MXPA01005967A - Pancreatic polypeptide zsig66 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig66, a novel secreted protein. The polynucleotides encoding zsig66, may, for example, be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

Description

PITUITARY POLYPEPTIDE ZSIG66 BACKGROUND OF THE INVENTION The proliferation and differentiation of multicellular organisms are controlled by hormones and polypeptide growth factors. These molecules that can diffuse allow the cells to communicate with each other and act in concert to regulate the cells and form organs, and to repair and regenerate damaged tissues. Examples of hormones and growth factors include steroid hormones (eg, estrogen, testosterone), parathyroid hormone, follicle-stimulating hormone, interleukins, platelet-derived growth factor (PDFG), factor epidermal growth (EGF), the stimulating factor of the granulocyte-macrophage colony (GM-CSF), erythropoietin (EPO) and calcitonin. Hormones and growth factors influence cellular metabolism by binding to proteins. These proteins can be integral membrane proteins that are linked to the signaling pathways within the cell, such as second messenger systems. Other kinds of proteins that influence hormones and Ref. No. 130025 growth factors are soluble molecules, such as transcription factors. Thus, there is a continuing need to discover new hormones, growth factors and the like. The in vivo activities of these cytokines illustrate the enormous clinical potential of, and the need for, other cytokines, cytokine agonists, and cytokine antagonists. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein. BRIEF DESCRIPTION OF THE INVENTION Within one aspect, the present invention provides an isolated polynucleotide encoding a zsig66 polypeptide comprising an amino acid residue sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), from the SEC. ID. NO: 2, where the percent identity of the amino acid is determined using a FASTA program with ktup = 1, penalty for opening the interval = 10, penalty for extension of the interval = 1, and substitution matrix = BLOSUM62, with other established parameters default. Within one embodiment, the isolated polynucleotide described above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 431 to nucleotide 472; (b) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 425 to nucleotide 472; (c) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 425 to nucleotide 475; (d) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 305 to nucleotide 475; (e) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 224 to nucleotide 475; and (f) a polynucleotide sequence complementary to (a) or (b). Within another embodiment, the isolated polynucleotide described above comprises nucleotide 1 through nucleotide 252 of SEQ. ID NO: 11. Within another embodiment, the isolated polynucleotide described above comprises an amino acid residue sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from the amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), of SEQ. ID. NO: 2. Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig66 polypeptide comprising an amino acid sequence as shown in SEQ. ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and a transcription terminator. Within one embodiment, the expression vector described above further comprises a secretory signal sequence operably linked to the DNA segment. Within a third aspect, the present invention provides a cultured cell into which an expression vector has been introduced as described above, wherein the cell expresses the polypeptide encoded by the DNA segment. Within a fourth aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first segment of DNA encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of : (a) the amino acid sequence of SEC. ID. NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in the structure, and encode the fusion protein. Within another aspect, the present invention provides a fusion protein produced by a method comprising: culturing a host cell into which a vector comprising the following operably linked elements has been introduced: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as described above, and (c) a transcriptional terminator, and recovering the protein encoded by the DNA segment. Within another aspect, the present invention provides an isolated polypeptide comprising an amino acid residue sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), of SEQ. ID. NO: 2, where the percent identity of the amino acid is determined using a FASTA program with ktup = 1, penalty for opening the interval = 10, penalty for extension of the interval = 1, and substitution matrix = BLOSUM62, with other established parameters default. Within one embodiment, the isolated polypeptide described above further comprises portions 1 to 4 separated from the N-terminal or C-terminal in an Ala28 ~ configuration. { 7 } -Ml-. { 3 } -M2-. { 5 } -M3-. { 6.} -M4-. { 10 } -Glys4, where Ml is "portion 1", an amino acid sequence as shown in amino acids 36 to 41 of the SEC. ID. NO: 2, M2 is "portion 2", an amino acid sequence as shown in amino acids 45 to 50 of the SEC. ID. NO: 2, M3 is "portion 3", an amino acid sequence as shown in amino acids 56 to 61 of the SEC. ID. NO: 2, M4 is "portion 4", an amino acid sequence as shown in amino acids 68 to 73 of the SEC. ID. NO: 2, Ala28 is the Alanine residue in amino acid number 28 in the SEC. ID. NO: 2, Gly84 is the Glycine residue at amino acid number 84 in the SEC. ID.

NO: 2, and. { #} denotes the number of amino acids between the portions. Within another embodiment, the isolated polypeptide described above comprises an amino acid residue sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), from the SEC. ID. NO: 2. Within another embodiment, the isolated polypeptide described above further comprises an amidated C-terminal Serine residue. Within another aspect, the present invention provides a method for producing a zsig66 polypeptide comprising: culturing a cell as described above; and isolating the zsig66 polypeptide produced by the cell.

Within another aspect, the present invention provides a method for detecting, in a test sample, the presence of a modulator of the activity of the zsig66 protein, which comprises: transfecting a cell expressing zsig66, with a reporter gene construct which is responsive or responds to a cellular path stimulated by zsigdd; and producing the zsigdd polypeptide by the method described above; and adding the zsig66 polypeptide to the cell, in the presence and absence of a test sample; and comparing the response levels with the zsig66 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of zsig66 activity in the test sample. Within another aspect, the present invention provides a method for producing an antibody to the zsig66 polypeptide comprising the following steps for: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous amino acid sequence as shown in SEQ ID NO: 2 of amino acid number 28 (Ala) to amino acid number 84 (Gly); (b) a polypeptide consisting of the amino acid sequence of SEQ. ID. NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); (c) a polypeptide as described above; (d) a polypeptide consisting of amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ. ID. NO: 2; (e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (lie) of SEC. ID. NO: 2; (f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid number 32 (Ser) of the SEC. ID. NO: 2; (g) a polypeptide consisting of amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEC. ID. NO: 2; and (h) a polypeptide consisting of amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEC. ID. NO: 2; and wherein the polypeptide generates an immune response in the animal to produce the antibody; and isolate the animal's antibody. Within another aspect, the present invention provides an antibody produced by the method described above, which binds to the zsigdd polypeptide. Within one embodiment, the antibody described above is a monoclonal antibody. Within another aspect, the present invention provides an antibody that binds to a polypeptide described above.

Within another aspect, the present invention provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ. ID.

NO: 1 or the complement of the SEC. ID. NO: 1, under conditions wherein said polynucleotide will hybridize to the complementary polynucleotide sequence; visualize the first reaction product; and comparing said first reaction product with a control reaction product from a native type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient. Within another aspect, the present invention provides a pharmaceutical composition comprising an isolated polypeptide as described above, wherein the polypeptide is in combination with a pharmaceutically acceptable carrier. These and other aspects of the invention will be apparent from the reference to the following detailed description of the invention and the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are hydrophobicity graphs of zsig66, determined from a hydrophilicity profile of Hopp / Woods based on a window of six sliding residues, with the residuals G, S, and T hidden and with the residues H, Y, and W exposed, ignored. DETAILED DESCRIPTION OF THE INVENTION Before setting forth the invention in detail, it will be helpful to define the following terms for the understanding thereof: The term "affinity tag" is used herein to denote a segment of the polypeptide that can be attached to a second polypeptide to provide purification or detection of the second polypeptide or provide binding sites of the second polypeptide to a substrate.Mostly, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag The affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985, Nilsson et al., Methods Enzymol 198: 3, '1991), glutathione S transferase (Smith and Johnson, Gene _67: 31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Nati. Acad.

Sci. USA 82: 7952-4, 1985), substance P, Flag ™ peptide (Hopp et al., Biotechnoloqy 6: 1204-10, 1988), the streptavidin binding peptide, or other antigenic epitope or binding domain . See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. The affinity tags encoding the DNAs are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene that occupies the same chromosomal location. Allelic variation becomes natural through a mutation, and can result in genotypic or phenotypic polymorphism within populations. Mutations of the genes can be either silent (without change in the encoded polypeptide) or they can be encoded polypeptides having the altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within the polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain carboxyl-terminal sequence positioned to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but not necessarily to the carboxyl terminus of the complete polypeptide. The term "complement / anti-complement pair" denotes non-identical portions that form a stable, associated pair, not covalently, under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen pairs (or incomplete antigen or epitope), sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity of <; 10 ^ M ~ A The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation when compared to the reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'. The term "contig" denotes a polynucleotide having a contiguous portion of sequence identical or complementary to other polynucleotides. The contiguous sequences are said to "overlap" to a given sequence portion of the polynucleotide sequence either in its entirety or along a partial portion of the polynucleotide. For example, the contiguous polynucleotide sequence 5 '-ATGGAGCTT-3' are 5'-AGCTTgagt-3 'and 3' -tcgacTACC-5 The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (when compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residues (ie, the GAU and GAC triplets each encode Asp). The term "expression vector" is used to denote a DNA molecule, linear or circular, comprising 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 the 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 environment and is therefore free from other unwanted or foreign coding sequences, and is in a form suitable for use within of protein production systems through genetic engineering. Such isolated molecules are those that are separated from their natural environment and include genomic and cDNA clones. The 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 apparent to one skilled 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 in a condition other than its natural environment, such as separated from blood and animal tissue. In a preferred form, the separated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, ie, greater than 95% pure, more preferably 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 derived or glycosylated forms. The term "operably linked" when referring to DNA segments, indicates that the segments are arranged to work in concert for their intended purposes, for example, the transcription starts at the promoter and proceeds through the coding segment up to the terminator The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation. "Paralogs" are distinct but structurally related proteins made by an organism.

It is believed that paralogs are generated through genetic duplication. For example, α-globin, β-globin and myoglobin are paralogs with each other. A "polynucleotide" is a double or single-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed in base pairs (abbreviated "pp") nucleotides ("nt"), or kilobases ("kb"). Where the context permits, the last two terms may describe polynucleotides, which are double-stranded or single-stranded. When the term applies to double-stranded molecules it is used to denote a full length and will be understood. which is 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 placed in stages as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. 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 with its recognized meaning in the art to denote a portion of a gene that contains the DNA sequences that provide the RNA polymerase linkage and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes. A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined here in terms of their basic amino acid structures; Substituents such as carbohydrate groups are generally unspecified, but nevertheless may be present. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (ie, a ligand) and that mediates the effect of the ligand in the cell. The membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand binding domain and an intracellular effector domain that is typically involved in signal transduction. The binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell. This interaction in turn generates an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include transcription of genes, phosphorylation, dephosphorylation, increases in the production of cyclic AMP, mobilization of calcium from cells, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be linked to the membrane, cytosolic or nuclear; monomeric (eg, thyroid-stimulating hormone receptor, beta-adrenergic receptor) or multimeric (the PDGF receptor, the growth hormone receptor, the receptor • IL-3, the GM-CSF receptor, the G-CSF receptor, erythropoietin receptor and IL-6 receptor). The term "secretory signal sequence" denotes a DNA sequence encoding 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 the which is synthesized. The larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. The term "splice variant" is used herein to denote the alternative forms of RNA transcribed from a gene. Splice variation is generated naturally through the use of alternative splice sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several transcribed mRNAs of the same gene. The splice variants can encode the polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splicing variant of a mRNA transcribed from a gene. The molecular weights and lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood as approximate values. When such values are expressed as "around" X or "approximately" X, the set value of X will be understood to have an accuracy of ± 10%. All references cited here are in their entirety incorporated by reference. The present invention is based in part on the discovery of a new DNA sequence encoding a polypeptide. The polypeptide and the corresponding polynucleotide are isolated from a human pituitary library. The polypeptide has been designated zsig66. The new zsigdd polypeptides of the present invention were initially identified by comparison with an EST database for proteins homologous to proteins having a secretory signal sequence. These proteins were characterized by a methionine initiation site, in the 5 'direction and a hydrophobic region of approximately 13 amino acids, followed by a cleavage site of the peptide signal of the peptide. An EST database was compared for the new DNA sequences whose translations would meet these search criteria. An EST was found and its corresponding cDNA was sequenced. It is believed that the nucleotide sequence of zsigdd encodes the complete coding sequence of the predicted protein. The zsigdd can be a new pituitary hormone, activation hormone that activates the pituitary hormone, a cell-cell signaling molecule, a growth factor, cytokine, the protein that modulates nerve cells, the protein associated with the extracellular matrix, secreted , with the activity of growth hormone hormone, or similar, and is a member of a family of novel proteins. The sequence of the zsigdd polypeptide was obtained from a single clone that is believed to contain its corresponding polynucleotide sequence. The clone was obtained from a pituitary library. Other libraries that could also be searched for such sequences include fetal, pancreatic, liver, ovarian, placental, and similar libraries. The nucleotide sequence of a DNA encoding the representative zsig66 is described in SEQ. ID NO: 1, and its deduced sequence of 84 amino acids is described in SEQ. ID NO: 2. In its entirety, the zsigdd polypeptide (SEQ ID NO: 2) represents a segment of the full-length polypeptide (residue 1 (Met) to residue 84 (Gly) of SEQ ID NO: 2). The domains and characteristic structures of the zsigdd are further described below. The analysis of the zsig66 polypeptide encoded by the DNA sequence of SEQ. ID NO: 1 revealed a sequence of 84 amino acids encoding the open reading frame (SEQ ID NO: 2) comprising a predicted signal peptide of 27 amino acid residues (residue 1 (Met) to residue 27 (Ser) of SEQ ID NO: 2), and a mature polypeptide of 57 amino acids (residue 28 (Ala) to residue 84 (Gly) of the SEC. ID NO: 2). In addition, post-translational processing can truncate zsigdd, for example in a cysteine link, and still provide a short active polypeptide. As such, the amino acid residues 69 (Phe) to 84 (Gly) of SEC. ID NO: 2, preferably amino acid residues 69 (Phe) to 83 (Ser) of SEC. ID NO: 2, and more preferably amino acid residues 70 (Cys) to 83 (Ser) of the SEC. ID NO: 2, comprise an active zsigdd polypeptide. In another embodiment, the Serine residue of the amino acid residues of short active polypeptide 69 (Phe) to 83 (Ser) of SEQ. ID NO: 2 or the residues of amino acids 70 (Cys) to 83 (Ser) of the SEC. ID NO: 2 can additionally be amidated in the C-terminal Serine residue. The structure of zsig66 that surrounds the C-C bond (see below), shows some similarity with vasopressin and oxytocin. As such, zsig66 may have vasodilator, cardiovascular or metabolic capacity. For general references in vasopressin and oxytocin, see Rehbein, M. et al., Biol. Chem. 367: 695-704, 1986; Sausville, E. et al., J. Biol. Chem. 260: 10236-10241, 1985; Ivell, R. et al., Endocrinol. 127: 2990-2996, 1990; Mohr, E et al., FEBS Lett. 193: 12-16, 1985; Chauvet, M.-T. et al., Proc. Nati Acad. Sci. USA 80: 2839-2843, 1983; Schlesinger, D.H. and Audhya, T.K., FEBS Lett. 128: 325-328, 1981; and Light, A. and DuVigneaud, V., Proc. Soc. Exp. Biol. Med. 9_8: 692-696, 1958. Additionally, the zsig66 polypeptide contains several regions of low variance or low degeneracy (see, Sheppard, P. et al., Gene 150: 163-167, 1994). . These regions exhibiting low degeneracy are described in the following portions of zsig66: 1) "portion 1" (corresponding to amino acids 36 (Trp) to 41 (Gly) of SEQ ID NO: 2); 2) "portion 2" (corresponding to amino acids 45 (Thr) to 50 (Phe) of SEQ ID NO: 2); 3) "portion 3" (corresponding to amino acids 56 (Glu) to 61 (Val) of SEQ ID NO: 2); 4) "portion 4" (corresponding to amino acids 68) (Lys) to 73 (Asn) of the SEC. ID. NO: 2); and Portions 1 through 4 are separated from the N-terminal to the C-terminal in a configuration represented by the following: Ala28-. { 7 } -Ml-. { 3 } -M2-. { 5 } -M3-. { 6 } -M4-. { 10 } -Gly84 wherein Ala28 is the initial residue of the mature polypeptide (as shown in SEQ ID NO: 2), Gly84 is the final residue or termination residue of the mature polypeptide (as shown in SEQ ID NO. : 2), M # denotes the specific portion described above (for example Ml is portion 1, etc.), and. { #} denotes the approximate number of amino acid residues between the portions, up to plus or minus 2 residues. The presence of conserved portions and low variance in general correlates with or defines important structural regions in proteins. Regions of low variance (eg, hydrophobic clusters) are usually present in regions of structural importance (Sheppard, P. et al., Gene 150: 163-167, 1994). Such regions of low variance often contain rare or infrequent amino acids, such as tryptophan. The flanking regions and between such conserved portions and those of low variance or variation may be more variable, but they are often functionally significant because they relate to or define important structures and activities such as the binding domains, the biological and enzymatic activity, the transduction of signal, tissue location domains and the like. In addition, there are two residues of Cysteine forming a disulfide bond located in the SEC. ID NO: 2 in amino acid number 70 and 74 that may be important for the structure or activity of the zsig66 polypeptide. In addition, there are 2 consensus phosphorylation sites in the zsig66 polypeptide: a protein kinase C (PKC) site at amino acid 53 (Ser) of the SEC. ID NO: 2; and a casein kinase II (CK2) at amino acid 43 (Thr) of SEC. ID NO: 2. Furthermore, the genomic structure of zsig66 is easily determined by one of skill in the art by comparing the sequence of the cDNA of the SEC. ID NO: 1 and the amino acid translated from the SEC. ID NO: 2 with the genomic DNA in which the gene is contained (Genbank Accession No. AC015783). For example, such analysis can be easily performed using the FASTA program as described here. As such, the intron and exon junctions in this region of genomic DNA can be determined for the zsig66 gene. The region of human genomic DNA in which zsig66 is located is shown in SEQ. ID NO: 24. The entire coding sequence appears to be contained in a single exon, as there are no putative introns within the SEC. ID NO: 24. Thus, the present invention includes the zsigdd gene as it is located in human genomic DNA. The corresponding polynucleotides that encode the regions, domains, portions, residues and sequences of the zsigdd polypeptide described above are as described in SEQ. ID NO: 1. The amino acids of low degeneracy in the zsigdd can be used as a tool to identify new members of the family. For example, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify the sequences encoding the low degeneracy portions of the RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designated from the zsigdß sequences are useful for this purpose. In particular, the degenerate oligonucleotide primers designated from the following zsigdd amino acid sequences of portions 1 through 5 are useful for this purpose: a) WIQGMG (portion 1); SEC. ID NO: 3), which corresponds to the degenerate polynucleotides of the SEC. ID NO: 7 and its complement; b) TDFNPF (portion 2; SEQ ID NO: 4), which corresponds to the degenerate polynucleotides of the SEC. ID NO: 8 and its complement; c) KFCVVN (portion 3; SEQ ID NO: 5), which corresponds to the degenerate polynucleotides of the SEC. ID NO: 9 and its complement; d) EIELFV (portion 4; SEQ ID NO: 6), corresponding to the degenerate polynucleotides of the SEC. ID NO: 10 and its complement. The present invention also provides polynucleotide molecules, including DNA and RNA molecules, which encode the zsigdd polypeptides described herein.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible between these polynucleotide molecules. The SEC. ID. NO: 11 is a degenerate DNA sequence that encompasses all of the DNAs encoding the zsig66 polypeptide of the SEC. ID. NO: 2. Those skilled in the art will recognize that the degenerate sequence of the SEC. ID. NO: 11 also provides all the RNA sequences encoding the SEC. ID. NO: 2 substituting U for T. Thus, the polynucleotides encoding the zsigdd polypeptide comprising from nucleotide 1 to nucleotide 252 of SEQ. ID. NO: 11 and its RNA equivalents are contemplated by the present invention. Table 1 establishes the one-letter codes used within the SEC. ID. NO: 11 to denote the positions of the degenerate nucleotide. The "resolutions" are the nucleotides denoted by the code of a letter. The "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 1 Base Code of the Nucleotide Base Code Nucleotide Resolution Complement A A C C G G G G C C T T A A RA | GYC | TYC | TRA | GMA | CKG | TKG | TMA | CSC | GSC | GWA | TWA | THA | C | TDA | G | TBC | G | TVA | C | GVA | C | GBC | G | TDA | G | THA | C | TNA | C | G | TNA | C | G | T The degenerate codons used in SEC. ID NO: , which encompass all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2 Codon Code Code of One Codon Synonyms Three Letter Degeneration Letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T AC ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC CAT GAY Glu E CAÁ GAG GAR Gln Q CAÁ CAG CAR His H CAC CAT CAY Arg R 'ACÁ AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG He 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 AND TAC TAT TAY Trp W TGG TGG Ter • TAA TAG TGA TRR Asn | Asp B RAY GluIGln Z SAR Any X NNN Someone with ordinary skill in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode each of the amino acids. For example, the degenerate codon for serine (SN) can, in some cases, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some cases, code for serine (AGY). A similar relationship exists between the codons that encode phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence can encode variant amino acid sequences, but only someone of ordinary skill in the art can easily identify such sequence variants by reference to the amino acid sequence of the SEC. ID NO: 2. Variant sequences can be tested for functionality as described here.

Someone with ordinary skill in the art will also appreciate that different species can show "preferential codon usage". In general, see, Grantham, et al., Nuc. Acids Res., 8: 1893-1912, 1980; Haas, et al. Curr. Biol., 6: 315-324, 1996; Wain-Hobson, et al., Gene, 13: 355-364, 1981; Grosjean, H., and Fiers,., Gene, 18: 199-209, 1982, Holm, L., Nuc. Acids Res., 14: 3075-3087, 1986; and Ikemura, T., J. Mol. Biol., 158: 573-597, 1982. As used herein, the terms "preferential codon usage" and "preferential codons" are terms in the art that refer to the translation codons of the proteins that are most frequently used. in cells of a certain species, thus favoring one or a few representatives of the possible codons that encode each amino acid (see Table 2). For example, the amino acid Threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells the ACC is the most commonly used codon.; In other species, for example, insect cells, yeasts, viruses or bacteria, different Thr codons may be preferential. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences in the recombinant DNA can, for example, improve the production of the protein making the translation of the protein more efficient within a particular cell type or species. Therefore, the degenerate codon sequence described in SEQ. ID NO: 11 serves as a template or model for optimizing the expression of polynucleotides in various cell types and species commonly used in the art and described herein. Sequences containing preferential codons can be tested and optimized for expression in several species, and can be tested for functionality as described herein. Within the preferred embodiments of the invention the isolated polynucleotides will hybridize to regions of similar size of the SEC. ID NO: 1, or a complementary sequence thereof, under severe conditions. In general, severe conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic concentration and pH) at which 50% of the target sequence is hybridized to a perfectly matched probe. Suitable, severe hybridization conditions are equivalent from about 5 hours to overnight incubation at about 42 ° C in a solution comprising: about 40-50% formamide, up to about 5X SSC, about 5X Denhardt solution , up to about 10% dextran sulfate, and about 10-20 μg / ml of commercially available, denatured carrier DNA; the hybridization is then carried out by washing the filters at up to about 2X SSC. For example, an adequate wash of severity is equivalent to 0.1X SSC to 2x SSC, 0.1% SDS, from 55 ° C to 65 ° C. The conditions of hybridization and washing, severe, depend on the length of the probe, reflected in the Tm, the solutions of hybridization and washing used, and are routinely determined empirically by someone with ordinary skills in the art. As previously noted, isolated polynucleotides of the present invention include DNA and RNA. Methods for the preparation of DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zsig66 RNA. Such tissues and cells are identified by staining or Northern blotting (Thomas, Proc. Nati, Acad. Sci. USA 77: 5201, 1980), and include the pituitary, pancreas, tissues of endocrine origin, and the like. Total RNA can be prepared using the extraction of guanidinium isothiocyanate followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is prepared from RNA using the method of Aviv and Leder (Proc Nati Acad Sci USA 69: 1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly (A) + using known methods. In an alternative, genomic DNA can be isolated. The polynucleotides encoding the zsigdd polypeptides are then identified and isolated by, for example, hybridization or PCR. A full-length clone encoding zsigdd can be obtained by conventional cloning procedures. Complementary DNA clones (cDNAs) are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or modify a cDNA clone to include at least one genomic intron. The methods for the preparation of cDNAs and genomic clones are well known and are within the level of someone with ordinary skills in the art., and include the use of the sequence described herein, or parts thereof, for generating probes or priming a library. Expression libraries can be converted into probes with antibodies to zsig66, receptor fragments or other specific binding moieties. The polynucleotides of the present invention can also be synthesized using machines for DNA synthesis. Currently, the method of choice is the phosphoramidite method. If chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically simple or uncomplicated and can be supplemented by the synthesis of the complementary strands and then pairing the strands by heating and cooling. For the production of longer genes (longer than about 300 bp), special strategies are usually employed, because the coupling efficiency of each cycle during chemical synthesis of DNA is rarely 100%. To overcome this problem, synthetic (double-stranded) genes are assembled in a modular fashion from single-stranded fragments that are from 20 to 100 nucleotides in length.

A method for constructing a synthetic gene requires the initial production of a group of complementary, overlapping oligonucleotides, each of which is between 20 to 60 nucleotides in length. Each internal section of the gene has complementary 3 'and 5' terminal extensions designed for the base pairs with precisely an adjacent section. Thus, after the gene is assembled, the process is completed by sealing the cuts or slits along the basic structures of the two strands with T4 DNA ligase. In addition to the protein coding sequence, synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector. An alternative way to prepare a full-length gene is to synthesize a specified set of partially overlapping oligonucleotides (40 to 100 nucleotides). After the overlapping 3 'and 5' complementary short regions are hybridized with heat and cold, the large spaces still remain, but the paired regions of short bases are long and stable enough to maintain the structure together . The separations or spaces are filled and the duplex DNA is completed via the enzymatic synthesis of the DNA by polymerase I of E. coli DNA. After the enzymatic synthesis is completed, the end portions are sealed. The double-stranded constructs are sequentially linked together to form the complete gene sequence that is verified by DNA sequence analysis. See Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1989 and Climie et al., Proc. Nati Acad. Sci. USA 87: 633-7, 1990). The zsigdd polynucleotide sequences described herein can also be used as probes or primers for the 5 'non-coding regions of a zsigdd gene. In view of tissue-specific expression observed for zsigdd by Northern blotting, it is expected that this region of the gene will provide specific expression of ovaries and pancreas. The promoter elements of a zsigdd gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. The cloning of the 5 'flanking sequences also facilitates the production of the zsig66 proteins by "gene activation" as described in US Patent No. 5,641,670. Briefly, the expression of an endogenous zsigdd gene in a cell is altered by introducing into the zsig66 site a DNA construct comprising at least one target sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The target sequence is a 5 'non-coding sequence of zsig66 that allows homologous recombination of the construct with the endogenous zsigdd site, whereby sequences within the construct are operably linked to the endogenous zsigdd coding sequence. In this manner, an endogenous zsig66 promoter can be replaced or supplemented with other regulatory sequences to provide specific, improved, or otherwise regulated tissue expression. The present invention also provides polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are human zsig66 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. The orthologs of human zsigdd can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type expressing zsigdd as described herein. Suitable sources of mRNA can be identified by Northern staining of the probes with the probes designed from the sequences described herein. A library is then prepared from the RNA of a positive tissue or cell line. The cDNA encoding the zsigdd can then be isolated by a variety of methods, such as the generation of probes with a complete or partial human cDNA with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zsigdd sequences described herein. Within a further method, a DNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the zsig66 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ. ID NO: 1 represents a single allele of the human zsigdd gene and polypeptide, and allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by generating cDNA probes or genomic libraries of different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEC. ID NO: 1, which includes those containing the silent mutations and those in which the mutations result in changes in the amino acid sequence, are within the scope of the present invention, as are the proteins that are the allelic variants of the SEC . ID NO: 2. The cDNAs generated from the alternatively spliced mRNAs, which retain the properties of the zsig66 polypeptide, are included within the scope of the present invention, as are the polypeptides encoded by such cDNAs and mRNAs. The allelic variants and splice variants of these sequences can be cloned by generating cDNA probes or genomic libraries for example a human thyroid cDNA library of different individuals or tissues according to standard procedures known in the art. The present invention also provides the zsigdd polypeptides that are substantially similar to the polypeptides of SEQ. ID NO: 2 and its orthologs. The term "substantially similar" is used herein to denote polypeptides having 70%, preferably 75%, more preferably at least 80%, of sequence identity to the sequences shown in SEQ. ID NO: 2 or your orthologs. Such polypeptides will more preferably be at least 90% identical, and more preferably 95% or more identical to SEC. ID NO: 2 or your orthologs. The percentage of 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. Nati Acad. Sci. USA 89: 19015-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment regions using a space opening penalty value of 10, a penalty value for the space extension of 1 , and the "blosum 62" registration matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The identity percentage is calculated as: Total number of identical matings x 100 [length of the longest sequence plus the number of spaces entered in the longest sequence to align the two sequences] 1 = H oí ro H E-? N CN OJ O 1 1 C? • H ro OJ J I 1 C r- H rH ro CN 1 1 1 1 Pu Lß OJ OJ rH ro rH 1 1 1 S in O OJ HHH rH rH 1 1 1 1 rH ro rH O rH rp CN OJ I 1 1 1 1 1 J OJ O R R OJ TH rH I 1 1 1 1 t OJ ro HO OJ rH n rH t * . 1 I 1 1 1 1 co rn ro H CN CN CN CN 1 1 1 1 1 1 1 I or U3 CN ** • J ro CN O OJ CN ro ro I 1 1 I 1 1 t 1 I? in OJ O ro ro rH J ro H r o r J OJ 1 I 1 I 1 1 I 1 1 a in OJ O R C H O R H OJ H OJ 1 1 or rH 1 1 1 1 1 u Q O R O OJ H ro • H ro ro rH O rH ro ro I 1 1 1 1 II 1 1 I «> r-l ro s O O R O R OJ H O CN fO 1 1 I 1 I t 1 1 The sequence identity of the polynucleotide molecules is determined by similar methods using a ratio as described above. Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity or similarity search algorithm of Pearson and Lipman is a suitable protein alignment method to examine the level of identity shared by an amino acid sequence described herein and the amino acid sequence of a putative zsigdd variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85: 2444 (1988), and by Pearson, Meth. Enzymol. 183: 63 (1990). Briefly, the FASTA first characterizes the sequence similarity by identifying the regions shared by the question sequence (eg, SEQ ID NO: 2) and a test sequence that has either the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2), without considering the substitutions, insertions or conservative deletions of amino acids. The ten regions with the highest density of identities are then re-recorded 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 record. If there are several regions with records greater than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with the separations or spaces. Finally, the highest recording regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444 (1970); Sellers, SIAM J. Appl. Math. 26: 787 (1974)), which allows amino acid insertions and deletions. The illustrative parameters for the FASTA analysis are: ktup = 1, penalty for opening the interval = 10, penalty for extension of the interval = 1, and substitution matrix = BLOSUM62. These parameters can be entered into a FASTA program by modifying the record matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183: 63 (1990). The FASTA can also be used to determine the sequence identity of the nucleic acid molecules using a ratio as described above. For comparisons of nucleotide sequences, the ktup value may be in the range of one to six, preferably three to six, more preferably three, with other parameters adjusted by default. The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared to the amino acid sequence of SEQ. ID. NO: 2. The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of the protein sequence segments, which represent highly conserved regions of more than 500 related protein groups (Henikoff and Henikoff, Proc. Nati, Acad. Sci. USA 89: 10915 (1992)). Accordingly, substitution frequencies BLOSUM62 can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Conservative substitutions of preferred amino acids are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2). or 3), whereas the most preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). Variant zsigdd polypeptides or substantially homologous polypeptides of zsig66 are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, which is conservative of the amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the activity of the polypeptide; small eliminations, typically from one to about 30 amino acids; and small terminal amino or carboxyl spreads, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes the polypeptides from about 35 to about 90 amino acid residues, or in some embodiments, 15 to 16 amino acid residues (eg, residues 69-83 of SEQ ID NO: 2 with or without a amide group), which comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of the SEC. ID NO: 2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsigdd polypeptide and the affinity tag. Such preferred sites include thrombin cleavage sites and factor Xa cleavage sites. Table 4 Conservative Substitutions of Basic Amino Acids: arginine lysine histidine Acids: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatics: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The present invention further provides a variety of other mergers of polypeptides and related multimeric proteins comprising one or more polypeptide fusions. For example, a zsigdd polypeptide can be prepared as a fusion for a dimerizing protein as described in U.S. Patent Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in relation to this include the domains of the constant region of the immunoglobulin. The immunoglobulin zsigdd polypeptide fusions can be expressed in engineered cells to produce a variety of multimeric zsigdd analogues. The helper domains are fused to the zsig66 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zsig66 polypeptide or protein can be made a target for a predetermined cell type by fusing a zsig66 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 made targets for therapeutic or diagnostic purposes. A zsigdd polypeptide can be fused to two or more portions, such as an affinity tag for purification and a target domain. Fusions of the polypeptide may also comprise one or more cleavage sites, particularly between the domains. See Tuan et al., Connective Tissue Research 3_4: 1-9, 1996. The proteins of the present invention may also comprise amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyltreonine, hydroxyethylcysteine, hydroxyethylhomocysteine. , nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods are known in the art for the incorporation of amino acid residues that do not occur naturally in proteins. For example, an in vi trc system can be employed where nonsense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for amino acid synthesis and aminoacylated tRNA are known in the art. The transcription and translation of the plasmids containing nonsense mutations is performed in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 1_13: 2722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 25_9: 806-809, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 90: 10145-10149, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271_: 19991-19998, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and in the presence of naturally occurring, desired amino acids ( example, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Amino acids that do not occur naturally are incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 33: 7470-7476, 1994. Amino acid residues that occur naturally can be converted into species that do not occur naturally by chemical modification in vi tro. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally, and unnatural amino acids can be replaced by amino acid residues zsig66. The essential amino acids in the zsigdd polypeptides of the present invention can be identified according to methods 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. Nati Acad. Sci. USA 88: 4498-502, 1991). In the latter technique, mutations of a single alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for their biological or biochemical activity as described below to identify the amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-4708, 1996. The ligand-receptor sites or other biological interactions can also be determined by physical analysis of the structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with the mutation of the amino acids of the putative contact site. See, for example, de Vos et al., Science 255: 306-312, 1992; Smith et al., J. Mol. Biol. 22: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identities of the essential amino acids can also be inferred from the analysis of the homologies with the related polypeptides. The determination of the amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined.

Within these regions one can determine the specific residues that will be more or less tolerant of the change and will maintain the complete tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, multiple sequence alignment with nucleotide or higher amino acid identity and computer analysis using available programs (for example, the Insight II® viewer and modeling tools) of homology, MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packaging, and hidden polar interactions (Barton, Current Opinion, Struct. Biol. 5: 372-376, 1995 and Cordes et al. al., Current Opin, Struct. Biol. 6: 3-10, 1996). In general, when modifications are designed to the molecules or identify the specific fragments, the determination of the structure will be accompanied by an evaluation activity of the modified molecules. Changes of the amino acid sequence are made in the zsigdd polypeptides to minimize the disruption of the higher order structure essential for biological activity. For example, when the zsigdd polypeptide comprises one or more helices, changes in amino acid residues will be made so that the geometry of the helix and other components of the molecule are not broken where changes in conformation decrease some critical function , for example, the link of the molecule to its binding partners. The effects of amino acid sequence changes can be predicted, for example, by computer modeling as described above or determined by analysis of crystal structure (see, for example, Lapthorn et al., Nat. Struct. Biol. 2: 266-268, 1995). Other techniques that are well known in the art compare the folding of a variant protein to a standard molecule (e.g., the native protein). For example, the comparison of the cysteine pattern in a variant and in standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods to determine the cysteine residues that are associated with disulfide bonds or that are free of such associations (Bean et al., Anal. Biochem. 201: 216- 226, 1992; Gray, Protein Sci. 2: 1732-1748, 1993; and Patterson et al., Anal. Chem. 66: 3727-3732, 1994). It is generally believed that if a modified molecule does not have the same pattern of disulfide bonds as the standard or reference molecule, the folding would be affected. Another well-known and accepted method for measuring bending is circular dichrosism (CD). The measurement and comparison of the CD spectrum generated by a modified molecule and the standard molecule is routine (Johnson, Proteins 7: 205-214, 1990). Crystallography is another well-known method for analyzing bending and structure. Nuclear magnetic resonance (NMR), the formation of the digestive peptide map and the formation of the epitope map are also well-known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257: 961-864, 1992). A Hopp / Woods hydrophilicity profile of the zsigdd protein sequence as shown in SEQ. ID NO: 2 can be generated (Hopp et al., Proc. Nati Acad. Sci. 78: 3824-3828, 1981, Hopp, J. Immun.Meth. 88: 1-18, 1986 and Triquier et al., Protein Engineering _11: 153-169, 1988). The profile is based on a sliding window of six residues. Residues G, S, and T hidden and the exposed residues H, Y, and W were ignored (see Figure 1). For example, in the hydrophilic regions of zsigdd, they include: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEC. ID NO: 2; (2) amino acid number 7 (Val) to amino acid number 12 (He) of SEC. ID NO: 2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEC. ID NO: 2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of the SEC. ID NO: 2; (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEC. ID NO: 2. Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing the modifications in the amino acid sequence of a zsig66 polypeptide, so as not to break the overall biological and structural profile. Of particular interest for replacement are the hydrophobic residues selected from the group consisting of Val, Leu and He or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, hydrophobic residues tolerant of substitution could include residues as shown in SEC. ID NO: 2. Cysteine residues are relatively intolerant of substitution. The identities of the essential amino acids can also be inferred from the analysis of the sequence similarity between other proteins with the zsigdd. Using methods such as the "FASTA" analysis described previously, regions of high similarity or similarity within a family of proteins are identified and used to analyze the amino acid sequence for the conserved regions. An alternative methodology for identifying a zsigdd variant polynucleotide is to determine whether a potential nucleic acid molecule encoding a zsigdd variant polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ. ID NO: 1, as discussed above. Other methods for identifying essential amino acids in the polypeptides of the present invention are methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081 (1989), Bass et al. ., Proc. Nat A Acad. Sci. USA 88: 4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), Pages 259-311 ( Academic Press, Inc. 1998).] In the latter technique, unique mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for their biological activity as described below to identify the amino acid residues that are critical to the activity of the molecule See also, Hilton et al., J. Biol. Chem. 271: 4699 (1996).

The present invention also includes functional fragments of zsigdd polypeptides and nucleic acid molecules that encode such functional fragments. A "functional" zsigdd or fragment thereof defined herein is characterized by its proliferative activity or differentiation, by its ability to induce or inhibit the specialized functions of the cell, or by its ability to specifically bind to an anti-zsigdd or zsig66 receptor antibody. (either soluble or immobilized). The present invention further provides fusion proteins that encompass: (a) polypeptide molecules comprising one or more of the portions described above; and (b) functional fragments comprising one or more of these portions. The other portion of the fusion protein polypeptide can be comprised of a non-native and / or unrelated secretory signal peptide that facilitates the secretion of the fusion protein. Routine removal assays of the nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule encoding a Zsig66 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ. ID NO: 1 can be digested with nuclease BaI31 to obtain a series of eliminations placed one on top of the other. These DNA fragments are then inserted into the expression vectors in the appropriate reading frame, and the expressed polypeptides are isolated and tested for zsig66 activity, or for their ability to bind anti-zsigdd or zsigdβ receptor antibodies. An alternative for exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons for the specific production of a desired zsigdd fragment. Alternatively, particular fragments of a zsigdd gene can be synthesized using the polymerase chain reaction. Standard methods for identifying functional domains are well known to those skilled in the art. For example, studies of truncation in either or both of the terminal ends of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66: 507 (1995). In addition, standard techniques for functional analysis are described, for example, by Treuter et al., Molec. Gen. Genet. 240: 113 (1993), Content et al., "Expression and preliminary deletion analysis of the ^ 2 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Inferieron Systems, Cantell ( ed.), pages 65-72 (Nijhoff 1987), Herschman, "The EGF Receiver," in Control of Animal Cell Proliferation, 1, Boynton et al., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270: 29270 (1995); Fukunaga et al., J. Biol. Chem. 270: 25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50: 1295 (1995), and Meisel et al., Plant Molec. Biol. 30: 1 (1996). Multiple substitutions of amino acids can also be made and tested using known methods of mutagenesis and selection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-57, 1988) or Bowie and Sauer (Proc. Nati. Acad. Sci. USA 86: 2152-2156, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include the phage sample (e.g., Lowman et al., Biochem., 30: 10832-10837, 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 4_6: 145, 1986; Ner et al., DNA 7: 127, 1988). The variants of the zsigdd DNA described and the polypeptide sequences can be generated through intermixing of the DNA as described by Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by homologous recombination in vi tro by random fragmentation of a generating DNA followed by reassembly using PCR, resulting in mutations at randomly introduced sites. This technique can be modified using a family of generating DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. The selection or separation for the desired activity, followed by the additional iterations of the mutagenesis and the assay provides a rapid "evolution" of the sequences by selecting the desirable mutations while a simultaneous selection against the negative changes is carried out. Mutagenesis methods are described herein and can be combined with high throughput automated screening methods to detect the activity of the mutagenized polypeptides, cloned, in the host cells. Mutagenized DNA molecules that encode active polypeptides (eg, secreted and detected by antibodies; or measured by a signal transduction type assay) 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. In addition, the proteins of the present invention (or fragments of the polypeptide thereof) can be linked to other bioactive molecules, particularly other cytokines, to provide multi-functional molecules. For example, one or more domains, hydrophilic regions, or regions containing the 1-4 portions of zsigdd can be linked to other proteins to improve their biological properties or production efficiency. The present invention thus provides a series of hybrid molecules in which a segment comprising one or more of the domains, hydrophilic regions, or regions containing the 1-4 portions of the zsig66 is fused to another polypeptide. The fusion is preferably done by splicing at the DNA level to allow the expression of the chimeric molecules in the recombinant production systems. The resulting molecules are then tested or assayed for such properties as improved solubility, improved stability, extended shelf life, improved expression and secretion levels, and pharmacodynamics. Such hybrid molecules may further comprise additional amino acid residues (eg, a polypeptide linker) between the component proteins or polypeptides. Using the methods described herein, one of ordinary skill in the art can identify and / or prepare a variety of polypeptides that are substantially homologous to the SEC. ID NO: 2 or the allelic variants thereof and retain the functional and structural properties of the native protein. For example, using the methods described above, someone could identify a receptor binding domain in the zsig66; a ligand-extracellular binding domain of a receptor for the zsigdd; the heterodimeric and homodimeric binding domains; other structural or functional domains; affinity marks; or other important domains for protein-protein or signal transduction interactions. Such polypeptides may also include additional polypeptide segments as described above in a general manner. For any zsig66 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can easily generate a complete degenerate polynucleotide sequence encoding this variant using the information set forth in Tables 1 and 2 above. The zsigdd polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in host cells engineered according to conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in a culture, and include bacterial cells, fungi, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cells culturing multicellular organisms, are preferred. Techniques for the manipulation of cloned DNA molecules and the introduction of exogenous into a variety of host cells are described 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 zsig66 polypeptide of the present invention is operably linked to others. genetic elements required for their expression, which generally include the transcription promoter and the terminator, inside an expression vector. The vector will 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 the selectable markers can be provided in separate vectors, and the replication of the exogenous DNA can be provided by integration in the genome of the host cell. The selection of promoters, terminators, selectable markers, vector 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 providers.

To direct a zsigdd polypeptide to the secretory pathway of a host cell, a secretory signal sequence (also known as a secretory peptide, leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the zsig66 polypeptide, or it may be derived from another secreted protein (eg, t-PA) or synthesized de novo. The secretory signal sequence is linked to the DNA sequence of zsig66, ie, the two sequences are linked in the correct reading structure and placed to direct the newly synthesized peptide towards the pathway or path of the host cell. The secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences can be placed anywhere in the DNA sequence of interest (see, for example, Welch et al. al., U.S. Patent No. 5,037,743, Holland et al., U.S. Patent No. 5,143,830). Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides in the secretory pathway. The present invention provides such signal fusion polypeptides. A signal fusion polypeptide can be generated wherein a secretory signal sequence derived from residue 1 (Met) to residue 27 (Ser) of SEQ ID NO: 2 is operably linked to another polypeptide using methods known in the art and described here. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused to the amino terminus to an additional peptide to direct the additional peptide to the secretory pathway. Such constructs have numerous applications known in the art. For example, these secretory signal sequence fusion constructs can direct the secretion of an active component of a protein not normally secreted. Such fusions can be used in vivo or in vi tro to direct peptides through the secretory pathway. Cultured mammalian cells are suitable as host cells 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 J. 1: 841-845, 1982), transfection mediated by DEAE-dextran (Ausubel et al., Ibid.), And transfection mediated by liposomes (Hawley-Nelson et al., Focus L5: 73, 1993; Ciccarone et al., Focus 1_5: 80, 1993), and viral vectors (A. Miller and G. Rosman, BioTechniques 7: 980-90, 1989).; Q. Wang and M. Finer, Nature Med. 2: 714-16, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., US Patent No. 4,784,950; Palmiter et al., US Patent No. 4,579,821; and Ringold, US Patent No. 4,656,134. Suitable cultured mammalian cells include COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. 10314), 293 (ATCC No. CRL 1373; Graham et al., J. Gen. Virol. 3_6: 59-72, 1977) and Chinese hamster ovary cell lines (eg, CHO-K1; ATCC No. CCL 61) . Suitable additional cell lines are known in the art and are available to the public from depositors such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as SV-400 or cytomegalovirus promoters. See, for example, U.S. Patent No. 4,956,288. Other suitable promoters include those of the metallothionein genes (US Patent No. 4, 579,821 and 4,601,978) and the major late promoter of the adenovirus. The selection of the drug is generally used to select the cells of mammals cultured within the foreign DNA that 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 capable of passing the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that encodes resistance to the antibiotic neomycin. The selection is carried out in the presence of a drug of the neomycin type, such as G-418 or the like. Selection systems can also be used to increase the level of expression of interest, a process referred to as "amplification." The amplification is carried out by culturing the transfectants in the presence of a low level of the selection agent and then increasing the amount of the selection agent to select the cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Alternate markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC Class I, the alkaline phosphatase of the placenta, can be used to classify the transfected cells of the cells not transfected by such means as FACS sorting or magnetic bead separation technology. Other higher eukaryotic cells can also be used as hosts, which include plant cells, insect cells and bird cells. The use of Agroba cterium rhizogenes as a vector for the expression of genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) JU: 47-58, 1987. The transformation of insect cells and the production of foreign polypeptides therein is described by Guarino et al., US Patent No. 5,162,222; and in the WIPO publication 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californi ca nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. The second method for manufacturing recombinant zsigdd baculovirus utilizes a system based on the transposons described by Luckow (Luckow, VA, et al., J. Virol 67: 4566-79 , 1993). This system, which uses the transfer vectors, is sold in the Bac-to-Bac equipment (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl ™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsigdd polypeptide to a baculovirus genome maintained in E. coli as a large plasmid called a "bacmido". The pFastBací ™ transfer vector uses the AcNPV polyhedrin promoter to drive expression of the gene of interest, in this case zsig66. However, the pFastBací ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the baculovirus basic protein promoter (also called the Peor promoter, p6.9 or MP) which is expressed earlier in baculovirus infection, and has been shown to be advantageous for expressing the secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen Virol 71: 971-6, 1990; Bonning, B.C. et al., J Gen Virol 75: 1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., J Biol Chem 270: 1543-9, 1995. In such constructs of the transfer vector, a short or long version of the basic protein promoter can be used. In addition, the transfer vectors can be constructed so as to replace the secretory signal sequences of native zsigdd with the secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from the Ecdysteroid Glucosyltransferase (EGT), honeybee Melitina (Invitrogen Corporation, Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the sequence of secretory signal of native zsigdd. In addition, the transfer vectors can include a fusion in the structure with DNA encoding an epitope tag on the C- or N-terminus of the expressed zsig66 polypeptide, eg, a tag of the Glu-Glu epitope (Grussenmeyer, T. et al., Proc. Nati, Acad. Sci. 82: 7952-4, 1985). Using a technique known in the art, a transfer vector containing zsig66 is transformed into E. coli, and is separated or selected with respect to the bacmides containing an interrupted lacZ gene indicative of the recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques and used to transfect Spodoptera frugiperda cells, for example, Sf9 cells. The recombinant virus expressing the zsigdd is produced subsequently. The recombinant viral material is manufactured by methods commonly used in the art. The recombinant virus is used to infect the host cells, typically a cell line derived from the soldier worm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology, Principies and Applications of Recornbinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the cell line High FiveO ™ (Invitrogen) derived from Trichopl usia ni (US Patent No. 5,300,435). The commercially available serum-free medium is used to grow and maintain the cells. The appropriate medium is Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and Ex-cellO405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for T. ni cells. The cells are grown from an inoculation density of about 2-5 x 10 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral material is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically about 3. The procedures used are generally described in available laboratory manuals (King, LA and Possee, RD ibid., O'Reilly, DR et al., ibid., Richardson, CD, ibid.). Subsequent purification of the zsig66 polypeptide from the supernatant can be achieved using the methods described herein. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in relation to this include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for the transformation of S. cerevisiae cells with the exogenous DNA and the production of the recombinant polypeptides thereof are described by, for example, Kawasaki, Patent North American No. 4,599,311; Kawasaki et al., Patent North American No. 4,931,373; Brake, North American Patent No. 4,870,008; Welch et al., North American Patent No. ,037,743; and Murray et al., US Patent No. 4,845,075. Transformed cells are selected by the phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in a medium containing qlucose. Suitable promoters and terminators for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092; ) and the alcohol dehydrogenase genes. See also US Patent 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 mayis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose, are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., US Patent No. 4,935,349. Methods for transforming Acremoni um chrysogenum are described by Sumino et al., US Patent No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as a host for the production of recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in the transformation of P. methanolica will commonly be prepared as double-stranded circular plasmids, which are preferably linearized prior to transformation. For the production of the polypeptide in P. methanolica, it is preferred that the promoter and the terminator in the plasmid be those of a P. methanoli ca gene such as an alcohol utilization gene of P. methanoli ca (AUG1 or AUG2). Other useful promoters include those of dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase genes (CAT). To facilitate integration of the DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A preferred selectable marker for use in Pichia methanolica is an ADE2 gene from P. methanolica, which codes for phosphoro-5-aminoimidazole carboxylase (AIRC); EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale industrial processes where it is desirable to minimize the use of methanol, the use of host cells in which both methanol utilization genes (AUG1 and AUG2) are eliminated is preferred. For the production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into the cells of P. methanoli ca. It is preferred to transform the P. methanoli ca cells by electroporation using a pulsed electric field, which decreases exponentially, having a field strength of 2.5 to 4.5 kV / cm, preferably around 3.75 kV / cm, and a time constant from 1 to 40 milliseconds, more preferably around 20 milliseconds.

Prokaryotic host cells, which include strains of the bacterium Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing the foreign DNA sequences cloned therein are well known in the art (see, for example, Sambrook et al., Ibid.). When a zsigdd polypeptide is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed into the periplasmic space by a bacterial secretion sequence. In the above case, the cells are used, and the granules are recovered and denatured using, for example, guanidino isothiocyanate or urea. The denatured polypeptide can be re-doubled and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of oxidized and reduced glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a functional and soluble form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, thus obviating the need for denaturing and re-bending. The transformed or transfected host cells were 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. The media may also contain such components as growth factors or serum, as required. The growth medium will generally be selected for cells that contain exogenously added DNA, for example, for drug selection or deficiency in an essential nutrient which is complemented by the selectable marker made in the expression vector or co-transfected into the host cell. The P. methanoli ca cells are grown in a medium comprising suitable sources of carbon, nitrogen and trace nutrients at a temperature of about 25 ° C to 35 ° C. Liquid cultures are provided with sufficient aeration by conventional means, such as stirring small flasks or spraying fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto ™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto ™ yeast extract (Difco Laboratories), 0.004% and L-leucine at 0.006%). It is preferred to purify the polypeptides of the present invention to a purity > 80%, more preferably up to a purity > 90%, still more preferably purity > 95%, and particularly preferred is a pharmaceutically pure state, which is greater than 99.9% pure with respect to the contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The expressed recombinant zsigdd polypeptides (which include zsigdd fusion or chimeric polypeptides) can be purified using conventional purification methods and means and / or fractionation. Precipitation with ammonium sulfate and extraction with acid or chaotrope can be used for the fractionation of the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high resolution liquid chromatography. Suitable chromatographic media include dextrans derivatives, agarose, cellulose, polyacrylamide, specialized silicas, and the like. Preferred are PEI, DEAE, QAE and Q derivatives. Exemplary chromatographic media include those media derived with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Mont eryville, PA ), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they are to be used. . These supports can be modified with reactive groups that allow the binding of the proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, and / or carbohydrate moieties. Examples of coupling chemistries include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding the ligand or receptor polypeptides to the support media are well known in the art. The selection of a particular method is a matter of routine design and will be determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, R. (ed.) Protein Purification: Principles and practice, Springer-Verlag, New York, 1987. The polypeptides of the present invention can be isolated by exploiting their structural and biological properties. For example, ion-adsorption chromatography of immobilized metals (IMAC) can be used to purify histidine-rich proteins, including those comprising the polyhistidine tags. Briefly, a first gel is charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem.3: 1-7, 1985). The proteins rich in histidine will be adsorbed to this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, pH decrease, or the use of strong chelating agents. Other purification methods include the purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp. 529-39). Within the additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, the maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. In addition, using the methods described in the art, polypeptide fusions, or hybrid zsigdd proteins, are constructed using regions or domains of zsigdd in combination with those of paralogs, orthologs, or heterologous proteins (Sambrook et al., Ibid. , Altschul et al., Ibid., Picard, D. Cur. Opin. Biology, 5: 511-515, 1994, and references thereof). These methods allow the determination of the biological importance of domains or large regions in a polypeptide of interest. Such hybrids can alter reaction kinetics, binding, constriction or expansion of substrate specificity, or alter the tissue and cellular location of a polypeptide, and can be applied to polypeptides of unknown structure. The fusion polypeptides can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide that encodes one or more components of the fusion protein in the proper reading frame can be generated using techniques known and expressed by the methods described herein. For example, part or all of the domains conferring a biological function can be trapped between the zsígdd of the present invention with the equivalent domains of functionality from another protein. Such domains include but are not limited to the secretory signal sequence, and motifs 1 to 4. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to the polypeptides of the present invention or other similar proteins (e.g., paralogs, or orthologs) or heterologous proteins, depending on the built merger. In addition, such fusion proteins can display other properties as described herein. Standard cloning and molecular biological techniques can be used to generate the equivalent domains between the zsigdd polypeptide and those polypeptides to which they are to be fused. Generally, a DNA segment, which encodes a domain of interest, eg, a zsigdd domain described herein, is operably linked in the structure to at least one other DNA segment encoding an additional polypeptide (e.g., a domain or analog region in a similar protein), and inserted into an appropriate expression vector, as described herein. The DNA constructs are generally made such that several DNA segments encoding the corresponding regions of a polypeptide are operably linked in the structure to make a single construct encoding the complete fusion protein, or a functional portion thereof. For example, a DNA construct would encode from the N-terminus to the C-terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct that would encode from the N-terminus to the C-terminus a fusion protein comprising a signal polypeptide followed by a mature protein. Such fusion proteins can be expressed, isolated, and assayed for activity as described herein. The zsigdd polypeptides or fragments thereof can be prepared through chemical synthesis. The zsigdd polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine residue. The polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing the polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; Kaiser et al., Anal. Biochem. 34: 595, 1970. After complete synthesis of the desired peptide on a solid support, the peptide-resin is reacted with a reagent that cleaves the polypeptide from the resin and removes most of the side chain protecting groups. Such methods are well established in the art. The activity of the molecules of the present invention can be measured using a variety of assays that measure for example, signal transduction, cell motility, steroidogenesis, mitogenesis or binding. Such assays are well known in the art. The zsigdd polypeptides of the present invention can be used to study the proliferation or differentiation of pancreatic cells. Such methods of the present invention generally comprise incubating the a cells, the b cells, the d cells, the F cells and the acinar cells in the presence and absence of the zsigdd polypeptide, the monoclonal antibody, agonist or antagonist thereof and observing the changes in the proliferation or differentiation of the cells in the islets. Similarly, the zsig66 polypeptides of the present invention can be used to study the proliferation or differentiation of pituitary cells. A further aspect of the invention provides a method for studying insulin. Such methods of the present invention comprise incubating the adipocytes in a culture medium comprising the zsigdd polypeptide, the monoclonal antibody, the same insulin agonist or antagonist and observing the changes in the secretion or differentiation of the adipocyte protein. The present invention also provides methods for studying the cellular metabolism of mammals. Such methods of the present invention comprise incubating the cells to be studied, for example, a line of appropriate human cells, ± the zsig66 polypeptide, the monoclonal antibody, the agonist or antagonist thereof, and observing the changes in adipogenesis, gluconeogenesis, lipogenesis, glucose uptake or the like Also, zsig66 polypeptides, agonists or antagonists thereof can be therapeutically useful to promote wound healing. For example, in the pancreas. To verify the presence of their capacity in the zsigdd polypeptides, the agonists or antagonists of the present invention, such zsig66 polypeptides, the agonists or antagonists are evaluated with respect to their ability to facilitate healing of the wounds according to the known procedures in The technique. If desired, the efficiency of the zsigdd polypeptide relative to this can be compared to the growth factor receptors, such as those for EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II, the growth factor of fibroblasts (FGF) and similar. In addition zsigdd polypeptides, agonists or antagonists thereof can be evaluated in combination with one or more growth factors to identify the synergistic effects. In addition, zsigdd polypeptides, agonists or antagonists thereof can be therapeutically useful for anti-microbial applications. To verify the presence of this ability in the zsig66 polypeptides, the agonists or antagonists of the present invention, such zsigdd polypeptides, agonists or antagonists are evaluated for their antimicrobial properties according to procedures known in the art. See for example, Barsum et al., Eur. Respir. J. 8 (5): 709-14, 1995; Sandovsky-Losica et al., J. Med. Vet. Mycol. (England) 28 (4): 279-87, 1990; Mehentee et al., J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, J__ Med. Vet. Mycol. 24: 477-479, 1986 and the like. If desired, the efficiency or functioning of the zsigdd polypeptide in relation to this can be compared to known proteins that are functional in this regard, such as proline-rich proteins, lysozyme, histatins, lactoperoxidases or the like. In addition, zsigdd polypeptides, agonists or antagonists thereof can be evaluated in combination with one or more antimicrobial agents to identify the synergistic effects. Anti-microbial protective agents can act directly or indirectly. Such antimicrobial agents operate via the mechanisms of membrane association or in the formation of pores of action by directly attaching to the offending microbe. The antimicrobial agents can also act via an enzymatic mechanism, breaking the protective substances of the microbes or the walls / membrane of the cells thereof. Antimicrobial agents capable of inhibiting the proliferation or action of microorganisms or of disrupting the integration of the microorganism by means of either the mechanisms established above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that antimicrobial activity . Such techniques involve the cultivation of cells in the presence of an effective amount of said zsig66 polypeptide, or an agonist or antagonist thereof. Also, the zsigdd polypeptides or agonists thereof can be used as reagents in cell cultures in studies in the infection with exogenous microorganisms, such as bacterial, viral or fungal infection. Such portions can be used in animal models of in vivo infection. Also, the adhesion properties to the microorganisms of the zsig66 polypeptides or agonists thereof can be studied under a variety of conditions in binding assays and the like. The proteins of the present invention are useful, for example, in the treatment of diseases in the ovaries, pancreatic, ocular, blood or bone, can be measured in vi tro using cultured cells or in vivo administering molecules of the invention claimed to the appropriate animal model. For example, host cells expressing a secreted form of the zsigdd polypeptide can be embedded in an alginate environment and injected (implanted) into recipient animals. Microencapsulation with alginate-poly-L-lysine, permoselective membrane encapsulation and diffusion chambers are a means to trap the transfected cells of mammals or the cells of major mammals to allow the diffusion of proteins and other macromolecules secreted or released by the cells captured to the receiving animal. More importantly, the capsules are masked and protect the encrusted, foreign cells from the immune response of the recipient animal. Such encapsulations can extend the life of the injected cells from a few hours or days (bare cells) to several weeks (embedded cells). Alginate strands provide a simple and quick means to generate the embedded cells and test, in vivo the proteins secreted from them. The materials necessary to generate the alginate strands are known in the art. In an exemplary procedure, 3% alginate is prepared in sterile H20, and filtered sterile. Just before the preparation of the alginate strands, the alginate solution is filtered again. A suspension of about 50% cells (containing about 5 x 10 5 to about 5 x 10 7 cells / mL) is mixed with the 3% alginate solution. One mL of the alginate / cell suspension is extruded in a filtered, sterile, 100mM CaCl2 solution for a period of about 15 minutes, forming a "strand". The extruded strand is then transferred to a 50 mM CaCl 2 solution and then to a 25 mM CaCl 2 solution. the strand is then rinsed with deionized water before coating the strand by incubation in a 0.01% solution of poly-L-lysine. Finally, the strand is rinsed with Lactated Ringer's Solution and extracted from the solution in a syringe plunger (without needle). A large-hole needle is then attached to the syringe, and the strand is injected intraperitoneally into a recipient in a minimum volume of the lactated Ringer's solution. An in vivo methodology for testing the proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retrovirus, vaccinia virus and adeno-associated virus (AAV). Adenoviruses, a double-stranded DNA virus, is currently the best-studied gene transfer vector for the delivery of heterologous nucleic acid (for review, see Becker et al., Meth Cell Biol. 43: 161-89 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine _4: 44-53, 1997). The adenovirus system offers several advantages: (i) the adenovirus can accommodate the relatively large DNA inserts; (ii) can grow to high titles; (iii) it infects a wide range of mammalian cell types; and (iv) can be used with a large number of different promoters including adjustable, tissue-specific, and ubiquitous promoters. Also, because the adenoviruses are established in the bloodstream, they can be administered by intravenous injection. Using the adenovirus vectors where the portions of the adenovirus genome are removed, the inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential gene has been removed from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When administered intravenously to human animals, the adenovirus has the liver as the main target. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, the host tissue (eg, the liver) will be expressed and processed (and secreted, if a secretory signal sequence is present) the heterologous protein. The secreted proteins will enter the circulation in the highly vascularized liver, and the effects on the infected animal can be determined. In addition, adenoviral vectors containing various deletions of viral genes can be used as an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses have eliminated El and furthermore contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72: 2022-2032, 1998; Raper, SE et al., Human Gene Therapy 9: 671-679). , 1998). In addition, it was reported that the elimination of E2b reduces the immune responses (Amalfitano, A. et al., J. Virol. 72: 926-933, 1998). In addition, by removing the complete adenovirus genome, large inserts of the heterologous DNA can be accommodated. The generation of the so-called "stomach-free" adenoviruses in which all the viral genes have been eliminated are particularly advantageous for the insertion of large inserts of heterologous DNA. For review, see Yeh, P. and Perricaudet, M., FASEB J. 11: 615-623, 1997. The adenovirus system can also be used for the production of in vi tro protein. By culturing the non-293 cells infected with adenovirus under conditions where the cells do not divide rapidly, the cells can produce proteins for extended periods of time. For example, 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 the infected cells to survive for several weeks without significant cell division. Alternatively, 293 cells infected with the adenovirus vector can be grown as adherent cells or in the suspension culture at relatively high densities to produce significant amounts of protein (see Garnier et al., Cytotechnol 1: 5: 145-55 (1994)). )). With any protocol, a secreted heterologous protein, expressed, can be rapidly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the arrangement of the protein expressed in the cells. Within the protocol of the production of infected 293 cells, non-secreted proteins can also be obtained effectively. In view of the observed distribution for zsigdd, agonists (including the natural ligand / substrate / cofactor / etc.) And antagonists have enormous potential in both in vivo and in vi tro applications. For example, zsigdd and agonist compounds are useful as components of the defined cell culture medium, and can be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. The agonists are thus useful in specifically promoting the growth and / or development of the cells in the culture. Considering the expression of zsigdd in the pituitary, zsig66 polypeptides and zsig66 agonists can be particularly useful as research reagents, particularly for the growth of pituitary and endocrine cell types, ovarian cell lines, human eggs, embryonic cells animals or primary cultures derived from these tissues. As such, the zsigdd polypeptide can be provided as a supplement in the cell culture medium. Antagonists are also useful as research reagents for characterizing ligand-receptor interaction sites. Inhibitors of activity of zsig66 (zsig66 antagonists) include anti-zsigdd antibodies and soluble proteins that bind to the zsigdd polypeptide. Inhibitors of zsigdd activity (zsigdd antagonists) include anti-zsig66 antibodies and soluble zsigdd receptors, as well as peptide and non-peptide agents (which include ribozymes). The zsig66 polypeptide can also be used to identify inhibitors (antagonists) of its activity. The test compounds are added to the assays described herein to identify compounds that inhibit zsigdd activity. In addition to those assays described herein, samples can be tested for inhibition of zsigdd activity within a variety of assays designed to measure binding or agglutination, oligomerization, or stimulation / inhibition of zsig66-dependent cellular responses. For example, cell lines expressing zsigdd can be transfected with a reporter gene construct that is responsive to a cell pathway stimulated with zsig66. Reporter gene constructs of this type are known in the art, and generally comprise a zsigdd DNA response element operably linked to a gene encoding the protein detectable in the assay, such as luciferase. DNA response elements may include, but are not limited to, AMP cyclic response elements (CRE), hormone response elements (HRE) insulin response elements (IRE) (Nasrin et al., Proc. Nati. Acad. Sci. USA £ [7: 5273-7, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). The AMP cyclic response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habener, Molec. Endocrinol 4 (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 5_6: 335-44; 1989. Candidate compounds that serve as test samples include solutions, mixtures or extracts, are tested for the response level of the zsig66 polypeptide. The ability of the test sample to inhibit the activity of the zsig66 polypeptide in the target cells is evidenced by a decrease in the zsigdd stimulation of reporter gene expression in the presence of a test sample relative to a control that was cultured in the absence of a test sample. Assays of this type will detect compounds that directly block the zsigdd binding to the cell's surface receptors, for example, dimerization, as well as compounds that block processes in the cell pathway subsequent to the receptor-ligand linkage. Alternatively, compounds or other samples compared to direct blockade of zsigdd, or blocking of zsigdd that binds to other cell surface molecules, which use zsigdd labeled with a detectable label (eg, 125I, biotin, horseradish peroxidase, FITC , and similar). Within assays of this type, the ability of a test sample to inhibit the binding of zsigdd tagged to another protein may be indicative of inhibitory activity, which can be confirmed through secondary assays. The receptors used within the binding assays can be cellular proteins or immobilized proteins, isolated. Alternatively, the above methodology can be used to identify agonists of zsigdd activity. Candidate compounds that serve as test samples include solutions, mixtures or extracts, and are tested for the ability to mimic the activity of the zsigdd polypeptide in the target cells as evidenced by stimulation of reporter gene expression in the presence of a test sample and in the absence of zsig66, relative to a control (cultured in the absence of a test sample and in the absence of a zsig66 polypeptide), using the assays as described above. A zsigdd polypeptide can be expressed as a fusion with a constant region of the immunoglobulin heavy chain, typically an Fc fragment, containing two domains of constant region and lacking the variable region. Methods for preparing such fusions are described in U.S. Patent Nos. 5,155,027 and 5,6567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfides linked together and two non-Ig polypeptides are arranged in close proximity to each other. Fusions of this type can be used for the affinity purification ligand, as an in vitro assay tool, or as a zsigdd antagonist. For use in assays, the chimeras are linked to a support via the Fc region and used in an ELISA format. A zsigdd polypeptide can be used for the purification of the receptor or polypeptides that bind thereto. The zsigdd polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide resins, or similar materials that are stable under the conditions of use. Methods for linking the polypeptides to solid supports are known in the art, and include chemistry with amines, activation by cyanogen bromide, activation by N-hydroxysuccinimide, activation by epoxide, activation by sulfhydryl, and activation by hydrazide. The resulting medium is generally configured in the form of a column, and fractions of the membrane containing the receptors are passed through the column one or more times to allow the receptor to bind to the zsigdd polypeptide of the ligand. The receptor is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl) or pH to break the ligand-receptor bond. A test system that uses the ligand-linker receptor (or an antibody, a member of a complement / anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia, Biosensor, Piscataway, NJ) can be used advantageously. Such a receptor, antibody, member of a complement / anti-complement pair or fragment is immobilized on the surface of a small receptor portion. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, member or fragment is covalently linked, using the chemistry of the amine or sulfhydryl, to the dextran fibers that bind to a gold film inside the cell of flow. A test sample is passed through the cells. If a ligand, epitope, or opposite member of the complement / anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in the surface plasmon resonance of the gold film. This system allows the determination of start and stop speeds, from which the link affinity can be calculated, and the link stoichiometry can be evaluated. The ligand binding receptor polypeptides can also be used within other systems known in the art. Such systems include Scatchard analysis for binding affinity determination (see Scatchard, Ann, NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253: 545-48, 1991; Cunningham et al., Science 245: 821-25, 1991). As a ligand, the activity of the zsig66 polypeptide can be measured by a silicon-based biosensor microphysiometer that measures the ratio of extracellular acidification or proton excretion associated with a binding receptor and subsequent physiological cellular responses. An exemplary device is the Cytosensor ™ Microfiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulator and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257: 1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228: 84-108, 1997; Arimilli, S. et al., A_ Immunol. Meth. 212: 49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346: 87-95, 1998. The microphysiometer can also be used for an adherent or non-adherent assay of eukaryotic or prokaryotic cells. By measuring the changes of extracellular acidification in the cell medium over time, the microphysiometer directly measures the cellular responses to various stimuli, which include the zsig66 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a eukaryotic cell responsive to zsig66, compared to a control eukaryotic cell that does not respond to the zsig66 polypeptide. Eukaryotic cells responsive to zsigdd comprise cells within which a receptor for zsigdd has been transfected creating a cell responsive to zsig66; or cells that respond naturally to zsigdd such as cells derived from pituitary tissue. The differences, measured by a change, for example, an increase or decrease in extracellular acidification, in the response of cells exposed to the zsig66 polypeptide, relative to a control not exposed to zsig66, are a direct measure of the modulated cellular responses by zsig66. In addition, such responses modulated by zsigdd can be tested under a variety of stimuli. Using the microphysiometer, a method is provided for identifying zsig66 polypeptide agonists, which comprises providing cells that respond to a zsigdd 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, for example, an increase or decrease, in a cellular response of the second portion of the cells compared to the first portion of the cells. The change in cellular response is shown as a measurable change in the proportion of extracellular acidification. In addition, culturing a third portion of the cells in the presence of the zsig66 polypeptide and the absence of a test compound can be used as a positive control for cells responsive to zsigdd, and as a control to compare agonist activity of a test compound with that of the zsig66 polypeptide. In addition, using the microphysiometer, a method is provided for identifying antagonists of the zsigdd polypeptide, which comprises providing cells that respond to a zsigdd polypeptide, culturing a first portion of the cells in the presence of zsig66 and in the absence of a zsigdd compound. test, culturing a second portion of the cells in the presence of zsig66 and in the presence of a test compound, and detecting a change, e.g., an increase or decrease in a cellular response of the second portion of the cells compared to the first portion of the cells. The change in cellular response is shown as a proportion of extracellular acidification that is change that can be measured. Antagonists and agonists, for the zsig66 polypeptide, can be quickly identified using this method. In addition, zsigdd can be used to identify cells, tissues, or cell lines that respond to a pathway stimulated by zsigdd. The microphysiometer, described above, can be used to rapidly identify the responsive cells of the ligand, such as the cells responsive to the zsigdd of the present invention. The cells can be cultured in the presence or absence of the zsig66 polypeptide. Those cells that generate a measurable change. in extracellular acidification in the presence of zsig66 are responsive to zsigdd. Such cell lines can be used to identify antagonists and agonists of the zsig66 polypeptide as described above. The polypeptides, the nucleic acid and / or antibodies of the present invention can be used in the treatment of conditions associated with the development of the gonads, pregnancy, changes in puberty, menopause, cancer in the ovary, fertility, ovarian function , polycystic ovarian syndrome, pancreas, diabetes, eye diseases, pituitary function, regulation of blood pressure, water balance, osteoporosis, and other bone diseases. The molecules of the present invention can be used to modulate or treat or prevent the development of pathological conditions in such diverse tissues as the pancreas and ovaries. In particular, certain syndromes or conditions may respond to such diagnoses, treatments or prevention. In addition, natural functions, such as ovulation, can be suppressed or controlled for use in birth control by the molecules of the present invention. For a review of the function and pituitary conditions that the zsigdd can effect or regulate, see Imura, H. (ed.) The Pituitary Gland, Raven Press, New York, 1994. As a polypeptide expressed in the pituitary, the zsig66 polypeptide of the present invention can act in the neuroendocrine / exocrine cellular development decision pathway in the organ systems in the body, and therefore be able to regulate the expansion of the neuroendocrine and exocrine cells in the pancreas. One such regulatory use is that of cell regeneration of the islands. Also, it has been hypothesized that the autoimmunity that triggers the IDDM starts in utero, and the zsigdd polypeptide is a developmental gene involved in cell division. Assays and animal models are known in the art to monitor the decision of the exocrine / neuroendocrine cell line, to observe the equilibrium of the pancreatic cells and to evaluate the zsigdd polypeptide, fragments, fusion proteins, antibodies, agonists or antagonists in the prevention or treatment of the conditions set forth above. The molecules of the present invention will be useful for treating growth disorders, adrenal diseases, hormonal and pubertal diseases, reproductive diseases and cancers, menopause, conditions associated with the production of milk in humans and animals, improved milk production in humans and animals, and pituitary diseases and cancers. The polypeptides, nucleic acids and / or antibodies of the present invention may be useful in the treatment of conditions and complications associated with these conditions. The molecules of the present invention can be used for the function of the pituitary, pituitary hormones, reproductive organs, improvement of fertility, birth control, and the like, or to treat or prevent the development of pathological conditions in such diverse tissue. as pancreas, other endocrine and pituitary tissues. In particular, certain syndromes and conditions may respond to such diagnoses, treatment or prevention. The zsig66 polypeptide is expressed in the pituitary and may have additional biological activity as described above. Oogenesis is the process in which a diploid stem cell proceeds through multiple stages of differentiation, culmination in the formation of a terminally differentiated cell with a unique function, an oocyte. Unlike spermatogenesis, which begins at puberty and continues during the life of a male human being, oogenesis begins during fetal development and by birth, a complete supply in the female primary oocytes is stored in the ovaries in the primordial follicles and await maturation and release. In the adult ovary, folliculogenesis begins when the follicles enter the growth phase. The follicles that grow early suffer a dramatic process of cell proliferation and differentiation. The classical control of ovarian function by luteinizing hormone (LH) and follicle stimulating hormone (FSH) is now thought to include the action of a variety of molecules that act to promote cell-cell interactions between cells of the follicle. For review, see Gougeon, A., Endocrine Rev. 17: 121-155, 1996. Hence, the mechanisms to control folliculogenesis of the ovaries and selection of the dominant follicle are still under investigation. As the zsigdd is expressed in the pituitary, it can serve as a role in the regulation of folliculogenesis and the selection of dominant follicles, affecting the proliferation or differentiation of follicular cells, affecting cell-cell interactions, modulating the hormones involved in the process , and similar.

The cycle of the ovaries in mammals includes the growth and maturation of the follicles, followed by the ovulation and transformation of the follicles in the corpus luteum. The physiological events in the ovarian cycle are dependent on the interactions between hormones and cells within the hypothalamic-pituitary-ovarian axis, which include the gonadotropin-releasing hormone (GnRH), LH, and FSH. In addition, estradiol, synthesized in the follicles, primes the hypothalamic-pituitary axis and is required for the middle cycle of the gonadotropin to arise that stimulates the re-initiation of oocyte meiosis and leads to ovulation and subsequent extrusion of an oocyte. the follicle This increase in gonadotropin also promotes the differentiation of follicle cells from the secretion of estradiol to progesterone. Progesterone, secreted by the corpus luteum, is needed for the uterine development required for the implantation of fertilized oocytes. The central role of hypothalamic-pituitary-gonadal hormones in the ovarian cycle and reproductive cascade, and the role of sex steroids in target tissues and organs, for example, uterus, breast, adipose tissue, bones and liver , has made its modular activity desirable for therapeutic applications. Such applications include treatments for precocious puberty, endometriosis, uterine leiomyomata, hirsutism, infertility, premenstrual syndrome (PMS), amenorrhea, and as contraceptive agents. The zsigdd polypeptides, agonists and antagonists that modulate the actions of such hormones can be of therapeutic value. In addition, zsig66 can act as a hormone by itself, and as such the zsigdd polypeptides, agonists and antagonists can be of therapeutic value. Such molecules may also be useful for modulating steroidogenesis, both in vivo and in vi tro, and modulate aspects of the ovarian cycle such as oocyte maturation, cell-cell interactions, follicular development and rupture, luteal function , and promote uterine implantation of fertilized oocytes. Molecules that modulate hormone action may have therapeutic benefits to be used before or during the development of puberty. For example, puberty in females is marked by an establishment of feedback loops to control hormone levels and the production of hormones. Abnormalities resulting from hormone imbalances during puberty have been observed and include precocious puberty, where changes in puberty occur in females prior to the age of 8. Molecules that modulate hormones can be used in this case, to suppress the secretion of hormones and to delay the onset of puberty. The level and proportion of the steroid hormones and the gonadotropin can be used to assess the existence of hormonal imbalances associated with conditions, as well as to determine if the normal hormonal balance has been restored after the administration of a therapeutic agent. Similarly, the zsigdd level can also be measured in relation to the hormonal imbalances. The determination of estradiol, progesterone, LH, and FSH, for example, of the serum is known to one skilled in the art. Such assays can be used to monitor hormone levels after administration of zsig66 in vivo, or in a transgenic mouse model where the zsigdd gene is expressed or the murine ortholog is removed. Thus, as a hormone modulating molecule, the zsig66 polypeptide may have therapeutic application for the treatment, for example, of early menopausal bleeding, as part of a therapeutic regimen for pregnancy support, or to treat the symptoms associated with the polycystic ovarian syndrome (PCOS), PMS and menopause. further, other in vivo rodent models are known in the art to test the effects of the zsigdd polypeptide on, for example, polycystic ovary syndrome (PCOS). The proteins of the present invention can also be used in applications to improve fertilization during assisted reproduction in humans and animals. Such assisted production methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer, and gamete intrafallopian transfer. Such methods are useful to help those who have had physiological or metabolic disorders that prevent or prevent natural conception. Such methods are also used in breeding programs of animals, for example, for livestock, race horses, domestic and wild animals, and could be used as methods for the creation of transgenic animals. The zsigdd polypeptides could be used in the induction of ovulation, either independently or in conjunction with a gonadotropin regimen or agents such as clomiphene citrate or bromocriptine (Speroff et al., Induction of ovulation, Clinical Gynecologic Endocrinology and Infertility, 5th ed., Baltimore, Williams &; Wilkins, 1994). As such, the proteins of the present invention can be administered to the recipient before fertilization or combined with sperm, an egg or an egg-sperm mixture before in vitro or in vivo fertilization. Such proteins can also be mixed with oocytes before cryopreservation to improve the viability of preserved oocytes for use in assisted reproduction. The zsig66 polypeptides, agonists and antagonists of the present invention can be used directly or incorporated into therapies for the treatment of reproductive disorders. Conditions such as luteal phase deficiency would benefit from such therapy (Soules, "Luteal phase deficiency: A subtle abnormality of ovulation" in, Infertility: Evaluation and Treatment, Keye et al., Eds., Philadelphia, WB Saunders, nineteen ninety five). In addition, the administration of the gonadotropin releasing hormone is shown to stimulate reproductive behavior (Riskin and Moss, Res. Bull. 11: 481-5, 1983; Kadar et al., Physiol. Behav. 51_: 601-5 , 1992 and Silver et al., J. Neuroendocrin 4: 4: 207-10, 199; King and Millar, Cell, Mol. Neurobiol., 15: 5-23, 1995). Given the high prevalence of sexual dysfunction and impotence in humans, molecules, such as zsig66, that can modulate or enhance the activity of gonadotropin may find application in treatments for these conditions. The zsigdd polypeptides of the present invention can be used to study the proliferation, maturation and differentiation of ovarian cells and other reproductive tissues, that is, by acting as a leutinizing agent that converts the granular cells of estradiol to the cells that produce progesterone. . For example, such methods of the present invention generally comprise incubating the granulosa cells, the theca cells, the oocytes or a combination thereof, in the presence and absence of the zsig66 polypeptide, the monoclonal antibody, the agonist and antagonist thereof. and observe changes in the proliferation, maturation and differentiation of cells. See, for example, Basini et al., (J. Rep. Immunol., 37: 139-53, 1998); Duleba et al., (Fert. Ster. 69: 335-40, 1998); and Campbell, B.K. et al., J. Reprod. and Fert. 112: 69-77, 1998). The molecules of the present invention are useful as components of the defined cell culture medium, as described herein, and can be used alone or in combination with other cytokines and hormones to replace the serum that is commonly used in cell culture. The molecules of the present invention are particularly useful in specifically promoting the growth, development, differentiation and / or maturation of ovarian cells in the culture, and may also prove useful in the study of the ovarian cycle, reproductive function, cell-cell interactions. ovarian cell, and fertilization. In addition, zsigdd can affect other lateral hormones of those in the reproductive tissues. In addition, the present invention also provides methods for studying steroidogenesis and secretion of the steroid hormone. Such methods generally comprise incubating the cells that produce the ovary or other hormone-producing cells in the culture medium comprising the zsig66 polypeptides, monoclonal antibodies, agonists or antagonists thereof with or without the gonadotropins and / or steroid hormones, and Subsequently observe the secretion of proteins and steroids. Exemplary gonadotropin hormones include luteinizing hormone and follicle-stimulating hormone (Rouillier et al., Mol. Reprod Dev. 50: 170-7, 1998). Exemplary steroid hormones include estradiol, androstenedione, and progesterone. The effects of zsig66 on steroidogenesis or steroid secretion can be determined by methods known in the art, such as radioimmunoassay (to detect estradiol levels, androstenedione and progesterone, and the like), and the immuno-radiometric assay (IRMA). Molecules expressed in the pituitary, such as zsigdd polypeptide, and which can modulate hormones, hormone receptors, growth factors, or cell-cell interactions, reproductive cascade, other organ systems or the cardiovascular system, or are involved in the oocytes or in the development of the ovaries or other developments, they will be useful as markers for cancer of the reproductive organs and other organ systems and as the therapeutic agents for hormone-dependent cancers, by inhibiting the growth and / or hormone-dependent growth of tumor cells. Cancers of the human reproductive system such as the ovaries, uterus, cervical, testicular and prostate cancer are common. In addition, the receptors for the steroid hormones involved in the reproductive cascade are found in the tumors and in the tumor cell lines (breast, prostate, endometrium, ovary, kidney, and pancreatic tumors) (Kakar et al., Mol. Cell. Endocrinol., 196: 145-49, 1994; Kakar and Jennes, Cancer Letts. , 98: 57-62, 1995). Thus, expression of zsigdd in the pituitary, with concomitant effects on reproductive tissues and other organ systems suggests that the polypeptides of the present invention would be useful in diagnostic methods for the detection and monitoring of reproductive tissues and other cancers . The diagnostic methods of the present invention involve the detection of zsigdd polypeptides in the serum or tissue biopsy of a patient who undergoes function analysis or reproductive evaluation for a possible reproductive or cancer condition or other . Such polypeptides can be detected using the immunoassay and antibody techniques, described herein, which are capable of recognizing the epitopes of the polypeptide. More specifically, the present invention contemplates methods for detecting zsig66 polypeptides comprising: displaying a test sample containing potentially zsigdd polypeptides to an antibody bound to a solid support, wherein said antibody binds to a first epitope of a polypeptide zsig66; wash the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsigdd polypeptide, wherein the second antibody is associated with a detectable tag; and detect the detectable label. Altered levels of the zsig66 polypeptides in a test sample such as serum, sweat, saliva, biopsy, and the like, can be monitored as an indication of reproductive or cancer function or condition, when compared against normal control. The additional methods use probes or primers derived, for example, from the nucleotide sequence described herein can also be used to detect the expression of zsigdd in a patient sample, such as a tumor, stomach, lung, blood, saliva, sample biopsy. of tissue, or similar. For example, the probes can be hybridized to the tumor tissues and the hybridized complex can be detected by hybridization in itself. The zsigdd sequences can also be detected by PCR amplification using the degenerate cDNA by reverse translation of the sample mRNA as a template (PCR Primer A Laboratory Manual, Diffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared to a normal control, both the increase or decrease in the expression of the zsigdd in a sample of the patient, relative to that of a control, can be monitored and used as an indicator or diagnosis of the condition. As a pituitary hormone, zsig66 can regulate blood pressure and water balance (osmotic) in the human body as discussed here, as such, zsigdd, agonists and antagonists have great therapeutic potential in the region of blood pressure . For example, zsig66, agonists and antagonists have a therapeutic potential in improving blood pressure, for example in the case of traumatic shock; or lower blood pressure, for example in the case of hypertension. In addition, water balance is important to maintain electrolytes as well as to affect overall health. As such, the zsigdd, agonists and antagonists have great therapeutic potential in regulating water balance, for example after major organ surgeries, nutritional trauma or conditions of conditions that alter such balance.

In addition, the zsig66 polypeptides can modulate the energy balance in mammals. The zsigdd thyroid expression pattern suggests that zsig66 may show effects on glucose uptake, for example through GLUT-1, and thermogenesis (thermoregulation). Among other methods known in the art or described herein, the energy balance in mammals can be evaluated by monitoring one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, utilization of oxygen or similar. These metabolic functions are monitored by techniques (assays or animal models) known to one of ordinary skill in the art, as will be more fully established below, for example, the glucoregulatory effects of insulin are predominantly exerted on the liver, muscle skeletal and adipose tissue. Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization. In the liver, insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis. In skeletal and adipose muscle tissue, insulin acts to stimulate the taking, storage and utilization of glucose. The zsigßd polypeptide is expressed in the thyroid but may show extrathyroidal activity in organs that affect metabolic functions. A) Yes, the pharmaceutical compositions of the present invention may be useful in the prevention or treatment of pancreatic diseases. For example, zsigdd can be associated with the pathological regulation of the expansion of neurocrine and exocrine cells in the pancreas as evident in IDDM, pancreatic cancer or the like. The pharmaceutical compositions of the present invention may also be involved in the prevention or treatment of pancreatic conditions characterized by dysfunction associated with the pathological regulation of blood glucose levels, insulin resistance or digestive function. There are recognized methods in the art for monitoring all the metabolic functions mentioned above. Thus, one of ordinary skill in the art will be able to evaluate the zsigdd polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists for the functions of metabolic modulation.

Exemplary modulation techniques are established below. Adipogenesis, gluconeogenesis and glycogenolysis are inter-related components of energy balance in mammals, which can be evaluated by known techniques using, for example, the ob / ob mouse or the db / db mouse. The ob / ob mouse is a generated mouse that can be homozygous for an inactivation mutation in the ob (obese) site. Such an ob / ob mouse is hyperphagic and hypometabolic and is thought to be deficient in the production of the circulating OB protein. The db / db mouse is generated so that it is homozygous to inactivate the mutation in the db (diabetes) site. The db / db mouse shows a phenotype similar to that of the ob / ob mouse, except that the db / db mouse shows a more severe diabetic phenotype. Such a db / db mouse is believed to be resistant to the effects of the circulating OB protein. Also, several in vi tro methods for assessing these parameters are known in the art. Insulin-stimulated lipogenesis, for example, can be monitored by measuring the incorporation of l4C-acetate into triglycerides (Mackall et al., J. Biol. Chem. 251: 6462-6464, 1976) or triglyceride accumulation (Kletzien et al. al., Mol.Pharmacol. 41: 393-398, 1992).

Glucose uptake can be evaluated, for example, in a test for insulin-stimulated glucose transport. Differentiated L6, non-transfected myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g / 1 glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After two to five hours of culture, the medium is replaced with fresh, glucose-free DMEM, containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine. Appropriate concentrations of insulin or IGF-1, or a serial dilution of the test substance, are added, and the cells are incubated for 20-30 minutes. Deoxyglucose-labeled with ^ H or L ^ C is added to approximately 1 final concentration of 1 M, and the cells are incubated for approximately 10-30 minutes. The cells are rapidly rinsed with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g., 1% SDS or 1 N NaOH). The cell lysate is then evaluated by accounting in a scintillation counter. The radioactivity associated with the cells is taken as a measure of glucose transport after subtracting non-specific bonds when determined by incubating cells in the presence of cytochalasin b, an inhibitor of glucose transport. Other methods include those described by, for example, Manchester et al., Am. J. Physiol. 266 (Endocrinol Metab 29): E326-E333, 1994 (glucose transport stimulated by insulin). Protein synthesis can be evaluated, for example, by comparing the precipitation of the 35s-methionine-tagged proteins followed by the incubation of the test cells with 35s-methionine and 35s-methionine and a putative modular protein synthesis. Thermogenesis can be evaluated as described by B. Stanley in The Biology of Neuropeptide and Related Peptides, W. Colmers and C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, p. 457-509; C. Billington et al., Am. J. Physiol. 260: R321, 1991; N. Zarjevski et al., Endocrinology A33: 1753, 1993; C. Billington et al., Am. J. Physiol. 2_66: R1765, 1994; Heller et al., Am. J. Physiol. 252 (4 Pt 2): R661-7, 1987; and Heller et al., Am. J. Physiol. 245 (3): R321-8, 1983. Also, the metabolic rate, which can be measured by a variety of techniques, is an indirect measurement of thermogenesis. Oxygen utilization can be evaluated as described by Heller et al., Pflugers Arch 369 (1): 55-9, 1977. This method also involves an analysis of hypothalamic temperature and metabolic heat production. Oxygen utilization and thermoregulation have been evaluated in humans as described by Haskell et al., J. Appl. Physiol. 51 (4): 948-54, 1981. The zsig66 polypeptides of the present invention can act in the decision path of the neuroendocrine / exocrine cells and therefore is able to regulate the expansion of the neuroendocrine and exocrine cells in the pancreas . A regulating use is that of the regeneration of the cells of the islets. Also, the hypothesis has been generated that the autoimmunity generated by the IDDM starts in the uterus and the zsig66 polypeptide is a developmental gene involved in cell partitioning. Assays in animal models are known in the art to monitor the decision of generation of exocrine / neuroendocrine cell lines to observe pancreatic cell balance and to evaluate the zsigdd polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention of the treatment of the conditions established above. Zsig66 polypeptides can also be used to prepare antibodies that specifically bind to zsigdd epitopes, peptides or polypeptides. The zsig66 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and generate an immune response. One skilled in the art will recognize that polypeptides carrying antigenic epitopes contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zsig66 polypeptide (eg, SEQ. ID NO: 2). Polypeptides comprising a larger portion of the zsigdd polypeptide, ie, from 30 to 10 residues up to the full length of the amino acid sequence are included. Immunogenic antigens or epitopes may also include attached labels, adjuvants and carriers, as described herein. Suitable antigens include the zsigdd polypeptide encoded by SEC. ID. NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly), or a fragment of contiguous amino acids 9 to 57, thereof. Other suitable antigens include portions 1 through 4, as described herein. Preferred peptides for use as antigens are the hydrophilic zsig66 peptides such as those predicted by someone skilled in the art from a hydrophobicity plot (See Figure). The hydrophilic zsigdd peptides include the peptides comprising the amino acid sequences selected from the group consisting of: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ. ID NO: 2; (2) amino acid number 7 (Val) to amino acid number 12 (He) of SEC. ID NO: 2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEC. ID NO: 2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of the SEC. ID NO: 2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEC. ID NO: 2. The antibodies of an immune response generated from inoculation with these antigens can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982. As will be apparent to one skilled in the art, antibodies can be generated from the inoculation of a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zsigdd polypeptide or fragment thereof. The immunogenicity of a zsig66 polypeptide can be increased through the use of an adjuvant such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig66 or a portion thereof with an immunoglobulin polypeptide or with the maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like", such a portion can be advantageously linked or bound to a macromolecular carrier (such as limpet closure hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization . As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as the proteolytic fragments F (ab '> 2 and' Pab. Antibodies or intact fragments by genetic engineering, such as chimeric antibodies, Fv fragments, and single chain antibodies and the like, as well as synthetic antigen binding peptides and polypeptides, are also included. can be humanized by grafting non-human CDRs into the human structure and constant regions, or by incorporating complete non-human variable domains (optionally by "wrapping" them with a human-like surface by replacing the exposed residues) , where the result is a "coated" antibody.) In some cases, humanized antibodies can etain non-human residues within the domains of the structure-of the human variable region to improve the appropriate binding characteristics. Through humanized antibodies, the biological half-life can be increased, and the potential for adverse immune reactions after administration to humans is reduced. In addition, human antibodies can be produced in non-human, transgenic animals, which have been engineered by genetic ingestion as described in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.

Alternative techniques for the generation or selection of antibodies useful herein include in vitro exposure of the lymphocytes to the zsig66 protein or peptide, and the selection of libraries that display the antibody in the phage or similar vectors (e.g. of the use of the peptide or protein zsigdd labeled or immobilized). The genes encoding the polypeptides potential zsig66 polypeptide binding domains can be obtained by random selection of the peptide libraries displayed on the phage (phage sample) or on the bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These libraries that display the random peptides can be used to select peptides that interact with a known target which can be a protein or a polypeptide, such as a ligand or a receptor, a synthetic or biological macromolecule, organic or inorganic substances. Techniques for creating and selecting such libraries of random peptide samples are known in the art (Ladner et al., US Patent No. 5,223,409; Ladner et al., U.S. Patent No. 4,946,778; Ladner et al., U.S. Patent No. 5,403,484 and Ladner et al., U.S. Patent No. 5,571,698) and libraries and randomized peptide sample kits for the selection of such libraries are commercially available, for example from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Libraries displaying the random peptide can be selected using the zsig66 sequences described herein to identify proteins that bind to zsigdd. These "binding polypeptides" that interact with the zsigdd polypeptides can be used to label the cells; for isolating the homologous polypeptides by affinity purification; they can be conjugated directly or indirectly to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for the selection of expression libraries and neutralizing activity, for example, for the blocking interaction between the ligand and the receptor, or as a viral link to a receptor. The binding polypeptides can also be used for diagnostic assays for the determination of circulating levels of polypeptides; for the detection or quantification of soluble polypeptides as markers of pathologies or conditions that are being studied. These binding polypeptides can also act as "antagonists" of zsig66 to block zsigdd binding and signal transduction in vi tro and in vivo. These anti-zsig66 binding polypeptides would be useful for inhibiting zsigdd protein or binding activity. The antibodies are considered to bind specifically if: 1) they show a threshold level of binding activity, and 2) they do not significantly cross-react with the molecules of the related polypeptide. A threshold level of binding is determined if the anti-zsigdd antibodies here specifically bind if they bind to a zsig66 polypeptide, peptide or epitope with an affinity at least 10 times greater than that of the binding affinity to the control polypeptide (which it is not zsigdd). It is preferred that the antibodies exhibit a binding affinity (Ka) of 10% or more, preferably 107%, greater, more preferably 10%, greater, and more preferably, 109% or more. of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949) Yes anti-zsigdd antibodies which do not significantly cross-react with the molecules of the polypeptides, for example, if they detect zsigdd but not the known related polypeptides using a standard Western blot or blot analysis (Ausubel et al., ibid.). Examples of polypeptides Known related are those described in the prior art, such as orthologs, and paralogs, and similar known members of a family of proteins, the selection can be made using zsig66 polypeptides mutan tes, and zsigdd nonhuman. In addition, the antibodies can be "separated" against known related polypeptides to isolate a population that specifically binds to the polypeptides of the invention. For example, the antibodies generated with respect to zsig66 are absorbed into the related polypeptides adhered to the insoluble matrix; antibodies specific to zsig66 will flow through the matrix under the proper buffering conditions. Such separation allows the isolation of polyclonal and monoclonal antibodies that do not cross-react with closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). The selection and isolation of antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 4_3: 1-98, 1988; Monoclonal Antibodies: Principies and Practice, Goding, J.W. (eds.), Academic Press, Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically, anti-binding zsig66 antibodies can be detected by a number of methods in the art, and are described below. A variety of assays known to those skilled in the art can be used to detect antibodies that specifically bind to the zsig66 proteins or peptides. 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: countercurrent immunoelectrophoresis, radioimmunoassay, radioimmunoassay, immunosorbent assay enzyme-linked (ELISA), spot or Western blot or dot blot tests, inhibition or competition assays, and sandwich assays. In addition, the antibodies can be selected to bind to the native type against the mutant zsigdd protein or polypeptide. The antibodies to the zsigdd and the zsigdd-binding polypeptides described herein can be used to target the cells expressing the zsigdd; to isolate zsig66 by affinity purification; for diagnostic tests to determine the circulation levels of zsig66 polypeptides; to detect or quantify soluble zsigdd as a marker of the underlying pathology or condition; in the analytical methods that use the FACS; for the selection of expression libraries; to generate the anti-idiotypic antibodies; to detect or quantify soluble zsig66 polypeptides as markers of the underlying pathology or conditions. These antibodies and binding polypeptides can also act as "antagonists" of zsigdd to block the binding of zsig66 and the transduction of the signal in vi tro and in vivo. These polypeptides that bind to the anti-zsig66 would be useful to inhibit the activity of zsig66 or the binding of the protein.

Antibodies to zsig66 can be used for marker cells that express zsigdd; to isolate zsigdd by affinity purification; for diagnostic assays to determine the circulation levels of zsigdd polypeptides; to detect or quantify soluble zsig66 as a marker of pathologies or conditions that are under study; in analytical methods that use FACS; for the selection of expression libraries; for the generation of anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block the activity of zsig66 in vi tro and in vivo. Suitable labels or direct labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect labels or tags can characterize the use of biotin-avidin or other complement / anti-complement pairs as intermediates. The antibodies of the present invention can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates can be used for in vivo diagnosis or therapeutic applications. In addition, antibodies to zsigdd or fragments thereof can be used in vi tro to detect the denatured zsig66 or fragments thereof in assays, eg, Spotted or Western Blot or other assays known in the art. The antibodies or polypeptides described herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates may be used for in vivo diagnosis or therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for example). More specifically, zsig66 polypeptides or anti-zsigdd antibodies, or fragments or bioactive portions thereof, can be coupled to detectable or cytotoxic molecules and can be delivered to mammals having cells, tissues or organs that express the anti-complementary molecule. Suitable detectable molecules can be linked directly or indirectly to the polypeptides or antibodies, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can be linked directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (e.g., diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly bound to the polypeptide or antibody, or indirectly linked by means of a chelating portion, for example). Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect binding of a cytotoxic or detectable molecule, the cytotoxic or detectable molecule can be conjugated with a member of a complementary / anti-complementary pair, wherein the other member is linked to the polypeptide or antibody portion. For these purposes, biotin / streptavidin is a complementary / anticomplementary pair. In another embodiment, the polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for the target cells or tissue of inhibition or ablation (eg, to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus an objective domain), a fusion protein that includes only the target domain may be suitable for targeting a molecule detectable, a cytotoxic molecule or a molecule complementary to a cell or tissue type of interest. In cases where the domain is only a fusion protein and includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic target vehicle for the cell / tissue-specific delivery of conjugates of generic anti-complementary-detectable / cytotoxic molecules. In another embodiment, the zsig66-cytokine fusion proteins or the antibody-cytokine fusion proteins can be used to improve the in vivo death of target tissues (e.g., cancers in the blood and in the bone marrow), if the zsig66 polypeptide or anti-zsig66 antibody targets hyperproliferative blood or bone marrow cells (See, in general, Hornick et al., Blood 89: 4437-47, 1997). It describes fusion proteins that are capable of targeting a cytokine from a desired site of action, thereby providing a high local concentration of cytokine. Suitable zsig66 polypeptides or anti-zsigdd antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and mediated fused cytokine improves target cell lysis by effector cells. Cytokines suitable for this purpose include interleukin 2 and the granulocyte macrophage colony stimulation factor (GM-CSF), for example. Differentiation is a dynamic and progressive process, which starts with the pluripotent stem cells and ends with the terminally differentiated cells. Pluripotent stem cells that can be regenerated without the need to express a line from a set of differentiation markers that are lost when they wish to express a cell line. The progenitor cells express a set of differentiation markers that may or may not be continuously expressed as well as cell progression towards the cell line route directed towards maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cellular products, enzymes that produce cell products, and receptors.

The stage of a differentiation of the cell population is monitored by identifying the markers present in the cell population. Myocytes, osteoblasts, adipocytes, crondrocytes, fibroblasts and reticular cells are considered to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn, Symp 136: 42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87: 731-38, 1987), so that identification is usually done in the stages of mature and progenitor cells . The existence of early-stage cardiac myocyte progenitor cells (often referred to as cardiac myocyte stem cells) has been speculated, but not demonstrated, in adult cardiac tissue. The novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and cardiac myocyte progenitor cells, both in vivo and ex vivo. There is evidence to suggest that the factors that stimulate specific cell types towards terminal differentiation or dedifferentiation affect the entire population of cells that originate from a common precursor or stem cell. Thus, the present invention includes the stimulation or inhibition of the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, crondrocytes and endothelial cells. The molecules of the present invention can, while stimulating the proliferation or differentiation of cardiac myocytes, inhibit the proliferation or differentiation of adipocytes, because they affect their common precursor / stem cells. Thus the molecules of the present invention can have a use in the inhibition of chondrosarcomas, atherosclerosis, restenosis and obesity. Tests that measure differentiation include, for example, the measurement of cell surface markers associated with the specific expression of tissue stages, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5: 281- 284, 1991; Francis, Differentiation 5: 7: 63-75, 1994; Raes, Adv., Cell Biol. Technol. Bioprocesses, 161-171, 1989, all are incorporated herein by reference). Alternatively, the zsigdd polypeptide itself can serve as an additional cell surface associated with the secreted marker associated with the specific expression of the stage of a tissue. As such, direct measurement of the zsig66 polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for tissue differentiation. Similarly, direct measurement of the zsig66 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression. Increases in cell invasion and motility or gain or loss of zsig66 expression in a pre-cancerous or cancerous condition, compared to normal tissue, can serve as a diagnosis for transformation, invasion and metastasis in progression of the tumor. As such, knowledge of the tumor stage of progression or metastasis will help the physician choose the most appropriate therapy, or the aggressiveness of the treatment for a given individual cancer patient. Methods of measuring gain and loss of expression (either mRNA or protein) are well known in the art and are described herein and can be applied to the expression of zsig66. For example, the appearance or disappearance of the polypeptides that regulate cell motility can be used to aid in the diagnosis and prognosis of prostate cancer (Banyard, J and Zetter, BR, Cancer and Metast, Rev. 17: 449-458, 1999).

As an effector of cell motility, the gain or loss of zsig66 expression can serve as a diagnosis for the prostate and other cancers. In addition, the activity and effect of zsigdd on tumor progression and metastasis can be measured in vivo. Several models of syngeneic mice have been developed to study the influence of polypeptides, compounds or other treatments in the progression of the tumor. In these models, the tumor cells passing in the culture are implanted in the mice of the same strain as the tumor donor. The cells will develop into tumors that have similar characteristics in the recipient mice, and metastasis will also occur in some of the models. The appropriate tumor models for our studies include Lewis lung carcinoma (ATCC No. CRL-1642) melanoma B16 (ATCC No. CRL-6323), among others. These are the most commonly used tumor lines, syngeneic to the C57BL6 mouse, which are easily cultured and manipulated in vi tro. Tumors that result from the implantation of any of these cell lines are capable of metastasis to the lung in the C57BL6 mouse. The Lewis lung carcinoma model in mice has recently been used to identify an inhibitor of angiogenesis (O'Reilly MS, et al., Cell 79: 315-328, 1994). C57BL6 / J mice are treated with an experimental agent either through the daily injection of the recombinant protein, agonist or antagonist or a simultaneous injection of the recombinant adenovirus. Three days after this treatment, 105 to 106 cells are implanted under the dorsal skin. Alternatively, the cells themselves can be infected with the recombinant adenovirus such as one that expresses zsig66, prior to implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically. Mice usually develop visible tumors in 5 days. Tumors are allowed to grow for a period of up to 3 weeks, during which time they can reach a size of 1500 - 1800 mm3 in the treated control group. The size of the tumor and the body weight are monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed together with the lungs and liver. It has been shown that the weight of the lung correlates quite well with the weight of the metastatic tumor. As an additional measure, the lung surface metastases were counted. The dissected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and hybridization in itself, using methods known in the art and described herein. The influence of the polypeptide expressed in question, for example, zsigdd, on the ability of the tumor to generate the vasculature and undergo metastasis can thus be assessed. In addition, along with the use of the adenovirus, the implanted cells can be transiently transfected with the zsig66. The use of stable zsigdd transfectants as well as the use of inducible promoters to activate in vivo expression of zsigdd are known in the art and can be used in this system to assess the zsigdd induction of metastasis. In addition, the conditioned medium of purified zsig66 or zsigdd can be injected directly into this mouse model, and hence can be used in this system. For general reference see, O'Reilly MS, et al., Cell 79: 315-328, 1994; and Ruscianno D. et al., Murine Models of Liver Metastasis. Metastasis Invasion 14: 349-361, 1995. In yet another embodiment, if the zsig66 polypeptide or the anti-zsig66 antibody targets vascular cells or tissues, such polypeptide can be conjugated to a radionuclide, and particularly to a beta-radionuclide. emitter to reduce restenosis. Such a therapeutic methodology has less danger for the doctors who administer the radioactive therapy. For example, strips impregnated with iridium-192 placed in the blood vessels with surgical grafts of patients until the required dose of radiation was delivered showed a decrease in tissue growth in the blood vessel and a larger luminal diameter than the group of control, which received the strips with placebo. In addition, revascularization and thrombosis in the surgical graft were significantly lower in the treatment group. Similar results are predicted with the objective condition of a bioactive conjugate containing a radionuclide, as described herein. The bioactive polypeptide or the antibody conjugates described herein may be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. The molecules of the present invention can be used to identify and isolate the receptors that bind to zsig66. For example, the proteins and peptides of the present invention can be immobilized on a column and membrane preparations and run on the column (Immobilized Affinity League Techniques, Hermanson et al., Eds., Academic Press, San Diego, CA, 1992, pp. 195-202). Proteins and peptides can also be radiolabelled (Methods in Enzymol., Vol.182, "Guide to Protein Purification", M. Deutscher ed., Acad. Press, San Diego, 1990, 721-37) or labeled by photoaffinity (Brunner et al., Ann. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem Pharmacol. 3_3: 1167-80, 1984) and specific cell surface proteins can be identified. The molecules of the present invention, although expressed in the pituitary, can exert their effects on either side and hence can be useful for treating diabetes, and pancreatic cancer. The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the treatment of conditions and complications associated with these conditions. The molecules of the present invention can be used to modulate insulin, glucagon, and the like, or to treat or prevent the development of pathological conditions in various tissues such as the pancreas and other endocrine tissues. In particular, certain syndromes and conditions can be treated or considered as objectives for such diagnoses, treatment or prevention. Since zsig66 appears to have a similarity to vasopressin and oxytocin, zsig66 could be useful as a modulator of blood pressure, muscle tension, and / or osmotic balance. For example, the modification of blood pressure in situations such as heart attacks, stroke, traumatic shock, surgery, and any number of bleeding complications. As a modulator of blood pressure, muscle tension or / and osmotic balance can modulate the contractility in the organ and tissue systems where it has an effect. Thus, the activity of the molecules of the present invention can be measured using a variety of assays that measure cell contractility. Such assays are well known in the art. As zsig66 is structurally related to vasopressin, it can be used to modulate the tissues that contract. For example, the contractile tissues on which the zsig66 can act include the skeletal muscle, the uterus, tissues in the testes, for example, the vas deferens; the tissues of the prostate; the gastrointestinal tissues, for example the colon and small intestine; and the heart. The effects of zsigdd polypeptide, its antagonists and agonists, on tissue contractility can be measured in vi tro using a tensiometer with or without electrical field stimulation.

Such assays are well known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ileum, samples of uterine tissues and other contractile tissues, as well as to organ systems, such as the atrium, and they can be used to determine whether the zsigdd polypeptide, its agonists or antagonists, improve or decrease contractility. The molecules of the present invention are useful herein for treating dysfunction associated with contractile tissues or can be used to improve or suppress contractility in vivo. As such, the molecules of the present invention have utility in the treatment of cardiovascular diseases, infertility, in vitro fertilization, birth control, treatment of impotence or other reproductive dysfunctions of men, as well as induced birth. The effect of zsig66 polypeptides, antagonists and agonists of the present invention on tissue contractility include the uterus, prostate, testes, gastrointestinal tissues, and heart can be measured on a tensiometer that measures contractility and relaxation in tissues. See, Dainity et al., J. Pharmacol. 100: 767, 1990; Rhee et al., Neurotox. 16: 179, 1995; Anderson, M.B., Endocrinol. H4: 364-368, 1984; and Downing, S.J. and Sherwood, O.D. Endocrinol 116: 1206-1214, 1985. For example, the measurement of vasodilatation of the aortic rings is well known in the art. Briefly, the aortic rings are taken from 4-month-old Sprague-Dawley rats and placed in a buffer solution, such as a modified Krebs solution (118.5 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO4.7H20, KH2P04 1.2 mM, 2.5 mM CaCl2.2H20, 24.8 mM NaHCO3 and 10 mM glucose). One skilled in the art will recognize that this method can be used with other animals, such as rabbits, other strains of rats, guinea pigs, and the like. The rings are then joined to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data is recorded with a Ponemah physiological platform (Gould Instrument Systems, Inc., Valley, View, OH) and placed in a low of oxygenated tissue (02 to 95%, C02 to 5%) that contains the buffer solution. The tissues are adjusted to a stress of 1 gram and allowed to stabilize approximately for one hour before the test. The integrity of the rings can be tested with norepineferin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After the integrity is verified, the rings are washed three times with fresh buffer and left to rest for about an hour. To test a sample for vasodilation, or tissue relaxation of the aortic rings, the rings contract to two grams of tension and are allowed to stabilize for fifteen minutes. A sample of the zsigdd polypeptide is then added to 1, 2 or 3 of the 4 baths, without washing, and the tension in the rings is recorded and compared with the control rings containing only buffer. The improvement or relaxation of contractility by zsigdd polypeptides, their agonists and antagonists is measured directly by this method, and can be applied to other contractile tissues such as the uterus, prostate, and testes. The activity of the molecules of the present invention can be measured using a variety of assays that measure the stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and / or secretion of digestive enzymes. Of particular interest are changes in contractility of smooth muscle cells. For example, the contractile response of the mammalian duodenum segments to their other gastrointestinal smooth muscle tissue (Depoortere et al., J. Gastrointestinal Motility _1: 150-159, 1989, incorporated herein by reference). An exemplary in vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between the commissures and the longitudinal portion to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60: S384-390, 1995). Gastric motility is generally measured in the clinical specifications as required for gastric emptying time and subsequent transit time to the gastrointestinal tract. Gastric emptying scans are well known to those skilled in the art, and briefly, comprise the use of an oral contrast agent, such as barium, or a radioactive food. Solids and liquids can be measured independently. A food or test liquid is radiolabelled with an isotope (eg 99Tc), and after ingestion or administration, the transit time through the gastrointestinal tract and gastric emptying is measured by visualization using the gamma cameras (Meyer et al. al., Am. J. Dig. Dis. 21: 296, 1976; Collins et al., Gut 24: 1117, 1983; Maughan et al., Diabet. Med. 13 9 Sup. 5.-S6-10, 1996 and Horowitz et al., Arch. Intern. Med. 145: 1467-1472, 1985). These studies can be performed before and after the administration of a PRO-motility agent to quantify the efficiency of the drug. The polypeptides, antagonists, nucleic acid agonists and / or antibodies of the present invention can also be used in the treatment of conditions associated with the contractility of gastrointestinal cells, the secretion of enzymes and digestive acids, gastrointestinal motility, the whole or recruitment of digestive enzymes; inflammation, particularly when it affects the gastrointestinal system, the condition of reflux and the regulation of absorption of nutrients. Specific conditions that will benefit from treatment with the molecules of the present invention include, but are not limited to, diabetic gastroparesis, post-surgical gastroparesis, vagotomy, idiopathic, chronic intestinal pseudo-obstruction, and gastroesophageal reflux disease. Additional uses include, gastric emptying for radiological studies, stimulation of contraction and bladder antrectomy. The motor and neurological effects of the molecules of the present invention make them useful for the treatment of obesity and other metabolic diseases where neurological feedback modulates nutritional absorption. The molecules of the present invention are useful for regulating satiety, glucose uptake and metabolism, and gastrointestinal conditions associated with neuropathy. The molecules of the present invention are also useful as additives for glucose-containing anti-hypoglycemic preparations and as adsorption enhancers for oral drugs that require rapid action of the nutrient. Additionally, the molecules of the present invention can be used to stimulate insulin release induced with glucose. The polynucleotides encoding zsigdd polypeptides are useful within the applications of gene therapy where it is desired to increase or inhibit the activity of zsigdd. If a mammal has a mutated or absent zsig66 gene, the zsig66 gene can be introduced into mammalian cells. In one embodiment, a gene encoding a zsigdd polypeptide is introduced in vivo into a viral vector. Such vectors include a defective or attenuated DNA virus, such as, but not limited to, herpes simplex virus (HSV); papillomavirus, Epstein Barr virus (EBV), adenovirus, retrovirus, adeno-associated virus (AAV), and the like. Defective viruses, which lack almost entirely or entirely viral genes, are preferred. A defective virus is not ineffective after introduction to the cell. The use of defective viral vectors allows 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 herpes simplex virus 1 vector (HSV1) (Kaplitt et al., Molec., Cell Neurosci.2: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987; Salmulski et al., J. Virol. 63: 3822-8, 1989). In another embodiment, a zsigdd gene can be introduced into a retroviral vector, for example, as described in 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., J. Virol. 6 ^ 2: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al .; and Kuo et al., Blood 82: 845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc Nati Acad Sci USA 84: 7413-7, 1987; Mackey et al., Proc. Nati Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The molecular objective of liposomes for specific cells represents an area of benefit. More particularly, the direction of transfection to particular cells represents an area of benefit. For example, the direction of transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidneys and brain. Lipids can be chemically coupled to other molecules for the purpose of becoming objective. Peptides that have become objective (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be chemically coupled to the liposomes.

It is possible to eliminate the target cells of the body; introduce the vector as a naked DNA plasmid; and then re-implant the transformed cells in the body. The naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, the use of a genetic pistol or the use of a DNA vector transporter. See, for example, Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263-14621-4, 1988. The antisense methodology can be used to inhibit the transcription of the zsig66 gene, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of the zsigdd-encoding polynucleotide (eg, a polynucleotide as set forth in SEQ ID NO: 1) are designed to bind to mRNA encoding zsigdd and to inhibit the translation of such mRNA. . Such antisense polynucleotides are used to inhibit the expression of the genes encoding the zsig66 polypeptide in the cell culture or in a subject.

The present invention also provides reagents that will find use in diagnostic applications. For example, the zsigdd gene, a probe comprising zsigdd DNA or RNA, or a subsequence thereof, can be used to determine whether the zsigdd gene is present on the chromosome or whether a mutation has occurred. Detectable chromosomal aberrations at the zsig66 gene site include, but are not limited to aneuploids, changes in gene copy number, insertions, deletions, changes in the restriction site, and rearrangements. Such aberrations can be detected using the polynucleotides of the present invention using molecular genetic techniques, such as restriction fragment length polymorphism analysis (RFLP), in-fluorescence hybridization methods, repetitive analysis in short series (STR) that employs PCR techniques, and other genetic binding techniques, known in the art (Sambrook et al., Ibid., Ausubel, et al., Ibid.; Marian, AJ, Chest, 108: 255-265, 1995 ). The formation of the hybrid map by radiation is a genetic technique of somatic cells, developed for the construction of contiguous high-resolution maps of mammalian chromosomes (Cox et al., Science 250: 245-50, 1990). The partial or complete knowledge of a gene sequence allows someone to design the PCR primers suitable for use with panels for hybrid mapping by chromosomal radiation. Panels for hybrid radiation mapping are commercially available and cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels allow rapid chromosomal localizations based on PCR and the ordering of genes, labeled sites of the sequence (STSs), and other polymorphic and non-polymorphic markers within a region of interest. This includes the establishment of directly proportional physical distances between the newly discovered genes of interest and the markers previously mapped. Accurate knowledge of the position of the gene can be useful for a number of purposes including: 1) determining whether a sequence is part of an existing contiguous or contiguous and obtaining additional genetic sequences that are surrounded in various ways, such as YACs clones, BACs or cDNA; 2) provide a possible candidate gene for a heritable condition which shows binding to the same chromosomal region; 3) cross-reference model organisms, such as the mouse, which can help determine what function a particular gene might have. The sites labeled of the sequence (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that use a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on the DNA sequence they can be fully described within an electronic database, for example, the Database of Tagged Sequence Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of health, Bethesda, MD http://www.ncbi.nlm.nih.gov) , and can be searched with a sequence of genes of interest for the map formation data contained within these STS sequences of short genomic tags.

The zsig66 gene can be used to detect the genes of the known function map for the same region of the human chromosome. In addition, probes of the zsig66 polynucleotide can be used to detect abnormalities or genotypes associated with human disease states as well as the map for the same region of the human genome. See the map of the Online Gene Medellian Inheritance of Man (OMIM), publicly available on a network server (WWW) (http://www3.ncbi.nlm.nih.gov/htbin-post/0mim). Candidate genes for a heritable condition that show the link to the same chromosomal region as the zsigdd gene can be identified by this. The zsig66 gene is located in the 4q28-q31 region of chromosome 4 (Example 3). Several known genes form maps for this place and are associated with the states of human suffering: the clustering of the alpha, beta and gamma genes of fibrinogen in 4q28, and mutations in it are associated with conditions such as dysfibrinogenemia, hypofibrinogenemia , and fibrinogenemia which result in malfunction in blood coagulation and other complications (See, for example, Berg, K and Keirulf, P. Clin Genet, 36: 229-235, 1989). Probes of the zsig66 polynucleotide can be used to detect abnormalities or genotypes associated with these markers associated with fibrinogen. In addition, the zsig66 polynucleotide probes can be used to detect abnormalities or genotypes associated with deletions and translocations of chromosome 4q28-q31 associated with human conditions, such as Reiger's syndrome (deletion in 4q); loss of heterozygosity, or translocation between 4q28-q31 and another chromosome; the translocations involved with malignant progressions of tumors; or mutations, which are expected to be involved in rearrangements of chromosomes in malignancies; or eliminations and translocations in other cancers. Similarly, the probes of the zsig66 polynucleotide to detect the abnormalities or genotypes associated with the trisomy of chromosome 4q28-q31 and the loss of chromosomes associated with human conditions such as Reiger's syndrome (above). In addition, among other genetic sites, those for type I autosomal dominant pseudohypoaldosteroidism (PHA) (4q31.1), mesenchymal dysgenesis of the anterior segment and Reiger syndrome (4q28-q31), the susceptibility of hepatocellular carcinoma associated with the Hepatitis B virus (4q32.1), and others, all manifest themselves in states of human suffering as well as for the map of this region of the human genome. See the gene map in Online Medellian Inheritance of Man (OMIM), and references therein, for this region of chromosome 4 on a publicly available WWW server (http://www3.ncbi.nlm.nih.gov/htbin -post / Omim / getmap? -chromosome = 4q28, and the surrounding regions through 4q31). All these serve as possible candidate genes for a heritable condition that shows the link to the same chromosomal region as the zsigdd gene. Thus, the probes of the zsig66 polynucleotide can be used to detect abnormalities or genotypes associated with these defects. Similarly, defects at the zsigdd site itself can result in a heritable human condition. As a pituitary hormone, the defects of the zsigdd molecules of the present invention can have far-reaching effects on the human body. The molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention could aid in the detection, prevention of diagnosis, and treatment associated with a genetic defect of zsig66. A diagnosis could help doctors determine the type of genetic condition and appropriate associated therapy, or assistance in genetic counseling. As such, inventive anti-zsig66 antibodies, polynucleotides and polypeptides can be used for the detection of zsigdd polypeptide, mRNA or anti-zsigdd antibodies, thus serving as markers and can be used directly to detect or diagnose known conditions or that still they will be discovered, or cancers, as described herein, using methods known in the art and described herein. In addition, probes of the zsigdd polynucleotide can be used to detect allelic differences between individuals suffering or without suffering at the chromosomal site of zsig66. As such, the zsigdd sequences can be used as diagnostics in the formation of the fornsic DNA profile. Such profile formation can be applied to commercial animals as well as for use in breeding programs. In general, diagnostic methods used in genetic linkage analysis to detect an abnormality or genetic aberration in a patient are known in the art. Most diagnostic methods include the steps of (a) obtaining a genetic sample from a potentially ill patient, the sick patient or the potential carrier without disease of an allele of recessive disease; (b) producing a first reaction product by incubating the genetic sample with a ZSMFld polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence, such as in the RFLP analysis or by incubating the genetic sample with the primers in the 5 'and 3' direction in a PCR reaction under appropriate PCR reaction conditions, (iii) Visualize the first reaction product by gel electrophoresis and / or other known method such as the visualization of the first reaction product with a ZSMFld polynucleotide probe wherein the polynucleotide will hybridize to the polynucleotide sequence complementary to the first reaction; and (iv) comparing the first reaction product, visualized, to a second control reaction product of a genetic sample of a natural type patient. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the individual with a condition or in the individual with a potential condition, or the presence of a heterozygous recessive carrier phenotype for an individual without a condition , or the presence of a genetic defect in a tumor of an individual with a condition, or the presence of a genetic abnormality in a pre-implanted fetus or embryo. For example, a difference in the pattern of the restriction fragment, the length of the PCR products, the length of the repetitive sequences in the genetic site ZSIG66, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference compared to control of the normal natural or native type. Controls can be from unaffected family members, or unrelated individuals, depending on the testing and availability of samples. Genetic samples for use within the present invention include genomic DNA, mRNA, and the isolated form of cDNA from any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells. , amniotic fluid, and the like. The probe or primer of the polynucleotide can be RNA or DNA, and will comprise a portion of the SEC. ID NO: 1, the complement of the SEC. ID NO: 1, or an RNA equivalent thereof. Such methods to show the analysis of genetic linkage to the phenotypes are well known in the art. For reference to PCR-based methods in diagnostics see, in general, Mathew (ed.), Protocols in Human Molecular Genetics (Human Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applica tions (Human Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Human Press, Inc. 1996), (Human Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols ( Human Press, Inc. 1998), Clinical Applications of PCR (Human Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Human Press, Inc. 1998). The aberrations associated with the Zsig66 site can be detected using nucleic acid molecules of the present invention employing molecular genetic techniques, such as restriction fragment length polymorphism analysis, PCR techniques employing the analysis of the refractory mutation system of amplification, detection of single-strand conformation polymorphism, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108: 255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Labora tory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al. , (eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al. (eds.), Curren t Protocols in Human Genetics (John Wiley &Sons 1998), and Richards and Ward, "Molecular Diagnostic Testing," in Principies of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998) ). Direct analysis of a zsig66 gene for a mutation can be performed using the genomic DNA of a subject. Methods for amplifying genomic DNA, obtained for example from peripheral blood lymphocytes, are well known to those skilled in the art (see, for example, by Dracopoli et al. (Eds.), Current Protocols in Human Genetics, on pages 7.1.6 to 7.1.7 (John Wiley &Sons 1998). Mice genetically engineered to express the zsigdd gene, referred to as "transgenic mice", and mice showing a complete absence of the function of the zsig66 gene, referred to as "aghenic mice", can also be generated (Snouwaert et al., Science 257: 1083, 1992; Lowell et al., Nature 366: 740-42, 1993; Capecchi, MR, Science 244: 1288- 1292, 1989; Palmiter, RD et al., Annu., Rev. Genet, 20: 465-499, 1986.) For example, transgenic mice overexpressing zsigdd, either anywhere or under a tissue-restricted or specific promoter. of the tissue can be used to ask whether overexpression causes a phenotype For example, overexpression of a native-type zsig66 polypeptide, the polypeptide fragment or a mutant thereof can alter normal cellular processes, resulting in a phenotype that identifies a tissue in which the expression of zsigdd is functionally relevant and it may indicate a therapeutic goal for zsigdd, its agonists or antagonists. For example, a preferred transgenic mouse to generate genetic engineering is one that overexpresses the mature zsigdd polypeptide (approximately amino acids 28 (Ala) to 84 (Gly) of the SEC. ID NO: 2). In addition, such overexpression can result in a phenotype that shows similarity with human conditions. Similarly, the mouse blocked with zsig66 can be used to determine if zsig66 is absolutely required in vivo. The phenotype of the blocked mouse is predictive of the in vivo effects of those that may have a zsig66 antagonist, such as those described herein. The cDNA of human zsig66 can be used to isolate genomic DNA, cDNA, and murine zsig66 mRNA, which is subsequently used to generate a blocked mouse. These mice can be used to study the zsig66 gene and the protein encoded therefor in an in vivo system, and can be used as in vivo models for the corresponding human diseases. In addition, expression of the transgenic mouse of the antisense zsig66 polynucleotides or ribozymes directed against the zsigdd, described herein, can be used analogously to the blocked mouse described above. For pharmaceutical use, the proteins of the present invention are formulated for parenteral delivery, particularly intravenous or subcutaneous, according to conventional methods. Intravenous administration will be by bolus injection or infusion during a typical period of one to several hours. In general, the pharmaceutical formulations will include a zsigdd polypeptide in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like. The formulations may also include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent the loss of proteins on the surfaces of the vials, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed. , 1995. Therapeutic doses will generally be determined in the range of 0.1 to 100 μg / kg of the patient's weight per day, preferably 0.5-20 g / kg per day, with the exact dose determined by the doctor according to accepted standards , taking into account the nature and severity of the condition that is going to be treated, the characteristics of the patient, etc. The determination of the dose is within the level of ordinary skill in the art. The proteins can be administered for an acute treatment, for a week or less, often for a period of one to three days or they can be used in the treatment of chronic diseases, for several months or years. The invention will be further illustrated by the following non-limiting examples.

EXAMPLES Example 1 Identification of the zsigdd A. Using an EST Sequence to Obtain the Full-length zsigdd The scan of a database of the translated cDNA library using a signal trap as a question resulting in an identification of a sequence of the mark of the expressed sequence (EST) of a pituitary library that was found to be homologous to a human secretory signal sequence. Confirmation of the EST sequence was made by sequence analysis of the cDNA from which the EST originated. this cDNA was contained in a plasmid of a human pituitary library, and sequenced using the following primers to generate the complete double-stranded sequence of this clone: ZC6,768 (SEQ ID NO: 12), ZC20,137 (SEQ. ID NO: 13), ZC 20,136 (SEQ ID NO: 14), ZC 20,132 (SEQ ID NO: 15), ZC 20,134 (SEQ ID NO: 16), ZC 19,964 (SEQ ID NO : 17), ZC19,965 (SEQ ID NO: 18), ZC694 (SEQ ID NO: 19).

Example 2 Tissue Distribution Analysis was performed by spotting or Northern blotting using Multi-Human Tissue Staining (MTN I, MTN II, and MTN III) (Clontech). The cDNA described in Example 1 was used in a PCR reaction using oligos ZC21,336 (SEQ ID NO: 20) and ZC21,338 (SEQ ID NO: 21) as primers. The PCR conditions were as follows: 94 ° C for 1.5 minutes; 35 cycles at 94 ° C for 15 seconds, 66 ° C for 30 seconds; 72 ° C for 30 seconds, one cycle at 72 ° C for 5 minutes, followed by a retention at 4 ° C. A sample of the PCR reaction product was run on a 4% agarose gel, a band of the expected size of approximately 220 bp was observed. The 220 bp PCR fragment was purified using commercially available equipment (QiaexII ™; Qiagen) and then radiolabelled with 3 P-dCTP using Rediprime ™ (Amersham), a random primer labeling system, in accordance with manufacturer's specifications. The probe was then purified using a Nuc-Trap ™ column (Stratagene), according to the manufacturer's specifications. The ExpressHyb ™ solution (Clontech) was used for the prehybridization and as a hybridization solution the Northern blots were used. Hybridization took place overnight at 55 ° C using 1-2 x 10 cpm / ml of the labeled probe. The spots were then washed for 30 minutes in 2X SSC / 1% SDS at room temperature, followed by a wash in 2X SSC / 0.1% SDS at 65 ° C, followed by a wash in 0.1X SSC / 0.1% SDS. at 65 ° C. The stains were exposed 7 days. No signals of the transcript were observed in the tissues represented in the stained. In addition, a produced, stored Northern blot containing the RNA of the pituitary gland was also probed, following the methods and conditions described above. Again, no signals were observed, suggesting that mRNA for zsigdd is relatively short and / or expressed in only specific physiological conditions. Spot Spotting was also performed using RNA in Master Blots ™ (Clontech). The methods and conditions for Spot Spotting are the same as for the Spot Tissues described above. A signal was observed in the genomic DNA.

Example 3 Assignment and Placement Chromosome of the zsigdd Gene The zsigdd map for chromosome 4 was formed using the commercially available version of the "Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc., Huntsville, AL). The "Stanford G3 RH Panel" contains the DNA of each of the 83 hybrid clones by radiation of the entire human genome, plus two control DNA (the MR donor and the A3 receiver). A publicly available network server (WWW) (http://shgc-www.stanford.edu) allows the chromosomal location of the markers For the formation of the zsig66 map with the "Stanford G3 RH Panel", 20 μl reactions were performed in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a thermal cycle former "RoboCycler Gradient 96" (Stratagene). Each of the 85 PCR reactions consists of 2 μl of the KlenTaq 10X PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 μl of dNTPs mixture (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl of the sense primer (address 5 '), ZC21,450 (SEQ ID NO: 22), 1 μl of the antisense primer (3'), ZC21,451 (SEQ ID NO: 23), 2 μl of "RediLoad" (Research Genetics , Inc. Huntsville, AL), 0.4 μl of Advantage KlenTaq 50X Polymerase Blend (Clontech Laboratories, Inc.), 25 ng of DNA from a single hybrid clone or control and ddH2? for a total volume of 20 μl. The reactions were carried out with an equal amount of mineral oil and sealed. The conditions of the PCR cycle former were as follows: 1 initial cycle of 5 minutes of denaturation at 94 ° C, 35 cycles of a denaturation of 45 seconds at 94 ° C, 45 seconds of heating and cooling at 60 ° C and 1 minute and 15 seconds extension at 72 ° C; followed by 1 final 7-minute extension cycle at 72 ° C. The reactions were separated by electrophoresis on a 2% agarose gel (Life 'Technologies, Gaithersburg, MD). The results showed that the binding of zsigdd to the marker SHGC-57096 of chromosome 4 with an LOD record of > 11 and at a distance of 0 cR_10000 from the marker. The use of surrounding genes, which have been cytogenetically mapped, places the zsigdd in the chromosomal region 4q28-q31. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > ZymoGenetics. Inc. < 120 > Pituitary polypeptide zsig66 < 130 > 98-81 < 150 > US 09 / 212,947 < 151 > 1998-12-16 < 160 > 24 < 170 > FastSEQ for Windows Version 3.0 < 210 1 < 211 > 2375 < 212 > DNA < 213 Homo Sapiens < 220 > < 221 > CDS < 222 > (224) ... (475) < 400 > 1 gttgcccagt ctggtctcaa actcctaggc tcaagtgacc ctcccacttc gacctcccaa 60 agtgctggga ttacaggtgt gagctaccat gcctggctga tgttggttat ttttagcaaa 120 ataaaaactc ttagatgtct gaaactttca ctatcactct atgatctatt ataaatcaat 180 aagaatcttt aggtaggctg tccccttttt ctctctttcc tec atg aaa gaa drops 235 Met Lys Glu His 1 aat gaa gtg agt aaa aaa gcc att tat ttg ccc ttc ctt cta gcc cea 283 Asn Glu Val Ser Lys Lys Wing He Tyr Leu Pro Phe Leu Leu Wing Pro 5 10 15 20 gtg ctg att cea gcc ttc agt gct gat cea gtt tet gga tea ctg tgg 331 Val Leu He Pro Ala Phe Ser Ala Asp Pro Val Ser Gly Ser Leu Trp 25 30 35 ata cag ggt atg ggg gcc act cta acc gac ttt aat ccc ttc ctg gat 379 lie Gln Gly Met Gly Ala Thr Leu Thr Asp Phe Asn Pro Phe Leu Asp 40 45 50 tea gct cgt gaa ata gaa ttg ttt gtt tta gtt gt ttg gct ttg aag 427 Be Wing Arg Glu He Glu Leu Phe Val Leu Val Val Leu Ala Leu Lys 55 60 65 ttt tgt gta gtg aat tgt cag agt ect gga ctg aac tea ate tet ggt 475 Phe Cys Val Val Asn Cys Gln Ser Pro Gly Leu Asn Ser He Ser Gly 70 75 80 taattcttga tgaaatgatt tctcctttct cacagaacca aatggacatc ctcagttgtt 535 ctctgttcag gaatctccct gtggaaagtc agtggaggtt taaccaggtt gggagagagg 595 acagtcactg ctggctgctg attcccttca cctactgtga gtatecaatt tttcacttcc 655 ctgcctattc ttctaatgag aaataaecca agaagaaaaa gaagtggaac caaaacccaa 715 aaccatcaaa caagettaag acaaattttg tcctttggct ttccagtctt etcatetaat 775 catattattt tcagcaactt ttgataacgt gttgcttgta taaaatcaat ttgtgtgtct 835 tttttctttc attctttctt ccttttttca caataagatc ttgggagagt gaatgctaat 895 gcagttctaa agtggaagct taaaactaag aacagtttgt tctccttcca tccctttaca 955 ctgtgatctt taacatcacc gtcaaggaga gtgtgtttgg ttttgtgatc aatttagctg 1015 tcaggggttt gtgattttct gagagctgac tcttgctgct aatgaacatg atcatttata 1075 cactgtcact gtagtatagg catgttaaat aattgatcag gccctggcag ttagagaatg 1135 gcaaatgcag tccctttgtt gctatggtaa tacatttggt taggggggct gtcatttgga 1195 ccacatggat ctggcacatg tgacataatt gcctgtggtt gaggcggttg caggagaaaa 1255 tcaacttaag aagaggtggc attttgaagt aaattaatgt atatttcagg gttttttttg 1315 ggettaacat tgtggagtga cttattcttt tctattaaaa ggctcattct cagetaatgg 1375 gaatgagtca taageataga agtgacagat ttgtttactt gctgacacag tggaatttgt 1435 tcatttattg tataaaatgg ttattgtgtg aagtttacat tcctccctct attatcccct 1495 ccataagtga ggagggctcc aattgatgcc gggcttatta gggatcccga agggaaagga caacatactc 1555 tcttttgttc tctgtttttt tttctttaaa caatcataag tttttaaagg 1615 taggggctat gtttttgtat tctttcaaca aatatttatt tgttgcaagg tgctcttagg 1575 ggttgggagc acagtgtgag caaagcagat atggtccttg ccctcagagg ttagactgtg 1735 gtggattatg tccatttgtc tttgagtttc tcctttagcc etatcettag cettatcaag 1795 aaccttctca atatgt GTTT ggtaagtgtt tagaatagtt tcttcctttc cagrtccagtc 1855 tettaatata tgtttcttcc aatgatctat taatactatt tgtgaacaac caacacaaaa 1915 gcttaattga gtaaatgaaa cataagtgea gcccttccat tgatccatgt ccttcttttc 1975 cttcatagtt tagecaagtt ctacctctgc tgtgatgcca tccaagccaa cagcaatgtc 2035 tttctggcta tttttttact cagttgatgt gacatatttt gttctgtttt ttaaaaatgt 2095 tattattggc caggcgtggt ggctcacacc tgtaatccca gcactttggg aggccgaggc 2155 gggcagatca tgaggtcagg agategagac catcctggct aacacagtga aaccctgtct 2215 ctacaaaaaa ttagctgggc gtggtggcac gcgcctgtaa tcccagctac ttgggaggct 2275 gaggcaggag aatcacttga acccgggagg cggaggttgc agtgagccaa gattgtgcca 2335 ctgctctcca gcctgggtga cagagtgaga atctgtctca '2375 < 210 2 < 2U > 84 < 212 > PRT < 213 > Homo Sapiens < 400 > 2 Met Lys Glu H1s Asn Glu Val Ser Lys Lys Wing He Tyr Leu Pro Phe 1 5 10 15 Leu Leu Pro Wing Val Leu Pro Pro Wing Phe Ser Wing Asp Pro Val Ser 25 30 Gly Ser Leu Trp He Gln Gly Met Gly Wing Thr Leu Thr Asp Phe Asn 40 45 Pro Phe Leu Asp Ser Ala Arg Glu He Glu Leu Phe Val Leu Val Val 50 55 60 Leu Ala Leu Lys Phe Cys Val Val Asn Cys Gln Ser Pro Gly Leu Asn 65 70 75 80 Be He Be Gly < 210 > 3 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Portion 1 of the Zsig66 Polypeptide < 400 > 3 Trp He Gln Gly Met Gly 1 5 < 210 > 4 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Portion 2 of the Zsig66 Polypeptide < 400 > 4 Thr Asp Phe Asn Pro Phe 1 5 210 > 5 < 211 > 6 < 212 > PRT Z13 > Artificial Sequence < 220 > < 223 > Portion 3 of the Zsigßß Polypeptide < 400 > 5 Lys Phe Cys Val Val Asn 1 5 < 210 > 6 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Portion 4 of the Zsigßß Polypeptide < 400 > 6 Glu He Glu Leu Phe Val 1 5 < 210 > 7 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Degenerate Oligonucleotide for Portion 1 of zsigßß < 221 > misc_brand < 222 > (D. Í 17) < 223 > n - A.T.C or G 400 > 7 tggathcarg gnatggg 17 < 210 > 8 < ai > 17 < 212 > DNA * 213 > Artificial Sequence < 220 > < 223 > Degenerate Oligonucleotide for Portion 2 of zsigßß < 221 > misc_brand 222 > (1) . . . (17) < 223 > n - A.T.C or G < 400 8 acngayttya ayccntt 17 < 210 > 9 < 211 > 17 < 212 DNA < 213 Artificial Sequence < 220 > < 223 > Degenerate Oligonucleotide for Lot 3 of zsigßß < 221 > misc_brand < 222 > (1) ... (17) < 223 > n - A.T.C or G 400 > 9 aarttytgyg tngtnaa 17 < 210 > 10 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Degenerate Oligonucleotide for Lot 4 of zsigßß < 221 > misc_brand < 222 (1) ... (17) < 223 > n - A.T.C or G < 400 > 10 garathgary tnttygt 17 < = 210 > 11 < 211 > 252 < 212 > DNA * 213 > Artificial sequence < 220 > < 223 > Degenerate Nucleotide Sequence for zsigßß < 221 > misc_brand < 222 > (D ... Í252) < 223 > n - A.T.C or G 400 > 11 atgaargarc ayaaygargt nwsnaaraar gcnathtayy tnccnttyyt gt pytngcnccn 60? Ytnathc cngcnttyws ngcngayccn gtnwsnggnw snytntggat hcarggnatg 120 ggngcnacny tnaengaytt yaayccntty ytngaywsng cnmgngarat gt hgarytntty 180? Ytngtng tnytngcnyt naarttytgy gtngtnaayt gycarwsncc nggnytnaay wsnathwsng gn 240 252 < 210 > 12 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer ZC6768 < 400 > 12 gcaattaacc ctcactaaag ggaac 25 < 210 > 13 < 211 > 20 < 212 > DNA ^ l ^ Artificial Sequence < 220 > < 223 > Oligonucleotide primer ZC20137 < 400 > 13 ttctcattag aagaataggc 20 < 210 > 14 < 211 > 20 < 212 DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer ZC20136 < 400 > 14 gaataagtca ctccacaatg 20 < 210 > 15 < 211 > 21 < 212 > DNA < 213 Artificial Sequence < 220 > < 223 > Oligonucleotide primer ZC20132 < 400 > 15 tagagaatgg caaatgcagt c 21 < 210 > 16 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer ZC20134 < 400 > 16 caaggtgctc ttaggggttg 20 < 210 > 17 < 211 > 20 < 212 > DNA < 213 Artificial Sequence < 220 > < 223 > ZC19964 primer of oligonucleotide 400 > 17 ggactgaact caatctctgg 20 < 210 > 18 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > ZC19965 primer of oligonucleotide 400 > 18 tagtattaat agatcattgg 20 210 > 19 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > ZC694 primer of the 40t 19 oligonucleotide taatacgact cactataggg 20 < 210 > 20 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer ZC21336 < 400 > 20 cagagattga gttcagtcca ggac 2 < 210 > 21 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > ZC21338 primer of oligonucleotide 400 > 21 cttccttcta gccccagtgc tga 23 < 210 > 22 < 211 > 18 < 212 > DNA 213 > Artificial Sequence < 220 > < 223 > ZC21450 primer of oligonucleotide 400 > 22 gggg ttgtg attttctg 18 < 210 > 23 < 211 > 18 < 212 DNA < 213 Artificial Sequence < 220 > < 223 Primer ZC21451 of oligonucleotide < 400 > 23 tttgccattc tctaactg 18 < 210 24 211 > 271 < 212 > DNA < 213 > Homo sapiens 400 > 24 ccatgaaaga tctctttcct gtgagtaaaa acacaatgaa aagccattta tttgcccttc 60 cttctagccc cagtgctgat tccagccttc agtgctgatc cagtttctgg atcactgtgg 120 atacagggta tgggggccac tctaaccgac tttaatccct tcctggattc agctcgtgaa 180 atagaattgt ttgttttagt tgtattggct ttgaagtttt gtgtagtgaa ttgtcagagt 240 271 t cctggactga actcaatctc tggttaattc

Claims (22)

185 CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. - An isolated polynucleotide encoding a polypeptide, characterized in that it comprises a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), of SEQ. ID. NO: 2, 186 where the percent identity of the amino acid is determined using a FASTA program with ktup = 1, penalty for interval opening = 10, penalty for extension of interval = 1, and substitution matrix = BLOSUM62, with other parameters established by default.

2. An isolated polynucleotide according to claim 1, characterized in that the polynucleotide is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 431 to nucleotide 472; (b) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 425 to nucleotide 472; (c) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 425 to nucleotide 475; (d) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 305 to nucleotide 475; (e) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 224 to nucleotide 475; and 187 (f) a polynucleotide sequence complementary to (a) or (b).

3. An isolated polynucleotide sequence according to claim 1, characterized in that the polynucleotide comprises nucleotide 1 to nucleotide 252 of SEQ. ID NO: 11.

4. An isolated polynucleotide according to claim 1, characterized in that the polynucleotide encodes a polypeptide comprising an amino acid residue sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and 188 (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), of SEQ ID NO: 2.

5.- The polynucleotide molecule isolated according to claim 1, characterized in that the polynucleotide encodes a polypeptide containing portions 1 to 4 separated from the N-terminal or C-terminal in an Ala28- configuration. { 7 } -Ml-. { 3 } -M2-. { 5 } -M3-. { 6 } -M4-. { 10 } -Gly84, where Ml is the "portion 1", an amino acid sequence as shown in amino acids 36 to 41 of the SEC. ID. NO: 2, M2 is "portion 2", an amino acid sequence as shown in amino acids 45 to 50 of the SEC. ID. NO: 2, M3 is "portion 3", an amino acid sequence as shown in amino acids 56 to 61 of the SEC. ID. NO: 2, M4 is "portion 4", an amino acid sequence as shown in amino acids 68 to 73 of the SEC. ID. NO: 2, Ala28 is the Alanine residue in amino acid number 28 in the SEC. ID. NO: 2, 189 Gly84 is the glycine residue at amino acid number '84 in the SEC. ID. NO: 2, and. { #} denotes the number of amino acids between the portions.

6. An expression vector characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig66 polypeptide comprising an amino acid sequence as shown in SEQ. ID.

NO: 2 of amino acid number 28 (Ala), to amino acid number 84 (Gly); and a transcription terminator. 1 . - An expression vector according to claim 6, characterized in that it comprises a secretory signal sequence operably linked to the DNA segment.

8. - A cultured cell into which an expression vector has been introduced according to claim 6, characterized in that the cell expresses a polypeptide encoded by the DNA segment.

9. A DNA construct that encodes a fusion protein, the DNA construct characterized in that it comprises: a first segment of DNA encoding a polypeptide comprising an amino acid residue sequence selected from the group consisting of: (a) the amino acid sequence of SEQ. ID. NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in the structure, and encode the fusion protein.

10. A fusion protein produced by a method characterized in that it comprises: culturing a host cell in which a vector comprising the following operably linked elements has been introduced: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein according to claim 9; and (c) a transcriptional terminator, and recover the protein encoded by the segment of DNA

11. An isolated polypeptide characterized in that it comprises a sequence of amino acid residues that is at 191 minus 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), of SEC. ID. NO: 2, where the percent identity of the amino acid is determined using a FASTA program with ktup = 1, penalty for opening the interval = 10, penalty for extension of the interval = 1, and substitution matrix = BLOSUM62, with other established parameters default. 192

12. - An isolated polypeptide according to claim 11, characterized in that the polypeptide further comprises portions 1 to 4 separated from the N-terminal or C-terminal in an Ala28- configuration. { } -Ml-. { 3 } -M2-. { 5} -M3-. { 6.} -M4-. { 10.}. -Gly84, where Ml is the "portion 1", an amino acid sequence as shown in amino acids 36 to 41 of the SEC. ID. NO: 2, M2 is "portion 2", an amino acid sequence as shown in amino acids 45 to 50 of the SEC. ID. NO: 2, M3 is "portion 3", an amino acid sequence as shown in amino acids 56 to 61 of the SEC. ID. NO: 2, M4 is "portion 4", an amino acid sequence as shown in amino acids 68 to 73 of the SEC. ID. NO: 2, and Ala28 is the Alanine residue at amino acid number 28 in the SEC. ID. NO: 2, Gly84 is the Glycine residue at amino acid number 84 in the SEC. ID. NO: 2, and. { #} denotes the number of amino acids between the portions. 193

13. - An isolated polypeptide according to claim 11, characterized in that it comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), of SEQ. ID. NO: 2.

14. An isolated polypeptide according to claim 11, characterized in that the polypeptide further comprises an amidated C-terminal serine residue. 194

15. - A method for producing a polypeptide, characterized in that it comprises: culturing a cell according to claim 8; and isolating the polypeptide produced by the cell.

16. A method for detecting, in a test sample, the presence of a modulator of the activity of the zsig66 protein, characterized in that it comprises: transfecting a cell responsive to the protein, with a reporter gene construct that is responsive or that responds to a pathway or cellular pathway stimulated by the protein; and producing the polypeptide by the method of claim 15; and adding the polypeptide to the cell, in the presence and absence of a test sample; and comparing the response levels with the polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of the activity of the protein in the test sample. 195

17. A method for producing an antibody to a polypeptide, characterized in that it comprises the following steps for: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous sequence of amino acids as shown in SEQ ID NO: 2 of amino acid number 28 (Ala) to amino acid number 84 (Gly); (b) a polypeptide consisting of the amino acid sequence of SEQ. ID. NO: 2 of residue number 28 (Ala) • to amino acid number 84 (Gly); (c) a polypeptide according to claim 11, 12 or 13; (d) a polypeptide consisting of amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ. ID. NO: 2; (e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (He) of SEQ. ID. NO: 2; (f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid number 32 (Ser) of the SEC. ID. NO: 2; (g) a polypeptide consisting of amino acid number 9 (Asp) to amino acid number 34 (Ser) of SEC. ID. NO: 2; and 196 (h) a polypeptide consisting of amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEC. ID. NO: 2; and wherein the polypeptide generates an immune response in the animal to produce the antibody; and isolate the animal's antibody.

18. An antibody produced by the method according to claim 17, characterized in that it binds to the polypeptide of SEQ. ID. N0: 2

19. The antibody according to claim 18, characterized in that the antibody is a monoclonal antibody.

20. An antibody that specifically binds to a polypeptide of claim 11, 12 or 13.

21. A method for detecting a genetic abnormality in a patient, characterized in that it comprises: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ. ID. NO: 1 or the complement of the SEC. ID. NO: 1, under conditions wherein said polynucleotide will hybridize to the complementary polynucleotide sequence; 197 visualize the first reaction product; and comparing said first reaction product with a control reaction product from a native type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.

22. A pharmaceutical composition comprising an isolated polypeptide according to claim 11, 12 or 13, characterized in that the polypeptide is in combination with a pharmaceutically acceptable carrier.