MXPA99003530A - Fibroblast growth factor homologs - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zFGF-5, a novel member of the FGF family. The polypeptides, and polynucleotides encoding them, are proliferative for muscle cells and may be used for remodelling cardiac tissue and improving cardiac function. The present invention also includes antibodies to the zFGF-5 polypeptides.

Description

HOMOLOGOUS OF THE FIBROBLASTOS GROWTH FACTOR BACKGROUND OF THE INVENTION The family of fibroblast growth factor (FGF) consists of at least nine different members (Basilico et al., Adv Cancer Res. 59: 115-165, 1992 and Fernig et al., Proa. Growth Factor Res. 5 (4): 353-377 , 1994) which generally act as mitogens for a broad spectrum of cell types. For example, basic FGF (also known as FGF-2) is mitogenic in vitro for endothelial cells, vascular smooth muscle cells, fibroblasts and generally for cells of mesodermal or neuroectodermal origin, including cardiac and skeletal myocytes (Gospodarowicz et al., J. Cell, Biol. 70: 395-405, 1976, Gospodarowicz et al., J. Cell. Biol. 89: 568-578, 1981 and Kardami, J. Mol. Cell. Biochem. 92: 124-134, 1990. ). It has been shown that bFGF in vivo plays a role in cardiac development in birds (Sugi et al., Dev. Biol. 168: 567-574, 1995 and Mima et al., Proc. Nat'l. Acad. Sci. 2: 467-471, 1995), and induces coronary collateral development in dogs (Lazarous et al., Circulation 94: 1074-1082, 1996). In addition, non-mitogenic activities have been demonstrated for various members of the FGF family. Non-proliferative activities associated with acidic and / or basic FGF include: increased endothelial release of plasminogen activator REF: 30017 tissue, stimulation of extracellular matrix synthesis, chemotaxis for endothelial cells, induced expression of fetal contractile genes in cardio iocytes (Parker et al., J. Clin. Invest. 85: 507-514, 1990) and a improved hormonal response capacity of the pituitary or pituitary (Baird et al., J. Cellular Phvsiol 5: 101-106, 1987). Several members of the FGF family do not have a signal sequence (aFGF, bFGF and possibly FGF-9) and therefore would not be expected to be secreted. Several members of the FGF family have the ability to migrate to the cell nucleus (Friesel et al., FASEB 9: 919-925, 1995). All members of the FGF family are bound to heparin based on their structural similarities. Structural homology occurs between species, suggesting a conservation of its structure / function relationship (Ornitz et al., J. Biol. Chem. 271 (25): 15292-15297, 1996). There are four known extracellular FGF receptors (FGFR) and all of them are also tyrosine kinase. In general, members of the FGF family bind to all known FGFRs, however, specific FGFs bind to specific receptors with higher degrees of affinity. Another means for specificity within the FGF family is the spatial and temporal expression of ligands and their receptors during embryogenesis. The evidence suggests that FGF most likely act only in an autocrine and / or paracrine manner, due to their binding affinity to heparin, which limits their diffusion from the release site (Flaumenhaft et al., J. Cell. Biol. (4): 1651-1659, 1990). The basic FGF lacks a signal sequence and therefore is restricted to paracrine or autocrine modes of action. It has been postulated that basic FGF is stored intracellularly and released in the face of tissue damage. It has been shown that basic FGF has two receptor binding regions that are different from the heparin binding site (Abraham et al., EMBO J. 5 (10: 2523-2528, 1986) .FGFR-3 has been shown to play a role in bone growth Mice that have become agénicos homozygous for FGFR-3 (- / -) result in postnatal skeletal abnormalities (Colvin et al., Nature Genet 12: 309-397, 1996 and Deng et al. ., Cell 84: 911-921, 1996.) The mutant phenotype suggests that in normal mice, FGFR-3 plays a role in the regulation of cell division of chondrocytes in the region of the bone growth plate (Goldfarb, Cytokine and Growth Factor Rev. 7 (4): 311-325, 1996.) The ligand for FGFR-3 has not been identified in the bone growth plate, although four FGFRs have been identified., all of which have been shown to have variants of functional division, it is very likely the possibility that there are novel receptors for FGF. For example, a receptor for the FGF-8a isoform has not been identified (MacArthur et al., J. Virol. 69 (4): 2501-2507, 1995.). FGF-8 is a member of the FGF family that was originally isolated from mammary carcinoma cells as an androgen-inducible mitogen. It has been mapped to the human chromosome 10q25-q26 (White et al., Genomics 30: 109-11, 1995). FGF-8 is involved in the development of embryonic members (Vogel et al., Development 122: 1737-1750, 1996 and Tanaka et al., Current Bioloory 5 (6): 594-597, 1995). The expression of FGF-8 during embryogenesis in cardiac, urogenital and neural tissue indicates that it may play a role in the development of these tissues (Crossley et al., Development 121: 439-451, 1995). There is some evidence that acrocephalosyndactyly, a congenital condition marked by a cone-shaped head and wedge-shaped fingers and heels, is associated with point mutations of FGF-8 (White et al., 1995, ibid.). FGF-8 has five exons, in contrast to the other known FGFs, which only have three exons. The first three exons of FGF-8 correspond to the first exon of the other FGFs (MacArthur et al., Development 121: 3603-3613, 1995). The human gene for FGF-8 codes for four isoforms which differ in their N-terminal regions: the FGF isoforms a, b, e and f; in contrast to the gene in mice which results in eight isoforms of FGF-8 (Crossley et al., 1995, ibid). Human FGF-8a and FGF-8b have 100% homology with mouse proteins, and FGF-8e and FGF-8f proteins are 98% homologous between human and mouse (Gemel et al., Genomics 35: 253-257 , nineteen ninety six) . Heart disease is the leading cause of death in the United States, which constitutes up to 30% of all deaths. Myocardial infarction (MI) constitutes 750,000 hospital admissions per year in the United States, with more than 5 million people diagnosed with coronary heart disease. Risk factors for MI include diabetes mellitus, hypertension, core obesity, smoking, high plasma low density lipoprotein concentrations, or genetic predisposition. Cardiac hyperplasia is an increase in the proliferation of cardiac myocytes and has been shown to occur with normal aging in the human and rat (Olivetti et al., J. Am. Coil, Cardiol 24 (l): 140-9, 1994 and Anversa et al., Circ. Res. 67: 871-885, 1990), and catecholamine-induced cardiomyopathy in rats (Desiher et al., Am. J. Cardiovasc Pathol.5 (l): 79-88, 1994). Whether the increase in myocytes originates with some parent, or that is the result of proliferation of a more terminally differentiated cell type, still remains controversial. However, because the infarction and other causes of myocardial necrosis seem to be irreparable, it seems that the normal mechanisms of cardiac hyperplasia can not compensate for the excessive death of myocytes and the need remains for exogenous factors that promote hyperplasia and that ultimately result in renewal of the heart's ability to function. Bone remodeling is a dynamic process by which tissue mass and skeletal architecture are maintained. The process is a balance between bone resorption and bone formation, where two types of cells are considered to be the main elements. These cells are the osteoblasts and the osteoclasts. Osteoblasts synthesize and deposit matrix that becomes new bone. The activities of osteoblasts and osteoclasts are regulated by many factors, systemic and local, including growth factors. Although the interaction between local and systemic factors has not been fully elucidated, there seems to be a consensus that growth factors play a key role in the regulation of both normal skeletal remodeling and in the repair of fractures. Some of the growth factors that have been identified in bone include: IFG-I, IGF-II, TGF-ßl t TGF-S2, bFGF, aFGF, PDGF and the family of bone morphogenic proteins (Baylink, et al., J Bone Mineral Res. 8 (Supp.2): S565-S572, 1993).

When bone resorption exceeds bone formation, there is a net loss in bone, and the propensity for fractures increases. A decreased bone formation is associated with aging and certain pathological conditions. In the United States alone, there are approximately 1.5 million fractures annually that are attributed to osteoporosis. The impact of these fractures on the quality of life of patients is immense. The costs associated with the health care system in the United States are estimated to be $ 5- $ 10 billion annually, excluding the costs of long-term care. Other therapeutic applications for growth factors that influence bone remodeling include, for example, the treatment of damage which requires the proliferation of osteoblasts to heal, such as fractures as well as the stimulation of mesenchymal cell proliferation and synthesis. of intramembranous bone which is indicated in aspects of fracture repair (Joyce et al 36th Annual Meeting, Orthopedic Research Society, February 5-8, 1990. New Orleans, LA). The present invention provides such polypeptides for these and other uses that are apparent to those familiar in the art from the teachings herein.

BRIEF DESCRIPTION OF THE INVENTION Within one aspect, the present invention provides an isolated polynucleotide molecule that encodes the homologous fibroblast growth polypeptide (FGF) that is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEC. FROM IDENT. NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that code for a polypeptide that is at least 60% identical to the amino acid sequence of SEQ. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6 from nucleotide 82 to nucleotide 621. In a modality, the isolated polynucleotide molecule comprises a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1 from nucleotide 1 to nucleotide 621, or a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6 from nucleotide 1 to nucleotide 621. In another embodiment, the isolated polynucleotide molecule comprises a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1 from nucleotide 82 to nucleotide 621. In another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment that is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that code for a polypeptide that is at least 60% identical to the amino acid sequence of SEQ. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala), and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6, from nucleotide 82 to nucleotide 621; and a transcription terminator. In another aspect, the present invention provides a cultured cell into which an expression vector comprising the following operably linked elements has been introduced: a transcription promoter; a DNA segment that is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants - lu of (a); c) polynucleotide molecules that code for a polypeptide that is at least 60% identical to the amino acid sequence of SEQ. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6, from nucleotide 82 to nucleotide 621; and a transcription terminator, wherein the cell expresses a polypeptide encoded by the DNA segment. In another aspect, the present invention provides a method for producing a homologous FGF polypeptide comprising: culturing a cell in which an expression vector comprising the following operably linked elements has been introduced: a transcription promoter; a DNA segment that is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1, from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that code for a polypeptide that is at least 60% identical to the amino acid sequence of SEQ. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6, from nucleotide 82 to nucleotide 621; and a transcription terminator, whereby the cell expresses a homologous FGF polypeptide encoded by the DNA segment; and recovering the homologous FGF polypeptide. In another aspect, the present invention provides a homologous polypeptide of isolated FGF, which is selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 175 (Met); b) allelic variants of (a); e) polypeptide molecules that are at least 60% identical to SEC. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 175 (Met). In another aspect, the. present invention provides an isolated FGF homologous polypeptide that is selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 196 (Lys); b) allelic variants of (a); and c) polypeptide molecules that are at least 60% identical to SEC. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 196 (Lys). In another embodiment, the present invention provides an isolated FGF homologous polypeptide that is selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 207 (Wing); b) allelic variants of (a); and c) polypeptide molecules that are at least 60% identical to the amino acids of SEC. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala). In a further embodiment, the present invention provides a homologous FGF polypeptide that further comprises a signal sequence. In another embodiment, the present invention provides a homologous FGF polypeptide that further comprises a signal sequence as shown in SEQ. FROM IDENT. NO: 2, from amino acid residue 1 (Met) to the amino acid residue 27 (Ala). The present invention also provides a pharmaceutical composition comprising a homologous polypeptide of purified FGF, in combination with a pharmaceutically acceptable carrier. In another aspect, the present invention provides an antibody that binds to an epitope or antigenic determinant of a polypeptide molecule comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from the residue (Met) to the residue 207 (Ala). In another embodiment, the present invention provides an antibody that binds to a polypeptide molecule comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 196 (Lys). In another aspect, the present invention provides a method for stimulating the proliferation of myocytes or myocyte progenitors which comprises administering to a mammal in need thereof, an amount of a FGF homologous polypeptide sufficient to produce a clinically significant increase in the number of myocytes or myocyte progenitors in the mammal. In another embodiment, the present invention provides a method for stimulating the proliferation of myocytes or progenitors of myocytes, wherein the myocytes or progenitors of myocytes are cardiac myocytes or progenitors of cardiac myocytes. In another aspect, the present invention provides a method for ex vivo stimulation of myocyte progenitor cells or myocytes comprising culturing cardiac tissue cells with an amount of a FGF homologous polypeptide sufficient to produce an increase in the number of progenitor cells of myocytes or myocytes in cardiac tissue cells cultured in the presence of a FGF homologous polypeptide, as compared to myocyte progenitor cells or cardiac tissue myocytes cultured in the absence of a FGF homologous polypeptide.

In another embodiment, the present invention provides a method for the ex vivo stimulation of progenitor cells of myocytes or myocytes, wherein the myocytes or progenitors of myocytes are cardiac myocytes or progenitors of cardiac myocytes. In another aspect, the present invention provides a method for delivering an agent or medicament selectively to cardiac tissue, comprising: attaching a first molecule comprising a FGF homologous polypeptide to a second molecule comprising an agent or medicament for forming a chimera; and administering the chimera to cardiac tissue.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 and Figure 2 illustrate a multiple alignment of factor 1 homolog of human fibroblast growth factor (FHF-1), human myocyte activating factor (FGF-10), factor 4 homologue of human fibroblast growth factor (FIG. FHF-4), factor 2 homolog of human fibroblast growth factor (FHF-2), factor 3 human fibroblast growth homologue (FHF-3), human FGF-4, human FGF-6, human FGF-2 ( basic), human FGF-1 (acid), human keratinocyte growth factor 2 (KGF-2), human keratinocyte growth factor precursor (FGF-7), human zFGF-5, human FGF-8, human FGF-5, human FGF-9 and human FGF-3. The symbol "*" designates conserved amino acids; the symbol ":" designates substituted amino acid substitutions; and the symbol "." designates amino acid substitutions conserved less strictly. Figure 3 is a family similarity matrix illustrating percent identity between human FGF-5, human FGF-6, human FGF-7, human FGF-8, human FGF-9, human zFGF-5, FGF- Human, human FGF-1, human FHF-1, human FGF-2, human FHF-2, human FHF-4, human FGF-3, human KGF-2, human FHF-3 and human FGF-4.

DETAILED DESCRIPTION OF THE INVENTION The term "ortholog" (or "homologous species") denotes a polypeptide or protein obtained from a species having homology to a polypeptide or analogous protein of a different species. The term "paralog" denotes a polypeptide or protein obtained from a given species having homology to a different polypeptide or protein of the same species. The term "allelic variant" denotes any of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. The genetic mutations can be silent (without change in the encoded polypeptide) or can encode polypeptides having an altered amino acid sequence. The term allelic variant is also used herein to indicate a protein encoded by an allelic variant of a gene. The term "expression vector" denotes a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that are provided for transcription. Such additional segments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, an extender or enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated" when applied to a polynucleotide molecule indicates that the polynucleotide has been extracted from its natural genetic environment and is therefore free of other foreign or unwanted coding sequences and is in a form suitable for use within systems of production of proteins subjected to genetic engineering. Such isolated molecules are those that are separated from their natural environment including cDNAs and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they usually associate, but which may include naturally occurring 5 'and 3' untranslated regions, such as promoters and thermometers. The identification of associated regions will be apparent to a person familiar with the art (see for example Dynan and Tijan, Nature 316: 774-78, 1985). When applied to a protein, the term "isolated" indicates that the protein is in a condition different from that which occurs in its native environment, for example as part of the blood or tissue of the animal. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the protein in a highly purified form, ie, more than 95% pure, preferably more than 99% pure. The term "operably linked", when referring to DNA segments, indicates that the segments are arranged in such a way that they work in concert for their intended purposes, for example, transcription starts at the promoter and proceeds through the coding segment to the terminator The term "polynucleotide" denotes a single or double chain polymer of deoxyribonucleotide or ribonucleotide bases that are read from the 5 'end to the 3' end. The polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vi tro or prepared from a combination of natural and synthetic molecules. The term "polynucleotide molecule complements" denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'. The term "degenerate nucleotide sequence" indicates a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that codes for a polypeptide). Degenerate codons contain different triplets of nucleotides, but code for the same amino acid residue (ie, the triplets GAU and GAC encode, each for Asp). The term "promoter" indicates a portion of a gene that contains DNA sequences that are provided for the binding of RNA polymerase and the initiation of transcription. The promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes. 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 largest peptide is commonly removed to remove a secretory peptide during its transit through the secretory pathway. 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 multiple domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. The binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and the other molecule or molecules in the cell. In turn, this interaction leads to an alteration in the metabolism of the cell. Metabolic events that are related to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, cellular calcium mobilization, membrane lipid mobilization, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. Most nuclear receptors also show a multiple domain structure, which includes an amino-terminal transactivating domain, a DNA binding domain and a ligand-binding domain. In general, receptors can be membrane, cytosolic or nuclear bound; monomeric (eg, thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric receptor (eg, the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM-CSF receptor, the G-CSF receptor, erythropoietin receptor and IL-6 receptor). The term "complement / anticomplement pair" denotes non-identical portions that form a stable, non-covalently associated pair under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototype members of a complement / anticomplement 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. When the subsequent dissociation of the complement / anticomplement pair is desirable, the complement / anticomplement pair or preferably has a binding affinity of < The present invention is based in part on the discovery of a novel DNA sequence encoding a homologous polypeptide of fibroblast growth factor (FGF) having homology to FGF-8. In weaving the mRNA corresponding to this novel DNA shows that the expansion is higher in fetal cardiac tissue and in adult cardiac tissue, followed by neither apparent but decreased expression concentrations in fetal lung, skeletal muscle, smooth muscle tissues such as small intestine, colon and trachea The homologous polypeptide of FGF has been termed as zFGF-5 The novel zFGF-5 polypeptides of the present invention were initially identified by requesting an EST database for growth factors. of EST and was predicted to be related to the FGF family.The novel FGF homologous polypeptide encoded by the full-length cDNA contains a motif of the formula: CXFXEX. { 6.} And, where X is any amino acid and X { } is the number of amino acids X greater than one. This motif occurs in all known members of the FGF family and is unique for these proteins. The nucleotide sequence of the cDNA for zFGF-5 is described in SEQ. FROM IDENT. NO: 1, and its deduced amino acid sequence is described in SEQ. FROM IDENT. NO: 2. When comparing amino acid residue 28 (Glu) with amino acid residue 181 (Gln) of SEC. FROM IDENT. NO: 2 with the corresponding region of FGF-8 (see Figures 1 and 2), the aligned and deduced amino acid sequence has approximately 56% identity. The novel polypeptide encoded by the polynucleotide described herein contains the CXFXE motif. { 6.} And in all the members of the FGF family. The CXFXE motifs. { 6.} And they are highly conserved. An amino acid consensus sequence of the CXFXEX domain. { 6.} And it includes factor 1 homologs of human fibroblast growth factor (FHF-1, Smallwood et al., Proc. Nati, Acad. Sci. USA 93_: 9850-9857, 1996), the human myocyte activating factor (FGF) -10; HSU76381, GENBANK identifier, http://www.ncbi.nlm.nih.gov/), factor 4 homologue of human fibroblast growth factor (FHF-4; Smallwood et al., 1996, ibid.), factor 2 homologue of human fibroblast growth factor (FHF-2; Smallwood et al., 1996, iJbid.), factor 3 homologue of human fibroblast growth factor (FHF-3; Smallwood et al., 1996, ibid. ), Human FGF-4 (Basilico et al., Adv. Cancer Res. 59: 115-165, 1992), human FGF-6 (Basilico et al., 1992, ibid.), Human FGF-2 (basic; Basilico et al., 1992, ibid. ), Human FGF-1 (acid, Basilico et al., 1992, iJbid.), Human keratinocyte growth factor 2 (KGF-2; HSU67918 GENBANK identifier, http://www.ncbi.nlm.nih.gov/) ), precursor of human keratinocyte growth factor (FGF-7, Basilico et al., 1992, ibid.), human zFGF-5, human FGF-8 (Gemel et al., Genomics 35: 253-257, 1996) , Human FGF-5 (Basilico et al., 1992, ibid.), Human FGF-9 (Miyamoto et al., Mol. Cell, Biol. 13: 4251-4259, 1993), and human FGF-3 (Basilico et al. al., 1992, ibid). Analysis of the cDNA encoding zFGF-5 polypeptide (SEQ ID NO: 1) shows an open reading frame for 207 amino acids (SEQ ID NO: 2) comprising a mature polypeptide of 180 amino acids (SEQ ID NO: 2). residue 28 to residue 207 of SEQ ID NO: 2). The multiple alignment of zFGF-5 with other known FGFs shows a block of a high percent identity corresponding to amino acid residue 127 (Cys) with amino acid residue 138 (Tyr), of SEQ. FROM IDENT. NO: 2 and shown in the figure. Several of the members of the FGF family do not have signal sequences. The members of the FGF family are characterized by heparin binding domains. A putative heparin binding domain for zFGF-5 has been identified in the region from amino acid residue 148 (Gly) to amino acid residue 169 (Gln) of the SEC. FROM IDENT. NO: 2. It has been postulated that receptor-mediated signaling starts before the binding of the FGF ligand that forms a complex with heparin sulfate proteoglycans on the surface of the cell. Many members of the FGF family can be placed in one of two related families in phase in their structures and functions. aFGF and bFGF consist of three exons separated by two introns of variable length. FGF-8 consists of five exons, the first three of which correspond to the first exon of aFGF and bFGF. All known members of the FGF family are divided to form unique polypeptides.

The SEC. FROM IDENT. NO: 6 is a degenerate polynucleotide sequence encompassing all nucleotides that can encode the zFGF-5 polypeptide of the SEC. FROM IDENT. NO: 2 (amino acids 1 or 28 to 207). Therefore, the polynucleotides encoding the zFGF-5 polypeptide vary from nucleotide 1 or 82 to nucleotide 621 of SEQ. FROM IDENT. NO: 6 are contemplated by the present invention. Fragments and fusions are also contemplated by the present invention as described above with respect to SEC. FROM IDENT. NO: 1 which are formed from analogous regions of the SEC. FROM IDENT. NO: 6, wherein nucleotides 82 to 621 of SEQ. FROM IDENT. NO: 6 correspond to nucleotides 82 to 621 of the SEC. FROM IDENT. NO: 1 for the coding of a mature molecule of zFGF-5. The symbols in the SEC. FROM IDENT. NO: 6 are summarized in table 1 below.

GABLA 1 Nucleotide Resolutions Complement Resolutions A A T T C C G G G G C C T T A A R A | G Y c | t Y C | T R A | G M A | C K G | T K G | T M A | C S C | G S C | G c | G A | -T W A | T H A | C T D A | G | T B C | G T V A | C | G V A | C G B C | G | T D A | G T H A | C | T N A | C G | T N A | C | G | T The degenerate codons used in SEC. FROM IDENT. NO: 6 encompass all possible codons for a given amino acid, as set forth in Table 2 below.

TABLE 2 AminoLetra Degenerated Acid Codon Codons Cys C TGC TGT TGY Ser S AGC AGT TCA TCG TCT TCN WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGC GGG GGG GGG 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 He I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr AND TAC TAT TAY Trp W TGG TGG Ter • TAA TAG TGA TRR AsnIAsp B RAY Glu | Gln Z SAR Any X NNN Separation - A person usually familiar with the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons coding for each amino acid. For example, in some circumstances, the degenerate codon for serine (WSN) encodes for arginine (AGR) and the degenerate codon for arginine (MGN) in some circumstances can be modified for serine (AGY). There is a similar relationship between the codons that code for phenylalanine and leucine. Therefore, some polynucleotides encompassed by the degenerate sequence may have some incorrect amino acids, but a person usually familiar with the art can easily identify such erroneous sequences with reference to the amino acid sequence of the SEC. FROM IDENT. NO: 2. The highly conserved amino acids in zFGF-5 can be used as a tool to identify new members of the family. To identify new members of the family in EST databases, you can use the conserved CXFXEX motif. { 6.} Y. In another method, using polynucleotide probes and hybridization methods, RNA obtained from a variety of tissue sources can be used to generate cDNA libraries and probe these libraries for new members of the family. In particular, the reverse transcription polymerase chain reaction (RT-PCR) can be used to amplify sequences that encode highly degenerate DNA primer primers designed from sequences corresponding to amino acid residue 127 (Cys) to the amino acid residue 138 (Tyr) of the SEC. FROM IDENT. NO: 2. Within the preferred embodiments of the present invention, the isolated polynucleotides will serve as a probe and will hybridize with regions of similar size of the SEC. FROM IDENT. NO: 1 or a sequence complementary to it, under conditions of restriction. In general, it is selected that the restriction conditions are from 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. Typical restriction conditions are those in which the salt concentration is at least about 0.02 M at pH 7 and the temperature is at least about 60 ° C.

As previously indicated, isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from cardiac tissue, although DNA can also be prepared using RNA from other tissues or isolated from genomic DNA. Total RNA can be prepared using extraction with guanidine hydrochloride followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Nati, Acad. Sci. USA 69: 1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. Then the polynucleotides encoding the zFGF-5 polypeptides are identified and isolated, for example, by hybridization or by PCR. The present invention also provides counterpart polypeptides and polynucleotides of other species (orthologs or paralogs). Among the zFGF-5 polypeptides of particular interest of other mammalian species include mouse, rat, porcine, ovine, bovine, canine, feline, equine and other primate proteins. The identification of the paralogs of the human sequence are particularly interesting because although 8 paralogs of mouse FGF-8 have been identified, only 4 human paralogs are known. Human paralogs or homologous species of human proteins 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 the protein. Suitable sources of mRNA can be identified by Northern blotting with probes designed from the sequences described herein. A library is then prepared from mRNA of a positive tissue or cell line. Then cDNA encoding zFGF-5 can be isolated by various methods, such as probing with whole or partial human cDNA with one or more sets of degenerate probes based on the described sequences. CDNA can also be cloned using the polymerase chain reaction or PCR (Mullis, US Pat. No. 4,683,202), using primers designed from the sequences described herein. Within a further 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 zFGF-5. Similar techniques can also be applied for the isolation of genomic clones. Those familiar with the art will recognize that the sequences described in SEQ. FROM IDENT. NO: 1 and SEC.

FROM IDENT. NO: 2 represent a single allele of the human zFGF-5 gene and polypeptide and that allelic variation and alternative division is expected to occur. Allelic variants can be cloned by probing cDNA or genomic libraries of different individuals, according to conventional or standard procedures. Allelic variants of the DNA sequence are shown in SEC. FROM IDENT. NO: 1, including those that contain silent mutations and those in which the mutations result in changes in the amino acid sequence, which are within the scope of the present invention insofar as they are proteins which are allelic variants of the SEC. FROM IDENT. NO: 2. The present invention also provides isolated polypeptides of zFGF-5 that are substantially homologous to polypeptides of SEQ. FROM IDENT. NO: 2 and its homologous / orthologous species. The term "substantially homologous" is used herein to mean polypeptides having 50%, preferably 60%, more preferably at least 80% sequence identity with the sequences shown in SEQ. FROM IDENT. NO: 2 or its orthologs or its paralogs. Such polypeptides will most preferably be at least 90% identical, and much more preferably 95% identical or more, with respect to SEC. FROM IDENT. NO: 2 or its orthologs or paralogs. The percent identity in the sequence is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616. 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915-10919. 1992. Briefly, two amino acid sequences are aligned to optimize their alignment qualifications using a separation gap penalty of 10, a separation extension penalty of 1 and the "blosu 62" rating matrix of Henikoff and Henikoff (ibid. ) as shown in Table 3 (amino acids are indicated by standard one-letter codes). The percent identity is then calculated as: > PU 2 LD L? Ís ro ro O ro in ro ro < o ro ro ro ro Ro H o ro P ro r-0 ro 1 55 Ro o o ro ro ro ro H o r H 1 rf; ^ H H or H H H H 1 < Pt¡ o s w o ¡u H w < F I H CN ro H 1 m CN CN O 1 1 ** ro CN CN 1 1 1 r r-l ro CN 1 1 1 1 KD CN CN H ro H I 1 1 or CN H H H H H 1 1 1 1 1 ro H O H ro CN CN 1 1 1 1 1 O ro CN t-t CN H H 1 1 1 1 1 o ro CN H ro H ro 1 1 1 1 1 H CN H CN CN CN ro 1 1 1 1 1 ro CN O CN CN ro ro 1 1 1 1 1 1 ro O ro CN CN 1 1 1 1 1 1 ro H O H CN r-l CN 1 1 1 1 1 1 CN ro H H CN CN H l 1 1 1 1 1 ro H o H ro ro 1 1 1 1 1 1 ro CN H O CN ro 1 1 1 1 1 ro H CN ro ro CN ro 1 1 1 l l 1 1 CN < -l r-l O ro CN O 1 1 1 1 LXJ OJ C? E- »Total number of identical matches x 100 [length of the largest sequence plus the number of separations entered in the largest sequence in order to align the two sequences] The sequence identity of polynucleotide molecules is determined by similar methods using a ratio as described above. The substantially homologous proteins and polypeptides are characterized in that they have 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 which do not significantly affect the legacy or activity of the protein or polypeptide; small deletions, typically from one to about 30 amino acids; and small extensions in the amino or carboxyl terminal portion, such as a methionine residue in the amino terminal part, a small linker peptide or up to about 20-25 residues, or a small extension that facilitates purification (affinity tag), such as a tract of poly-histidine, protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Method Enzvmol 198: 3, 1991), glutathione S transferase (Smith and Johnson, Gene 6731. 1988), maltose binding protein (Kellerman and Ferenci, Methods Enzvmol, 90: 459-463, 1982).; Guan et al., Gene 67: 21-30. 1987), or another antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991, which is incorporated herein by reference. DNAs encoding affinity tags are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ, New England Biolabs, Beverly, MA).

Table 4 Conservative amino acid substitutions Basic: argmma lysine histidine Acid: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatics: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The proteins of the present invention may also comprise, in addition to the standard or conventional 20 amino acids, amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine, allo-threonine, methyltreonine, hydroxyethyl cysteine, hydroxyethyl homocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, 4-fluorophenylalanine, 4-hydroxyproline, 6-N-methyl-lysine, 2- aminoisobutyric, isovaline and a-methylserine. Various methods are known in the art for incorporating amino acid residues into proteins which do not occur naturally. For example, an in vi tro system where nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs can be used. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. The transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113: 2722. 1991; Ellman et al., Meth. Enzymol. 202: 301. 1991; Chung et al. , Science 259: 806-09, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 90: 10145-49. 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: 19991-98, 1996). Within a third method, E. coli cells are activated in the absence of a natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired amino acid that does not occur naturally (e.g., 2-azaphenylalanine, -azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). The amino acid that does not occur naturally is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 33.:74,470-76, 1994. Naturally occurring amino acid residues can be converted to species that do not occur naturally by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993). The essential amino acids in the zFGF-5 polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine analysis mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989 ). In the latter technique, unique mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for biological activity (eg proliferation of cardiac myocytes or fibroblasts, or stimulation of bone formation) to identify the residues amino acids that are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-4708, 1996. Ligand-receptor interaction sites can also be determined by physical analysis of the structure, determined by techniques such as nuclear magnetic resonance, crystallography , electron diffraction, or photoaffinity labeling, together with mutation of putative amino acids from the contact site. See, for example, de Vos et al., Science 255: 306-312. 1992; Smith et al., J. Mol. Biol. 224: 899-904. 1992; Wlodaver et al., FEBS Lett. 309.-59-64, 1992. The identities of the essential amino acids can also be inferred from analysis of homologies with related FGFs and are shown in Figures 1 and 2. The analysis of the amino acid sequence of zFGF-5 shows a dibasic site in the C-terminal part of the polypeptide (amino acid residue 196-197 (Lys-Arg)). It has been shown that a truncated C-terminal polypeptide comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 196 (Lys), has biological activity. Dibasic amino acids such as Arg-X-X-Arg (wherein X is any amino acid residue), Arg-Arg or Lys-Arg; they are subject to separation by several enzymes including, but not limited to, thrombin and carboxypeptidases. Therefore, it is within the scope of the claims to make conservative changes in the dibasic amino acid residues, in particular, the dibasic residues in amino acid residues 196 and 197 (Lys and Arg, respectively) of the SEC. FROM IDENT. NO: 2. Based on the analysis of the FGF family, a molecule truncated in the C-terminal part comprising from amino acid residue 28 (Glu) to residue 175 (Met) of SEC. FROM IDENT. NO: 2 can be biologically active. It is predicted that an intramolecular disulfide bond occurs between amino acid residue 109 (Cys) and residue 129 (Cys) of the SEC. FROM IDENT. NO: 2 Based on the homology alignments with the crystal structures FGF-1 and FGF-2 (Eriksson et al., Prot. Sci. 2: 1274. 1993), the predictions of the secondary structure for the structure of the beta chain of zFGF -5 correlates with amino acid residues 56-59, 64-69, 73-76, 85-92, 96-102, 106-111, 115-119, 128-134, 138-144, 149-155, and 173 -177 of the SEC. FROM IDENT. NO: 2. Critical amino acids for the binding of zFGF-5 to receptors can be identified by site-directed mutagenesis of the entire zFGF-5 polypeptide. More specifically, it can be identified using site-directed mutagenesis of amino acids in the zFGF-5 polypeptide which corresponds to the amino acid residues in FGF acid (FGF1) and basic FGF (FGF2) identified as critical for the binding of these FGFs to their receptors. (Blaber et al., Biochem. 35: 2086-2094, 1996). These amino acids include Tyr33, Arg53, ASN110, Tyrll2, Lysll9, Trpl23, Leul49 and Metl51 in human FGF2, and Tyr30, Arg50, Asnl07, Tyrl09, Lysll6, Trpl22, Leul48 and Leul50 in human FGF1, as shown in Figure 1 and in Figure 2. The corresponding amino acids in zFGF-5, as shown in Figure 1 and Figure 2, would be Tyr58, Gly77, Asnl36, Tyrl38, Lysl45, Trpl49, Met175 and Argl77. A person familiar with the art will recognize that other members, in whole or in part of the FGF family, may exhibit structural or biochemical similarities with zFGF-5 and may be substituted for such analysis. Such regions would be important for biological functions of the molecule. Multiple amino acid substitutions can be performed and tested using known methods of mutagenesis and analysis such as those described by Reidhaar-Olson and Sauer (Science 241: 53-57, 1988) or Bowie and Sauer (Proc. Nat. Acad. Sci. USA 86: 2152-2156, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selected 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 (eg, Lowman et al., Biochem 30: 10832-10837, 1991, Ladner et al., U.S. Patent No. 5,223,409, Huse, WIPO publication WO 92/06204) and Site-directed mutagenesis Derbyshire et al., Gene 46: 145.1986: Ner et al., DNA 7127, 1988). Mutagenesis methods as described above can be combined with high throughput automated analysis methods to detect the activity of the cloned and mutagenized polypeptides in host cells. Mutagenized DNA molecules that code for active polypeptides (eg, cell proliferation) can be recovered from the host cells and can be rapidly sequenced using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods described above, a person usually familiar with the art can identify and / or prepare a variety of polypeptides that are substantially homologous to residues 28 (Glu) to 196 (Lys) or residues 28 (Glu) to 207 ( Ala) of the SEC. FROM IDENT. NO: 2, the allelic variants thereof or the biologically active fragments thereof, and retain the proliferative properties of the wild-type protein. Such polypeptides may also include additional polypeptide segments, as generally described above. The polypeptides of the present invention include full-length proteins, fragments thereof and fusion proteins, can be produced in host cells subject to genetic engineering according to conventional techniques. Suitable host cells are those types of cells which can be transformed or transfected with exogenous DNA and which can be grown in culture, which include bacteria, fungal cells and cultured higher eukaryotic cells. Eukaryotic cells are preferred, particularly cultured cells of multicellular organisms. Techniques for manipulating cloned DNA and introducing exogenous DNA into various host cells are described by Sambrook et al. , Molecular Clonincr: A Laboratory Manual, 2a. 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, which are incorporated herein by reference. In general, a DNA sequence encoding a zFGF-5 polypeptide of the present invention is operably linked to other genetic elements necessary for its expression, which generally include a transcription promoter and a 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 familiar with the art will recognize that within certain systems selectable markers can be provided on separate vectors and exogenous DNA replication can be provided by integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a usual matter of design within the skill level usual in the art. Many such elements are described in the literature and are available through commercial providers. To direct a zFGF-5 polypeptide within the secretory pathway of a host cell, a secretory signal sequence (also known as leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be a native sequence or a chimera comprising a signal sequence derived from another secreted protein (e.g. t-PA and secretory leader a-pre-pro) or synthesized de novo. The secretory signal sequence binds to the DNA sequence for zFGF-5 in the correct reading frame. Secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest. Although certain signal sequences may be placed elsewhere in the DNA sequence of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743, Holland et al., U.S. Patent No. 5,143,830). A universal acceptor plasmid that can be used to clone a DNA encoding any polypeptide of interest, and including fusion polypeptides, is disclosed. The acceptor plasmid is useful within a method for preparing a double stranded circular DNA molecule. The method comprises the steps of: (a) providing a double-stranded donor DNA fragment encoding a polypeptide of interest, - (b) providing a linear, double-stranded acceptor plasmid having a first and second blunt ends and comprising a selectable marker and a replication sequence that is functional in Saccharomyces cerevisiae, wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest; (c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to the first region of the acceptor plasmid and a second segment identical in sequence to the first region of the donor DNA fragment, wherein each of the first and second segments of the first linker is at least 10 bp in length; (d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to the second region of the acceptor plasmid and a second segment identical in sequence to the second region of the donor DNA fragment, wherein each of the first and second segments of the second linker is at least 10 bp in length; and (e) combining the donor DNA fragment, the acceptor plasmid, the first DNA linker and the second DNA linker in a host cell of Saccharomyces cerevisiae whereby the donor DNA fragment binds to the acceptor plasmid by homologous recombination of the Donor DNA, the acceptor plasmid and the linkers to form a closed circular plasmid. The acceptor plasmid further comprises a transcription promoter proximal to the first end, and the donor DNA fragment is operably linked to the transcription promoter within the closed circular plasmid. The acceptor plasmid further comprises a DNA segment encoding a leader peptide and / or one or more DNA segments encoding a peptide tag, positioned such that these DNA segments are operably linked to the donor DNA fragment within a plasmid circular closed. Within a preferred embodiment, the acceptor plasmid further comprises: (a) a promoter, a DNA segment encoding a leader peptide, and a DNA segment encoding a first peptide tag, wherein the DNA segment encoding for a leader peptide is placed between the promoter and the DNA segment encoding a first peptide tag proximal to the first terminus of the acceptor plasmid, wherein the promoter, the DNA segment encoding a leader peptide and the DNA segment which encodes for a first peptide tag, are linked operably; and (b) a DNA segment encoding a second peptide tag proximal to the end segment of the acceptor plasmid. A method for preparing a circular, double-stranded DNA molecule comprising the steps of (a) providing a plurality of overlapping, double-stranded donor DNA fragments which encode collectively for a polypeptide of interest; (b) providing a linear double-stranded acceptor plasmid having a first and second blunt ends and comprising a selectable marker and a replication sequence that is functional in Saccharomyces cerevisiae, wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest; (c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to the first region of the acceptor plasmid, and a second segment identical in sequence to a first region of one of the donor DNA fragments, wherein each of the first and second segments of the first linker is at least 10 bp long; (d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to a second region of the acceptor plasmid and a second segment identical in sequence to a region of another of the donor DNA fragments, wherein each of the first and second segments of the linker is at least 10 bp in length; and (e) combining the donor DNA fragments, the acceptor plasmid, the first DNA linker and the second DNA linker in a host cell of Saccharomyces cerevisiae whereby the donor DNA fragments are bound to the acceptor plasmid by homologous recombination for forming a closed circular plasmid comprising a region encoding the polypeptide of interest. The acceptor plasmid further comprises one or more of a transcription promoter, a segment of DNA encoding a leader peptide and one or more segments of DNA encoding a peptide tag as described above. Mycotic cells, including yeast cells, and particularly cells of the genera Saccharomyces or Pichia, are particularly preferred cells for hosts for the production of zFGF-5 fragments or polypeptide fusions. Other methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, by 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, which are incorporated by reference herein. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An alternative preferred vector system for use in yeast is the POT1 vector system described by Kawasaki et al (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in medium containing glucose. Suitable promoters and terminators for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S. Patent No. 4,615,947, and Bitter, U.S. Patent No. 4,977,092; , which are incorporated by reference in this document) and genes for alcohol dehydrogenase. See also U.S. Patent Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, which are incorporated herein by reference. Transformation systems are known in the art for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maya, Pichia pastoris, Pichia guillermondii and Candida maltose. A particularly preferred system uses Pichia methanolica (see, PCT application WO 9717450). For alternative transformation systems see, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465. 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by reference. The methods for transforming Acremonium chrysogenum are described by Sumino et al., U.S. Patent No. 5,162,228, which is incorporated by reference herein. Methods for transforming Neurospora are described by Lambowitz, U.S. Patent No. 4,486,533, which is incorporated herein by reference. Cultured mammalian cells are also preferred 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, Soma ic Cell Genetics 7: 603. 1981; Graham and Van der Eb, Virology : 2: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley et al. Sons, Inc., NY, 1987), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993), which are incorporated herein by reference. The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., 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, which are incorporated herein by reference. Preferred cultured mammalian cells include cell lines COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), 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., CH0-K1; ATCC No. CCL 61). Additional suitable cell lines are known in the art and are available from public depositaries such as American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as the SV-40 or cytomegalovirus promoters. See, for example, U.S. Patent No. 4,956,288. Other suitable promoters include those of metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978, which are incorporated by reference herein) and the adenovirus major late promoter. Drug selection is generally used to select 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 that are capable of passing the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene that codes for resistance to the antibiotic neomycin. The selection is carried out in the presence of a neomycin-type medicament, such as G-418 or the like. Selection systems can also be used to increase the level of expression of the gene of interest, a process termed "amplification". The amplification is carried out by culturing transfectants in the presence of a low concentration of the selective agent and then increasing the amount of selective agent to select the cells that produce high concentrations of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Other higher eukaryotic cells, including insect cells, plant cells and poultry cells, can also be used as hosts. The transformation of insect cells and the production of foreign polypeptides therein are described by Guarino et al., U.S. Patent No. 5,162,222; Bang et al., U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463, which are incorporated by reference herein. The use of Agrobacterium rhizogenes as a vector for expression in genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components necessary for the growth of the chosen host cells. A variety of suitable means are known in the art that include defined means and complex media and generally include a carbon source, a source of nitrogen, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, as required. The growth medium will generally be selected for cells that contain the exogenously added DNA, for example, selection by drug or deficiency in an essential nutrient which is supplemented by the selectable marker carried on the expression vector or cotransfected in the host cell. The recombinant zFGF-5 polypeptides expressed (or chimeric polypeptides) can be purified using methods and means of fractionation and / or conventional purification. Ammonium sulfate precipitation and catropo acid extraction can be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high resolution liquid chromatography. The suitable anion exchange medium includes derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas and the like. PEI, DEAE, QAE and Q derivatives are preferred (particularly DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ)) 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 spheres, resins based in silica, cellulosic resins, agarose spheres, cross-linked agarose spheres, polystyrene spheres, cross-linked polyacrylamide resins and the like which are insoluble under the conditions in which they are to be used .These supports can be modified with reactive groups that allow the protein binding by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. of coupling include activation by cyanogen bromide, activation by N-hydroxysuccinimide, activation by epoxide, activation by sulfhydryl, activation by hydrazide and carboxyl and amino derivatives for coupling chemistry with carbodiimide. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for attaching receptor polypeptides to support media are well known in the art. The selection of a particular method is a matter of usual design and is determined in part by the properties of the support chosen. See, for example, Affinity Chromatography: Principies & Methods. Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988. The polypeptides of the present invention can also be isolated by taking advantage of their heparin binding properties. For a review see, Burgess et al., Ann. Rev. of Biochem. 58: 575-606, 1989. Members of the FGF family can be purified to apparent homogeneity by affinity chromatography on heparin-Sepharose (Gospodarowicz et al., Proc. Nati, Acad. Sci. 81: 6963-6967, 1984). and are eluted using linear NaCl gradients (Ron et al., J. Biol. Chem. 268 (4): 2984-2988, 1993; Chromatocrraphy: Principies &Methods, pp. 77-80, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1993, in "Immobilized Affinity Ligand Techniques", Hermanson et al., Eds., Pp. 165-167, Academic Press, San Diego, 1992; Kjellen et al., Ann. Rev. Biochem. Ann. Biochem 60: 443-474, 1991; and Ke et al., Protein Expr. Purif. 3 (6): 497-507, 1992). Other purification methods include the use of ionic absorption chromatography with immobilized metals (IMAC) to purify histidine rich proteins. Briefly, a gel is first charged with divalent metal ions to form a chelate (E. Sulkowski, Trends in Biochem.3: 1-7, 1985). The proteins rich in histidine will be absorbed into this matrix with different affinities, based on the metal ion used, and eluted by competitive elution, by lowering the pH or by the use of strong chelating agents. Other purification methods 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). Alternatively, a fusion of the polypeptide of interest and an affinity tag (eg, polyhistidine, maltose-bound protein, immunoglobulin domain) can be constructed to facilitate purification. Protein renaturation processes (and optionally reoxidation) can be advantageously used. It is preferred to purify the protein up to >80% purity, more preferably up to > 90% purity, even more preferably > 95% and is particularly preferred as a pharmaceutically pure state, that is greater than 99.9% with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious or pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. ZFGF-5 polypeptides or fragments thereof can also be prepared through chemical synthesis. The zFGF-5 polypeptides may be monomers or ultimers; glycosylated or non-glycosylated, pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue. The activity of the molecules of the present invention can be measured by using a variety of assays that measure, for example, neogenesis or hyperplasia (i.e., proliferation) of cardiac cells based on the tissue specificity of the adult heart. Additional reactivities 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 chemotactic factor for endothelial cells, fibroblasts and / or phagocytic cells; osteogenic factor and factor to expand mesenchymal pluripotent cells and precursor populations. Proliferation can be measured using cultured cardiac cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. Generally, the proliferative effects are observed as an increase in the number of cells and therefore may include the inhibition of apoptosis as well as mitogenesis. Cultured cells include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, endothelial cells of the human umbilical vein of primary cultures. Established cell lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), chum CHH-1 heart cells (ATCC No. CRL-1680), rat heart myoblasts H9c2 (ATCC No. CRL-1446), Shionogi mammary carcinoma (Tanaka et al., Proc. Nati, Acad. Sci. 89: 8928-8932, 199) and adenocarcinoma cells LNCap.FGC (ATCC No. CRL-1740). Assays to measure cell proliferation are well known in the art. For example, assays that measure proliferation include assays such as chemosensitivity neutral red dye (Cavanaugh et al., Investigational New Drug 8: 347-354, 1990, incorporated herein by reference), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989, incorporated by reference herein), the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J Immunol. Methods 82: 169-179, 1985, incorporated by reference herein), and the 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 Rea 5: 69-84, 1995, and Scudiero et al., Cancer Res. 48: 4827-4833, 1988, all incorporated by reference herein. ). Differentiation is a dynamic and progressive process that begins with pluripotent cells and ends with terminally differentiated cells. Pluripotent cells that can regenerate without damage to a line express a set of differentiation markers that are lost when binding to a cell line is made. The progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress to the cell line path to maturity. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cellular products, enzymes to produce cellular products and receptors. The stage of differentiation of a population of cells is monitored by identification of markers present in the cell population. It is considered that myocytes, osteoblasts, adipocytes, chondrocytes, fibroblasts and reticular cells originate from a pluripotentially common mesenchymal cell (Owen et al., 5 Ciba Fdn, Symp 136: 42-46, 1988). Markers for mesenchymal pluripotent cells have not yet been well defined (Owen et al., J. of Cell Sci. 87: 731-738, 1987), so that identification is usually performed, in the stages of the progenitor cell and mature The existence of cells early myocyte cardiac progeny (often referred to as cardiac myocyte pluripotent cells) has been speculated, but has not been demonstrated, in adult cardiac tissue. The novel polypeptides of the present invention are useful for studies to isolate cells mesenchymal pluripotent cells and cardiac progeny of cardiac myocytes, both in vivo and ex vivo. There is evidence to suggest that the factors that stimulate specific types of cells down a path to terminal differentiation or dedifferentiation, affects the total population of cells that originates from a common precursor or pluripotential cell. Therefore, the present invention includes the stimulating inhibition or proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chondrocytes and endothelial cells.

The molecules of the present invention, while stimulating the proliferation or differentiation of cardiac myocytes, can inhibit the proliferation or differentiation of adipocytes by virtue of affecting their common precursor / pluripotent cells. Therefore, the molecules of the present invention have use in inhibition of chondrosarcomas, atherosclerosis, restenosis and obesity. Tests that measure differentiation include, for example, measuring cell surface markers associated with specific expression of a tissue cap, 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 as reference in this document). In vivo assays to evaluate cardiac neogenesis or hyperplasia include treating immature neonatal rats with the molecules of the present invention. The cardiac function of the animals is measured as the heart rate, the blood pressure the cardiac output to determine the left ventricular function. Post-mortem methods to determine cardiac improvement include: increased cardiac weight, core / cytoplasmic volume, staining of cardiac histological slices to determine nuclear antigen concentrations in proliferating cells (PCNA) compared to cytoplasmic actin concentrations (Quaini et al. ., Circulation Res. 75: 1050-1063, 1994 and Resis et al., Proc. Nati, Acad. Sci. 93: 8630-8635, 1996). In vivo tests to measure change in bone formation rates include performing bone histology (see Recker, R., eds, Bone Histomorphometry: Techniques and Interpretation, Boca Raton: CRC Press, Inc., 1983) and quantitative computer tomography (QCT).; Ferreti, J. Bone 17: 353S-364S, 1995; Orphanoludakis et al., Investig. Radiol. 14: 122-130 .. 1979 and Durand et al., Medical Phvsics 19: 569-573. 1992). An ex vivo assay for measuring changes in bone formation would, for example, be a calavarial or cranial test (Gowen et al., J. Immunol., 136: 2478-2482, 1986). With respect to the modulating energy balance, particularly as it relates to the metabolism, proliferation and differentiation of adipocytes, zFGF-5 polypeptides modulate the effects on metabolic reactions. Such metabolic reactions include adipogenesis, gluconogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization and the like. Among other methods known in the art or described herein, the energy balance of mammals can be evaluated by monitoring one or more of the metabolic functions mentioned above. These metabolic functions are monitored by techniques (tests or animal models) known to those usually familiar in the art, as more fully set forth in the following. For example, the effects of glucose-regulating insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue. In skeletal muscle and adipose tissue, insulin acts to stimulate the uptake, storage and utilization of glucose. There are recognized methods in the art for monitoring all of the metabolic functions mentioned above. Therefore, a person usually familiar with the art will be able to evaluate zFGF-5 polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists for metabolic modulating functions. Exemplary modulating techniques are set forth below. For example, insulin-stimulated lipogenesis can be monitored by measuring the incorporation of 14C-acetate into the triglyceride (Mackall et al., J. Biol. Chem. 251: 6462-6464, 1976) or triglyceride accumulation (Klet et al. , Mol, Pharmacol, 41: 393-398, 1992). The uptake stimulated by zFGF-5 can be evaluated, for example, in a test for glucose transport stimulated by insulin. Primary adipocytes or NIH 3T3 Ll cells (ATCC No. CCL-92.1) are placed in DMEM containing 1 g / 1 glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After 2 to 5 hours of culture, the medium is replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate and 2 mM glutamine. The appropriate concentrations of zFGF-5, insulin or IGF-1 or a series of dilutions of the test substance are added, and the cells are incubated for 20-30 minutes. Deoxyglucose labeled with 3 H or 14 C is added to "50 μM final concentration and the cells are incubated for approximately 10-30 minutes. The cells are then moistened with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g., 1% SDS or 1 N NaOH). The cell lysate is then evaluated by counting in a scintillation counter. Cell-associated radioactivity is taken as a measure of glucose transport after subtracting the non-specific binding determined by incubating cells in the presence of quitocalasin b, an inhibitor of glucose transport. Other methods include those previously described, for example, by Manchester et al. , Am. J. Physiol. 266 (Endocrinol Metab 29): E326-E333, 1994 (glucose transport stimulated by insulin). Insulin synthesis can be evaluated, for example, by comparing the precipitation of 35S-methionine labeled proteins followed by incubation of the test cells with 35S-methionine and 35S-methionine and a putative modulator of protein synthesis.

Thermogenesis can be assessed as described by B. Stanley in The Biology of Neuropeptide and and Related Peptides, W. Colmers and Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C. Billington et al., Am. J. Physiol. 260: R321, 1991; N. Zarjevski et al., Endocrinolocry 133: 1753, 1993; C. Billington et al., Am. J. Physiol. 266: R1765. 1994; Heller et al., Am. J. Physiol. 252 (4 Pt 2): R661-7. 1987; and Heller et al., Am. J. Physiol. 245 (3): R321-8, 1983. In addition, the metabolic rate which can be measured by a variety of techniques, is an indirect measurement of thermogenesis. Oxygen utilization can be evaluated as described by Heller et al., Pflugers Arch 369 (1): 55-9, 1977. This method also involves an analysis of the hypothalmic temperature and the metabolic production of heat. Oxygen utilization and thermoregulation have also been evaluated in humans as described by Haskell et al. , J. Appl. Physiol. 51 (4): 948-54, 1981. The zFGF-5 polypeptides can also be used to prepare antibodies that specifically bind epitopes, peptides or polypeptides of zGFG-5. Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989, and Hurrell, JGR, Ed. , Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, which are incorporated herein by reference). And as would be evident to a person usually familiar with the technique, polyclonal antibodies can be generated from a variety of homeothermic animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats. The immunogenicity of a zFGF-5 polypeptide can be increased by the use of an adjuvant, such as alumina (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zFGF-5 or a portion thereof with an immunoglobulin polypeptide or with a maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such a portion may be advantageously linked or bound 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" include polyclonal antibodies, polyclonal antibodies purified by affinity, monoclonal antibodies and antigen-binding fragments, such as F (ab ') 2 and proteolytic fragments of Fab. Also included are intact antibodies or genetically engineered fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as peptides and polypeptides that bind to synthetic antigens. Non-human antibodies can be immunized by grafting only non-human CDRs onto a human backbone and constant regions or by incorporating all of the non-human variable domains (optionally by hiding them with a human-like surface by substitution of exposed residues). , where the result is a "coated" antibody). In some cases, humanized antibodies can retain non-human residues within the human variable region structure domains to improve the appropriate binding characteristics. Through humanized antibodies, the biological average duration can be increased, and the potential for adverse immune reactions before administration to humans is reduced. Alternatively, techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zFGF-5 protein or peptide., and the selection of antibody display libraries in phage vectors or the like (e.g., by use of immobilized or labeled zFGF-5 protein or peptide). Antibodies are defined to be specifically bound if they bind to a zFGF-5 polypeptide with a binding affinity (K of 10"M or greater, preferably 107 M" 1 or greater, more preferably 108 M "1 or greater , and much more preferably 109 M "1 or greater.The binding affinity of an antibody can be easily determined by a person usually familiar with the art (eg, by Scatchard analysis) .A variety of known assays can be used. by those familiar with the art for detecting antibodies which bind specifically to zFGF-5 proteins or peptides .. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), transfer of m wide or Western blot test, inhibition of the competition test and interposition test (sandwich). In addition, the antibodies can be analyzed for binding to wild-type zFGF-5 protein or peptide compared to a mutant. Antibodies to zFGF-5 can be used to label cells that express zFGF-5; to target another protein, a small molecule or a chemical for cardiac tissue; for isolating zFGF-5 by affinity purification, for diagnostic tests to determine the circulating concentrations of zFGF-5 polypeptides; to detect or quantify soluble zFGF-5 as a marker of underlying pathology or disease; in analytical methods that use FACS; for analysis of expression libraries; to generate anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zFGF-5 mediated proliferation in vitro or in vivo. Suitable labels or address labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect labels or brands can make use of biotin-avidin pairs or another complement / anticomplement pair as intermediaries. The antibodies herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates can be used for in vivo diagnosis or other therapeutic applications. The molecules of the present invention can be used to identify and isolate receptors involved in proliferation of cardiac myocardium. For example, the proteins and peptides of the present invention can be immobilized on a column and membrane preparations can be run on the column (Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., Academic Press, San Diego, CA, 1992). , pp.195-202). Proteins and peptides can also be radiolabelled (Methods in Enzymol .. vol.182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or can be labeled by photoaffinity (Brunner et al., Ann. Rev. Biochem., 62: 483-514, 1993 and Fedan et al., Biochem Pharmacol 33: 1167-1180, 1984) and proteins specific for the cell surface can be identified. Antagonists will be useful for inhibiting the proliferative activities of zFGF-5 molecules, in cell types such as cardiac cells, which include myocytes, fibroblasts and endothelial cells; osteoblasts and chondrocytes. Genes coding for the zFGF-5 polypeptide that bind domains can be obtained by analysis of random peptide libraries displayed on phages (display in phage) or on bacteria such as E. coli. The nucleotide sequences coding for polypeptide can be obtained in numerous ways, for example by random mutagenesis and random synthesis of polynucleotides. These random peptide display libraries can be used for analyzes in search of 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 analyzing such random peptide display libraries are known in the art (Ladner et al., U.S. Patent No. 5,223,409, Ladner et al., U.S. Patent No. 4,946,778, Ladner et al., U.S. Patent No. 5,403,484 and Ladner et al., U.S. Patent No. 5,571,698) and random peptide display libraries and kits for analysis of such libraries are commercially available, for example, from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be analyzed using zFGF-5 sequences described herein to identify proteins which bind to zFGF-5. These "binding proteins" which interact with zFGF-5 polypeptides can be used for cell labeling; to isolate homologous polypeptides by affinity purification; they can be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like. These binding proteins can also be used in analytical methods such as analysis expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays to determine circulating polypeptide concentrations; to detect or quantify soluble polypeptides as markers of pathology or underlying disease. These binding proteins can also act as "antagonists" of zFGF-5 to block the binding of zFGF-5 and the transduction of an in vi tro and in vivo signal. These zFGF-5 binding proteins can be useful for inhibiting the expression of genes which result in proliferation or differentiation. Such zFGF-5 binding proteins can be used for the treatment, for example, of rhabdomyosarcoma, cardiac myxoma, bone cancers of osteoblastic origin and dwarfism, arthritis, ligament and cartilage repair, either alone or in combination with other therapies. The molecules of the present invention will be useful for the proliferation of cardiac tissue cells, such as myocytes or cardiac myoblasts; myocytes or skeletal myoblasts and smooth muscle cells; chondrocytes; endothelial cells; adipocytes and osteoblasts in vi tro. For example, the molecules of the present invention are useful as components of defined cell culture media and can be used alone or in combination with other cytokines and hormones to substitute serum that is commonly used in cell culture. The molecules of the present invention are particularly useful in specifically promoting the growth and / or development of myocytes in culture, and may also prove useful in the study of cardiac myocyte hyperplasia and regeneration. The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the treatment of disorders associated with myocardial infarction, congestive heart failure, hypertrophic cardiomyopathy and dilated cardiomyopathy. The molecules of the present invention may also be useful for limiting the size of the infarction subsequent to heart attack, promoting angiogenesis and wound healing after angioplasty or endythectomy, to develop coronary collateral circulation, for revascularization in the eye, for complications related to a poor circulation such as diabetic foot ulcer, for attack, after coronary reperfusion using pharmacological methods and other indications where angiogenesis is beneficial. The molecules of the present invention may be useful for improving cardiac function, either by inducing neogenesis and / or hyperplasia of cardiac myocytes, by inducing coronary collateral formation, or by inducing remodeling of the necrotic myocardial area. Other therapeutic uses for the present invention include induction of neogenesis and / or skeletal muscle hyperplasia, kidney regeneration and / or for the treatment of systemic and pulmonary hypertension. Coronary collateral development induced by zFGF-5 is measured in rabbits, dogs or pigs using models of chronic coronary occlusion (Landau et al., Amer. Heart J. 29: 924-931, 1995; Sellke et al., Surgerv 120 (2): 182-188. 1996 and Lazarous et al., 1996, ibid.). The benefits of zFGF-5 for treating an attack are tested in vivo in rats using bilateral occlusion of the carotid artery and by measuring histological changes as well as maze performance (Gage et al., Neurobiol., Anging 9: 645-655 1988). The efficacy of zFGF-5 in hypertension is tested in vivo using spontaneous hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl 1: S114-116, 1995). The molecules of the present invention can be used to target the delivery of agents or drugs to the heart. For example, the molecules of the present invention will be useful for limiting expression to the heart, by virtue of the tissue-specific expression directed by the zFGF-5 promoter. For example, specific expression for the heart can be obtained using an adenoviral discistronic construct for zFGF-5 (Rothmann et al., Gene Therapy 3: 919-926, 1996). In addition, zFGF-5 polypeptides can be used to restrict other cardiac tissue agents or drugs by binding zFGF-5 polypeptides to another protein (Franz et al., Circ.Res. 73: 629-638, 1993) by linking a first molecule that is constituted of a polypeptide homologue of zFGF-5 with a second agent or drug to form a chimera. Proteins, for example antibodies, can be used to form chimeras with zFGF-5 molecules of the present invention. Examples of agents or medicaments include, but are not limited to, bioactive polypeptides, genes, toxins, radionuclides, small molecule pharmaceuticals and the like. The linkage can be direct or indirect (for example liposomes) or it can be produced by recombinant means, chemical binding, strong non-covalent interaction and the like. In one embodiment of the present invention, a composition comprising zFGF-5 protein is used as a therapeutic agent to improve bone formation mediated by osteoblasts. The compositions and methods using the compositions of the invention can be applied to promote the repair of bone defects and deficiencies, such as those that occur in closed, open and non-union fractures, - to promote bone healing in plastic surgery, - to stimulate internal osseous growth within non-cemented prosthetic joints and dental implants; in the treatment of diseases and periodontal defects; to increase bone formation during osteogenesis by distraction; and in the treatment of other skeletal disorders that can be treated by stimulation of osteoblastic activity, such as osteoporosis and arthritis. The de novo bone formation provided by the methods of the present invention will have use in the repair of a congenital, trauma-induced, oncological or bone-healing resection subsequent to irradiation-induced osteonecrosis.CR.

(Hart et al, Cancer 37: 2580-2585, 1976). The methods of the present invention also find use in plastic surgery. For pharmaceutical use, the proteins of the present invention are formulated for parenteral administration, particularly intravenous or subcutaneous, according to conventional methods. Intravenous administration will be in bolus injection or infusion during a typical period of one to several hours. In general, the pharmaceutical formulations will include a zFGF-5 protein in combination with a pharmaceutically acceptable carrier such as saline, buffered saline, 5% dextrose in water or the like. The formulations may additionally include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on bottle surfaces, etc. Formulation methods are well known in the art and are described, for example, in Regmington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton PA, 1990, which is incorporated herein by reference. Therapeutic doses will generally be in the range of 0.1 to 100 μg / kg patient weight per day, preferably 0.5-20 μg / kg per day, with the exact dose determined by the physician according to accepted standards, when taking in consideration the nature and severity of the condition to be treated, the patient's features, etc. The determination of the dose is within the usual skill level of a person familiar with the technique. The proteins can be administered for acute treatment, for a week or less, often for a period of one to three days, or they can be used in chronic treatment, for several months or years. In general, a therapeutically effective amount of zFGF-5 is an amount sufficient to produce a clinically significant change in myocyte proliferation, cardiac function, bone formation or increases in specific cell types associated with mesenchymal stem cells and progenitors for myocytes, osteoblasts and chondrocytes. In particular, a significant clinical increase in the number of myocytes or myocyte progenitor cells can be determined by measuring the left ventricular ejection fraction before, and after administration of zFGF-5 molecules, and determining at least an increase in 5%, preferably 10% or more, in the total ejection fraction. Tests to determine the ejection fraction, measured by blood ejected per beat, are well known to those usually familiar in the art. The invention is further illustrated by the following non-limiting examples.

EXAMPLES Use 1 Extension of the EST sequence Scanning of a translated DNA database using a search for growth factors resulted in the identification of an expressed sequence tag sequence (EST) which is a novel member of the FGF family and was designated zFGF-5. The initiating oligonucleotides were designed ZC11,676 (SEQ ID NO: 3) and ZC11, 677 (SEQ ID NO: 4) from the sequence of an expressed sequence tag (EST). The primers were used to prime internally within EST, and then PCR was performed using MARATHON READY cDNA (Clontech, Palo Alto, CA) from adult cardiac tissue as a template in polymerase chain reaction (PCR). The conditions used for PCR were one cycle at 94 ° C for 90 seconds, 35 cycles at 94 ° C for 15 seconds, 68 ° C for 1 minute, - followed by 1 cycle for 10 minutes at 72 ° C and 4 ° C incubation period. The PCR reaction again created 160 bp of the EST sequence, and confirmed that the EST sequence is correct.

Other libraries that can be amplified with the oligonucleotide primers include skeletal muscle, lung, stomach, small intestine and thyroid. 2 Fabric distribution The Northern blot was performed using multiple human tissue spots from Clontech (Palo Alto, CA). The 160 bp DNA fragment described in Example 1 was electrophoresed on a 1% agarose gel, the fragment was electroeluted and then radioactively labeled using a random-start labeling system MEGAPRIME DNA (Amersham, Arlington Heights, IL) according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA). EXPRESSHYB solution was used (Clontech, Palo Alto, CA) for prehybridization and as a hybridizing solution for Northern blots. Hybridization took place overnight at 68 ° C and the spots were subsequently washed in 2X SSC and 0.05% SDS at room temperature, followed by a wash at 0. IX SSC and 0.1% SDS at 50 ° C. observed a single band at approximately 2.0 kb.The intensity of the signal was the highest for the adult heart with relatively less intense signals in skeletal muscle and stomach.

Example 3 Assay for in vitro activity of zFGF-5 A. The mitogenic activity of zFGF-5 was assayed using cell lines and cells from a primary culture. The conditioning medium of the cells expressing the recombinant protein and / or the purified protein is added to the cultures of the following cell lines: NIH 3T3 fibroblasts (ATCC No. CRL-1658), crushed heart CHH-1 cells (ATCC No CRL-1680), rat cardiac myoblasts (H9c2 (ATCC No. CRL-1446), Shionogi mammary cardioma cells (Tanaka et al., 1992, ibid.) And adenocarcinoma cells LNCaP.FGC. to test the proliferative capacity of zFGF-5 include: cardiac fibroblasts, cardiac myocytes, skeletal myocytes and endothelial cells of the human umbilical vein.Mytogenic activity was assayed by measurement of 3H-thymidine incorporation in base in Raines and Ross method (Meth. Enzymology 109: 749-773, 1985) Briefly, cells at rest are plated cells at a density of 3 x 12.4 cells / ml in an appropriate medium.A typical growth medium is a growth medium. Dulbecco (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal bovine serum (FCS). Cells were grown in 96-well plates and allowed to grow for 3-4 days. The growth medium was removed and 180 μl of DFC (Table 5) containing 0.1% FCS were added per well. Half of the wells have zFGF-5 protein added to them and the other half are a negative control, without zFGF-5. The cells are incubated for up to 3 days at 37 ° C in 5% C02, and the medium is recovered. 100 microliters of DFC containing 0.1 of FCS and 2 μCi / ml of 3H-thymidine are added to each well, and the plates are incubated an additional 1-24 hours at 37 ° C. The medium is removed by aspiration, and 150 μl of trypsin are added to each well. The plates are incubated at 37 ° C until the cells detach (at least 10 minutes). Detached cells are harvested on filters using a LKB Walllac 1295-001 cell harvester (LKB Wallac, Pharmacia, Gaithersburg, MD). The filters are dried by heating in a microwave oven for 10 minutes and counted in a LKB Betaplate 1250 scintillation counter (LKB Wallac) as described by the supplier.

TABLE 5 250 ml of Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL) 250 ml of Ham's F12 medium (Gibco-BRL) 0.29 mg / ml of L-glutamine (Sigma, St. Louis, MO) 1 mM sodium pyruvate (Sigma, St. Louis, MO) 25 mM Hepes (Sigma, St. Louis, MO) 10 μg / ml fetuin (Aldrich, Milwaukee, Wl) 50 μg / ml insulin (Gibco-BRL) 3 ng / ml selenium (Aldrich, Milwaukee, Wl) 20 μg / ml transferrin (JRH, Lenexa, KS).

B. The hearts of neonatal mice were isolated from 1 day of age and then discontinued by repeated digestion with collagenase, following the protocol of Brand et al., (J. Biol. Chem. 268: 11500-11503, 1993). Individual myocytes were isolated in a Percoll gradient and plated in 2 ml plates in 6-well tissue culture plates. 0. 5 X 106 cells / ml. Three days later, the layers were washed three times with PBS without calcium or magnesium, and they were fed again with 1 ml of serum-free medium.

(Table 6). The wells were inoculated with 1011 particles of AdCMV-zFGF5 per well in AdCVM-GFP (green fluorescent protein) as a control, and incubated at 37 ° C for 8 hours. The wells were then washed again 3 times with PBS without calcium or magnesium, and then re-fed with 2 ml of serum-free medium. In the following 48 hours after inoculation with AdCMV-zFGF5 the cultured myocytes stopped beating and had undergone a morphological alteration, whereas the wells inoculated with AdCMV-GFP continued to pulse spontaneously and were not affected morphologically by the inoculation. The wells inoculated with AdCMV-zFGF5 also contained, after 48 hours, a confluent layer of nonadherent, viable cells, without any loss in confluence of the layers of adherent myocytes, indicating the proliferative activity of adCMV-zFGF5 on mouse myocytes. cultivated.

Table 6 DMEM Ham's F12 nutrient mixture (Gibco-BRL; 1-l mix with DMEM) 17 mM NaHC03 (Sigma) 2 mM L-glutamine (Sigma) 1% PSN (Sigma) 1 μg / ml insulin 5 μg / ml transferrin 1 nM LiCl (Sigma) Selenium 1 nM 25 μg / ml ascorbic acid (Sigma) 1 nM thyroxine (Sigma) C. Fused zFGF-5 is added to maltose binding protein (MBP), as described in Example 9A and purified as described in Example 10, to myocytes (Example 3B) at a concentration of 0.1 ng / ml of MBP-zFGF5 which shows that they stimulate myocyte profusion too.

Example 4 Assay for ex vivo activity of zFGF-5 Cardiac mitogenesis is measured ex vivo by removing whole hearts from neonatal or 8-week-old mice or rats. The excised heart is placed in the middle of Joklik (Sigma, St. Louis, MO) or Dulbecco at 37 ° C, 5% C02 for 4-24 hours. During the incubation period, the zFGF-5 polypeptide is added at a concentration range of 1 pg / ml to 100 μg / ml. Negative controls only use shock absorber. 3H-thymidine is added and the samples are incubated for 1-4 hours after which the heart is divided and mitogenesis is determined by autoradiography. The sections are used for histomorphometry to determine the nucleus / cytoplasmic volume (McLaughlin, Am. J. Physiol. 271: R122-R129, 1996). Alternatively, the heart is lyophilized and resuspended in 1 ml of 0.1 N NaOH. DNA is precipitated using ice-cold 10% trichloroacetic acid (TCA). The supernatant is added to 9 ml of scintillation fluid to measure non-specific incorporation of 3 H-thymidine. The resulting pellet is resuspended in 1 ml of BTS-450 tissue solubilizer (Beckman, Fullerton, CA) and added to 9 ml of scintillation fluid to measure the specific DNA incorporation of 3H-thymidine. The left and right ventricles are isolated from 1-day-old CD-1 mice (Jackson Labs, Bar Harbor, ME), and incubated for 4 hours with 3 ng / ml of zFGF5Hep2 (n = 13, see Example 10 ) or control (n = 10). 3H-thymidine is added for 1 hour. The ventricles are washed several times and then homogenized in 1 ml of Joklik's medium. The resulting homogenate is added to 9 ml of scintillation mixture and analyzed for total 3 H-thymidine uptake and DNA incorporation. zFGF5-Hep2 increases uptake of 3H-thymidine and incorporation into DNA 2.068 + 0.489 times with respect to the control, indicating that zFGF5 is mitogenic for cardiac cells.

Example 5 Assay for in vivo activity of zFGF-5 The proliferative effects of zFGF-5 were tested in vivo using two-week-old neonatal rats and / or two-month-old adult rats. The rats were injected intraperiocardially either acutely or chronically.

A. Neonatal rats were treated with zFGF-5 for 1 to 14 days over a dose range of 50 ng / day to 100 μg / day. After treatment, the effects of zFGF-5 versus false-treated animals were evaluated by measuring the increased cardiac weight, improvement of left ventricular function in vivo and ex vivo, and by increment in cardiac nuclear fractions with respect to cytosolic volume, which were determined histomorphometrically.

B. Rats with cardiomyopathy induced by chronic infusion of catecholamine by coronary ligation or for models of cardiomyopathy such as the Syrian cardiomyopathic hamster (Solé et al., Amer. J. Cardiol. 62 (11): 20G-24G, 1988) were also used. ) to evaluate the effects of zFGF-5 on cardiac function and tissue. To induce cardiomipathy using catecholamine, 7-8 week old rats were continuously infused epinephrine for 2 weeks via osmotic minipumps implanted subcutaneously between their shoulder blades. Infusion of epinephrine results in an increase in the rating of left ventricular fibrotic lesion from 0.005 ± 0.005 to 2.11 ± 0.18, scale of 0-3); increased the width of the left ventricular myocyte cell from 17.36 + 0.46 μm to 23.05 + 0.62 μm, - and contractile responses of the left ventricular papillary muscle negligible to isoproterenol (0.2 vs 1.1 grams of tension, in comparison with rats that were subjected to by infusion saline solution). After a two-week treatment period, the rats were injected intraperiocardially every day with either vehicle, zFGF-5, bFGF, IGF-I or IGF-II for up to 14 days. The rats were sacrificed and histomorphometry and histochemistry were performed. Rats treated as above, were also evaluated at the end of catecholamine treatment, and again after treatment with growth factor, where cardiac regeneration was measured as decreased ratings of ventricular fibrotic lesion, reduced myocytic cell width and increased in left ventricular papillary contractile responses to isoproterenol.

Example 6 Chromosomal mapping of zFGF-5 We mapped zFGF-5 to chromosome 5 using the commercially available version of the Whitehead Institute / MIT Center for Genome Research "GeneBridge 4 Radiation Hybrid Panel" (Research Genetics, Inc., Huntsville, AL). The GeneBridge 4 Radiation Hybrid Panel contains DNA suitable for use in PCR of each of the 93 hybrid radiation clones, plus two control DNAs (the donor HFL and the A23 receptor). A publicly available server on the WWW (http://www-genome.wi.mit .edu / cgi-bin / contig / rhmapper.pl) allows mapping to the Whitehead Institute / MIT Center for hybrid map genome research by radiation of the human genome (the hybrid radiation map "WICGR") which was built with the GeneBridge 4 Radiation Hybrid Panel. For the mapping of zFGF-5 with the "GeneBridge 4 RH Panel", reactions of 25 μl were adjusted in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used for PCR in a thermal cycler "RoboCycler Gradient 96"(Stratagene). Each of the 95 PCR reactions consisted of 2.5 μl 50X of Advantage KlenTaq Polymerase Mix (Clontech), 2 μl of dNTP mixture (2.5 mM each, Perkin-Elmer, Foster City, CA), 1.25 μl of direct primer, ZC11,677 (SEQ ID NO: 4), 1.25 μl of antisense primer, ZC12,053 (SEQ ID NO: 5). 2.5 μl of "RediLoad" (Research Genetics, Inc.), 0.5 μl of "Advantage KlenTaq Polymerase Mix" (Clontech Laboratories, Inc.), 0.25 ng of DNA from a single hybrid clone or a control and ddH20 for a total volume of 25 μl. The reactions were coated with an equal amount of mineral oil and sealed. The conditions of the recycler for PCR were the following: 1 initial cycle of 4 minutes at 94 ° C, 35 cycles of 1 minute at 94 ° C, 1.5 minutes of linearization and reattachment at 66 ° C and 1.5 minutes of extension at 72 ° C C, followed by 1 final 7-minute extension cycle at 72 ° C. The reactions were separated by electrophoresis on a 3% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME). The results showed that zFGF-5 map 541.12 cR from the top of human chromosome 5 of the binding group on the hybrid WICGR radiation map. In relation to the centromere, its closest final marker is WI-16922 and its closest marker is WI-14692. The use of surrounding CHLC map markers also helps position zFGF-5 in the 5q34-q35 region on chromosome 5 CHLC version v8c7 of the integrated marker map (The Cooperative Human Linkage Center, WWW server-http: // www. chlc.org/ChlcIntegratedMaps .html).

Example 7 Effects of zFGF-5 on bone A. An adenovirus vector containing the cDNA for zFGF-5 was constructed using methods described by Becker et al.

(Methods in Cell Biology 43: 161-189, 1994). Briefly, he cDNA was cloned for zFGF-5 (as shown in SEQ ID NO.

NO: l) as an Xba I-Sal I fragment in pACCMV (Gluzman et al., In Eucaryotic Viral Vectors. Gluzman (eds.) Pp. 187-192, Cold Spring Harbor Press, Cold Spring Harbor NY, 1982). The vector pACCMV contains part of the adenovirus genome 5, the CMV promoter and an SV40 terming sequence. The plasmid containing the vector and the cDNAd insert is cotransfected with a plasmid containing the adenovirus genome 5, designated pJM17 (McGrory et al., Virology 163: 614-617, 1988) in 293 cells (ATCC No. CRL- 1573; American Type Culture Collection, Rockville, MD), leading to a recombination event and the production of a recombinant adenovirus containing zFGF-5, termed AdCMV-zFGF5. The presence of the cDNA for zFGF-5 is confirmed by PCR.

The adenovirus vector AdCMV-zFGF5 is used for gene transfer in vivo by intravenous injection of between 1 x 1011 and 5 x 10X1 particles / mouse. It has been shown that after intravenous injection, most viruses target the liver and transduce hepatocytes very efficiently (Herz et al., Proc. Nati, Acad. Sci. USA 90: 2812-2816, 1993). It has been shown that the cells that produce protein encoded by the cDNA in the case of secreted proteins, secrete them to circulation. High concentrations of expression and physiological effects have been demonstrated (Ohwada et al., Blood 88: 768-774, 1996, Stevenson et al., Arteriosclerosis, Thrombosis and Vascular Biology, 15: 479-484, 1995; Setoguchi et al., Blood 84: 2946-2953, 1994; and Sakamoto et al., Proc. Nati, Acad. Sci. USA 91: 12368-12372, 1994). Six-week-old CD-l mice were treated (Jackson Labs, Bar Harbor, ME) with adenoviruses not containing the cDNA insert (AdCMV-null) or AdCMV-zFGF5 either via the tail vein or intrapericardially (IPC). A total of 5 x 10 11 viral particles / 100 μl / mouse were administered. 14 days after the injection, the animals were sacrificed, and the tibias and femurs were removed without being separated to examine any potential inflammatory response. The bones were fixed in 10% neutral buffered formalin and processed. They were decalcified in 5% formic acid with 10% sodium citrate, washed with water, dehydrated in a series of 70% -100% ethanol, rinsed in xylene and embedded in paraffin. Samples were cut longitudinally through both the metaphyses of the tibia and the femur and stained with hematoxylin and neocin for identification of bone cells. The osteoblasts were identified by the negative Golgi area and the eccentric nucleus, while the osteoclasts were identified by multinucleation, non-uniform shape and the Howship gaps associated with these resorbent cells. For bone histomorphometry, samples of femur were taken. No cancellous bone volume was measured due to variation in sample size (ie, the femur samples were not cut exactly in the same plane). Three bone parameters were evaluated for histomorphometric changes. 1. The number of endosteal osteoblasts: measured along the endosteal surface of cancellous bone at a magnification of 180 X in a 1.22 mm area proximal to the growth plate. 2. The number of endothelial osteoclasts: measured along the endosteal surface of cancellous bone at a magnification of 180 X in an area of 1.22 mm proximal to the growth plate. 3. Width of the growth plate: measured every 72 μm at a magnification of 90 X across the entire width of the plate except at the peripheral ends to determine the growth activity of the plate. Analysis of the data (mean ± standard deviation), n = 4-7 / group) showed the following: 1. There seems to be no detectable inflammatory response at the junction between the tibia and the femur. 2. AdCMV-zFGF5 administered IV or IPC in mice significantly increases osteogenic activity in the distal femural metaphysis when examined at 2 weeks. This stimulation of osteogenic activity is indicated by: a) significant increases in the amount of endosteal osteoblasts in cancellous bone of distal femurs after IV infusion or IPC injection of AdCMV-zFGF5, 530% and 263%, respectively, when compare with their controls where only a relative vector; and b) observation of increased osteogenic tissues at the bone surface, suggesting increased differentiation of bone marrow stromal cells towards the osteoblast line. 3. The number of endosteal osteoclasts is not significantly affected by the IV or IPC administration of AdCMV-zFGF5 when compared to its control with relative vector only. 4. The width of the growth plate is significantly decreased by IV infusion, but not by IPC injection, of AdCMV-zFGF5, suggesting decreased growth plate activity after IV infusion. The differential effects of administration of AdCMV-zFGF5 have not been elucidated. These results suggest that zFGF-5 is a strong mitogen for stimulation of osteoblast proliferation and that zFGF-5 has the ability to induce new bone formation.

B. Using essentially the same procedures described above in 7.A. QCT was performed on female CD-1 (Jackson Labs) to which 1 x 1011 particles of AdCMV-zFGF5 were injected per mouse. Mice were sacrificed 30 days after injection and the heart / tibial length ratios were increased compared to controls (injected with empty adenovirus or saline). There were no differences between the groups in the tibia lengths to justify the change, nor was there any difference in any other organ weight between the groups. Therefore, the indication is that the adenovirus and zFGF-5 selectively increase total bone density, trabecular bone density and cortical thickness in the femur, as measured by QCT.

Example 8 Effect of zFGF-5 on the heart As described in 7.B. it was administered to the mice CD-1, a single IV injection of AdCMV-zFGF5, was sacrificed after four weeks, and the heart / length ratios of tibia were found to be increased compared to mice treated with empty adenovirus or with saline. The results showed that there are no differences between the groups in the tibia lengths to justify this change, nor are there differences in any other weight of the organs between the groups. This result suggests that AdCMV-zFGF5 selectively increases cardiac growth when administered as an IV adenoviral construct.

Example 9 Expression of zFGF-5 A. Construction of plasmids encoding zFGF-5 zFGF5, a homologue of fibroblast growth factor, is expressed in E. coli using the MBP (maltose binding protein) fusion system of New England Biolabs (NEB; Beverly, MA). In this system, the cDNA for zFGF5 binds to the 3 'end of the malE gene to form a fusion protein MBP-zFGF5. The expression of the fusion protein is given by the tac promoter. The expression is "inactivated" until the promoter is induced by the addition of 1 mmol of IPTG (isopropyl b-thiogalactosylpyranoside). Three variations of this fusion protein were made, which differ only at their separation site to liberate zFGF5 from MBP. One construct had a thrombin cleavage site genetically engineered between the MBP and zFGF5 domains. The second construct had a factor Xa cleavage site, rather than a thrombin cleavage site. The third construct had an enterokinase cleavage site, rather than a thrombin cleavage site. The constructs were constructed in frame fusions with MBP according to the multiple cloning site (MCS) of the vector pMAL-c2 (NEB), and according to the manufacturer's instructions. ZFGF5 was amplified via PCR using primers which introduced convenient cloning sites, as well as cleavage sites using the following oligonucleotide primers: 1) for the thrombin construct: zcl2,652 (SEQ ID NO: 7) and zcl2 631 (SEQ ID NO: 8); 2) for the factor Xa construct: zcl5,290 (SEQ ID NO: 9) and zcl2,631 (SEQ ID NO: 8); and 3) for the enterokinase construct: zcl5,270 (SEQ ID NO: 10) and zcl2,631 (SEQ ID NO: 8). In each case, the relative signal sequence for zFGF5 was not amplified; zFGF5 as expressed begins at amino acid residue 26 of the SEC. FROM IDENT. NO: 2 (Val changes to an Ala). The thrombin construct is constructed by inserting a fragment of zFGF5 Xba I-Sal into the Xba I-Sal sites of pMAL-c2. The factor Xa construct is constructed by inserting the blunt-Sal I fragment into the Xmn I-Sal I sites of MCS. The enterokinase construct is constructed by inserting an Xba-Sal I fragment into the Xba-Sal I sites of pMAL-c2. Once the constructs were constructed, they were transformed into a variety of E. coli host strains and analyzed for high level expression. The thrombin construct (designated pSDH90.5) was transfected into DH10B cells (GIBCO-BRL), while both the factor Xa construct (designated pSDH117.3) and the enterokinase construct (designated pSDH116.3) were transfected into TOP10 cells (Invitrogen, San Diego, CA). The three MBP mergers are approximately 63kD (43kD in the MBP domain and approximately 20kD in the zFGF5 domain).

B. Homologous recombination / zFGF5 The expression of zFGF5 in Pichia ethanolica utilizes the expression system described in the co-assigned PCT WO 9717450, incorporated herein by reference. An expression plasmid is constructed which contains all or part of a polynucleotide encoding zFGF5 via homologous recombination. The expression vector is constructed from pCZR204, which contains the AUG1 promoter, followed by the c-Fpp leader sequence, followed by an aminoterminal peptide tag, a blunt end Smal restriction site, a terminal carboxy peptide tag , a translational high codon, followed by the terminator AUG1, the selectable marker ADE2 and finally the untranslated region AUG13 '. Also included in this vector are the URA3 and CEN-ARS sequences necessary for selection and replication in S. cerevisisiae, and the AmpR and colEl ori sequences necessary for selection and replication in E. coli. The zFGF5 sequence inserted in this vector starts at residue 27 (Ala) of the amino acid sequence of zFGF5. To construct pSDH114, a plasmid for the expression of zFGF5 in P. methanolica, the following DNA fragments were transformed into S. cerevisisae: 100 ng of the "acceptor vector" pCZR204 which has been digested with Smal; 1 μg of a Xbal-Sall restriction fragment released from pSDH90.5 and spanning the sequence encoding zFGF5; 1 μg of a double-stranded linker segment, generated by synthetic PCR, spanning 70 base pairs of the sequence encoding aFpp at one end and binding to the 70 base pairs of the amino-terminal coding sequence of the mature sequence of zFGF5 in the other, which is generated from the four Oligonucleotides acl3,497 (SEQ ID NO: 11); zcl5,131 (SEQ ID NO: 12), zcl5,132; (SEQ ID NO: 18), acl5,134 (SEQ ID NO: 13) of which the sense or direct strand of a double strand sequence is shown in SEQ. FROM IDENT. NO: 19 (5 'linker sequence (aFpp - > zFGF5 N-terminal)) and 1 μg of a synthetic double-stranded linker segment spanning 70 base pairs of the coding sequence in the carboxy terminal part from zFGF5 at one end with 70 base pairs of the terminator sequence AUG1 which was generated from four oligonucleotides 13.529 (SEQ ID NO: 14); zcl3,525 (SEQ.

FROM IDENT. NO: 15) zcl3,526 (SEQ ID NO: 16), zcl3,528 (SEQ ID NO: 17) of which the direct or sense string of a double-stranded sequence in the SEC is shown. FROM IDENT. NO: 20 (linker sequence 3 '(zFGF5 C-terminal -> terminal terminator AUG1)). The Ura + colonies were selected, and the DNA from the resulting yeast colonies was extracted and transformed into E. coli. Individual clones harboring the correct expression construct were identified by PCR analysis with oligonucleotides zcl3,497 (SEQ ID NO: 11) and zcl3,528 (SEQ ID NO: 12) followed by restriction digestion to verify the presence of the zFGF5 insert and the DNA sequencing to confirm that the desired DNA sequences have been linked together. Plasmid DNA was isolated on a larger scale for one of the correct clones, and the DNA is digested with Sfi I to release the Pichia-zFGF5 expression cassette from the vector backbone. DNA cut with Sfi I is transformed into the Pichia methanolica expression host and is called PMAD16, and plated on ADE D plates for selection. Various clones are taken and analyzed by means of Western blotting for high level expression of zFGF5. More specifically, for small-scale protein production (eg, plate or shake flask production), P. methanolica transformants carrying an expression cassette comprising a methanol regulator promoter (such as the AUG1 promoter) are made grow in the presence of methanol and the absence of interference amounts from other carbon sources (eg glucose). For small-scale experiments, which include preliminary analysis of expression concentrations, transformants can grow at 30 ° C in solid medium containing, for example 20 g / 1 Bacto-agar (Difco), 6.7 g / 1 base of nitrogen and yeast without amino acids (Difco), 10 g / 1 of methanol, 0.4 mg / 1 of biotin and 0.56 g / 1 of powder -Ade -Thr -Trp. Because methanol is a volatile source of carbon, it is easily lost in prolonged incubation. A continuous supply of methanol can be provided by placing a 50% solution of methanol in water in the inverted plate covers, whereby the methanol is transferred to the growing cells by evaporation transfer. In general, more than 1 ml of methanol is used per 100 mm plate. Slightly larger scale experiments can be carried out using cultures grown in shake flasks. In a typical procedure, the cells are cultured for 2 days in minimal methanol plates, as described above, at 30 ° C and then the colonies are used to inoculate a small volume of methanol minimum medium (6.7 g / 1 base). of nitrogen and yeast without amino acids, 10 g / 1 of methanol, 0.4 mg / 1 of biotin) at a cell density of approximately 1 x 10 cells / ml. The cells are grown at 30 ° C. Cells growing in methanol have a high oxygen yield, which requires vigorous agitation during cultivation. The methanol is re-supplied daily (typically, 1/100 volumes of 50% methanol per day). For large-scale crop production, fresh cultures of highly productive clones are prepared in shake flasks. The resulting cultures are then used to inoculate culture medium in a fermentor. Typically, a 500 ml culture in YEPD that has grown at 30 ° C for 1-2 days with vigorous shaking is used to inoculate a 5 liter fermenter. The cells are grown in a suitable medium containing salts, glucose, biotin and trace elements at 28 ° C, pH 5.0, and >30% of 02 dissolved. After the initial glucose load (as indicated by a decrease in oxygen consumption) is consumed, a glucose / methanol feed supplied into the container to induce the production of the protein of interest. Because large-scale fermentation is carried out under carbon limiting conditions, the presence of glucose in the feed does not repress the methanol-inducible promoter.

Example 10 Purification of zFGF5 E.coli fermentation medium is obtained from a strain expressing zFGF5 as a maltose binding protein fusion (pSDH90.5, as described above). The MBPzFG5 fusion is solubilized during sonication in a French rupture press, using a buffer containing 20 mM Hepes, 0.4 M NaCl, 0.01 M EDTA, 10 mM DTT at pH 7.4. The extraction buffer also includes amounts of 5 μg / ml of Pepstatin, Leupeptin, Aprotinin and Bestatin. Phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 0.5 mM is also included. The extract is centrifuged at 18,000 x g for 30 minutes at 4 ° C. The resulting supernatant is processed in an Amylose resin (Pharmacia LKB Biotechnology, Piscataway, NJ) which binds the MBP domain of the fusion. Upon washing the column, the bound MBPzFGFS fusion is eluted in the same buffer as the extraction buffer without DTT and protease inhibitors but containing 10 mM maltose. The accumulated eluate of MBPzFGF5 is treated with 1: 100 (w / w) of bovine thrombin for fusion of MBPzFGFS. The disruption reaction is allowed to proceed for 6 to 8 hours at room temperature, after which the reaction mixture is passed over a bed of bezamidine Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ) to remove the thrombin using the same Elution buffer as described above for amylose affinity chromatography. The spent fraction containing the separated product zFGF5 and the free MBP domain are applied to a heparin affinity matrix Toso Haas (Toso Haas, Montgomeryville, PA) equilibrated in 0.5 M NaCl, 20 mM Hepes, 0.01 M EDTA at pH 7.4. Both MBP and zFGF5 both bind to heparin under these conditions. The bound proteins are eluted with a column with 2 to 3 gradient volumes formed between 0.5 M NaCl and 2.0 M NaCl in column buffer. MBP elutes first, at 0.7 M NaCl, and separated zFGF5 elutes at approximately 1.3 M NaCl. The accumulated zFGF5 fractions are passed through an amylose stage once to remove any MBPzfgfS that is a minor contaminant. The purified material is designated zFGF5-Hep2, and shows a unique highly pure species at -20 kDa by analysis on reducing SDS-PAGE. The amino acid sequencing of the N-terminal part provides the active N-terminal sequences but mass spectrophotometry data show molecular masses indicating that the C-terminal part must be truncated at residue 196 (Lys) of SEQ. FROM IDENT. NO: 2, where a "dibasic site" is present. The zFGF5 protein is very stable in 1.3 M NaCl.

When dialysis is performed in PBS, the added zFGF5 is added and left in phase in solution. Therefore, formulations that include heparin and other "polyanions" can be used to prevent the aggregation of pure zFGF5. 11 Production of antibodies Antibodies to zFGF5, were produced, using standard techniques known in the art and previously described, by immunization of guinea pigs, rabbits and mice with the peptides QTRARDDVSRKQLRLYC (SEQ ID NO: 2 amino acid residue 40 to residue 56), named zFGF-1; YTTVTKRSRRIRPTHRAC (SEQ ID NO: 2 amino acid residue 191 to residue 207, with an additional Cys in the C-terminal part), designated zFGF-5 or the full-length zFGF5 polypeptide as shown in SEQ. FROM IDENT. NO: 2, plus the MPB fusion protein, which is designated MBP-FGF5. The peptides were conjugated through the Cys residues using maleimide-activated KLH (Pierce Chemical Co., Rockford, IL). Table 7 is a description of the animals, immunization concentrations and antibody separations.

Table 7 Peptide or animal level Antibody protein immunization produced ZFGF5-1 guinea pig 50 / ug / initial animal purified by 25 μg / animal booster affinity and fractionated by rabbit IgG 100 μg / initial animal purified by 50 μg / animal affinity booster and fractionated with IgG ZFGF5-2 guinea pig 50 μg / initial animal purified by 25 μg / animal affinity booster and fractionated with 100 μg rabbit IgG / initial animal purified by 50 μg / animal affinity booster and fractionated with mouse IgG ZFGF5-MBP 20 μg / initial animal 10 μg Animal / reinforcement rabbit 200 μg / initial animal purified per 100 μg / animal affinity booster Example 12 Effects of zFGF5 on ob / ob mice The effects of zFGF-5 on adipocytes and fat metabolism were examined using female ob / ob mice (C57B1 / 6J, Jackson Labs, Bar Harbor, ME). The mice were obese insulin resistant and have "fatty bones". The animals were weighed and found to be all of the same weight, and IV injected with 1011 particles per mouse of AdCMVzFGF-5 either. in saline or Ad5CMV-GFP for controls, as described in example 7. 17 days after injection, control mice injected with Ad5CMV-GFP had gained 5342 ± 0.5 grams of body weight compared to the day of injection , while mice treated with AdCMVzFGF-5 lost 3.183 + 0.743 grams of body weight.

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 by the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: ZymoGenetics, Inc. 1201 Eastlake Avenue East Seattle, Washington 98102 United States of America (ii) TITLE OF THE INVENTION: HOMOLOGOS NOVEDOSOS FGF (iii) NUMBER OF SEQUENCES 20 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: ZymoGenetics, Inc. (B) STREET: 1201 Eastlake Avenue East (C) CITY: Seattle (D) STATE: WA (E) COUNTRY: USA (F) ZIP: 98102 (v) READILY FORM OF THE COMPUTER: (A) TYPE OF MEDIA: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: TWO (D) SOFTWARE OR PROGRAM: FastSEQ for Windows Version 2.0 (vi) CURRENT REQUEST DATA : (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Sawislak, Deborah A (B) REGISTRATION NUMBER: 37,438 (C) REFERENCE NUMBER / FILE: 96-20 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 206-442-6672 (B) TELEFAX: 206-442-6678 (C) TÉLEX: (2) INFORMATION FOR SEC. FROM IDENT. NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 917 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: Coding sequence (B) LOCATION: 1 ... 621 (D) OTHER INFORMATION: (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 1: ATG TAT TCA GCG CCC TCC TCC GCC TGC ACT CTG TGT TTC CTC TTC CTG 48 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 CTG CTG TGC TTC CAG GTA CAG GTG CTG GTT GCC GAG GAG AAC GTG GAC 96 Leu Leu Cys Phe Gln Val Gln Val Leu Val Ala Glu Glu Asn Val Asp 20 25 30 TTC CGC ATC CAC GTG GAG AAC CAG ACG CGG GCT CGG GAC GAT GTG AGC 144 Phe Arg He His Val Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45 CGT AAG CAG CTG CGG CTG TAC CAG CTC TAC AGC CGG ACC AGT GGG AAA 192 Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 CAC ATC CAG GTC CTG GGC CGC AGG ATC AGT GCC CGC GGC GAG GAT GGG 240 His He Gln Val Leu Gly Arg Arg He Ser Wing Arg Gly 'Glu Asp Gly 65 70 75 80 GAC AAG TAT GCC CAG CTC CTA GTG GAG ACA GAC ACC TTC GGT AGT CAA 288 Asp Lys Tyr Wing Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 GTC CGG ATC AAG GGC AAG GAG ACG GAA TTC TAC CTG TGC ATG AAC CGC 336 Val Arg He Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 AAA GGC AAG CTC GTG GGG AAG CCC GAT GGC ACC AGC AAG GAG TGT GTG 384 Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 TTC ATC GAG AAG GTT CTG GAG AAC AAC TAC ACG GCC CTG ATG TCG GCT 432 Phe He Glu Lys Val Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Wing 130 135 140 AAG TAC TCC GGC TGG TAC GTG GGC TTC ACC AAG AAG GGG CGG CCG CGG 480 Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 AAG GGC CCC AAG ACC CGG GAG AAC CAG CAG GAC GTG CAT TTC ATG AAG 528 Lys Gly Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170"175 CGC TAC CCC AAG GGG CAG CCG GAG CTT CAG AAG CCC TTC AAG TAC ACG 576 Arg Tyr Pro Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 ACG GTG ACC AAG AGG TCC CGT CGG ATC CGG CCC ACA CAC CCT GCC TAGGC 626 Thr Val Thr Lys Arg Ser Arg Arg He Arg Pro Thr His Pro Wing 195 200 205 CACCCCGCCG CGGCCCTCAG GTCGCCCTGG CCACACTCAC ACTCCCAGAA AACTGCATCA 686 GAGGAATATT TTTACATGAA AAATAAGGAT TTTATTGTTG ACTTGAAACC CCCGATGACA 7 6 AAAGACTCAC GCAAAGGGAC TGTAGTCAAC CCACAGGTGC TTGTCTCTCT CTAGGAACAG 806 ACAACTCTAA ACTCGTCCCC AGAGGAGGAC TTGAATGAGG AAACCAACAC TTTGAGAAAC 866 CAAAGTCCTT TTTCCCAAAG GTTCTGAAAA AAAAAAAAAA AAAAACTCGA G 917 (2) INFORMATION FOR SEC. FROM IDENT. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 207 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 Leu Leu Cys Phe Gln Val Gln Val Leu Val Wing Glu Glu Asn Val Asp 20 25 30 Phe Arg He His Val Glu Asn Gln Thr Arg Wing Arg Asp Asp Val Ser 35 40 45 Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 His He Gln Val Leu Gly Arg Arg He Ser Wing Arg Gly Glu Asp Gly 65 70 75 80 Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 Val Arg He Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 Phe He Glu Lys Val Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala '130 135 140 Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175 Arg Tyr Pro Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 Thr Val Thr Lys Arg Ser Arg Arg He Arg Pro Thr His Pro Wing 195 200 205 (2) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC11676 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3 GGACTTGACT ACCGAAGGTG TCTG 24 (2) INFORMATION FOR SEC. FROM IDENT. NO: 4 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC11677 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 4: GTCGATGTGA GCCGTAAGCA GCT 23 (2) INFORMATION FOR SEC. FROM IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC12053 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5: GCATACTTGT CCCCATCCTC GCCGCG 26 (2) INFORMATION FOR SEC. FROM IDENT. NO 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 621 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear ATGTAYWSNG CNCCNWSNGC NTGYACNTGY YTNTGYYTNC AYTTYYTNYT NYTNTGYTTY 60 CARGTNCARG TNYTNGTNGC NGARGAAAY GTNGAYTTYM GNATHGAYGT NGARAARCAR 120 ACNMGNGCNM GNGAYGAYGT NWSNMGNAAR CARYTNMGNY TNTAYCARYT NTAYWSNMGN 180 ACNWSNGGNA ARCAYATHCA RGTNYTNGGN MGNMGNATHW SNGCNMGNGG NGARGAYGGN 240 GAYAARTAYG CNCARYTNYT NGTNGARACN GAYACNTTYG GNWSNCARGT NMGNATHAAR 300 GGNAARGARA CNGARTTYTA YYTNTGYATG AAYMGNAARG GNAARYTNGT NGGNAARCCN 360 GAYGGNACNW SNAARGARTG YGTNTTYATH GARAARGTNY TNGARAAYAA YTAYACNGCN 420 YTNATGWSNG CNAARTAYWS NGGNTGGTAY GTNGGNTTYA CNAARAARGG NMGNCCNMGN 480 AARGGNCCNA ARACNMGNGA RAAYCARCAR GAYGTNCAYT TYATGAARMG NTAYCCNAAR 540 GGNCARCCNG ARYTNCARAA RCCNTTYAAR TAYACNACNG TNACNAARMG NWSNMGNMGN 600 ATHMGNCCNA CNCAYCCNGC N 621 (2) INFORMATION FOR SEC. FROM IDENT. NO: 7 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC12652 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 7 TATTTATCTA GACTGGTTCC GCGTGCCGCC GAGGAGAACG TGGACTT 47 (2) INFORMATION FOR SEC. FROM IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC12631 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8 GTATTTGTCG ACTCAGGCAG GGTGTGTGGG CCG 33 (2) INFORMATION FOR SEC. FROM IDENT. NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC15290 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: GCCGAGGAGA ACGTGGACTT CC 22 (2) INFORMATION FOR SEC. FROM IDENT. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC15270 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10 TATTTATCTA GAGATGACGA TGACAAGGCC GAGGAGAACG TGGACTT 47 (2) INFORMATION FOR SEC. FROM IDENT. NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC13497 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 11: AGCATTGCTA AAGAAGAAGG TGTAAGCTTG GACAAGAGAG A 41 (2) INFORMATION FOR SEC. FROM IDENT. NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 63 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONE: ZC15131 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12: GGTGTAAGCT TGGACAAGAG AGAGGAGAAC GTGGACTTCC GCATCCACGT GGAGAACCAG50 ACG 63 (2) INFORMATION FOR SEC. FROM IDENT. NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC15134 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 13 CCGGCTGTAG AGCTGGTACA GCCGCAGCTG CTTACGGCT 39 (2) INFORMATION FOR SEC. FROM IDENT. NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC13529 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 14: CTTCAGAAGC CCTTCAAGTA CACGACGGTG ACCAAGAGGT CC 42 (2) INFORMATION FOR SEC. FROM IDENT. NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC13525 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 15: ACGACGGTGA CCAAGAGGTC CCGTCGGATC CGGCCCACAC ACCCTGCCTA GGGGGAATTC60 G 61 (2) INFORMATION FOR SEC. FROM IDENT. NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC13526 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 16: CAAACAGGCA GCCCTAGAAT ACTAGTGTCG ACTCGAGGAT CCGAATTCCC CCTAGGCAGG 60 G 61 (2) INFORMATION FOR SEC. FROM IDENT. NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: ADNC (vii) IMMEDIATE SOURCE: (B) CLONA: ZC13528 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 17: CTCAAAAATT ATAAAAATAT CCAAACAGGC AGCCCTAGAA TACT 44 (2) INFORMATION FOR SEC. FROM IDENT. NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE: (B) CLONA: ZC15132 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 18: CAGCCGCAGC TGCTTAGCGC TCACATCGTC CCGAGCCCGC GTCTGGTTCT CCACGTGGAT GC 62 (2) INFORMATION FOR SEC. FROM IDENT. NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 141 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 19: AGCATTGCTG CTAAAGAAGA AGGTGTAAGC TTGGACAAGA GAGAGGAGAA CGTGGACTTC 60 CGCATCCACG TGGAGAACCA GACGCGGGCT CGGGACGATG TGAGCCGTAA GCAGCTGCGG 120 CTGTACCAGC TCTACAGCCG G 141 (2) INFORMATION FOR SEC. FROM IDENT. NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 144 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 20: CTTCAGAAGC CCTTCAAGTA CACGACGGTG ACCAAGAGGT CCCGTCGGAT CCGGCCCACA 60 CACCCTGCCT AGGGGGGAATT CGGATCCTCG AGTCGACACT AGTATTCTAG GGCTGCCTGT 120 TTGGATATTT TTATAATTTT TGAG 144 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (20)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An isolated polynucleotide molecule characterized in that it encodes a homologous fibroblast growth factor (FGF) polypeptide, characterized in that it is selected from the group consisting of of: a) polynucleotide molecules comprising a nucleotide sequence, as shown in SEQ. FROM IDENT. NO: 1 from nucleotide 82 to nucleotide 621; b) naturally occurring variants that are at least 80% identical to (a), - c) polynucleotide molecules that code for a polypeptide that is at least 60% identical to the amino acid sequence of SEQ. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising the nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6 from nucleotide 82 to nucleotide 621.
2. 2. The isolated polynucleotide molecule according to claim 1, characterized in that the polynucleotide molecule comprises a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1, from nucleotide I to nucleotide 621 or a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6, from nucleotide 1 to nucleotide 621.
3. 3. The isolated polynucleotide molecule, according to claim 1, characterized in that the polynucleotide molecule comprises a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1, from nucleotide 82 to nucleotide 621.
4. 4. The isolated polynucleotide molecule, according to claim 1, characterized in that the polynucleotide is DNA.
5. 5. An expression vector, characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment that is selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 1, from nucleotide 82 to nucleotide 621, - b) naturally occurring variants that are at least 80% identical to (a), - c) polynucleotide molecules that encode a peptide that is at least 60% identical to the amino acid sequence of the SEC. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ. FROM IDENT. NO: 6, from nucleotide 82 to nucleotide 621; and a transcription terminator.
6. 6. A cultured cell characterized in that within which an expression vector according to claim 5 has been introduced, characterized in that the cell expresses a polypeptide encoded by the DNA segment.
7. A method for producing a FGF homologous polypeptide, characterized in that it comprises: culturing a cell in which an expression vector according to claim 5 has been introduced, characterized in that the cell expresses a homologous polypeptide of FGF encoded by the segment of DNA; and recovering the homologous FGF polypeptide.
8. 8. A homologous polypeptide of isolated FGF, characterized in that it is selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 175 (Met); b) variants that occur naturally that are at least 80% identical to (a); and c) polypeptide molecules that are at least 60% identical to SEC. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 175 (Met).
9. 9. an isolated FGF homologous polypeptide, characterized in that it is selected from the group consisting of a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2 from residue 28 (Glu) to residue 196 (Lys), b) naturally occurring variants that are at least 80% identical to (a), - and c) polypeptide molecules that are at least 60% identical to SEC. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 196 (Lys).
10. 10. A homologous polypeptide of isolated FGF, characterized in that it is selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 207 (Ala), - b) naturally occurring variants that are at least 80% identical to (a), - and c) polypeptide molecules that are at least 60% identical to the amino acids of the SEC. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 207 (Ala).
11. 11. The homologous polypeptide of FGF, according to claim 8, characterized in that it also comprises a signal sequence.
12. 12. The homologous polypeptide of FGF, according to claim 8, characterized in that it further comprises a signal sequence, as shown in SEQ. FROM IDENT. NO: 2, from amino acid residue 1 (Met) to amino acid residue 27 (Ala).
13. 13. A pharmaceutical composition, characterized in that it comprises a purified FGF homologous polypeptide, according to claim 8, in combination with a pharmaceutically acceptable carrier.
14. 14. An antibody that binds to an epitope or antigenic determinant of a polypeptide consisting of an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 1 (Met) to residue 207 (Ala).
15. 15. The antibody according to claim 14, characterized in that it binds to a polypeptide molecule comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 196 (Lys).
16. 16. A method for stimulating the proliferation of myocytes or progenitors of myocytes, characterized in that it comprises administering to a mammal in need thereof a quantity of. a FGF homologous polypeptide that is selected from the group consisting of: (a) polypeptide molecules comprising an amino acid sequence as shown in SEQ. FROM IDENT. NO: 1, from residue 28 (Glu) to residue 175 (Met), - (b) naturally occurring radiants that are at least 80% identical to (a), - and (c) molecules of polypeptides that are at least 60% identical to SEC. FROM IDENT. NO: 2, from amino acid residue 28 (Glu) to amino acid residue 175 (Met) as shown in SEQ. FROM IDENT. NO: 2; in an amount sufficient to produce a clinically significant increase in the amount of myocytes or progenitors of myocytes in the mammal.
17. 17. The method according to claim 16, characterized in that the myocytes or progenitors of myocytes are cardiac myocytes or progenitors of cardiac myocytes.
18. 18. A method for ex vivo stimulation of myocyte progenitor cells, characterized in that it comprises culturing cardiac tissue cells with an amount of a FGF homologous polypeptide that is selected from the group consisting of: (a) polypeptide molecules comprising a amino acid sequence as shown in SEC. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 175 (Met); (b) naturally occurring variants that are at least 80% identical to (a), - and (c) polypeptide molecules that are at least 60% identical to SEC. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to amino acid residue 175 (Met) as shown in SEQ. FROM IDENT. NO: 2; in an amount sufficient to produce an increase in the number of myocyte progenitor cells or in myocytes in cardiac tissue cells cultured in the presence of the FGF homologous polypeptide, as compared to cardiac tissue in progenitor cells of myocytes or myocytes cultured in absence of the FGF homologous polypeptide.
19. 19. The method according to claim 18, characterized in that the myocytes or myocyte progenitors are cardiac myocytes or progenitors of cardiac myocytes.
20. 20. A method for delivering a selectively agent or medicament to cardiac tissue, characterized in that it comprises: linking a first molecule comprising an FGF polypeptide that is selected from the group consisting of: (a) polypeptide molecules comprising an amino acid sequence as shown in the SEC. FROM IDENT. NO: 2, from residue 28 (Glu) to residue 175 (Met); (b) naturally occurring variants that are at least 80% identical to (a), - and (c) polypeptide molecules that are at least 60% identical to SEC. FROM IDENT. NO: 2 from amino acid residue 28 (Glu) to residue 175 (Met) as shown in SEQ. FROM IDENT. NO: 2; with a second molecule comprising an agent or medicament to form a chimera, - and supplying the chimera to cardiac tissue.