WO1998013493A2 - A family of genes encoding apoptosis-related peptides, peptides encoded thereby and methods of use thereof - Google Patents

Abstract

An isolated polynucleotide at leat 60 % homologous to SEQ ID NO: 1, 3, 5 or 18 encoding a SARP polypeptide; vectors comprising a polynucleotide sequence encoding at least 11 consecutive amino acids of αSARP polypeptide; a host cell transformed with an isolated polynucleotide or vector; antibodies specific for SARP and use of such polynucleotides and antibodies in diagnostic and therapeutic method. Therapeutic uses of antibodies and polynucleotides of sarp. Methods for treating diseases related to the regulation of SARP expression in tissue and bodily fluid samples, including cancers.

Description

A FAMILY OF GENES ENCODING APOPTOSIS-RELATED PEPTIDES, PEPTIDES ENCODED THEREBY AND METHODS OF USE THEREOF

This Application Claims Priority To U.S. Provisional Application Serial

Numbers 60/026,603 Filed September 24, 1996 And 60/028,363 Filed October 1 1. 1996.

TECHNICAL FIELD The present invention relates to the field of diagnosing and treating conditions related to apoptosis, or programmed cell death. More specifically, it relates to the identification and characterization of a novel gene family, the expression of which is associated with apoptosis.

BACKGROUND OF THE INVENTION Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many ofthe conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death can be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and infection by human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun. 775:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al.

(1988) Biochem. Biophys. Res. Commun. 155:324-331 ; Kruman et al. (1991) ./ Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunology Today 12:102; and Sheppard and Ascher ( 1992) J. AIDS 5:143. Agents that modulate the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions. Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267. The blebs, small, membrane-encapsulated spheres that pinch off of the surface of apoptotic cells, may continue to produce superoxide radicals which damage surrounding cell tissue and may be involved in inflammatory processes.

While apoptosis is a normal cellular event, it can also be induced by pathological conditions and a variety of injuries. Apoptosis is involved in a wide variety of conditions including, but not limited to, cardiovascular disease; cancer regression; immunoregulation; viral diseases; anemia; neurological disorders; gastrointestinal disorders, including but not limited to, diarrhea and dysentery; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; and aging. Genes which have been shown to activate the apoptosis pathway in tumor cells include the FAS antigen, TNFα and TNFβ. See, e.g., Tomei and Cope et al in Apoptosis II: The Molecular Basis of Apoptosis in Disease (1994) Cold Spring Harbor Laboratory Press. In the nematode C. elegans, mutations in the genes ced- 3 and ced-4 prevent autonomous cell death during development. Yuan and Ilorvitz (1990) Dev. Biol. 138:33. A mutation which activates the nematode gene ced-9 prevents cell death during development, whereas mutations that inactive this gene promote programmed cell death. In mammalian cells, the p-53 gene has been shown to induce apoptosis in some cells, but not others.

Apoptosis-inhibiting genes under investigation include bcl-2 which was isolated from B-cell lymphomas and blocks apoptosis without affecting cell proliferation. See, e.g., Tsujimoto et al. Science 226: 1087; Hockenberry et al. (1990) Nature 348:334. The mechanism by which bcl-2 inhibits apoptosis is not known. Mcl-1, expressed in myeloid cells, exhibits sequence similarity to bcl-2 and is believed to be involved in regulating apoptosis. Kozopas et al. (1993) Proc. Natl. Acad. Sci. USA 90:3516.

Members of a large family of putative transmembrane receptors related to the Drosophila melanogaster tissue polarity g ne frizzled have been cloned recently. See, Wang et al. (1995) J. Biol. Chem. 271 :4468. Frizzled family members are found in organisms as diverse as nematodes and humans and are expressed in a variety of tissues and during embryonic development. In Drosophila, frizzled mutations affect the polarity of structures, such as sensory bristles, on the body surface. The precise functions and clinical significance of t e frizzled family in other species remains largely unknown.

All references cited herein, both supra and infra, are hereby incorporated by reference herein.

SUMMARY OF THE INVENTION The present invention encompasses isolated polynucleotides, polypeptides and antibodies derived from or reactive with the products ofthe novel apoptosis- related genes. The invention also encompasses uses of these compositions.

Accordingly, one aspect of the invention is polynucleotides encoding polypeptides of the SARP family. Representative polypeptides are those having the amino acid sequence of SEQ. ID. NO: 2, 4, 6 or 7. The invention likewise encompasses polynucleotides encoding peptides having substantial homology to the amino acid sequence of SEQ. ID. NO: 2, 4, 6 or 7.

In another aspect, the invention provides isolated polynucleotides that are comprised of a region of at least 15 contiguous nucleotides, where these nucleotides are capable of forming a stable duplex with a polynucleotide encoding sequence of SEQ. ID. NO: 1 , 3, 5 or 18.

Another aspect ofthe invention is cloning and expression vectors comprising the polynucleotides ofthe invention. Also included are host cells comprising the polynucleotides of the invention. In another aspect, the invention comprises polypeptides of at least 1 1 amino acid residues of SEQ. ID. NO: 2, 4, 6 or 7 and further comprises polypeptides substantially homologous to 1 1 amino acid residues of SEQ. ID. NO: 2, 4, 6 or 7. The invention also provides fusion polypeptides comprising a polypeptide ofthe present invention.

The invention also provides for polyclonal or monoclonal antibodies which specifically bind to the polypeptides of the invention. There are termed αSARP antibodies.

In another aspect, methods of detecting the polynucleotides of the invention are provided. These methods comprise contacting a biological sample under conditions that permit the formation of a stable complex, and detecting any stable complexes formed.

Another aspect ofthe invention is methods of detecting the SARP family of proteins. These methods entail the steps of contacting a biological sample obtained from an individual with an αSARP antibody of the invention under conditions that permit the stable antigen-antibody complex and detecting stable complex formed, if any.

Also provided are methods for treatment of apoptosis by administration of a therapeutically effective amount ofthe polynucleotides and/or polypeptides of the invention to a patient in need of such treatment. The methods include making a composition for treatment of conditions related to apoptosis. Other methods using these compositions include preventing apoptosis in cultured cells, methods of increasing organ preservation for subsequent organ transplantation and in situ preservation for bypass operations, e.g., heart, liver, lungs, brain, etc., and methods of treating dermatological conditions in which apoptosis is implicated.

Also provided are methods for the detection of disease by providing a test sample of bodily fluid; assaying the test sample for the presence of a gene product of an hsarp gene; and comparing the amount of gene product detected in the test sample to the amount of gene product detected in a non-diseased sample of the same tissue type as the test sample. Assaying encompasses, but is not limited to, nucleic acid hybridization and antibody - antigen interactions.

In an additional embodiment ofthe present invention, a method of treatment of a patient is provided, comprising administering to the patient a therapeutical ly effective amount of a pharmaceutically acceptable composition comprising a component selected from the group comprising a sarp or antisense- sarp polynucleotide or a SARP polypeptide or SARI³ antibody. The method can be a method of treating apoptosis related conditions. In a specific embodiment, the patient is suffering from a condition related to cancer, including, but not limited to, cancer ofthe mammary tissue, the prostate or the prostate epithelial tissue. In an additional embodiment, the composition contains a sarp polynucleotide or the gene product of that polynucleotide, a SARP polypeptide.

The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description and figures, wherein only the preferred embodiments ofthe invention are shown and described, simply by way of illustration ofthe best mode of carrying out the invention. As is readily recognized, the invention is capable of modifications within the skill ofthe relevant art without departing from the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A shows alignment of hSARP2 predicted amino acid sequence to frizzled proteins. [SEQ. ID. NOS: 7-9].

Figure IB shows a comparison ofthe amino acid sequence of mSARPl (SEQ. ID. NO: 2) to various frizzled proteins (SEQ. ID. NOS: 10-14).

Figure 2 is a Northern blot depicting tissue specific expression of sarpl in various mouse tissues. RNAs were isolated from different tissues resolved on 1.2% formaldehyde-agarose gel, transferred to nylon membrane and probed by msarpl at high stringency. Figure 3 A depicts the results of a Northern blot analysis of multiple human tissues with a probe specific for hsarp2.

Figure 3B is a compilation of Northern blots depicting tissue specific expression of hsarpl and hsarp3 in various human tissues. Multiple tissue northern blots were probed at high stringency conditions.

Figure 4 depicts the results of a Northern blot analysis of normal and transformed cell lines with a probe specific for hsarp2.

Figure 5 is a Northern blot depicting expression o msarpl in 10T1/2 quiescent cells after reseeding at low density. Figure 6, panels (A) through (C) show the percentage of viable transformed MCF7 cell lines after different treatments. MCF7 cells were transformed with either an expression vector (pcDNA3) or with pcDNΛ3 carrying the hsarp2 gene. Panel (A) shows the percentage of living cells after seven days of serum deprivation. Panel (B) shows the percentage of living cells after 24 hour treatment with adriamycin at 1 μg/ml. Panel (C) shows the percentage of living cells after 24 hour treatment with hTNF at 50 ng/ml. Panel (D) shows the relative amounts of hsarp2 expression in each of the MCF7 clones used in the experiments described in the Examples presented herein.

Figure 7 is a Northern blot of RNA isolated from rat cardiac myocytes after various treatments probed with msarpl cDNA fragment.

Figure 8 is 2 bar graphs depicting viability of the control, β-galactosidase, and msarpl transfected neonatal rat cardiac myocytes subjected for 24 hour to serum free medium or adriamycin treatment. The amount of infections virus particles per cell are shown in parentheses. Figure 9 is a series of graphs depicting (A) the effect of cycloheximide on

10T1/2 log and quiescent cell death induced by serum deprivation and (B) the effect of conditioned medium from quiescent cells on cells subjected to serum deprivation and cycloheximide treatment. Figure 10 depicts (A) graphs, (B) a Northern blot, and (C) a Western analysis. The graphs depict the effects of TNF and Ceramide on cell viability in the presence of SARPs. The Northern blot depicts control RNA from cells transfected by pcDNA3, RNA from cells transfected by msarpl or hsarpl recombinant vectors. The proteins of serum free conditioned media from 10T1/2 and MCF7 cells were concentrated by filtration and subjected to western analysis using anti-GST-mSARPl antisera (1 :5000 dilution).

Figure 1 1 depicts the comparison of hsarpl expression in human normal and neoplastic prostate epithelial cells at 1 OX and 40X magnifications. Figure 12 depicts the comparison of hsarp2 expression in human normal and neoplastic mammary epithelial cells at lOx and 40x magnifications.

Figure 13 depicts the detection by Western analysis of β-catenin in MCF7

cells transfected with pcDNA3, msarpl and hsarp2.

MODE(S) FOR CARRYING OUT THE INVENTION

Disclosed herein is a new gene family, the expression of which is associated with apoptosis. The genes are termed "sarp" (secreted apoptosis related protein), sarp genes are derived from murine sources whereas hsarp genes are derived from human sources. These genes, including msarpl, hsarpl, hsarpl and hsarp3, encode novel proteins which belong to the family of proteins termed "SARP". The hsarp2 gene is expressed in a variety of tissues. When hsarp2 was inserted into an expression vector and transfected into human cell lines, it increased the percentage of cells undergoing apoptosis in culture. The hsarp2 gene is expressed in exponentially growing non-transformed cell lines, and repressed in quiescent ones. Increased expression of hsarpl has been shown to increase programmed cell death in a breast carcinoma cell line in a dose dependent manner. A BLAST search of Gene Bank revealed significant homology between the novel gene family and members ofthe Frizzled Like" gene family (see Fig. IB, SEQ. ID. NOS: 10-14). Thefrizzled-like gene family encodes cell membrane proteins having seven transmembrane domains with unknown functions. It was previously shown that Wnt and frizzled proteins interact. Bhanot et al. (1996) Nature 382:225-230. Multiple sequence alignment to human frizzled- ike proteins showed that the novel family is most homologous in the extracellular N-terminal domains oifrizzled-lϊke proteins, with little homology in the transmembrane region. SARPs have now been shown to interfere with the Wnt-frizzled protein interaction and modify apoptosis by effecting cell-cell and cell-extracellular matrix signaling.

We have cloned a family of novel genes from mouse cells and from human heart and pancreas cDNA libraries. The expression of these genes is associated with the early stages of apoptosis. The mouse gene, termed msarpl, contains a single open reading frame which encodes a predicted protein product of 295 amino acids which is secreted, msarpl is expressed at high levels in heart, lung and is upregulatcd in cardiomyocytes subjected to injuries which trigger apoptosis. Transcription of msarpl is also significantly induced in 10T1/2 cells which reached quiescence, a state of arrested cell growth which is characterized by increased resistance to apoptotic stimuli.

The novel gene family also includes three human genes, termed hsarpl, hsarpl and hsarp3. hsarpl is closely homologous to msarpl and has one open reading frame (ORF) which encodes a 212 amino acid polypeptide, termed hSARPl . hsarpr. encodes a protein of 316 amino acids, termed hSARP3, which is homologous to hSARP2 and mSARPl . hSARPl is expressed at highest levels in colon, small intestine, pancreas and prostate. hSARP3 is expressed predominately in pancreas. The hsarpl cDNA sequence contains 1302 nucleotides and encodes a polypeptide of 314 amino acids having an N-terminal methionine and C-terminal lysine amino acid residues. The full length cDNA sequence includes 301 nucleotides ofthe 5' untranslated region and 62 nucleotides of 3' untranslated region. The hsarpl cDNA contains one major open reading frame (ORF) (hSARP2). The ATG start site is found at position 303, and the termination site is at position 1248. When hsarpl is inserted into an expression vector and transfected into human cell lines, it increases the percentage of cells that undergo apoptosis in culture. As used herein, "sarp" including msarpl hsarpl , hsarpl and hsarp3, refer to the nucleic acid molecules encoding the SARPs, and derivatives and complementary nucleotides thereof. "SARP" including mSARP, hSARPl, hSARP2 and hSARP3 refer to the proteins encoded thereby. Other members of the family can be obtained by the methods described in the Examples presented herein.

The present invention encompasses nucleotide sequences ofthe new gene family. The nucleotides include, but are not limited to, the cDNA, genome- derived DNA and synthetic or semi -synthetic DNA or RNA. The nucleotide sequence of msarpl is contained in SEQ. ID. NO: 1 ; the nucleotide sequence of hsarpl is contained in SEQ. ID. NO: 3, the sequence of hsarp3 is contained in

SEQ. ID. NO: 5, and the nucleotide sequence of hsarpl is contained in SEQ. ID. NO: 18. As described in the examples herein, the mRNA of this gene family has been detected in a variety of human organs and tissues by Northern blot analysis. Expression of hsarpl mRNA, for example, was detected in most human tissues probed; in exponentially growing human mammary nontransformed cells and in exponentially growing human normal diploid fibroblast cells.

The term "polynucleotide" is used to mean a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. The terms "polynucleotide" and "nucleotide" as used herein are used interchangeably. Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown. The term "polynucleotide" includes double-stranded, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment ofthe invention described herein that is a polynucleotide encompasses both the double- stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can be comprised of modified nucleotides, such as methylated nucleotides and nucleotide analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1 -methyladeninc, 1 -methylpseudouracil, 1- methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5- pentynyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a dcoxyribonucleic acid is also considered an analogous form of pyrimidine.

If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition arc, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms ofthe polynucleotide(s).

Further, any ofthe hydroxyl groups ordinarily present in the sugars can be replaced by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5' and 3' terminal hydroxy groups can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.

Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, but not limited to, 2^,-O- methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.

As noted above, one or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR₂ ("amidate"), P(O)R, P(O)OR\ CO or CH₂

("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing and ether (-O-) linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Although conventional sugars and bases will be used in applying the method ofthe invention, substitution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing a final product, as can alternative backbone structures like a polyamide backbone. An "antisense" polynucleotide is a sequence complementary to all or part of a functional RNA or DNA. For example, antisense RNA is complementary to sequences of the mRNA copied from the gene.

A "fragment" (also called a "region") of a polynucleotide (i.e., a polynucleotide encoding a sarp) is a polynucleotide comprised of at least 9 contiguous nucleotides of the novel genes. Preferred fragments are comprised of a region encoding at least 5 contiguous amino acid residues, more preferably, at least 10 contiguous amino acid residues, and even more preferably at least 15 contiguous amino acid residues. The term "recombinant" polynucleotide as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic in origin which, by virtue of its origin or manipulation: is not associated with all or a portion of a polynucleotide with which it is associated in nature; is linked to a polynucleotide other than that to which it is linked in nature; or does not occur in nature. The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The polymer can be linear or branched, it can comprise modified amino acid residues, and it can be interrupted by non-amino acid residues. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition arc, for example, polypeptides containing one or more analogs of an amino acid residue (including, for example, unnatural amino acid residues, etc.), as well as other modifications known in the art.

A polypeptide "fragment" (also called a "region") of a SARP is a polypeptide comprising an amino acid sequence of a SARP that has at least 5 contiguous amino acid residues of a sequence of a SARP, more preferably at least 8 contiguous amino acid residues, and even more preferably at least about 10 contiguous amino acid residues. For purposes of this invention, a fragment of a SARP can be identified and characterized by any of the following functions: (a) homology to a SARP; (b) ability to change a percentage of cells undergoing apoptosis; or (c) effect cell death. A SARP fragment can have any, more than one, or all ofthe above identified functions. Methods for determining these functions (a) through (c) will be described below.

A "fusion polypeptide" is a polypeptide comprising regions in a different position in the sequence than occurs in nature. The regions can normally exist in separate proteins and are brought together in the fusion polypeptide; or they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide.

A "functionally equivalent fragment" of a SARP polypeptide or sarp polynucleotide preserves at least one property and/or function of the SARP polypeptides or sarp polynucleotides. For example, the sequences can be varied by adding additional nucleotides or peptides as known in the art, such that the functionality ofthe sequence is not altered. Other examples are deletion and/or substitution of sequences. Alternatively, the sequences can be varied by substituting nucleotides or amino acid residue, or a combination of addition, deletion, or substitution. As is evident to one of skill in the art, functionality of a polypeptide sequence includes characteristics and/or activities ofthe sequence, such as antigenicity and effect on the apoptotic pathway. It is also clear that functionality of a polynucleotide sequence depends in part upon its intended use, and any functionality that is preserved in a fragment of a polynucleotide satisfies this definition. For instance, a "functionally equivalent fragment" of a sarp polynucleotide can be one in which an ability to hybridize is preserved, as the desired polynucleotide can be used as a probe. Alternatively, a "functionally equivalent fragment" of a sarp polynucleotide can mean that the polynucleotide encodes a fragment of a SARP that has a function associated with an intact SARP, and preferably a function associated with apoptosis modulation. A functionally equivalent fragment of the novel polypeptides or polynucleotide can have the same, enhanced, or decreased function when compared to the SARP polypeptides or polynucleotides. Other functions of SARP have been listed above. A functionally equivalent fragment has at least 9 nucleotides or at least 5 amino acids, preferably has at least 15 nucleotides or at least 10 amino acids, even more preferably has at least 25 nucleotides or at least 20 amino acids.

"Stringent conditions" for hybridization of both DNA/DNA and DNA/RNA are as described in Sambrook et al. (1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. Ed., Cold Spring Harbor Laboratory Press. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25°C, 37°C, 50°C, and 68°C; buffer concentrations of 10xSSC, 6xSSC, lxSSC (where SSC is 0.15M NaCl and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50% and 75%; incubation times from 5 minutes to 24 hours; 1 , 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6xSSC, lxSSC, O. lxSSC, or deionized water.

A "stable duplex" of polynucleotides, or a "stable complex" formed between any two or more components in a biochemical reaction, refers to a duplex or complex that is sufficiently long-lasting to persist between formation of the duplex or complex and subsequent detection, including any optional washing steps or other manipulation that can take place in the interim.

The term "antibody" refers to an immunoglobulin protein or antigen binding fragment that recognizes a particular antigen. Preferably, the antibodies of the present invention (termed αSARP) arc not specific to members of the

Frizzled family of proteins. Antibodies can be monoclonal or polyclonal. The generation and characterization of antibodies is within the skill of an ordinary artisan. The term "antibody" further encompasses proteins which have been coupled to another compound by chemical conjugation, or by mixing with an excipient or an adjuvant. The term antigen binding fragment includes any peptide that binds to the SARP in a specific manner. Typically, these derivatives include such immunoglobulin fragments as Fab, F(ab')2, Fab', scfv (both monomeric and polymeric forms) and isolated H and L chains. The term αSARP encompasses antigen binding fragments. An antigen binding fragment retains the specificity of the intact immunoglobulin, although avidity and/or affinity can be altered.

The antigen binding fragments (also termed "derivatives" herein) are typically generated by genetic engineering, although they can alternatively be obtained by other methods and combinations of methods. This classification includes, but is not limited to, engineered peptide fragments and fusion peptides.

Preferred compounds include polypeptide fragments ofthe CRDs, antibody fusion proteins comprising cytokine effector components, antibody fusion proteins comprising adjuvants or drugs, and single-chain V region proteins. Additionally, the antigen binding fragments of this invention can be used as diagnostic and imaging reagents.

Scfv can be produced either recombinantly or synthetically. For synthetic production of scfv, an automated synthesizer can be used. For recombinant production of scfv, a suitable plasmid containing polynucleotide that encodes the scfv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as Escherichia coli, and the expressed protein can be isolated using standard protein purification techniques. A particularly useful system for the production of scfvs is plasmid pET- 22b(+) (Novagen, Madison, WI) in E. coli. pET-22b(+) contains a nickel ion binding domain consisting of 6 sequential histidine residues, which allows the expressed protein to be purified on a suitable affinity resin. Another example of a suitable vector is pcDNA3 (Invitrogen, San Diego, CA), described above.

Conditions of expression should ensure that the scfv assumes optimal tertiary structure. Depending on the plasmid used (especially the activity ofthe promoter) and the host cell, it may be necessary to modulate the rate of production. For instance, use of a weaker promoter, or expression at lower temperatures, may be necessary to optimize production of properly folded scfv in prokaryotic systems; or, it may be preferably to express scfv in eukaryotic cells. The invention also encompasses antibodies conjugated to a chemically functional moiety. Typically, the moiety is a label capable of producing a detectable signal. These conjugated antibodies are useful, for example, in detection and imaging systems. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds substrate cofactors and inhibitors. See, for examples of patents teaching the use of such labels, U.S.

Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moieties can be covalently linked to the antibodies, recombinantly linked, or conjugated to the antibodies through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex. Methods of antibody production and isolation arc well known in the art.

See, for example, Harlow and Lane (19SS) Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Purification methods include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and cluomatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti- immunoglobulin. The antibodies can also be purified on affinity columns comprising a SARP protein; for example, in the form of a purified Abl or Ab3. Preferably, the antibodies can be purified using Protein- A-CL-Sepharose™ 4B chromatography followed by chromatography on a DEAE-Sepharose™ 4B ion exchange column.

A "cell line" or "cell culture" denotes higher eukaryotic cells grown or maintained in vitro. It is understood that the descendants of a cell may not be completely identical (either moφhologically, genotypically, or phenotypically) to the parent cell.

A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incoφoration of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in moφhology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention. A "vector" is a self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication of vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation ofthe DNA or RNA. Also included are vectors that provide more than one ofthe above functions. Suitable cloning vectors are known in the art e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are discussed for instance in Galesa and Ramji Vectors, John Wiley & Sons (1994). Examples of prokaryotic host cells appropriate for use in this invention include, but are not limited to, E. coli and Bacillus subtilis.

Examples of eukaryotic host cells include, but are not limited to, avian, insect, plant and animal cells such as C057, HeLa and CHO cells.

"Expression vectors" are defined as polynucleotides which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An "expression system" usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

A "signal sequence" is a short amino acid sequence that directs newly synthesized secretory or membrane proteins to and through cellular membranes such as the endoplasmic reticulim. Signal sequences are typically in the N- terminal portion of a polypeptide and are cleaved after the polypeptide has crossed the membrane.

A "gene product" encompasses any product or products of transcription or translation of a gene, including without limitation mRNAs, tRN As and proteins.

"Heterologous" means derived from (i.e., obtained from) a genotypically distinct entity from the rest ofthe entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, thus becoming a heterologous polynucleotide. A promoter which is linked to a coding sequence with which it is not naturally linked is a heterologous promoter.

The heterologous polynucleotide can comprise a sequence of interest for puφoses of therapy, and can optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors for the replication of a polynucleotide, and expression vectors for translation of a polynucleotide encoding sequence. Also included are viral vectors, which comprise a polynucleotide encapsidated or enveloped in a viral particle.

Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. While the cloning vector selected can vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self- replicate, can possess a single target for a particular restriction endonuclease, or can carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUCl 8, mpl8. mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Stratagene, and Invitrogen. Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding a polypeptide of interest. The polynucleotide encoding the polypeptide is operatively linked to suitable transcriptional controlling elements, such as promoters, enhancers and terminators. For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons. These controlling elements (transcriptional and translational) can be derived from the sarp genes, or they can be heterologous (i.e., derived from other genes or other organisms). A polynucleotide sequence encoding a signal peptide can also be included to allow a polypeptide to cross or lodge in cell membranes or be secreted from the cell.

A number of expression vectors suitable for expression in eukaryotic cells including yeast, avian, and mammalian cells are known in the art. One example of an expression vector is pcDNA3 (Invitrogen, San Diego, CA, in which transcription is driven by the cytomegalovirus (CMV) early promoter/enhancer.

This vector also contains recognition sites for multiple restriction enzymes for insertion ofthe polynucleotide of interest. Another example of an expression vector (system) is the baculovirus/insect system.

A vector of this invention can contain one or more polynucleotides encoding a polypeptide. It can also contain polynucleotide sequences encoding other polypeptides that enhance, facilitate, or modulate the desired result, such as lymphokines, including, but not limited to, IL-2, IL-4 and GM-CSF. A preferred lymphokine is GM-CSF. Preferred GM-CSF constructs are those which have been deleted for the AU-rich elements from the 3 ^* untranslated regions and sequences in the 5' untranslated region that are capable of forming a haiφin loop.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as vaccinia virus, which is discussed below). The choice of means of introducing vectors or polynucleotides will often depend features ofthe on the host cell. Once introduced into a suitable host cell, expression of a polypeptide can be determined using any assay known in the art. For example, presence of polypeptide can be detected by RIA or ELISA ofthe culture supernatant (if the polypeptide is secreted) or cell lysates.

An "isolated" or "purified" polynucleotide, polypeptide or antibody is one that is substantially free ofthe materials with which it is associated in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free ofthe materials with which it is associated in nature.

A biological "sample" encompasses a variety of sample types obtained from an individual and is typically used in a diagnostic procedure or assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimens or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term encompasses various kinds of clinical samples obtained from any species, and also includes, but is not limited to, cells in culture, cell supernatants, cell lysates, scrum, plasma, biological fluid, and tissue samples.

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For puφoses of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread (i.e., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

"Apoptosis-associated" refers to any condition in which the apoptosis pathway leading to cell death is involved. These conditions can be normal or pathogenic biological events and can be initiated by a wide variety of signals, including, but not limited to, hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and human immunodeficiency virus (HIV) infection.

Infarctions are caused by a sudden insufficiency of arterial or venous blood supply due to e boli, thrombi, or pressure that produces a macroscopic area of necrosis; the heart, brain, spleen, kidney, intestine, lung and testes are likely to be affected. Apoptosis occurs to tissues surrounding the infarct upon reperfusion of blood to the area; thus, modulation by a biological modifier-induced change in endogenous production or by in vivo transfection, could be effective at reducing the severity of damage caused by heart attacks and stroke.

Chemotherapeutic agents, ionizing radiation, and infection by HIV also initiate the apoptosis pathway. Currently, a variety of food supplements have been used in an attempt to ameliorate the gastrointestinal disorders that accompany chemotherapy, radiation and AIDS. These supplements generally contain carbohydrates, fats and plant protein hydrolysates. See, e.g. , Tomei and

Cope et al. in Apoptosis: The Molecular Basis of Cell Death (1991) Cold Spring Harbor Laboratory Press. PCT Publication No. WO 95/15173 describes plant- derived delipidated extracts capable of producing anti-apoptotic effect. Thus, affecting the molecular basis of apoptosis-associated conditions has therapeutic utility in numerous clinical situations.

"Antisense therapy" is a method of attenuating gene expression using a therapeutic polynucleotide. The therapeutic polynucleotide comprises a sequence or complementary sequence that is capable of forming a stable hybrid with either the target gene itself, or more typically the heteronuclear or messenger RNA transcribed therefrom. Typically, the therapeutic polynucleotide is operatively linked to a suitable promoter. The antisense polynucleotide need not be the exact complement ofthe target polynucleotide to be effective, so long as stable hybrids form under physiological conditions. A moderate number of mutations, insertions or deletions can be present, depending on the length ofthe antisense polynucleotide. The antisense polynucleotide need not hybridize with the entire target gene-coding sequence, although longer hybridizing regions are preferred over shorter ones. An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more doses, ln terms of treatment, an "effective amount" of polynucleotide, and/or polypeptide is an amount sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of apoptosis-associated disease states or otherwise reduce the pathological consequences of the disease. Detection and measurement of these indicators of efficacy are discussed below. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage. These factors include age, sex and weight of the patient, the condition being treated, the severity ofthe condition and the form of the antibody being administered. For instance, the concentration of scfv need not be as high as that of native antibodies in order to be therapeutically effective.

An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include farm and sport animals, and pets.

The invention thus includes isolated nucleotide encoding (or complementary thereto) polypeptides substantially identical to (i.e. having at least 90% sequence identity to) SARPs as exemplified by SEQ ID NOS: 2, 4, 6 and 7, with any amino acid substitutions preferably being conservative, or an allelic variant thereof, or to a homologue of SARP from a species other than man. The invention therefore includes, for example, either or both strands of a cDNA encoding a SARP or an allelic variant thereof; a recombinant DNA which is incoφorated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic cell; or genomic DNA fragments (e.g. produced by PCR or restriction endonuclease treatment of human or other genomic DNA). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide.

The isolated DNA can be incoφorated into a vector (e.g., a virus, phage or plasmid) which can be introduced by transfection or infection into a cell. Suitable vectors include any known in the art, including, but not limited to, those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors are known in the art and need not be described in detail herein. The vector can include one or more expression control sequences, in which case the cell transfected with the vector is capable of expressing the polypeptide. The vectors can also provide inducible promoters for expression of sarps. Inducible promoters are those which do not allow constitutive expression of the gene but rather, permit expression only under certain circumstances. Such promoters can be induced by a variety of stimuli including, but not limited to, exposure of a cell containing the vector to a ligand, metal ion, other chemical or change in temperature.

These promoters can also be cell-specific, that is, inducible only in a particular cell type and often only during a specific period of time. The promoter can further be cell cycle specific, that is, induced or inducible only during a particular stage in the cell cycle. The promoter can be both cell type specific and cell cycle specific. Any inducible promoter known in the art is suitable for use in the present invention.

Polynucleotides comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. Amplified DNA can be isolated from the host cell by standard methods. See, e.g., Sambrook et al. (1989). RNA can also be obtained from transformed host cell, it can be obtained by using an DNA- dependent RNA polymerase.

The invention includes modifications to sarp DNA sequences such as deletions, substitutions and additions particularly in the non-coding regions of genomic DNA. Such changes are useful to facilitate cloning and modify gene expression. Various substitutions can be made within the coding region that either do not alter the amino acid residues encoded or result in conservatively substituted amino acid residues. Nucleotide substitutions that do not alter the amino acid residues encoded are useful for optimizing gene expression in different systems. Suitable substitutions are known to those of skill in the art and are made, for instance, to reflect preferred codon usage in the particular expression systems. The invention encompasses functionally equivalent variants and derivatives of sarps which can enhance, decrease or not significantly affect the properties of SARPs. For instance, changes in the DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect its properties.

Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threoninc; lysine/arginine; and phenylalanine/tyrosine. Any conservative amino acid substitution which does not significantly affect the properties of SARPs is encompassed by the present invention.

Techniques for nucleic acid manipulation useful for the practice of the present invention are described in a variety of references, including but not limited to, Molecular Cloning: A Laboratory Manual, 2nd ed.. Vol. 1-3, eds. Sambrook et al. Cold Spring Harbor Laboratory Press (1989); and Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987) and periodic updates. Also within the invention is an isolated polynucleotide of at least 15 nucleotides in length, preferably at least 30, more preferably at least 100, and most preferably at least 500, including (a) DNA encoding a SARP, (b) the complement thereof; or a double stranded DNA including both (a) and (b). Multiple copies of this isolated DNA (useful, for example, as a hybridization probe or PCR primer) can be produced synthetically or by recombinant means, by transfecting a cell with a vector containing this DNA.

The invention also includes a purified preparations of SARP peptides, or fragments of these peptides that comprise an antigenic polypeptide containing at least 10 amino acid residues of the peptide (preferably at least 1 1, more preferably at least 14, and most preferably at least 18), which polypeptide fragment contains an epitope ofthe peptide such that an antibody raised against the fragment (or against a conjugate of the polypeptide and, if necessary, a carrier molecule) forms an immune complex with the peptide itself. Purification or isolation of SARPs expressed either by the recombinant DNA or from biological sources can be accomplished by any method known in the art. Generally, substantially purified proteins are those which are free of other, contaminating cellular substances, particularly proteins. Preferably, the purified peptides are more than eighty percent pure and most preferably more than ninety-five percent pure.

Suitable methods of protein purification are known in the art and include, but are not limited to, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, HPLC and FPLC. Any purification scheme that does not result in substantial degradation ofthe protein is suitable for use in the present invention. The invention further comprises suitable antibodies are generated by using a SARP as an antigen or, preferably, peptides encompassing regions of SARPs that lack substantial homology to the other gene products such as the Frizzled proteins. Such an antibody can either be polyclonal or monoclonal, and is generated by standard methods including the step of immunizing an animal with an antigen containing an antigenic portion of at least one SARP.

Also encompassed within the invention are hybrid polypeptides containing: (1 ) SARP or an antigenic fragment thereof, covalently attached to (2) a second polypeptide. Such hybrid polypeptides can be made by a number of standard techniques well known to those of ordinary skill, including recombinant methods, in which case the covalent attachment is a peptide bond, or chemical conjugation in which case the covalent attachment is another type of bond, such as a disulfide bond. Linking a SARP or an antigenic fragment thereof to a second polypeptide provides a means for readily isolating the hybrid from a mixture of proteins, by the use of an affinity column to which the second polypeptide (e.g. glutathione transferase) binds directly. Such hybrid polypeptides can also have the advantage of increased immunogenicity relative to SARP or a fragment thereof, so that antibodies are more readily obtained.

Both the isolated nucleotides of the invention and the antibodies ofthe invention are useful in detecting SARP expression. Any method for detecting specific mRNA species is suitable for use in this method. This is easily accomplished using PCR. Preferably, the primers chosen for PCR correspond to the regions ofthe sarp genes that lack substantial homology to other genes. Alternatively, Northern blots can be utilized to detect sarp mRNA by using probes specific to these genes. Methods of utilizing PCR and Northern blots are known in the art and are not described in detail herein.

Transgenic animals containing the sarp nucleotides are also encompassed by the invention. Methods of making transgenic animals are known in the art and need not be described in detail herein. For a review of methods used to make transgenic animals, see, e.g. PCT publication no. WO 93/04169. Preferably, such animals express recombinant sarps under control of a cell-specific and, even more preferably, a cell cycle specific promoter.

In another embodiment, diagnostic methods are provided to detect the expression ofthe novel gene family either at the protein level or the mRNA level.

Abnormal levels of SARPs are likely to be found in the tissues of patients with diseases associated with inappropriate apoptosis; diagnostic methods are therefore useful for detecting and monitoring biological conditions associated with such apoptosis defects. Detection methods are also useful for monitoring the success of SARP- related therapies. Both the isolated sarp nucleotides and the antibodies ofthe invention are useful in diagnostic methods. One such diagnostic method includes the steps of providing a test cell (e.g. in the form of a tissue section or a cell preparation) from a given type of tissue; contacting the mRNA of the test cell with a nucleic acid probe containing a sequence antisense (i.e. complementary to the sense strand of) a segment of a sarp gene. The segment is at least 15 nucleotides in length, preferably at least 20, more preferably at least 30, even more preferably at least 40 and most preferably at least 100 nucleotides in length. The amount of hybridization of the probe to the mRNA ofthe test cell is compared to the amount of hybridization ofthe probe to the mRNA of a normal control (i.e. non-apoptotic) cell from the same type of tissue. An increased amount of hybridization in the test cell is an indication that the test cell will have an increased incidence of apoptosis. The assay can be conveniently carried out using standard techniques of in situ hybridization or Northern analysis. The antibody-based assays ofthe invention are comparable to the above.

The proteins ofthe test cell, or from a fluid bathing the test cell, are contacted with an antibody (polyclonal or monoclonal) specific for a SARP, and the amount of immunocomplex formed with such proteins is compared with the amount formed by the same antibody with the proteins of a normal control cell (or fluid bathing a normal control cell) from the same type of tissue as the test cell.

In another embodiment, treatment of apoptosis-associated conditions are provided. The invention thus encompasses ex vivo transfection with the sarp gene family, in which cells removed from animals including man are transfected with vectors encoding SARPs or antisense sarps and reintroduccd into animals. Suitable transfected cells include individual cells or cells contained within whole tissues. In addition, ex vivo transfection can include the transfection of cells derived from an animal other than the animal or human subject into which the cells are ultimately introduced. Such grafts include, but are not limited to, allografts, xenografts, and fetal tissue transplantation.

The present invention also encompasses antisense therapy to attenuate levels of SARP. Antisense polynucleotides need not be the exact complement of the target polynucleotide to be effective, so long as stable hybrids form under physiological conditions. A moderate number of mutations, insertions or deletions can be present, depending on the length ofthe antisense polynucleotide. Preferably, the complementary sequence ofthe antisense polynucleotide is 50% identical to that ofthe target, including base differences, insertions, and deletions. More preferably, the sequences are about 75% identical; even more preferably they are about 85% identical; still more preferably they are about 95% identical; and most preferably, they are completely identical. The antisense polynucleotide need not hybridize with the entire SARP encoding sequence, although longer hybridizing regions are preferred over shorter ones. Preferably, the hybridizing region is at least about 30 bases in length; more preferably it is at least about 60 bases; even more preferably it is at least about 100 bases; more preferably it is at least about 200 bases or more.

Essentially any cell or tissue type can be treated in this manner. Suitable cells include, but are not limited to, cardiomyocytes and lymphocytes. As an example, in treatment of HIV-infected patients by the above-described method, the white blood cells are removed from the patient and sorted to yield the CD4 cells. The CD4 cells are then transfected with a vector encoding either SARP or antisense to sarp and reintroduced into the patient. Alternatively, the unsorted lymphocytes can be transfected with a recombinant vector having at least one •swyj-modulator under the control of a cell-specific promoter such that only CD4 cells express or down-regulate the sarp genes. In this case, an ideal promoter would be the CD4 promoter; however, any suitable CD4⁺ T cell-specific promoter can be used.

The practice ofthe present invention employs, unless otherwise indicated, conventional molecular biological techniques, which are within the skill ofthe art. See e.g.. "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed„ 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Wei & CC. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds.,

1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds.. 1994); "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991).

The following examples are provided to illustrate but not limit the present invention.

Example 1 Identification and Cloning of the sarp family cDNAs Cells and Tissues

All cell lines were obtained from the American Type Culture Collection (ATCC) and grown and maintained according to the supplier's recommendations.

Tissue specimens for an RNA isolation were taken from male 20 g BALB/c mice (Babko). The primary cardiomyocytes were prepared from hearts of a day-old Sprague Dawley rats according to a technique described by Simpson (1985). The ischemia was performed in a serum and glucose free RPMI media by incubating the cells during 8 hours at 37°C in an atmosphere of 95% N₂/5% CO₂. The postischemic reperfusion was stimulated by adding of fetal bovine serum (FBS) to 10%), glucose to 2g/L and placing the cells in 5% CO₂ at 37°C for 16 hours. For viral infection, the cells were incubated with appropriate amount ofthe infectious particles in serum free media at 37°C 2 hour. Then the medium was replaced by the regular growth medium (RPMI/10% FBS). The adenovirus titers were determined by limiting dilution and plaque assay using 293 cells exposed to the virus dilutions. The number of viruses capable to infect 80-90%) of cells was determined with the β-galactosidase virus infected cells and X-Gal (5-bromo-4- chloro-3-indolyl β-D-galactoside) staining.

Oligonucleotide Synthesis

Primers for DNA sequencing and PCR, adapters were synthesized on an Applied Biosystems model 394, gel purified and desalted using Sep-Pak C18 cartridges (Water Associates). A 14-mer (5' CCTGTAGATCTCCC 3', SEQ. ID. NO: 15) and an 18-mer (5' ATTTCGGAGATCTACAGG 3', SEQ. ID. NO: 16) oiigonucleotides were used with the EcoRI-Bglll adapter. For differential display reactions an arbitrary d(N10) and an anchored oligo(T) such as _TTTTTTTTTTTTTTTNS (SEQ. ID. NO: 17) were used. RNA isolation RNΛ from different cell lines and tissues was isolated using the guanidine- isothiocyanate method of Chomezinski and Sacchi (1987). RNA concentration was determined by spectrophotometry (Sambrook et al., 1989). 20 μg samples of total RNA were subjected to electrophoresis in a 1.2% agarose-formaldehyde gel (Sambrook et al., 1989) and visualized using ethidium bromide. RNA was then transferred using 10X SSC (lxSSC is 0.15M NaCl/0.015M Na-citrate) by diffusion onto a nylon membrane (Hybond N+, Amersham) according to the method of Lichtenstein et al. (1990). Membrane-bound RNA was crosslinked by UV-irradiation as recommended by the manufacturers. Differential display For differential display reactions the first strand cDNA was synthesized using 2 μg of total RNA isolated from either logarithmically growing or quiescent 10T1/2 cells. First strand synthesis was primed using an anchored oligo(dT) with Superscript Reverse Transcriptase (Gibco) according to the manufacturer's protocol. In PCR reactions, arbitrary d( 10) and anchored oligo(dT) primers were used. PCR conditions were essentially the same as published originally in Liang & Pardee, 1992. The PCR-amplified cDNA products were resolved on a 6% DNA sequencing gel (Sambrook et al., 1989). Differentially displayed bands were excised from the gel, reamplified using the same primers and conditions, and inserted into pCRScript (Stratagene).

Construction ofthe cDNA library

The mouse 10T1/2 fibroblast λZAP II based cDNA library was constructed essentially as described in (Zapf et al. 1990) with some modifications. Two 40 μl reaction mixtures were prepared containing 10 μg heat denatured poly(A+)RNA, 1 x First Strand Buffer (Gibco BRL), 10 mM DTT, 50 units of

RNase Block (Stratagene), 2 mM of each dATP, dCTP, dGTP and dTTP, 10 μCi [a- P]dCTP, 400 U Superscript Reverse Transcriptase II (Gibco). 2.5 μg oligo(dT) was added to one reaction mixture and 25 μg d(N6) to the other mixture. Both reaction mixtures were incubated for 1 hour at 42°C and terminated by heating at 65°C for 10 min. Second strand synthesis was performed by first adding 362 μL H₂0.80 μL of 5x second strand reaction buffer (100 mM Tris-HCl pH(7.5), 500 mM KCl, 25 mM MgCl₂, 50 mM DTT), and 1.5 μL of 15 mg/mL BSA to the first strand reactions. Second strand synthesis was initiated by- adding 12 μL of 10 U/μL E. coli DNA polymerase I (NEB) and 2.5 μL of 1 U/μL RNase H (Pharmacia). Reactions were incubated for 1 hour at 15°C, and 1 hour at room temperature. The two reactions, now double stranded cDNA, were combined and ligated to the EcoRI-Bglll adapters (Zapf et al. 1990). Low molecular weight cDNA species and unligated adapters were separated using Bio- Gel A- 15m chromatography (Bio Rad). The ligation of the cDNA to λZAP II/EcoRJ/CIAP (Stratagene) was carried out according to the manufacturer's instructions. Packaging and titration were performed essentially following to the supplier's instructions (Stratagene). A library of 8x10 independent recombinant clones was obtained. Cloning ofthe differentially displaced gene from mouse cells.

To isolate msarpl cDNA, the quiescent 10T1/2 cell library was screened using the PCR insert as a probe. Approximately 2.5x10 to 3.0x10 recombinant phages were plated in E. coli XL-Blue (Stratagene) and. transferred onto nitrocellulose filters (Millipore) according to the manufacturer's instructions. The DNA fragments were ³²P-labeled according to the method described in Feinberg and Vogelstein ( 984) Anal. Biochem. 137:266-267 and used to screen the library according to the method described in Keifer et al. (1991 ).

The largest clone, msarpl, was then chosen for further analysis. DNA sequencing of msarpl was performed by the Sanger & Nicholson dideoxynucleotide method, using M13 forward and internally specific primers.

The msarpl gene contains a single extended open reading frame encoding a predicted protein product of 295 amino acids (mSARPl). 252 bp of 5'- untranslated sequence and 891 bp of 3' -untranslated sequence with two putative polyadenylation signals positioned 637 bp and 234 bp from the 3'-end. Interestingly the 3' -untranslated region contains eleven conserved 3'-UTR/HMG motifs thought to be involved in posttranscriptional degradation of mRNA (Reeves et al., 1987). Global alignment of the msarpl sequence to Entrez (14.0) using the Mac Vector package revealed homology to genes encoding for the seven- transmembrane rat proteins homologs ofthe Drosophila melanogaster frizzled (fz) gene product. The msarpl gene does not have any transmembrane regions, and the C- terminal region is rich in basic amino acids, msarpl has one hydrophobic stretch, which may represent a signal sequence. Multiple alignments using Entrez and the NCBI gene sequence data banks showed strong homology between the N-terminal region of mSARPl and the extracellular parts of mouse (Figure IB), rat and human genes products. The C-terminal region of mSARPl contains several short polypeptide stretches which show homology to the sites of frizzled proteins positioned between the transmembrane regions. The EST database revealed a 400 bp DNA sequence isolated from a human breast cDNA library which showed 75% identity to msarp 1.

Cloning of human cDNAs

A human pancreas and human heart cDNA libraries were obtained from Clontech and screened using msarpl cDNA as a probe. Two cDNA clones, hsarpl and hsarp3, were recovered from the pancreas library and subjected to further analysis. One clone, hsarpl, was obtained from the human heart cDNA.

The hsarp2 cDNA sequence [SEQ ID NO: 18] contains 1302 nucleotides. The full length sequence includes 301 nucleotides ofthe 5' untranslated region and 62 nucleotides of 3' untranslated region. The hsarp2 cDNA contains one major ORF (1.SARP2). The ATG start site is found at position 303, and the termination site is at position 1248. The hsarp2 gene encodes a polypeptide of 314 amino acid residues with an N-terminal methionine and C-terminal lysine. Clone hsarpl is 890 nucleotides in length and encodes a polypeptide having about 95% homology to msarpl. The ATG of hsarpl is at position 203 and there is a putative signal peptide recognition site 23 amino acids downstream ofthe N-terminus. The hsarp3 clone is 1923 nucleotides and encodes a polypeptide 316 amino acids including a putative 28 amino acid secretion signal at the N-terminus. Example 2 Expression of novel genes in tissue types

Isolated DNA fragments were labeled with [³²P]dCTP (3000 Ci/mmol, Amersham) in a random priming reaction according to Feinberg and Vogelstein, (1982), supra. Hybridization was carried out according to the standard protocol described in Sambrook et al. (1989), supra. The membranes were washed two times with 2x SSC at room temperature for 30 minutes. Following two additional washes at 56°C in 0.1 x SSC, 0.1 % SDS, the membranes were autoradiographed onto a Kodak X-Omat films. Expression of msarpl in mouse tissue

To analyze msarpl expression in mouse tissues, Northern blots of various mouse tissues were prepared according to the standard protocol. The results are shown in Figure 2. High levels of expression were detected in mouse heart and lung. Detectable amounts of transcript were revealed also in kidney. No other mouse tissues expressed the RNA corresponding to msarpl . No expression of msarpl was detected in transformed cell lines FL5.12; WI-L2; S49; HT29; MCF7. Expression ofthe novel genes in human tissue

To determine expression ofthe sarp gene family in human tissues, Clontech human multiple tissue Northern blots were probed with labeled hsarpl , hsarp2, and hsarp3, as described above. Figures 3Λ (hsarpl) and 3B (hsarpl and hsarp3) show the tissue specific expression of hsarpl , hsarp2, and hsarp3.

The results indicate that hsarp2 is expressed in almost all tissue types analyzed (FIGURE 3A). Hybridization showed an RNA band sized approximately 5.0 kb. The highest levels of hsarpl expression were found in pancreas, colon, prostate and small intestine. Figure 3B. Lower levels of expression were detected in heart, brain, lung, skeletal muscle and prostate. Thymus. spleen, peripheral blood leukocytes, testis, ovary, placenta, liver, kidney and all fetal human tissues have faint or no signals. Hybridization to all tissue types except brain revealed two transcripts of 2.1 kb and 1.6 kb in length, probably reflecting an alternative utilization ofthe two polyadenylation signals identified in 3 '-UTR. hsaφ3 is expressed predominantly in pancreas, and has only one RNA transcript of 2.1 kb in size (Figure 3B). Expression of hsarp2 in several transformed and non transformed cell lines was analyzed. No hsarp2 expression was observed in all transformed cell line analyzed. The expression of hsarp2 is detectable in exponentially growing human mammary nontransformed cells and suppressed when the cells reach quiescent conditions (FIGURE 4). The same expression pattern of hsarp2 was seen in normal human diploid fibroblast cells.

Example 3 Expression of msarpl in 10T1/2 cells To determine differential expression of msarpl, transcription of the gene was evaluated in 10T1/2 cells. Significant induction of msarpl transcription was seen as the 10T1/2 cells reached quiescence (see Figure 5). Cells grown to quiescence were reseeded at low density in three plates. At different time points after reseeding, the cells from one of the plates were extracted for RNA isolation, the cells of second plate were used for cell cycle analysis and the third plate of cells deprived of serum for 24 hours to estimate the number of dead cells.

Figure 5 represents Northern hybridization ofthe differentially displayed DNA fragment to the RNA samples isolated from the 10T1/2 cells at different phases of growth: 1-3 - exponentially growing, 90 to 95%) confluent and quiescent (G₀) cells respectively; 4-6 - the quiescent cells were replated at lower density and harvested after 0, 2 and 6 hours, respectively. Figure 5 indicates that the message corresponding to msarpl disappears shortly after reseeding. Analysis ofthe second plate indicated that reseeded cells enter the cell cycle 16 hours after reseeding. No significant change in the number of dead cells was observed in the serum-deprived plates. These results suggest in the first 2-3 hours after low density reseeding quiescent cells produce an antiapoptotic factor or factors, in sufficient amounts to maintain typical quiescent cell resistance to serum deprivation.

Since it has previously been shown that media conditioned with exponentially growing 10T1/2 cells also prevents apoptosis, we also analyzed msarpl expression in serum deprived exponentially growing cells. RNA was isolated at different time points after removal of serum. Hybridization revealed significant induction of the msarpl message by the 16th hour after serum removal. No induction of msarpl was observed in cells grown in serum free media supplemented with TPA.

Example 4 Expression of msarpl after Ischemic injury to cardiomyocytes We had previously shown that ischemic injury to myocardial cells triggers apoptosis during repertusion. Further, we have also shown that the human clone, hsarpl , is expressed in adult heart tissue and not in fetal heart tissue. To determine msarpl expression relating to ischemic injury and apoptosis, cardiomyocyte cells were subjected to a variety of stressing stimuli. RNA isolated from these cells was electrophoresed and transferred to a membrane for hybridization. Blots probed with msarpl showed upregulation of msarpl in all stressed cells. As in the case of human fetal heart tissue, no RNA species corresponding to msarpl were found in unstressed, primary cardiomyocytes obtained from newborn rats.

Example 5 mSARPl peptide interacts with cell surface proteins mSARPl was stably transfected into MCF7 cells by first introducing a SacI fragment of msarpl into the EcoRV/Notl sites in pcDNA3. The pcDNA3 construct was then transfected into MCF7 cells using LipofectAMINE reagent (Gibco BRL) according to the manufacturer's instructions.

For indirect immunostaining, trypsinized cells were incubated with rabbit anti-mSARPl antisera at a 1 : 100 dilution for 1 hour at 4°C. The cells were washed three times with PBS supplemented with 1 % BSA and then incubated with 20 μg/mL FITC-labeled secondary antibodies (Boehringer Mannheim). The cells were analyzed on Becton-Dickinson FACS system, and the resulting data analyzed using CellQuest™ software (Becton Dickinson).

Example 6

Apoptotic Effects of hSARP2 The Notl/Xbal fragment of hsarp2 was inserted into the Notl/Xbal sites of the mammalian expression vector pcDNA3 (Invitrogen). MCF7 breast carcinoma cells were transfected with this construct using LipofectAMINE reagent (Gibco BRL) according to manufacturer^'s protocol. The percentage of living cells was estimated by counting the relative amount of adherent cells using a Coulter Counter (NZ). As shown in Figure 6, hsarp2 expression causes decrease in the percentage of viable cells. The cells were also treated with hTNF (50 ng/ml) and adriamycin (1 μg/ml). The results obtained are depicted in Figure 6.

Example 7 Effect of mSARPl on cardiomyocyte death RNA from rat neonatal primary cardiomyocytes was isolated after treatments inducing cell death, such as glucose, serum , or serum and glucose deprivation. Ischemia was simulated by placing the cells in oxygen and growth factor deprived condition for 8 hours followed by 16 hours of incubation in normal environment (referred to as a "reperfusion"). The Northern hybridization presented in Figure 7 show that saφl expression in the cells surviving these treatments is upregulated. In a second experiment, cardiomyocytes plated at high density were infected with recombinant viruses at a multiplicities of 50 and 100 infectious particles per cell. The msaφl containing recombinant adenovirus was constructed by subcloning ofthe corresponding cDNA Sad fragment into the Notl/EcoRV site of pAdLXR-1 adenoviral replication-deficient vector. The virus bearing β-galactosidase gene was used as a control. After the infection cells were subjected for 24 hours to serum deprivation or treatment with adriamycin. The cell viability was calculated as a percentage ofthe adherent cells, in experimental conditions, taken from those of control samples. The results presented in Figure 8 show that after serum deprivation or adriamycin treatment the amount of viable msaφl -virus infected cells is significantly higher than that for β-galactosidase infected or control, non infected cells.

Example 8 Effect of SARP expression on Apoptosis

C3H/10T1/2 cells were grown in Eagle's basal medium (BME) supplemented with 1 % heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified 5% CO₂ atmosphere without antibiotics. Cells were plated at 2x10 cells/mL and fed every 3-4 days. Approximately 2 weeks after the initial seeding, the cells were completely quiescent and few if any mitotic cells were present. To analyze the effect of serum deprivation or cycloheximide treatment, the exponentially proliferating (approximately 75% confluent) or quiescent cultures were transferred to serum-free medium or medium supplemented with 10 μg/mL cycloheximide. At 24 hours, the apoptotic (i.e. non-adherent) cells and the non-apoptotic (i.e. adherent) cells were collected separately and their amounts were evaluated using a cell counter (Coulter Counter ZM). Serum free conditioned medium was obtained after 24 hour incubation of quiescent lOT^'1/2 cells in BME. The RNA was isolated by the guanidine-isothiocyanate method described in Chomezinski and Sacchi (1987) Anal. Biochem. 162:156-59. 20 μg samples of total RNA were subjected to electrophoresis in a 1.1% agarose formaldehyde gel. Sambrook et al. (eds) (1989).

It has previously been shown that exponentially proliferating 10T1/2 cells are especially sensitive to scrum deprivation and die by apoptosis. Tomei et al. (1993) Proc. Natl. Acad. Sci. USA 90:853-857. Figure 9A shows that after 24 hours in a serum free medium, about 50% of the cells detach and are found to be apoptotic. When cell cultures reach density dependent quiescence, cells become resistant to withdrawal of growth factors and other serum components.

Similarly, quiescent cells are significantly more resistant to the cytotoxic effects of staurosporine, menadione and cis-platinum. These are pro-apoptotic agents that have differing mechanisms of action. During exponential proliferation apoptosis is delayed by the addition of cycloheximide. In contrast, inhibition of protein synthesis rapidly induces death in quiescent cells arrested in G₀ (Figure 9A). Apoptosis of G₀ is also induced by puromycin, as well as inhibition of RNA synthesis by actinomycin D or α-amanitin. These results imply that in quiescent

10T1/2 cultures, cells possess all components ofthe apoptotic pathway but activation is suppressed by quiescent state specific protein(s). This viewpoint is consistent with the observation that conditioned medium from quiescent 10T1/2 cells can inhibit apoptotic death of both serum deprived exponentially growing and cycloheximide treated quiescent 10T1/2 cells (Figure 9B). These results strongly suggest that the anti-apoptotic protein(s) is secreted from quiescent 10T1/2 cells and influences the response of neighboring cells.

To clone cDNA corresponding to this mRNA species, the 10T1/2 quiescent cells, human heart and pancreas cDNA libraries were screened using the differentially displayed DNA fragment as a probe. Four different recombinants were identified. Two of them screened from 10T1/2 and human pancreas were orthologous and designated as msarpl and hsarpl . The other two clones hsarpl and hsarp3, were obtained from the human heart and pancreas libraries, respectively. With the exception of hsarpl, these cDNA clones have a single extended open reading frame predicting full length proteins which share several common structural properties. Starting from the N-terminus, the hydrophobic putative signal peptides are followed by the mature protein sequences, 270-300 amino acids in length with 16 invariant cysteines. Of these, 10 cysteines are located in the N-terminal 110 to 120 amino acids segments which are 15-30% identical to the extracellular cysteine rich domain ("CRD") offrizzled-lϊke proteins. None ofthe hsarp group contains transmembrane regions which are characteristic of frizzled- ke proteins. Wang et al. (1996) J. Biol. Chem 271 :4468-4476. The partial polypeptide sequencing of hSARPl has revealed about 95%o identity with the mS ARP 1.

The MCF7 breast adenocarcinoma cell line was chosen as a model to study the involvement of SARP proteins in the processes of apoptosis. The programmed cell death of these cells induced by different agents has been well characterized. Zyed et al. (1994) Cancer Res. 54:825-831. This cell type does not express either sarpl or sarpl. MCF7 cells were stably transfected with a pcDNA3 mammalian expression vector bearing full length msarpl or hsarpl. The transfectants expressing msarpl and hsarpl were selected by Northern hybridization. The growth rate and cell cycle of transfected MCF7 cells were not significantly different from the parental cells; however, the results presented in Figure 10 (A) demonstrate that the expression of mSARPl and hSARP2 had opposite effects on cell sensitivity to cytotoxic stimuli. The expression of mSARPl resulted in higher resistance, expression of hSARP2 sensitized the cells to apoptosis induced by TNF and by ceramide, a secondary messenger in apoptotic pathways caused by various agents. Ilannun and Obeid (1995) T. Biochem. Sci. 20:73-7; and Kolesnick and Fuks (1995) J Exp. Med. 181 :1949-52.

Due to the fact that SARPs have the signal sequences but no transmembrane domains, it was believed that they are secreted proteins. This theory was tested as follows. Polyclonal anti-mSARPl antibodies were raised against the GST-mSARPl recombinant protein and affinity purified using MBP-mSARPl affinity column. Bacterial expression of GST-mSARPl and MBP-mSARPl fusion proteins was carried out using the pGEX-5X-2 (Pharmacia) and pMAL (NEB) vectors, respectively. For anti-hSARP2 antibodies a polypeptide derived from non-Frizzled-like C-terminal domain (167-185aa) (SEQ. ID. NO: 19) ofthe protein was used as an immunogen. Using the resultant affinity purified anti-mSARPl or anti-hSARP2 antibodies, the secreted proteins were detected in the conditioned media from both the transformed MCF7 cells and untransformed quiescent 10T1/2 (Figure 10 (C)). Notably, the mSARP antibodies fail to interact with hSARP2. The experiments described identify a new family of genes capable of modulating cellular apoptotic response to cytotoxic signals. It is important to note the high degree of sequence similarity between SARP CRDs and the similar regions of the frizzled proteins, a class of cellular membrane receptors with seven transmembrane domains. In Drosophila melanogaster, frizzled proteins are involved in regulation of bristle and hair polarity. Adler (1992) Cell 69: 1073-

1087. Recently, the ability of Dfz2, a. frizzled protein family member, to function as a receptor for Wingless protein was reported. Bhanot et al. (1996) Nature 382:225-230. Wingless is a member of Wnt gene family whose products are involved in cell-cell and cell-extracellular matrix interaction. Nussc and Varmus (1992) Cell 69:1073-1087. Secreted proteins SARPs are involved with regulation of Wntfrizzled protein interaction. From this viewpoint it is interesting that expression ofthe members of all three gene families,/πzz/e_ , Wnt and sarp, is tissue specific. Wang et al. (1996); Nusse and Varmus (1992); Gavin et al. (1990) Genes and Devel 4:2319-2332; and Chan et al. (1992) J. Biol Chem. 267:25202- 25207. The role of cell-cell and cell -extracellular matrix interaction in regulation of apoptosis is well documented. Rouslahti and Reed (1994) Cell 77:477-478; Bates et al. (1994) Cell. Biol. 125:403-415; and Boudreau et al. (1995) Science 267:891-893. Thus, among other functions all three families of genes are involved in the regulation of programmed cell death. Example 9 Comparison of hsarp expression in human normal and neoplastic cells

In this example, human normal and neoplastic tissues were evaluated for their expression of hsarp genes. Normal and neoplastic prostate epithelial tissues were assessed for hsarpl expression, and normal and neoplastic mammary tissues were assessed for hsarpl expression.

Experiments were performed as follows: First, digoxigenin (DIG) labeled hsarp RNA probes were obtained using RNA DIG labeling kit (Boerhinger Mannheim GmbH, Concord, CA) according to the protocol given in

Nonradioactive in Situ Hybridization Application Manual, Second Edition, 1996, p. 44. Then, 5 μm formalin-fixed, paraffin-embedded cancer tissue (prostate epithelial or mammary) sections were hybridized with the appropriate DIG labeled hsaφl or hsarp2 RNA probe. Finally, detection of mRNA was performed using a Genius kit (Boerhinger Mannheim GmbH, Concord, CA) according to the protocol given in Nonradioactive in Situ Hybridization Application Manual, Second Edition, 1996, p. 127.

Figures 11 (prostate epithelial tissue) and 12 (mammary tissue) show the results. Expression of hsaφl is elevated in prostate tumor cells as compared to the normal tissue control, as evidenced by the pervasive dark area in the 10X and

40X cancer sample as compared to the normal sample. Expression of hsaφ2 is suppressed in mammary tumor cells as compared to the normal tissue control. These results support the anti- and pro- apoptotic activity of hSARPl and hSARP2, respectively. This example shows that detection of saφ gene products in tissues can be used to diagnose a variety of diseases associated with the modulation of hsaφ expression, including cancers. Further, because hSARPs are secreted proteins, bodily fluid samples can also be used for such diagnostic puφoses. While this example specifically demonstrates the use of in situ hybridization using an mRNA probe for detection of saφ gene products, alternative methods of detecting the presence of amino acids or nucleic acids in both tissue and bodily fluid are well known in the art. Further, one skilled in these fields is capable of selecting appropriate probes for use in methods of the present invention based on the sequences disclosed herein or incoφorated by reference.

Example 10 Expression of SARPs modifies the intracellular levels of β-catenin. In the previous examples, it was shown that the sarp genes encode secreted proteins capable of modifying cell response to pro-apoptotic stimuli. This experiment evaluates the ability of SARP proteins to interfere with the Wnt- frizzled proteins signaling pathway. Recently, it was shown that frizzled proteins function as receptors for members ofthe Wnt protein family. Yang-Snyder et al. (1996) Curr Biol 6: 1302-6; Bhanot et al. (1996) Nature 382:225-30; Orsulic et al.

(1996) Current Biology 6: 1363-1267; and Perrimon (1996) Cell 86:513-516.

Interaction of Wnt family members with their respective frizzled receptor causes inactivation of glycogen synthase kinase 3β (GSK-3) or its Drosophila homologue Zw-3. Pai et al. (1997) Development 124:2255-66; Cook ct al. (1996) EMBO J. 15:4526-4536; and Siegfried et al. (1994) Nature 367:76-80. In the absence of Wnt, GSK-3B phosphorylates β-catenin (Armadillo is its Drosophila homologue). Phosphorylated β-catenin or Armadillo are degraded more rapidly than non-phosphorylated forms ofthe proteins. Perrimon (1996) Cell 86:513-516; Siegfried et al. (1994) Nature 367:76-80; Rubinfeld et al. (1996) Science 272: 1023-6; and Yost et al. (1996) Genes and Development 10: 1443-1454. As a result, Wnt signaling causes changes in intracellular concentration of β-catenin or Armadillo and this parameter has been used to register Wnt-frizzled proteins interaction and signal transduction. Bhanot et al. (1996) Nature 382:225-30. Because SARPs are soluble proteins possessing a domain homologous to CRD of frizzled proteins it was hypothesized that they functioned by interference with Wnt-frizzled protein interaction.

Recently it was shown that β-catenin accumulated in colon cancer (Korinek et al. (1997) Science 275:1784-7; and Morin et al. (1997) Science 275:1787-90); and melanomas (Rubinfeld et al. ( 1997) Science 275 : 1790-2), that had mutations in tumor suppressor APC. Moreover regulation of β-catenin is critical to APC's tumor suppressive effect. Morin ct al. (1997) Science l '5: 1787 '- 90. The results herein described show a correlation between the levels of β- catenin and the expression ofthe SARP family members which possess pro- or anti-apoptotic activity. A higher level of β-catenin in tumors is associated with a reduction in apoptotic cell death, a feature characteristic of carcinogenesis. Thompson (1995) Science 267:1456-1462.

To determine whether SARPs interfered with Wnt-frizzled protein interaction, the expression of β-catenin in MCF7-transfectants was compared. The experiment was performed as follows. Cell Cultures. MCF7 human breast adenocarcinoma cells were plated at 2x10^" cells/ml and cultured in Modified Eagle Medium (MEM) supplemented with 10% FBS. Serum free conditioned medium was obtained after 24 hour incubation of quiescent MCF7 cells in MEM. Transfection of MCF7. MCF7 cells were transfected with the pcDNA3 mammalian expression vector (Invitrogen), containing either no insert, msaφl, or hsarp2 cDNAs, using LipofectAMINE reagent (Gibco) according to manufacturer's protocol. Stable transfectants and two-three weeks later single cell originated clones were selected with 1 mg/ml G418 and expression ofthe respective genes was confirmed by Northern hybridization. Immunohistochemistry. Paraformaldehyde-fixed transfected MCF7 cells grown on 4-well Lab-Tek chamber slides were probed by anti-^β-catenin monoclonal IgG (Transduction Laboratories). Staining was performed by avidin- biotin-peroxydase system (Vector Laboratories) using diaminobenzidine as a substrate. IgG isolated from preimmune serum was used as a negative control. Western Immunoblot. For Western analysis the samples of conditioned media were concentrated using CENTRIPREP-10 concentrators (AMICON). Cells were harvested in extraction buffer consisting of 20 mM tris-HCl (pH 7.8), 5 mM MgCl₂, 250 mM sucrose, 1%> NP40. After 1 hour incubation on ice extracts were clarified by centrifugation. Protein concentrations ofthe cellular extracts were determined using DC Protein Assay kit (Bio Rad). Equal amount of proteins were subjected to SDS/PAGE (Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual (Second ed.) (CSHL Press), transferred onto nitrocellulose membranes and probed with the anti-GST-mSARPl polyclonal affinity purified IgG (1 μg/mL) or anti -β-catenin monoclonal IgG (Transduction Laboratories) .

The results appear in Figure 13, an image of a Western immunoblot which shows that expression of SARP2 decreases the intracellular concentration of β- catenin. The effect of SARP 1 on the levels of β-catenin is more complicated. Western blot was not sensitive enough to discern a significant difference between SARP 1 and the control, but immunohistochemical data revealed a higher concentration of β-catenin in the SARP1 transfectants. It is clear from these results that the expression of SARPs modifies the intracellular levels of β-catenin. supporting that SARPs interfere with Wnt-frizzled proteins signaling pathway. This example supports that sarp genes and their products can be used not only to diagnose a variety of diseases associated with the modulation of hsaφ expression, including cancers, but also to actively interfere with the action of these diseases on an intracellular level, and therefor to treat these diseases.

Further, the present invention encompasses methods of screening for potential therapeutic agents that modulate the interaction between SARP and Wnt- frizzled proteins by comparing the effect of SARPs on the Wnt-frizzled signaling pathway in the presence or absence ofthe therapeutic agent in question. Generally, such a drug screening assay can be performed by (a) combining a Wnt protein and a SARP protein under conditions in which they interact, to form a test sample; (b) exposing said test sample to a potential therapeutic agent and; (c) monitoring the interaction of the SARP protein and the frizzled protein; wherein, a potential therapeutic agent is selected for further study when it modifies the interaction compared to a control test sample to which no potential therapeutic agent has been added.

Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the descriptions and examples should not be construed as limiting the scope ofthe invention, which is delineated by the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(1) APPLICANT: Umansky, Samuil

Melkonyan , Hovsep

(ii) TITLE OF INVENTION: A FAMILY OF GENES ENCODING

APOPTOSIS-RELATED PEPTIDES; PEPTIDES ENCODED THEREBY AND METHODS OF USE THEREOF

(iil) NUMBER OF SEQUENCES: 19

(lv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: MORRISON & FOERSTER

(B) STREET: 755 Page Mill Road

(C) CITY: Palo Alto

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(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION-

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lehnhard , Susan K.

(B) REGISTRATION NUMBER: 33,943

(C) REFERENCE/DOCKET NUMBER: 23647-20018.00 dx) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (650) 813-5600

(B) TELEFAX: (650) 494-0792

(2) INFORMATION FOR SEQ ID NO : 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2030 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) ( ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 253..1137

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

AATTCGGAGA TCTACAGGCC TGTAGATCTC CGGCTCACTC TGCTCCCCCG GGTCGGAGCC 60

CCCCGGAGCT GCGCGCGGGC TTGCAGTGCC TTGCCCGCGC CGACCTCCCG GCGCCCGGCT 120

TCGCGCGTTC GGCCGCCCGC TGTCCAGAGC CCCCACGAGC AGAGCGAGGG AGTCCCGGAC 180

GAGCTCGAGC TCCGGCCGCC TCTCGCTTCC CCCGCTCGGC TCCCTCCGCC CCCCGGGGGT 240

CGCTAGTCCA CG ATG CCG CGG GGC CCT GCC TCG CTG CTG CTG CTA GTC 288

Met Pro Arg Gly Pro Ala Ser Leu Leu Leu Leu Val 1 5 10

CTC GCC TCG CAC TGC TGC CTG GGC TCG GCG CGT GGG CTC TTC CTC TTC 336 Leu Ala Ser His Cys Cys Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe 15 20 25

GGC CAG CCC GAC TTC TCC TAC AAG CGC ACG AAC TGC AAG CCC ATC CCC 384 Gly Gin Pro Asp Phe Ser Tyr Lys Arg Thr Asn Cys Lys Pro lie Pro 30 35 40

GCC AAC CTG CAG CTG TGC CAC GGC ATC GAG TAC CAG AAC ATG CGG CTG 432 Ala Asn Leu Gin Leu Cys His Gly lie Glu Tyr Gin Asn Met Arg Leu 45 50 55 60

CCC AAC CTG CTG GGC CAC GAG ACC ATG AAG GAG GTG CTG GAG CAG GCG 480 Pro Asn Leu Leu Gly His Glu Thr Met Lys Glu Val Leu Glu Gin Ala 65 70 75

GGC GCC TGG ATT CCG CTG GTC ATG AAG CAG TGC CAC CCG GAC ACC AAG 528 Gly Ala Trp lie Pro Leu Val Met Lys Gin Cys His Pro Asp Thr Lys 80 85 90

AAG TTC CTG TGC TCG CTC TTC GCC CCT GTC TGT CTC GAC GAC CTA GAT 576 Lys Phe Leu Cys Ser Leu Phe Ala Pro Val Cys Leu Asp Asp Leu Asp 95 100 105

GAG ACC ATC CAG CCG TGT CAC TCG CTC TGC GTG CAG GTG AAG GAC CGC 624 Glu Thr lie Gin Pro Cys His Ser Leu Cys Val Gin Val Lys Asp Arg 110 115 120

TGC GCC CCG GTC ATG TCC GCC TTC GGC TTC CCC TGG CCA GAC ATG CTG 672 Cys Ala Pro Val Met Ser Ala Phe Gly Phe Pro Trp Pro Asp Met Leu 125 130 135 140

GAG TGC GAC CGT TTC CCG CAG GAC AAC GAC CTC TGC ATC CCC CTC GCT 720 Glu Cys Asp Arg Phe Pro Gin Asp Asn Asp Leu Cys lie Pro Leu Ala 145 150 155 AGT AGC GAC CAC CTC CTG CCG GCC ACA GAG GAA GCT CCC AAG GTG TGT 768 Ser Ser Asp His Leu Leu Pro Ala Thr Glu Glu Ala Pro Lys Val Cys 160 165 170

GAA GCC TGC AAA ACC AAG AAT GAG GAC GAC AAC GAC ATC ATG GAA ACC 816 Glu Ala Cys Lys Thr Lys Asn Glu Asp Asp Asn Asp He Met Glu Thr 175 180 185

CTT TGT AAA AAT GAC TTC GCA CTG AAA ATC AAA GTG AAG GAG ATA ACG 864 Leu Cys Lys Asn Asp Phe Ala Leu Lys He Lys Val Lys Glu He Thr 190 195 200

TAC ATC AAC AGA GAC ACC AAG ATC ATC CTG GAG ACA AAG AGC AAG ACC 912 Tyr He Asn Arg Asp Thr Lys He He Leu Glu Thr Lys Ser Lys Thr 205 210 215 220

ATT TAC AAG CTG AAC GGC GTG TCC GAA AGG GAC CTG AAG AAA TCC GTG 960 He Tyr Lys Leu Asn Gly Val Ser Glu Arg Asp Leu Lys Lys Ser Val 225 230 235

CTG TGG CTC AAA GAC AGC CTG CAG TGC ACC TGT GAG GAG ATG AAC GAC 1008 Leu Trp Leu Lys Asp Ser Leu Gin Cys Thr Cys Glu Glu Met Asn Asp 240 245 250

ATC AAC GCT CCG TAT CTG GTC ATG GGA CAG AAG CAG GGC GGC GAA CTG 1056 He Asn Ala Pro Tyr Leu Val Met Gly Gin Lys Gin Gly Gly Glu Leu 255 260 265

GTG ATC ACC TCC GTG AAA CGG TGG CAG AAG GGC CAG AGA GAG TTC AAG 1104 Val He Thr Ser Val Lys Arg Trp Gin Lys Gly Gin Arg Glu Phe Lys 270 275 280

CGC ATC TCC CGC AGC ATC CGC AAG CTG CAA TGC TAGTTTCCCA GTGGGGTGGC 1157 Arg He Ser Arg Ser He Arg Lys Leu Gin Cys 285 290 295

TTCTCTCCAT CCAGGCCCTG AGCTCTGTAG ACCACTTGCC TCCGGACCTC ATTTCCGGTT 1217

TCCCAAGCAC AGTCCGGGAA AGCTACAGCC CCAGCTTGGA GCCGCTTGCC CTGCCTCCTG 1277

CATGTGTGTA TCCCTAACAT GTCCTGAGTT ATAAGGCCCT AGGAGGCCTT GGAAACCCAT 1337

AGCTGTTTTC ACGGAAAGCG AAAAGCCCAT CCAGATCTTG TACAAATATT CAAACTAATA 1397

AAATCATGAC TATTTT ATG AAGTTTTAGA ACAGCTCGTT TTAAGGTTAG TTTTGAATAG 1457

CTGTAGTACT TTGACCCGAG GGGCATTTTC TCTCTTTGGT CAGTCTGTTG GCTTATACCG 1517

TGCACTTAGG TTGCCATGTC AGGCGAATTG TTTCTTTTTT TTTTTTTTTT TCCCTCTGTG 1577

GTCTAAGCTT GTGGGTCCCA GACTTAGTTG AGATAAAGCT GGCTGTTATC TCAAAGTCTT 1637 CCTCAGTTCC AGCCTGAGAA TCGGCATCTA AGTCTTCAAA CATTTCGTTG CTCGTTTTAT 1697

GCCCTCATGA GCTCTGACCA TTGCATGCGT TCCCATCCCA GCTACAGAAC TTCAGTTTAT 1757

AAGCACACAG TAACCATTCC TCATTGCATG ATGCCCTCAA ATAAAAAGTG AATACAGTCT 1817

ATAAATTGAC GAGTATTTTA AGCTTTGTTT AAAACATCTT TTAATTCAAT TTTTTAATCA 1877

TTTTTTTTGC AAACTAAATC ATTGTAGCTT ACCTGTAATA TACGTAGTAG TTGACCTGGA 1937

AAAGTTGTAA AAATATTGCT TTAACCGACA CTGTAAATAT TTCAGATAAA CATTATATTC 1997

TTTGTATATA AACTCCTGTA GATCTCCGAA TTC 2030 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 295 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Pro Arg Gly Pro Ala Ser Leu Leu Leu Leu Val Leu Ala Ser His 1 5 10 15

Cys Cys Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe Gly Gin Pro Asp 20 25 30

Phe Ser Tyr Lys Arg Thr Asn Cys Lys Pro He Pro Ala Asn Leu Gin 35 40 45

Leu Cys His Gly He Glu Tyr Gin Asn Met Arg Leu Pro Asn Leu Leu 50 55 60

Gly His Glu Thr Met Lys Glu Val Leu Glu Gin Ala Gly Ala Trp He 65 70 75 80

Pro Leu Val Met Lys Gin Cys His Pro Asp Thr Lys Lys Phe Leu Cys 85 90 95

Ser Leu Phe Ala Pro Val Cys Leu Asp Asp Leu Asp Glu Thr He Gin 100 105 110

Pro Cys His Ser Leu Cys Val Gin Val Lys Asp Arg Cys Ala Pro Val 115 120 125

Met Ser Ala Phe Gly Phe Pro Trp Pro Asp Met Leu Glu Cys Asp Arg 130 135 140 Phe Pro Gin Asp Asn Asp Leu Cys He Pro Leu Ala Ser Ser Asp His 145 150 155 160

Leu Leu Pro Ala Thr Glu Glu Ala Pro Lys Val Cys Glu Ala Cys Lys 165 170 175

Thr Lys Asn Glu Asp Asp Asn Asp He Met Glu Thr Leu Cys Lys Asn 180 185 190

Asp Phe Ala Leu Lys He Lys Val Lys Glu He Thr Tyr He Asn Arg 195 200 205

Asp Thr Lys He He Leu Glu Thr Lys Ser Lys Thr He Tyr Lys Leu 210 215 220

Asn Gly Val Ser Glu Arg Asp Leu Lys Lys Ser Val Leu Trp Leu Lys 225 230 235 240

Asp Ser Leu Gin Cys Thr Cys Glu Glu Met Asn Asp He Asn Ala Pro 245 250 255

Tyr Leu Val Met Gly Gin Lys Gin Gly Gly Glu Leu Val He Thr Ser 260 265 270

Val Lys Arg Trp Gin Lys Gly Gin Arg Glu Phe Lys Arg He Ser Arg 275 280 285

Ser He Arg Lys Leu Gin Cys 290 295

(2) INFORMATION FOR SEQ ID NO : 3 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 870 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(lx) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 235..870

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

GGCTCATTCT GCTCCCCCGG GTCGGAGCCC CCCGGAGCTG CGCGCGGGCT TGCAGCGCCT 60

CGCCCGCGCT GTCCTCCCGG TGTCCCGCTT CTCCGCGCCC CAGCCGCCGG CTGCCAGCTT 120

TTCGGGGCCC CGAGTCGCAC CCAGCGAAGA GAGCGGGCCC GGGACAAGCT CGAACTCCGG 180 CCGCCTCGCC CTTAACCAGC TCCGTCCCTC TACCCCCTAG GGGTCGCGCC CACG ATG 237

Met

CTG CAG GGC CCT GGC TCG CTG CTG CTG CTC TTC CTC GCC TCG CAC TGC 285 Leu Gin Gly Pro Gly Ser Leu Leu Leu Leu Phe Leu Ala Ser His Cys 300 305 310

TGC CTG GGC TCG GCG CGC GGG CTC TTC CTC TTT GGC CAG CCC GAC TTC 333 Cys Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe Gly Gin Pro Asp Phe 315 320 325

TCC TAC AAG CGC AGC AAT TGC AAG CCC ATC CCG GCC AAC CTG CAG CTG 381 Ser Tyr Lys Arg Ser Asn Cys Lys Pro He Pro Ala Asn Leu Gin Leu 330 335 340

TGC CAC GGC ATC GAA TAC CAG AAC ATG CGG CTG CCC AAC CTG CTG GGC 429 Cys His Gly He Glu Tyr Gin Asn Met Arg Leu Pro Asn Leu Leu Gly 345 350 355 360

CAC GAG ACC ATG AAG GAG GTG CTG GAG CAG GCC GGC GCT TGG ATC CCG 477 His Glu Thr Met Lys Glu Val Leu Glu Gin Ala Gly Ala Trp He Pro 365 370 375

CTG GTC ATG AAG CAG TGC CAC CCG GAC ACC AAG AAG TTC CTG TGC TCG 525 Leu Val Met Lys Gin Cys His Pro Asp Thr Lys Lys Phe Leu Cys Ser 380 385 390

CTC TTC GCC CCC GTC TGC CTC GAT GAC CTA GAC GAG ACC ATC CAG CCA 573 Leu Phe Ala Pro Val Cys Leu Asp Asp Leu Asp Glu Thr He Gin Pro 395 400 405

TGC CAC TCT CGN TGC GTG CAG GTG AAG GAT CGC TGC GCC CCG GTC ATG 621 Cys His Ser Xaa Cys Val Gin Val Lys Asp Arg Cys Ala Pro Val Met 410 415 420

TCC GCC TTC GGC TTC CCC TGG CCC GAC ATG CTT GAG TGC GAC CGT TTC 669 Ser Ala Phe Gly Phe Pro Trp Pro Asp Met Leu Glu Cys Asp Arg Phe 425 430 435 440

CCC CAG GAC AAC GAC CTT TGC ATC CCC CTC GCT AGC AGC GAC CAC CTC 717 Pro Gin Asp Asn Asp Leu Cys He Pro Leu Ala Ser Ser Asp His Leu 445 450 455

CTG CCA GCC ACC GAG GAA GCT CCA AAG GTA TGT GAA GCC TGC AAA AAT 765 Leu Pro Ala Thr Glu Glu Ala Pro Lys Val Cys Glu Ala Cys Lys Asn 460 465 470

AAA AAT GAT GAT GAC AAC GAC ATA ATG GAA ACG CTT TGT AAA AAT GAT 813 Lys Asn Asp Asp Asp Asn Asp He Met Glu Thr Leu Cys Lys Asn Asp 475 480 485 TTT GCA CTG AAA ATA AAA GTG AAG GAG ATA ACC TAC ATC AAC CGT CGA 861 Phe Ala Leu Lys He Lys Val Lys Glu He Thr Tyr He Asn Arg Arg 490 495 500

CGC GGC CGC 870

Arg Gly Arg

505

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 212 ammo acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Leu Gin Gly Pro Gly Ser Leu Leu Leu Leu Phe Leu Ala Ser His 1 5 10 15

Cys Cys Leu Gly Ser Ala Arg Gly Leu Phe Leu Phe Gly Gin Pro Asp 20 25 30

Phe Ser Tyr Lys Arg Ser Asn Cys Lys Pro He Pro Ala Asn Leu Gin 35 40 45

Leu Cys His Gly He Glu Tyr Gin Asn Met Arg Leu Pro Asn Leu Leu 50 55 60

Gly His Glu Thr Met Lys Glu Val Leu Glu Gin Ala Gly Ala Trp He 65 70 75 80

Pro Leu Val Met Lys Gin Cys His Pro Asp Thr Lys Lys Phe Leu Cys 85 90 95

Ser Leu Phe Ala Pro Val Cys Leu Asp Asp Leu Asp Glu Thr He Gin 100 105 110

Pro Cys His Ser Xaa Cys Val Gin Val Lys Asp Arg Cys Ala Pro Val 115 120 125

Met Ser Ala Phe Gly Phe Pro Trp Pro Asp Met Leu Glu Cys Asp Arg 130 135 140

Phe Pro Gin Asp Asn Asp Leu Cys He Pro Leu Ala Ser Ser Asp His 145 150 155 160

Leu Leu Pro Ala Thr Glu Glu Ala Pro Lys Val Cys Glu Ala Cys Lys 165 170 175 Asn Lys Asn Asp Asp Asp Asn Asp He Met Glu Thr Leu Cys Lys Asn 180 185 190

Asp Phe Ala Leu Lys He Lys Val Lys Glu He Thr Tyr He Asn Arg 195 200 205

Arg Arg Gly Arg 210

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1984 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 216..1166

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

AAGCTTGATA TCGAATTCGC GGCCGCGTCG ACGGGAGGCG CCAGGATCAG TCGGGGCACC 60

CGCAGCGCAG GCTGCCACCC ACCTGGGCGA CCTCCGCGGC GGCGGCGGCG GCGGCTGGGT 120

AGAGTCAGGG CCGGGGGCGC ACGCCGGAAC ACCTGGGCCG CCGGGCACCG AGCGTCGGGG 180

GGCTGCGCGG CGCGACCCTG GAGAGGGCGC AGCCG ATG CGG GCG GCG GCG GCG 233

Met Arg Ala Ala Ala Ala

215

GCG GGG GGC GTG CGG ACG GCC GCG CTG GCG CTG CTG CTG GGG GCG CTG 281 Ala Gly Gly Val Arg Thr Ala Ala Leu Ala Leu Leu Leu Gly Ala Leu 220 225 230

CAC TGG GCG CCG GCG CGC TGC GAG GAG TAC GAC TAC TAT GGC TGG CAG 329 His Trp Ala Pro Ala Arg Cys Glu Glu Tyr Asp Tyr Tyr Gly Trp Gin 235 240 245 250

GCC GAG CCG CTG CAC GGC CGC TCC TAC TCC AAG CCG CCG CAG TGC CTT 377 Ala Glu Pro Leu His Gly Arg Ser Tyr Ser Lys Pro Pro Gin Cys Leu 255 260 265

GAC ATC CCT GCC GAC CTG CCG CTC TGC CAC ACG GTG GGC TAC AAG CGC 425 Asp He Pro Ala Asp Leu Pro Leu Cys His Thr Val Gly Tyr Lys Arg 270 275 280 ATG CGG CTG CCC AAC CTG CTG GAG CAC GAG AGC CTG GCC GAA GTG AAG 473

Met Arg Leu Pro Asn Leu Leu Glu His Glu Ser Leu Ala Glu Val Lys 285 290 295

CAG CAG GCG AGC AGC TGG CTG CCG CTG CTG GCC AAG CGC TGC CAC TCG 521

Gin Gin Ala Ser Ser Trp Leu Pro Leu Leu Ala Lys Arg Cys His Ser 300 305 310

GAT ACG CAG GTC TTC CTG TGC TCG CTC TTT GCG CCC GTC TGT CTC GAC 569

Asp Thr Gin Val Phe Leu Cys Ser Leu Phe Ala Pro Val Cys Leu Asp 315 320 325 330

CGG CCC ATC TAC CCG TGC CGC TCG CTG TGC GAG GCC GTG CGC GCC GGC 617

Arg Pro He Tyr Pro Cys Arg Ser Leu Cys Glu Ala Val Arg Ala Gly 335 340 345

TGC GCG CCG CTC ATG GAG GCC TAC GGC TTC CCC TGG CCT GAG ATG CTG 665

Cys Ala Pro Leu Met Glu Ala Tyr Gly Phe Pro Trp Pro Glu Met Leu 350 355 360

CAC TGC CAC AAG TTC CCC CTG GAC AAC GAC CTC TGC ATC GCC GTG CAG 713

His Cys His Lys Phe Pro Leu Asp Asn Asp Leu Cys He Ala Val Gin 365 370 375

TTC GGG CAC CTG CCC GCC ACC GCG CCT CCA GTG ACC AAG ATC TGC GCC 761

Phe Gly His Leu Pro Ala Thr Ala Pro Pro Val Thr Lys He Cys Ala 380 385 390

CAG TGT GAG ATG GAG CAC AGT GCT GAC GGC CTC ATG GAG CAG ATG TGC 809

Gin Cys Glu Met Glu His Ser Ala Asp Gly Leu Met Glu Gin Met Cys 395 400 405 410

TCC AGT GAC TTT GTG GTC AAA ATG CGC ATC AAG GAG ATC AAG ATA GAG 857

Ser Ser Asp Phe Val Val Lys Met Arg He Lys Glu He Lys He Glu 415 420 425

AAT GGG GAC CGG AAG CTG ATT GGA GCC CAG AAA AAG AAG AAG CTG CTC 905

Asn Gly Asp Arg Lys Leu He Gly Ala Gin Lys Lys Lys Lys Leu Leu 430 435 440

AAG CCG GGC CCC CTG AAG CGC AAG GAC ACC AAG CGG CTG GTG CTG CAC 953

Lys Pro Gly Pro Leu Lys Arg Lys Asp Thr Lys Arg Leu Val Leu His 445 450 455

ATG AAG AAT GGC GCG GGC TGC CCC TGC CCA CAG CTG GAC AGC CTG GCG 1001

Met Lys Asn Gly Ala Gly Cys Pro Cys Pro Gin Leu Asp Ser Leu Ala 460 465 470

GGC AGC TTC CTG GTC ATG GGC CGC AAA GTG GAT GGA CAG CTG CTG CTC 1049

Gly Ser Phe Leu Val Met Gly Arg Lys Val Asp Gly Gin Leu Leu Leu 475 480 485 490 ATG GCC GTC TAC CGC TGG GAC AAG AAG AAT AAG GAG ATG AAG TTT GCA 1097 Met Ala Val Tyr Arg Trp Asp Lys Lys Asn Lys Glu Met Lys Phe Ala 495 500 505

GTC AAA TTC ATG TTC TCC TAC CCC TGC TCC CTC TAC TAC CCT TTC TTC 1145 Val Lys Phe Met Phe Ser Tyr Pro Cys Ser Leu Tyr Tyr Pro Phe Phe 510 515 520

TAC GGG GCG GCA GAG CCC CAC TGAAGGGCAC TCCTCCTTGC CCTGCCAGCT 1196

Tyr Gly Ala Ala Glu Pro His 525

GTGCCTTGCT TGCCCTCTGG CCCCGCCCCA ACTTCCAGGC TGACCCGGCC CTACTGGAGG 1256

GTGTTTTCAC GAATGTTGTT ACTGGCACAA GGCCTAAGGG ATGGGCACGG AGCCCAGGCT 1316

GTCCTTTTTG ACCCAGGGGT CCTGGGGTCC CTGGGATGTT GGGCTTCCTC TCTCAGGAGC 1376

AGGGCTTCTT CATCTGGGTG AAGACCTCAG GGTCTCAGAA AGTAGGCAGG GGAGGAGAGG 1436

GTAAGGGAAA GGTGGAGGGG CTCAGGGCAC CCTGAGGCGG AGGTTTCAGA GTAGAAGGTG 1496

ATGTCAGCTC CAGCTCCCCT CTGTCGGTGG TGGGGCCTCA CCTTGAAGAG GGAAGTCTCA 1556

ATATTAGGCT AAGCTATTTG GGAAAGTTCT CCCCACCGCC CCTGTACGCG TCATCCTAGC 1616

CCCCCTTAGG AAAGGAGTTA GGGTCTCAGT GCCTCCAGCC ACACCCCCTG CCTTCCCCAG 1676

CTTGCCCATT TCCCTGCCCC AAGGCCCAGA GCTCCCCCCA GACTGGAGAG CAAGCCCAGC 1736

CCAGCCTCGG CATAGACCCC CTTCTGGTCC GCCCGTGGCT CGATTCCCGG GATTCATTCC 1796

TCAGCCTCTG CTTCTCCCTT TTATCCCAAT AAGTTATTGC TACTGCTGTG AGGCCATAGG 1856

TACTAGACAA CCAATACATG CAGGGTTGGG TTTTCTAATT TTTTTAACTT TTTAATTAAA 1916

TCAAAGGTCG ACGCGCGGCC GCGGAATTCC TGCAGCCCGG GGGATCCCCG GGTACCGAGC 1976

TCGAATTC 1984

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 317 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Met Arg Ala Ala Ala Ala Ala Gly Gly Val Arg Thr Ala Ala Leu Ala 1 5 10 15 Leu Leu Leu Gly Ala Leu His Trp Ala Pro Ala Arg Cys Glu Glu Tyr 20 25 30

Asp Tyr Tyr Gly Trp Gin Ala Glu Pro Leu His Gly Arg Ser Tyr Ser 35 40 45

Lys Pro Pro Gin Cys Leu Asp He Pro Ala Asp Leu Pro Leu Cys His 50 55 60

Thr Val Gly Tyr Lys Arg Met Arg Leu Pro Asn Leu Leu Glu His Glu 65 70 75 80

Ser Leu Ala Glu Val Lys Gin Gin Ala Ser Ser Trp Leu Pro Leu Leu 85 90 95

Ala Lys Arg Cys His Ser Asp Thr Gin Val Phe Leu Cys Ser Leu Phe 100 105 110

Ala Pro Val Cys Leu Asp Arg Pro He Tyr Pro Cys Arg Ser Leu Cys 115 120 125

Glu Ala Val Arg Ala Gly Cys Ala Pro Leu Met Glu Ala Tyr Gly Phe 130 135 140

Pro Trp Pro Glu Met Leu His Cys His Lys Phe Pro Leu Asp Asn Asp 145 150 155 160

Leu Cys He Ala Val Gin Phe Gly His Leu Pro Ala Thr Ala Pro Pro 165 170 175

Val Thr Lys He Cys Ala Gin Cys Glu Met Glu His Ser Ala Asp Gly 180 185 190

Leu Met Glu Gin Met Cys Ser Ser Asp Phe Val Val Lys Met Arg He 195 200 205

Lys Glu He Lys He Glu Asn Gly Asp Arg Lys Leu He Gly Ala Gin 210 215 220

Lys Lys Lys Lys Leu Leu Lys Pro Gly Pro Leu Lys Arg Lys Asp Thr 225 230 235 240

Lys Arg Leu Val Leu His Met Lys Asn Gly Ala Gly Cys Pro Cys Pro 245 250 255

Gin Leu Asp Ser Leu Ala Gly Ser Phe Leu Val Met Gly Arg Lys Val 260 265 270

Asp Gly Gin Leu Leu Leu Met Ala Val Tyr Arg Trp Asp Lys Lys Asn 275 280 285 Lys Glu Met Lys Phe Ala Val Lys Phe Met Phe Ser Tyr Pro Cys Ser 290 295 300

Leu Tyr Tyr Pro Phe Phe Tyr Gly Ala Ala Glu Pro His 305 310 315

( 2 ) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

Met Gly He Gly Arg Ser Glu Gly Gly Arg Arg Gly Ala Ala Leu Gly 1 5 10 15

Val Leu Leu Ala Leu Gly Ala Ala Leu Leu Ala Val Gly Ser Ala Ser 20 25 30

Glu Tyr Asp Tyr Val Ser Phe Gin Ser Asp He Gly Pro Tyr Gin Ser 35 40 45

Gly Arg Phe Tyr Thr Lys Pro Pro Gin Cys Val Asp He Pro Ala Asp 50 55 60

Leu Arg Leu Cys His Asn Val Gly Tyr Lys Lys Met Val Leu Pro Asn 65 70 75 80

Leu Leu Glu His Glu Thr Met Ala Glu Val Lys Gin Gin Ala Ser Ser 85 90 95

Trp Val Pro Leu Leu Asn Lys Asn Cys His Ala Gly Thr Gin Val Phe 100 105 110

Leu Cys Ser Leu Phe Ala Pro Val Cys Leu Asp Arg Pro He Tyr Pro 115 120 125

Cys Arg Trp Leu Cys Glu Ala Val Arg Asp Ser Cys Glu Pro Val Met 130 135 140

Gin Phe Phe Gly Phe Tyr Trp Pro Glu Met Leu Lys Cys Asp Lys Phe 145 150 155 160

Pro Glu Gly Asp Val Cys He Ala Met Thr Pro Pro Asn Pro Thr Glu 165 170 175

Ala Ser Lys Pro Gin Gly Thr Thr Val Cys Pro Pro Cys Asp Asn Glu 180 185 190 Leu Lys Ser Glu Ala He He Glu His Leu Cys Ala Ser Glu Phe Ala 195 200 205

Leu Arg Met Lys He Lys Glu Val Lys Lys Glu Asn Gly Asp Lys Lys 210 215 220

He Val Pro Lys Lys Lys Lys Pro Leu Lys Leu Gly Pro He Lys Lys 225 230 235 240

Lys Asp Leu Lys Lys Leu Val Leu Tyr Leu Lys Asn Gly Ala Asp Cys 245 250 255

Pro Cys His Gin Leu Asp Asn Leu Ser His His Phe Leu He Met Gly 260 265 270

Arg Lys Val Lys Ser Gin Tyr Leu Leu Thr Ala He His Lys Trp Asp 275 280 285

Lys Lys Asn Lys Glu Phe Lys Asn Phe Met Lys Lys Met Lys Asn His 290 295 300

Glu Cys Pro Thr Phe Gin Ser Val Phe Lys 305 310

(2) INFORMATION FOR SEQ ID NO : 8 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 565 amino acids

(B) TYPE: araino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 ^•

Met Arg Pro Arg Ser Ala Leu Pro Arg Leu Leu Leu Pro Leu Leu Leu 1 5 10 15

Leu Pro Ala Ala Gly Pro Ala Gin Phe His Gly Glu Lys Gly He Ser 20 25 30

He Pro Asp His Gly Phe Cys Gin Pro He Ser He Pro Leu Cys Thr 35 40 45

Asp He Ala Tyr Asn Gin Thr He Met Pro Asn Leu Leu Gly His Thr 50 55 60

Asn Gin Glu Asp Ala Gly Leu Glu Val His Gin Phe Tyr Pro Leu Val 65 70 75 80

Lys Val Gin Cys Ser Pro Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr 85 90 95 Ala Pro Val Cys Thr Val Leu Glu Gin Ala He Pro Pro Cys Arg Ser 100 105 110

He Cys Glu Arg Ala Arg Gin Gly Cys Glu Ala Leu Met Asn Lys Phe 115 120 125

Gly Phe Gin Trp Pro Glu Arg Leu Arg Cys Glu His Phe Pro Arg His 130 135 140

Gly Ala Glu Gin He Cys Val Gly Gin Asn His Ser Glu Asp Gly Ala 145 150 155 160

Pro Ala Leu Leu Thr Thr Ala Pro Pro Pro Gly Leu Gin Pro Gly Ala 165 170 175

Gly Gly Thr Pro Gly Gly Pro Gly Gly Gly Gly Ala Pro Pro Arg Tyr 180 185 190

Ala Thr Leu Glu His Pro Phe His Cys Pro Arg Val Leu Lys Val Pro 195 200 205

Ser Tyr Leu Ser Tyr Lys Phe Leu Gly Glu Arg Asp Cys Ala Ala Pro 210 215 220

Cys Glu Pro Ala Arg Pro Asp Gly Ser Met Phe Phe Ser Gin Glu Glu 225 230 235 240

Thr Arg Phe Ala Arg Leu Trp He Leu Thr Trp Ser Val Leu Cys Cys 245 250 255

Ala Ser Thr Phe Phe Thr Val Thr Thr Tyr Leu Val Asp Met Gin Arg 260 265 270

Phe Arg Tyr Pro Glu Arg Pro He He Phe Leu Ser Gly Cys Tyr Thr 275 280 285

Met Val Ser Val Ala Tyr He Ala Gly Phe Val Leu Gin Glu Arg Val 290 295 300

Val Cys Asn Glu Arg Phe Ser Glu Asp Gly Tyr Arg Thr Val Val Gin 305 310 315 320

Gly Thr Lys Lys Glu Gly Cys Thr He Leu Phe Met Met Leu Tyr Phe 325 330 335

Phe Ser Met Ala Ser Ser He Trp Trp Val He Leu Ser Leu Thr Trp 340 345 350

Phe Leu Ala Ala Gly Met Lys Trp Gly His Glu Ala He Glu Ala Asn 355 360 365 Ser Gin Tyr Phe His Leu Ala Ala Trp Ala Val Pro Ala Val Lys Thr 370 375 380

He Thr He Leu Ala Met Gly Gin He Asp Gly Asp Leu Leu Ser Gly 385 390 395 400

Val Cys Phe Val Gly Leu Asn Ser Leu Asp Pro Leu Arg Gly Phe Val 405 410 415

Leu Ala Pro Leu Phe Val Tyr Leu Phe He Gly Thr Ser Phe Leu Leu 420 425 430

Ala Gly Phe Val Ser Leu Phe Arg He Arg Thr He Met Lys His Asp 435 440 445

Gly Thr Lys Thr Glu Lys Leu Glu Arg Leu Met Val Arg He Gly Val 450 455 460

Phe Ser Val Leu Tyr Thr Val Pro Ala Thr He Val He Ala Cys Tyr 465 470 475 480

Phe Tyr Glu Gin Ala Phe Arg Glu His Trp Glu Arg Ser Trp Val Ser 485 490 495

Gin His Cys Lys Ser Leu Ala He Pro Cys Pro Ala His Tyr Thr Pro 500 505 510

Arg Met Ser Pro Asp Phe Thr Val Tyr Met He Lys Tyr Leu Met Thr 515 . 520 525

Leu He Val Gly He Thr Ser Gly Phe Trp He Trp Ser Gly Lys Thr 530 535 540

Leu His Ser Trp Arg Lys Phe Tyr Thr Arg Leu Thr Asn Ser Arg His 545 550 555 560

Gly Glu Thr Thr Val 565

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 585 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Ala Arg Pro Asp Pro Ser Ala Pro Pro Ser Leu Leu Leu Leu Leu 1 5 10 15 Leu Ala Gin Leu Val Gly Arg Ala Ala Ala Ala Ser Lys Ala Pro Val 20 25 30

Cys Gin Glu He Thr Val Pro Met Cys Arg Gly He Gly Tyr Asn Leu 35 40 45

Thr His Met Pro Asn Gin Phe Asn His Asp Thr Gin Asp Glu Ala Gly 50 55 60

Leu Glu Val His Gin Phe Trp Pro Leu Val Glu He Gin Cys Ser Pro 65 70 75 80

Asp Leu Arg Phe Phe Leu Cys Thr Met Tyr Thr Pro He Cys Leu Pro 85 90 95

Asp Tyr His Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110

Lys Ala Gly Cys Ser Pro Leu Met Arg Gin Tyr Gly Phe Ala Trp Pro 115 120 125

Glu Arg Met Ser Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135 140

Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro 145 150 155 160

Arg Pro Phe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly Ala Pro 165 170 175

Ala Ser Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe Val Cys Lys Cys 180 185 190

Arg Glu Pro Phe Val Pro He Leu Lys Glu Ser His Pro Leu Tyr Asn 195 200 205

Lys Val Arg Thr Gly Gin Val Pro Asn Cys Ala Val Pro Cys Tyr Gin 210 215 220

Pro Ser Phe Ser Ala Asp Glu Arg Thr Phe Ala Thr Phe Trp He Gly 225 230 235 240

Leu Trp Ser Val Leu Cys Phe He Ser Thr Ser Thr Thr Val Ala Thr 245 250 255

Phe Leu He Asp Met Asp Thr Phe Arg Tyr Pro Glu Arg Pro He He 260 265 270

Phe Leu Ser Ala Cys Tyr Leu Cys Val Ser Leu Gly Phe Leu Val Arg 275 280 285 Leu Val Val Gly His Ala Ser Val Ala Cys Ser Arg Glu His Asn His 290 295 300

He His Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr He Val Phe Leu 305 310 315 320

Leu Val Tyr Phe Phe Gly Met Ala Ser Ser He Trp Trp Val He Leu 325 330 335

Ser Leu Thr Trp Phe Leu Ala Ala Ala Met Lys Trp Gly Asn Glu Ala 340 345 350

He Ala Gly Tyr Gly Gin Tyr Phe His Leu Ala Ala Trp Leu He Pro 355 360 365

Ser Val Lys Ser He Thr Ala Leu Ala Leu Ser Ser Val Asp Gly Asp 370 375 380

Pro Val Ala Gly He Cys Tyr Val Gly Asn Gin Asn Leu Asn Ser Leu 385 390 395 400

Arg Arg Phe Val Leu Gly Pro Leu Val Leu Tyr Leu Leu Val Gly Thr 405 410 415

Leu Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg He Arg Ser Val 420 425 430

He Lys Gin Gly Gly Thr Lys Thr Asp Lys Leu Glu Lys Leu Met He 435 440 445

Arg He Gly He Phe Thr Leu Leu Tyr Thr Val Pro Ala Ser He Val 450 455 460

Val Ala Cys Tyr Leu Tyr Glu Gin His Tyr Arg Glu Ser Trp Glu Ala 465 470 475 480

Ala Leu Thr Cys Ala Cys Pro Gly His Asp Thr Gly Gin Pro Arg Ala 485 490 495

Lys Pro Glu Tyr Trp Val Leu Met Leu Lys Tyr Phe Met Cys Leu Val 500 505 510

Val Gly He Thr Ser Gly Val Trp He Trp Ser Gly Lys Thr Val Glu 515 520 525

Ser Trp Arg Arg Phe Thr Ser Arg Cys Cys Cys Arg Pro Arg Arg Gly 530 535 540

His Lys Ser Gly Gly Ala Met Ala Ala Gly Asp Tyr Pro Glu Ala Ser 545 550 555 560

Ala Ala Leu Thr Gly Arg Thr Gly Pro Pro Gly Pro Ala Ala Thr Tyr 565 570 575 His Lys Gin Val Ser Leu Ser His Val 580 585

(2) INFORMATION FOR SEQ ID NO: 10-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 666 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Ala Val Ser Trp He Val Phe Asp Leu Trp Leu Leu Thr Val Phe 1 5 10 15

Leu Gly Gin He Gly Gly His Ser Leu Phe Ser Cys Glu Pro He Thr 20 25 30

Leu Arg Met Cys Gin Asp Leu Pro Tyr Asn Thr Thr Phe Met Pro Asn 35 40 45

Leu Leu Asn His Tyr Asp Gin Gin Thr Ala Ala Leu Ala Met Glu Pro 50 55 60

Phe His Pro Met Val Asn Leu Asp Cys Ser Arg Asp Phe Arg Pro Phe 65 70 75 80

Leu Cys Ala Leu Tyr Ala Pro He Cys Met Glu Tyr Gly Arg Val Thr 85 90 95

Leu Pro Cys Arg Arg Leu Cys Gin Arg Ala Tyr Ser Glu Cys Ser Lys 100 105 110

Leu Met Glu Met Phe Gly Val Pro Trp Pro Glu Asp Met Glu Cys Ser 115 120 125

Arg Phe Pro Asp Cys Asp Glu Pro Tyr Pro Arg Leu Val Asp Leu Asn 130 135 140

Leu Val Gly Asp Pro Thr Glu Gly Ala Pro Val Ala Val Gin Arg Asp 145 150 155 160

Tyr Gly Phe Trp Cys Pro Arg Glu Leu Lys He Asp Pro Asp Leu Gly 165 170 175

Tyr Ser Phe Leu His Val Arg Asp Cys Ser Pro Pro Cys Pro Asn Met 180 185 190 Tyr Phe Arg Arg Glu Glu Leu Ser Phe Ala Arg Tyr Phe He Gly Leu 195 200 205

He Ser He He Cys Leu Ser Ala Thr Leu Phe Thr Phe Leu Thr Phe 210 215 220

Leu He Asp Val Thr Arg Phe Arg Tyr Pro Glu Arg Pro He He Phe 225 230 235 240

Tyr Ala Val Cys Tyr Met Met Val Ser Leu He Phe Phe He Gly Phe 245 250 255

Leu Leu Glu Asp Arg Val Ala Cys Asn Ala Ser Ser Pro Ala Gin Tyr 260 265 270

Lys Ala Ser Thr Val Thr Gin Gly Ser His Asn Lys Ala Cys Thr Met 275 280 285

Leu Phe Met Val Leu Tyr Phe Phe Thr Met Ala Gly Ser Val Trp Trp 290 295 300

Val He Leu Thr He Thr Trp Phe Leu Ala Ala Val Pro Lys Trp Gly 305 310 315 320

Ser Glu Ala He Glu Lys Lys Ala Leu Leu Phe His Ala Ser Ala Trp 325 330 335

Gly He Pro Gly Thr Leu Thr He He Leu Leu Ala Met Asn Lys He 340 345 350

Glu Gly Asp Asn He Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Val 355 360 365

Asp Ala Leu Arg Tyr Phe Val Leu Ala Pro Leu Cys Leu Tyr Val Val 370 375 380

Val Gly Val Ser Leu Leu Leu Ala Gly He He Ser Leu Asn Arg Val 385 390 395 400

Arg He Glu He Pro Leu Glu Lys Glu Asn Gin Asp Lys Leu Val Lys 405 410 415

Phe Met He Arg He Gly Val Phe Ser He Leu Tyr Leu Val Pro Leu 420 425 430

Leu Val Val He Gly Cys Tyr Phe Tyr Glu Gin Ala Tyr Arg Gly He 435 440 445

Trp Glu Thr Thr Trp He Gin Glu Arg Cys Arg Glu Tyr His He Pro 450 455 460

Cys Pro Tyr Gin Val Thr Gin Met Ser Arg Pro Asp Leu He Leu Phe 465 470 475 480 Leu Met Lys Tyr Leu Met Ala Leu He Val Gly He Pro Ser He Phe 485 490 495

Trp Val Gly Ser Lys Lys Thr Cys Phe Glu Trp Ala Ser Phe Phe His 500 505 510

Gly Arg Arg Lys Lys Glu He Val Asn Glu Ser Arg Gin Val Leu Gin 515 520 525

Glu Pro Asp Phe Ala Gin Ser Leu Leu Arg Asp Pro Asn Thr Pro He 530 535 540

He Arg Lys Ser Arg Gly Thr Ser Thr Gin Gly Thr Ser Thr His Ala 545 550 555 560

Ser Ser Thr Gin Leu Ala Met Val Asp Asp Gin Arg Ser Lys Ala Gly 565 570 575

Ser Val His Ser Lys Val Ser Ser Tyr His Gly Ser Leu His Arg Ser 580 585 590

Arg Asp Gly Arg Tyr Thr Pro Cys Ser Tyr Arg Gly Met Glu Glu Arg 595 600 605

Leu Pro His Gly Ser Met Ser Arg Leu Thr Asp His Ser Arg His Ser 610 615 620

Ser Ser His Arg Leu Asn Glu Gin Ser Arg His Ser Ser He Arg Asp 625 630 635 640

Leu Ser Asn Asn Pro Met Thr His He Thr His Gly Thr Ser Met Asn 645 650 655

Arg Val He Glu Glu Asp Gly Thr Ser Ala 660 665

(2) INFORMATION FOR SEQ ID NO: 11

(l) SEQUENCE CHARACTERISTICS -

(A) LENGTH: 537 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Ala Trp Pro Gly Thr Gly Pro Ser Ser Arg Gly Ala Pro Gly Gly 1 5 10 15 Val Gly Leu Arg Leu Gly Leu Leu Leu Gin Phe Leu Leu Leu Leu Arg 20 25 30

Pro Thr Leu Gly Phe Gly Asp Glu Glu Glu Arg Arg Cys Asp Pro He 35 40 45

Arg He Ala Met Cys Gin Asn Leu Gly Tyr Asn Val Thr Lys Met Pro 50 55 60

Asn Leu Val Gly His Glu Leu Gin Thr Asp Ala Glu Leu Gin Leu Thr 65 70 75 80

Thr Phe Thr Pro Leu He Gin Tyr Gly Cys Ser Ser Gin Leu Gin Phe 85 90 95

Phe Leu Cys Ser Val Tyr Val Pro Met Cys Thr Glu Lys He Asn He 100 105 110

Pro He Gly Pro Cys Gly Gly Met Cys Leu Ser Val Lys Arg Arg Cys 115 120 125

Glu Pro Val Leu Arg Glu Phe Gly Phe Ala Trp Pro Asp Thr Leu Asn 130 135 140

Cys Ser Lys Phe Pro Pro Gin Asn Asp His Asn His Met Cys Met Glu 145 150 155 160

Gly Pro Gly Asp Glu Glu Val Pro Leu Pro His Lys Thr Pro He Gin 165 . 170 175

Pro Gly Glu Glu Cys His Ser Val Gly Ser Asn Ser Asp Gin Tyr He 180 185 190

Trp Val Lys Arg Ser Leu Asn Cys Val Leu Lys Cys Gly Tyr Asp Ala 195 200 205

Gly Leu Tyr Ser Arg Ser Ala Lys Glu Phe Thr Asp He Trp Met Ala 210 215 220

Val Trp Ala Ser Leu Cys Phe He Ser Thr Thr Phe Thr Val Leu Thr 225 230 235 240

Phe Leu He Asp Ser Ser Arg Phe Ser Tyr Pro Glu Arg Pro He He 245 250 255

Phe Leu Ser Met Cys Tyr Asn He Tyr Ser He Ala Tyr He Val Arg 260 265 270

Leu Thr Val Gly Arg Glu Arg He Ser Cys Asp Phe Glu Glu Ala Ala 275 280 285

Glu Pro Val Leu He Gin Glu Gly Leu Lys Asn Thr Gly Cys Ala He 290 295 300 He Phe Leu Leu Met Tyr Phe Phe Gly Met Ala Ser Ser He Trp Trp 305 310 315 320

Val He Leu Thr Leu Thr Trp Phe Leu Ala Ala Gly Leu Lys Trp Gly 325 330 335

His Glu Ala He Glu Met His Ser Ser Tyr Phe His He Ala Ala Trp 340 345 350

Ala He Pro Ala Val Lys Thr He Val He Leu He Met Arg Leu Val 355 360 365

Asp Ala Asp Glu Leu Thr Gly Leu Cys Tyr Val Gly Asn Gin Asn Leu 370 375 380

Asp Ala Leu Thr Gly Phe Val Val Ala Pro Leu Phe Thr Tyr Leu Val 385 390 395 400

He Gly Thr Leu Phe He Ala Ala Gly Leu Val Ala Leu Phe Lys He 405 410 415

Arg Ser Asn Leu Gin Lys Asp Gly Thr Lys Thr Asp Lys Leu Glu Arg 420 425 430

Leu Met Val Lys He Gly Val Phe Ser Val Leu Tyr Thr Val Pro Ala 435 440 445

Thr Cys Val He Ala Cys Tyr Phe Tyr Glu He Ser Asn Trp Ala Leu 450 455 460

Phe Arg Tyr Ser Ala Asp Asp Ser Asn Met Ala Val Glu Met Leu Lys 465 470 475 480

He Phe Met Ser Leu Leu Val Gly He Thr Ser Gly Met Trp He Trp 485 490 495

Ser Ala Lys Thr Leu His Thr Trp Gin Lys Cys Ser Asn Arg Leu Val 500 505 510

Asn Ser Gly Lys Val Lys Arg Glu Lys Arg Gly Asn Gly Trp Val Lys 515 520 525

Pro Gly Lys Gly Asn Glu Thr Val Val 530 535

(2) INFORMATION FOR SEQ ID NO : 12 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 709 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Glu Arg Ser Pro Phe Leu Leu Ala Cys He Leu Leu Pro Leu Val 1 5 10 15

Arg Gly His Ser Leu Phe Thr Cys Glu Pro He Thr Val Pro Arg Cys 20 25 30

Met Lys Met Thr Tyr Asn Met Thr Phe Phe Pro Asn Leu Met Gly His 35 40 45

Tyr Asp Gin Gly He Ala Ala Val Glu Met Gly His Phe Leu His Leu 50 55 60

Ala Asn Leu Glu Cys Ser Pro Asn He Glu Met Phe Leu Cys Gin Ala 65 70 75 80

Phe He Pro Thr Cys Thr Glu Gin He His Val Val Leu Pro Cys Arg 85 90 95

Lys Leu Cys Glu Lys He Val Ser Asp Cys Lys Lys Leu Met Asp Thr 100 105 110

Phe Gly He Arg Trp Pro Glu Glu Leu Glu Cys Asn Arg Leu Pro His 115 120 125

Cys Asp Asp Thr Val Pro Val Thr Ser His Pro His Thr Glu Leu Ser 130 135 140

Gly Pro Gin Lys Lys Ser Asp Gin Val Pro Arg Asp He Gly Phe Trp 145 150 155 160

Cys Pro Lys His Leu Arg Thr Ser Gly Asp Gin Gly Tyr Arg Phe Leu 165 170 175

Gly He Glu Gin Cys Ala Pro Pro Cys Pro Asn Met Tyr Phe Lys Ser 180 185 190

Asp Glu Leu Asp Phe Ala Lys Ser Phe He Gly He Val Ser He Phe 195 200 205

Cys Leu Cys Ala Thr Leu Phe Thr Phe Leu Thr Phe Leu He Asp Val 210 215 220

Arg Arg Phe Arg Tyr Pro Glu Arg Pro He He Tyr Tyr Ser Val Cys 225 230 235 240

Tyr Ser He Val Ser Leu Met Tyr Phe Val Gly Phe Leu Leu Gly Asn 245 250 255 Ser Thr Ala Cys Asn Lys Ala Asp Glu Lys Leu Glu Leu Gly Asp Thr 260 265 270

Val Val Leu Gly Ser Lys Asn Lys Ala Cys Ser Val Val Phe Met Phe 275 280 285

Leu Tyr Phe Phe Thr Met Ala Gly Thr Val Trp Trp Val He Leu Thr 290 295 300

He Thr Trp Phe Leu Ala Ala Gly Arg Lys Trp Ser Cys Glu Ala He 305 310 315 320

Glu Gin Lys Ala Val Trp Phe His Ala Val Ala Trp Gly Ala Pro Gly 325 330 335

Phe Leu Thr Val Met Leu Leu Ala Met Asn Lys Val Glu Gly Asp Asn 340 345 350

He Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Leu Asp Ala Ser Arg 355 360 365

Tyr Phe Val Leu Leu Pro Leu Cys Leu Cys Val Phe Val Gly Leu Ser 370 375 380

Leu Leu Leu Ala Gly He He Ser Leu Asn His Val Arg Gin Val He 385 390 395 400

Gin His Asp Gly Arg Asn Gin Glu Lys Leu Lys Lys Phe Met He Arg 405 410 415

He Gly Val Phe Ser Gly Leu Tyr Leu Val Pro Leu Val Thr Leu Leu 420 425 430

Gly Cys Tyr Val Tyr Glu Leu Val Asn Arg He Thr Trp Glu Met Thr 435 440 445

Trp Phe Ser Asp His Cys His Gin Tyr Arg He Pro Cys Pro Tyr Gin 450 455 460

Ala Asn Pro Lys Ala Arg Pro Glu Leu Ala Leu Phe Met He Lys Tyr 465 470 475 480

Leu Met Thr Leu He Val Gly He Ser Ala Val Phe Trp Val Gly Ser 485 490 495

Lys Lys Thr Cys Thr Glu Trp Ala Gly Phe Phe Lys Arg Asn Arg Lys 500 505 510

Arg Asp Pro He Ser Glu Ser Arg Arg Val Leu Gin Glu Ser Cys Glu 515 520 525

Phe Phe Leu Lys His Asn Ser Lys Val Lys His Lys Lys Lys His Gly 530 535 540 Ala Pro Gly Pro His Arg Leu Lys Val He Ser Lys Ser Met Gly Thr 545 550 555 560

Ser Thr Gly Ala Thr Thr Asn His Gly Thr Ser Ala Met Ala He Ala 565 570 575

Asp His Asp Tyr Leu Gly Gin Glu Thr Ser Thr Glu Val His Thr Ser 580 585 590

Pro Glu Ala Ser Val Lys Glu Gly Arg Ala Asp Arg Ala Asn Thr Pro 595 600 605

Ser Ala Lys Asp Arg Asp Cys Gly Glu Ser Ala Gly Pro Ser Ser Lys 610 615 620

Leu Ser Gly Asn Arg Asn Gly Arg Glu Ser Arg Ala Gly Gly Leu Lys 625 630 635 640

Glu Arg Ser Asn Gly Ser Glu Gly Ala Pro Ser Glu Gly Arg Val Ser 645 650 655

Pro Lys Ser Ser Val Pro Glu Thr Gly Leu He Asp Cys Ser Thr Ser 660 665 670

Gin Ala Ala Ser Ser Pro Glu Pro Thr Ser Leu Lys Gly Ser Thr Ser 675 680 685

Leu Pro Val His Ser Ala Ser Arg Ala Arg Lys Glu Gin Gly Ala Gly 690 695 700

Ser His Ser Asp Ala 705

(2) INFORMATION FOR SEQ ID NO .13 :

(l) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 572 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

Met Arg Gly Pro Gly Thr Ala Ala Ser His Ser Pro Leu Gly Leu Cys 1 5 10 15

Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Pro Thr Asp Thr Arg Ala 20 25 30 Gin Pro Tyr His Gly Glu Lys Gly He Ser Val Pro Asp His Gly Phe 35 40 45

Cys Gin Pro He Ser He Pro Leu Cys Thr Asp He Ala Tyr Asn Gin 50 55 60

Thr He Leu Pro Asn Leu Leu Gly His Thr Asn Gin Glu Asp Ala Gly 65 70 75 80

Leu Glu Val His Gin Phe Tyr Pro Leu Val Lys Val Gin Cys Ser Pro 85 90 95

Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110

Leu Asp Gin Ala He Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120 125

Gin Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gin Trp Pro Glu 130 135 140

Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu He Cys 145 150 155 160

Val Gly Gin Asn Thr Ser Asp Gly Ser Gly Gly Ala Gly Gly Ser Pro 165 170 175

Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Pro Pro Phe Thr Ala 180 185 190

Met Ser Pro Ser Asp Gly Arg Gly Arg Leu Ser Phe Pro Phe Ser Cys 195 200 205

Pro Arg Gin Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe Leu Gly 210 215 220

Glu Arg Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn Gly Leu 225 230 235 240

Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu Trp Val Gly 245 250 255

Val Trp Ser Val Leu Ser Cys Ala Ser Thr Leu Phe Thr Val Leu Thr 260 265 270

Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu Arg Pro He He 275 280 285

Phe Leu Ser Gly Cys Tyr Phe Met Val Ala Val Ala His Val Ala Gly 290 295 300

Phe Leu Leu Glu Asp Arg Ala Val Cys Val Glu Arg Phe Ser Asp Asp 305 310 315 320 Gly Tyr Arg Thr Val Ala Gin Gly Thr Lys Lys Glu Gly Cys Thr He 325 330 335

Leu Phe Met Val Leu Tyr Phe Phe Gly Met Ala Ser Ser He Trp Trp 340 345 350

Val He Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly 355 360 365

His Glu Ala He Glu Ala Asn Ser Gin Tyr Phe His Leu Ala Ala Trp 370 375 380

Ala Val Pro Ala Val Lys Thr He Thr He Leu Ala Met Gly Gin Val 385 390 395 400

Asp Gly Asp Leu Leu Ser Gly Val Cys Tyr Val Gly Leu Ser Ser Val 405 410 415

Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe Val Tyr Leu Phe 420 425 430

He Gly Thr Ser Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg He 435 440 445

Arg Thr He Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu Glu Lys 450 455 460

Leu Met Val Arg He Gly Val Phe Ser Val Leu Tyr Thr Val Pro Ala 465 470 475 480

Thr He Val Leu Ala Cys Tyr Phe Tyr Glu Gin Ala Phe Arg Glu His 485 490 495

Trp Glu Arg Thr Trp Leu Leu Gin Thr Cys Lys Ser Tyr Ala Val Pro 500 505 510

Cys Pro Pro Arg His Phe Ser Pro Met Ser Pro Asp Phe Thr Val Phe 515 520 525

Met He Lys Tyr Leu Met Thr Met He Val Gly He Thr Thr Gly Phe 530 535 540

Trp He Trp Ser Gly Lys Thr Leu Gin Ser Trp Arg Arg Phe Tyr His 545 550 555 560

Arg Leu Ser His Ser Ser Lys Gly Glu Thr Ala Val 565 570

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 685 amino acids (B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: peptide

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu

1 5 10 15

Ala Val Leu Gin Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys Glu 20 25 30

Leu Ala Cys Gin Glu He Thr Val Pro Leu Cys Lys Gly He Gly Tyr 35 40 45

Asn Tyr Thr Tyr Met Pro Asn Gin Phe Asn His Asp Thr Gin Asp Glu 50 55 60

Ala Gly Leu Glu Val His Gin Phe Trp Pro Leu Val Glu He Gin Cys 65 70 75 80

Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro He Cys 85 90 95

Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105 110

Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gin Tyr Gly Phe Ala 115 120 125

Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gin Gly Asn Pro 130 135 140

Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala 145 150 155 160

Pro Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Pro Gly Glu Gin 165 170 175

Pro Pro Ser Gly Ser Gly His Ser Arg Pro Pro Gly Ala Arg Pro Pro 180 185 190

His Arg Gly Gly Ser Ser Arg Gly Ser Gly Asp Ala Ala Ala Ala Pro 195 200 205

Pro Ser Arg Gly Gly Lys Ala Arg Pro Pro Gly Gly Gly Ala Ala Pro 210 215 220

Cys Glu Pro Gly Cys Gin Cys Arg Ala Pro Met Val Ser Val Ser Ser 225 230 235 240 Glu Arg His Pro Leu Tyr Asn Arg Val Lys Thr Gly Gin He Ala Asn 245 250 255

Cys Ala Leu Pro Cys His Asn Pro Phe Phe Ser Gin Asp Glu Arg Ala 260 265 270

Phe Thr Val Phe Trp He Gly Leu Trp Ser Val Leu Cys Phe Val Ser 275 280 285

Thr Phe Ala Thr Val Ser Thr Phe Leu He Asp Met Glu Arg Phe Lys 290 295 300

Tyr Pro Glu Arg Pro He He Phe Leu Ser Ala Cys Tyr Leu Phe Val 305 310 315 320

Ser Val Gly Tyr Leu Val Arg Leu Val Ala Gly His Glu Lys Val Ala 325 330 335

Cys Ser Gly Gly Ala Pro Gly Ala Gly Gly Arg Gly Gly Ala Gly Gly 340 345 350

Ala Ala Ala Ala Gly Ala Gly Ala Ala Gly Arg Gly Ala Ser Ser Pro 355 360 365

Gly Ala Arg Gly Glu Tyr Glu Glu Leu Gly Ala Val Glu Gin His Val 370 375 380

Arg Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr Val Val Phe Leu Leu 385 390 395 400

Val Tyr Phe Phe Gly Met Ala Ser Ser He Trp Trp Val He Leu Ser 405 410 415

Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly Asn Glu Ala He 420 425 430

Ala Gly Tyr Ser Gin Tyr Phe His Leu Ala Ala Trp Leu Val Pro Ser 435 440 445

Val Lys Ser He Ala Val Leu Ala Leu Ser Ser Val Asp Gly Asp Pro 450 455 460

Val Ala Gly He Cys Tyr Val Gly Asn Gin Ser Leu Asp Asn Leu Arg 465 470 475 480

Gly Phe Val Leu Ala Pro Leu Val He Tyr Leu Phe He Gly Thr Met 485 490 495

Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg He Arg Ser Val He 500 505 510

Lys Gin Gin Gly Gly Pro Thr Lys Thr His Lys Leu Glu Lys Leu Met 515 520 525 He Arg Leu Gly Leu Phe Thr Val Leu Tyr Thr Val Pro Ala Ala Val 530 535 540

Val Val Ala Cys Leu Phe Tyr Glu Gin His Asn Arg Pro Arg Trp Glu 545 550 555 560

Ala Thr His Asn Cys Pro Cys Leu Arg Asp Leu Gin Pro Asp Gin Ala 565 570 575

Arg Arg Pro Asp Tyr Ala Val Phe Met Leu Lys Tyr Phe Met Cys Leu 580 585 590

Val Val Gly He Thr Ser Gly Val Trp Val Trp Ser Gly Lys Thr Leu 595 600 605

Glu Ser Trp Arg Ala Leu Cys Thr Arg Cys Cys Trp Ala Ser Lys Gly 610 615 620

Ala Ala Val Gly Ala Gly Ala Gly Gly Ser Gly Pro Gly Gly Ser Gly 625 630 635 640

Pro Gly Pro Gly Gly Gly Gly Gly His Gly Gly Gly Gly Gly Ser Leu 645 650 655

Tyr Ser Asp Val Ser Thr Gly Leu Thr Trp Arg Ser Gly Thr Ala Ser 660 665 670

Ser Val Ser Tyr Pro Lys Gin Met Pro Leu Ser Gin Val 675 680 685

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CCTGTAGATC TCCC 14

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:16:

ATTTCGGAGA TCTACAGG 18 (2) INFORMATION FOR SEQ ID NO: 17:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(XI ) SEQUENCE DESCRIPTION: SEQ ID NO: 17: _{ττχτττττττ TTTTTNS 17}

(2) INFORMATION FOR SEQ ID NO: 18:

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 1308 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GAATTCCGGT CCGGAGTCAG TGCCGCGCGC CCGCCGCCCC GCGCCTTCCT GCTCGCCGCA 60

CCTCCGGGAG CCGGGGCGCA CCCAGCCCGC AGCGCCGCCT CCCCGCCCGC GCCGCCTCCG 120

ACCGCAGGCC GAGGGCCGCC ACTGGCCGGG GGGACCGGGC AGCAGCTTGC GGCCGCGGAG 180

CGGGCAACGC TGGGGACTGC GCCTTTTGTC CCCGGAGGTC CCTGGAAGTT TGCGGCAGGA 240

CGCGCGCGGG GAGGCGGCGG AGGCAGCCCC GACGTCGCGG AGAACAGGGC GCAGAGCCGG 300

CATGGGCATC GGGCGCAGCG AGGGGGGCCG CCGCGGGGCA GCCCTGGGCG TGCTGCTGGC 360

GCTGGGCGCG GCGCTTCTGG CCGTGGGCTC GGCCAGCGAG TACGACTACG TGAGCTTCCA 420

GTCGGACATC GGCCCGTACC AGAGCGGGCG CTTCTACACC AAGCCACCTC AGTGCGTGGA 480

CATCCCCGCG GACCTGCGGC TGTGCCACAA CGTGGGCTAC AAGAAGATGG TGCTGCCCAA 540

CCTGCTGGAG CACGAGACCA TGGCGGAGGT GAAGCAGCAG GCCAGCAGCT GGGTGCCCCT 600

GCTCAACAAG AACTGCCACG CCGGCACCCA GGTCTTCCTC TGCTCGCTCT TCGCCCCCGT 660

CTGCCTGGAC CGGCCCATCT ACCCGTGTCG CTGGCTCTGC GAGGCCGTGC GCGACTCGTG 720

CGAGCCGGTC ATGCAGTTCT TCGGCTTCTA CTGGCCCGAG ATGCTTAAGT GTGACAAGTT 780 CCCCGAGGGG GACGTCTGCA TCGCCATGAC GCCGCCCAAT CCCACCGAAG CCTCCAAGCC 840

CCAAGGCACA ACGGTGTGTC CTCCCTGTGA CAACGAGTTG AAATCTGAGG CCATCATTGA 900

ACATCTCTGT GCCAGCGAGT TTGCACTGAG GATGAAAATA AAAGAAGTGA AAAAAGAAAA 960

TGGCGACAAG AAGATTGTCC CCAAGAAGAA GAAGCCCCTG AAGTTGGGGC CCATCAAGAA 1020

GAAGGACCTG AAGAAGCTTG TGCTGTACCT GAAGAATGGG GCTGACTGTC CCTGCCACCA 1080

GCTGGACAAC CTCAGCCACC ACTTCCTCAT CATGGGCCGC AAGGTGAAGA GCCAGTACTT 1140

GCTGACGGCC ATCCACAAGT GGGACAAGAA AAACAAGGAG TTCAAAAACT TCATGAAGAA 1200

AATGAAAAAC CATGAGTGCC CCACCTTTCA GTCCGTGTTT AAGTGATTCT CCCGGGGGCA 1260

GGGTGGGGAG GGAGCCTCGG GTGGGGTGGG AGCGGGGGGC CGGAATTC 1308 (2) INFORMATION FOR SEQ ID NO: 19:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

He Ala Met Thr Pro Pro Asn Pro Thr Glu Ala Ser Lys Pro Gin Gly 1 5 10 15

Thr Thr Val

Claims

CLAIMS What is claimed is:

1. An isolated polynucleotide encoding a polypeptide having at least 90% identity to SEQ ID NO: 2, 4. 6 or 7.

2. An isolated polynucleotide at least 15 nucleotides in length from the coding region of SEQ ID NO: 1 , 3, 5 or 18, or complement thereof.

3. An isolated polypeptide encoded by the polynucleotide of claim 1.

4. An isolated polypeptide fragment or functionally equivalent polypeptide fragment to a sequence shown in SEQ ID NO: 2, 4, 6 or 7.

5. A fusion polypeptide comprising ( 1 ) a linear sequence of at last 1 1 amino acid residues essentially identical to a sequence shown in SEQ ID NO: 2, 4. 6 or 7, covalently attached to (2) a second polypeptide.

6. A recombinant expression vector comprising a polynucleotide sequence encoding a polypeptide of at least 1 1 consecutive amino acid residues shown in

SEQ ID NO: 2, 4, 6 or 7.

7. A recombinant cloning vector comprising a linear sequence of at least 18 nucleotides identical to a linear sequence within SEQ ID NO: 1 , 3, 5 or 18.

8. A host cell transformed by the polynucleotide of claim 1 , or by the vector of claim 7.

9. The host cell of claim 8 wherein the cell expresses said polypeptide from said vector.

10. A monoclonal or isolated polyclonal antibody specific for a protein encoded in coding region ofthe polynucleotides of claim 1.

1 1. The antibody of claim 10, which is a monoclonal antibody.

12. The antibody of claim 10, which is an isolated polyclonal antibody.

13. A method of detecting SARP protein expression comprising the steps of:

(a) providing a test cell;

(b) contacting the proteins of the test cell with the antibody of claim 10 under conditions that permit formation of a stable complex between the proteins ofthe test cell and the antibody; and

(c) comparing the amount of immunocomplex formed with the proteins ofthe test cell to the amount of immunocomplex formed with the proteins of a non-apoptotic cell ofthe same tissue type as the test cell.

14. A method of detecting SARI¹ protein expression comprising the steps of:

(a) providing a test cell;

(b) contacting the mRNA of the test cell with a nucleic acid probe containing a sequence antisense to a segment at least 15 nucleotides in length of

SEQ ID NO: 1 , 3, 5 or 18 under conditions that permit formation of a stable complex between the mRNA ofthe test cell and the nucleic acid probe; and (c) comparing the amount of hybridization ofthe probe to the mRNA ofthe test cell to the amount of hybridization ofthe probe to the mRNA of a non-apoptotic cell ofthe same tissue type as the test cell.

15. A method of diagnosing a disease associated with the modulation of

SARP expression, comprising:

(a) providing a test sample of tissue;

(b) assaying said test sample for the presence of a gene product of an hsarp gene; and (c) comparing the amount of gene product detected in said test sample to the amount of gene product detected in a non-diseased sample of the same tissue type as the test sample.

16. The method of claim 15, wherein said gene product is a protein.

17. The method of claim 16, wherein assaying comprises contacting said test sample with an antibody to said protein under conditions that permit formation of a stable complex between said antibody and any of said protein present in said test sample.

18. The method of claim 15, wherein said gene product is an hsarp mRNA.

19. The method of claim 18, wherein assaying comprises contacting said test sample with a nucleic acid probe containing a sequence antisense to a segment at least 15 nucleotides in length of an hsarp mRNA under conditions that permit formation of a stable complex between the nucleic acid probe and any complementary mRNA present in said test sample.

20. The method of claim 15, wherein said hsarp gene is hsarpl .

21. The method of claim 20, wherein said disease is a cancer ofthe prostate epithelial tissue.

22. The method of claim 15, wherein said hsarp gene is hsarpl.

23. The method of claim 22, wherein said disease is a cancer of the mammary tissue.

24. A method of diagnosing a disease associated with the modulation of SARP expression, comprising:

(a) providing a test sample of bodily fluid;

(b) assaying said test sample for the presence of a SARP protein; and

(c) comparing the amount of SARP protein detected in said test sample to the amount of SARP protein detected in a non-diseased sample ofthe same fluid type as the test sample.

25. The method of claim 24, wherein assaying comprises contacting said test sample with an antibody to said SARP protein under conditions that permit formation of a stable complex between said antibody and any of said SARP protein present in said test sample.

26. The method of claim 24, wherein said SARP protein is hSARPl .

27. The method of claim 26, wherein said disease is a cancer ofthe prostate epithelial tissue.

28. The method of claim 24, wherein said SARP protein is hSARP2.

29. The method of claim 28, wherein said disease is a cancer ofthe mammary tissue.

30. A method of treatment of a patient comprising administering to the patient a therapeutical ly effective amount of a pharmaceutically acceptable composition comprising a component selected from the group comprising a sarp or antisense-h p polynucleotide or a SARP polypeptide or SARP antibody.

31. The method of claim 30, wherein the patient is suffering from a condition related to cancer.

32. The method of claim 31 , wherein the condition related to cancer is cancer of the mammary tissue.

33. The method of claim 31 , wherein the condition related to cancer is cancer of the prostate.

34. The method of claim 31 , wherein said condition related to cancer is a cancer of the prostate epithelial tissue.

35. The method of claim 30, wherein said polynucleotide is hsarpl.

36. The method of claim 30, wherein said polypeptide is SARP2

37. A method of treating an apoptosis related condition comprising administering a therapeutically effective amount of a pharmaceutically acceptable composition comprising a sarp or antisense-Iisαrp polynucleotide or a SARI* polypeptide or SARP antibody, to a patient in need of such therapy.

38. The method of claim 37, wherein said apoptosis related condition is a cancer.

39. The method of claim 38, wherein said cancer is cancer ofthe mammary tissue.

40. The method of claim 38, wherein said cancer is cancer of the prostate.

41. The method of claim 37, wherein said apoptosis related condition is a cancer of the prostate epithelial tissue.

42. The method of claim 37, wherein said polynucleotide is hsarp2.

43. The method of claim 37, wherein said polypeptide is SARP2.

44. A method for screening potential therapeutic agents that modulate the effect of SARP proteins on the Wnt-frizzled protein interaction comprising the steps of:

(a) combining a Wnt protein and a SARP protein under conditions in which they interact, to form a test sample;

(b) exposing said test sample to a potential therapeutic agent and; (c) monitoring the interaction ofthe SARP protein and the frizzled protein: wherein, a potential therapeutic agent is selected for further study when it modifies the interaction compared to a control test sample to which no potential therapeutic agent has been added.