MXPA01004975A - Novel human wnt gene - Google Patents

Abstract

The Wnt proteins are a family of secreted glycoproteins which, in many organisms, have a role in the morpholigical development of tissues in both embryonic and adult contexts. To date, about 13 human Wnt genes have been identified. The present invention provides a new form of human Wnt, designated"Zwnt1".

Description

NEW GENE WNT HUMAN TECHNICAL FIELD The present invention relates generally to a new gene that codes for a cell signaling molecule. In particular, the invention relates to a novel Wnt, designated as "Zwntl", and to nucleic acid molecules encoding Zwntl.

BACKGROUND OF THE INVENTION The cellular differentiation of multicellular organisms is controlled by the hormones and growth factors of the polypeptides. These molecules that can spread allow cells to communicate with each other, to act in concert to form tissues and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include steroid hormones, parathyroid hormone, follicle stimulating hormone, interferons, interleukins, platelet-derived growth factor, epidermal growth factor, and granulocyte-acrophage colony stimulating factor. , among others . Ref: 129603 Hormones and growth factors influence cellular metabolism by binding to receptor proteins. Certain receptors are integral membrane proteins that bind to the hormone or growth factor outside the cell, and which are linked to the signaling pathways within the cell, such as second messenger systems. Another class of receptors are soluble intracellular molecules. Wnt proteins are emerging as one of the pre-existing families of signaling molecules in animal development. To date, murine Wnt genes include Wnt-1, Wnt-2, Wnt-2B / 13, Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, WntSB-Wnt-6, Wnt-7A, Wnt-IB, Wnt-8A, Wnt-8B, Wnt-10A, Wnt-IOB, Wnt-11 and Wnt-15, while the following human Wnt genes have been described: Wnt-1, Wnt-2, Wnt-2B / 13, Wnt-3, Wnt-4, Wnt-5A, Wnt-7A, Wnt-8A, Wnt-8B, Wnt-IOB, Wnt-11, Wnt-14, and Wnt-15. See for example, Nusse and Varmus, Cel 31:99 (1982), van Ooyen et al., EMBO J. 4: 2905 (1985), Wainwright et al., EMBO J. 7: 1743 (1988), McMahon and MacMahon. , Development 107: 643 (1989) Gavin et al., Genes Dev. 4: 2319 (1990), Roelin et al., Proc. Nati 'Acad. Sci. USA 87: 4519 (1990). Roelink et al., Genomics 17: 790 (1993-), Adamson et al., Genomics 24: 9 (1994), Huguet et al., Cancer Res. 54: 2615 (1994) Bouillet, Mech Dev. 58: 141 ( 1996), Ikegawa et al., Cytogenet. Cell Genet. 74: 149 (1996), Katoh et al., Oncogene 13: 813 (1995), Lako et al., Genomics 35: 386 (1996), Wang and Schakleford, Oncogene 13: 1531 (1996), Bergstein, Genomics 46: 450 (1997), Bui et al., Oncogene 14: 1249 (1997), Grove et al., Development 125: 2315 (1988), and The Wnt Gene Home Pay (http: // w -leland. Standford. EduArnusse / wnt indo .html). Wnt genes typically encode secreted glycoproteins having 350-400 amino acids, and proteins often include a conserved pattern of 23-24 cysteine residues in addition to other invariant residues (Cadigan and Nusse, Genes &Dev. 11: 3286 ( 1997)). After cellular secretion, Wnt proteins are thought to reside mainly in the extracellular matrix at or associated with the cell surface. In accordance with the classic Wnt signaling path model, Wnt proteins induce the expression of the gene by de-repressing a signal pathway via a so-called "Frizzled" transmembrane receptor (see for example, Brown and Moon). , Curr, Opin, Cell, Biol. 10: 182 (1998)). In the absence of Wnt, the kinase-3β activity of glycogen synthase results in degradation of the free cytosolic group of β-catenin. The association of cognate Wnt proteins and Frizzled receptors leads to the activation of a signaling path. The closest intracellular component of this pathway is the Disheveled protein, which becomes phosphorylated and inhibits glycogen synthase kinase-3b. Consequently, the pooling of intracellular β-catenin is increased, and β-catenin can interact with elements of the lymphoid enhancer family / T cell factor (LEF / TCF) of architectural transcriptional factors in the nucleus. These complexes bind to the LEF / TCF consensus sites, in promoters and induce the transcription of Wtn-responsive genes. Several secreted factors inhibit 'Wnt signaling (see, for example Finch et al., Proc. Natl' Acad. Sci. USA 94: 6110 (1997); Moon et al. , Cell 88: 125 (1997); Luyten et al., WO 98/16641); Brown and Moon, Curr. Opin. Cell. Biol. 10: 182 (1998)). Frzb proteins, for example, bind to secreted Wnt proteins and prevent productive interactions between Wnt and Frizzled proteins. These proteins contain a region that is homologous to a putative Wnt binding domain of the Frizzled proteins. Wnt proteins are multipotent, and proteins are capable of inducing different biological responses in both embryonic and adult contexts (see for example, Ingham, TIG 12: 382 (1996)). This type of broad activity is shared with the fibroblast growth factors, ß transforming growth factors, and nerve growth factors (Nusse and Varmus, Cell 69: 1013 (1992)). When expressed, Wnt proteins can promote tumor formation (Erdreich-Epstein and Shackleford, Growth Factors 15: 149 (1998)). Deleted mutations in mice have shown Wnt proteins to be essential for brain development, and growth outside of the embryonic promise for the kidney, germinative ends and germinative legs (McMahon and Bradley, Cell 62: 1013 (1990), Tho as and Capecchi, Na ture 346: 841 (1990), Stark et al., Nature 372: 619 (1994), Takada et al., Genes Dev. 8: 174 (1994), and Parr and McMahon, Na ture 374: 350 (1995) Although new uses of Wnt proteins can be discovered, there is a need for the provision of new Wnt proteins for biopharmaceuticals.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a novel Wnt protein, designated "Zwntl". The present invention also provides Zwntl variant polypeptides and Wwntl fusion proteins, as well as nucleic acid molecules that encode such polypeptides and proteins, and methods for using these nucleic acid molecules and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION 1. Compendium The present invention provides nucleic acid molecules that encode a new human Wnt protein, designated as "Zwntl". An illustrative nucleotide sequence encoding Zwntl is provided by SEQ ID NO: 1. The encoded polypeptide has the following amino acid sequence: MHPHLRSKHK LPHSFLQLSP RSAPNDILDL RLPPEPVLNA NTVCLTLPGL SRRQMEVCVR HPDVAASAIQ GIQIAIHECQ HQFRDQRWNC SSLETRNKIP YESPIFSRGF RESAFAYAIA AAGWHAVSN ACALGKLKAC GCDASRRGDE EAFRRKLHRL QLDALQRGKG LSHGVPEHPA LPTASPGLQD SWEWGGCSPD MGFGERFSKD FLDSREPHRD IHARMRLHNN RVGRQAVMEN MRRKCKCHGT SGSCQLKTCW QVTPEFRTVG ALLRSRFHRA TLIRPHNRNG GQLEPGPAGA PSPAPGAPGP RRRASPADLV YFEKSPDFCE REPRLDSAGT VGRLCNKSSA GSDGCGSMCC GRGHNILRQT RSERCHCRFH WCCFWCEEC RITEWVSVCK (SEQ ID NO: 2). Thus, the Zwntl gene described herein encodes a polypeptide of 400 amino acids, as shown in SEQ ID NO: 2. The amino acid residues 20-400 of SEQ ID NO: 2, which are encoded by nucleotides 264 -1406 of SEQ ID NO: 1, represent the mature Zwntl polypeptide. The mature Zwntl amino acid sequence (SEQ ID NO: 4) is approximately 96% identical to the amino acid sequence of murine Wnt-lOA (Wang and Shackleford, Oncogene 13: 1531; GenBank Access No. U61969; 'SEQ ID NO: 7 here), approximately 75% identical to a WntlOA newt, designated "PWnt-lOa" (Caubit et al., Dev. Dyn. 208: 139 (1997); GenBank Access No. U65428), and 62% identical to the sequence of Wnt-IOB amino acid (Bui et al., Oncogene 14: 1249 (1997); GenBank Access No. 000744). The Zwntl polypeptide contains cysteine residues, which are more conserved among most Wnt proteins (amino acid residues 44, 79, 90, 132, 140, 142, 197, 245, 247, 254, 259, 329, 345, 355, 359, 360, 375, 377, 382, 383, 387, 390 and 399 of SEQ ID NO: 2), and potential glycosylation sites linked to asparagine are located at amino acids 89 and 346 of the SEC ID NO: 2 A chromosomal localization study revealed that the Zwntl gene resides on human chromosome 2 to 2q36.3.

Other genes associated with this locus or site include the intestinal alkaline phosphatase gene, and the 2B hydroxytryptamine receptor gene. Northern analysis indicates that the Zwntl gene is strongly expressed in the spleens, prostate, small intestine, spinal cord, lymph node and fetal kidney, and to a lesser extent in the adrenal glands, thymus, testes, uterus, colon and lymphocytes of the peripheral blood. In contrast, the little or no expression of the Zwtnl gene was detectable in tissues such as the heart, placenta, lung, brain, liver, muscle skeleton, kidney, pancreas, stomach, thyroid, trachea, spinal cord, fetal brain, fetal lung and fetal liver. The expression of the Zwntl gene was also detectable in CD4 + lymphocytes in RNA isolated from a mixed lymphocyte reaction, but not in lymphocytes CD8 +, CD19 + lymphocytes, or monocytes. The CD19 surface marker is the most ubiquitously expressed surface marker in the B lymphocyte lineage, and therefore, the results indicate that the Zwntl gene is not expressed by B cells. Although both CD4 and CD8 surface markers are expressed by lymphocytes designated as T cells, CD4 is expressed by helper T cells, while CD8 is expressed by cytotoxic T cells. Functionally, a difference between two types of T cells is that the CD4 + cells, sensitized by the cells that present the antigen, carry the largest histocompatibility complex of the peptides linked to class II, produce lymphocytes that participate in the activation of B cells, macrophages, natural killer cells and several subsets of T cells. In contrast, sensitized CD8 + cells increase in number in response to lymphocytes, and are capable of destroying any cell that expresses the specific antigenic fragments associated with the class I molecules of the largest histocompatibility complex. In sum, hybridization studies show that differentiated Zwntl sequences can be used between various cell and tissue types. As detailed below, the present invention provides isolated peptides having an amino acid sequence that is at least 70%, at least 80% or at least 90% identical to the amino acid sequence of SEQ ID NO: 4, in wherein said asylated polypeptides specifically bind to an antibody that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 4, provided that such isolated polypeptides do not include the murine Wnt-lOA protein, which is identified by GenBank Access No. U61969, and the Wnt-lOA newt protein, which is identified by GenBank Access No. U65428. An illustrative polypeptide is a polyeptide comprising the amino acid sequence of SEQ ID NO: 4. The present invention further provides antibodies and antibody fragments that specifically bind to such polypeptides. Exemplary antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from antibodies, murine monoclonal antibodies and human monoclonal antibodies. Exemplary antibody fragments include F (ab ') 2, F (ab) 2, Fab', Fab, Fv, scFv, and minimal recognition units. The present invention also provides isolated nucleic acid molecules, which encode a Zwntl polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule having the nucleotide sequence of SEQ. NO: 3, (b) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 4, and (c) a nucleic acid molecule that remains hybridized after stringent washing conditions to an acid molecule nucleic acid sequence having the nucleotide sequence of nucleotides 264-1406 of SEQ ID NO: 1, or the complement of nucleotides 264-1406 of SEQ ID NO: 1, with the proviso that such isolated nucleic acid molecules do not include the murine Wnt-lOA gene, which is identified by GenBank Access No. U61969, and the Wnt-lOA newt gene, which is identified by GenBank Access No. U65428. Nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution. The present invention further contemplates isolated nucleic acid molecules comprising a nucleotide sequence of nucleotides 264 to 1406 of SEQ ID NO: 1. The present invention also includes vectors and expression vectors comprising such nucleic acid molecules. Such expression vectors may comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked to the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked to the transcription terminator. The present invention also includes recombinant host cells comprising these vectors and expression vectors. Exemplary host cells include bacterial, fungal, insect, mammalian and plant cells. Recombinant host cells comprising such expression vectors can be used to prepare the Zwntl polypeptides by culturing such recombinant host cells comprising the expression vector and producing the Zwntl protein., and the isolation of the Zwntl protein from the cultured recombinant host cells. The present invention also contemplates methods for detecting the presence of Zwntl RNA in a biological sample, comprising the steps of (a) contacting a Zwntl nucleic acid probe under hybridization conditions with either (i) RNA molecules of test isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of nucleotides 264 to 1406 of SEQ ID NO: 1, or its complement, and (b) detecting hybrid formation of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of Zwntl RNA in the biological sample. As an illustration, the biological sample can be a human biological sample. The present invention further provides methods for detecting the presence of a Zwntl polypeptide in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or an antibody fragment that specifically binds to a polypeptide that has the amino acid sequence of SEQ ID NO: 4, wherein the contact is made under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibodies or antibody fragments linked. Such antibodies or antibody fragments may comprise a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label and colloidal gold. An exemplary biological sample is a human biological sample. The present invention also comprises equipment for carrying out these detection methods. For example, a kit for detecting the expression of the Zwntl gene may comprise a container comprising a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 264 to 1406 of SEQ ID NO: 1, (b) a nucleic acid molecule comprising the complement of nucleotides 264 to 1406 of the nucleotide sequence of SEQ ID NO: 1, ( c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, and (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides. Such a device may also comprise a second container comprising one or more reagents capable of indicating the presence of the nucleic acid molecule. On the other hand, a kit for the detection of the Zwntl protein may comprise a container comprising an antibody, or an antibody fragment, that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 4. These and other aspects of the invention, will become apparent from the reference to the following detailed description. 2. Definitions In the following description, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention. As used herein, "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments degenerated by the polymerase chain reaction (PCR) ), and fragments generated by any ligation, separation, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or nucleotide analogs that originate naturally (e.g., α-enantiomeric forms of naturally occurring nucleotides), or a combination of both. The modified nucleotides may have alterations in their sugar portions and / or in the pyrimidine or purine-based portions. Sugar modifications include, for example, the replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars that can be functionalized with ethers or esters. However, the entire sugar portion can be replaced with sterically or electronically similar structures, such as carbocyclic sugar analogs and aza-sugars. Examples of modifications in a base portion include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorotoates, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which comprise nucleic acid bases bound to a polyamide structure. The nucleic acids can be either single-stranded or double-stranded. The term "complement of a nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation compared to a reference nucleotide sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to the 5'CCCGTGCAT 3 '. The term "contiguous" denotes a nucleic acid molecule having a contiguous portion of the sequence identical or complementary to another nucleic acid molecule. The contiguous sequences are said to "overlap" to a given portion of the sequence of the nucleic acid molecule. The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons, when compared to a reference nucleic acid molecule encoding a polypeptide. Degenerate codons contain different triplets of nucleotides, but they encode the same amino acid residues (ie, the GAU and GAC triplets each encode Asp).

The term "structural gene" refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide. An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated into the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome of such species. A "nucleic acid molecule construct" is a nucleic acid moleculeeither single stranded or double stranded, which has been modified through human intervention to contain combined and juxtacolocated nucleic acid segments in an arrangement that does not exist in nature. "Linear DNA" denotes non-circular DNA molecules that have free 5 'and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or by physical breakdown. "Complementary DNA (cDNA)" is a molecule of Single-stranded DNA that is formed from an mRNA pattern by the reverse transcriptase of the enzyme. Typically, a primer complementary to the portions of the mRNA is used for the initiation of reverse transcription. Those skilled in the art can also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA standard. A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5 'non-coding region of a gene, near the transcriptional start site of a structural gene. Sequence elements within the promoters that function at the initiation of transcription are often characterized by consensual nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, specific elements of differentiation (DSEs, McGehee et al., Mol Endocrinol 7: 551 (1993)), cyclic AMP response elements ( CREs), serum response elements (SREs, Treisman, Seminars in Cancer Biol. 1:41 (1990)), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE / ATF (O'Reilly et al., J. Biol. Chem. 267: 19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269: 25128 (1994)), protein binding to the element of response cAMP, SP1, (CREB; Loeken, Gene Expr. 3: 253 (1993)) and octamer factors (see generally, Watson et al., eds., Molecular Biology of the Gene 4th Ed. (The Benjamin / Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303: 1 (1994). )). If a promoter is an inducible promoter, then the rate of transcription increases in response to an induction agent. In contrast, the proportion of transcription is not regulated by an induction agent if the promoter is a constitutive promoter. Promoters are also known to be repressive. A "core promoter" contains nucleotide sequences essential for the function of the promoter, including the TATA block and the start of transcription. By this definition, a central promoter may or may not have detectable activity in the absence of specific sequences that may increase activity or confer tissue-specific activity. A "regulatory element" is a nucleotide sequence that modulates the activity of a central promoter. For example, a regulatory element may contain a nucleotide sequence that binds to cellular factors that allow transcription exclusively or preferentially in particular cells, tissues or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific", "tissue-specific" or "organelle-specific" manner, for example, the regulatory element Zwntl preferentially induces the expression of the spleens, thymus, chordal spine and lymphatic node, as opposed to tissues of placenta, lung and liver.An "enhancer" is a type of regulatory element that can increase the efficiency of transcription, with respect to distance or increment orientation, relative to the transcription start site. "Heterologous DNA" refers to a DNA molecule, or a population of DNA molecules, that do not exist naturally within a given host cell. heterologous to a particular host cell may contain DNA derived from host cell species (ie, endogenous DNA), as soon as the host DNA it is combined with non-host DNA (ie, exogenous DNA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter, is considered a heterologous DNA molecule. Conversely, a heterologous DNA molecule may comprise an endogenous .gen operably linked with an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a native-type cell is considered to be heterologous DNA if such a DNA molecule is introduced into a mutant cell that lacks the wild type or native gene. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined here in terms of their basic amino acid structures; Substituents such as carbohydrate groups are not generally specified, but nevertheless may be present. A peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide. An "integrated genetic element" is a segment of DNA that has been incorporated into a chromosome of a host cell after the element is introduced into the cell through human manipulation. Within the present invention, the integrated genetic elements are most commonly derived from linearized plasmids that are introduced into cells by electroporation or other techniques. The integrated genetic elements are passed from the original host cell to their progeny. A "cloning vector" is a nucleic acid molecule such as a plasmid, cosmid, or bacteriophage, which has the ability to replicate autonomously in a host cell. Cloning vectors typically contain one or more small numbers of restriction endonuclease recognition sites that allow the insertion of a nucleic acid molecule in a determinable manner without loss of an essential biological function of the vector, as well as nucleotide sequences that encode a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. An "expression vector" is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene and a transcription terminator. The expression of the gene is usually placed under the control of a promoter, and such a gene is said to be "operably linked" to the promoter. Similarly, a regulatory element and a central promoter are operably linked if the regulatory element modulates the activity of the central promoter. A "recombinant host" is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or an expression vector. In the present context, an example of a recombinant host is a cell that produces Zwntl from an expression vector. In contrast, Zwntl can be produced by a cell that is a "natural source" of Zwntl and that lacks an expression vector. "Integrative transformers" are recombinant host cells, in which heterologous DNA becomes integrated into the genomic DNA of the cells. A "fusion protein" is a hybrid protein that is expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes. For example, a fusion protein may comprise at least part of a Zwntl polypeptide fused to a polypeptide that binds to an affinity matrix. Such a fusion protein provides a means to isolate large amounts of Zwntl using affinity chromatography.

The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule called a "ligand". This interaction mediates the effect of the ligand on the cell. The receptors can be linked to the membrane, cytosolic or nuclear; monomeric (for example, the thyroid-stimulating hormone receptor, the beta-adrenergic receptor) or multimeric (the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM-CSF receptor, the receptor G-CSF, the erythropoietin receptor and the IL-6 receptor). The membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand binding domain and the intracellular effector domain are located on separate polypeptides that comprise the entire functional receptor. In general, the binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell, which, in turn, leads to an alteration in the cell's metabolism. Metabolic events that are often linked to receptor ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, cellular calcium mobilization, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. The term "secretory signal sequence" denotes a DNA sequence encoding a peptide (a "secretory peptide") that, as a component of a larger polypeptide directs the larger polypeptide through a secretory pathway of a cell in the which is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. An "isolated polypeptide" is a polypeptide that is essentially free of contaminating cellular components, such as carbohydrates, lipids and other proteinaceous impurities associated with the polypeptide in nature. Typically, an isolated polypeptide preparation contains the polypeptide in a highly purified form, ie, at least about 80% pure, at least 90% pure, at least 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide, is by the appearance of a single band after polyacrylamide gel electrophoresis (SDS) with sodium dodecyl sulfate protein preparation and staining Blue Brilliant Coomassie of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively derivatized or glycosylated forms. The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within the polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain carboxyl-terminal sequence positioned to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but not necessarily to the carboxyl terminus of the entire polypeptide. The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, the expression involves the transcription of the structural gene in the mRNA and the translation of the mRNA into one or more polypeptides. The term "splice variant" is used herein to denote the alternative forms' of the RNA transcribed from a gene. Splice variation is generated naturally through the use of alternative splice sites within a transcribed RNA molecule, or less commonly between RNA molecules transcribed separately, and can result in several mRNAs transcribed from the same gene. The splice variants can encode the polypeptides having altered amino acid sequence. The term "splice variant" is also used herein to denote a polypeptide encoded by a splicing variant of a mRNA transcribed from a gene. As used herein, the term "immunomodulatory" includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules. The term "complement / anti-complement pair" denotes non-identical portions that form a stable, associated pair, not covalently, under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen pairs (or incomplete antigen or epitope), sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity of < 109 M-1. An "anti-idiotype antibody" is an antibody that binds to the variable region domain of an immunoglobulin. In the present context, an anti-idiotype antibody binds to the variable region of an anti-Zwntl antibody, and thus, an anti-idiotype antibody mimics an epitope of Zwntl. An "antibody fragment" is a portion of an antibody such as F (ab ') 2 / F (ab) 2, Fab', Fab, and the like. With respect to the structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. For example, a fragment of anti-Zwntl monoclonal antibody binds to a Zwntl epitope. The term "antibody fragment" also includes a genetically or synthetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv" fragments that consist of the variable regions of the chains heavy and light, recombinant single-chain polypeptide molecules in which the heavy and light chain variable regions are connected by a peptide linker ("scFv proteins"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable region. A "chimeric antibody" is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the rest of the antibody molecule is derived from a human antibody. "Humanized antibodies" are recombinant proteins in which the murine complementary determinant regions of a monoclonal antibody have been transferred from the heavy and light variable chains of murine immunoglobulin to a human variable domain. As used herein, a "therapeutic agent" is a molecule or atom which is conjugated to a portion of antibody to produce a conjugate, which is used for therapy. Examples of therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes. A "detectable label" is a molecule or atom which may be conjugated to a portion of the antibody to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions or other marker portions. The term "affinity tag" is used herein to denote a segment of the polypeptide that can bind to a second polypeptide to provide purification or detection of the second polypeptide or provide binding sites of the second polypeptide to a substrate. Primarily, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985, Nilsson et al., Methods Enzymol 198: 3, 1991), glutathione S transferase. (Smith and Johnson, Gene 67: 31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82: 1952 (1985)), substance P, FLAG peptide (Hopp et al. al., Biotechnology 6: 1204 (1988)), the streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. Affinity tags encoding nucleic acid molecules are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ ). A "discovered antibody" is a complete antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. The antibodies discovered include both monoclonal and polyclonal antibodies, as well as certain recombinant antibodies, such as humanized and chimeric antibodies. How it is used here, the term "antibody component" includes both a whole antibody and an antibody fragment. An "immunoconjugate" is a conjugate of an antibody component with a therapeutic agent or a detectable label. As used herein, the term "antibody fusion protein" refers to a recombinant molecule comprising an antibody component and a therapeutic agent. Examples of suitable therapeutic agents for such fusion proteins include immunomodulators ("antibody-immunomodulatory fusion protein") and toxins ("antibody-toxin fusion protein"). An "objective polypeptide" or an "objective peptide" is an amino acid sequence that comprises at least one epitope, and that is expressed in a target cell, such as a tumor cell, or a cell carrying an infectious agent antigen . T cells recognize peptide epitopes presented by a molecule of the highest histocompatibility comlejo to a target polypeptide or target peptide and tickly lyse the cell or recruit other immune cells to the site of the target cell, thereby killing the target cell. An "antigenic peptide" is a peptide which will bind to a molecule of the highest histocompatibility complex to form an MCH-peptide complex, which is recognized by a T cell, thereby inducing a response of the cytotoxic lymphocyte after the presentation to the T cell. Thus, the antigenic peptides are capable of binding to a higher-histocompatibility complex molecule and inducing a cytotoxic response to T cells, such as lysing the cells or releasing the specific cytokine against the cells target which bind or express the antigen. The antigenic peptide can be linked in the context of a complex molecule of higher class I or class II histocompatibility, or in a cell presenting antigen or in a target cell. In eukaryotes, RNA polymerase II catalyses the transcription of a structural gene to produce mRNA. A nucleic acid molecule can be designed to contain a polymerase II RNA standard, in which the RNA transcript has a sequence that is complementary to that of a specific RNA. The RNA transcript is called "an anti-sense RNA" and a nucleic acid molecule that encodes anti-sense RNA is called an "anti-sense gene." Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation. A "anti-sense oligonucleotide specific for Zwntl" or an "antisense oligonucleotide Zwntl" is an oligonucleotide having a sequence (a) capable of forming a stable triplet with a portion of the Zwntl gene, or (b) capable of forming a stable duplex with a portion of a mRNA transcript of the Zwntl gene. A "ribozyme" is a nucleic acid molecule that contains a catalytic center. The term includes RNA envelopes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions. A nucleic acid molecule that codes for a ribozyme is called a "ribozyme gene." An "external leader sequence" is a nucleic acid molecule that directs the endogenous ribozyme, P RNase, to a particular species of intracellular mRNA, resulting in cleavage of the mRNA by the P RNase. A nucleic acid molecule that encodes an external leader sequence is called an "external leader sequence gene". The term "variant Zwntl gene" refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO: 2 or a modification of SEQ ID NO: 4. Such variants include polymorphisms that originate naturally from Zwntl genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The additional variant forms of the Zwntl genes are nucleic acid molecules containing insertions or deletions of the nucleotide sequences described herein. A Zwntl variant gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or its complement, under rigorous conditions. The term "Zwntl variant" does not, however, include murine WntlOA, which is identified by GenBank Access No. U61969, and does not include 1 Wnt-lOa newt protein (GenBank Access No. U65428). Alternatively, the Zwntl variant genes can be identified by comparison of the sequence. Two amino acid sequences have "100% amino acid sequence identity", if the amino acid residues of the two amino acid sequences are the same when they are aligned for maximum correspondence. Similarly, two nucleotide sequences have "100 percent nucleotide sequence identity" if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximum correspondence. Sequence comparisons can be made using standard software programs such as those included in the LASERGENE bioinformatics computing suites, which is produced by DNASTAR (Madison, Wisconsin). Other methods for comparing two amino acid or nucleotide sequences by determining the optimal alignment are well known to those skilled in the art (see for example, Peruski and Peruski, The Internet and the New Bioligy: Tools for Genome c and Mol ecular Research (ASM Press, Inc. 1997), Wu et al (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins", in Methods in Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bioshop (ed.), Guide to Human Genome Computing, 2nd Edition (Academic Press Inc., 1998)). The particular methods for determining the sequence identity are described below. With respect to the particular method used to identify a variant Zwntl gene or a variant Zwntl polypeptide, a variant gene or polypeptide encoded by a variant gene, can be characterized by the ability to specifically bind to an anti-Zwntl antibody. The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene that occupies the same chromosomal location. Allelic variation becomes natural through a mutation, and can result in phenotypic polymorphism within populations. Mutations of the genes can be either silent (without change in the encoded polypeptide) or they can be encoded polypeptides having the altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation. "Paralogs" are distinct but structurally related proteins made by an organism. It is believed that paralogs are generated through genetic duplication. For example, α-globin, β-globin and myoglobin are paralogs with each other. The present invention includes functional fragments of Zwntl genes. Within the context of this invention, "a functional fragment" of a Zwntl gene refers to a nucleic acid molecule that encodes a portion of a Zwntl polypeptide which specifically binds to an anti-Zwntl antibody. For example, a functional fragment of a Zwntl gene described herein, comprises a portion of the nucleotide sequence of SEQ ID NO: 1, and encodes a polypeptide that specifically binds to an anti-Zwntl antibody. Due to the impression of the standard analytical methods, the molecular weights and lengths of the polymers are understood as approximate values. When such value is expressed as "around" X or "approximately" X, the declared value of X will be understood as accurate to + 100%. 3. P roduction of a human Zwntl gene The nucleic acid molecules encoding a human Zwntl gene can be obtained by the selection of a human cDNA or a library using polynucleotide probes based on SEQ ID NO: 1. These techniques are standard and well established. As an illustration, a nucleic acid molecule encoding a human Zwntl gene can be isolated from a human cDNA library. In this case, the first step may be prepared as the cDNA library is prepared by RNA isolation from the spleen, thymus or uterine tissue, using methods well known to those skilled in the art. In general, RNA isolation techniques may provide a method for cell disruption, means for inhibiting RNase-directed degradation of RNA, and a method for separating RNA from DNA, protein, and polysaccharide contaminants. For example, total RNA can be isolated by freezing the tissue in liquid nitrogen, grinding the frozen tissue with a mortar and crushing it to lysis the cells, extraction of the ground tissue with a phenol / chloroform solution removes the proteins , and separation of the RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et al (eds.), Short Protocols in Molecular Biology, 3rd Edition, pages 4-1 a 4-6 (Joh Wiley &Son 1995) ["Ausubel (1995)"] Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)"] ). Alternatively, the total RNA can be isolated from the spleen, thymus, or uterine tissue by extraction of the ground tissue with guanidinium isothiocyanate, extraction with organic solvents, and separation of the RNA from contaminants using differential centrifugation (see for example, Chirgwin et al. al., Biochemistry 18: 52 (1979), Ausubel (1995) on pages 4-1 to 4-6, Wu (1997) on pages 33-41). For a construct, poly (A) + RNA, a DNA library, can be isolated from a total RNA preparation. Poly (A) + ANR can be isolated from total RNA using the standard technique of olig (dT) cellulose chromatography (see for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 63: 1408 (1972) Ausubel (1995) on pages 4-11 to 4-12). Double-stranded cDNA molecules are synthesized from poly (A) + RNA using techniques well known to those in the art, (see for example, Wu (1997) on pages 41-46). However, commercially available equipment can be used. to synthesize double-stranded DNA molecules. For example, such equipment is available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA). Several cloning vectors are suitable for the construction of a cDNA library. For example, a cDNA library can be prepared in a vector derived from a bacteriophage, such as a gtlO vector. See, for example, Huynh et al., "Constructing and Screening cDNA Libraries in? GtlO y? Gtll", in DNA Cloning: A Practical Approach Vol. I, Glover (ed.), Page 49 (IRL Press, 1985); Wu (1997) on pages 47-52. Alternatively, the double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE, La Jolla CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors. Suitable cloning vectors can also be obtained from the American Type Culture Collection (Manassas, VA). To amplify the cloned cDNA molecules, the cDNA library was inserted into a prokaryotic host, using standard techniques. For example, a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD). A human genomic library can be prepared by means well known in the art (see for example, Ausubel (1995) on pages 5-1 to 5-6; Wu (1997) on pages 307-327). Genomic DNA can be isolated by lysing the tissue with the Sarkosyl detergent, digestion of the lysate with proteinase K, purification of the insoluble waste from the lysate by centrifugation, precipitation of the nucleic acid from the lysate using isopropanol, and purification of the DNA resuspended in a cesium chloride density gradient. DNA fragments that are suitable for the production of a library can be obtained by randomization or by partial digestion of the genomic DNA with the restriction endonucleases. Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide the appropriate term, the use of the phosphatase treatment alkaline to avoid undesirable binding of the DNA molecules, and ligation with the appropriate ligases. Techniques for such manipulation are well known in the art (see, for example, Ausubel (1995) on pages 5-1 to 5-5; Wu (1997) on pages 307-372). Nucleic acid molecules encoding a human Zwint gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having the nucleotide sequences that are based on the nucleotide sequences of the Zwintl gene human as described here. General methods for the selection of libraries with PCR are provided by for example, Yu et al., "Use of the Polymerase Chain Reaction to Screen Phage Librarles", in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applicants, White (ed.), Pages 211-215 (Humana Press, Inc., 1993). However, the techniques for using PCR to isolate related genes is described, for example, by Preston, "Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members", in Methods in Mol ecular Biology, Vol. 15: PCR Protocols: Current Methods and Application, White (ed.), Pages 317-337 (Humana Press, Inc. 1993). Alternatively, human libraries can be obtained from commercial sources such as Research Genetics (Hunstville, AL) the American Type Culture Collection (Manassas, VA). A library containing cDNA or genomic clones, can be selected with one or more polynucleotide probes based on SEQ ID NO: 1, using standard methods (see for example, Ausubel (1995) on pages 6-1 to 6-11 ). Anti-Zwntl antibodies, produced as described below, can also be used to isolate DNA sequences encoding human Zwntl genes from cDNA libraries. For example, antibodies can be used to select the expression libraries? Gtll, or the antibodies can be used for immuno selection after hybrid selection and translation (see for example, Ausubel (1995) on pages 6-12). at 6-16, Margolis et al., "Screening? expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2n?. Edi tion, Glover et al., (eds.), pages 1-14. (Oxford University Press 1995)). As an alternative, a Zwintl gene can be obtained by synthesizing the nucleic acid molecules using long oligonucleotides from mutual primers and the nucleotide sequences described herein. (see for example, Ausubel (1995) on pages 8-8 to 8-9). The techniques established using the polymerase chain reaction, provide the ability to synthesize DNA molecules of at least two kilobases in length (Adang et al., Plant Molec., Biol. 21: 1131 (1993), Bambot et al., PCR Methods and Applications 2: 266 (1993), Dillon et al., "Use of the Polymerase Chain Reaction for the Rapid Construction. of Synthetic Genes ", in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuck et al., PCR Methods App. 4: 299 (1995)). The nucleic acid molecules of the present invention can also be synthesized with "genetic machines" using protocols such as the phosphoramidite method. If the chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically directed and can be accompanied by the synthesis of complementary strands and then the alignment of these. For the production of longer genes (> 300 base pairs), however, special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is rarely 100%. To overcome this problem, the synthetic genes (double strand) are assembled in modular form from the fragments of a single strand that are from 20 to 100 nucleotides in length. A method for constructing a synthetic gene requires the initial production of a series of complementary, overlapping oligonucleotides, each of which is between 20 to 60 nucleotides in length. The sequences of the strands are planned so that, after alignment, the two final segments of the gene are aligned to give truncated ends. Each internal section of the gene has 3 'and 5' terminal extensions that are designated as a base pair with precisely an adjacent section. Thus, after the gene is assembled, the only remaining requirement to complete the process is to seal the ruptures together with the base structures of the two strands with a T4 DNA ligature. In addition to the protein coding sequence, synthetic genes can be designated with terminal sequences that facilitate insertion into restriction endonuclease sites of a cloning vector and other sequences should be added, such that they contain the signals for self-initiation and the termination of the transcription and translation. An alternative way to prepare full size genes is to synthesize a specified series of overlapping oligonucleotides (40 to 100 nucleotides). After the extensions 3 'and 5' (6 to 10 nucleotides) are aligned, large openings still remain, but the paired extensions at the base are both sufficiently stable and sufficiently long to maintain the structure together. The duplo is complete and the openings are filled by enzymatic DNA synthesis with polymerase I of E. coli DNA. These enzymes use the 3'-hydroxyl groups as replication initiation sites and the single-stranded regions as standards. After the enzymatic synthesis is complete, the ruptures are sealed with T4 DNA ligase. For longer genes, the entire gene sequence is usually assembled from double-stranded fragments that are each placed together by the binding of four to six overlapping oligonucleotides (20 to 60 base pairs each). If there is a sufficient quantity of the double-stranded fragments after each synthesis, and stage of alignment, they are simply attached to each other. Otherwise, each fragment is cloned into a vector to apply the amount of available DNA. In both cases, the double-stranded constructs are sequentially raised to the others to form the complete gene sequence. Each fragment of double strand and the complete sequence, should be characterized by DNA sequence analysis to verify that the chemically synthesized gene has the correct nucleotide sequence. For reviews in the synthesis of polynucleotides, see for example, Glick and pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA (ASM Press 1994), Itakura et al. , Annu. Rev. Biochem. 53: 323 (1984), and Climie et al. , Proc. Nat 'l Acad. Sci. USA 87: 633 (1990). The Zwntl cDNA sequence or Zwntl genomic fragment can be determined using standard methods. The Zwntl polynucleotide sequences described herein can also be used as probes or primers to clone the 5 'uncloned regions of a Zwntl gene. Promoter elements of a Zwntl gene can be used to direct the expression of heterologous genes in e.g. peripheral blood lymphocytes of transgenic animals or patients undergoing gene therapy. The identification of genomic fragments containing a Zwntl promoter or regulatory element can be achieved using well-established techniques, such as deletion analysis (see generally, Ausubel (1995)). The cloning of the 5 'flanking sequences also facilitates the production of Zwntl proteins by "gene activation", as described in U.S. Patent No. 5,461,670. Briefly, the expression of an endogenous Zwntl gene in a cell is delayed by the introduction into the Zwntl site, a DNA construct comprising at least one target sequence, a regulatory sequence, an exon, and an unpaired enpair donor site. . The target sequence is a 5 'non-coding sequence of Zwntl that allows homologous recombination of the construct with the endogenous Zwntl site, thereby, the sequences within the construct become operably linked with the endogenous Zwntl coding sequence. In this way, an endogenous Zwntl promoter or supplement can be replaced with other regulatory sequences to provide tissue-specific, increased, or otherwise regulated expression. 4. Production of Zwntl Gene Variants The present invention provides a variety of nucleic acid molecules, including DNA molecules and RNA, which encodes the Zwntl polypeptides described herein.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible between these polynucleotide molecules. SEQ ID NO: 3 is a degenerate nucleotide sequence encompassing all the nucleic acid molecules encoding the Zwntl polypeptide of SEQ ID NO: 2, while SEQ ID NO: 6 is a degenerate nucleotide sequence encompassing all the nucleic acid molecules encoding the mature Zwntl polypeptide of SEQ ID NO: 4. Those skilled in the art will recognize that the degenerate sequences of SEQ ID NOs: 3 and also provide all of the RNA sequences encoding SEQ ID NOs: 3. NOs: 2 and 4, respectively, by the substitutions of U by T. Thus, the present invention contemplates the nucleic acid molecules encoding the Zwntl polypeptide comprising nucleotides 207 to 1406 of SEQ ID NO: 1, acid molecules nucleic that encode al. Zwntl polypeptide comprising nucleotides 264 through nucleotides 1406 of SEQ ID NO: 1, nucleic acid molecules encoding mature Zwntl polypeptides, comprising nucleotides 264 through 1406 of SEQ ID NO: 1, and their RNA equivalents . Table 1 discloses one letter codes used within SEQ ID NO: 3 to denote degenerate positions. "Resolutions" are the nucleotides denoted by a one letter code. "Complement" indicates the code for the complementary nucleotides. For example, the code Y denotes either C or T, and its complement R denotes A or G. A is complementary to T, and G is complementary to C.

The degenerate codons used in SEQ ID NOs: 3 and 6, encompass all possible codons for a given amino acid, are set forth in Table 2.

Table 2 One of ordinary skill in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode an amino acid. For example, the degenerate codon for serine (WSN), in some circumstances, can encode arginine (AGR) and the degenerate codon for arginine (MNG) can, in some circumstances, encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence can encode variant amino acid sequences, but one ordinarily skilled in the art can easily identify such variant sequences by reference to the amino acid sequences of SEQ ID NOs: 2 and 4. and easily test the variant sequences for their functionality as described here. Different species may exhibit "preferential codon employment". In general, see, - Grantham et al., Nuc. Acids, Res. 8: 1893 (1980), Haas et al. , Curr. Biol. 6: 315 (1996), Wain-Hobson et al. , Gene 13: 355 (1981), Grosjean and Fiers, Gene 18: 199 (1982), Holm, Nuc. Acids Res. 14: 3075 (1986), Ikemura, J. Mol. Bi ol. 158: 513 (1982), Sharp and Matassi, Curr. Opin. Genet Dev. 4: 851 (1994), Kane, Curr. Opin. Biotechnol. 6: 494 (1995), and Makrides, Microbiol. Rev. 60: 512 (1996). As used herein, the term "preferential codon usage" or "preferential codons" is a term of the art that refers to the translation codons of the protein that are most frequently used in the cells of certain species, thus favoring one or a few of the possible representative codons that encode each amino acid (see Table 2). For example, the amino acid Trenonine (Thr) can be encoded by ACA, ACC, ACG or ACT, but in ACC mammalian cells, the codon is most commonly used; in other species, for example, yeast cells, viruses or bacteria, different Thr codons may be preferential. The preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of the preferential codon sequences in the recombinant DNA can, for example, increase the production of the protein by making the translation of the protein more efficient with a particular type or species of cell. Therefore, the degenerate codon sequences described in SEQ ID NOs: 3 and 6, serve as a standard for optimizing the expression of polynucleotides in various cell types and species commonly used in the art and described herein. Sequences containing preferential codons can be tested and optimized for expression in several species, and tested for their functionality as described herein. The present invention further provides variant polypeptides and nucleic acid molecules that represent the counterparts of other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are polypeptides of other mammalian species, including porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. The orthologs of human Zwintl can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zwintl as described herein. Suitable sources of mRNA can be identified by Northern staining of the probes with the probes designed from the sequences described herein. A library is then prepared from the RNA of a positive tissue or cell line. The cDNA encoding Zwintl can then be isolated by a variety of methods, such as the generation of probes with a complete or partial human cDNA with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction, with primers designed from the representative human Zwntl sequences described herein. Within a further method, a DNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the Zwntl polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ. ID NO: 1 represent a single allele of the human gene and Zwntl, and allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by generating cDNA probes or genomic libraries of different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEC. ID NO: 1, which includes those containing the silent mutations and those in which the mutations result in changes in the amino acid sequence, are within the scope of the present invention, as are the proteins that are the allelic variants of the SEC . ID NO: 2 or SEQ ID NO: 4. The cDNA molecules generated from the alternatively spliced mRNAs, which retain the properties of the Zwntl polypeptide, are included within the scope of the present invention, as are the polypeptides encoded by such cDNAs and ARNms. The allelic variants and splice variants of these sequences can be cloned by generating cDNA probes or genomic libraries for example a human thyroid cDNA library of different individuals or tissues according to standard procedures known in the art. Within certain embodiments of the invention, the isolated nucleic acid molecules can be hybridized under stringent conditions to the nucleic acid molecules comprising nucleotide sequences described herein. For example, such nucleic acid molecules can be hybridized under stringent conditions to the nucleic acid molecules comprising the nucleotide sequences of SEQ ID NO: 1, to the molecules. of nucleic acid consisting of the nucleotide sequences of nucleotides 264-1460 of SEQ ID NO: 1, or to nucleic acid molecules consisting of a nucleotide sequence complementary to SEQ ID NO: 1, to nucleotides 264- 1406 of SEQ ID NO: l. In general, stringent conditions are selected to be between about 5 ° C lower than the melting point term (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which, 50% of the target sequence hybridizes to a perfectly matched probe. A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can be hybridized if the nucleotide sequences have some degree of complementarity. Hybrids can tolerate unequal base pairs in the double helix, but the stability of the hybrid is influenced by the degree of equality. The Tm of the unequal hybrid decreases by 1 ° C for each 1-1.5% of base pair mismatch. By varying the stringency of the hybridization conditions, control over the degree of inequality that will be present in the hybrid is allowed. The degree of stringency increases as the hybridization temperature increases and the ionic extension of the hybridization buffer increases. The stringent hybridization conditions encompass temperatures of about 5-25 ° C below the Tm of the hybrid and a hybridization buffer having up to 1 M Na +. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the Tm of the hybrid by approximately 1 ° C per 1% formamide in the buffer solution. In general, such stringent conditions include temperatures of 20-70 ° C and a hybridization buffer containing up to 6xSSC and 0-50% formamide. A higher degree of stringency can be achieved at temperatures from 40-70 ° C with a hybridization buffer having up to 4xSSC and from 0-50% formamide. Highly stringent conditions typically encompass temperatures of 42-70 ° C with a hybridization buffer having up to IxSSC and 0-50% formamide. Different degrees of stringency can be used during hybridization and washing to achieve the maximum specific binding to the target sequence. Typically, washings after hybridization are performed at increased degrees of stringency to remove probes from unhybridized polynucleotides from hybridized complexes. The above conditions are suggested to serve as a guide and are also within the abilities of a person skilled in the art to adapt these conditions for use with a particular polypeptide hybrid. The Tm for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence. Those conditions which influence the Tm include the size and content of base pairs of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of the destabilizing agents in the hybridization solution. Numerous equations for calculating Tm are known in the art, and are specific for DNA, RNA, and DNA-RNA hybrids and sequences of polynucleotide probes of varying lengths (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al., (Eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 221 (1990). Sequence analysis software, such as OLIGO 6-0 (LRS, Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International, Palo Alto, CA), as well as Internet sites, are tools available for analysis a given sequence and calculate the Tm based on the user's defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of the longer polynucleotide sequences, > 50 base pairs, it is carried out at temperatures of approximately 20-25 ° C below the calculated Tm. For smaller probes, < 50 base pairs, hybridization is typically done at Tm or below 5-10 ° C. This allows the maximum hybridization ratio for the DNA-DNA and DNA-RNA hybrids. The length of the polynucleotide sequence influences the relationship and stability of the hybrid formation. The sequences of smaller probes, < 50 base pairs, reach equilibrium with the complementary sequences quickly, but can form fewer stable hybrids. Incubation times of any minute to hours can be used to achieve the hybrid formation. Longer probe sequences reach equilibrium more slowly, but form more stable complexes even at lower temperatures. Incubations are typically allowed to proceed overnight or remain. In general, the incubations are carried out for a period equal to three times the calculated Cot time. The Cot time, is the time taken to reassociate the polynucleotide sequences, can be calculated by a particular sequence by methods known in the art. The base pair composition of the polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the selection of the hybridization temperature and the ionic strength of the hybridization buffer. The A-T pairs are less stable than the G-C pairs in aqueous solutions containing sodium chloride. Therefore, if the G-C content is higher, the hybrid is more stable. Even the distribution of residues G and C within the sequence also contribute positively to the stability of the hybrid. In addition, the composition of base pairs can be manipulated to alter the Tm of a given sequence. For example, 5-methyldeoxycytidine can be replaced by deoxycytidine and 5-bromodeoxyuridine can be replaced by thymidine to increase Tm while 7-deazz-2'-deoxyguanosine can be replaced by guanosine to reduce the Tm dependence. The ionic concentration of the hybridization buffer also affects the stability of the hybrid. Hybridization buffers generally contain blocking agents such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), Denatured salmon sperm DNA, tRNA, milk powder (BLOTTO), heparin or SDS, and a source of Na +, such as SSC (IX SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl, 10 M NaH2P04, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid increases. Typically, hybridization buffers contain between 10 mM - 1 M Na +. The addition of destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the Tm of a hybrid. Typically, formamide is used at a concentration of up to 50% to allow incubations carried out at lower and more convenient temperatures. Formamide also acts to reduce non-specific background when using RNA probes. As an illustration, a nucleic acid molecule encoding a Zwintl variant polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) at 42 ° C overnight in a solution that comprises 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 M sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (Denhardt solution lOOx: 2% (p / v) Ficoll 400, 2% (w / v) polyvinylpyrrolidone and 2% (w / v) bovine serum albumin), 10% dextran sulfate, and 20 μg / ml denatured, trimmed salmon sperm DNA. One skilled in the art can discern variations of these hybridization conditions. For example, the hybridization mixture can be incubated at a higher or lower temperature, such as about 65 ° C in a solution that does not contain formamide. However, premixed hybridization solutions are available. { for example, EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed in accordance with the manufacturer's instructions. After hybridization, the nucleic acid molecules can be washed to remove unhybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions. Typical stringent washing conditions include washing in a 0.5x-2x SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 55-65 ° C. That is, the nucleic acid molecules encode a Zwnt-1 variant permenecen polypeptide hybridized after stringent washing conditions, with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) or with a nucleic acid molecule having the nucleotide sequence of nucleotides 264 to 1406 of SEQ ID NO: 1 (or its complement), in which the stringency of washing is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65 ° C, including 0.5x SSC with 0.1% SDS at 55 ° C, or 2x SSC with 0.1% SDS at 65 ° C. One skilled in the art can easily see equivalent conditions, for example, by replacing SSPE for SSC in the washed solution. Typically, highly stringent washing conditions include washing in a 0.1x-0.2x SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 50-65 ° C. In other words, the nucleic acid molecules encoding a variant Zwnt-1 polypeptide, remain hybridized after stringent ford conditions, with a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 1 (or its complement ), or with a nucleic acid molecule having the nucleotide sequence of nucleotides 264-1406 of SEQ ID NO: 1 (or its complement), in which, the stringency of the wash is equivalent to 0.1x-0.2x SSC with 0.1% SDS at 50-65 ° C, including 0.1 x SSC with 0.1% SDS at 50 ° C, or 0.2x SSC with 0.1% SDS at 65 ° C. The present invention also provides the isolated Zwntl polypeptides having a sequence identity substantially similar to the polypeptides of SEQ. ID NO: 2 or SEQ ID NO: 4, or your orthologs. The term "substantially similar" is used herein to denote polypeptides having 70%, 80%, 90%, 95%, 96%, 97%, 98% and 9%, sequence identity to the sequences shown in the SEC . ID NO: 2 or SEQ ID NO: 4, with the proviso that such polypeptides do not include the murine Wnt-lOA protein, which is identified by GenBank Access No. U61969, or the Wnt-lOA protein newt, which is identified by GenBank Access No. U65428.

The present invention also contemplates Zwntl variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the polypeptide encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and a Hybridization assay, as described above. Such Zwnt-1 variants include nucleic acid molecules (1) that remain hybridized after stringent washing conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) or with a nucleic acid molecule having the nucleotide sequence of the nucleotides 264-1406 of SEQ ID NO: 1 (or its complement), in which, the stringency of the wash is equivalent to 0.5x-2x SSC with 0.1% SDS a 50-65 ° C, and (2) which encodes a polypeptide having 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the SEQ ID NO: 2 or SEQ ID NO: 4, with the proviso that such polypeptides do not include the murine Wnt-lOA protein. (GenBank Access No. U61969) and the Wnt-lOA newt protein (GenBank Access No. U65428).

Alternatively, the Zwntl variants can be characterized as nucleic acid molecules (1) that remain hybridized after highly stringent washing conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) or with a nucleic acid molecule having the nucleotide sequence of the nucleotides 264-1406 of SEQ ID NO: 1 (or its complement) in which, the stringency of the wash is equivalent to 0.1x0.2x SSC with 0.1% of SDS at 50-65 ° C, and (2) which encodes a polypeptide having 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of amino acid of SEQ ID NO: 2 or SEQ ID NO: 4, with the proviso that such polypeptides do not include the murine Wnt-lOA protein. (GenBank Access No. U61969) and the Wnt-lOA newt protein (GenBank Access No. U65428). The percentage of sequence identity is determined by conventional methods. See, for example, Altschul et al. , Bull. Ma th. Bio. 48: 603 (1986), and Henikoff and Henikoff, Proc. Nat 'l Acad. Sci. USA 89: 10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment regions using a space aperture penalty value of 10, a penalty value for the space extension of 1, and the "blosum 62" registration matrix of Henikoff and Henikoff (ibid.) As shown in Table 3 (amino acids are indicated by the standard one-letter codes). The identity percentage is calculated as: ([Total number of identical matings] / [length of the long sequence plus the number of spaces entered in the longest sequence to align the two sequences]) (100). i * r »rH 1 It's been 1 E- in OJ OJ O & t rH t-l «a < ro OJ s tn O J H H rH 1 1 t-i L? rH CO O rH Co OJ Ol I 1 1 1 1 > 1 • 4 «3» J o o l r-l Ol t-l rH 1 1 1 1 1 1 • * J co H o ro l rH C rH CO 1 1 1 1 1 1 X oo o co rH OJ t-l J H OJ! J CO 1 1 1 1 1 1 or VJD J • 4 JO ol O OJ J ro CO 1 1 1 1 1 1 1 1 w Lfl Í O r tH OJ CO RH O R OJ 1 1 I 1 1 1 1 1 a CS JO ro il oo HO rf JH OJ 1 1 1 1 1 1 1 1 1 uf? m ro co rH CO «H Ol o H H Ol O I 1 1 1 t 1 1 1 1 1 1 1 1 Q VD O OL H fO H O co H O r-l co ro 1 1 1 1 1 1 1 1 1 1 S VD r-1 CO oo O i H ro ro OJ ol rH O l co 1 1 t 1 r 1 1 1 co O in in o fO i O O O O C OJ O Ol o Ol ro 1 1 1 1 1 1 1 1 1 1 1 1 1 (you H < c • tf H Oí or rH H s JH rH l HHO or OJ O i 1 1 1 1 1 1 1 1 1 1 1 ctí E- < od ss Q us? OS tH *? tt £ fu CU CO E- S ¡* > Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity or similarity search algorithm of Pearson and Lipman is a suitable protein alignment method to examine the level of identity shared by an amino acid sequence described herein and the amino acid sequence of a putative Zwntl variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Na t 'l Acad. Sci. USA 85: 2444 (1988), and by Pearson, Meth. Enzymol. 183: 63 (1990). Briefly, the FASTA first characterizes the sequence similarity by identifying the regions shared by the interrogation or search sequence (eg, SEQ ID NO: 2) and a test sequence that has either the highest density of identities ( if the variable ktup is 1) or the pairs of identities (if ktup = 2), without considering the substitutions, insertions or deletions of conservative amino acids. The ten regions with the highest density of identity are then re-recorded or re-evaluated by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score or record.

If there are several regions with records greater than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined together to form an approximate alignment with intervals. Finally, the highest recording regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444 (1970); Sellers, SIAM J. Appl. Ma th. 26: 181 (1974)), which allows the insertions and deletions of amino acids. The illustrative parameters for the FASTA analysis are: ktup = 1, penalty for opening the interval = 10, penalty for extension of the interval = 1, and substitution matrix = BL0SUM62. These parameters can be entered into a FASTA program by modifying the score matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183: 63 (1990). The FASTA can also be used to determine the sequence identity of the nucleic acid molecules using a ratio as described above. For comparisons of nucleotide sequences, the ktup value may be in the range of one to six, preferably three to six, more preferably three, with other parameters adjusted as described above. The present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared to the amino acid sequence of SEQ. ID NO: 2, or SEC. ID NO: 4. That is, the variants can be obtained to contain one or more amino acid substitutions of the SEC. ID NO: 2, or SEC. ID NO: 4, in which an alkyl amino acid is replaced by an alkyl amino acid in an amino acid sequence of Zwntl, an aromatic amino acid is replaced by an aromatic amino acid in an amino acid sequence of Zwntl, an amino acid containing sulfur is replaced by an amino acid containing sulfur in an amino acid sequence of Zwntl, a hydroxy-containing amino acid is replaced by an amino acid containing hydroxy in an amino acid sequence of Zwntl, an acidic amino acid is replaced by an acidic amino acid in an amino acid sequence of Zwntl, a basic amino acid is substituted by a basic amino acid in a Zwntl amino acid sequence, or a dibasic monocarboxylic amino acid is substituted by a dibasic monocarboxylic amino acid in an amino acid sequence of Zwntl. Among the common amino acids, for example, a "conservative amino acid substitution" is illustrated by a substitution between the amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine , tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate (5) glutamine and asparagine, and (6) usin, arginine, and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local alignments of the protein sequence segments, representing highly conserved regions of more than 500 related protein groups (Henikoff and Henikoff, Proc. Na tl. Sci. USA 89: 10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define the conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely on chemical properties (as discussed above), the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2 or 3. According to this system, conservative substitutions of preferred amino acids are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), while most preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g. , 2 or 3) . Particular variants of the Zwntl are characterized by being greater than 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding human amino acid sequences (ie, SEQ ID NO. : 2), wherein the variation in the amino acid sequence is due to one or more conservative amino acid substitutions. Conservative amino acid changes in a Zwntl gene can be introduced by substituting the nucleotides for the nucleotides recited in the SEC. ID NO: 1. Such "conservative amino acid" variants can be obtained, for example, by oligonucleotide-directed mutagenesis, mutagenesis by linker scanning, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) in Pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991). The proteins of the present invention can also comprise the amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyltreonin, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine. Several methods are known in the art to incorporate amino acid residues that do not occur naturally within proteins. For example, an in vi tro system in which non-sense (non-5 ') mutations can be suppressed using chemically monoacylated suppressor RNAs can be employed. Methods for amino acid synthesis and aminocilant tRNA are known in the art. The transcription and translation of plasmids containing non-sense mutations is typically carried out in a cell-free system comprising an S30 extract of E. coli and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al. , J. Am. Chem. Soc. 113: 2122 (1991), Ellman et al. , Methods Enzymol. 202: 301 (1991), Chung et al. , Science 259: 806 (1993), and Chung et al. , Proc. Nafl Acad. Sci. USA 90: 10145 (1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of the Mutated mRNA and the chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991 (nineteen ninety six) ) . Within a third method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the amino acid (s) that does not occur. naturally, desired (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The amino acid that does not occur naturally is incorporated in the protein instead of its natural counterpart. See, Koide et al. , Biochem. 33: 1410 (1994). Naturally occurring amino acid residues can be converted to species that do not occur naturally by chemical modification in vi tro. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395 (1993)). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally, and unnatural amino acids can be replaced by the amino acid residues of Zwntl. Particular variants of Zwntl can be designed by modifying the human sequence to include one or more amino acid substitutions corresponding to the murine Wnt-lOA (GenBank Access No. U61969; SEQ ID NO: 7 here). For example, Table 4 shows differences between the SEC. ID NO: 2 and the murine Wnt-lOA, which were identified following the alignment of the amino acid sequences. Using the illustrative table, the Zwntl variants can be designed by introducing one or more amino acid substitutions from the Wnt-lOA sequence. Alternatively, murine Wnt-lOA sequences can be designed by introducing one or more amino acid substitutions of the Zwntl sequence. In this way, it is possible to obtain a "humanized" variant of the murine Wnt-lOA. Although Zwntl variants can be designed with any number of amino acid substitutions, certain variants will include at least about X amino acid substitutions, wherein X is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

Table 4 The essential amino acids in the polypeptides of the present invention can be identified according to the methods in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081 (1989), Bass et al. ., Proc. Na t '1 Acad. Sci. USA 88: 4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), Pages 259- 311 (Academic Press, Inc. 1998).] In the latter technique, unique mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for their biological activity as described below to identify residues of amino acids that are critical to the activity of the molecule See also, Hilton et al., J. Biol. Chem. 271: 4699 (1996) Identities of essential amino acids can also be interfered with by analyzing homologies with or after Wnt proteins.

The location of the binding domains of the Zwntl receptor can be determined by physical analysis of the structure, as determined by techniques such as magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with the amino acid mutation of the putative contact site. See, for example, de Vos et al. , Science 255: 306 (1992), Smith et al. , J. Mol. Biol. 224: 899 (1992), and Wlodaver et al. , FEBS Lett. 309: 59 (1992). In addition, Zwntl labeled with biotin or FITC can be used for the expression of cloning of Zwntl receptors. Multiple amino acid substitutions can be performed and tested using known methods of mutagenesis and selection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53 (1988)) or Bowie and Sauer (Proc. Na t r l Acad. Sci. USA 86: 2152 (1989)). Briefly, these authors describe methods for the simultaneous randomization of two or more positions in a polypeptide, selecting the functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include the phage sample (e.g., Lowman et al., Biochem 30: 10832 (1991), Ladner et al., U.S. Patent No. 5,223,409, Huse, international publication No. WO 92/06204. , and region-directed mutagenesis (Derbyshire et al., Gene 46: 145 (1986), and Ner et al., DNA 7: 127, (1988)). The variants of the Zwntl polypeptide and nucleotide sequences described they can also be generated by stirring or shuffling the DNA as described by Stemmer, Na ture 370: 389 (1994), Stemmer, Proc. Na tr 1 Acad. Sci. USA 91: 10141 (1994), and the international publication No. WO 97/20078 Briefly, variant DNAs are generated by homologous recombination in vi tro by the random fragmentation of an original DNA followed by reassembly using PCR, resulting in mutations at randomly introduced points. family of original DNAs, such as alleles or DNAs of different species, to introduce the additional variability in the process. The selection or separation of the desired activity, followed by additional iterations of mutagenesis and assays provides a rapid "evolution" of the sequences by selecting the desirable mutations while simultaneously selecting against deleterious changes.

Mutagenesis methods as described herein can be combined with automated, high efficiency screening methods to detect the activity of mutagenized polypeptides cloned in the host cells. Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind to anti-Zwntl antibodies, can be recovered from host cells and rapidly sequenced using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. The present invention also includes the "functional fragments" of Zwntl polypeptides and nucleic acid molecules that encode such functional fragments. Routine removal assays of the nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule encoding a Zwntl polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ. ID NO: 1 can be digested with nuclease BaI31 to obtain a series of eliminations placed one on top of the other. An alternative for exonuclease digestion is to use oligonucleotide directed mutagenesis to introduce deletions or stop codons for the specific production of a desired fragment. Alternatively, particular fragments of a Zwntl gene can be synthesized using the polymerase chain reaction. As an illustration, studies of truncation in either or both of the terminal ends of the interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66: 507 (1995). In addition, standard techniques for functional analysis are described, for example, by Treuter et al. , Molec. Gen Genet 240: 113 (1993), Content et al. , "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), Pages 65-72 (Nijhoff 1987) , Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Vol. 1, Boynton et al., (Eds.) Pages 169-199 (Academic Press 1985), Coumailleau et al. , J. Biol. Chem. 270: 29210 (1995); Fukunaga et al., J. Biol. Chem. 270: 25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50: 1295 (1995), and Meisel et al., Plant Molec. Biol. 30: 1 (1996).

The present invention also contemplates the functional fragments of a Zwntl gene having amino acid changes, compared to the amino acid sequences of SEC. ID NO: 2 or SEC. ID NO: 4. A variant Zwntl gene can be identified based on the structure of determination of identity level with the amino acid and nucleotide sequences of SECs. ID NOS: 1, 2, and 4, as discussed above. An alternative approach to identifying a variant gene in the structure base is to determine whether a nucleic acid molecule encoding a potential variant Zwntl gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ. ID NO: 1, as discussed above. The present invention also provides fragments of polypeptides or peptides comprising a portion carrying a Zwntl polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope", which is a part of a protein that generates an antibody response when the entire protein is used as an immunogen. Peptides carrying the immunogenic epitope can be identified using standard methods (see, for example, Geysen et al., Proc. Na t'l Acad. Sci. USA 81: 3998 (1983)).

In contrast, fragments of the polypeptide or peptides may comprise an "antigenic epitope," which is a region of a molecule of the protein to which an antibody can specifically bind. Certain epitopes consist of a contiguous or linear extension of amino acids, and the antigenicity of such an epitope is not broken by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al., Science 219: 660 (1983)). Accordingly, the peptides carrying the antigenic epitope and the polypeptides of the present invention are useful for generating antibodies that bind to the polypeptides described herein. Peptides carrying the antigenic epitope and the polypeptides preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or approximately 15 to about 30 amino acids of SEQ. ID NO: 2 or SEC. ID NO: 4. Such epitope-bearing peptides and polypeptides can be produced by fragmentation of a Zwntl polypeptide, or by chemical synthesis of the peptide, as described herein. In addition, the epitopes can be selected by the phage sample from the random libraries of the peptide (see, for example, Lane and Stephen, Curr Opin. Immunol., 5: 268 (1993), and Cortese et al., Curr. Opin. Biotechnol 7: 616 (1996)). Standard methods for the identification of epitopes and the production of small peptide antibodies comprising an epitope are described, for example, by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson (ed.), Pages 105-116 (The Humana Press, Inc. 1992), Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), Pages 60-84 (Cambridge University Press 1995), and Coligan et al. , (eds.), Current Protocols in Immunology, pages 9.3.1 - 9.3.5 and pages 9.4.1 - 9.4.11 (John Wiley & amp; amp;; Sons 1997). For any Zwntl polypeptide, including variants and fusion proteins, one of ordinary skill in the art can easily generate a totally degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above. In addition, those skilled in the art can use a set of standard programs to design Zwntl variants based on the nucleotide or amino acid sequences described herein. Accordingly, the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the SEC. ID NO: 1, nucleotides 264-1406 of the SEC. ID NO: 1, amino acid residues 20-400 of SEC. ID NO: 2, SEC. ID NO: 2, SEC. ID NO: 3, and SEC. ID NO: 4. Suitable forms of computer-readable media include a magnetic medium and an optically readable medium. Examples of magnetic media include a fixed or hard disk, a random access memory (RAM) microcircuit, a diskette, a digital linear tape (DLT), a disk cache, and a ZIP disk. Optically readable media is exemplified by compact disks (for example, CD-read-only memory (ROM), CD-rewritable (RW), and CD-rewritable), and versatile discs / digital video (DVD) (for example, DVD-ROM, DVD-RAM, and DVD + RW).

. Production of Zwntl Fusion Proteins Zwntl fusion proteins can be used to express Zwntl in a recombinant host and to isolate the expressed Zwntl. As described below, the particular Zwntl fusion proteins also have uses in diagnosis and therapy. One type of fusion protein comprises a peptide that guides a Zwntl polypeptide from a recombinant host cell. To direct a Zwntl towards the secretory pathway or path of a eukaryotic host cell, a secretory signal sequence (also known as a signal peptide, a leader sequence, a prepro sequence or a pre sequence) is provided in the expression vector of the Zwntl. While the secretory signal sequence can be derived from the Zwntl, a suitable signal sequence can also be derived from another protein secreted or synthesized de novo. The secretory signal sequence is operably linked to a sequence encoding Zwntl such that the two sequences are joined in the correct reading structure and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. The secretory signal sequences are positioned common 5 'to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences can be positioned anywhere in the nucleotide sequence of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Although the secretory signal sequence of Zwntl or another protein produced by mammalian cells (e.g., tissue-type plasminogen activator signal sequence, as described, for example, in U.S. Patent No. 5,641,655) is useful for Expression of Zwntl in recombinant mammalian hosts, a yeast signal sequence is preferred for expression in yeast cells. Examples of suitable yeast signal sequences are those derived from the a-factor of the yeast coupling pheromone (encoded by the MFal gene), the invertase (encoded by the SUC2 gene), or the acid phosphatase (encoded by the gene). PH05). See, for example, Romanos et al. , "Expression of Cloned Genes in Yeast," in DNA Cloning 2: A Practical Approach, 2nd Edition, Glover and Hames (eds.), Pages 123-167 (Oxford University Press 1995). In bacterial cells, it is often desirable to express a heterologous protein as a fusion protein to decrease toxicity, increase stability, and improve recovery of the expressed protein. For example, Zwntl can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide. The glutathione S-transferase fusion proteins are typically soluble, and easily purifiable from E lysates. coli on immobilized glutathione columns. In similar approaches or methodologies, a Zwntl fusion protein comprises a polypeptide of the maltose-binding protein that can be isolated with an amylose resin column, while a fusion protein comprising the C-terminus of A truncated Protein A gene can be purified using IgG-Sepharose. The techniques established to express a heterologous polypeptide as a fusion protein in a bacterial cell are described, for example, by Williams et al. , "Expression of Foreign Proteins in E.coli, Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical Approach, 2nd Edition, Glover and Hames (Eds.), Pages 15-58 (Oxford University Press 1995). In addition, expression systems are commercially available. For example, the PINPOINT protein Xa purification system (Promega Corporation, Madison, WI) provides a method for isolating a fusion protein comprising a polypeptide that becomes biotinylated during expression with a resin comprising avidin. Peptide labels or tags that are useful for isolating the heterologous polypeptides expressed by either eukaryotic or prokaryotic cells include the polyHistidine tags (which have an affinity for the nickel chelating resin), the c-myc labels or tags, the calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds to anti-RYIRS antibodies), the Glu tag -Glu, and the FLAG tag (which binds to anti-FLAG antibodies). See, for example, Luo et al. , Arch. Biochem. Biophys. 329: 215 (1996), Morganti et al. , Biotechnol. Appl. Biochem. 23: 61 (1996), and Zheng et al. , Gene 186: 55 (1997). Nucleic acid molecules encoding such labels or peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO). Another form of the fusion proteins comprises a Zwntl polypeptide and a constant region of the immunoglobulin heavy chain, typically an Fc fragment, which contains two or three constant region domains and a binding region but lacks the variable region. As an illustration, Chang et al. , US Patent No. 5,723,125, discloses a fusion protein comprising a human interferon and an Fc fragment of human immunoglobulin. The C-terminus of the interferon is linked to the N-terminus of the Fc fragment by a linker portion of the peptide. An example of a peptide linker is a peptide comprising primarily an inert sequence of T cells, which is immunologically inert. An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO: 5). In this fusion protein, a preferred Fc portion is a human α4 chain, which is stable in the solution and has little or no complementary activation activity. Accordingly, the present invention contemplates a Zwntl fusion protein comprising a portion of Zwntl and a human Fc fragment, wherein the C-terminus of the Zwntl portion binds to the N-terminus of the Fc fragment via the peptide linker , such as a peptide consisting of the amino acid sequence of SEC. ID NO: 5. The Zwntl portion can be a Zwntl molecule or a fragment thereof. In another variation, a Zwntl fusion protein comprising an IgG sequence, a Zwntl portion covalently attached to the amino terminal terminus and the IgG sequence, and a signal peptide that is covalently linked to the amino terminal of the Zwntl portion, wherein the sequence of IgG consists of the following elements in the following order: a binding region, a CH2 domain, a CH3 domain. In accordance with this, the IgG sequence lacks a CHi domain. This general approach to producing fusion proteins comprising both antibody and non-antibody portions have been described by LaRochelle et al. , EP 742830 (WO 95/21258). Fusion proteins comprising a portion of Zwntl and an Fc portion can be used, for example, as an in vitro testing tool. For example, the presence of a Zwntl receptor in a biological sample can be detected using a Zwntl-antibody fusion protein, in which the Zwntl portion is used to target the like receptor or the like, and a macromolecule, such as Protein A or an anti-Fc antibody, are used to detect the bound complex fusion protein-receptor. In addition, such fusion proteins can be used to identify agonists and antagonists that interfere with the binding of Zwntl to its receptor. In addition, using the methods described in the art, Zwntl hybrid proteins can be constructed using regions or domains of the inventive Zwntl in combination with those of other Wnt family proteins (e.g., Wnt-1, Wnt-2, Wnt- 2B / 13, Wnt-3, Wnt-4, Wnt-5A, Wnt-7A, Wnt-8A, Wnt-8B, Wnt-IOB, Wnt-11, Wnt-14, and human Wnt-15), or proteins heterologous (see, for example, Picard, Cur. Opin. Biology 5: 511 (1994)). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids can alter the kinetics of the reaction, binding, constricting, or expanding the substrate specificity, or altering the tissue and cellular location or tissue of a polypeptide, and can be applied to polypeptides of the unknown structure. For example Horisberger and Di Marco, Pharmac. Ther. 66: 501 (1995), describes the construction of the fusion protein hybrids comprising different interferon subtypes, as well as the hybrids comprising the interferon α domains from different species. The fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide that encodes both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. General methods for the enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) on pages 16-19 to 16-25.

Production of Zwntl polypeptides in Cultured Cells Polypeptides of the present invention that include the full-length polypeptides, functional fragments, and fusion proteins can be produced in recombinant host cells following conventional techniques. To express a Zwntl gene, a nucleic acid molecule encoding the polypeptide must be operalinked to the regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene that is suitable for the selection of cells that carry or carry the expression vector. Expression vectors that are suitable for the production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements that encode a bacterial origin of replication and a marker of resistance to the antibiotic to provide growth and selection of the vector of expression in a bacterial host; (2) eukaryotic DNA elements that control the initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of the transcripts, such as a transcription termination / polyadenylation sequence. As discussed above, expression vectors can also include nucleotide sequences that encode a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell. For example, a Zwntl expression vector may comprise a Zwntl gene and a secretory sequence derived from a Zwntl gene or other secreted gene. The Zwntl proteins of the present invention can be expressed in mammalian cells. Examples of mammalian host cells include African green monkey kidney cells (Vero, ATCC CRL 1587), human embryonic kidney cells (293-HEK, ATCC CRL 1573), baby hamster kidney cells (BHK -21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell.

Molec. Genet 12: 555 (1986)]), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548 ) monkey kidney cells transformed with SV40 (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658). For a mammalian host, the transcriptional or translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with the particular gene having a high level of expression. Suitable transcriptional and translational regulatory sequences can also be obtained from mammalian genes, such as actin, collagen, myosin and metallothionein genes.

The transcriptional regulatory sequences include a sufficient promoter region to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hammer et al., J. Molec. Appl.

Genet 1: 273 (1982)), the Herpes virus TK promoter (McKnight, Cell 31: 355 (1982)), the SV40 early promoter (Benoist et al., Nature 290: 304 (1981)), the promoter of the Rous sarcoma virus (Gorman et al., Proc. Nat '1 Acad. Sci. USA 79: 6111 (1982)), the promoter of cytomegalovirus (Foecking et al., Gene 45: 101 (1980)), and the promoter of mouse mammary tumor virus (see, generally, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture, "in Protein Engineering: Principles and Practice, Cleland et al. (Eds.), Pages 163-181 (John Wiley &Sons, Inc. 1996).) Alternatively, a prokaryotic promoter, such as the promoter of the RNA polymerase of bacteriophage T3 can be used to control the expression of the Zwntl gene in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al., Mol.Cell.Biol.10: 4529 (1990) , and Kaufman et al., Nucí Acids Res. 19: 4485 (1991)).

An expression vector can be introduced into the host cells, using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like. Preferably, the transfected cells are selected and propagated to provide recombinant host cells comprising the expression vector stably integrated into the genome of the host cell. Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991). For example, a suitable selectable marker is a gene that provides resistance to antibiotic neomycin. In this case, the selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by culturing the transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to select the cells that produce high levels of the products of the introduced genes. A selectable, amplifiable, preferred marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Alternatively, markers that introduce an altered phenotype, such as a green fluorescent protein or cell surface proteins such as CD4, CD8, MHC Class I, placental alkaline phosphatase, can be used to classify the transfected cells from the cells not transfected by such means as the FACS classification or the separation technology by magnetic beads. Zwntl polypeptides can also be produced by culturing mammalian cells using a viral delivery system. Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV). Adenoviruses, a double-stranded DNA virus, is currently the best-studied gene transfer vector for the delivery of heterologous nucleic acid (for review, see Becker et al., Meth Cell Biol. 43: 161 (1994), and Douglas and Curiel, Science &Medicine 4:44 (1997)). The advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow them to a high titer, the ability to infect a wide range of mammalian cell types, and the flexibility that allows use with a number Large vector available containing different promoters. By removing portions of the adenovirus genome, larger inserts (up to 7 kb) of the heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. One option is to eliminate the essential gene from the viral vector, which results in the inability to replicate unless the El gene is provided by the host cell. Human 293 cells infected with the adenovirus vector (ATCC Nos. CLR-1573, 45504, 45505), for example, can be grown as adherent cells or in the suspension culture at relatively high densities to produce significant amounts of protein (see Garnier et al., Cytotechnol 15: 145 (1994)).

Zwntl genes can also be expressed in other higher eukaryotic cells, such as bird cells, fungi, insects, yeasts, or plants. The baculovirus system provides an efficient means to introduce Zwntl genes into insect cells. Suitable expression vectors are based on Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as the Drosophila heat shock protein (Hsp) promoter 70, the immediate early gene promoter of Autographa californica nuclear polyhedrosis virus (ie-1) and the delayed early 39K promoter, the baculovirus promoter PL O, and the Drosophila metallothionein promoter.

A second method for manufacturing the recombinant baculovirus uses a transposon-based system described by Luckow (Luckow, et al., J. Virol. 67: 4566 (1993)). This system, which uses transfer vectors, is sold in the BAC-to-BAC equipment (Life Technologies, Rockville, MD). This system utilizes a transfer vector, PFASTBAC (Life Technologies) which contains a Tn7 transposon to move the DNA encoding the Zwntl polypeptide to a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid". See, Hill-Perkins and Possee, J.

Gen Virol. 71: 911 (1990), Bonning, et al. , J. Gen. Virol. 75: 1551 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem. 270: 1543 (1995). In addition, the transfer vectors can include a fusion in the structure with a DNA encoding an epitope tag or label at the C- or N-terminus of the expressed Zwntl polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. USA 82: 1952 (1985)). Using a technique known in the art, a transfer vector containing a Zwntl gene is transformed into E. coli and selected for bacmides containing an interrupted lacZ gene indicative of the recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques. The illustrative pBASTBAC vector can be modified to a considerable degree. For example, the polyhedrin promoter can be removed and replaced with the baculovirus basic protein promoter (also called the Peor promoter, p6.9 or MP) which is expressed earlier in baculovirus infection, and has been shown to be advantageous for expressing the secreted proteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol. 71: 911 (1990), Bonning, et al., J. Gen. Virol. 75: 1551 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem. 270: 1543 (1995) In such constructs of the transfer vector, a short or long version of the basic protein promoter can be used. construct to replace the secretory signal sequences of native Zwntl with the secretory signal sequences derived from insect proteins, eg, a secretory signal sequence of Ecdysteroid Glucosyltransferase (EGT), honey bee Melitin (Invitrogen Corporation; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the secretory signal sequence of native Zwntl. The recombinant or bacmid virus is used to transfect the host cells. Suitable insect host cells include IPLB-Sf-21 cell lines, an ovarian cell line from the pupa of Spodoptera frugiperda, such as Sf9 (ATCC CRL 1711), 5f2lAE and Sf21 (Invitrogen Corporation, San Diego CA ), as well as the Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (US Patent No. 5,300,435). A commercially available serum-free medium can be used to grow and maintain the cells. Suitable media are Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and Ex-cellO405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for the T. ni cells. When the recombinant virus is used, the cells are typically grown from an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral material is added to a multiplicity of infection (MOI) from 0.1 to 10, more typically close to 3. Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al., "Manipulation of Baculovirus Vectors," in Methods in Molecular Biology, Volume 7: Gene Transfer and Expression Protocols, Murray (ed.), Pages 147-168 (The Humana Press, Inc. 1991), by Patel et al. , "The baculovirus expression system," in DNA Cloning 2: Expression Systems, 2nd Edi tion, Glover et al. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel (1995) on pages 16-37 to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995 ), and by Lucknow, "Insect Cell Expression Technology," in Protein Engineering: Principies and Practice, Cleland et al. (eds.), pages 183-218 (John Wiley &Sons, Inc. 1996). Mushroom cells, which include yeast cells, can also be used to express the genes described here. Yeast species of particular interest in relation to this include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Suitable promoters for the expression of yeasts include the promoters of GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available. These vectors include vectors based on YIp, such as YIp5, vectors YRp, such as YRpl7, vectors YEp such as vectors YEpl3 and YCp, such as YCpl9. The methods to transform S cells. Cerevisiae with the exogenous DNA and to produce the recombinant polypeptides thereof are described by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al. , U.S. Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008, Welch et al. , U.S. Patent No. 5,037,743, and Murray et al. , U.S. Patent No. 4,845,075. Transformed cells are selected by the phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the P0T1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in a medium containing glucose. Suitable additional promoters and terminators for use in yeasts include those of the glycolytic enzyme genes (see, for example, Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092) and the alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454. Transformation systems for other yeasts include Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose are known in the art. See, for example, Gleeson et al. , J. Gen. Microbiol. 132: 3459 (1986), and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be used according to the methods of Mcknight et al. , US Patent No. 4,935,349. The methods for transforming Acremonium chrysogenum are described by Sumino et al. , United States Patent No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Patent No. 4,486,533. For example, the use of Pichia methanolica for the production of recombinant proteins is described by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al. , Yeast 14: 11-23 (1998), and in international publications Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules that are used in the transformation of P. methanolica will be prepared in a common manner as double-stranded circular plasmids, which are preferably linearized before transformation. For the production of the polypeptide in P. methanolica it is preferred that the promoter and the terminator in the plasmid be that of a P. methanolica gene, such as an alcohol utilization gene of P. methanolica (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A preferred selectable marker for use in Pichia methanolica is an AD £ 2 gene from P. methanolica, which codes for phosphoribosyl-5-aminoimidazole carboxylase carboxylase (AIRC).; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine. For large-scale industrial processes, where it is desirable to minimize the use of methanol, it is preferred to use the host cells in which both the methanol utilization genes (AUG1 and AUG2) are removed. For the production of secreted proteins, host cells deficient in vacuolar protease genes are preferred (PEP4 and PRB1). Electroporation is used to facilitate the introduction of a plasmid containing the DNA encoding a polypeptide of interest into the cells of P. methanolica. The P. methanolica cells can be transformed by electroporation using a pulse electric field, with exponential decay, having a field strength of 2.5 to 4.5 kV / cm, preferably approximately 3.75 kV / cm, and a constant of time (t) from 1 to 40 milliseconds, more preferably approximately 20 milliseconds. Expression vectors can also be introduced into the protoplasts of plants, the tissues of intact plants, or isolated plant cells. Methods for introducing expression vectors into plant tissues include direct infection or co-cultivation or tissue of plants with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al. , Science 227: 1229 (1985), Klein et al. , Biotechnology 10: 268 (1992), and Miki et al. , "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993). Alternatively, the Zwntl genes can be expressed in prokaryotic host cells. Suitable promoters that can be used to express the Zwntl polypeptides in a prokaryotic host are well known to those skilled in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the PR and PL promoters of the bacteriophage lambda. , the promoters trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ from E. coli, the promoters of B. subtilis, the promoters of bacteriophages of Bacillus, the promoters of Streptomyces, the promoter int of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene. Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol. 1: 211 (1987), Watson et al. , Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (nineteen ninety five) . Preferred prokaryotic hosts include E. coli and Bacillus subtilus. Suitable strains of E. coli include BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, DH1, DH4I, DH5, DH5I, DH5IF ', DH51MCR, DH10B, DH10B / p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains of Bacillus subtilus include BR151, YB886, MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)) .

When a Zwntl polypeptide is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a bacterial secretion sequence. In the above case, the cells are lysed and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can be refolded or redoubled and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter almost, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, thus obviating the need for denaturing and refolding. Methods for expressing proteins in prokaryotic hosts are well known to those skilled in the art (see, for example, Williams et al. , "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995), Ward et al. , "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou, "Expression of Proteins in Bacteria," in Protein Engineering: Principies and Practice, Cleland et al. (eds.), page 101 (John Wiley & amp; amp; amp; amp; amp.; Sons, Inc. 1996)). Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are provided, for example, by Ausubel (1995). General methods for the expression and recovery of foreign proteins produced by the system of mammalian cells are provided by, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al. (eds.) pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for the recovery of proteins produced by a bacterial system are provided by, for example, Grisshammer et al. , "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 59-92 (Oxford University Press 1995). Established methods for isolating recombinant proteins from a baculovirus system are described by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995). As an alternative, the polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well known to those skilled in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85: 2149 (1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd Edition). ), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem Pept. Prot. 3: 3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989), Fields and Colowick, " Solid-Phase Peptide Synthesis, "Methods in Enzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc. 1997). Variations in the strategies of the total chemical synthesis, such as "native chemical ligation" and "ligation of the expressed protein" are also standard methods (see, for example, Dawson et al., Science 266: 1-16 (1994)). , Hackeng et al, Proc. Nat r 1 Acad Sci. USA 94: 7845 (1997), Dawson, Methods Enzymol 287: 34 (1997), Muir et al, Proc. Na t 'l Acad Sci. USA 95: 6705 (1998), and Severinov and Muir, J. Biol. Chem. 273: 16205 (1998)). 7. Isolation of Zwntl Polypeptides The polypeptides of the present invention can be purified to at least 80% purity, more preferably at least 90% purity, even more preferably up to at least about 95% purity, or even higher than 95% of purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and freeing them from infectious and pyrogenic agents. The polypeptides of the present invention can also be purified to a pharmaceutically pure state, which is greater than 99.9% pure. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides or animal origin. Conventional fractionation and / or purification methods can be used to obtain purified Zwntl preparations from natural sources (eg, spleen, thymus, spinal cord, and lymph node tissue), and recombinant Zwntl polypeptides and Zwntl polypeptides for fusion of recombinant host cells. In general, precipitation with ammonium sulfate can be used and extraction by acid or chaotrope can be used for the fractionation or fractionation of the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high resolution liquid chromatography. Suitable chromatographic medium includes dextrans derivatives, agarose, cellulose, polyacrylamide, special silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. The exemplary chromatographic medium includes those media derived with phenyl, butyl or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toxopearl butyl 650 (Toso Haas, Montgomeryville, PA ), Octyl-Sepharose (Pharmacia) and the like, or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they are to be used. . These supports can be modified with the reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of coupling chemistries include activation with cyanogen bromide, activation of N-hydroxysuccinimide, activation by epoxide, activation by sulfhydryl, activation by hydrazide, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. The selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affini and Chroma tography: Principies & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996). Additional variations in the isolation and purification of the Zwntl can be designed by those skilled in the art. For example, anti-Zwntl antibodies, obtained as described below, can be used to isolate large amounts of protein by immunoaffinity purification. In addition, methods for linking ligands, such as Zwntl, to receptor polypeptides linked to support media are well known in the art. The polypeptides of the present invention can be isolated by exploitation of particular properties. For example, immobilized metal ion adsorption chromatography (IMAC) can be used to purify histidine rich proteins, including those comprising the polyhistidine tags or labels. Briefly, a gel is charged first with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem 3: 1 (1985)). Rich proteins in histidine will be absorbed into this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, lowering the pH, or using strong chelating agents. Other purification methods include the purification of the glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (M. Deutscher, (ed.), Meth. Enzymol, 182: 529 (1990)).

Within the additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., the maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. Zwntl polypeptides or fragments thereof can also be prepared through chemical synthesis, as described below. The Zwntl polypeptides can be monomers or multimers; glycosylated or non-glycosylated; PEGylated and not PEGylated; and may or may not include an initial methionine amino acid residue. The present invention also contemplates chemically modified Zwntl compositions, in which the Zwntl polypeptide is linked to a polymer. Typically, the polymer is soluble in water so that the Zwntl conjugate does not precipitate in an aqueous environment, such as a physiological environment. An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled. An example of a reactive aldehyde is propionaldehyde of polyethylene glycol, or monoalkoxy- (C1-C10), or aryloxy derivatives thereof (see, for example, Harris, et al., US Patent No. 5,252,714). The polymer can be branched or unbranched. In addition, a mixture of polymers can be used to produce the Zwntl conjugates. The Zwntl conjugates used for the therapy should preferably comprise pharmaceutically acceptable water soluble polymer portions. Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, (C1-C10) monoalkoxy-PEG, aryloxy-PEG, poly- (N-vinylpyrrolidone) PEG, tresyl monomethoxy PEG, propionaldehyde PEG, bis-succinimidyl carbonate PEG , propylene glycol homopolymers, a copolymer of polypropylene oxide / ethylene oxide, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEGs can have a molecular weight of about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A Zwntl conjugate can also comprise a mixture of such water soluble polymers. Anti-Zwntl antibodies or anti-idiotype antibodies can also be conjugated with a water soluble polymer.

The present invention contemplates compositions comprising a peptide or polypeptide described herein. Such compositions may additionally comprise a carrier. The carrier can be a conventional organic or inorganic carrier. Examples of the carriers include water, buffer, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like. The peptides and polynucleotides of the present invention comprise at least six, preferably at least nine, and more preferably at least 15 contiguous amino acid residues of SEQ. ID NO: 2. Illustrative molecules comprise the following amino acid residues of SEC. ID NO: 26 'to 35, 94 to 103, 178 to 187, 196 to 205, 270 to 279, 305 to 314, 312 to 321, and 347 to 356. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of these amino acid sequences. The nucleic acid molecules encoding such peptides and polypeptides are useful as primers and probes of the polymerase chain reaction. 8. Production of Antibodies for Zwntl Proteins Antibodies to Zwntl can be obtained, for example, by using the product of a Zwntl or Zwntl expression vector isolated from a natural source as an antigen. Particularly useful are anti-Zwntl "antibodies that specifically bind" to Zwntl. The antibodies are considered specifically bound if the antibodies exhibit at least one of the following two properties: (1) the antibodies bind to the Zwntl with a threshold level of binding activity, and (2) the antibodies do not cross-react significantly with the polypeptides related to Zwntl. With respect to the first feature, antibodies bind specifically if they bind to a Zwntl polypeptide, peptide or epitope with a binding affinity (Ka) of 106 M "1 or greater, preferably 107 M 1 or more, more preferably 108 M- 1 or greater, and more preferably 108 M "1 or greater. The binding affinity of an antibody can be easily determined by one of ordinary skill in the art, for example, by the Scatchard analysis [Scatchard, Ann. NY Acad. Sci. 51: 660 (1949)]. Regarding the second feature, the antibodies do not cross-react significantly with respect to the related molecules of the polypeptide, for example, if they detect the Zwntl protein, but not the known related polypeptides using a standard Western blot analysis. Examples of the known, related polypeptides are orthologs and proteins from the same species that are members of the protein family. For example, anti-Zwntl antibodies that bind specifically bind to Zwntl, but not to polypeptides such as Wnt-1, Wnt-2, Wnt-2B / 13, Wnt-3, Wnt-4, Wnt-5A , Wnt-7A, Wnt-8A, Wnt-8B, Wnt-IOB, Wnt-11, Wnt-14, and human Wnt-15. In addition, the present invention contemplates anti-Zwntl antibodies that specifically bind to Zwntl, but do not bind to the murine Wnt-lOA, or those that do not bind to the new Wnt-lOA ("PWnt-lOa"). Suitable antibodies include antibodies that bind to Zwntl in regions that have low sequence similarity to other Wnt proteins. Anti-Zwntl antibodies can be produced using the peptides and polypeptides carrying the antigenic Zwntl epitopes. The peptides and polypeptides carrying the antigenic epitope of the present invention contain a sequence of at least nine, preferably from 15 to about 30 amino acids contained within SEQ. ID NO: 2 or within the SEC. ID NO: 4. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the complete amino acid sequence of a polypeptide of the invention, are also useful for inducing antibodies that bind to Zwntl. It is desirable that the amino acid sequence of the epitope-bearing peptide be selected to provide substantial solubility in aqueous solvents (ie, the sequence includes relatively hydrophilic residues, whereas hydrophobic residues are preferably avoided). In addition, amino acid sequences containing proline residues may also be desirable for the production of antibodies. Polyclonal antibodies to the recombinant Zwntl protein or Zwntl isolated from natural sources can be prepared using methods well known to those skilled in the art. For example, Busse and Séguin, Biochem. Mol. Biol. Int. 30: 607 (1993), describe the production of polyclonal antibodies against a synthetic peptide corresponding to an epitope exposed on the surface of a Wnt-1 homologue. The antibodies can also be generated using a fusion protein of Zwntl-glutathione transferase, which is similar to a method described by Burrus and McMahon, Exp. Cell. Res. 220: 363 (1995). General methods for the production of polyclonal antibodies are described, for example, by Green et al. , "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), Pages 1-5 (Humana Press 1992), and Williams et al. , "Expression of foreign proteins in E.coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edi tion, Glover et al., (eds.), page 15 (Oxford University Press 1995). The immunogenicity of a Zwntl polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as Zwntl fusions or a portion thereof or an immunoglobulin polypeptide or with a maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like" such a portion can be advantageously attached or bonded to a macro olecular carrier (such as the limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. Although polyclonal antibodies are typically generated in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep, an anti-Zwntl antibody of the present invention can also be derived from a primate antibody. subhuman General techniques for generating diagnostic and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al. , International Patent Publication No. WO 91/11465, and Losman et al. , In t. J. Cancer 46: 310 (1990). Alternatively, monoclonal anti-Zwntl antibodies can be generated. Rodent monoclonal antibodies to specific antigens can be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Na ture 256: 495 (1975), Coligan et al., (Eds. ), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & amp; amp; amp;; Sons 1991) ["Coligan"], Picksley et al. , "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edi tion, Glover et al. (eds.), page 93 (Oxford University Press 1995)). Briefly, the monoclonal antibodies can be obtained by injecting the mice with a composition comprising a product of the Zwntl gene, verifying the presence of antibody production by removing the serum sample, removing the spleen to obtain the B lymphocytes. , by fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting the positive clones that produce the antibodies to the antigen, cultivating the clones that produce the antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. In addition, an anti-Zwntl antibody of the present invention can be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been genetically engineered to produce specific human antibodies in response to the antigenic immunity test. In this technique, the elements of the human light and heavy chain sites are introduced into the strains of mice derived from embryonic stem cell lines that contain disruptions that are considered target sites of the light chain and heavy chain , endogenous The transgenic mouse can synthesize human antibodies specific for human antigens, and mice for use to produce the hybridomas that secrete human antibodies. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al. , Na ture Genet. 7:13 (1994), Lonberg et al. , Na ture 368: 856 (1994), and Taylor et al. , In t. Immun. 6: 579 (1994). Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and on pages 2.9.1. -2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). For particular uses, it may be desirable to prepare anti-Zwntl antibody fragments. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by digestion of pepsin or papain from whole antibodies by conventional methods. As an illustration, fragments of antibodies can be produced by enzymatic cleavage of the antibodies with pepsin to provide a 5S fragment denoted F (ab ') 2- This fragment can be further cleaved using a thiol reducing agent to produce monovalent fragments of 3.5S Fab '. Optionally, the cleavage or cleavage reaction can be performed using a blocking group for the sulfhydryl groups which results in the cleavage of the disulfide bonds. As an alternative, cleavage or enzymatic cleavage using pepsin produces two monovalent Fab fragments and one Fc fragment directly. These methods are described, for example, by Goldenberg, US Patent No. 4,331,647, Nisohoff et al. , Arch Biochem. Biophys. 89: 230 (1960), Porter, Biochem. J. 73: 119 (1959), Edelman et al. , in Methods in Enzymolgy Vol. 1, page 422 (Academic Press 1967), and by Coligan et al. , on pages 2.8.1-2.8.10 and 2.10.-2.10.4. Other antibody cleavage methods, such as heavy chain separation to form monovalent fragments of light-heavy chains, in addition to excision of the fragments, or other enzymatic, chemical or genetic techniques can also be used, while fragments are bind to the antigen that is recognized by the intact antibody. For example, the Fv fragments comprise an association of VH and VL chains. This association may be non-covalent, as described by Inbar et al. , Proc. Wat '1 Acad. Sci. USA 69: 2659 (1972). Alternatively, the variable chains can be linked by an intramolecular or crosslink disulfide bond by chemicals such as glutaraldehyde (see, eg, Sandhu, Cri t Rev. Biotech, 12: 431 (1992)). The Fv fragments can comprise the VH and VL chains which are connected by a peptide linker. These single chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising the DNA sequences encoding the VH and VL domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector that is subsequently introduced into a host cell, such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide by forming a bridge of the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al. , Methods: A Companion to Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science 242: 423 (1988), Ladner et al., U.S. Patent No. 4,946,778, Pack et al., Bio / Technology 11: 1211 (1993), and Sandhu, supra). As an illustration, a scFV can be obtained by exposing the lymphocytes to the Zwntl in vi tro polypeptide, and selecting the sample libraries of the antibody in the phage or similar vectors (e.g., through the use of the Zwntl protein or peptide). tagged or immobilized). The genes encoding the polypeptides having potential Zwntl polypeptide binding domains can be obtained by random selection of the peptide libraries displayed on the phage (phage sample) or on the bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in various ways, such as through random mutagenesis and random polynucleotide synthesis. These random sample libraries of the peptide can be used to select peptides that interact with the known target which can be a protein or polypeptide, such as a ligand or receptor, a synthetic or biological macromolecule, or organic or inorganic substances. Techniques for creating and selecting such random, peptide sample libraries are known in the art (Ladner et al., US Patent No. 5,223,409, Ladner et al. , U.S. Patent No. 4,946,778, Ladner et al. , U.S. Patent No. 5,403,484, Ladner et al. , U.S. Patent No. 5,571,698, and Kay et al. , Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and randomized libraries and sample kits for selecting such libraries are commercially available, for example from CLONTECH Laboratories, Inc. (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Peptide sample libraries, randomized, can be selected using the Zwntl sequences described herein to identify proteins that bind to Zwntl. Another form of an antibody fragment is a peptide encoding a single region that determines complementarity (CDR). CDR peptides ("minimal recognition units") can be obtained by the construction of genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region from the RNA of the cells that produce the antibody (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 ( 1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995), and Ward et al. , "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principies and Applications, Birch et al. , (eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zwntl antibody can be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring the mouse complementarity determining regions from the light and heavy variable chains of the mouse immunoglobulin to a human variable domain. The typical residues of the human antibodies are then replaced in the regions of the working structure of the murine counterparts. The use of the antibody components derived from the humanized monoclonal antibodies obviates the potential problems associated with the immunogenicity of the murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 431 (1992), Singer et al., J. Immun. 150: 2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principies and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley &Sons, Inc. 1996), and by Queen et al. , U.S. Patent No. 5,693,762 (1997). Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zwntl antibodies or antibody fragments, using standard techniques. See, for example, Green et al. , "Production of Polyclonal Antisera," in Methods in Molecular Biology: Immunochemi cal Protocols, Manson (ed.), Pages 1-12 (Humana Press 1992). Also, see Coligan on pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using the anti-Zwntl antibodies or the antibody fragments as the immunogens with the techniques described above. As another alternative, humanized anti-idiotype antibodies or anti-idiotype antibodies of subhuman primates can be prepared using the techniques described above. Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Patent No. 5,208,146, Greene, et al. , U.S. Patent No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol. 77: 1875 (1996).

Zwntl anti-idiotype antibodies, as well as Zwntl polypeptides, can be used to identify and isolate Zwntl ligands. For example, the proteins and peptides of the present invention can be immobilized on a column and used to bind the receptor proteins of the membrane preparations that are run on the column (Hermanson et al. (Eds.), Immobili. zed Affini and Ligand Techniques, pages 195-202 (Academic Press 1992)). Affinity labeled or radiolabelled Zwntl polypeptides can also be used to identify or localize the Zwntl ligands in a biological sample (see, eg, Deutscher (ed.), Methods in Enzymol., Vol.182, pages 721- 37 (Academic Press 1990), Brunner et al., Ann. Rev. Biochem. 62: 483 (1993), Fedan et al., Biochem Pharmacol 33: 1167 (1984)). Also see, Varthakavi and Minocha, J. Gen. Virol. 77: 1875 (1996), who describe the use of anti-idiotype antibodies for receptor identification. Among other uses described below, anti-Zwntl antibodies can be used to isolate CD4 + cells. Standard methods for the selection of T cell subpopulations with antibodies are described, for example, by Saxton et al. , Blood 89: 2529 (1997). 9. Use of the Zwntl Nucleotide Sequences to Detect the Zwntl Gene Expression and to Examine the Structure of the Zwntl Gene The nucleic acid molecules can be used to detect the expression of a Zwntl gene in a biological sample. Such probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ. ID NO: 1, or a fragment thereof, as well as the single-stranded nucleic acid molecules that have the complement of the SEC nucleotide sequence. ID NO: 1, or a fragment thereof. The probe molecules can be DNA, RNA, oligonucleotides, and the like. Certain probes are linked to the regions of a Zwntl gene that has a low sequence similarity to comparable regions in other Wnt proteins. For example, suitable probes that bind to at least one of the following portions of the SEC nucleotide sequence. ID NO: 1 is linked to regions of low sequence similarity to human Wnt-IOB: nucleotides 714 to 758, and nucleotides 1090 to 1134. As used, the term "portion" refers to at least eight nucleotides up to at least 20 or more nucleotides. In a basic assay, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic concentration that promote base pairing between the probe and target Zwntl RNA species . After separation of the unbound probe from the hybridized molecules, the number of hybrids is detected. Well-established hybridization methods of RNA detection include northern analysis and hybridization of spot / groove staining (see, for example, Ausubel (1995) on pages 4-1 to 4-27, and Wu et al. (Eds. .), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997). Nucleic acid probes can be detectably labeled with radioisotopes such as 32P or 35S. Alternatively, Zwntl RNA can be detected with a non-radioactive hybridization method (see, eg, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Humana Press, Inc. 1993)).

Typically, non-radioactive detection is achieved by the enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative non-radioactive portions include biotin, fluorescein, and digoxigenin. The Zwntl oligonucleotide probes are also useful for in vivo diagnosis. As an illustration, 18F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Na ture Medicine 4: 461 (1998)). The numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase the sensitivity of the detection methods. Standard techniques for performing PCR are well known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applica tions (Humana Press, Inc. 1993) Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo ( ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)). Preferably, the PCR primers are designed to amplify a portion of the Zwn tl gene that has a low sequence similarity to a comparable region in other Wnt sequences. As an illustration, suitable primers are designed to amplify the regions within nucleotides 714 to 758, and nucleotides 1090 to 1134 of SEQ. ID NO: 1. Those regions correspond to regions of low sequence similarity within the human Wnt-IOB. A variation of the PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, the RNA is isolated from a biological sample, reverse transcribed to the cDNA and the cDNA is incubated with the Zwntl primers (see, for example, Wu et al., (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR , "in Methods in Gene Biotechnology, pages 15-28 (CRC Press, 1997)). Then the PCR is performed and the products are analyzed using standard techniques. As an illustration, the RNA is isolated from a biological sample using, for example, the guanidinium thiocyanate cell lysis procedure described above. Alternatively, the solid phase technique can be used to isolate the mRNA from a cell lysate. The reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short dT homopolymers, or Zwntl anti-sense oligomers. The oligo-dT primers offer the advantage that several mRNA nucleotide sequences are amplified which can provide the target control sequences. The Zwntl sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers are typically 20 bases in length. The products of PCR amplification can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by staining with ethidium bromide. Alternatively, the fractionated PCR products can be transferred to a membrane, hybridized with a detectably labeled Zwntl probe, and examined by autoradiography. Additional alternative approaches or methodologies include the use of deoxyribonucleic acid triphosphates labeled with digoxigenin to provide chemiluminescent detection, and the colorimetric assay with C-TRAK. Another approach or methodology for the expression of Zwntl is a cyclization probe (CPT) technology, in which a single-stranded target DNA joins with an excess of chimeric DNA-RNA-DNA probe to form a complex, the RNA portion is cleaved with RNAase H, and the presence of the excised chimeric probe is detected (see, for example, Beggs et al., J. Clin Microbiol 34: 2985 (1996), Bekkaoui et al., Biotechniques 20 : 240 (1996)). Alternative methods for the detection of sequences of Zwn tl can use methodologies such as amplification based on nucleic acid sequence (NASBA), the cooperative amplification of templates or patterns by cross-hybridization (CATCH), and the ligase chain reaction (LCR) (see, for example, Marshall et al. , US Patent No. 5,686,272 (1997), Dyer et al. , J. Virol. Methods 60: 161 (1996), Ehricht et al. , Eur. J. Biochem. 243: 358 (1997), and Chadwick et al. , J. Virol. Methods 70:59 (1998)). Other standard methods are known to those skilled in the art. Zwntl probes and primers can also be used to detect and localize the expression of the Zwntl gene in tissue samples. Methods for such hybridization in itself are well known to those skilled in the art (see, for example, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc. 1994), Wu et al. (Eds. ), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Situ Hybridization (RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al. (Eds.), "Localization of DNA or Abundance of mRNA by Fluorescence in If your Hybridization (RISH)," in methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc. 1997). Several additional diagnostic methods are well known to those skilled in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996).) A study of the chromosomal map formation showed that the Zwntl gene res ide on human chromosome 2 in 2.q36.3. The nucleic acid molecules comprising the Zwntl nucleotide sequences can be used to determine whether the chromosomes of a subject contain a mutation in the Zwntl gene. The detectable chromosomal aberrations in the Zwntl gene site include, but are not limited to, aneuploidy, changes in the number of copies of genes, insertions, deletions, changes in restriction sites and rearrangements. Such aberrations can be detected using nucleic acid molecules of the present invention using molecular, such as analysis length polymorphism restriction fragment (RFLP), PCR techniques employing analyzing short tandem repeat genetic (STR) analysis system mutation refractory amplification (ARMS), detection conformation polymorphism single stranded (SSCP), the cleaving methods RNase gel electrophoresis of denaturing gradient analysis of mismatching assisted fluorescence (FAMA ), and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108: 255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Labora tory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al. , (eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley &Sons 1998), and Richards and Ward, "Molecular Diagnostic Testing," in Principies of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998)). The protein truncation test is also useful for detecting the inactivation of a gene in which translation-termination mutations produce only portions of the encoded protein (see, for example, Stoppa-Lyonnet et al., Blood 91: 3920 (1998)). According to this methodology, the RNA is isolated from a biological sample and used to synthesize the cDNA. PCR is then used to amplify the target sequence of Zwntl and to introduce an RNA polymerase promoter, and a translation initiation sequence, and an ATG triplet in the structure. The PCR products are transcribed using an RNA polymerase and the transcripts are translated in vi tro with a reticulocyte lysate system coupled to T7. The translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products. The truncation test of the protein is described, for example, by Dracopoli et al. (eds.), Curren t Protocols in Human Genetics, pages 9.11.1 - 9.11.18 (John Wiley & amp; amp; amp;; Sons 1998). The present invention also contemplates the equipment to perform a diagnostic assay for the expression of the Zwntl gene or detect mutations in the Zwn tl gene. Such kits comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ. ID NO: 1, or a fragment thereof, as well as single-stranded nucleic acid molecules containing the complement of the SEC nucleotide sequence. ID NO: 1, or a fragment thereof. Illustrative fragments reside within nucleotides 264-1406 of the SEC. ID NO: 1. The probe molecules can be DNA, RNA, oligonucleotides, and the like. The kits can comprise the nucleic acid primers to perform the PCR. Such equipment contains all the necessary elements to perform a nucleic acid diagnostic assay described above. A kit will comprise at least one container comprising a Zwntl probe or primer. The equipment may also comprise a second container comprising one or more reagents capable of indicating the presence of Zwntl sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like. A kit may also comprise a means to indicate to the user that the Zwntl probes and primers are used to detect the expression of the Zwntl gene. For example, the written instructions may state that the attached nucleic acid molecules can be used to detect either a nucleic acid molecule encoding the Zwntl, or a nucleic acid molecule having a nucleotide sequence that is complementary to a sequence. of nucleotides encoding the Zwntl, or to analyze the chromosomal sequences associated with the Zwntl site. The written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.

. Use of Anti-Zwntl Antibodies to Detect Zwntl Protein The present invention contemplates the use of anti-Zwntl antibodies to select biological samples in vitro for the presence of Zwntl. In a type of in vitro assay, anti-Zwntl antibodies are used in the liquid phase. For example, the presence of Zwntl in a biological sample can be tested by mixing the biological sample with a trace amount of the labeled Zwntl and an anti-Zwntl antibody under conditions that promote the binding between Zwntl and its antibody. The Zwntl and anti-Zwntl complexes in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein that binds to the antibody, such as an Fc antibody or Staphylococcus protein A. The concentration of Zwntl in the biological sample will be inversely proportional to the amount of labeled Zwntl bound to the antibody and directly related to the amount of free labeled Zwntl. Alternatively, in vitro assays can be performed in which the anti-Zwntl antibody binds to a solid phase carrier. For example, the antibody can be attached to a polymer, such as aminodextran, to bind the antibody to an insoluble support such as a polymer-coated bead, plate or tube. Other suitable tests in vi tro will be readily apparent to those skilled in the art. In another methodology, anti-Zwntl antibodies can be used to detect Zwntl in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zwntl and to determine the distribution of Zwntl in the tissue examined. General immunochemistry techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application," in Mammalian Development: A Practical Approach, Monk (ed.), Pages 115-38 (IRL Press 1987), Coligan on pages 5.8.1-5.8.8, Ausubel (1995) on pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press, Inc. 1992)). Immunochemical detection can be performed by contacting a biological sample with an anti-Zwntl antibody, and then contacting the biological sample with a detectably labeled molecule that binds to the antibody. For example, the detectably labeled molecule may comprise a portion of the antibody that binds to an anti-Zwntl antibody. Alternatively, the anti-Zwntl antibody can be conjugated with avidin / streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin / streptavidin). Numerous variations of this basic technique are well known in the art to those skilled in the art. Alternatively, an anti-Zwntl antibody can be conjugated to a detectable label to form an anti-Zwntl immunoconjugate. Suitable detectable labels inc, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, or colloidal gold. Methods for making and detecting such detectably labeled immunoconjugates are well known to those of ordinary skill in the art, and are described in more detail below. The detectable label can be a radioisotope that is detected by auto-raphiography. Isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 I, 131 I, 35 S and 1 C. The anti-Zwntl immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently labeled antibody is determined by exposing the immunoconjugate in light of a suitable wavelength and detecting the resulting fluorescence. Fluorescent labeling compounds inc fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescein. Alternatively, the anti-Zwntl immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound. The presence of the immunoconjugate is determined by detecting the presence of the luminescence that arises during the course of a chemical reaction. Examples of luminescent labeling compounds include luminol, isoluminiol, an aromatic acridium ester, an imidazole, an acridium salt and an oxalate ester. Similarly, a bioluminescent compound can be used to label the anti-Zwntl immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of the luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.

Alternatively, the anti-Zwntl immunoconjugates can be detectably labeled by linking an anti-Zwntl antibody component to an enzyme. When the anti-Zwntl-enzyme conjugate is incubated in the presence of an appropriate substrate, the portion of the enzyme reacts with the substrate to produce a chemical moiety that can be detected, for example, by spectrometric, fluorometric, or visual means. Examples of the enzymes that can be used to detectably label the polyspecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase. Those of skill in the art will know other suitable labels that may be employed in accordance with the present invention. The binding of the marker portions to the anti-Zwntl antibodies can be complemented by standard techniques known in the art. The typical methodology in relation to this is described by Kennedy et al, Clin. Chim. Acta 70: 1 (1976), Schurs et al. , Clin. Chim. Acta 81: 1 (1977), Shih et al. , Int'l J. Cancer 46: 1101 (1990), Stein et al. , Cancer Res. 50: 1330 (1990), and Coligan, supra. In addition, the convenience and versatility of immunochemical detection can be improved by using anti-Zwntl antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al. (Eds.), "Avidin-Biotin Technology , "Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.," Immunochemical Applications of Avidin-Biotin Technology, "in Methods In Molecular Biology, Vol 10, Manson (ed.), On pages 149 -162 (The Humana Press, Inc. 1992) Methods for performing immunoassays are well established, see, for example, Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoassays," in Monoclonal Antibodies: Production Engineering and Clinical Application, Ritter and Ladyman (eds.), Pages 180-208, (Cambridge University Press, 1995), Perry, "The Role of Monoclonal Antibodies in the Advancement of Technology, "in Monoclonal Antibodies Principies and Applications, Birch and Lennox (eds.), Pages 107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (Academic Press, Inc. 1996) In a related methodology, biotin-tagged Zwntl or FITC can be used to identify cells that bind to Zwntl, such linkage can be detected, for example, using flow cytometry.The present invention also contemplates equipment for the performance of an immunological diagnostic assay for the expression of the Zwntl gene Such kits comprise at least one container comprising an anti-Zwntl antibody, or an antibody fragment.An equipment may also comprise a second container comprising one or more reagents capable of indicating the presence of anti-Zwntl antibody or antibody fragments Examples of such indicator reagents include detectable labels such as an radioactive iquette, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold and the like. A kit may also comprise a means for transmitting to the user that Zwntl antibodies or antibody fragments are used to detect the Zwntl protein. For example, written instructions can establish that the attached antibody or antibody fragment can be used to detect Zwntl. The written material can be applied directly to a container, or the written material can be provided in the form of an insert in the package. 22. Therapeutic Uses of Polypeptides Having Zwntl Activity The present invention includes the use of proteins, polypeptides, and peptides having Zwntl activity (such as Zwntl polypeptides, anti-idiotype anti-Zyntl antibodies, and Zwntl fusion proteins) to a subject that lacks an adequate amount of this Wnt polypeptide. For example, molecules that have Zwntl activity can be used to improve proliferation, differentiation or maintenance of a hematopoietic stem / progenitor cell. Generally, the dosage of the polypeptide, protein or peptide administered will vary depending on factors such as the age, weight, height, sex, general medical condition and medical history of the patient. Typically, it is desirable to provide the receptor with a dosage of a molecule having Zwntl activity which is in the range of about 1 pg / kg to 10 mg / kg (amount of agent / patient's body weight), although a lower dose or higher can also be administered as circumstances dictate. The administration of a molecule having Zwntl activity to a subject can be intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, intrathecally, by perfusion through the regional catheter, or by direct intralesional injection. When therapeutic proteins are administered by injection, administration can be by continuous infusion or by single or multiple boluses. A pharmaceutical composition comprising a protein, polypeptide, or peptide having a Zwntl activity can be formulated according to known methods for preparing the pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is considered to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile, phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995). For the purposes of therapy, molecules having Zwntl activity and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of a protein, polypeptide, or peptide having Zwntl activity and a pharmaceutically acceptable carrier is considered to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiological of a recipient patient. A pharmaceutical composition comprising molecules having Zwntl activity can be provided in a liquid form, or in solid form. Liquid forms include formulations encapsulated in liposomes, illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled release forms, such as miniosmotic pumps or an implant. Other dosage forms can be designed by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Read &; Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).

As an illustration, the pharmaceutical compositions of Zwntl can be supplied as a kit comprising a container comprising the Zwntl. The Zwntl can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted prior to injection. Such equipment may additionally comprise written information or indications and use of the pharmaceutical composition. In addition, such information may include a statement that the composition of Zwntl is contraindicated in patients with known hypersensitivity to Zwntl. 12. Therapeutic Uses of Zwntl Nucleotide Sequences The present invention includes the use of Zwntl nucleotide sequences to provide Zwntl to a subject in need of such treatment. In addition, a therapeutic expression vector that inhibits the expression of the Zwntl gene, such as an anti-sense molecule, a ribozyme, or an outer leader sequence molecule, can be provided. The provision of molecules having Zwntl activity can be used to improve the proliferation, differentiation or maintenance of a hematopoietin / progenitor stem cell. There are numerous methodologies for introducing a Zwntl gene into a subject, including the use of recombinant host cells expressing Zwntl, the provision of naked nucleic acids encoding Zwntl, the use of a cationic lipid carrier with a nucleic acid molecule that encodes Zwntl, and the use of viruses expressing Zwntl, such as recombinant retroviruses, retroviruses, recombinant adeno-associated viruses, recombinant adenoviruses, and recombinant herpes Simplex virus [HSV] (see, e.g., Mulligan, Science 260: 926 (1993), Rosenberg et al., Science 242: 1575 (1988), LaSalle et al., Science 259: 988 (1993), Wolff et al., Science 247: 1465 (1990), Breakfield and Deluca, The New Biologist 3: 203 (1991)). In an ex vivo methodology, for example, cells are isolated from a subject, transfected with a vector expressing a Zwntl gene, and then transplanted into the subject. To effect the expression of a Zwntl gene, an expression vector is constructed in which a nucleotide sequence encoding a Zwn tl gene that operably links to a core promoter, and optionally to a regulatory element, to control the transcription of the gen. The general requirements of an expression vector are described above. Alternatively, a Zwntl gene can be delivered using recombinant viral vectors, including, for example, adenoviral vectors (eg, Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA 90: 11498 (1993), Kolls. et al., Proc. Nat'l Acad Sci. USA 91: 215 (1994), Li et al., Hum Gene Ther 4: 403 (1993), Vincent et al., Nat. Genet 5: 130 (1993) , and Zabner et al., Cell 75: 201 (1993)), viral vectors associated with the adenovirus (Flotte et al., Proc. Nat'l Acad Sci. USA 90: 10613 (1993)), alphaviruses such as Virus Semliki Forest and the Sindbis Virus (Hertz and Huang, J. Vir. 66: 851 (1992), Raju and Huang, J. Vir. 65: 2501 (1991), and Xiong et al., Science 243: 1188 (1989) ), viral herpes vectors (eg, US Patent Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors (Koering et al., Hum The Gene 5: 457 (1994)), virus vectors pox (Ozaki et al., Biochem. Biophys. Res. Comm. 193: 653 (1993), Panicali and Paoletti, Proc. Nat'l Acad Sci. USA 79: 4927 (1982)), pox virus, such as pox virus from canaries or vaccine virus (Fisher-Hoch et al., Proc. Nat'l Acad Sci. USA 86: 311 (1989), and Flexner et al., Ann. N.Y. Acad Sci. 569: 86 (1989)), and retroviruses (eg, Baba et al., J. Neurosurg 79: 129 (1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al. al., J. Neurosci Res 33: 493 (1992), Vile and Hart, Cancer Res. 53: 962 (1993), Vile and Hart, Cancer Res. 53: 3860 (1993), and Anderson et al., Patent North American No. 5,399,346). Within several embodiments, either the viral vector itself, or a viral particle containing the viral vector can be used in the methods and compositions described. As an illustration of a system, the adenovirus, a double-stranded DNA virus, is a well-characterized gene transfer vector for delivering a heterologous nucleic acid molecule (for review, see Becker et al., Meth. Cell Biol. 43: 161 (1994); Douglas and Curiel, Science & Medicine 4:44 (1997)). The adenovirus system offers several advantages including: (1) the ability to accommodate relatively large DNA inserts, (ii) the ability to grow to a high titer, (iii) the ability to infect a broad range of cell types, mammals; and (iv) the ability to be used with many different promoters including ubiquitous, tissue-specific, and regulatable promoters.

In addition, adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream. Using the adenovirus vectors where the portions of the adenovirus genome are eliminated, the inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential gene is removed from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell. When administered intravenously to intact animals, the main target of the adenovirus is the liver. Although an adenoviral delivery system with a deletion of the El gene can not replicate in the host cells, the host tissue will express and process a heterologous encoded protein. The host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence. The secreted proteins will enter the circulation from the tissue that expresses the heterologous gene (for example, the highly vascularized liver).

In addition, adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector. Such adenoviruses have eliminated El and also contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72 ^: 2022-2032, 1998; Raper, SE et al., Human Gene Therapy 9: 611- 619, 1998). In addition, it was also reported that the elimination of E2b reduces the immune responses (Amalfitano, A. et al., J. Virol. 72.:926~933 '1998). By removing the genome from the complete adenovirus, large inserts of the heterologous DNA can be accommodated. The generation of so-called "stomach-free" adenoviruses in which all viral genes have been eliminated are particularly advantageous for the insertion of large inserts of heterologous DNA (for review, see Yeh, P. and Perricaudet, M., FASEB J. 11 : 615-623, 1997)). The high titer materials of the recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using the standard methods. For example, recombinant HSV can be prepared in Vero cells, as described by Brandt et al. , J. Gen. Virol. 72: 2043 (1991), Herold et al. , J. Gen. Virol 75: 1211 (1994), Visalli and Brandt, Virology 185: 419 (1991), Grau et al. , Invest. Oph thalmol. Vis. Sci. 30: 2474 (1989), Brandt et al. , J. Virol. Meth. 36: 209 (1992), and by Brown and MacLem (eds.), HSV Virus Protocols (Humana Press 1997). Alternatively, an expression vector comprising a Zwntl gene can be introduced into the subject's cells by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare the liposomes for an in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Na t'l Acad Sci. USA 84: 7413 (1987); Mackey et al. Proc. Na t 'l Acad Sci. USA 85: 8027 (1988)). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. The lipids can be chemically coupled to other molecules for the purpose of being considered objective. Target peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be chemically coupled to the liposomes.

Electroporation is another alternative mode of administration of a Zwntl nucleic acid molecule.

For example, Aihara and Miyazaki, Na ture Biotechnology 1 6: 861 (1998), have demonstrated the use of in vivo electroporation for the transfer of genes to muscle. In an alternative methodology to gene therapy, a therapeutic gene can encode a Zwntl antisense RNA that inhibits the expression of Zwntl. The methods for preparing the Wnt anti-sense constructs are known to those skilled in the art. See, for example, Erickson et al. , Dev. Genet. 14: 214 (1993) [transgenic mouse], Augustine et al., Dev. Genet. 14: 500 (1993) [cultivation of murine full embryos], and Olson and Gibo, Exp. Cell Res. 241: 134 (1998) [cultured cells]. Suitable sequences for the anti-sense Zwntl molecules can be derived from the Zwntl nucleotide sequences described herein. Alternatively, an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence encoding a ribozyme. Ribozymes can be designed to express the activity of the endonuclease that is directed to a certain target sequence in an mRNA molecule (see, for example, Draper "and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337) In the context of the present invention, ribozymes include nucleotide sequences that bind to Zwntl mRNA. Expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of the mRNA molecules encoding a Zwn tl gene.According to this methodology, a leader sequence external can be constructed to direct the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Altman et al. , U.S. Patent No. 5,168,053, Yuan et al. , Science 263: 1269 (1994), Pace et al. , International Publication No. WO 96/18733, George et al. , International Publication No. WO 96/21731, and Werner et al, International Publication No. WO 97/33991). Preferably, the external leader sequences comprise a ten to fifteen nucleotide sequence complementary to the Zwntl mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine. Transcripts from the outer guide sequence are linked to the target mRNA species by base pair formation between the mRNA and complementary external leader sequences, thereby promoting cleavage of the mRNA by the RNase P at the nucleotide located at the 5 nd place. 'of the region of paired bases. In general, the dosage of a composition comprising a therapeutic vector having a Zwntl nucleic acid sequence, such as a recombinant virus, will vary depending on factors such as age, weight, height, sex, general medical condition and prior medical history. of the subject. Suitable routes of administration of the therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into the cavity containing a tumor. A composition comprising vectors, non-viral vectors, or a combination of viral and non-viral vectors of the present invention can be formulated according to known methods to prepare the pharmaceutically useful compositions, whereby the vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier. As noted above, a composition, such as phosphate buffered saline is considered to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient subject. Other suitable carriers are well known to those in the art (see, for example, Remington's Pharmaceutical Sciences, 19th Ed (Mack Publishing Co. 1995), and Gilman's The Pharmacological Basis of Therapeutics, Ith Ed. (MacMillan Publishing Co. 1985)). For therapy purposes, a therapeutic gene expression vector, or a recombinant virus comprising such vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount. A combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is considered to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiological of a recipient subject. When the subject treated with an expression vector of the therapeutic gene or a recombinant virus is a human, then the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with an expression vector of the therapeutic gene or a recombinant virus does not involve introducing into the cells a nucleic acid molecule that can be part of a human germline and can be passed on to successive generations ( that is, human germline gene therapy).

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (17)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated polypeptide, comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 4, characterized in that the isolated polypeptide specifically binds to an antibody that specifically binds to a polypeptide consisting of of an amino acid sequence of SEQ ID NO: 4, with the proviso that said isolated polypeptide does not include the murine Wnt-lOA protein, which is identified by GenBank Accession No. U61969, and the Wnt-lOA protein newt, which is identified by GenBank Access No. U65428.

2. The isolated polypeptide of claim 1, characterized in that the isolated polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 4.

3. The isolated polypeptide of claim 1, characterized in that the isolated polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4.

4. The isolated polypeptide of claim 1, characterized in that any difference between the amino acid sequence of the polypeptide and the amino acid sequence corresponding to SEQ ID NO: 4, is due to a conservative amino acid substitution.

The isolated polypeptide of claim 1, characterized in that the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 4,

6. An isolated nucleic acid molecule, characterized in that the nucleic acid molecule is either a molecule of nucleic acid, comprising the nucleotide sequence of SEQ ID NO: 3, or a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 4.

7. An isolated nucleic acid molecule, characterized in that it comprises a nucleotide sequence of nucleotides 264 to 1406 of SEQ ID NO: 1.

8. A vector, characterized in that it comprises a molecule of claim 7.

9. An expression vector, characterized in that it comprises the nucleic acid molecule isolated from the claim 7, a transcription promoter, and a transcription terminator, wherein the promoter is operably linked to a nucleic acid molecule, and wherein the molecule of nucleic acid is operably linked to the transcription terminator.

10. A recombinant host cell comprising the expression vector of claim 9, characterized in that the host cell is selected from the group consisting of a bacterial, yeast, fungal, insect, mammalian, and plant cell.

11. A method for using the expression vector of claim 9 to produce a polypeptide comprising amino acid residues from 20 to 400 of SEQ ID NO: 2, characterized in that it comprises the expression vector and that it produces the polypeptide.

12. The method of claim 11, characterized in that it further comprises isolation of the polypeptide from the cultured recombinant host cells.

13. An antibody or antibody fragment, characterized in that it binds especially to the polypeptide of claim 5.

The antibody of claim 13, characterized in that the antibody is selected from the group consisting of: (a) a polyclonal antibody, (b) a murine monoclonal antibody, (c) a humanized antibody derived from (b), and (d) a human monoclonal antibody.

15. A method for detecting in a biological sample, the presence of RNA encoding the amino acid sequence of SEQ ID NO: 4, characterized in that it comprises: (a) contacting a Zwntl nucleic acid probe under hybridization conditions with either (i) the test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe consists of at least eight nucleotides to at least 20 nucleotides of the nucleotide sequence of nucleotides 264 to 1406 of SEQ ID NO: 1, or its complement, and (b) detect the hybrid formation of the nucleic acid probe and either test RNA molecules or acid molecules nucleic acid synthesized, wherein the presence of hybrids indicates the presence of RNA encoding the amino acid sequence of SEQ ID NO: 4, in the biological sample.

16. A method for detecting in a biological sample, the presence of a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, characterized in that it comprises: (a) contacting the biological sample with an antibody, or an antibody fragment , of claim 13, wherein the contact is transformed under conditions that allow binding of the antibody or fragment to the biological sample, and (b) detecting any bound antibody or bound antibody fragment. The method of claim 16, characterized in that it comprises the antibody or antibody fragment, further comprising a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold.