The glpQ enzyme is a dimeric enzyme, activated by Ca ions, that hydrolyzes deacylated phospholipids to an alcohol plus G3P. The G3P is subsequently transported into the cell via the GIpT transport system. Glycerophosphoryl diesters (deacylation products of phospholipids) are also high-affinity substrates for ugpQ which are also hydrolyzed to alcohol plus G3P. G3P can be utilized by E. coli as a carbon source, but is also an essential precursor for phospholipids, which are important components for cell membranes. Thus, amongst other functions, these molecules appear to be involved in membrane biosynthesis or maintenance. For review, see Brzoska, P. and Boos, W., J. Bacteriol. 170: 4125-4135,1988; Tommassen, J. et al., Mol. Gen. Genet. 226: 321- 327,1991; Kasahara, M. et al., Nuc. Acids Res. 17: 2854,1989; Ryffel, C., J. Bacteriol.

173 : 7416-7422, 1991.

Mammalian phosphodiesterases (PDEs) are a family of enzymes present in several different tissues in the human body. These PDEs known to regulate the intracellular second messenger cyclic nucleotide monophosphates cAMP and cGMP by breaking them down. Inhibitors of PDEs have proven useful as therapeutics in a wide variety of human conditions such as asthma, allergy, pulmonary disease, heart conditions, suppression of other inflammatory conditions, and erectile dysfunction.

Several different PDE isoenzymes have been shown to be involved in regulating different cell functions. Moreover, PDEs differ in substrate specificity, affinity, sensitivity to endogenous cofactors and modulators, as well as localization to specific

organ systems or tissues. As such, inhibitors that selectively affect specific PDE isoenzymes have been isolated and are therapeutically useful for various conditions. Although several PDE gene families have been isolated, there are probably more yet to be discovered. For review, see Schmidt et al., Clin. Exper. Allergy 29: 99-109,1999; Wallis, R. M. et al., Am. J. Cardiol. 83: 3C-12C, 1999; and Pette, M. et al., J.

Neuroimmunol. 98: 147-156,1999.

Considering the importance of this family of proteins, there is a continuing need to discover new phosphodiesterase proteins that modulate lipid biosynthesis and degradation, regulate transport of macromolecules, regulate intracellular second messengers such as cyclic nucleotide monophosphates, affect proliferation, differentiation, and apoptotic pathways. The in vivo activities of both inducers and inhibitors of these pathways illustrates the enormous clinical potential of, and need for, such novel proteins, their agonists and antagonists. The present invention addresses this need by providing 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 DRAWINGS The Figure 1 is a hydrophobicity plot of zcytorl3 using a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored.

DETAILED DESCRIPTION OF THE INVENTION Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms: The term"affinity tag"is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly- histidine tract, protein A (Nilsson et al., EMBO J. 4 : 1075,1985; Nilsson et al., Methods Enzymol. 198: 3,1991), glutathione S transferase (Smith and Johnson, Gene 67: 31,

1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82: 7952- 4,1985), substance P, Flag peptide (Hopp et al., Biotechnology 6 : 1204-10,1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107,1991. DNAs encoding affinity tags 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 occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The terms"amino-terminal" (also,"N-Terminal") and"carboxyl- terminal" (also"C-terminal") are used herein to denote positions within polypeptides.

Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

The term"complement/anti-complement pair"denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.

For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M-1.

The term"complements of a polynucleotide molecule"denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5'ATGCACGGG 3' is complementary to 5'CCCGTGCAT 3'.

The term"contig"denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to"overlap"a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3'are 5'- AGCTTgagt-3'and 3'-tcgacTACC-5'.

The term"degenerate nucleotide sequence"denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i. e., GAU and GAC triplets each encode Asp).

The term"expression vector"is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term"isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5'and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78,1985).

An"isolated"polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated 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, i. e. 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 glycosylated or derivatized forms.

The term"operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e. g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

The term"ortholog"denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

"Paralogs"are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a- globin, p-globin, and myoglobin are paralogs of each other.

A"polynucleotide"is a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'to the 3'end.

Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

Sizes of polynucleotides are expressed as base pairs (abbreviated"bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term"base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired.

A"polypeptide"is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as"peptides".

The term"promoter"is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5'non-coding regions of genes.

A"protein"is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell.

Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term"receptor"denotes a cell-associated protein that binds to a bioactive molecule (i. e., a ligand) and mediates the effect of the ligand on the cell.

Membrane-bound receptors are characterized by a multi-peptide structure, for example, comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e. g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e. g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

The term"secretory signal sequence"denotes a DNA sequence that encodes a polypeptide (a"secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term"splice variant"is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by imprecise analytical methods (e. g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as"about"X or"approximately"X, the stated value of X will be understood to be accurate to 10%.

All references cited herein are incorporated by reference in their entirety.

Within one aspect, the present invention provides an isolated polynucleotide that encodes a polypeptide comprising 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 25 (Cys), to amino acid number 230 (Leu); (b) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 231 (Ile), to amino acid number 605 (Arg); (c) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); and (d) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 1 (Met) to amino acid number 605 (Arg). Within one embodiment, the isolated polynucleotide disclosed above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 229 to nucleotide 846; (b) a polynucleotide sequence as shown in SEQ ID NO : I from nucleotide 847 to nucleotide 1971; (c) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 229 to nucleotide 1971 ; and (d) a polynucleotide sequence as shown in SEQ ID NO : 1 from nucleotide 157 to nucleotide 1971. Within another embodiment, the isolated polynucleotide disclosed above comprises nucleotide 1 to nucleotide 1815 of SEQ ID NO : 3. Within another embodiment, the isolated polynucleotide disclosed above encodes a polypeptide

that 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 25 (Cys), to amino acid number 230 (Leu); (b) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 231 (Ile), to amino acid number 605 (Arg); (c) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); and (d) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 1 (Met) to amino acid number 605 (Arg). Within another embodiment, the isolated polynucleotide disclosed above encodes a polypeptide that has phosphodiesterase activity.

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 polypeptide as shown in SEQ ID NO : 2 from amino acid number 22 (Cys), to amino acid number 605 (Arg); and a transcription terminator, wherein the promoter is operably linked to the DNA segment, and the DNA segment is operably linked to the transcription terminator. In one embodiment, the expression vector disclosed above further comprising a secretory signal sequence operably linked to the DNA segment.

Within a third aspect, the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.

Within another aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO : 2 from amino acid number 1 (Met), to amino acid number 24 (Gly); (b) the amino acid sequence of SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 230 (Leu); (c) the amino acid sequence of SEQ ID NO : 2 from amino acid number 231 (Ile) to amino acid number 605 (Arg); (d) the amino acid sequence of SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); (e) the amino acid sequence of SEQ ID NO : 2 from amino acid number 1 (Met), to amino acid number 605 (Arg); and at least one other DNA segment encoding an additional polypeptide, wherein the first and

other DNA segments are connected in-frame; and wherein the first and other DNA segments encode the fusion protein.

Within another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein as disclosed above; and a transcription terminator, wherein the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcription terminator.

Within another aspect, the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA construct.

Within another aspect, the present invention provides a method of producing a fusion protein comprising: culturing a cell as disclosed above; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolated polypeptide comprising 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 25 (Cys), to amino acid number 230 (Leu); (b) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 231 (le), to amino acid number 605 (Arg); (c) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); and (d) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 1 (Met) to amino acid number 605 (Arg).

In one embodiment, the isolated polypeptide disclosed above 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 25 (Cys), to amino acid number 230 (Leu); (b) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 231 (Ile), to amino acid number 605 (Arg); (c) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); and (d) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 1 (Met) to amino acid number 605 (Arg). In another embodiment, the isolated polypeptide disclosed above has phosphodiesterase activity.

Within another aspect, the present invention provides a method of producing a phosphodiesterase polypeptide comprising: culturing a cell as disclosed above; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides a method of producing an antibody comprising: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 580 amino acids, wherein the polypeptide is identical to a contiguous sequence of amino acids in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); (b) a polypeptide as disclosed above; (c) a polypeptide consisting of the amino acid sequence of SEQ ID NO : 2 from amino acid number 31 (Gln) to amino acid number 36 (Asp); (d) a polypeptide consisting of the amino acid sequence of SEQ ID NO : 2 from amino acid number 238 (Pro) to amino acid number 333 (Glu); (e) a polypeptide consisting of the amino acid sequence of SEQ ID NO : 2 from amino acid number 360 (Arg) to amino acid number 365 (Glu); (f) a polypeptide consisting of the amino acid sequence of SEQ ID NO : 2 from amino acid number 540 (Arg) to amino acid number 545 (Asp); and (g) a polypeptide consisting of the amino acid sequence of SEQ ID NO : 2 from amino acid number 600 (Ile) to amino acid number 605 (Arg); and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.

Within another aspect, the present invention provides an antibody produced by the method as disclosed above, which binds to a polypeptide comprising 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 25 (Cys), to amino acid number 230 (Leu); (b) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 231 (De), to amino acid number 605 (Arg); (c) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); and (d) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 1 (Met) to amino acid number 605 (Arg). In one embodiment, the antibody disclosed above is a monoclonal antibody.

Within another aspect, the present invention provides an antibody that specifically binds to a polypeptide comprising 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 25 (Cys), to amino acid number 230 (Leu); (b) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 231 (De), to amino acid number 605 (Arg); (c) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg); and (d) the amino acid sequence as shown in SEQ ID NO : 2 from amino acid number 1 (Met) to amino acid number 605 (Arg).

Within another aspect, the present invention provides a method of detecting, in a test sample, the presence of a modulator of zcytorl3 protein activity, comprising: culturing a cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the protein encoded by the DNA segment in the presence and absence of a test sample; and comparing levels of activity of zcytorl3 in the presence and absence of a test sample, by a biological or biochemical assay; and determining from the comparison, the presence of modulator of zcytorl3 activity in the test sample.

These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and attached drawings.

The present invention is based in part upon the discovery of a novel DNA sequence that encodes a protein having sequence similarity to a glycerophosphoryl diester phosphodiesterase. Analysis of the tissue distribution of the mRNA corresponding to this novel DNA showed expression in testis, spleen, heart and skeletal muscle. The polypeptide has been designated zcytorl3.

The novel polypeptide, encoded by the isolated cDNA, showed homology with glycerophosphoryl diester phosphodiesterases. The zcytorl3 polynucleotide sequence encodes the entire coding sequence of the predicted protein. Zcytorl3 is a novel glycerophosphoryl diester phosphodiesterase that may be involved in lipid biosynthesis or degradation, as an intracellular signaling molecule, transport protein, tissue contractility, involved in an apoptotic cellular pathway, or the like.

The sequence of the zcytorl3 polypeptide was deduced from a single clone that contained its corresponding polynucleotide sequence. The clone was

obtained from a human pituitary library. Other libraries that might also be searched for such sequences include testis, spleen, heart and skeletal muscle, and the like.

The nucleotide sequence of a representative zcytorl3-encoding DNA is described in SEQ ID NO : 1, and its deduced 605 residue amino acid sequence is described in SEQ ID NO : 2. In its entirety, the zcytorl3 polypeptide (SEQ ID NO : 2) represents a full-length polypeptide segment (residue 1 (Met) to residue 605 (Arg) of SEQ ID NO : 2). The domains and structural features of the zcytorl3 polypeptide are further described below.

Analysis of the zcytorl3 polypeptide encoded by the DNA sequence of SEQ ID NO : 1 revealed an open reading frame encoding 605 amino acids (SEQ ID NO : 2) comprising a predicted secretory signal peptide of 24 amino acid residues (residue 1 (Met) to residue 24 (Gly) of SEQ ID NO : 2), and a mature polypeptide of 581 amino acids (residue 25 (Cys) to residue 605 (Arg) of SEQ ID NO : 2). Zcytorl3 comprises a hydrophobic N-terminal domain of approximately 200 amino acid residues (residues 25 (Cys) to 230 (Leu) of SEQ ID NO : 2); and a C-terminal phosphodiesterase domain of approximately 375 amino acid residues (residues 231 (Ile) to 605 (Arg) of SEQ ID NO : 2). Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding. The corresponding polynucleotides encoding the zcytorl3 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO : 1.

The presence of transmembrane regions, and conserved and low variance motifs generally correlates with or defines important structural regions in proteins. Regions of low variance (e. g., hydrophobic clusters) are generally present in regions of structural importance (Sheppard, P. et al., supra.). Such regions of low variance often contain rare or infrequent amino acids, such as Tryptophan. The regions flanking and between such conserved and low variance motifs may be more variable, but are often functionally significant because they may relate to or define important structures and activities such as binding domains, biological and enzymatic activity, signal transduction, cell-cell interaction, tissue localization domains and the like.

The regions of conserved amino acid residues in zcytorl3, described above, can be used as tools to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved regions from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zcytorl3 sequences are useful for this purpose. Designing and using such degenerate primers may be readily performed by one of skill in the art.

The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zcytorl3 polypeptides disclosed herein.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO : 3 is a degenerate DNA sequence that encompasses all DNAs that encode the zcytorl3 polypeptide of SEQ ID NO : 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO : 3 also provides all RNA sequences encoding SEQ ID NO : 2 by substituting U for T. Thus, zcytorl3 polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 1815 of SEQ ID NO : 3 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NO : 3 to denote degenerate nucleotide positions."Resolutions"are the nucleotides denoted by a code letter."Complement" indicates the code for the complementary nucleotide (s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M AjC K GIT K G|T M AjC S C|G S C|G W AIT W AIT H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T The degenerate codons used in SEQ ID NO : 3, encompassing all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Codons Degenerate Acid Code Codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V TGA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter. TAATAGTGA TRR <BR> <BR> Asn|Asp B RAY<BR> <BR> Glu|Gln Z SAR<BR> <BR> Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO : 2. Variant sequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that different species can exhibit"preferential codon usage."In general, see, Grantham, et al., Nuc. Acids Res. 8: 1893-912,1980; Haas, et al. Curr. Biol. 6: 315-24,1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3075-87,1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. As used herein, the term"preferential codon usage"or"preferential codons"is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO : 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO : 1, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Numerous equations for calculating Tm are known in the art, and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al., Molecular Cloning : A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 227,1990). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4. 0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating Tm based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences (e. g., >50 base pairs) is performed at temperatures of about 20-25°C below the calculated Tm. For smaller probes (e. g., <50 base pairs) hybridization is typically carried out at the Tm or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the Tm of the hybrid about 1°C for each 1% formamide in the buffer solution. Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 p. g/ml denatured commercially-available carrier DNA. Generally, such stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC. For example, a suitable wash stringency is equivalent to 0. 1X SSC to

2X SSC, 0.1% SDS, at 55°C to 65°C. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes. Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zcytorl3 RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201,1980), and include testis, spleen, heart and skeletal muscle. Northern blots indicate that human zcytorl3 is highly expressed specifically in human testis and spleen, and only moderately to faintly expressed in other tissues examined (Example 2). Moreover, in mouse, zcytorl3 is highly expressed in heart, skeletal muscle and liver in the mouse (Example 2). Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCI gradient (Chirgwin et al., Biochemistry L8 : 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl.

Acad. Sci. USA 69: 1408-12,1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zcytorl3 polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length clone encoding zcytorl3 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e. g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.

Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zcytorl3, ligand fragments, or other specific binding partners.

zcytorl3 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5'non-coding regions of a zcytorl3 gene. In view of the tissue-specific expression observed for zcytorl3 by Northern blotting, this gene region is expected to provide for testis-, spleen-, heart-, and skeletal muscle-specific expression. Promoter elements from a zcytorl3 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. Cloning of 5'flanking sequences also facilitates production of zcytorl3 proteins by"gene activation"as disclosed in U. S. Patent No.

5,641,670. Briefly, expression of an endogenous zcytorl3 gene in a cell is altered by introducing into the zcytorl3 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zcytorl3 5'non-coding sequence that permits homologous recombination of the construct with the endogenous zcytorl3 locus, whereby the sequences within the construct become operably linked with the endogenous zcytorl3 coding sequence. In this way, an endogenous zcytorl3 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.

The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a DNA or a DNA fragment, then each complementary strand is made separately, for example via the phosphoramidite method known in the art. The production of short polynucleotides (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. However, for producing longer polynucleotides (longer than about 300 bp), special strategies are usually employed.

For example, synthetic DNAs (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. One method for building a synthetic DNA involves producing a set of overlapping, complementary oligonucleotides. Each internal section of the DNA has complementary 3'and 5' terminal extensions designed to base pair precisely with an adjacent section. After the DNA is assembled, the process is completed by ligating the nicks along the backbones

of the two strands. In addition to the protein coding sequence, synthetic DNAs can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector. Alternative ways to prepare a full-length DNA are also known in the art. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D. C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56,1984 and Climie et al., Proc. Natl. Acad. Sci.

USA 87: 633-7,1990.

The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zcytorl3 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zcytorl3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zcytorl3 as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A zcytorl3-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U. S. Patent No.

4,683,202), using primers designed from the representative human zcytorl3 sequence disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zcytorl3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO : 1 represents a single allele of human zcytorl3 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to

standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO : 1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO : 2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zcytorl3 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

The present invention also provides isolated zcytorl3 polypeptides that are substantially similar to the polypeptides of SEQ ID NO : 2 and their orthologs. The term"substantially similar"is used herein to denote polypeptides having 70%, preferably 80%, more preferably at least 85%, sequence identity to the sequences shown in SEQ ID NO : 2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16,1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-9,1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the"blosum 62"scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one- letter codes). The percent identity is then calculated as: Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] Table 3<BR> A R N D C Q E G H I L K M F P S T W Y V<BR> A 4<BR> R -1 5<BR> N -2 0 6<BR> D -2 -2 1 6<BR> C 0 -3 -3 -3 9<BR> Q -1 1 0 0 -3 5<BR> E -1 0 0 2 -4 2 5<BR> G 0 -2 0 -1 -3 -2 -2 6<BR> H -2 0 1 -1 -3 0 0 -2 8<BR> I -1 -3 -3 -3 -1 -3 -3 -4 -3 4<BR> L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4<BR> K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5<BR> M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5<BR> F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6<BR> P 1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7<BR> S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4<BR> T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5<BR> W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11<BR> Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7<BR> V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The"FASTA"similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zcytorl3. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'1 Acad. Sci. USA 85: 2444,1988; and by Pearson, Meth. Enzymol. 183: 63,1990.

Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e. g., SEQ ID NO : 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are"trimmed"to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff'value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444,1970; Sellers, SIAM J. Appl. Math. 26: 787,1974), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol., supra..

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons,

the ktup value can range between one to six, preferably from three to six, most preferably three, with other FASTA program parameters set as default.

The BLOSUM62 table (Table 3) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'1 Acad. Sci. USA 89: 10915,1992). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language"conservative amino acid substitution"preferably refers to a substitution represented by a BLOSUM62 value of greater than-1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0,1,2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e. g., 1,2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e. g., 2 or 3).

Variant zcytorl3 polypeptides or substantially similar zcytorl3 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino-or carboxyl-terminal extensions, 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 polypeptides of from about 550 to about 610 amino acid residues that comprise a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ ID NO : 2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zcytorl3 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4 Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.

For example, a zcytorl3 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U. S. Patents Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.

Immunoglobulin-zcytorl3 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zcytorl3 analogs. Auxiliary domains can be fused to zcytorl3 polypeptides to target them to specific cells, tissues, or macromolecules (e. g., collagen). For example, a zcytorl3 polypeptide or protein could be targeted to a predetermined cell type by fusing a zcytorl3 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way,

polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zcytorl3 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34: 1-9,1996.

The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3-and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation. of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.

113: 2722,1991; Ellman et al., Methods Enzymol. 202: 301,1991; Chung et al., Science 259: 806-9,1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-9, 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: 19991-8,1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e. g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (s) (e. g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart.

See, Koide et al., Biochem. 33: 7470-6,1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.

Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403,1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zcytorl3 amino acid residues.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5,1989; Bass et al., Proc. Natl. Acad. Sci. USA 88: 4498-502,1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708,1996. Sites of ligand-receptor or other biochemical interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255: 306-12,1992; Smith et al., J. Mol. Biol. 224: 899-904,1992; Wlodaver et al., FEBS Lett. 309: 59-64,1992. The identities of essential amino acids can also be inferred from analysis of homologies with related phosphodiesterases.

Determining amino acid residues that are within regions or domains that are critical to maintaining structural integrity is within the skill of one in the art. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e. g., the Insight T) viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5: 372-376,1995 and Cordes et al., Current Opin. Struct. Biol. 6: 3-10,1996). In general, when designing

modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.

Amino acid sequence changes are made in zcytorl3 polypeptides so as to minimize disruption of higher order structure essential to biological activity. For example, when the zcytorl3 polypeptide comprises one or more helices or functional domains, changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners, or enzymatic function. The effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e. g., Lapthorn et al., Nat. Struct. Biol. 2: 266-268,1995). Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e. g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or 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 disulfide bonding pattern as the standard molecule folding would be affected. Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7: 205-214,1990). Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257: 961-964,1992).

A Hopp/Woods hydrophilicity profile of the zcytorl3 protein sequence as shown in SEQ ID NO : 2 can be generated (Hopp et al., Proc. Natl. Acad.

Sci. 78: 3824-3828,1981; Hopp, J. Immun. Meth. 88: 1-18,1986 and Triquier et al., Protein Engineering 11 : 153-169, 1998). The profile is based on a sliding six-residue

window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored (See, Figure 1). For example, in zcytorl3, hydrophilic regions include: (1) amino acid number 31 (Gln) to amino acid number 36 (Asp) of SEQ ID NO : 2; (2) amino acid number 238 (Pro) to amino acid number 333 (Glu) of SEQ ID NO : 2; (3) amino acid number 360 (Arg) to amino acid number 365 (Glu) of SEQ ID NO : 2; (4) amino acid number 540 (Arg) to amino acid number 545 (Asp) of SEQ ID NO : 2; and (5) amino acid number 600 (Ile) to amino acid number 605 (Arg) of SEQ ID NO : 2.

Those skilled in the art will recognize that hydrophilicity or hydrophobicity is taken into account when designing modifications in the amino acid sequence of a zcytorl3 polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp; for example, residues tolerant of substitution could include such residues as shown in SEQ ID NO: 2. Cysteine residues will be relatively intolerant of substitution.

The identities of essential amino acids can also be inferred from analysis of sequence similarity between known phosphodiesterase family members with zcytorl3. Using methods such as"FASTA"analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An alternative approach to identifying a variant zcytorl3 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zcytorl3 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1, as discussed above.

Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081,1989; Bass et al., Proc. Natl Acad. Sci. USA 88: 4498,1991; Coombs and Corey,"Site-Directed Mutagenesis and Protein Engineering,"in Proteins: Analysis and Design, Angeletti (ed.), Academic Press, Inc., pp. 259-311,1998). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and

the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699, 1996.

The present invention also includes functional fragments of zcytorl3 polypeptides and nucleic acid molecules encoding such functional fragments. A "functional"zcytorl3 or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-zcytorl3 antibody or zcytorl3 substrate or binding partner (either soluble or immobilized).

Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zcytorl3 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO : 1 or fragments thereof, can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zcytorl3 activity, or for the ability to bind anti-zcytorl3 antibodies or zcytorl3 substrate or binding partner. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zcytorl3 fragment. Alternatively, particular fragments of a zcytorl3 polynucleotide can be synthesized using the polymerase chain reaction.

Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther.

66: 507,1995. Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240: 113,1993; Content et al.,"Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon,"in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), Nijhoff, pp. 65-72,1987; Herschman,"The EGF Receptor,"in Control of Animal Cell Proliferation 1 Boynton et al. (eds.), Academic Press, pp. 169-199,1985; 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.

In addition, the proteins of the present invention (or polypeptide fragments thereof) can be joined to other bioactive molecules, particularly other phosphodiesterases, to provide multi-functional molecules. For example, one or more domains or sub-fragments from zcytorl3 can be joined to other phosphodiesterases to enhance their biological properties or efficiency of production.

The present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the domains of zcytorl3 is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics. Such hybrid molecules may further comprise additional amino acid residues (e. g. a polypeptide linker) between the component proteins or polypeptides.

Multiple amino acid substitutions can be made and tested using known routine methods of mutagenesis and screening, such as those disclosed by Reidhaar- Olson and Sauer (Science 241: 53-7,1988) or Bowie and Sauer (Proc. Natl. Acad. Sci.

USA 86: 2152-6,1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e. g., Lowman et al., Biochem. 30: 10832-7,1991; Ladner et al., U. S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis (Derbyshire et al., Gene 46: 145,1986; Ner et al., DNA 7: 127, 1988).

Variants of the disclosed zcytorl3 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-51,1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous

recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid"evolution"of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e. g., those with phosphodiesterase activity, that induce signal transduction or bind anti-trypl antibodies, and the like) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO : 2 or that retain, for example, phosphodiesterase-like properties, substrate binding activity, mitogenic activity, induce cytokine secretion, binding, cell-cell communication, or signal transduction activity of the wild-type zcytorl3 protein. For example, using the methods described herein, one could identify a substrate binding domain on zcytorl3; heterodimeric and homodimeric binding domains; enzymatically active domains; other functional or structural domains; or other domains important for protein-protein interactions or signal transduction. Such polypeptides may also include additional polypeptide segments, such as affinity tags, as generally disclosed herein.

For any zcytorl3 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.

The zcytorl3 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host

cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zcytorl3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

To direct a zcytorl3 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zcytorl3, or may be derived from another secreted protein (e. g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the zcytorl3 DNA sequence, i. e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e. g., Welch et al., U. S.

Patent No. 5,037,743; Holland et al., U. S. Patent No. 5,143,830).

Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the

secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from residue 1 (Met) to residue 24 (Gly) of SEQ ID NO : 2 is operably linked to a DNA sequence encoding another polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non- secreted protein. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725,1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981: Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1 : 841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15: 73,1993; Ciccarone et al., Focus 15 : 80,1993, and viral vectors (Miller and Rosman, BioTechniques 7: 980-90,1989; Wang and Finer, Nature Med. 2: 714-6,1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U. S. Patent No.

4,713,339; Hagen et al., U. S. Patent No. 4,784,950; Palmiter et al., U. S. Patent No.

4,579,821; and Ringold, U. S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72,1977) and Chinese hamster ovary (e. g.

CHO-K1 ; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e. g., U. S. Patent No.

4,956,288. Other suitable promoters include those from metallothionein genes (U. S.

Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as"stable transfectants."A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as"amplification."Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e. g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore 11 : 47-58,1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U. S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica 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 of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V. A, et al., J Virol 67: 4566- 79,1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zcytorl3 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a"bacmid."The pFastBacl transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zcytorl3. However, pFastBaclTM can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing 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 transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native zcytorl3 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zcytorl3 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C-or N-terminus of the expressed zcytorl3 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82: 7952-4,1985). Using a technique known in the art, a transfer vector containing zcytorl3 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.

The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e. g. Sf9 cells.

Recombinant virus that expresses zcytorl3 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology : Principles and Applications of Recombinant DNA, ASM Press, Washington, D. C., 1994. Another suitable cell line is the High FiveO cell line (Invitrogen) derived from Trichoplusia ni (U. S. Patent No. 5,300,435).

Commercially available serum-free media are used to grow and maintain the cells.

Suitable media are Sf900 ItrM (Life Technologies) or ESF 92 ITM (Expression Systems) for the Sf9 cells; and Ex-cell0405' (JRH Biosciences, Lenexa, KS) or Express FiveO (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the zcytorl3 polypeptide from the supernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U. S. Patent No. 4,599,311; Kawasaki et al., U. S. Patent No. 4,931,373; Brake, U. S. Patent No. 4,870,008; Welch et al., U. S. Patent No. 5,037,743; and Murray et al., U. S. Patent No. 4,845,075.

Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e. g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTI vector system disclosed by Kawasaki et al. (U. S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.

Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e. g., Kawasaki, U. S. Patent No. 4,599,311; Kingsman et al., U. S.

Patent No. 4,615,974; and Bitter, U. S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U. S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and

4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol.

132: 3459-65, 1986 and Cregg, U. S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U. S. Patent No. 4,935,349.

Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U. S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U. S. Patent No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-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, it is preferred to use host cells in which both methanol utilization genes (AUGl and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRBI) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,

preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention.

Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e. g., Sambrook et al., ibid.). When expressing a zcytorl3 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate 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 shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto"M Peptone (Difco Laboratories,

Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

It is preferred to purify the polypeptides of the present invention to 280% purity, more preferably to 290% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

Expressed recombinant zcytorl3 polypeptides (or chimeric zcytorl3 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, 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 receptor polypeptides to support media are well known in the art.

Selection of a particular method is a matter of routine design and is determined in part

by the properties of the chosen support. See, for example, Affinitv Chromatographv : Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploitation of their structural or biochemical properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3: 1-7,1985).

Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include 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 additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e. g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification. Moreover purification methods used to purify mammalian, or bacterial phosphodiesterases polypeptides can be used to purify human zcytorl3 polypeptides. See, for example, Larson, TJ, and van Loo-Bhattacharya, A. T.

Arch. Biochem. Biophys. 260: 577-584,1988.

Moreover, using methods described in the art, polypeptide fusions, or hybrid zcytorl3 proteins, are constructed using regions or domains of the inventive zcytorl3 in combination with those of other phosphodiesterase family proteins (e. g. mammalian phosphodiesterases (PDEs), glycerophosphoryl diester phosphodiesterases, and the like), or heterologous proteins (Sambrook et al., ibid.; Altschul et al., ibid. ; Picard, Cur. Opin. Biology, 5: 511-5,1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating

them. Alternatively, a polynucleotide encoding various components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain (s) conferring a biological function may be swapped between zcytorl3 of the present invention with the functionally equivalent domain (s) from another family member.

Such domains include, but are not limited to, the secretory signal sequence, conserved motifs, and the N-terminal hydrophobic domain or C-terminal phosphodiesterase domain. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known phosphodiesterase family proteins (e. g. cleaving phospholipids or cyclic mononucleotide phosphates, and the like) depending on the fusion constructed.

Moreover, such fusion proteins may exhibit other properties as disclosed herein.

Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zcytorl3 polypeptide and those polypeptides to which they are fused. Generally, a DNA segment that encodes a domain of interest, e. g., a zcytorl3 active polypeptide or motif described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide and inserted into an appropriate expression vector, as described herein. Generally DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof. For example, a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a N-terminal hydrophobic domain and phosphodiesterase domain; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a phosphodiesterase domain; or, for example, any of the above as interchanged with equivalent regions from another protein. Such fusion proteins can be expressed, isolated, and assayed for activity, such as phosphodiesterase activity, as described herein.

Zcytorl3 polypeptides or fragments thereof may also be prepared through chemical synthesis. Zcytorl3 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

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 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 the entire synthesis of the desired peptide on a solid support, the peptide-resin is 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 molecules of the present invention can be measured using a variety of assays that measure lipid biosynthesis or degradation, cyclic nucleotide monophosphate cleavage, or other phosphodiesterase activity. Of particular interest are assays that measure changes in cell proliferation, apoptosis, generation of nucleotide monophosphates and lipidolysis products. Moreover phosphodiesterase activity can be measured using radioactive phospholipid substrates; cAMP assays described herein; substrates such as deacylation products of phosphatidylethanolamine, phosphatidylcholine, phosphatidyl glycerol, and the like by measuring hydrophilic alcohols; hydrolyzation of glycerol glycerophosphocholine and other glycerophosphodiesters possessing hydrophobic alcohols. Such assays are well known in the art. For a general reference, see Larson, TJ, and van Loo-Bhattacharya, A. T. ibid.; and Larson, TJ et al., J. Biol. Chem. 258: 5428-5432,1983.

Several tissues in which zcytorl3 is highly expressed are tissues that contract. For example contractile tissues in which zcytorl3 is expressed include testis, heart and skeletal muscle. Moreover, several human PDEs are found in contractile tissues such as smooth muscle, heart, lung, testicular tissues, and the like. The effects of zcytorl3 polypeptide, its antagonists and agonists, on tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation. Such assays are known in the art and can be applied to tissue samples, such as aortic rings,

muscle tissue, and other contractile tissue samples, as well as to organ systems, such as atria, and can be used to determine whether zcytorl3 polypeptide, its agonists or antagonists, enhance or depress contractility. Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo. As such, molecules of the present invention have utility in treating cardiovascular disease, muscle relaxants or stimulants, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as inducing birth.

The effect of the zcytorl3 polypeptides, antagonists and agonists of the present invention on contractility of tissues including skeletal and smooth muscle tissues, testis, heart, and the like, can be measured in a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100: 767,1990; Rhee et al., Neurotox. 16: 179,1995; Anderson, M. B., Endocrinol. 114: 364-368, 1984; and Downing, S. J. and Sherwood, O. D, Endocrinol. 116: 1206-1214,1985. For example, measuring vasodilatation of aortic rings is well known in the art. Briefly, aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCl, 4.6 mM KCI, 1.2 mM MgS04. 7H20, 1.2 mM KH2PO4, 2.5 mM CaCl2. 2H20, 24.8 mM NaHCO3 and 10 mM glucose). One of skill in the art would recognize that this method can be used with other animals, such as rabbits, other rat trains, Guinea pigs, and the like. The rings are then attached to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data recorded with a Ponemah physiology platform (Gould Instrument systems, Inc., Valley View, OH) and placed in an oxygenated (95% 02, 5% C02) tissue bath containing the buffer solution. The tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing. The integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour. To test a sample for vasodilatation, or relaxation of the aortic ring tissue, the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes. A zcytorl3 polypeptide, antagonist or agonist sample is then added to 1,2 or 3 of the 4 baths, without flushing,

and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of contractility by zcytorl3 polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as skeletal and smooth muscle tissue, gastrointestinal tissues, uterus, prostate, and testis.

The activity of molecules of the present invention can be measured using a variety of assays that measure 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 segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Mot lit incorporated herein by reference). An exemplary in vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between commissures and longitudinally 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 setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract. Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently. A test food or liquid is radiolabeled with an isotope (e. g. 99mTc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Meyer et al., Am. J. Dig. Dis. 21: 296,1976; Collins et al., Gut 24: 1117,1983; Maughan et al., Diabet. Med. 13 9 Supp. 5 : S6-10,1996 and Horowitz et al., Arch.

Intern. Med. 145: 1467-1472, 1985). These studies may be performed before and after the administration of a promotility agent to quantify the efficacy of the drug.

An in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).

Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T. C. 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) adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters.

Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

Using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential E1 gene has been deleted from the viral vector, and the virus will not replicate unless the E1 gene is provided by the host cell (the human 293 cell line is exemplary). When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an E1 gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e. g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

Moreover, adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72: 2022-2032,1998; Raper, S. E. et al., Human Gene Therapy 9 : 671- 679,1998). In addition, deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72: 926-933,1998). Moreover, by deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated.

Generation of so called"gutless"adenoviruses where all viral genes are deleted are particularly advantageous for 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 protein production in vitro.

By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For

instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Garnier et al., Cytotechnol. 15: 145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.

The activation of zcytorl3 polypeptide can be measured by a silicon- based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary device is the Cytosensor Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H. M. et al., Science 257: 1906-1912,1992; Pitchford, S. et al., Meth. Enzymol. 228: 84-108,1997; Arimilli, S. et al., J. Immunol. Meth. 212: 49-59, 1998; Van Liefde, 1. Et al., Eur. J. Pharmacol. 346: 87-95,1998. The microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including agonists, ligands, or antagonists of the zcytorl3 polypeptide. Preferably, the microphysiometer is used to measure responses of a zcytorl3-expressing eukaryotic cell, compared to a control eukaryotic cell that does not express zcytorl3 polypeptide.

Zcytorl3-expressing eukaryotic cells comprise cells into which zcytorl3 has been transfected, as described herein, creating a cell that is responsive to zcytorl3- modulating stimuli; or cells naturally expressing zcytorl3, such as zcytorl3-expressing cells derived from spleen, testis, muscle or heart tissue. Differences, measured by a change in extracellular acidification, for example, an increase or diminution in the

response of cells expressing zcytorl3, relative to a control, are a direct measurement of zcytorl3-modulated cellular responses. Moreover, such zcytorl3-modulated responses can be assayed under a variety of stimuli. Also, using the microphysiometer, there is provided a method of identifying agonists and antagonists of zcytorl3 polypeptide, comprising providing cells expressing a zcytorl3 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 diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Antagonists and agonists, including the natural ligand for zcytorl3 polypeptide, can be rapidly identified using this method.

In view of the tissue distribution observed for zcytorl3, agonists (including the natural ligand/substrate/cofactor/etc.) and antagonists have enormous potential in both in vitro and in vivo applications. Compounds identified as zcytorl3 agonists and antagonists are useful for modulating vasoconstriction and vasodilatation, modulating muscle tension and relaxation, muscle spasms, inflammation, modulating proliferation, modulating digestion, modulating heart conditions, modulating testicular function and fertility, and the like in vitro and in vivo. For example, zcytorl3 and agonist or antagonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Agonists or antagonists are thus useful in specifically promoting the growth and/or development of, for example, heart and muscle cells lineages in culture. Alternatively, zcytorl3 polypeptides and zcytorl3 agonist or antagonist polypeptides are useful as a research reagent, such as for the expansion of heart or muscle cells, or useful as an amino acid source for cell culture.

The activity of molecules of the present invention can be measured using a variety of assays that measure proliferation and/or differentiation of specific cell types, chemotaxis, adhesion, changes in ion channel influx, pH flux, regulation of second messenger levels and neurotransmitter release, cell motility, protein binding, apoptosis, or the like. Such assays are well known in the art. See, for example, in

"Basic & Clinical Endocrinology Ser., Vol. 3,"Cytochemical Bioassays: Techniques & Applications, Chayen; Chayen, Bitensky, eds., Dekker, New York, 1983.

The activity of molecules of the present invention can also be measured using a variety of assays that measure, for example, signal transduction upon binding a ligand or substrate, or antibody binding. For example, zcytorl3 polypeptides, complementary binding polypeptides, or anti-zcytorl3 antibodies can be labeled and tested for specific and saturating binding to specific substrates, cell lines or cells.

Identification of positive cells to which zcytorl3 polypeptides, complementary binding polypeptides, or anti-zcytorl3 antibodies binds can be achieved by injecting a radioactively or fluorescently-labeled zcytorl3 polypeptide, polypeptide fragments, complementary binding polypeptides, or anti-zcytorl3 antibodies and using art- recognized immunohistochemistry methods to visualize a cell or tissue in vivo where zcytorl3 binds. After identification of bound positive cells, activity can be tested for zcytorl3-mediated activation of a signal transduction pathway using methods known in the art. For instance, vector constructs containing a reporter (e. g. SRE-luciferase, STAT-luciferase, thyroid hormone response element (THRE)-luciferase or the like) can be introduced into the positive cell lines expressing zcytorl3; such cell lines, when exposed to conditioned media containing secreted zcytorl3 activating proteins will demonstrate zcytorl3-mediated signal transduction activity through activation of the measurable reporter. Such assays are well known in the art. Specific assays include, but are not limited to, bioassays measuring signal transduction.

Moreover, zcytorl3 phosphodiesterase activity can be measured using labeled phospholipids, deacylation products of phosphatidylethanolamine, phosphatidylcholine, phosphatidyl glycerol, and the like; hydrolyzation of glycerol glycerophosphocholine and other glycerophosphodiesters. Moreover, glucose-3- phosphate, or cyclic mononucleotide phosphate substrates can be used to asses transport of zcytorl3 substrates into zcytorl3-expressing cells. Moreover such substrates can be assessed for cleavage by zcytorl3 using assays in the art and described herein. In addition, use of anti-zcytorl3 antibodies and inhibitors of known phosphodiesterases, can be utilized as well to determine specificity of the zcytorl3 activity on these substrates. For a general reference, see Larson, TJ, and van Loo-Bhattacharya, A. T.

Arch. Biochem. Biophys. 260: 577-584,1988; and Larson, TJ et al., J. Biol. Chem.

258:5428-5432,1983.

Zcytorl3 can also be used to identify modulators (e. g, agonists or antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit or stimulate the activity of zcytorl3. In addition to those assays disclosed herein, samples can be tested for inhibition/stimulation of zcytorl3 activity within a variety of assays designed to measure zcytorl3 binding, dimerization, heterodimerization, DNA binding or the stimulation/inhibition of zcytorl3-dependent cellular responses. For example, zcytorl3-expressing cell lines can be transfected with a reporter gene construct that is responsive to a zcytorl3-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zcytorl3-DNA response element operably linked to a gene encoding an assay detectable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl.

Acad. Sci. USA 87: 5273-7,1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72,1989). Cyclic AMP response elements are reviewed in Roestler et al., J.

Biol. Chem. 263: 9063-6; 1988 and Habener, Molec. Endocrinol. 4 : 1087-94; 1990.

Hormone response elements are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts or conditioned media from various cell types are tested for the ability to enhance the activity of zcytorl3 signal transduction as evidenced by an increase in zcytorl3 stimulation of reporter gene expression. Assays of this type will detect compounds that directly stimulate zcytorl3 signal transduction activity through binding the upstream receptor or by otherwise stimulating part of the signal cascade in which zcytorl3 is involved. As such, there is provided a method of identifying agonists of zcytorl3 polypeptide, comprising providing cells expressing zcytorl3 responsive to a zcytorl3 pathway, 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 increase in a cellular response of the second portion of the cells as compared to the first portion of the cells. Moreover a third cell, containing the reporter gene construct described above, but not expressing zcytorl3 polypeptide,

can be used as a control cell to assess non-specific, or non-zcytorl3-mediated, stimulation of the reporter. Agonists are useful to stimulate or increase zcytorl3 polypeptide function.

Moreover, compounds or other samples can be tested for direct blocking of zcytorl3 binding to another protein or substrate, e. g., a heterodimer described below, using zcytorl3 tagged with a detectable label (e. g., 125I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zcytorl3 to the other protein or substrate is indicative of inhibitory activity, which can be confirmed through secondary assays. Proteins used within binding assays may be cellular proteins or isolated, immobilized proteins.

Zcytorl3 activation can be detected by: (1) measurement of adenylate cyclase activity (Salomon et al., Anal. Biochem. 58: 541-48,1974; Alvarez and Daniels, Anal. Biochem. 187: 98-103, 1990); (2) measurement of change in intracellular cAMP levels using conventional radioimmunoassay methods (Steiner et al., J. Biol. Chem.

247: 1106-13,1972; Harper and Brooker, J. Cyc. Nucl. Res. 1: 207-18,1975); or (3) through use of a cAMP scintillation proximity assay (SPA) method (Amersham Corp., Arlington Heights, IL). These methods provide sensitivity and accuracy.

An alternative assay system involves selection of polypeptides that are able to induce expression of a cyclic AMP response element (CRE)-luciferase reporter gene, as a consequence of elevated cAMP levels, in cells expressing a zcytorl3 polypeptide, but not in cells lacking zcytorl3 expression, analogous to such assays employing calcitonin receptor as described in U. S. patent No. 5,622,839, U. S. Patent No. 5,674,689, and U. S. patent No. 5,674,981.

In addition, polypeptides of the present invention can be assayed and used for their ability to modify inflammation. Methods to determine proinflammatory and anti-inflammatory qualities of zcytorl3 polypeptide, its agonists or antagonists, are known in the art and discussed herein. For example, suppression of cAMP production is an indication of anti-inflammatory effects (Nihei, Y., et al., Arch. Dermatol. Res., 287: 546-552,1995). Suppression of cAMP and inhibition of ICAM and HLA-Dr induced by IFN-y in keratinocytes can be used to assess inhibition of inflammation.

Alternatively, enhancement of cAMP production and induction of ICAM and HLA-Dr

in this system can be an measurement of proinflammatory effects of a protein. As a phosphodiesterase, cytorl3, likewise can exhibit similar inflammatory effects, and may exert these effects in tissues in which it is expressed, or indirectly in other tissues. For example, zcytorl3 is expressed in the heart and skeletal muscle, and can be useful in promoting wound healing in this tissue, or exhibit anti-bacterial or anti-viral effects.

Moreover, zcytorl3, its agonists or antagonists can be useful in treatment of inflammatory heart or cardiovascular conditions, muscle inflammation, inflammation during and after surgery, arthritis, asthma, inflammatory bowel disease, diverticulitis, and the like. Moreover, direct measurement of zcytorl3 polypeptide and anti-zcytorl3 antibodies can be useful in diagnosing inflammatory diseases such as reperfusion ischemia, inflammatory bowel disease, diverticulitis, asthma, pelvic inflammatory disease (PID), psoriasis, arthritis, melanoma, and other inflammatory diseases.

Moreover zcytorl3 antagonists can be useful in treatment of myocarditis, atherosclerosis, pelvic inflammatory disease, (PID), psoriasis, arthritis, eczema, scleroderma, and other inflammatory diseases.

As such, zcytorl3 polypeptide, agonists or its antagonists, have potential uses in inflammatory diseases such as asthma and arthritis. For example, if zcytorl3 is proinflammatory, antagonists would be valuable in asthma therapy or other anti- inflammatory therapies where migration of lymphocytes is damaging. In addition, zcytorl3 can serve other important roles in lung function, for instance, bronchodilation, tissue elasticity, recruitment of lymphocytes in lung infection and damage. Assays to assess the activity of zcytorl3 in lung cells are discussed in Laberge, S. et al., Am. J.

Respir. Cell Mol. Biol. 17: 193-202, 1997; Rumsaeng, V. et al., J. Immunol., 159: 2904- 2910,1997; and Schluesener, H. J. et al., J. Neurosci. Res. 44: 606-611, 1996. Methods to determine proinflammatory and antiinflammatory qualities of zcytorl3 its agonists or its antagonists are known in the art. Moreover, other molecular biological, immunological, and biochemical techniques known in the art and disclosed herein can be used to determine zcytorl3 activity and to isolate agonists and antagonists.

The activity of molecules of the present invention may be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (i. e., proliferation) of cardiac cells based on the potential effects of activity of zcytorl3 in

heart. Additional activities likely associated with the polypeptides of the present invention include proliferation of endothelial cells, cardiomyocytes, fibroblasts, skeletal myocytes directly or indirectly through other growth factors; action as a chemotaxic factor for endothelial cells, fibroblasts and/or phagocytic cells; osteogenic factor; and factor for expanding mesenchymal stem cell and precursor populations.

Proliferation can be measured in vitro using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model.

Generally, proliferative effects are seen as an increase in cell number, and may include inhibition of apoptosis as well as stimulation of mitogenesis. Cultured cells for use in these assays include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, and human umbilical vein endothelial cells from primary cultures. Suitable established cell lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma cells (Tanaka et al., Proc. Natl. Acad. Sci. 89: 8928-8932,1992), and LNCap. FGC adenocarcinoma cells (ATCC No. CRL-1740.) Assays measuring cell proliferation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8: 347-354,1990), incorporation of radiolabeled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989), incorporation of 5-bromo-2'- deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol.

Methods 82: 169-179,1985), and use of tetrazolium salts (Mosmann, J. Immunol.

Methods 65: 55-63,1983; Alley et al., Cancer Res. 48: 589-601,1988; Marshall et al., Growth Reg. 5: 69-84,1995; and Scudiero et al., Cancer Res. 48: 4827-4833, 1988).

Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made.

Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation.

Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and

receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed 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 : 731-738,1987), so identification is usually made at the progenitor and mature cell stages. 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 factors that stimulate specific cell types down a pathway towards terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor or stem cell. Thus, the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes and endothelial cells. Molecules of the present invention may, while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells. Thus molecules of the present invention may have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.

Assays measuring differentiation include, for example, measuring cell- surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5: 281-284,1991; Francis, Differentiation 57 : 63-75,1994; Raes, Adv. Anim. Cell Biol. Technol.

Bioprocesses, 161-171,1989; all incorporated herein by reference). Alternatively, zcytorl3 polypeptide itself can serve as an additional cell-surface marker associated with stage-specific expression of a tissue. As such, direct measurement of zcytorl3 polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for differentiation of heart, skeletal muscle tissue, or other tissues.

In vivo assays for evaluating cardiac neogenesis or hyperplasia include treating neonatal and mature rats with the molecules of the present invention. The

animals'cardiac function is measured as heart rate, blood pressure, and cardiac output to determine left ventricular function. Post-mortem methods for assessing cardiac decline or improvement include: increased or decreased cardiac weight, nuclei/cytoplasmic volume, and staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res. 75: 1050-1063,1994 and Reiss et al., Proc. Natl. Acad. Sci. 93: 8630- 8635,1996.) Zcytorl3 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zcytorl3. In addition to those assays disclosed herein, samples can be tested for inhibition of zcytorl3 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zcytorl3- dependent cellular responses. For example, zcytorl3-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zcytorl3-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zcytorl3-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci.

USA 87: 5273-7,1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563- 72,1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol.

Chem. 263: 9063-6 ; 1988 and Habener, Molec. Endocrinol. 4 : 1087-94 ; 1990. Hormone response elements are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zcytorl3 on the target cells as evidenced by a decrease in zcytorl3 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block effectors that bind zcytorl3, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zcytorl3 binding to receptor using zcytorl3 tagged with a detectable label (e. g., 125I, biotin, horseradish peroxidase, FITC, and the like).

Within assays of this type, the ability of a test sample to inhibit the binding of labeled

zcytorl3 to the receptor is indicative of inhibitory activity, which can be confirme through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.

The tissue specificity of zcytorl3 expression suggests a role in spermatogenesis, a process that is remarkably similar to the development of blood cells (hematopoiesis). Briefly, spermatogonia undergo a maturation process similar to the differentiation of hematopoietic stem cells. In view of the tissue specificity observed for zcytorl3, agonists and antagonists have enormous potential in both in vitro and in vivo applications. Zcytorl3 polypeptides, agonists and antagonists may also prove useful in modulating spermatogenesis and thus aid in overcoming infertility.

Antagonists are useful as research reagents for characterizing sites of ligand-receptor interaction. In vivo, zcytorl3 polypeptides, agonists or antagonists may find application in the treatment of male infertility or as a male contraceptive agents.

The zcytorl3 polypeptides, antagonists of agonists, of the present invention can also modulate sperm capacitation. Before reaching the oocyte or egg and initiating an egg-sperm interaction, the sperm must be activated. The sperm undergo a gradual capacitation, lasting up to 3 or 4 hours in vitro, during which the plasma membrane of the sperm head and the outer acrosomal membrane fuse to form vesicles that facilitate the release of acrosomal enzymes. The acrosomal membrane surrounds the acrosome or acrosomal cap which is located at the anterior end of the nucleus in the sperm head. In order for the sperm to fertilize egg the sperm must penetrate the oocyte.

To enable this process the sperm must undergo acrosomal exocytosis, also known as the acrosomal reaction, and release the acrosomal enzymes in the vicinity of the oocyte.

These enzymes enable the sperm to penetrate the various oocyte layers, (the cumulus oophorus, the corona radiata and the zona pellucida). The released acrosomal enzymes include hyaluronidase and proacrosin, in addition to other enzymes such as proteases.

During the acrosomal reaction, proacrosin is converted to acrosin, the active form of the enzyme, which is required for and must occur before binding and penetration of the zona pellucida is possible. A combination of the acrosomal lytic enzymes and sperm tail movements allow the sperm to penetrate the oocyte layers. Numerous sperm must reach the egg and release acrosomal enzymes before the egg can finally be fertilized.

Only one sperm will successfully bind to, penetrate and fertilize the egg, after which the zona hardens so that no other sperm can penetrate the egg (Zaneveld, in Male Infertility Chapter 11, Comhaire (Ed.), Chapman & Hall, London, 1996). Peptide hormones, such as insulin homologs are associated with sperm activation and egg-sperm interaction.

For instance, capacitated sperm incubated with relaxin show an increased percentage of progressively motile sperm, increased zona penetration rates, and increased percentage of viable acrosome-reacted sperm (Carrell et al., Endocr. Res. 21: 697-707,1995).

Similarity of the zcytorl3 polypeptide structure with peptide hormones and localization of Zcytorl3 to the testis, prostate and uterus suggests that the zcytorl3 polypeptides described herein play a role in these and other reproductive processes.

Accordingly, proteins of the present invention can have applications in enhancing fertilization during assisted reproduction in humans and in animals. Such assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer and gamete intrafallopian transfer. Such methods are useful for assisting men and women who have physiological or metabolic disorders preventing natural conception or can be used to enhance in vitro fertilization. Such methods are also used in animal breeding programs, such as for livestock breeding and could be used as methods for the creation of transgenic animals. Proteins of the present invention can be combined with sperm, an egg or an egg-sperm mixture prior to fertilization of the egg. In some species, sperm capacitate spontaneously during in vitro fertilization procedures, but normally sperm capacitate over an extended period of time both in vivo and in vitro. It is advantageous to increase sperm activation during such procedures to enhance the likelihood of successful fertilization. The washed sperm or sperm removed from the seminal plasma used in such assisted reproduction methods has been shown to have altered reproductive functions, in particular, reduced motility and zona interaction. To enhance fertilization during assisted reproduction methods sperm is capacitated using exogenously added compounds. Suspension of the sperm in seminal plasma from normal subjects or in a"capacitation media"containing a cocktail of compounds known to activate sperm, such as caffeine, dibutyl cyclic adenosine monophosphate (dbcAMP) or theophylline, have resulted in improved reproductive function of the sperm, in particular, sperm motility and zonae penetration (Park et al.,

Am. J. Obstet. Gynecol. 158: 974-9, 1988; Vandevoort et al., Mol. Repro. Develop.

37: 299-304,1993; Vandevoort and Overstreet, J. Androl. 16: 327-33, 1995). The presence of immunoreactive relaxin in vivo and in association with cryopreserved semen, was shown to significantly increase sperm motility (Juang et al., Anim. Reprod.

Sci. 20: 21-9,1989; Juang et al., Anim. Reprod. Sci. 22: 47-53, 1990). Porcine relaxin stimulated sperm motility in cryopreserved human sperm (Colon et al., Fertil. Steril.

46: 1133-39,1986; Lessing et al., Fertil. Steril. 44: 406-9,1985) and preserved ability of washed human sperm to penetrate cervical mucus in vitro (Brenner et al., Fertil. Steril.

42: 92-6,1984). Polypeptides of the present invention can used in such methods to enhance viability of cryopreserved sperm, enhance sperm motility and enhance fertilization, particularly in association with methods of assisted reproduction.

In cases where pregnancy is not desired, zcytorl3 polypeptide or polypeptide fragments may function as germ-cell-specific antigens for use as components in"immunocontraceptive"or"anti-fertility"vaccines to induce formation of antibodies and/or cell mediated immunity to selectively inhibit a process, or processes, critical to successful reproduction in humans and animals. The use of sperm and testis antigens in the development of immunocontraceptives have been described (O'Hern et al., Biol Reprod. 52: 311-39,1995; Diekman and Herr, Am. J. Reprod.

Immunol. 37: 111-17,1997; Zhu and Naz, Proc. Natl. Acad. Sci. USA 94: 4704-9, 1997).

A vaccine based on human chorionic gonadotrophin (HCG) linked to a diphtheria or tetanus carrier was in clinical trials (Talwar et al., Proc. Natl. Acad. Sci. USA 91: 8532- 36,1994). A single injection resulted in production of high titer antibodies that persisted for nearly a year in rabbits (Stevens, Am. J. Reprod. Immunol. 29: 176-88, 1993). Such methods of immunocontraception using vaccines would include a zcytorl3 testes-specific protein or fragment thereof. The Zcytorl3 protein or fragments can be conjugated to a carrier protein or peptide, such as tetanus or diphtheria toxoid.

An adjuvant, as described above, can be included and the protein or fragment can be noncovalently associated with other molecules to enhance intrinsic immunoreactivity.

Methods for administration and methods for determining the number of administrations are known in the art. Such a method might include a number of primary injections over

several weeks followed by booster injections as needed to maintain a suitable antibody titer.

Regulation of reproductive function in males and females is controlled in part by feedback inhibition of the hypothalamus and anterior pituitary by blood-borne hormones. Testis proteins, such as activins and inhibins, have been shown to regulate secretion of active molecules including follicle stimulating hormone (FSH) from the pituitary (Ying, Endodcr. Rev. 9 : 267-93,1988; Plant et al., Hum. Reprod. 8: 41- 44,1993). Inhibins, also expressed in the ovaries, have been shown to regulate ovarian functions (Woodruff et al., Endocr. 132: 2332-42,1993; Russell et al., J. Reprod. Fertil.

100: 115-22,1994). Relaxin has been shown to be a systemic and local acting hormone regulating follicular and uterine growth (Bagnell et al., J. Reprod. Fertil. 48: 127-38, 1993). As such, the polypeptides of the present invention may also have effects on female gametes and reproductive tract. These functions may also be associated with zcytorl3 polypeptides and may be used to regulate testicular or ovarian functions.

A zcytorl3 polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U. S. Patents Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other.

Fusions of this type can be used to affinity purify ligand, as an in vitro assay tool, or a zcytorl3 ligand antagonist. For use in assays, the chimeras are bound to a support via the Fc region and used in an ELISA format.

A zcytorl3 polypeptide can also be used for purification of ligand, biomolecular substrates, or other proteins or antibodies that bind it. The zcytorl3 polypeptide or a ligand-binding polypeptide fragment thereof can be used. The 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, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide

activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing ligand, membrane preparations, or lipid preparations, are passed through the column one or more times to allow ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCI), or pH to disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed 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 attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. 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 surface plasmon resonance of the gold film. This system allows the determination of on-and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (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).

Zcytorl3 polypeptides can also be used to prepare antibodies that bind to zcytorl3 epitopes, peptides or polypeptides. The zcytorl3 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One of skill in the art would recognize that antigenic, epitope-bearing

polypeptides 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 zcytorl3 polypeptide (e. g., SEQ ID NO : 2). Polypeptides comprising a larger portion of a zcytorl3 polypeptide, i. e., from 30 to 10 residues up to the entire length of the amino acid sequence are included. Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the zcytorl3 polypeptide encoded by SEQ ID NO : 2 from amino acid number 25 (Cys) to amino acid number 605 (Arg), or a contiguous 9 to 580 amino acid fragment thereof. Other suitable antigens include the n-terminal hydrophobic domain and the C-terminal phosphodiesterase domain, as disclosed herein. Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot, determined, for example, from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored (See, Figure 1). Zcytorl3 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 31 (Gln) to amino acid number 36 (Asp) of SEQ ID NO : 2; (2) amino acid number 238 (Pro) to amino acid number 333 (Glu) of SEQ ID NO : 2; (3) amino acid number 360 (Arg) to amino acid number 365 (Glu) of SEQ ID NO : 2; (4) amino acid number 540 (Arg) to amino acid number 545 (Asp) of SEQ ID NO : 2; and (5) amino acid number 600 (Ile) to amino acid number 605 (Arg) of SEQ ID NO : 2. Antibodies from an immune response generated by inoculation of an animal 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 would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zcytorl3

polypeptide or a fragment thereof. The immunogenicity of a zcytorl3 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zcytorl3 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is"hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine 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 F (ab') 2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally"cloaking"them with a human-like surface by replacement of exposed residues, wherein the result is a"veneered"antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed 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.

Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules. A threshold level of binding is determined if anti- zcytorl3 antibodies herein bind to a zcytorl3 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zcytorl3)

polypeptide. It is preferred that the antibodies exhibit a binding affinity (Ka) of 106 M- 1 or greater, preferably 107 M-1 or greater, mor epreferably 108 M-1 or greater, and most preferably 109 M-1 or greater. The binding affinity 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).

Whether anti-zcytorl3 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zcytorl3 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family, Screening can also be done using non-human zcytorl3, and zcytorl3 mutant polypeptides. Moreover, antibodies can be"screened against" known related polypeptides, to isolate a population that specifically binds to the zcytorl3 polypeptides. For example, antibodies raised to zcytorl3 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zcytorl3 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known 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). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology. Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1- 98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101,1984.

Specifically binding anti-zcytorl3 antibodies can be detected by a number of methods in the art, and disclosed below.

A variety of assays known to those skilled in the art can be utilized to detect antibodies which bind to zcytorl3 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays

include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zcytorl3 protein or polypeptide.

Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zcytorl3 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zcytorl3 protein or peptide). Genes encoding polypeptides having potential zcytorl3 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the zcytorl3 sequences disclosed herein to identify proteins which bind to zcytorl3. These"binding polypeptides"which interact with zcytorl3 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e. g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of zcytorl3 polypeptides; for detecting or quantitating soluble zcytorl3

polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as zcytorl3"antagonists"to block zcytorl3 binding and signal transduction in vitro and in vivo. These anti-zcytorl3 binding polypeptides would be useful for inhibiting zcytorl3 activity or protein-binding.

Antibodies to zcytorl3 may be used for tagging cells that express zcytorl3; for isolating zcytorl3 by affinity purification; for diagnostic assays for determining circulating levels of zcytorl3 polypeptides; for detecting or quantitating soluble zcytorl3 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zcytorl3 activity in vitro and in vivo. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to zcytorl3 or fragments thereof may be used in vitro to detect denatured zcytorl3 or fragments thereof in assays, for example, Western Blots or other assays known in the art.

Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, zcytorl3 polypeptides or anti-zcytorl3 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.

Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the

like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, 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 attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/anticomplementary pair.

In another embodiment, polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, 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 a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein 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 targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, zcytorl3-cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the zcytorl3 polypeptide or anti-zcytorl3 antibody targets the hyperproliferative blood or bone marrow cell (See, generally, Hornick et al., Blood 89: 4437-47,1997). Hornick et al. described fusion proteins that enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable zcytorl3 polypeptides or anti-zcytorl3 antibodies target an undesirable cell or tissue (i. e., a tumor or a leukemia), and the fused

cytokine mediate improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.

In yet another embodiment, if the zcytorl3 polypeptide or anti-zcytorl3 antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.

The polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with vasoconstriction, heart arrhythmia, heart inflammation, congestive heart disease, muscle spasms and fatigue, inflammation, testicular function, fertility, birth control, and the like. The molecules of the present invention can be used to modulate contractility or inflammation or to treat or prevent development of pathological conditions in such diverse tissue as heart, testis, and skeletal muscle. In particular, certain syndromes/diseases may be amenable to such diagnosis, treatment or prevention.

Diagnostic methods of the present invention involve the detection of zcytorl3 polypeptides in the serum or tissue biopsy of a patient undergoing analysis of heart, spleen, testicular or muscle function or evaluation for possible cancers. Such polypeptides can be detected using immunoassay techniques and antibodies, described herein, that are capable of recognizing polypeptide epitopes. More specifically, the

present invention contemplates methods for detecting zcytorl3 polypeptides comprising: exposing a test sample potentially containing zcytorl3 polypeptides to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a zcytorl3 polypeptide; washing the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zcytorl3 polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label. Altered levels of zcytorl3 polypeptides in a test sample, such as serum sweat, saliva, biopsy, and the like, can be monitored as an indication of heart, spleen, testicular or muscle function or of cancer or other disease, when compared against a normal control.

Additional methods using probes or primers derived, for example, from the nucleotide sequences disclosed herein can also be used to detect zcytorl3 expression in a patient sample, such as a blood, saliva, sweat, tissue sample, or the like.

For example, probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization. Zcytorl3 sequences can also be detected by PCR amplification using cDNA generated by reverse translation of sample mRNA as a template (PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared with a normal control, both increases or decreases of zcytorl3 expression in a patient sample, relative to that of a control, can be monitored and used as an indicator or diagnostic for disease. Similarly, anti-zcytorl3 antibodies or zcytorl3 binding polypeptides and the like can be used to detect zcytorl3 polypeptides as described above.

Moreover, as human zcytorl3 is specifically expressed in testis and spleen tissues, it is useful for detection of those tissues in a human biological sample, tissue sample, biopsy or histologic sample for example, to assess whether spleen or testis tissue is present. Zcytorl3 polynucleotide probes, anti-zcytorl3 antibodies and binding polypeptides, and detection of the presence of zcytorl3 polypeptides in tissue

can be used to assess whether spleen or testis tissue is present, for example, after surgery involving the excision of a diseased or cancerous spleen or testis. As such, the polynucleotides, polypeptides, and antibodies of the present invention can be used as an aid to determine whether all spleen or testis tissue is excised after surgery, for example, after surgery for spleen or testicular cancer. In such instances, it is especially important to remove all potentially diseased tissue to maximize recovery from the cancer, and to minimize recurrence. Preferred embodiments include fluorescent, radiolabeled, or calorimetrically labeled anti-zcytorl3 antibodies or binding partners, that can be used in situ.

As discussed above, the stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Assays measuring differentiation include, for example, measuring cell markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5: 281-284,1991; Francis, Differentiation 57: 63-75,1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171,1989; all incorporated herein by reference). Alternatively, zcytorl3 polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue. As such, direct measurement of zcytorl3 polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for differentiation of tissues. Such markers can also be used to assess the progress of cancers and metastasis.

Similarly, direct measurement of zcytorl3 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression. Increases in invasiveness and motility of cells, or the gain or loss of expression of zcytorl3 in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in tumor progression. As such, knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient. Methods of measuring gain and loss of expression (of either mRNA or protein) are well known in the art and described herein and can be applied to zcytorl3 expression. For example, appearance or disappearance of polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of

prostate cancer (Banyard, J. and Zetter, B. R., Cancer and Metast. Rev. 17: 449-458, 1999). As an effector of cell motility, zcytorl3 gain or loss of expression may serve as a diagnostic for spleen, testicular, lymphoid, B-cell, endothelial, hematopoietic and other cancers. Moreover, as zcytorl3 expression appears to be restricted to testis and spleen in normal human tissues, lack of zcytorl3 expression in these tissues or strong zcytorl3 expression in non-testis or non-spleen tissue would serve as a diagnostic of an abnormality in the cell or tissue type, and could aid a physician in directing further testing or investigation, or aid in directing therapy.

Moreover, the activity and effect of zcytorl3 on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice 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 daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 105 to 106 cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant adenovirus, such as one expressing zcytorl3, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically. The mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1500-1800 mm3 in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed

along with the lungs and the liver. The lung weight has been shown to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e. g., zcytorl3, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with zcytorl3. Use of stable zcytorl3 transfectants as well as use of induceable promoters to activate zcytorl3 expression in vivo are known in the art and can be used in this system to assess zcytorl3 induction of metastasis. Moreover, purified zcytorl3 or zcytorl3-conditioned media can be directly injected in to this mouse model, and hence be used in this system. For general reference see, O'Reilly MS, et al. Cell 79: 315-328,1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis 14: 349-361, 1995.

The activity of zcytorl3 and its derivatives (conjugates) on growth and dissemination of tumor cells derived from human hematologic malignancies can also be measured in vivo in a mouse Xenograft model Several mouse models have been developed in which human tumor cells are implanted into immunodeficient mice, collectively referred to as xenograft models. See Cattan, AR and Douglas, E Leuk. Res.

18: 513-22, 1994; and Flavell, DJ, Hematological Oncology 14: 67-82,1996. The characteristics of the disease model vary with the type and quantity of cells delivered to the mouse. Typically, the tumor cells will proliferate rapidly and can be found circulating in the blood and populating numerous organ systems. Therapeutic strategies appropriate for testing in such a model include antibody induced toxicity, ligand-toxin conjugates or cell-based therapies. The latter method, commonly referred to adoptive immunotherapy, involves treatment of the animal with components of the human immune system (i. e., lymphocytes, NK cells) and may include ex vivo incubation of cells with zcytorl3 or other immunomodulatory agents.

Polynucleotides encoding zcytorl3 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zcytorl3 activity. If a mammal has a mutated or absent zcytorl3 gene, the zcytorl3 gene can be introduced

into the cells of the mammal. In one embodiment, a gene encoding a zcytorl3 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), retroviruses, papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (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; Samulski et al., J. Virol. 63: 3822-8, 1989).

In another embodiment, a zcytorl3 gene can be introduced in a retroviral vector, e. g., 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. 62: 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. Natl. Acad. Sci. USA 84: 7413-7,1987; Mackey et al., Proc. Natl.

Acad. Sci. USA 85: 8027-31,1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides (e. g., hormones or neurotransmitters), proteins such as antibodies, or non- peptide molecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e. g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e. g., Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4,1988.

Antisense methodology can be used to inhibit zcytorl3 gene transcription, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zcytorl3-encoding polynucleotide (e. g., a polynucleotide as set froth in SEQ ID NO : 1) are designed to bind to zcytorl3-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zcytorl3 polypeptide-encoding genes in cell culture or in a subject.

The present invention also provides reagents which will find use in diagnostic applications. For example, the zcytorl3 gene, a probe comprising zcytorl3 DNA or RNA or a subsequence thereof can be used to determine if the zcytorl3 gene is present on chromosome 11 or if a mutation has occurred. Zcytorl3 is located at the llql3-ql4 region of chromosome 11 (see, Example 3). Detectable chromosomal aberrations at the zcytorl3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, translocations, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid. ; Marian, Chest 108 : 255-65,1995).

The precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing

model organisms, such as mouse, which may aid in determining what function a particular gene might have.

The zcytorl3 gene is located at the 11q13-q14 region of chromosome 11. Several genes of known function map to this region. For example, zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with growth hormone secreting (ghs+) adenomas and acromagaly, where a susceptibility marker maps to l 1q13 (Cho, J. H. et al, Proc. Nat. Acad. Sci. 95: 7502-7507,1998). In fact, some mutations in ghs+ pituitary adenomas are associated with increased phosphodiesterase activity, in part by PDE4 and possibly other phosphodiesterases (Lania et al., J. Clin. Endocr. Metab. 83: 1624-1628,1998). Zcytorl3, localized to the llql3-ql4, could also be a phosphodiesterase involved in pituitary adenomas or other tumors. As such, use of inventive anti-zcytorl3 antibodies, polynucleotides, and polypeptides can be used for the detection of zcytorl3 polypeptide, mRNA or anti- zcytorl3 antibodies, thus serving as markers and be directly used for detecting or diagnosing ghs+ adenomas or other cancers using methods known in the art and described herein. Moreover heart abnormalities can also be associated with 11ql3 acromegaly (Lopez-Velasco, R. et al., J. Clin. Endocr. Metab. 82: 1047-1053,1997). As zcytorl3 is expressed in heart, as well as localized to llql3, it may be involved in manifestation of this phenotype. Moreover, the IDDM4 susceptibility gene, conferring susceptibility to type 1 diabetes, maps to l lql3. Zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with IDDM4, such as those that are implicated in type 1 diabetes (Hashimoto, L. et al., Nature 371: 161-164,1994; Hey, P. J. et al., Gene 216: 103-111,1998). Further, zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome l 1q13 deletions and translocations, for example those associated with cervical carcinomas (Jedusan, R. A. et al., Am. J. Hum. Genet. 56: 705-715,1995). Moreover, amongst other genetic loci, those for Bardet-Biedl Syndrome, type 1, (llql3), Best disease (macular dystrophy) (llql3), vitreoretinopathy (llql3), fibrosis of extraocular muscles (llql3. 1), Alexander Disease (llql3), and an autosomal dominant deafness marker (DFNA11) (llql3. 5), Delta-7-dehydrcolesterol reductase (Smith-Lender-Opitz Syndrome) (llql2-ql3), and Phospholipase C (beta 3; phosphatidylinositol specific)

(llql3), all manifest themselves in human disease states as well as map to this region of the human genome. See the Online Mendellian Inheritance of Man (OMIMTM, National Center for Biotechnology Information, National Library of Medicine.

Bethesda, MD) gene map, and references therein, for this region of chromosome 11 on a publicly available WWW server (http ://www3. ncbi. nlm. nih. gov/htbin- post/Omim/getmap ? chromosome=llql3). All of these serve as possible candidate genes for an inheritable disease that show linkage to the same chromosomal region as the zcytorl3 gene. Thus, zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.

One of skill in the art would recognize that of zcytorl3 polynucleotide probes are particularly useful for diagnosis of gross chromosomal abnormalities associated with loss of heterogeneity (LOH), chromosome gain, translocation, DNA amplification, and the like. Translocations within chromosomal locus 11q13-14 wherein the zcytorl3 gene is located are known to be associated with human disease.

For example, llql3 deletions and translocations, are associated with cervical carcinomas (Jedusan, R. A. et al., Am. J. Hum. Genet. 56: 705-715,1995), and chronic lymphocytic leukemia (CLL) (Tsujimoto, Y et al., Nature 315: 340-343,1985).

Amplification of the l lql3 locus is frequently amplified in breast cancer (Szepetowski, P. et al., Genomics 16: 745-750, 1993). Moreover, amplification in human tumors involves a stretch of DNA much larger that the selected gene that is amplified. Thus, since the zcytorl3 gene maps to this critical region, zcytorl3 polynucleotide probes of the present invention can be used to detect abnormalities or genotypes associated with 1 lq 13 translocation and amplification described above.

Similarly, defects in the zcytorl3 gene itself may result in a heritable human disease state. Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zcytorl3 genetic defect. As such, zcytorl7 polynucleotides, polypeptides, and anti-zcytorl7 antibodies serve an important use as a diagnostic to detect defects in the zcytorl7 gene or protein, or defects in surrounding chromosomal regions at the 1 lql3-ql4 region of chromosome 11.

A diagnostic could assist physicians in determining the type of disease and appropriate associated therapy, or assistance in genetic counseling. As such, the inventive anti-zcytorl3 antibodies, polynucleotides, and polypeptides can be used for the detection of zcytorl3 polypeptide, mRNA or anti-zcytorl3 antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein. Further, zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome llql3-ql4 deletions and translocations associated with human diseases, such as cervical carcinomas and CLL as described above, other translocations involved with malignant progression of tumors or other llql3-ql4 mutations, which are expected to be involved in chromosome rearrangements in malignancy; or in other cancers, or in spontaneous abortion. Similarly, zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 1 lql3-ql4 trisomy and chromosome loss associated with human diseases or spontaneous abortion. Thus, zcytorl3 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.

As discussed above, defects in the zcytorl3 gene itself may result in a heritable human disease state. Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zcytorl3 genetic defect. In addition, zcytorl3 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zcytorl3 chromosomal locus. As such, the zcytorl3 sequences can be used as diagnostics in forensic DNA profiling.

In general, the diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art.

Analytical probes will be generally at least 20 nt in length, although somewhat shorter probes can be used (e. g., 14-17 nt). PCR primers are at least 5 nt in length, preferably 15 or more, more preferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, a zcytorl3 polynucleotide probe may comprise an entire exon or more. Exons are readily determined by one of skill in the art by comparing zcytorl3 sequences (SEQ

ID NO : 1) with the genomic DNA for zcytorl3 (e. g., Genbank Accession No's.

AP000744, AP002354, AP002815). Most diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a zcytorl3 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a zcytorl3 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing the visualized first reaction product to a second control reaction product of a genetic sample from wild type patient. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre-implantation embryo. For example, a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the zcytorl3 genetic locus, and the like, are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples. Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form 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 polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO : 1, the complement of SEQ ID NO : 1, or an RNA equivalent thereof. Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols : Current Methods and

Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc.

1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Mutations associated with the zcytorl3 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, 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 (Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al. (eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998), and Richards and Ward,"Molecular Diagnostic Testing,"in Principles of Molecular Medicine, pages 83- 88 (Humana Press, Inc. 1998)). Direct analysis of an zcytorl3 gene for a mutation can be performed using a subject's genomic DNA. Methods for amplifying genomic DNA, obtained for example from peripheral blood lymphocytes, are well-known to those of skill in the art (see, for example, Dracopoli et al. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).

Mice engineered to express the zcytorl3 gene, referred to as"transgenic mice,"and mice that exhibit a complete absence of zcytorl3 gene function, referred to as"knockout mice,"may also be generated (Snouwaert et al., Science 257: 1083,1992; Lowell et al., Nature 366: 740-42,1993; Capecchi, M. R., Science 244: 1288-1292, 1989; Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499,1986). For example, transgenic mice that over-express zcytorl3, either ubiquitously or under a tissue-

specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype. For example, over-expression of a wild-type zcytorl3 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zcytorl3 expression is functionally relevant and may indicate a therapeutic target for the zcytorl3, its agonists or antagonists. For example, a preferred transgenic mouse to engineer is one that over- expresses the zcytorl3 mature polypeptide (approximately residue 25 (Cys) to residue 605 (Arg) of SEQ ID NO : 2). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases. Similarly, knockout zcytorl3 mice can be used to determine where zcytorl3 is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zcytorl3 antagonist, such as those described herein, may have. The human zcytorl3 cDNA can be used to isolate murine zcytorl3 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. Transgenic and knockout mice may be employed to study the zcytorl3 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases.

Moreover, transgenic mice expression of zcytorl3 antisense polynucleotides or ribozymes directed against zcytorl3, described herein, can be used analogously to transgenic mice described above.

Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of zcytorl3 can be used as standards or as"unknowns"for testing purposes. For example, zcytorl3 polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein zcytorl3 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of zcytorl3 polynucleotides in tissues (i. e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.

Zcytorl3 polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed zcytorl3 polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i. e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo. Zcytorl3 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution. For example, a kit containing the zcytorl3 can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the specific protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of zcytorl3 would be unique unto itself.

Moreover, since zcytorl3 is a glycerophosphoryl diester phosphodiesterase activity can be measured using radioactive phospholipid substrates; cAMP assays described herein; substrates such as deacylation products of phosphatidylethanolamine, phosphatidylcholine, phosphatidyl glycerol, and the like by measuring hydrophilic alcohols; hydrolyzation of glycerol glycerophosphocholine and other glycerophosphodiesters possessing hydrophobic alcohols as described herein.

Such assays are well known in the art, and can be used in an educational setting to teach students about phosphodiesterases and examine different properties, such as enzyme kinetics on varying phospholipids substrates, and the like, between zcytorl3 and other glycerophosphoryl diester phosphodiesterases in the art..

The antibodies which bind specifically to zcytorl3 can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify zcytorl3, cloning and sequencing the polynucleotide that encodes an antibody

and thus as a practicum for teaching a student how to design humanized antibodies. The zcytorl3 gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology.

Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum. Such educational kits containing the zcytorl3 gene, polypeptide or antibody are considered within the scope of the present invention.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 Isolation and Cloning of the Human Zcytorl3 Receptor A. Using an EST Sequence to Obtain Full-length zcytorl3 Scanning of a translated murine cDNA database resulted in identification of an expressed orthologous sequence tag (OST) sequence which was used to identify an expressed sequence tag (EST) sequence in a translated human cDNA database.

A mouse probe, based on the identified OST was generated by PCR using oligos ZC20386 (SEQ ID NO : 22) and ZC20389 (SEQ ID NO : 23), and mouse 15- day embryo mcDNA (Clontech) as a template under the following reaction conditions: 94°C for 2 minutes, 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds; followed by 72°C for 5 minutes. The PCR fragment was gel purified using QIAquick gel extraction kit (Qiagen) and sequenced. A contig (SEQ ID NO : 24) of the 376 bp mouse probe with the mouse OST sequence was used to search an EST database and identify a human EST. Oligonucleotide primers were designed from the sequence of the identified human EST. The primers were used for priming within the EST to isolate a full-length clone from a human placental cDNA library (see, below). The full length cDNA was designated zcytorl3.

B. Isolation of full length zcvtorl3 cDNA : To obtain a full-length cDNA, A panel of several cDNA libraries was screened by PCR. PCR reactions were set up using EX-Taq plus antibody (Panvera, Madison, WI; Clontech) and oligo primers ZC21763 (SEQ ID NO : 4) and ZC21762 (SEQ ID NO : 5) under the following reaction conditions: 94°C for 2 min.; 35 cycles of 94°C for 30 sec., 62°C for 30 sec., 72°C for 30 sec.; followed by 72°C for 5 min.

Positive samples included human fetal brain library total pool cDNA, human pituitary library total pool cDNA, two human placenta library total pool cDNAs, human salivary gland library total pool cDNA, and three human testis library total pool cDNAs. Since the PCR panel gave a strong positive in placenta, the human placenta cDNA library was further screened by PCR to obtain a full-length cDNA clone.

The placenta library was an arrayed library representing 1.0 X 106 clones. The working pools (designated"hplc pools") contained 10 pools of 100,000 clones/pool. The hplc pools were screened by PCR, as described above, for the presence of a full-length cDNA. Of 9 positives from the hplc pools, one designated "hplc pool #1"which contained 100,000 clones, was further screened. The Hplc pool #1 contains 10 sub-pools of 10,000 clones/pool, designated"hplb pools."Three positives were obtained from the hplb pools which were further subjected to 5'RACE.

5'RACE was performed on the three positive hplb pools. PCR using oligos ZC13006 (SEQ ID NO : 6) and ZC21762 (SEQ ID NO : 5) was performed as follows: 94°C for 2 min.; 5 cycles of 94°C for 30 sec., 61°C for 30 sec., and 72°C for 1 min.; followed by 30 cycles 94°C for 30 sec, 56°C for 30 sec., and 72°C for 1 min.; followed by 72°C for 5 min. PCR product from one hplb pool (Hplb pool #7) was gel isolated (see, below) and sequenced. The DNA sequence contained an initiating Met codon confirming the 5'end of the cDNA. Hplb pool #7 is divided further into 10 sub- sub-pools containing 1000 clones/pool, designated"hpla pools."These hpla pools were then screened the same manner by PCR, as described above. One positive hpla pool was identified. 5'RACE was then performed on the positive pool to verify that the pool was indeed positive. PCR was performed using oligos ZC13006 (SEQ ID NO : 6) and ZC21762 (SEQ ID NO : 5) as follows: 94°C for 2 min.; 35 cycles of 94°C for 30 sec., 62°C for 30 sec., and 72°C for 1 min.; followed by 72°C for 5 min.

C. Isolation of full length zcvtorl3 cDNA : Human placental cDNA library hpla positive pool screen The positive Hpla pool (above) was plated and filter-lifted using Hybond-N filters (Amersham, England). A total of about 4800 colonies were screened on 4 filters lifted from plates of about 1200 colonies per plate. The filters were marked with a hot needle for orientation, then denatured for 6 minutes in 0.5 M NaOH and 1.5 M Tris-HCL pH 7.2. The filters were then neutralized in 1.5 M NaCI and 0.5 M Tris- HCL pH 7. 2 for 6 minutes. The DNA was affixed to the filters using a Stratalinker UV crosslinker (Stratagene, La Jolla, Ca.) at 1200 joules. The filters were prewashed at 65 degrees C in prewash buffer consisting of 0.25X SSC, 0.25% SDS and 1mM EDTA.

The solution was changed a total of three times over a 45-minute period to remove cell debris. Filters were prehybridized overnight at 65°C in 25ml Expresshyb (Clontech, Palo Alto, Ca.). The probe was generated by PCR using oligos ZC21763 (SEQ ID NO : 4) and ZC21762 (SEQ ID NO : 5), as described above. The PCR fragment was gel purified using QIAquick gel extraction kit (Qiagen, Santa Clarita, Ca.). The probe was radioactively labeled with 32p using the Rediprime U DNA Labeling system (Amersham, UK) according to Manufacturer's specifications. The probe was purified using a Nuctrap push column (Stratagene cloning system, La Jolla, Ca). Expresshyb (Clontech, Palo Alto, Ca) solution was used for the hybridizing solution for the filters.

Hybridization took place overnight at 65°C. Filters were rinsed 2X in 65°C in pre-wash buffer (0. 25X SSC, 0.25% SDS and lmM EDTA). Then the filters were washed 2X in pre-wash solution at 65°C. Filters were exposed to film for 3 days at-80°C. There was 3 positive clones on the filters. These 3 clones were picked and streaked out to obtain individual colonies. 11 individual colonies were subsequently screened by PCR, using oligos ZC21763 (SEQ ID NO : 4) and ZC21762 (SEQ ID NO : 5) as described above.

Two clones were sequenced using the following primers: vector primer ZC6583 (SEQ ID NO : 7), vector primer ZC5020 (SEQ ID NO : 8), polyA primer ZC7764b (SEQ ID NO : 9), ZC20387 (SEQ ID NO : 10), ZC21292 (SEQ ID NO : 11), ZC22993 (SEQ ID NO : 12), ZC22994 (SEQ ID NO : 13), ZC22995 (SEQ ID NO : 14), ZC22996 (SEQ ID NO : 15), ZC23250 (SEQ ID NO : 16), ZC23251 (SEQ ID NO : 17), ZC23252 (SEQ ID

NO : 18), and ZC23253 (SEQ ID NO : 19). The cDNA sequence for zcytorl3 was full length and double stranded, as shown in SEQ ID NO : 1. The corresponding amino acid sequence for zcytorl3 is shown in SEQ ID NO : 2.

Example 2 Tissue Distribution of zcytorl3 in Human and Mouse Tissues A. Human tissue blots probed with a human zcytorl3 probe Northern blot analysis was performed using Human Multiple Tissue Blots (MTN I, MTN II, and MTN E) (Clontech, Palo Alto, Ca) and Human Fetal Multiple Tissue Blots (Clontech, Palo Alto, Ca). A 204 bp human probe, based directly on the identified EST was generated by PCR using oligos ZC21763 (SEQ ID NO : 4) and ZC21762 (SEQ ID NO : 5), under the following reaction conditions: 94°C for 2 minutes; 35 cycles of 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds; followed by 72°C for 5 minutes. The PCR fragment was gel purified using QIAquick gel extraction kit (Qiagen, Santa Clarita, Ca.). The probe was radioactively labeled with 32p using the Rediprime II DNA Labeling system (Amersham, UK) according to Manufacturer's specifications. The probe was purified using a Nuctrap push column (Stratagene cloning system, La Jolla, Ca). Expresshyb (Clontech) solution was used for the hybridizing solution for the blots. Hybridization took place overnight at 65°C. The blots were then washed 4X in 2X SCC and 0.05% SDS at RT, followed by two washes in 0. 1X SSC and 0.1% SDS at 50° C. One transcript size was detected at approximately 4kb. Signal intensity was highest for testis and spleen. Moderate signal in placenta. Faint signal in fetal brain, fetal liver, fetal kidney, heart, brain, liver, skeletal muscle, pancreas, thymus, prostate, ovary, small intestine, colon, PBL's, spinal cord, lymph node, trachea, and adrenal gland. Two larger transcripts of 6 kb and 10.5 kb were seen in placenta and PBL's.

A Dot Blot was also performed using Human RNA Master BlotsTM (Clontech). The methods and conditions for the Dot Blot were the same as for the Multiple Tissue Blots disclosed above. Strong signal intensity was present in cerebellum, thalamus, testis, pancreas, salivary gland, mammary gland, spleen,

placenta, fetal kidney and fetal spleen. Less intense signals were indicated in all other tissues.

B. Mouse blots probed with a mouse zcytorl3 probe Northern blot analysis was performed using Murine Multiple Tissue Northern Blots (Mouse, Mouse Embryo, Clontech, Palo Alto, Ca; MB 1012, MB 1002 Origene, Rockville, Maryland). Blots were probed to determine the tissue distribution of murine zcytorl3 expression. A 376 bp mouse probe, based on the identified OST (Example 1) was generated by PCR using oligos ZC20386 (SEQ ID NO : 22) and ZC20389 (SEQ ID NO : 23), and mouse 15-day embryo mcDNA (Clontech) as a template under the following reaction conditions: 94°C for 2 minutes, 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds; followed by 72°C for 5 minutes.

The PCR fragment was gel purified using QIAquick gel extraction kit (Qiagen). The probe was radioactively labeled with 32p using the Rediprime II DNA Labeling system (Amersham, UK) according to Manufacturer's specifications. The probe was purified using a Nuctrap push column (Stratagene). Expresshyb (Clontech) solution was used for the hybridizing solution for the blots. Hybridization took place overnight at 65°C.

The blots were then washed 4X in 2X SCC and 0.05% SDS at RT, followed by two washes in 0. 1X SSC and 0.1% SDS at 50°C and a final wash in. 1X SSC and 0.1% SDS at 56° C. Three transcripts of approximately 1.0 kb, 1.35 kb and 4.0 kb were seen, showing strong expression in heart, skeletal muscle and liver. Weak but detectable expression was seen in lung and 17-day embryo, and only a 4.0 kb band was seen in brain, and only a 1.0 kb band seen in liver.

A Dot Blot was also performed using Murine RNA Master Dot Blot (Clontech). The methods and conditions for the Dot Blot were the same as for the Multiple Tissue Blots disclosed above. Expression was seen in brain, eye, liver, heart, skeletal muscle, and 7-day embryo. Expression was detectable but faintly seen in lung, kidney, smooth muscle, pancreas, thyroid, thymus, submaxillary gland, testis, ovary, prostate, epididymus, uterus, 11-day embryo, 15-day embryo and 17-day embryo.

C. Human blots probed with a mouse zcytorl3 probe Northern blot analysis was performed using Human Multiple Tissue Blots (MTN I, MTN II, and MTN (Clontech, Palo Alto, Ca) and Human Fetal Multiple Tissue Blots (Clontech, Palo Alto, Ca). A 376 bp mouse probe, based directly on the identified OST was generated by PCR and purified as described in Example 2B above. Expresshyb (Clontech, Palo Alto, Ca) solution was used for the hybridizing solution for the blots. Hybridization took place overnight at 65° C. The blots were then washed 4X in 2X SCC and 0.05% SDS at RT, followed by three washes in 1. OX SSC at 50° C. Two transcripts of approximately 1.8 kb and 4.0 kb were prominently seen in heart, and skeletal muscle, and also seen in spleen, testis, spinal cord, lymph node and trachea. Only a 4.0 kb band seen in brain, placenta, pancreas, and testis. Only a 1.8 kb band seen in thymus, prostate, ovary, thyroid, stomach and bone marrow. Signal intensity was strongest in heart and skeletal muscle.

D. Zoo blot with a mouse zcytorl3 probe Southern blot analysis was performed using a Zoo Blot (Clontech). A 376 bp mouse probe, based directly on the identified OST was generated by PCR, and purified as described in Example 2B above. Expresshyb (Clontech, Palo Alto, Ca) solution was used for the hybridizing solution for the blots. Hybridization took place overnight at 65° C. The blots were then washed 4X in 2X SCC and 0.05% SDS at RT, followed by three washes in 1. OX SSC at 50° C. A signal was seen in mouse, rat, cow, and potential in yeast and rabbit. A smear was seen in human.

Example 3 Chromosomal Assignent and Placement of Zcvtorl3 Zcytorl3 was mapped to chromosome 11 using the commercially available version of the"Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc., Huntsville, AL). The"Stanford G3 RH Panel"contains DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient). A publicly available WWW server (http ://shgc-

www. stanford. edu, Stanford Human Genome Center, Stanford University, CA) allows chromosomal localization of markers.

For the mapping of Zcytorl3 with the"Stanford G3 RH Panel", 20 ßl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a"RoboCycler Gradient 96"thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2 Fl 10X KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 Al dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 1 sense primer, ZC24, 347, (SEQ ID NO : 20), 1 1 antisense primer, ZC24, 348 (SEQ ID NO : 21), 2 til"RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 Ill 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH20 for a total volume of 20 Rl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 70°C and 1 minute AND 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72oC.

The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).

The results showed linkage of Zcytorl3 to the chromosome 11 marker SHGC-22371 with a LOD score of >14 and at a distance of 0 cR 10000 from the marker.

The use of surrounding genes, which have been cytogenetically mapped, positions Zcytorl3 in the llql3-ql4 chromosomal region.

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 deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.