HELICAL POLYPEPTIDE ZALPHA29

Abstract: Novel cytokine polypeptides, materials and methods for making them, and method of use are disclosed. The polypeptides comprise at least 15 contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4, and may be prepared as polypeptide fusions comprise heterologous sequences, such as affinity tags. The polypeptides and polynucleotides encoding them may be used within a variety of therapeutic, diagnostic, and research applications.

Description: Description HELICAL POLYPEPTIDE ZALPHA29 BACKGROUND OF THE INVENTION Cytokines are polypeptide hormones that are produced by a cell and affect the growth or metabolism of that cell or another cell. In multicellular animals, cytokines control cell growth, migration, differentiation, and maturation. Cytokines play a role in both normal development and pathogenesis, including the development of solid tumors. Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF-α) to 140 kDa (Mullerian-inhibiting substance). They include single polypeptide chains, as well as disulfide-linked homodimers and heterodimers. Cytokines influence cellular events by binding to cell-surface receptors. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors. Cell differentiation and maturation are also under control of cytokines. For example, the hematopoietic factors erythropoietin, thrombopoietin, and G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow. Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors. The role of cytokines in controlling cellular processes makes them likely candidates and targets for therapeutic intervention; indeed, a number of cytokines have been approved for clinical use. Interferon-alpha (IFN-α), for example, is used in the treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and Puri, "Common and Uncommon Features of Cytokines and Cytokine Receptors: An Overview", in Aggarwal and Puri, eds., Human Cytokines: Their Role in Disease and Therapy. Blackwell Science, Cambridge, MA, 1995, 3-24). Platelet-derived growth factor (PDGF) has been approved in the United States and other countries for the treatment of dermal ulcers in diabetic patients. The hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683). G- CSF, GM-CSF, IFN-β, IFN-γ, and IL-2 have also been approved for use in humans (Aggarwal and Puri, ibid.). Experimental evidence supports additional therapeutic uses of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in injured baboon arteries (Giese et al., Restenosis Summit VIII, Poster Session #23, 1996; U.S. Patent No. 5,620,687). Vascular endothelial growth factors (VEGFs) have been shown to promote the growth of blood vessels in ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and have been proposed for use as wound-healing agents, for treatment of periodontal disease, for promoting endothelialization in vascular graft surgery, and for promoting collateral circulation following myocardial infarction (WTPO Publication No. WO 95/24473; U.S. Patent No. 5,219,739). A soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News 16(17):5-6, 1996). Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. I. Cancer 76:1410-1415, 1997). More recently, thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al., Bailliere's Clin. Haematol. JJ . :427-445, 1998). In view of the proven clinical utility of cytokines, there is a need in the art for additional such molecules for use as both therapeutic agents and research tools and reagents. Cytokines are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media. SUMMARY OF THE INVENTION Within one aspect of the invention there is provided an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of residues 48-62 of SEQ ID NO:2, residues 47-61 of SEQ ID NO:4, residues 63-104 of SEQ ID NO:2, residues 62-103 of SEQ ID NO:4, residues 105-119 of SEQ ID NO:2, residues 104-118 of SEQ ID NO:4, residues 120-137 of SEQ ID NO:2, residues 119-136 of SEQ ID NO:4, residues 138-152 of SEQ ID NO:2, residues 137-151 of SEQ ID NO:4, residues 153-170 of SEQ ID NO:2, residues 152-169 of SEQ ID NO:4, residues 171-185 of SEQ ID NO:2, and residues 170-184 of SEQ ID NO:4. Within one embodiment, the isolated polypeptide has from 15 to 1500 amino acid residues. Within a related embodiment, the sequence of amino acid residues is operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:5. Within another embodiment, the isolated polypeptide comprises at least 30 contiguous residues of SEQ ID NO:2 or SEQ ID NO:4. Within other embodiments, the isolated polypeptide comprises residues 48-185 or residues 27-190 of SEQ ID NO:6. Within further embodiments, the isolated polypeptide comprises residues 48-185 of SEQ ID NO:2, residues 47-184 of SEQ ID NO:4, residues 27-190 of SEQ ID NO:2, or residues 26-188 of SEQ ID NO:4. Within a second aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as disclosed above; and a transcription terminator. Within one embodiment, the DNA segment comprises nucleotides 79 to 570 of SEQ ID NO:7. Within another embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment. Within a third aspect the invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the DNA segment. Within one embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell. Within a fourth aspect the invention provides a method of making a protein comprising culturing a cell into which has been introduced an expression vector as disclosed above under conditions whereby the DNA segment is expressed and the polypeptide is produced, and recovering the protein. When the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, the polypeptide is secreted by the cell and recovered from a medium in which the cell is cultured. Within a fifth aspect the invention provides a protein produced by the method disclosed above. Within a sixth aspect of the invention there is provided an antibody that specifically binds to the protein disclosed above. Within a seventh aspect of the invention there is provided method of detecting, in a test sample, the presence of an antagonist of zalpha29 activity. The method comprises the steps of (a) culturing a cell that is responsive to zalρha29; (b) exposing the cell to a zalpha29 polypeptide in the presence and absence of a test sample; (c) comparing levels of response to the zalpha29 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and (d) determining from the comparison the presence of an antagonist of zalpha29 activity in the test sample. These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A-1D are a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters. Fig. 2 is an alignment of representative human (SEQ ID NO:2) and mouse (SEQ ID NO:4) zalpha29 amino acid sequences. DETAILED DESCRIPTION OF THE INVENTION Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms: The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, 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), maltose binding protein (Kellerman and Ferenci, Methods Enzymol. 90:459-463, 1982; Guan et al., Gene 67:21-30, 1987), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985; see SEQ ID NO:5), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding peptide, thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; and Eastman Kodak, New Haven, CT). The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having 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 terms "ammo-terminal" and "carboxyl-terminal" are used herein to denote positions within 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 sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide. "Angiogenic" denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation. Angiogenesis can also be measured using any of several in vivo assays as disclosed herein. A "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'. The term "corresponding to", when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned. The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp). The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. The isolated polypeptide may be substantially free of other polypeptides, particularly other polypeptides of animal origin. The polypeptides may be prepared in a highly purified form, i.e. greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. "Operably linked" means that two or more entities are joined together such that they function in concert for their intended purposes. When referring to DNA segments, the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator. When referring to polypeptides, "operably linked" includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained. The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation. A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length. 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". The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. A "secretory signal sequence" is a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A "segment" is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide. Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. All references cited herein are incorporated by reference in their entirety. The present invention provides novel cytokine polypeptides and proteins. This novel cytokine, termed "zalpha29", was identified by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoeitin, thrombopoietin, G-CSF, IL-2, IL-4, leptin, and growth hormone). Analysis of the human zalpha29 amino acid sequence shown in SEQ ID NO:2 indicates the presence of four amphipathic, alpha-helical regions. These regions include at least amino acid residues 48 through 62 (helix A), 105 through 119 (helix B), 138 through 152 (helix C), and 171 through 185 (helix D). Within these helical regions, residues that are expected to lie within the core of the four-helix bundle occur at positions 48, 51, 52, 55, 58, 59, 62, 105, 108, 109, 112, 115, 116, 119, 138, 141, 142, 145, 148, 149, 152, 171, 174, 175, 178, 181, 182, and 185 of SEQ ID NO:2. Residues 49, 50, 53, 54, 56, 57, 60, 61, 106, 107, 110, 111, 113, 114, 117, 118, 139, 140, 143, 144, 146, 147, 150, 151, 172, 173, 176, 177, 179, 180, 183, and 184 are expected to lie on the exposed surface of the bundle. Inter-helix loops comprise approximately residues 63 through 104 (loop A-B), 120 through 137 (loop B-C), and 153 through 170 (loop C-D). The human zalpha29 cDNA (SEQ ID NO:l) encodes a polypeptide of 190 amino acid residues. While not wishing to be bound by theory, this sequence is predicted to include a secretory peptide of 26 residues. Cleavage after residue 26 will result in a mature polypeptide (residues 27-190 of SEQ ID NO:2) having a calculated molecular weight (exclusive of glycosylation) of 18,558 Da. Those skilled in the art will recognize, however, that some cytokines (e.g., endothelial cell growth factor, basic FGF, and IL-lβ) do not comprise conventional secretory peptides and are secreted by a mechanism that is not understood. There is a single consensus N-linked glycosylation site in SEQ ID NO:2 at residues 111-113. The cDNA also includes a clear polyadenylation signal. The mouse zalpha29 polypeptide (SEQ JO NO:4) is predicted to include helices and loops at analogous positions, including helices at residues 47-61, 104-118, 137-151, and 170-184; and loops at residues 62-103, 119-136, and 152-169. See Fig. 2. Those skilled in the art will recognize that predicted domain boundaries are somewhat imprecise and may vary by up to ± 5 amino acid residues. Polypeptides of the present invention comprise at least 15 contiguous amino acid residues of SEQ ID NO:2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up to the entire predicted mature polypeptide (residues 27 to 190 of SEQ ID NO:2) or the primary translation product (residues 1 to 190 of SEQ ID NO:2). Corresponding mouse zalpha29 polypeptides (see SEQ ID NO:4) are also provided by the invention. As disclosed in more detail below, these polypeptides can further comprise additional, non-zalpha29, polypeptide sequence(s). Within the polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2 or SEQ ID NO:4. An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666. 1983. Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zalpha29, such as might occur in body fluids or cell culture media. Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha29 protein. Antigenic, epitope-bearing polypeptides contain a sequence of at least six, often at least nine, commonly from 15 to about 30 contiguous amino acid residues of a zalpha29 protein (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zalpha29 protein, i.e. from 30 to 50 residues up to the entire sequence, are included. It is preferred that the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided. Such regions include the interdomain loops of zalpha29 and fragments thereof, in particular loop B-C (residues 120-137 of SEQ ID NO:2), which is markedly hydrophilic (see Fig. IC). Polypeptides in this regard include those comprising residues 99-104, 129-134, and 162-167 of SEQ ID NO:2. Of particular interest within the present invention are polypeptides that comprise the entire four-helix bundle of a zalpha29 polypeptide (e.g., residues 48-185 of SEQ ID NO:2). Such polypeptides may further comprise all or part of one or both of the native zalpha29 amino-terminal (residues 27-47 of SEQ ID NO:2) and carboxyl- terminal (residues 186-190 of SEQ ID NO:2) regions, as well as non-zalpha29 amino acid residues or polypeptide sequences as disclosed in more detail below. Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO:2. These changes will usually be of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, an amino or carboxyl-terminal cysteine residue to facilitate subsequent linking to maleimide-activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination. Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zalpha29 polypeptide and the affinity tag. Exemplary cleavage sites include thrombin cleavage sites and factor Xa cleavage sites. The present invention further provides a variety of other polypeptide fusions. For example, a zalpha29 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Suitable dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-zalpha29 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zalpha29 analogs. In addition, a zalpha29 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multi-functional molecule. One or more helices of a zalpha29 polypeptide can be joined to another cytokine to enhance or otherwise modify its biological properties. Auxiliary domains can be fused to zalpha29 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zalpha29 polypeptide or protein can be targeted to a predetermined cell type by fusing a zalpha29 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zalpha29 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996. Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, often not more than about 1,200 residues, usually not more than about 1,000 residues, and will in many cases be considerably smaller. For example, a zalpha29 polypeptide of 164 residues (residues 27-190 of SEQ ID NO:2) can be fused to E. coli -galactosidase (1,021 residues; see Casadaban et al., I. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site to yield a polypeptide of 1,199 residues. In a second example, residues 27-190 of SEQ ID NO:2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag. As disclosed above, the polypeptides of the present invention comprise at least 15 contiguous residues of SEQ ID NO:2 or SEQ ID NO:4. These polypeptides may further comprise additional residues as shown in SEQ ID NO:2, a variant of SEQ ID NO:2, or another protein as disclosed herein. "Variants of SEQ ID NO:2" includes polypeptides that are at least 85%, at least 90%, or at least 95% identical to the corresponding region of SEQ ID NO:2. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as: Table 1 The level of identity between amino acid sequences can be determined using the "FASTA" similarity search algorithm disclosed by Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and by Pearson (Meth. Enzymol. 183:63, 1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored 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. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, I. Mol. Biol. 48:444, 1970; Sellers, SIAM I. Appl Math. 26:787, 1974), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 (ibid.). FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range from one to six, preferably from three to six, most preferably three, with other parameters set as default. The present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO:2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, ibid.). Thus, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the term "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of 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. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3). The proteins of the present invention can also comprise non-naturally occuring amino acid residues. Non-naturally occuring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRΝAs. Methods for synthesizing amino acids and aminoacylating tRΝA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., I. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-10149, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRΝA and chemically aminoacylated suppressor tRΝAs (Turcatti et al., I. Biol. Chem. 271:19991-19998. 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occuring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occuring amino acid is incoφorated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occuring amino acid residues can be converted to non-naturally occuring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395- 403, 1993). Amino acid sequence changes are made in zalpha29 polypeptides so as to minimize disruption of higher order structure essential to biological activity. For example, changes in amino acid residues will be made so as not to disrupt the four- helix bundle characteristic of the protein family. The effects of amino acid sequence changes can be predicted by computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., ibid.). A hydrophilicity profile of SEQ ID NO:2 is shown in Figs. 1A-1D. Those skilled in the art will recognize that this hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zalpha29 polypeptide, so as not to disrupt the overall profile. Residues within the core of the four-helix bundle can be replaced with other residues as shown in SEQ ID NO: 6. The residues predicted to be on the exposed surface of the four-helix bundle will be relatively intolerant of substitution. Other candidate amino acid substitutions within human zalpha29 are suggested by alignment of the human (SEQ ID NO:2) and mouse (SEQ ID NO:4) sequences as shown in Fig. 2, which sequences are approximately 85% identical overall. The cysteine residue at position 160 of SEQ ID NO:2 (position 159 of SEQ ID NO:4) lies in loop C-D, suggesting its participation in an interchain disulfide bond. This residue is thus expected to be relatively intolerant of substitution. One skilled in the art may employ many well known techniques, independently or in combination, to analyze and compare the structural features that affect folding of a variant protein or polypeptide to a standard molecule to determine whether such modifications would be significant. One well known and accepted method for measuring folding is circular dichroism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule are routine in the art (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known and accepted method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992). Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for 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 phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7: 127, 1988). Variants of the disclosed zalpha29 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 9J . : 10747- 10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zalpha29 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide. For example, expressing the complete zalpha29 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved. Differential processing of individual chains may result in heterogeneity of expressed polypeptides. Zalpha29 proteins of the present invention are characterized by their activity, that is, modulation of the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types. Biological activity of zalpha29 proteins is assayed using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion; or changes in cellular metabolism (e.g., production of other growth factors or other macromolecules). Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions. However, in view of the complexity of developmental processes (e.g., angiogenesis, wound healing), in vivo assays will generally be employed to confirm and further characterize biological activity. Certain in vitro models, such as the three-dimensional collagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm. 189:824-831. 1992), are sufficiently complex to assay histological effects. Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest. Assays can be conducted using zalpha29 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below. Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zalpha29 variant polypeptides. Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format. Mutagenized DNA molecules that encode active zalpha29 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptide fragments or variants of SEQ ID NO:2 or SEQ ID NO:4 that retain the activity of wild-type zalpha29. The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha29 polypeptides disclosed above. A representative DNA sequence encoding the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:l, and a representative DNA sequence encoding the amino acid sequence of SEQ ID NO:4 is shown in SEQ ID NO:3. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:7 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha29 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:7 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, zalpha29 polypeptide-encoding polynucleotides comprising nucleotides 1-534 or nucleotides 52-534 of SEQ ID NO:7, and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO:7 encoding other zalpha29 polypeptides disclosed herein. Table 2 sets forth the one-letter codes used within SEQ ED NO:7 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C. TABLE 2 Nucleotide Resolutions Complement Resolutions A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G w A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T The degenerate codons used in SEQ ID NO:7, encompassing all possible codons for a given amino acid, are set forth in Table 3, below. TABLE 3 Amino One-Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gin Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG lie I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Ter TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap - — One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ED NO: 2. Variant sequences can be readily tested for functionality as described herein. One of ordinary skill in the art will also appreciate that different species can exhibit preferential codon usage. See, in general, Grantham et al., Nuc. Acids Res. 8:1893-912, 1980; Haas et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; and U emura, I. Mol. Biol. 158:573-97, 1982. Introduction of preferred codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ED NO: 7 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Within certain embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ED NO:l, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zalpha29 RNA. Zalpha29 transcripts have also been detected in numerous tissues as disclosed below. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly(A) + RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zalpha29 polypeptides are then identified and isolated by, for example, hybridization or PCR. Full-length clones encoding zalpha29 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are commonly used within protein production systems, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. A partial human genomic zalpha29 sequence is shown in SEQ ED NO: 14. This sequence comprises an exon from nucleotide 1885 to nucleotide 2112 (corresponding to nucleotides 483-710 of SEQ ED NO:l). Partial mouse genomic sequences are shown in SEQ ED NO: 15 and SEQ ED NO: 16. Within SEQ ED NO: 15, nucleotides 6-165 are an exon corresponding to nucleotides 40-199 of SEQ ED NO:3. Within SEQ ED NO: 16, nucleotides 175-295 are an exon corresponding to nucleotides 200-320 of SEQ ID NO:3. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zalpha29, receptor fragments, or other specific binding partners. Zalpha29 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zalpha29 gene. Promoter elements from a zalpha29 gene can be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zalpha29 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zalpha29 gene in a cell is altered by introducing into the zalpha29 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zalpha29 5' non- coding sequence that permits homologous recombination of the construct with the endogenous zalpha29 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha29 coding sequence. In this way, an endogenous zalpha29 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression. Those skilled in the art will recognize that the sequences disclosed in SEQ ED NOS: 1-2 and 3-4 represent single allele of human and mouse zalpha29, respectively. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. The present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs"). Of particular interest are zalpha29 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zalpha29 can be cloned using 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 zalpha29 as disclosed above. A library is then prepared from mRNA of a positive tissue or cell line. A zalpha29-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human and mouse zalpha29 sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to a zalpha29 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. For any zalpha29 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that polypeptide using the information set forth in Tables 3 and 4, above. Moreover, those of skill in the art can use standard software to devise zalpha29 variants based upon the nucleotide and amino acid sequences described herein. The present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ D NO:l, SEQ ED NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:6, SEQ ED NO:7, and portions thereof. Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZEP disk. Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW). The zalpha29 polypeptides of the present invention, including full- length polypeptides, biologically active fragments, and fusion polypeptides can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide. As used herein, a "cell into which has been introduced an expression vector" includes both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory- Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a zalpha29 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. To direct a zalpha29 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zalpha29, or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo. The secretory signal sequence is operably linked to the zalpha29 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly sythesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Expression of zalpha29 polypeptides via a host cell secretory pathway is expected to result in the production of multimeric proteins. Such multimers include both homomultimers and heteromultimers, the latter including proteins comprising only zalpha29 polypeptides and proteins including zalpha29 and heterologous polypeptides (e.g., a second four-helix-bundle cytokine polypeptide). If a mixture of proteins results from expression, individual species are isolated by conventional methods. Monomers, dimers, and higher order multimers are separated by, for example, size exclusion chromatography. Heteromultimers can be separated from homomultimers by immunoaffinity chromatography using antibodies specific for individual dimers or by sequential immunoaffinity steps using antibodies specific for individual component polypeptides. See, in general, U.S. Patent No. 5,094,941. Multimers may also be assembled in vitro upon incubation of component polypeptides under suitable conditions. In general, in vitro assembly will include incubating the protein mixture under denaturing and reducing conditions followed by refolding and reoxidation of the polypeptides to from homodimers and heterodimers. Recovery and assembly of proteins expressed in bacterial cells is disclosed below. Cultured mammalian cells can be used as hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO I. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 1_5:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHO DG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. Promoters for use in cultured mammalian cells include promoters from SV-40 or cytomegalovirus (see, e.g., U.S. Patent No. 4,956,288), metallothionein gene promoters (U.S. Patent Nos. 4,579,821 and 4,601,978), and the adenovirus major late promoter. Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derivatives thereof. Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." An exemplary selectable marker is a gene encoding resistance to the antibiotic neomycin. 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 expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. The adenovirus system (disclosed in more detail below) can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. In an alternative method, adenovirus vector-infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. L5: 145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins can also be effectively obtained. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) according to methods known in the art. Within one method, recombinant baculovirus is produced through the use of a transposon-based system described by Luckow et al. (I. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-Bac™ kit; Life Technologies, Rockville, MD). The transfer vector (e.g., pFastBacl™; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551- 1556, 1994; and Chazenbalk and Rapoport, I. Biol. Chem. 270:1543-1549, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above. Using techniques known in the art, a transfer vector containing a zalpha29-encoding sequence is transformed into E. coli host cells, and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus that expresses zalpha29 protein is subsequently produced. Recombinant viral stocks are made by methods commonly used the art. For protein production, the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High Five™ cells; Invitrogen, Carlsbad, CA). See, for example, U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers. The cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally known in the art. Other higher eukaryotic cells can also be used as hosts, including plant cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., I. Biosci. (Bangalore) 11:47- 58, 1987. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming 5. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed 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 phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POT I vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., 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 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, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., I. Gen. Microbiol. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents No. 5,716,808, 5,736,383, 5,854,039, and 5,888,768. Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha29 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded 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 case, 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 recovering the protein, thereby obviating the need for denaturation and refolding. Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. Depending upon the intended use, the polypeptides and proteins of the present invention can be purified to >80% purity, >90% purity, >95% purity, or to a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. A purified polypeptide or protein can be prepared substantially free of other polypeptides or proteins, particularly those of animal origin. Expressed recombinant zalpha29 proteins (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Gmssenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art. Zalpha29 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, EL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach. ERL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides. Using methods known in the art, zalpha29 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue. Target cells for use in zalpha29 activity assays include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, pericytes, chondrocytes, osteoblasts, and prostate epithelial cells. Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Choi et al., Development 125:725-732, 1998). Activity of zalpha29 proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzymol 109:749-773, 1985; Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988). Differentiation can be assayed using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference). Zalpha29 activity may also be detected using assays designed to measure zalpha29-induced production of one or more additional growth factors or other macromolecules. Such assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), interleukin-6 (EL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules produced by the liver. Suitable assays include mitogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art. Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes. The relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a zalpha29 protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990). Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using 3 H-proline incorporation into nascent secreted collagen. 3 H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG) secretion from dermal fibroblasts and chondrocytes is measured using a 1,9- dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are also carried out in the presence of EL-lβ or TGF-β to examine the ability of zalpha29 protein to modify the established responses to these cytokines. Monocyte activation assays are carried out (1) to look for the ability of zalpha29 proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha29 proteins to modulate attachment-induced or endotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol. E38: 3799-3802, 1987). EL-lβ and TNFα levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA). Monocyte/macrophage cells, by virtue of CD14 (LPS receptor), are exquisitely sensitive to endotoxin, and proteins with moderate levels of endotoxin- like activity will activate these cells. Hematopoietic activity of zalpha29 proteins can be assayed on various hematopoietic cells in culture. Suitable assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WEPO Publication WO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix) are incubated in the presence of test polypeptide, then examined microscopically for colony formation. Known hematopoietic factors are used as controls. Mitogenic activity of zalpha29 polypeptides on hematopoietic cell lines can be measured as disclosed above. Cell migration is assayed essentially as disclosed by Kahler et al. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). A protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration. A typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (e.g., Transwell®; Coming Costar Corp.). The test sample, diluted in medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C. Non-migrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1% crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997). Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (I. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 10 4 - 10 5 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate). Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin. The activity of zalpha29 proteins can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary such device is the Cytosensor™ Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli et al., I. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. I. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha29 proteins, their agonists, and antagonists. The microphysiometer can be used to measure responses of a zalpha29-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zalpha29 polypeptide. Zalpha29-responsive eukaryotic cells comprise cells into which a receptor for zalpha29 has been transfected creating a cell that is responsive to zalpha29, as well as cells naturally responsive to zalpha29. Differences, measured by a change in extracellular acidification, in the response of cells exposed to zalpha29 polypeptide relative to a control not exposed to zalpha29, are a direct measurement of zalpha29-modulated cellular responses. Moreover, such zalpha29-modulated responses can be assayed under a variety of stimuli. The present invention thus provides methods of identifying agonists and antagonists of zalpha29 proteins, comprising providing cells responsive to a zalpha29 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change in extracellular acidification rate. Culturing a third portion of the cells in the presence of a zalpha29 protein and the absence of a test compound provides a positive control for the zalpha29-responsive cells and a control to compare the agonist activity of a test compound with that of the zalpha29 polypeptide. Antagonists of zalpha29 can be identified by exposing the cells to zalpha29 protein in the presence and absence of the test compound, whereby a reduction in zalpha29- stimulated activity is indicative of antagonist activity in the test compound. Expression of zalpha29 polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo. Zalpha29-encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced. One in vivo approach for assaying proteins of the present invention utilizes viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia vims, and adeno-associated vims (AAV). Adenovims, a double-stranded DNA vims, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997. The adenovims system offers several advantages. Adenovims can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenovimses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovims genome, larger inserts (up to 7 kb) of 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. In an exemplary system, the essential El gene is deleted from the viral vector, and the vims will not replicate unless the El gene is provided by the host cell (e.g., the human 293 cell line). When intravenously administered to intact animals, adenovims primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the vims cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined. An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., I. Biol. Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994. Transgenic mice, engineered to express a zalpha29 gene, and mice that exhibit a complete absence of zalpha29 gene function, referred to as "knockout mice" (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the zalpha29 gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of zalpha29 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997. Promoters for transgenic expression include promoters from metallothionein and albumin genes. Antisense methodology can be used to inhibit zalpha29 gene transcription to examine the effects of such inhibition in vivo. Polynucleotides that are complementary to a segment of a zalpha29-encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ YD NO:l) are designed to bind to zalpha29- encoding mRNA and to inhibit translation of such mRNA. Such antisense oligonucleotides can also be used to inhibit expression of zalpha29 polypeptide - encoding genes in cell culture. Most four-helix bundle cytokines as well as other proteins produced by activated lymphocytes play an important biological role in cell differentiation, activation, recmitment and homeostasis of cells throughout the body. Zalpha29 and inhibitors of zalpha29 activity are expected to have a variety of therapeutic applications. These therapeutic applications include treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, and diabetes. Zalpha29 may be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis. There may be a role of zalpha29 in mediating tumorgenesis, whereby a zalpha29 antagonist would be useful in the treatment of cancer. Zalpha29 may be useful in modulating the immune system, whereby zalpha29 and zalpha29 antagonists may be used for reducing graft rejection, preventing graft-vs-host disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g., HEV + patients), or in improving vaccines. Zalpha29 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF. When using zalpha29 in combination with an additional agent, the two compounds can be administered simultaneously or sequentially as appropriate for the specific condition being treated. For pharmaceutical use, zalpha29 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. In general, pharmaceutical formulations will include a zalpha29 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Zalpha29 will preferably be used in a concentration of about 10 to 100 μg/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 μg/ml may be used. For topical application, such as for the promotion of wound healing, the protein will be applied in the range of 0.1-10 μg/cm 2 of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or intermittently over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed. In general, a therapeutically effective amount of zalpha29 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological score. Zalpha29 proteins, agonists, and antagonists are useful for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which include both primary cells and cultured cell lines. Of particular interest in this regard are hematopoietic cells (including stem cells and mature myeloid and lymphoid cells), endothelial cells, smooth muscle cells, fibroblasts, and hepatocytes. Zalpha29 polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml. Those skilled in the art will recognize that zalpha29 proteins can be advantageously combined with other growth factors in culture media. Within the laboratory research field, zalpha29 proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or under-production of zalpha29 protein or in the analysis of cell phenotype. Zalpha29 proteins can also be used to identify inhibitors of their activity. Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zalpha29 protein. In addition to those assays disclosed above, samples can be tested for inhibition of zalpha29 activity within a variety of assays designed to measure receptor binding or the stimulation inhibition of zalpha29-dependent cellular responses. For example, zalpha29-responsive cell lines can be transfected with a reporter gene constmct that is responsive to a zalpha29- stimulated cellular pathway. Reporter gene constmcts of this type are known in the art, and will generally comprise a zalpha29-activated semm response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha29 on the target cells as evidenced by a decrease in zalpha29 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zalpha29 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. En the alternative, compounds or other samples can be tested for direct blocking of zalpha29 binding to receptor using zalpha29 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FEEC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zalpha29 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors. As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains. Antibodies are defined to be specifically binding if they bind to a zalpha29 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non- zalpha29) polypeptide or protein. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, which is incoφorated herein by reference). As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a zalpha29 polypeptide may be increased through the use of an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zalpha29 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine semm albumin (BSA) or tetanus toxoid) for immunization. Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zalpha29 polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled zalpha29 polypeptide). Human antibodies can be produced in transgenic, non- human animals that have been engineered to contain human immunoglobulin genes as disclosed in WEPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination. A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zalpha29 polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays. Antibodies to zalpha29 may be used for affinity purification of the protein, within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zalpha29 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vitro and in vivo. Antibodies to zalpha29 may also be used for tagging cells that express zalpha29; for affinity purification of zalpha29 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies. Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targetting of those compounds to cells expressing receptors for zalpha29. For certain applications, including in vitro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates. Antibodies of the present invention may also be directly or indirectly conjugated to dmgs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications (e.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996. Polypeptides and proteins of the present invention can be used to identify and isolate receptors. Zalpha29 receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes. For example, zalpha29 proteins and polypeptides can be immobilized on a column, and membrane preparations mn over the column (as generally disclosed in Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be radiolabeled (Methods Enzymol, vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Academic Press, San Diego, 1990, 721-737) or photoaffinity labeled (Bmnner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and used to tag specific cell-surface proteins. In a similar manner, radiolabeled zalpha29 proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library. The present invention also provides reagents for use in diagnostic applications. For example, the zalpha29 gene, a probe comprising zalpha29 DNA or RNA, or a subsequence thereof can be used to determine the presence of mutations at or near the zalpha29 locus at chromosome 2pl5. Detectable chromosomal aberrations at the zalpha29 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, often 15 or more nt, and frequently 20-30 nt. Short polynucleotides can be used when a small region of the gene is targetted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more. Probes will generally comprise a polynucleotide linked to a signal-generating moiety such as a radionucleotide. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (c) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ED NO: l, the complement of SEQ ED NO:l, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymoφhism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A.J. Marian, Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion. Within PCR assays, a patient genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is single strand conformational polymoφhism (SSCP) analysis (Hayashi, PCR Methods and Applications L34-38, 1991). The polypeptides, nucleic acids and/or antibodies of the present invention may be used in diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation (including cancer). Labeled zalpha29 polypeptides may be used for imaging tumors or other sites of abnormal cell proliferation. Inhibitors of zalpha29 activity (zalpha29 antagonists) include anti- zalpha29 antibodies and soluble zalpha29 receptors, as well as other peptidic and non- peptidic agents (including ribozymes). Such antagonists can be used to block the effects of zalpha29 on cells or tissues. Of particular interest is the use of antagonists of zalpha29 activity in cancer therapy. As early detection methods improve it becomes possible to intervene at earlier times in tumor development, making it feasible to use inhibitors of growth factors to block cell proliferation, angiogenesis, and other events that lead to tumor development and metastasis. Inhibitors are also expected to be useful in adjunct therapy after surgery to prevent the growth of residual cancer cells. Inhibitors can also be used in combination with other cancer therapeutic agents. In addition to antibodies, zalpha29 inhibitors include small molecule inhibitors and inactive receptor-binding fragments of zalpha29 polypeptides. Inhibitors are formulated for pharmaceutical use as generally disclosed above, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated. The relevant determinations are within the level of ordinary skill in the formulation art. Polynucleotides encoding zalpha29 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha29 activity. If a mammal has a mutated or absent zalpha29 gene, a zalpha29 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zalpha29 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA vims, such as, but not limited to, heφes simplex vims (HSV), papillomavirus, Epstein Barr vims (EBV), adenovims, adeno-associated vims (AAV), and the like. Defective vimses, which entirely or almost entirely lack viral genes, are preferred. A defective vims is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective heφes simplex vims 1 (HSVl) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 1991); an attenuated adenovims vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630, 1992; and a defective adeno-associated vims vector (Samulski et al., J. Virol. 61:3096-3101, 1987; Samulski et al., J. Virol. 63:3822-3888, 1989). Within another embodiment, a zalρha29 gene can be introduced in a retroviral vector as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33: 153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., I. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WEPO Publication WO 95/07358; and Kuo et al., Blood 82:845, 1993. Alternatively, the vector can be introduced by liposome-mediated transfection ("lipofection"). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027- 8031, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the puφose of targeting. Peptidic and non-peptidic molecules can be coupled to liposomes chemically. Within another embodiment, cells are removed from the body, a vector is introduced into the cells as a naked DNA plasmid, and the transformed cells are re-implanted into the body as disclosed above. Antisense methodology can be used to inhibit zalpha29 gene transcription in a patient as generally disclosed above. Zalpha29 polypeptides and anti-zalpha29 antibodies can be directly or indirectly conjugated to dmgs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, zalpha29 polypeptides or anti-zalpha29 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues, or organs that express the anti-complementary molecule. Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic molecules can be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90. These can be either directly attached to the polypeptide or antibody, or indirectly attached according to known methods, such as through a chelating moiety. Polypeptides or antibodies can also be conjugated to cytotoxic dmgs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these puφoses, biotin streptavidin is an exemplary complementary/anticomplementary pair. Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy. Of particular interest in this regard are conjugates of a zalpha29 polypeptide and a cytotoxin, which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization. Target cells (i.e., those displaying the zalpha29 receptor) bind the zalpha29-toxin conjugate, which is then internalized, killing the cell. The effects of receptor-specific cell killing (target ablation) are revealed by changes in whole animal physiology or through histological examination. Thus, ligand-dependent, receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand. One such toxin is saporin. Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular. In another embodiment, zalpha29-cytokine fusion proteins or antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447. 1997). In general, cytokines are toxic if administered systemically. The described fusion proteins enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zalpha29, thereby providing an elevated local concentration of cytokine. Suitable cytokines for this puφose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Such fusion proteins may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization. The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action. The invention is further illustrated by the following non-limiting examples. Examples Example 1 Zalpha29 Northern blot analysis was performed using commercially prepared blots of human RNA (Human Multiple Tissue Northern Blots I, I , and Ed; Human Fetal Multiple Tissue Northern Blot II; and Human RNA Master Blot; Clontech Laboratories, Inc., Palo Alto, CA). The zalpha29 hybridization probe was generated as a gel purified PCR amplification product. The amplification product was made using oligonucleotides ZC21,720 (SEQ ID NO:8) and ZC21,721 (SEQ ED NO:9) as PCR primers and a cloned zalpha29 cDNA (see SEQ ED NO: l) as template. The PCR amplification was performed as follows: 1 μl of zalpha29 cDNA (~2 ng) and 40 pmoles each of oligonucleotide primers ZC21,720 and ZC21,721 were added to a reaction mixture containing commercially available reagents (Advantage™ KlenTaq Polymerase Kit, Clontech Laboratories, Inc.) following the manufacturer's recommended protocol. The reaction was mn as follows: 94°C for 30 seconds, 25 cycles of 94°C for 5 seconds, 55°C for 5 seconds, and 68°C for 1 minute, followed by 68°C for 3 minutes and a hold at 4°C. The 422 bp PCR amplified fragment was gel purified and recovered using silica gel particles (QIAEX® II gel extraction kit; Qiagen, Valencia, CA) according to the manufacturer's recommended protocol. The probe was a radioactively labeled using a commercially available kit (Rediprime™ II random-prime labeling system; Amersham Coφ., Arlington Heights, EL) according to the manufacturer's protocol. The probe was purified using a a commercially available push column (NucTrap® column; Stratagene, La Jolla, CA; see U.S. Patent No. 5,336,412). A hybridization solution (ExpressHyb™ Hybridization Solution; Clontech Laboratories, Inc.) solution was used for the prehybridization and hybridization solutions for the Northern blots. Hybridization took place overnight at 65 °C. Following hybridization, the blots were washed in 2X SSC, 0.1% SDS at room temperature, followed by a wash in 0.1X SSC and 0.1% SDS at 50°C. The blots were exposed to film (BIOMAX, Eastman Kodak, New Haven, CT). An overnight exposure showed an approximately 870 base band in every lane on all of the blots. Every RNA sample on the RNA Master Blot was positive while the negative controls were negative. The positive tissues on the Northerns included heart, ovary, fetal lung, brain, small intestine, fetal liver, placenta, colon (mucosal lining), fetal kidney, lung, peripheral blood leukocyte, liver, stomach, skeletal muscle, thyroid, kidney, spinal cord, pancreas, lymph node, spleen, trachea, thymus, adrenal gland, prostate, bone marrow, testis, fetal brain. Positive tissues on the RNA Master Blot that were not also on the Northerns included amygdala, aorta, caudate nucleus, bladder, cerebellum, utems, cerebral cortex, pituitary gland, frontal lobe, salivary gland, hippocampus, mammary gland, medulla oblongata, appendix, occipital lobe, trachea, putamen, fetal heart, substantia nigra, fetal spleen, thalamus, fetal thymus, and subthalamic nucleus. The Northern blots were reprobed for human transferrin receptor. The resulting signal generated from the transferrin receptor probe was used to normalize the zalpha29 signal. The tissues with the greatest ratio of zalpha29 signal to transferrin receptor signal were heart, liver, and testis. Example 2 Zalpha29 was mapped to chromosome 2 using the commercially available version of the Stanford G3 Radiation Hybrid Mapping Panel (Research Genetics, Inc., Huntsville, AL). This panel contains PCRable DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient). A publicly available WWW server (http://shgc- www.stanford.edu) allows chromosomal localization of markers. For the mapping of Zalpha29 with the Stanford G3 RH Panel, 20-μl reaction mixtures were set up in a PCRable 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a thermal cycler (RoboCycler® Gradient 96; Stratagene). Each of the 85 PCR reactions consisted of 2 μl buffer (10X KlenTaq PCR reaction buffer; (Clontech Laboratories, Inc., Palo Alto, CA), 1.6 μl dNTPs mix (2.5 mM each, Perkin- Elmer, Foster City, CA), 1 μl sense primer ZC22,737 (SEQ ED NO: 10), 1 μl antisense primer ZC22,738 (SEQ ED NO: 11), 2 μl of a density increasing agent and tracking dye (RediLoad, Research Genetics, Inc., Huntsville, AL), 0.4 μl of a commercially available DNA polymerase/antibody mix (50X Advantage™ KlenTaq Polymerase Mix; Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x μl ddH2O for a total volume of 20 μl. The mixtures were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 5-minute denaturation at 94°C, 35 cycles of a 45-second denaturation at 94°C, 45 seconds annealing at 64°C and 75 seconds extension at 72°C; followed by a final extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (obained from Life Technologies, Gaithersburg, MD). The results showed linkage of Zalpha29 to the chromosome 2 framework marker SHGC-30949 with a LOD score of >11 and at a distance of 0 cR_ 10000 from the marker. The use of surrounding genes that have been physically mapped positions Zalpha29 in the 2pl6-pl5 region on chromosome 2. Example 3 The protein coding region of mouse zalpha29 was amplified by PCR using primers that added Fsel and Ascl restriction sties at the 5' and 3' termini respectively. PCR primers ZC23019 (SEQ ED NO: 12) and ZC23018 (SEQ ED NO: 13) were used with a template plasmid (pT7T3D-Pac) containing the full-length murine zalpha29 cDNA in a PCR reaction as follows: one cycle at 95 °C for 5 minutes; followed by 15 cycles at 95°C for 0.5 min., 58°C for 0.5 min., and 72°C for 0.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak. The PCR reaction product was loaded onto a 1.2% (low melt) agarose (SeaPlaque® GTG; FMC Coφ., Rockland, ME) gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The zalpha29 PCR product was excised from the gel. The gel slice was melted at 65°, and the DNA was extracted twice with phenol and precipitated with ethanol. The PCR product was then digested with Fsel + Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20μl TE (Tris/EDTA pH 8). The 567-bp zalpha29 fragment was then ligated into the Fsel-Ascl sites of a modified pAdTrack CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998). This constmct also contained the green fluorescent protein (GFP) marker gene. The CMV promoter driving GFP expression was replaced with the SV40 promoter and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal. In addition, the native polylinker was replaced with Fsel, EcoRV, and Ascl sites. This modified form of pAdTrack CMV was named pZyTrack. Ligation was performed using a DNA ligation and screening kit (Fast-Link™; Epicentre Technologies, Madison, WI). Clones containing the zalpha29 cDNA were identified by standard mini-prep procedures. To linearize the plasmid, approximately 5 μg of the pZyTrack zalpha29 plasmid was digested with Pmel. Approximately 1 μg of the linearized plasmid was cotransformed with 200ng of supercoiled pAdEasy (He et al., ibid.) into BJ5183 cells. The co-transformation was done using an electroporator (Gene Pulser®; Bio-Rad Laboratories, Inc., Hercules, CA) at 2.5 kV, 200 ohms, and 25 μFa. The entire co- transformation mixture was plated on 4 LB plates containing 25 μg/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin, and recombinant adenovims DNA was identified by standard DNA miniprep procedures. Digestion of the recombinant adenovims DNA with Fsel + Ascl confirmed the presence of the zalpha29 sequence. The recombinant adenovims miniprep DNA was transformed into E. coli host cells (DH10B™; Life Technologies, Gaithersburg, MD), and DNA was prepared using a commercially available plasmid isolation kit (QIAGEN® Plasmid Maxi Kit; Qiagen, Inc., Valencia, CA) as directed by the supplier. Approximately 5 μg of recombinant adenoviral DNA was digested with PacI enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 μl containing 20-30U of Pad. The digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol. The DNA pellet was resuspended in 5 μl distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Canada), inoculated the day before and grown to 60- 70% confluence, was transfected with the PacI-digested DNA. The Pad-digested DNA was diluted to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 25 μl of lmg/ml N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethyl-ammonium salts (DOTAP) (Boehringer Mannheim, Indianapolis, EN) was diluted to a total volume of 100 μl with HBS. The DNA was added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes. The media was removed from the 293A cells, and the cells were washed with 5 ml semm-free MEMalpha containing 1 mM sodium pyruvate, 0.1 mM MEM non- essential amino acids, and 25 mM HEPES buffer (media components obtained from Life Technologies, Gaithersburg, MD). 5 ml of semm-free MEM was added to the 293 A cells and held at 37°C. The DNA lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently and incubated at 37°C for 4 hours. After 4 hours the media containing the DNA lipid mixture was aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine semm. The transfected cells were monitored for GFP expression and formation of foci (viral plaques). Seven days after transfection of 293A cells with the recombinant adenoviral DNA, the cells expressed GFP and started to form foci. The cmde viral lysate was collected with a cell scraper and transferred to a 50-ml conical tube. To release most of the vims particles from the cells, three freeze/thaw cycles were done in a dry ice/ethanol bath and a 37° waterbath. The cmde lysate was amplified (primary (1°) amplification) to obtain a working "stock" of zalpha29 recombinant adenovims (rAdV) lysate. Ten 10-cm plates of nearly confluent (80-90%) 293A cells were set up 20 hours in advance. 200 ml of cmde rAdV lysate was added to each 10-cm plate, and the plates were monitored for 48 to 72 hours for CPE (cytopathic effect) under the white light microscope and expression of GFP under the fluorescent microscope. When all of the 293A cells showed CPE, the 1 ° stock lysate was collected, and freeze/thaw cycles were performed as above. For secondary (2°) amplification, 20 15-cm tissue culture dishes of 293A cells were prepared so that the cells were 80-90% confluent. All but 20 ml of 5% MEM media was removed, and each dish was inoculated with 300-500 ml 1° amplified rAdv lysate. After 48 hours the cells were lysed from vims production. This lysate was collected into 250-ml polypropylene centrifuge bottles. To purify the rAdV, NP-40 detergent was added to a final concentration of 0.5% to the bottles of cmde lysate to lyse all cells. Bottles were placed on a rotating platform for 10 minutes, agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The supernatants were transferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG-8000/2.5 M NaCl solution was added. The bottles were shaken overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes, and supernatants were discarded into a bleach solution. Using a sterile cell scraper, the precipitate from 2 bottles was resuspended in 2.5 ml PBS. The vims solution was placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G for 10 minutes to remove any additional cell debris. The supernatant from the 2-ml microcentrifuge tubes was transferred to a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCl. The volume of the vims solution was estimated, and 0.55 g/ml of CsCl was added. The CsCl was dissolved, and 1 ml of this solution weighed 1.34 g. The solution was transferred to polycarbonate thick-walled centrifuge tubes (3.2ml; Beckman #362305) and spun at 348,000 X G for 3-4 hours at 25°C in a Beckman Optima TLX micro-ultracentrifuge with a TLA- 100.4 rotor. The vims formed a white band. Using wide-bore pipette tips, the vims band was collected. The vims preparation was desalted by gel filtration using commercially available columns and cross-linked dextran media (PD-10 columns prepacked with Sephadex® G-25M; Pharmacia, Piscataway, NJ). The column was equilibrated with 20 ml of PBS. The vims was loaded and allow it to mn into the column. 5 ml of PBS was added to the column, and fractions of 8-10 drops were collected. The optical densities of 1:50 dilutions of each fraction was determined at 260 nm on a spectrophotometer. A clear absorbance peak was present between fractions 7-12. These fractions were pooled, and the optical density (OD) of a 1:25 dilution determined. Vims concentration was determined by the formula: (OD at 260nm)(25)(l.l x 10 12 ) = virions/ml. The OD of a 1:25 dilution of the zalpha29 rAdV was 0.059, giving a vims concentration of 3.3 X 10 12 virions/ml. To store the vims, glycerol was added to the purified vims to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C. A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada) was followed to measure recombinant vims infectivity. Briefly, two 96- well tissue culture plates were seeded with 1X10 293 A cells per well in MEM containing 2% fetal bovine semm for each recombinant vims to be assayed. After 24 hours, 10- fold dilutions of each vims from 1X 10 -2 to 1X 10 -14 were made in MEM containing 2% fetal bovine semm. 100 μl of each dilution was placed in each of 20 wells. After 5 days at 37°C, wells were read either positive or negative for CPE, and a value for plaque forming units/ml (PFU) was calculated. TCED 50 formulation used was as per Quantum Biotechnologies, Inc., above. The titer (T) was determined from a plate where vims used was diluted from 10 -2 to 10 -14 , and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells was determined. To calculate titer of the undiluted vims sample: the factor, "F" = l+d(S-0.5); where "S" is the sum of the ratios (R); and "d" is LoglO of the dilution series, for example, "d" is equal to 1 for a ten-fold dilution series. The titer of the undiluted sample is T = 10 = TCED 50 /ml. To convert TCED 50 /ml to pfu/ml, 0.7 is subtracted from the exponent in the calculation for titer (T). The zalpha29 adenovims had a titer of 1.3 X 10 1 pfu/ml. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims: CLAIMS What is claimed is: 1. An isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of residues 48-62 of SEQ ID NO:2, residues 47-61 of SEQ ED NO:4, residues 63-104 of SEQ ID NO:2, residues 62-103 of SEQ ID NO:4, residues 105- 119 of SEQ ED NO:2, residues 104-118 of SEQ ID NO:4, residues 120-137 of SEQ ED NO:2, residues 119-136 of SEQ ID NO:4, residues 138-152 of SEQ ID NO:2, residues 137-151 of SEQ ED NO:4, residues 153-170 of SEQ ID NO:2, residues 152-169 of SEQ ED NO:4, residues 171-185 of SEQ ID NO:2, and residues 170-184 of SEQ ID NO:4. 2. The isolated polypeptide of claim 1 which is from 15 to 1500 amino acid residues in length. 3. The isolated polypeptide of claim 2, wherein said sequence of amino acid residues is operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ED NO:5. 4. The isolated polypeptide of claim 1 comprising at least 30 contiguous residues of SEQ ID NO:2 or SEQ ED NO:4. 5. The isolated polypeptide of claim 1 comprising residues 48-185 of SEQ ED NO:6 or residues 27-190 of SEQ ED NO:6. 6. The isolated polypeptide of claim 1 comprising residues 48-185 of SEQ ED NO:2, residues 47-184 SEQ ED NO:4, residues 27-190 of SEQ ID NO:2, or residues 26-188 of SEQ ED NO:4. 7. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of residues 48-62 of SEQ ED NO:2, residues 47- 61 of SEQ ID NO:4, residues 63-104 of SEQ ED NO:2, residues 62-103 of SEQ ID NO:4, residues 105-119 of SEQ ED NO:2, residues 104-118 of SEQ ED NO:4, residues 120-137 of SEQ ID NO:2, residues 119-136 of SEQ ED NO:4, residues 138-152 of SEQ ED NO:2, residues 137-151 of SEQ ED NO:4, residues 153-170 of SEQ ID NO:2, residues 152-169 of SEQ ED NO:4, residues 171-185 of SEQ ED NO:2, and residues 170-184 of SEQ ED NO:4; and a transcription terminator. 8. The expression vector of claim 7 wherein the DNA segment comprises nucleotides 79 to 570 of SEQ ED NO:7. 9. The expression vector of claim 7 wherein the polypeptide comprises residues 48-185 of SEQ ED NO:6 or residues 27-190 of SEQ ID NO:6. 10. The expression vector of claim 7 wherein the polypeptide comprises residues 48-185 of SEQ ED NO:2, residues 47-184 of SEQ ED NO:4, residues 27-190 of SEQ ID NO:2, or residues 26-188 of SEQ ED NO:4. 11. The expression vector of claim 7 further comprising a secretory signal sequence operably linked to the DNA segment. 12. A cultured cell into which has been introduced the expression vector of any of claims 7-11, wherein the cell expresses the DNA segment. 13. A method of making a polypeptide comprising: culturing the cell of claim 12 under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the polypeptide. 14. A polypeptide produced by the method of claim 13. 15. An antibody that specifically binds to the polypeptide of claim 14. 16. A method of detecting, in a test sample, the presence of an antagonist of zalpha29 activity, comprising: culturing a cell that is responsive to zalpha29; exposing the cell to a zalpha29 polypeptide in the presence and absence of a test sample; comparing levels of response to the zalpha29 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison the presence of an antagonist of zalpha29 activity in the test sample.

Inventors: CONKLIN DARRELL C, GAO ZEREN, CONKLIN, DARRELL, C., GAO, ZEREN
Applicants: ZYMOGENETICS INC, ZYMOGENETICS, INC.
Application year: 2000
Main classification: C12N15
IPC codes: C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1