin Pro Thr 1410 1415 1420 Ala Thr Met His Gly Leu Phe Lys Phe Glu Val Thr Ala Thr Aso Thr 1425 1430 1435 1440 Ala Gly Ala Gin Asp Arg Thr Asp Val Thr Val Tyr Val Val Ser Ser 1445 1450 1455 Gin Asn Arg Val Tyr Phe Val Phe Vai Asn Thr Leu Gin Gin Val Giu 1460 1465 1470 Asp Asn Arg Asp Phe lie Ala Asp Thr Phe Ser Ala Gly Phe Asn Met 1475 1480 1485 Thr Cys Asn lie Asp Gin Val Val Pro Ala Asn Asp Pro Val Thr Gly 1490 1495 1500 R Ala Leu Glu His Ser Thr Gin Met Ala Ala Thr Ser Tyr Gly Thr 1 1510 115 1520 Thr Tyr Pro Tyr Ser Leu M- 152 7 NFOR@.,ITION FOR SEQ 7D NO: 3: Ci) SEQUENCE CHAPACTERI STICS: ()LENGTH: 107 amino acids TYPE: amino acid STPANDEDNESS: single CD) TOPOLOGY: li-'near (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Glu Trp Val Met Pro Pro Ile Phe Val Pro Glu Asn Gly Lys Gly Pro 1 5 10 Phe Pro GIn Arg Leu Asn Gin Leu Lys Ser Asn Lys Asp Arg Gly Thr 25 Lys Ile Phe Tyr Tyr Ser Ile Thr Gly Pro Gly Al1a Asp Ser Pro Pro 40 *Glu Gly Val Phe Thr Ile Glu Lys Glu Ser Gly Trz Leu Leu Leu His 9.*50 55 4D f *Met Pro Leu Aso Arg G u Lys le Val Lys Tyr Giu Leu Tyr Gly His 65 70 75 *Ala Val Ser Glu Asn Gly Ala Ser Val Glu Glu ProD Met Asn Ile Ser 90 le Ile Val Thr Asp Gin Asn Asp Asn. Lys Pro 100 105 INFORMA~TION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 105 amino acids TYPE: amino acid CC) STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTON: SEQ ID NO:4: Glu Aso Tbhr Vai Tyr Ser Phe Asp Ile Aso Glu Pksn Ala Gin Arg Gly 10 Tyr Gin Val Gly Gin Ile Val Ala Arg Aso Ala Aso Leu Gly Gin Asn 25 RA Al1a GIn Leu Ser Tyr Gly Vai Val Ser Aso Tro Ala Asn Aso Val Phe 40 4 01j 48 Ser Leu Asn Pro Gin Thir G-ly Tyr C-lu Gla Val Gin His Tyr 70 Gl'-v Gin Pro Ser Lea Ser Thr Aso Leu Asn Asp Asn Ala Pro 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICE LENGTH: 93 amino ac TYPE: amino acid STRANDEDNESS: singi TOPOLOGY: linear Mez Lea L-ea,- Ttr Ala Arg Lea Aso 7- 1 e ie, Valj Ginr Ala Gin Asp Asn 75 Th r Ile Tblr Val Tyr Cys Asn Val Leu 90 le Phe 105 :00 Ir 0 0 f *0 4 04 J 00 Ba.

as** 0 (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Ser Pro Val Ile Thr Lea Ala Ile Pro 1 5 10 Ser Lea Phe Pro l Te Pro Lea Ala Ser Asp 20 25 Gin Val Ala Glu Asp Gin Gla Gla Lys Gln 35 Cly Asn Leu Asp Arg Glu Arg Tr-p Asp Ser 55 Val Gin Asp Gly Gly Ser Pro Pro Arg Ala 70 Val Thr Val Leu Asp Thr Asn Aso Asn Ala 85 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Glu Arg Pro Tyr Thr 75 Pro As n Asp Gin Asp Ser Lys As n As n le Thr Lea Ile is Gla Val1 Ile Leu Gly Le a Mlet L ys Ar g s0 SEQUENCE DESCRITI-1ON: SEQ ID 'NO:6: Ile Val Thr Gilu Asn le Tro Lys Ala Pro Lys Pro Val Gia Mez Val 10 is Glu Asn Ser Thr Pro His Pro lie Lys 25 Asp Pro Gly Ala Gin Tyr Ser Leu Val 40 Phe Pro Phe Ser Ile Asp Gin Glu Gly 55 Asp Arg Glu Glu Lys Asp Ala Tyr Val 70 Glu Tyr Gly Lys Pro Leu Ser Tyr Pro Lys Asp Ile Asn Asp Asn Pro Pro Thr 100 105 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 105 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Ile Thr Gin Val Arg Asp Lys Giu Lys Leu Asp lie Tyr Val Thr Phe Tyr Ala Val Ala 75 Leu Glu Ile His Val 90 Cys Trp Asn Pro Arg Pro Leu Lys Asp Lys Val OS eg

S

0@ C C

S.

S. 0

S.

0

C

e g.

C

C

0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ile Thr Ala Asn Leu Gly Thr Gin Val ile 1 5 10 Glu Asp Glu Ile Thr Ile Ala lie Leu Asn 25 Pro Phe Ile Glu Leu Pro Phe Leu Ser Gly 35 Pro Val Ile Arg Arg Val Asp Asn Gly Ser 50 55 Arg Gin His Tyr Glu Leu Pro Gly Met Gin 65 70 Arg Val Asp Gly Gin Ser Leu Val Ala Gly 90 Asn Ile Asp Asp Asn Ala Pro Ile Ile 100 105 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Tyr Tyr Ser Ala Gin 75 Val Me Asn Tyr Ser Tyr Ser Glu Pro Leu His Phe Ala Glu Ser Leu His Asn Ile Ile Thr Met Ala Val Val (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Gin Asn ?he C-lu Pro Cys Arg Val Pro Glu 1 5 Thr Glu Cys Thr Tyr Gln Val Ser Aso Ala 25 Clu The Met Thr Phe Arg Ile Asp Ser Val 40 The Tyr lie Glu Arg Thr Asn Ile Pro Asn 55 Met Thr Ile Gly Val Asn Thr Ser Leu Asn 70 His Ile Phe Ser Val Thr Ala Leu Asp Ser 90 Val Thr Met Met Val Gin Val Ala Asn Val 100 105 Tro INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acias TYPE: amino acid STP-A.NDEDNESS: single TOPOLOGY: linear Leu Asp Arg Gin Phe 75 Leu Asn Gly Gly Gly Trp Va1 Pro Ser Clu Arg Aso Met Thr Asn Arg Pro Ile Clu Trp Ser Thr Pro 110 C-I y Ser Clu Leu Pro His Pro Leu Thr Thr Asn Leu Thr Arg 00 @6 0 0 06 0 00 00 S 0 00

S.

S.

@5

S

000 0000 0 *000 0000

S

0000 0 60000 0q 00 0 j i (xi) Leu 1 The Asn Ala As 0 Tyr SEQUENCE DESCRIPTION: SEQ ID NO:9: Clu Ile Phe Ala Val Gin Gin The Clu 5 10 Thr Val Arg Ala Ile Asp Gly Aso Thr 25 Tyr Arg Leu lIe Thr Asn C-lu Clu asp 40 Leu Pro Gly Gly Lys Ser Gly Ala Val 50 5D Thr Leu Gin Arc Glu Val The Pro Leu 70 Aso C-lu C-li Ala The Ser Th-,r Ser Thr Clu Clu Thr The Thn r 75 Asn Lys Ile The Leu Ile Val Ser Asn The VaI Val Val Tyr Met- Ser Ile Ala Ile Gin Pro Ile Asp Tyr Ile Asn lIe C-lu Arg Lvs Val Thr Asp Ile Asn Asp Gin Arg Pro Glu Pro 100 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear o 0..

o* **0 6 *5 *e (2) 0

S

(xi) SEQUENCE DESCRIPTION: SEQ ID Ile His Lys Glu Tyr Arg Leu Ala Ile Met 1 5 10 Leu Asn Phe Asp Lys Glu Phe Gly Phe His 25 Asn Ala Gin Tyr Thr Val Arg Leu Glu Ser 40 Ala Glu Ala Phe Tyr Ile Ala Pro Glu Val 50 55 Phe Ile Met Gly Thr Leu Asn His Ser Met 65 70 Glu Phe Gin Ser Ile Thr Ile Arg Val Val 85 90 Thr Arg His Val Gly Val Ala Leu Val His 100 105 Asn Asp Glu Gin Pro Ile Phe 115 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Glu Asp Val Gly Leu 75 Ala Ile Glu Lys Asp Tyr Asp Thr AsD Thr Asp Pro Gin Tyr Asp Leu Pro Leu Pro Arg Glu Asn Ile 110 Leu Gly Gly Gin Val Asn Asn Thr Gin Ala Thr Pro Asp Trp (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Glu His Ala Val Gin Thr Val Thr Phe Asp Glu Thr Glu Gly Glu Gly 1 5 10 Phe Phe Val Ala Lys Ala Val Ala His Asp Arg Asp Ile Gly Asp Val Val C-lu His Thr Leu Leu Gly Asn 40 Lys Leu Thr G-iy Aso le Ar; Val s0 55 Ar; Glu Ser C-lu Leu Phe Val G In 70 Gin Pro Phe His Thr Ala Thr Ser Ile Asn Asn Thr Pro Pro Thr Leu 100 INFORMATION FOR SEQ ID NO:i2: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid STRANDEDNESS: singie TOPOLOGY: iinear 253 Ala Val 2SF Pre Leu Thr Ile Aso Ser Ala Asn Aso Ser P he Tyr His Val Arg Ala T'hr As o Thr Leu Gly 75 Gin Leu Val Ile Ar; Leu Asn Aso 90 *0 se 0 *000a 0* 00: 0 0 0.@0 0 (xi) SEQUENCE DESCR7PTION: SEQ ID NO:12: Ar; Leu Pro Arg Gly Ser Pro Gin Val Glu Glu Asn Val Pro Aso Ala is His s o Gly Thr Ile Gl1u Val1 Le u Ar; Ile Gly G Iu ValI Ile Thr Ar; Phe 35 Gin Ala Phe Phe Arg Val Phe Glu 100 Tyr Gly Gin Giu As n Pro Val1 Val Aso Giu Ile Pro0 Giu 70 Al a L eu Aso Leu Asn Asp Ile Ar; Ser Tvr Ar; T ro 40 Glu Asnr Glu Leu A-so0 120 Al a 25 Asp Phe As n Ile Thr 105 G 1u Thr Thr Ar; Ile Ar.; 90 Val Ser Asp Ser Asn Asn 7 5 H is Arg Met Pro Phie Cvs As n As n ValI Leu Asp Al a Val1 Ar; Thr Ar; Thr 125 Thr T hr Glu Gly Ile A--so 110 Ile Th~r Lys Ile Leu Aso Leu Al a G in Giu Tyr As n Ile Tie A-so Met Asn Asp Asn Ala Pro Val Tro 130 135 INFORMA7TON FO0R SEQ !D NO:13: ()SEQUENCE CHARACTERISTICS: LN7C7: 124 anir aais TYPE: arn acId S7ZP N_1DEDNE siole TOPOLOGY: linear Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Val Giu Gly Thr Leu Giu Gin Asn Phe Arg Val Arq Glu Met Ser Ala 1 5 10 Gly Gly Leu Val Val Gly Ser Val Arg Ala Asp Asp Ile Asp Gly Pro 25 Leu Tyr Asn Gin Val Arg Tyr Thr Ile Phe Pro Arg Glu Asp Thr Asp 40 Lys Aso Leu Ile Met Ile Glu Leu Pro His Gly Ser Asn Phe Arg Glu 55 His Lys Arg Arq Ile Asp Ala Asn Thr Pro Pro Arg Phe His Leu Tyr 70 75 Tyr Thr Val Val Ala Ser Asp Arg Cys Ser Thr Glu Asp Pro Ala Asp 90

S

Cys Pro Pro Asp Pro Tyr Tyr Trp Gla Thr Glu Gly Asn Ile Thr Ile 100 105 110 00 His Ile Thr Asp Thr Asn Asn Lys Val Pro Gin Ala 115 120 INFORMATION FOR SEQ ID NO:14: 0O es. i) SEQUENCE CHARACTERISTICS: LENGTH: 122 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 0000 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Gla Tr Thr Lys Phe Asp Thr Val Val Tyr 7ie Tyr Glu Asn Ala Thr 10 His Lea Aso Cla Val Val Thr Leu lIe Ala Ser Asp Lei-, AspArg Aso 25 GiL lie Tyr is Me: Val Ser Tvr Val 7Ie asn Tyr Ala Val Asn Pro 40 Ara Lea Met Asn Phe Phe Ser Val Asn Arg Gla Thr Gly Lea Val Tyr 55 Nz- 54 Val Asp Tyr Glu Thr Gin Gly Ser 70 Pro T.hr His Ara Tl- Phe Phe Asn Gly Glu C-ly Asn Arg Asn C-in Asn 100 Leu Aso Val Asn Asp Asn Ala Pro 115 120 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 146 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Leu Aso Arc Aso Cly Aso C 75 so Leu le Aso Asn Pl-e Met Giy C>u Asp Thr Glu Val Leul Val Leu 105 1110 Giu Leu (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Pro Pro Ser Glu Leu Ser Trp Thr le Ser Glu Asn Leu 0* Ow 0 0- 0 0 00 0O a a.

S.

0e 0 0 0@ 0

C..

*000 0 0 4* 00 0 S 0 0 *00000 Gly Val Aro Leu Clu Pro His Ile Phe 20 25 Asp Thr Asp Asn Ser Arg Val Gly Tyr 35 40 Glu Arg Asp Ile Glu Val Pro Giu Lea 50 55 Ala Asn Val Thr Gly Tyr Giu Ile Lea 70 Ile Glu Val Pro Glu Lea Phe Val Met 85 Gly Gia Lea Glu Thr Ala Met Asp Lea 100 105 Ala Ilie Tyr le Lea Ala Phe Aso His 115 120 A-s n Ciau Thr Tyr C Iu Lea lie Ile His 130 135 Cia Phe 145 TINFORMAT:O0N FOR SEQ ID NO:16: Si SEQ U ENCE CH A tC T P S T 1CS: LENGTH: 120 amino acids TYPE: anino acid Al a Glu Phe Asn Ile 90 Lys Clv Pro Pro Tie Val1 Leu Gin Gly :le Aso Le u Met- Ser Ile T yr Pro A s n, 140 Arq As n Ile Thr Al a T rp Gin 125 Tyr Aso Lea Gin G la As n Gly 110 Met: T yr Lys Gin Glu Pro Ser T r Arc Aso Val Thr Th~r Tyr Ser M et Ala Pro STP-aNDEDN,1ESS: Si4-ngle TOPOLOGY: 1na SEQUENCE DESCRPTION: SEQ ID NO:16: Val Phe Pro Thr Asn Asp Ala Val ile Arg 1 10 Val le Asn Gly Val Leu Ala Thr Val Asn 25 Ile Ser Ala Thr Asp Pro Asp Gly Leu His 40 Gln Val Gly Asp Glu Glu Ser Gin Arg Tyr 55 Aso G' -y Glu Asn Leu Gly Ser Leu Arg Leu 70 Glu le Arg Glu Phe Arg Ile Thr Ile Arg 90 Asp Pro Gly Pro Leu Ser Thr Aso Met Thr 100 105 Pro Thr Gin Gly Glu Pro Arg Phe 115 120 INFORIMATION FOR SEQ ID NO: 17: SEQUENCE CHARA.CTERISTICS: LENGTH: 112 amino acids TYPE: amino acid STPANDEDNESS: single TOPOLOGY: linear Leu Gly Ala Phe Leu Ala Phe Al a Glu Gly Gin Gin Thr Ar g A rq Phe Val1 ValI Al a As o ValI Clii Leui Val1 Val1 Val Gin Val1 A rq Ghu Thr Asp Pro Glv 93 Phe he Os

C

0 6O C a t 4~, at a a a ~e a a.

S

'-S

a.

a.

eta see 0 0 a-C (xi) Ala Glii C ys Ser Lys SEQUENCE DESCRIPTION: SEQ ID NO:17: Ser Ser Clii His Ala Val Ala Phe Ile 10 Glu Ser His Gin Leu Pro Leu Ala Gin 25 Glu Asp Asp Cys His Ser 7le Tyr Tyr 40 Clii Cly HsPh-e Civ Let- Asp Pro Va I 5z Lys Clu Leii le A7,rg Ciii GIn Ser Ala 70 Clii Lys Ser Ala Cly Asp le Lys Asn H is Ara ile Ile Aso Cly Arc As n Arg Leu Phe Ser His Thr Leu Gin 7

I,

Ala Ala Ser Asn Ser Pro Asp Gl y Gly T1. Pro Leu Pro Ala Ser lie 90 Leu Thr Val Thi-r Val Thr Val Arg 100 105 Ala Aso Pro Arg Pro Val Phe INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i3: Met 1 Leu Aso Tyr Glu Val Pro Glu Phe Gin 10 Ser Ile Thr Ile Arg Val *0 eg 0 S

S

S 0

OS

0@ 0 0 0 0@

S.

S. C, 00 0* 0

C..

Val Ala Thr Asp Asn Asn Asp Thr INFORMATION FOR, SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Ar g His Val Gly Val (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 000S 5 *005 0 e.g.

@00g..

0 @0 SO 0 0 0

S

C

Met Xaa Glu Thr Tyr Glu Leu Ile Ile His Pro Phe Asn Tyr Tyr Ala 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear

RA

(xiJ) SEQUE"NCE DESCRIPTION: SEQ ID Met Xaa Xaa Xaa His Gin Leu Pro Leu Ala Gin Asp Ile Lys Asn His ff INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 2 OTHER INFORMATION: /note= "This (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 3 OTHER INFORMATION: /note= "This (ix) FEATURE: NAME/KEY: Modified-site LOCATION: OTHER INFORMATION: /note= "This position is Phe/Pro" position is Asn/Ile" position is Arg/Tyr" 0 0 0 00

S.

00S 0 0

S

@0 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 8 OTHER INFORMATION: /note= "This position is Ile/Gly" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Xaa Xaa Val Xaa Val Asp Xaa 1 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear s 0 00*

S

0 oo

S

(ix) FEATURE: NAME/KEY: Modified-site LOCATION: 4 OTHER INFORMATION: /note= (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 8 OTHER INFORMATION: /note= "This cosition is Phe/His" "This position is Arg/Asp" SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Asn Phe Xaa Ser Val Asn Xaa Giu INFORM--!ATION FOR SEQ ID NO:23: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 5 am-.inro acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: inear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Ala Xaa Asp Xaa Asp 1 INFORMATION FOR SEQ ID NO:24: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STPANDEDNESS: single CD) TOPOLOGY: l1inear 0: 00S (2)xi SEQUE ,IINC DECRPON SEQ ID NO:2 (C STADDES:snl CD) TOPOLOGY: linear 00000: (ix) FEATURE: CA) NAME/KEY: Modified-site LOCATION: 1 CD) OTHER INFORMATION: /note= "Alanine CA) or Valine(V)" xiJ) SEQUENCE DESCRIPTION: SEQ ID Xaa Xaa Asp Xaa Asp INFORMA.TION FO0R SEQ ID NO:26:

I.

Ci) SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Aso Xaa Asn Asp Asn 1 0. 0 00 0 0 0 so *00 0 0 0 0 00 0 00 6000 0 9009 *0S :00,00 0

Claims (22)

1. A method to identify an agent that binds to a BT-toxin receptor, said method comprising the steps of: i) contacting an agent with a BT-toxin receptor fragment selected from the group consisting of 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, 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:1 under high stringency and that binds to a BT-toxin, an isolated BT-toxin receptor fragment which binds to a BT-toxin and is encoded by the Bam-Sac nucleic acid fragment, an isolated BT-toxin receptor fragment which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the Bam-Sac 15 nucleic acid fragment under high stringency, an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin having an amino acid sequence found within SEQ ID NO:2, and an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high 20 stringency, and ii) determining whether said agent binds to said BT-toxin receptor fragment.

2. The method of claim 1, wherein said method further comprises the step of determining whether said agent blocks the binding of a BT-toxin to said BT-toxin receptor fragment.

3. The method of claim 1 or claim 2, wherein said cell that has been altered is a eukaryotic cell.

4. The method of claim 3, wherein eukaryotic cell is an insect cell.

The method according to any one of claims 1 to 4, wherein the BT-toxin receptor fragment is encoded by the Bam-Sac nucleic acid fragment.

6. The method according to any one of claims 1 to 4, wherein the BT-toxin receptor fragment is the isolated unglycosylated BT-toxin receptor which binds to a BT-toxin having an amino acid sequence found within SEQ ID NO:2.

7. 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, in the presence and absence of a BT-toxin, with a BT-toxin binding receptor fragment selected from the group consisting of 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, 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:1 under high stringency and that binds to a BT- 15 toxin, an isolated BT-toxin receptor fragment which binds to a BT-toxin and is encoded by the Bar-Sac nucleic acid fragment, an isolated BT- toxin receptor fragment which binds to a BT-toxin encoded by a nucleic acid i* molecule that hybridizes to the Bar-Sac nucleic acid fragment under high stringency, an isolated unglycosylated BT-toxin receptor which binds to a 20 BT-toxin having an amino acid sequence found within SEQ ID NO:2, and (f) an isolated unglycosylated BT-toxin receptor which binds to a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid Ssequence of 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 fragment.

8. The method of claim 7, wherein said BT-toxin is a member of the BT-cry(1) family of toxins.

9. The method of claim 7 or claim 8, wherein said cell that has been altered is a eukaryotic cell.

The method of claim 9, wherein eukaryotic cell is an insect cell.

11. The method according to any one of claims 7 to 10, wherein the BT-toxin receptor fragment is encoded by the Bar-Sac nucleic acid fragment. 63

12. The method according to any one of claims 7 to 10, wherein the BT-toxin receptor fragment is the isolated unglycosylated BT-toxin receptor which binds to a BT-toxin having an amino acid sequence found within SEQ ID NO:2.

13. An isolated antibody, wherein said antibody selectively binds to a BT-toxin receptor fragment selected from the group consisting of a) a BT- toxin receptor fragment which binds a BT-toxin encoded by the Bam-Sac fragment, b) a BT-toxin receptor protein fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes to the Bam-Sac nucleic acid sequence under high stringency, c) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin having an amino acid sequence found *.,within SEQ ID NO:2, and d) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes 15 to the nucleic acid sequence of SEQ ID NO:1 under high stringency.

14. The antibody of claim 13, wherein said antibody selectively binds to said BT-toxin receptor fragment and blocks the binding of a BT-toxin to said receptor fragment.

15. The antibody of claim 13, wherein said antibody selectively binds to an epitope located within the 234 C-terminal amino acids of the BT-toxin receptor depicted in SEQ ID NO:2.

16. An isolated BT-toxin receptor protein selected from the group consisting of a) a BT-toxin receptor fragment which binds a BT-toxin encoded by the Bam-Sac fragment, b) a BT-toxin receptor protein fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes to the Bam-Sac nucleic acid sequence under high stringency, c) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin having an amino acid sequence found within SEQ ID NO:2, and d) an unglycosylated BT-toxin receptor fragment which binds a BT-toxin encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO:1 under high stringency.

17. The method of claim 16, wherein the BT-toxin receptor fragment is encoded by the Bam-Sac nucleic acid fragment.

18. The method of claim 16, wherein the BT-toxin receptor fragment is the unglycosylated BT-toxin receptor which binds to a BT-toxin having an amino acid sequence found within SEQ ID NO:2.

19. A method to produce BT-toxin receptor protein fragment, said method comprising the steps of: i) culturing a cell that has been altered to contain a nucleic acid Smolecule that encodes a BT-toxin receptor protein fragment, wherein said cell has been altered to contain a nucleic acid molecule selected from the group consisting of a) the Bam-Sac nucleic acid, b) a nucleic acid molecule which Sencodes a BT-toxin receptor fragment which binds a BT-toxin having an 15 amino acid sequence found within SEQ ID NO:2, c) a nucleic acid molecule encoding a BT-toxin receptor fragment which binds a BT-toxin receptor that hybridizes to the Bam-Sac nucleic acid under high stringency, and d) a nucleic acid molecule which encodes a BT-toxin receptor fragment which binds a BT-toxin that hybridizes to the nucleic acid sequence of SEQ ID NO:1 20 under high stringency, under conditions in which said nucleic acid molecule is expressed and ii) isolating said BT-toxin receptor protein fragment.

The method of claim 19, wherein said cell that has been altered is a eukaryotic cell.

21. The method of claim 20, wherein eukaryotic cell is an insect cell.

22. The method according to any one of claims 19 to 21, wherein the nucleic acid is the Bam-Sac nucleic acid. Dated this nineteenth day of November 2001 University of Wyoming Patent Attorneys for the Applicant: F B RICE &CO a a. a a a a. a. '1