MXPA97007897A - Factor-11 fibroblas growth - Google Patents

Abstract

A growth factor-11 polypeptide of human fibroblasts and DNA (RNA) encoding such polypeptide is disclosed. A method for producing such polypeptide by recombinant techniques is also provided. Methods for using such a polypeptide to promote wound healing, for example as a result of burns and ulcers, to prevent neuronal damage associated with shock due to neuronal disorders and promote neuronal growth, and to prevent aging of the skin and hair loss, to stimulate angiogenesis, mesodermal induction in early embryos and the regeneration of limbs. Antagonists against such polypeptides and their use as a therapeutic or to prevent abnormal cell proliferation, hypervascular diseases and cell proliferation of epithelial lenses are also described. Diagnostic methods for detecting mutations in the coding sequence and alterations in the concentration of polypeptides in a sample derived from a host are also disclosed.

Description

FACTOR-11 FIBROBLASTOS GROWTH This invention relates to polynucleotides, newly identified, polypeptides encoded by such polynucleotides, the use of these polynucleotides and polypeptides, as well as the production of these polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been purportedly identified as a fibroblast growth factor / growth factor that binds to heparin, and is referred to hereafter as "FGF-11". The invention also relates to the inhibition of the action of these polypeptides. The growth factors of fibroblasts are a family of proteins characteristic of heparin binding and are, therefore, also named as growth factors that bind to heparin (HBGF). The expression of different members of these proteins is found in several tissues and they are under particular control, temporal and spatial. These proteins are potent mitogens for a variety of cells of mesodermal, ectodermal and endoermal origin, including fibroblasts, corneal and vascular endothelial cells, adrenal cortical cells, chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts and hematopoietic cells. Each member has functions that are performed along with others and also have their unique spectrum of functions. In addition to its ability to stimulate the proliferation of vascular endothelial cells, both FGF-1 and 2 with chemotactic endothelial cells and FGF-2 have been shown to penetrate the basement membrane. Consistent with these properties, both FGF-1 and 2 have the ability to stimulate angiogenesis. Another important characteristic of these growth factors is their ability to promote wound healing. Many other members of the FGF family share similar activities with FGF-1 and 2, such as promoting angiogenesis and wound healing. Several members of the FGF family have been shown to induce the formation of mesoderm and modulate the differentiation of neuronal cells, adipocytes and skeletal muscle cells. In addition to these biological activities in normal tissues, FGF proteins have been implicated in promoting tumorigenesis in carcinomas and sarcomas, promoting tumor vascularization and as transformation proteins, when their expression is deregulated. The FGF family currently consists of eight structurally related polypeptides: basic FGF, acid FGF, int 2, hst 1 / k-FGF, FGF-5, FGF-6, keratinocyte growth factor, AIGH (FGF-8) and recently The factor that activates the glia has been shown to be novel growth factors that bind to heparin, which are purified from the culture supernatant of a human glioma cell line (Miyamoto, M ET AL., Mol. and Cell. Biol., 13 (7): 4251-4259 (1993) .The genes for each have been cloned and sequenced.Two of the members, FGF-1 and FGF-2, have been characterized under many names, but, more often, as growth factors of acidic and basic fibroblasts, respectively.The products of normal gene have influence on the capacity to generate proliferation of most of the cells derived from mesoderm and neuroectoderm.They are able to induce angiogenesis in vivo and can play important roles in the early development (Burgess, W. H. and Maciag, T., Annu. Rev. Biochem., 58: 575-606 (1989)). Many of the previously identified members of the FGF family also join the same recipients and produce a second message through the union to these recipients. A eukaryotic expression vector encoding a secreted form of FGF-1 has been introduced by gene transfer into porcine arteries. This model defines the function of the gene in the arterial wall in vivo. The expression of FGF-1 induced intimal thickening in the porcine arteries 21 days after gene transfer (Nabel, E. G., et al. , Na ture, 362: 844-6 (1993)). It has further been shown that the basic growth factor of fibroblasts can regulate glioma growth and independent progression of its role in tumor angiogenesis and that release or secretion of the fibroblast basic growth factor may be required for these actions (Morrison , RS et al., J.Neurosci. Res., 34: 502-9 (1993)). Fibroblast growth factors, such as basic FGF, have been further implicated in the growth of Kaposi's sarcoma cells in vitro (Huang, YQ, et al., J. Clin. Invest., 91: 1191-7 ( 1993)). Similarly, the sequence of the cDNA encoding the basic growth factor of fibroblasts has been cloned downstream of the transcription promoter, recognized by the RNA polymerase of bacteriophage T7. The basic growth factors of fibroblasts, thus obtained, have been shown to have a biological activity indistinguishable from human placental fibroblast growth factor in mitogenicity, synthesis of plasminogen activator and angiogenesis assays (Squires, CH et al., J. Biol. Chem., 263: 16297-302 (1938)).

The U. A. Patent No. 5,155,214 discloses basic growth factors of substantially pure mammalian fibroblasts and their production. The amino acid sequences of basic growth factors of bovine and human fibroblasts are revealed, as is the DNA sequence encoding the polypeptide of bovine species. The newly discovered FGF-9 has about 30% sequence similarity to other members of the FGF family. Two residues of the cysteine and other consensual sequences in the family members are conserved well in the sequence of FGF-9. FGF-9 has been found to have no typical signal sequence at its N-terminus, such as that in the acidic and basic FGF. However, FGF-9 was found to be secreted from cells after synthesis, despite its lack of a typical signal sequence FGF (Miyamoto, M et al., Mol. And Cell. Biol., 13 (7). ): 4251-4259 (1993) In addition, FGF-9 was found to stimulate cell growth of progenitor cells from type 2 oligodendrocyte astrocytes, BALB / c3T3, and PC-12 cells, but not umbilical vein endothelial cells human (Naruo, K., et al., J. Biol. Chem., 268: 2857-2864 (1993).) Basic FGF and acid FGF are potent modulators of cell proliferation, cell motility, differentiation and survival and They act on cell types of ectoderm, mesoderm and endoderm.These two FGFs, together with KGF and AIGF, were identified by protein purification.However, the other four members were isolated as oncogenes whose expression was restricted to embryo- genesis and certain types of cancers.FGF-9 was shown to be a mitogen against Equal cells The members of the FGF family are reported to have oncogenic potency. FGF-9 has shown transforming potency when transformed into BALB / c3T3 cells (Miyamoto, M., et al., Mol Cell. Biol., 13 (7): 4251-4259 (1993). Androgen induced (AIGF) also known as FGF-8, was purified from a conditioned medium of mouse mammary carcinoma cells (SC-3), stimulated with testosterone.AIGF is a distinctive growth factor, similar to FGF, that it has putative signal peptides and that it shares 30-40% of the homology with known members of the FGF family.The cells of mammals transformed with the AIGF showed a remarkable stimulatory effect on the growth of SC-3 cells in the absence Androgen, therefore, AIGF is a mediator of androgen-induced growth of SC-3 cells, and perhaps other cells, since it is secreted by the tumor cells themselves.

The polypeptide of the present invention has been purportedly identified as a member of the FGF family as a result of the homology of the amino acid sequence with other members of the FGF family. In accordance with one aspect of the present invention, novel mature polypeptides are provided, as well as their fragments, analogs and biologically active derivatives and useful diagnostically and therapeutically. The polypeptides of the present invention are of human origin. According to another aspect of the present invention, isolated nucleic acid molecules are provided, which encode the polypeptides of the present invention, which include mRNA, DNA, cDNA, genomic DNA, as well as their antisensitive and biologically active analogues and their useful fragments diagnostically and therapeutically. According to yet another aspect of the present invention, processes are provided to produce such polypeptides by recombinant techniques through the use of recombinant vectors, such as the cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides of the present invention, as well as recombinant prokaryotic and / or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.

According to a further aspect of the present invention, a process is provided for using such polypeptides, or polynucleotides encoding such polypeptides, for the classification of agonists and antagonists and for therapeutic purposes, for example, promoting wound healing. , such as due to burns and ulcers, to prevent neuronal damage associated with shocks and due to neuronal disorders and promote neuronal growth, and prevent skin aging and hair loss, to stimulate angiogenesis, mesodermal induction in early embryos and the regeneration of limbs. According to yet a further aspect of the present invention, antibodies against such poly-peptides are provided. According to still another aspect of the present invention, antagonists against such polypeptides and processes are provided for their use in inhibiting the action of these polypeptides, for example, in the treatment of cell transformation, such as tumors, to reduce scars. and treat hyper-vascular diseases. According to another aspect of the present invention, nucleic acid probes, comprising nucleic acid molecules of different length, are provided to specifically hybridize a polynucleotide encoding a polypeptide of the present invention. According to yet another aspect of the present invention, diagnostic assays are provided to detect diseases or susceptibility to diseases related to mutations in a nucleic acid sequence of the present invention and to detect overexpression of the polypeptides that they encode. such sequences. According to yet another aspect of the present invention, a process is provided for using such polypeptides, or polynucleotides encoding those polypeptides, for purposes related to scientific research, DNA synthesis and manufacture of DNA vectors. These and other aspects of the present invention will be apparent to those skilled in the art of the teachings of the present invention. The following drawings are only illustrations of the specific embodiments of the present invention and do not mean limitations in any way. Figure 1 illustrates the sequence of the cDNA and the corresponding deduced amino acid sequence of FGF-11. The amino acid sequence shown represents the mature form of the protein. The one-letter standard abbreviation for amino acids is used. The sequence was performed with the use of an automatic apparatus that forms DNA sequences (Applied Biosystems, Inc.). Figure 2 illustrates the homology of the amino acid sequence between FGF-11 and the other members of the FGF family. The conserved amino acids are easily verifiable. According to one aspect of the present invention, isolated nucleic acid molecules (polynucleotides) are provided which code for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or for the mature polypeptide coded by the cDNA of the clone deposited in the Deposit No. 97150 of the ATCC, -the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America, 12 May 1995. The polynucleotide encoding FGF-11 of this invention was initially discovered in the cDNA collection derived from early stage human tissue of 9 weeks of age. The FGF-11 polypeptide is structurally related to all members of the fibroblast growth factor family and contains an open reading frame that encodes a 255 amino acid polypeptide. Among the main correspondences are: 1) 42% identity and 65% sequence similarity to FGF-9 over an extension of 127 amino acids; 2) identity of 37% and similarity of 64% with FGF-7 (keratinocyte growth factor) in a region of 87 amino acids; 3) identity of 38% and 64% similarity with FGF-1 (acidic FGF) in an extension of 120 amino acids. The characteristic of the family FGF / HBGF, GXLX (S, T, A, G) X6 (D, E) CXFXE is conserved in the polypeptide of the present invention, (X means any amino acid residue; (D, E) means residue D or E; X6 means any of 6 amino acid residues). The polynucleotide of the present invention can be in the form of the RNA or in the DNA form, this DNA includes the cDNA, genomic DNA and synthetic DNA. The DNA can be double-stranded or single-stranded. The coding sequence encoding the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO: 1) or that of the deposited clone or may be a different coding sequence, as a result of redundancy or degeneration of the genetic key, which encodes the same, the mature polypeptide, such as the DNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA. The polynucleotides encoding the mature polypeptide of Figure 1 (SEQ ID NO: 2) or the mature polypeptide encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and the additional coding sequence, such as a guiding or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and, optionally, the additional coding sequence) and the non-coding sequence, such as the introns or the 5 'and / or 3' non-coding sequence of the coding sequence for the mature polypeptide. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only the coding sequence for the polypeptide as well as a polynucleotide which includes the additional coding and / or the non-coding sequence. The present invention furthermore relates to variants of the polynucleotides described above, which encode the fragments, analogues and derivatives of the polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or the polypeptides that they encode. the cDNA of the deposited clones. Variants of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a variant that occurs not naturally of the polynucleotide. Thus, the present invention includes polynucleotides that encode the same mature polypeptide, as shown in Figure 1 (SEQ ID NO: 2) or the same mature polypeptides that code for the cDNAs of the deposited clones, as well as variants of such polynucleotides. , which encode a fragment, derivative or analogue of the poly-peptide of Figure 1 (SEQ ID NO: 2) or the polypeptides encoded by the cDNAs of the deposited clones. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. As indicated above, the polynucleotide may have a coding sequence, which is an allelic variant that naturally occurs from the coding sequence, shown in Figure 1 (SEQ ID NO: 1) or the coding sequence of the deposited clones. As is known in the art, an allelic variant is an alternative form of a polynucleotide sequence that can have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides. The present invention also includes polynucleotides, in which the coding sequence for mature polypeptides can be fused in the same reading frame to a polynucleotide sequence that aids in the expression and secretion of a polypeptide from a host cell, for example, a leader sequence that functions as a secretory sequence, to control the transport of a polypeptide from the cell. The polypeptide, which has a guiding sequence, is a preprotein and may have this guide sequence split by the host cell to give the mature form of the polypeptide. The polynucleotides can also encode a proprotein, which is a mature protein plus the additional 5 'amino acid residues. A mature protein that has a pro-sequence is a proprotein and is an inactive form of the protein. Once the pro-sequence unfolds, an active mature protein remains. Thus, for example, the polynucleotides of the present invention can encode a mature protein, or for a protein having a pro-sequence or for a protein having both a pro-sequence and a pre-sequence (leader sequence). The polynucleotides of the present invention may also have a fused coding sequence in the frame or a marker sequence that allows purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag supplied by a pQE-9 vector, to provide for purification of the mature fused polypeptide to the tag in the case of a bacterial host or, for example, the tag sequence may be a tag of the tag. hemagglutinin (HA) when a mammalian host is used, for example COS-7 cells. The HA tag corresponds to an epitope derived from the influenza hemoglutinin protein (Wilson, I., et al., Cell, 37: 767 (1984)). The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions that precede and follow the coding region (guide and drag) as well as intervening sequences (introns) between the individual coding segments (exons). Fragments of the full-length FGF-11 gene can be used as a hybridization probe for the cDNA library to isolate the full-length gene and to isolate other genes having a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe can also be used to identify a clone of the cDNA that corresponds to a full-length transcript of a clone or genomic clones containing the complete FGF-11 gene, which includes the regulatory and promoter regions, exons and instrons. An example of a classification comprises isolating the coding region of the FGF-11 gene, using the known DNA sequence, to synthesize an oligonucleotide probe. The labeled oligonucleotides, which have a sequence complementary to that of the gene of the present invention, are used to classify a collection of the human cDNA, the genomic DNA or mRNA, to determine which members of the probe collection are hybridized. The present invention further relates to polynucleotides that hybridize to the sequences described above, when there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention relates particularly to polynucleotides, which hybridize under stringent conditions to the polynucleotides described above. As used herein, the term "stringent conditions" means the hybridization that occurs only if there is at least 95% and preferably at least 97% identity between the sequences. Polynucleotides that hybridize to the polynucleotides described above, in a preferred embodiment, encode polypeptides that either retain substantially the same function or biological activity as that of the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO: l) or of the deposited cDNAs. Alternatively, the polynucleotide can have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 bases, which hybridize to a polynucleotide of the present invention and which have an identity, as described above, and which can or not retain the activity. For example, such polynucleotides can be used as probes for the polynucleotide of SEQ ID NO: 1, for example, for the recovery of the polynucleotide or as a diagnostic probe or as a PCR primer. Thus, the present invention is directed to polynucleotides having at least 70% identity, preferably at least 90% and more preferably at least 95% identity to a polynucleotide encoding the polypeptide of SEQ ID NO: 2 , like their fragments, these fragments have at least 30 bases and preferably at least 50 bases, and polypeptides encoded by these polynucleotides. The deposits, mentioned here, will be maintained, according to the Budapest Treaty, in the International Recognition of the Deposit of Microorganisms, for the purposes of the Patent Procedure. These deposits are provided merely as convenience and not as an admission that a deposit is required under Title 35 of the US Code, Section 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the encoded polypeptides here, they are incorporated as reference and are controlled, in the case of any conflict, with the description of sequences of the present. A license to use or sell the deposited materials may be required and no license is granted hereby. The present invention furthermore relates to a FG polypeptide, which has the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or which has the amino acid sequence encoded by the deposited cDNAs, like the fragments, analogs and derivatives of these polypeptides. The terms "fragment", "derivative" and "analog", when referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or those encoded by the deposited cDNAs, mean polypeptides that retain essentially the same function or biological activity as such polypeptides Thus, an analog includes a proprotein that can be activated by cleavage of the proprotein portion, to produce an active mature polypeptide The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably they are polypeptides The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNAs can be (i) one in which one or more amino acid residues are substituted with a residue of conserved or non-conserved amino acid (preferably a conserved amino acid residue) and any amino acid residue substituted may or may not be one encoded by the genetic key or (ii) one in which one or more of the amino acid residues includes a substituent group or (iii) one in which the natural polypeptide is fused with another compound, such as a compound for increasing the half-life of the polypeptide, for example, polyethylene glycol) or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a guiding or secretory sequence or a sequence which is employed for the purification of the mature polypeptide or a sequence of proproteins. Such fragments, derivatives and the like are considered within the scope of those skilled in the art of the teachings herein. The polypeptides and polynucleotides of the present invention are preferably delivered in an isolated form and are preferably purified to homogeneity. The term "isolated" means that the material is removed from its original environment (for example the natural environment, if this occurs naturally). For example, a naturally occurring polynucleotide or a polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from it or from all materials coexisting in the natura system, is isolated. Such a polynucleotide can be part of a vector and / or such a polynucleotide or polypeptide can be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptides of the present invention include the polypeptide of SEQ ID NO: 2 (in particular, the mature polypeptide) as well as polypeptides having at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO: 2 and more preferably at least 90 of similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO: 2 and even more preferably at least 95% similarity ( even more preferably at least 95% identity) to the polypeptide of SEQ ID NO: 2 and also includes portions of such polypeptides with such a portion of the poly-peptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. As is known in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence and its amino acid substitutes conserved from a polypeptide to the sequence of a second polypeptide. Fragments or portions of the polypeptides of the present invention can be employed in producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments can be used as intermediates to produce the full-length polypeptides. The fragments or portions of the polynucleotides of the present invention can be used for the synthesis of the full-length polynucleotides of the present invention. The present invention also relates to vectors that include the polynucleotides of the present invention, host cells that are genetically treated with the vectors of the invention and the production of polypeptides of the invention by recombinant techniques. The host cells can be genetically treated (transduced or transformed or transfected) with the vectors of this invention, which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. The treated host cells can be cultured in a conventional nutrient medium, modified, as appropriate, to activate promoters, select transformants or amplify the FGF genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to an artisan with ordinary skill in the art. The polynucleotide of the present invention can be used to produce a polypeptide by recombinant techniques. Thus, for example, the polynucleotide sequence can be included in any of a variety of expression vehicles, in particular vectors or plasmids, to express a polypeptide. Such vectors include chromosomal, non-cormosomal and synthetic DNA sequences, eg, SV40 derivatives, bacterial plasmids; Phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccines, adenoviruses, smallpox and pseudo-rabies viruses. However, any other vector or plasmid can be used as long as it is duplicable and viable in the host. The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art. Such procedures and others are considered within the scope by those skilled in the art. The DNA sequence in the expression vector is operably linked to one or more appropriate expression control (promoter) sequences for the direct synthesis of the mRNA. As representative examples of these promoters, there may be mentioned: the LTR or SV40 promoter, the E. coli, lac or trp, the phagolambda PL promoter and other promoters known in the expression of gene control in pro-cariotic cells or eukaryotes or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences to amplify expression. In addition, expression vectors preferably contain a gene for delivering a phenotypic treatment for the selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as resistance to tetracycline or ampicillin in E. coli. The vector containing the appropriate DNA sequence, as described hereinabove, as well as the promoter or the appropriate control sequence, can be employed to transform an appropriate host to allow the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: battery cells, such as E. coli, Salmonella typhimurium, Streptomyces; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or Bowes melanoma; adenovirus, plant cells, etc. The selection of an appropriate host is considered within the scope of those skilled in the art of the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as described broadly above. The builders comprise a vector, such as a plasmid or a viral vector, in which a sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, which include, for example, a promoter, operably linked to the sequence. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are provided, by way of example. Bacterials: pQE70, pQE60, pQE-9 (qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNHlda, pNH18a, pNH46a, (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo,? SV2cat, pOG44, pXTl, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, other plasmids or vectors can be used, as long as they are duplicable and viable in the host. The promoter regions can be selected from any desired gene, using CAT vectors (chloramphenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include: lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include immediate early CMV, HSV thymidine kinase, early and late SV40, retrovirus LTR, and mouse metallo-tionein-I. The selection of appropriate vectors and promoters is "within the level of one of ordinary skill in the art." In a further embodiment, the present invention relates to host cells that contain the above-described construct.The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.The introduction of the construct into the host cell can be effected by transfection of calcium phosphate, transfection mediated by DEAS-Detran, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, 1986).) Builders in host cells can be used in in a conventional manner, to produce the gene product encoded by the recombinant sequence Alternatively, the polypeptides of the invention can be produced synthetically by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems can also be used to produce such proteins using the RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Labora tory Manual, Second Edition (Cold Spring Harbor, N.Y., 1989). whose description is incorporated herein by reference. The transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by the insertion of an enhancer sequence into the vector. Increasing sequences are cis-acting elements of DNA, usually 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer or the late side of the replication origin (bp 100 to 270), an early promoter enhancer of cytomegalovirus, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. In general, recombinant expression vectors will include replication origins and selectable markers, which allow the transformation of the host cell, for example the ampicillin resistance gene of E. coli and the TRPI gene of S. cerevisiae, and a promoter derived from a highly expressed gene to direct the transcription of a downstream structural sequence. Such promoters may be derived from operons encoding glycolytic enzymes, such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase or heat shock proteins, among others. The heterologous structural sequence is assembled in an appropriate phase with the translation, initiation and termination sequences and preferably, a guiding sequence capable of directing the secretion of the translated protein in the periplasmic space or the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes a N-terminal identification peptide., which imparts the desired characteristics, for example stabilization or simplified purification of the expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding the desired protein together with the appropriate translation, initiation and termination signals in the operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and a replication origin to ensure maintenance of the vector and, if convenient, to provide amplification within the host. Prokaryotic hosts suitable for transformation include: E. coli, Bacillus subtilis, Salmonella typhimurium and several species within the genera Pseudomonas, Streptomyces and Staphylococcus, although others may also be used as a selection material.

As a representative, but non-limiting example, useful expression vectors for bacterial use may comprise a selectable marker and replication bacterial origin derived from commercially available plasmids, comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI (promete Biotec, Madison I, USA). These "skeleton" sections of pBR322 are combined with an appropriate promoter and the structure sequence is to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is removed from the oppression by appropriate means (e.g., temperature shift or chemical induction) and Cells are grown for an additional period. The cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. The microbial cells used in the expression of proteins can be disorganized by any convenient method, including the freeze-thaw cycle, sonication, mechanical interruption or use of cell lysis agents. Several mammalian cell culture systems can also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman in Cell, 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example C127 cell lines. , 3T3, CHO, HeLa and BHK. The mammalian expression vectors will comprise a suitable replication origin, promoter and enhancer, and also any necessary ribosome binding site, polyadenylation site, donor and acceptor splice sites, transcription termination sequences and non-transcribed sequences of the flank 5 '. DNA sequences derived from the SV40 viral genome, eg, the SV40 origin, the early promoter, the splicer of the enhancer and the polyadenylation sites can be used to deliver the required non-transcribed genetic elements. The polypeptide of the present invention can be recovered and purified from the recombinant cell cultures by the methods used here before, which include the precipitation of ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, chromatography of phosphocellulose, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification stages. The polypeptide of the present invention can be a naturally purified product, or a product of synthetic chemical processes or produced by recombinant techniques from a prokaryotic or eukaryotic host (e.g., by bacterial, yeast, higher plant, insect and of mammals, in the crop). Depending on the host employed in a recombinant production method, the polypeptides of the present invention may be glycosylated with mammalian carbohydrates or other eukaryotic carbohydrates, or may not be glycosylated. The polypeptides of the invention may also include an amino acid residue of methionine. The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, can be employed in the treatment to stimulate the revascularization of ischemic tissues due to various disease conditions., such as thrombosis, arteriosclerosis and other cardiovascular conditions. These polypeptides can also be used to stimulate angiogenesis and regeneration of limbs. The polypeptides can also be used in tissue repair, and in ulcers, since they are mitogenic to several cells of different origins, such as fibroblast cells and skeletal muscle cells, and, therefore, facilitate repair or replacement of damaged or diseased tissues. The polypeptides of the present invention can also be used to stimulate neuronal growth and thus treat and prevent the neuronal damage associated with shocks and occurring in certain neuronal disorders or neurodegenerative conditions, such as Alzheimer's disease, Parkinson's disease and complexes related to AIDS. FGF-11 has the ability to stimulate the growth of chondrocytes, therefore, it can be used to increase bone and periodontal regeneration and aid in tissue transplants or bone grafts. The polypeptide of the present invention can also be used to prevent skin aging due to sunburn, by stimulating the growth of keratinocytes. The FGF-11 polypeptide can also be employed to prevent hair loss, since members of the FGF family activate the hair-forming cells and promote the growth of melanocytes. Along with the same lines, the polypeptides of the present invention can be used to stimulate the growth and differentiation of hematopoietic cells and cells of the bone marrow, when used in combination with other cytokines. The FGF-11 polypeptide can also be used to maintain organs after transplantation or to support cell cultures of primary tissues. The polypeptide of the present invention can also be used to induce tissue of mesodermal origin to differentiate in early embryos. According to yet another aspect of the present invention, a process is provided for using such polypeptides, or polynucleotides encoding such polypeptides, for purposes related to scientific research, DNA synthesis, manufacture of DNA vectors and for to provide diagnostics and therapeutic agents for the treatment of human diseases. This invention provides a method for the identification of the polypeptide receptors of the present invention. The genes encoding the receptor can be identified by numerous methods known to those skilled in the art, for example, obtaining the ligand and the FACS classification (Coligan, et al., Current Protocols in Immun., 1 (2), Chapter 5, (1991)). Preferably, expression cloning is employed in which the polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example NIH3T3 cells, which is known to contain multiple receptors for the proteins of the FGF family, and SC-3 cells, and a collection of the cDNA created from this RNA is divided into groups and used to transfect COS cells or other cells that are not sensitive to the polypeptides. Transfected cells growing in glass cursors are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of resources, including the iodization or inclusion of a recognition site for a site-specific protein kinase. Following the fixation and incubation, the cursors are subjected to a self-radiographic analysis. The positive sets are identified and the subsets are prepared and re-transfected using an iterative subset and a reclassification process, eventually providing simple clones that encode the assumed receiver. As an alternative approach for receiver identification, labeled polypeptides can be linked by photoaffinity with the cell membrane or extract preparations expressing the receptor molecule. The interlaced material is resolved by PAGE analysis and exposed to an X-ray film. The labeled complex, which contains the polypeptide receptors, can be cut, resolved into peptide fragments and subjected to the protein micro-sequence. The amino acid sequence obtained from the microsequence will be used to design a set of degenerate oligonucleotide probes to classify a cDNA library to identify the genes encoding the presumed receptors. This invention provides a method for classifying compounds to identify those that modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a polypeptide of the present invention, the compound to be classified and 3 [H] -thymidine under cell culture conditions, where the fibroblast cell will normally proliferate . A control assay can be performed in the absence of the compound to be classified and compared to the amount of proliferation of the fibroblast, in the presence of the compound, to determine whether the compound stimulates proliferation, determining the uptake of the 3 [H ] -thymidine in each case .. The amount of proliferation of fibroblast cells is measured by liquid scintillation chromatography, which measures the incorporation of 3 [H] -thymidine. Both agonist and antagonist compounds can be identified by this procedure. In another method, a mammalian cell or membrane preparation, which expresses a receptor for a polypeptide of the present invention, is incubated with a labeled polypeptide of the present invention, in the presence of the compound. The ability of the compound to increase or block this interaction can then be measured. Alternatively, the response of the second known messenger system following the interaction of a compound to be classified and the FGF-11 receptor was measured and the ability of the compound to bind to the receptor and produce a second response of the messenger is measured for determine whether the compound is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, tyrosine phosphorylation, ion channels or hydrolysis of the phosphoinositide. Examples of antagonist compounds include antibodies, or, in some cases, oligonucleotides, which bind to the polypeptide receptor of the present invention, but do not produce a second messenger response or bind to the FGF-11 polypeptide itself. Alternatively, a potential antagonist may be a mutant form of the polypeptide that binds to the receptors, however, no second response of the messenger is produced and, therefore, the action of the polypeptide is effectively blocked. Another antagonist compound to the gene of FGF-11 and the product of the gene is an antisensitive builder prepared with the use of antisensitive technology. This technology can be used to control the expression of the gene through the formation of a triple helix or the antisensible DNA or RNA, both methods are based on the binding of a poly-nucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence encoding the mature polypeptides of the present invention is used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. An oligonucleotide of DNA is designed to be complementary to a region of the gene involved in transcription (triple helix, see Lee et al., Nucí Acids Res., 6: 3073 (1979); Cooney et al. , Science, 241: 456 (1988); and Dervan et al. , Science, 251: 1360 (1991)), thus preventing the transcription and production of the polypeptides of the present invention. The antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of the mRNA molecule into the polypeptide (Antisense-Okano, J.- Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligo-nucleotides described above can also be delivered to the cells so that the antisensible RNA or DNA can be expressed in vivo to inhibit the production of the polypeptide. Potential antagonist compounds also include small molecules that bind to and occupy the binding site of the receptors, thereby rendering the receptor inaccessible to its polypeptide, such that normal biological activity is impeded. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules. Antagonist compounds can be used to inhibit cell growth and the effects of the proliferation of the polypeptides of the present invention on neoplastic cells and tissues, ie the stimulation of tumor angiogenesis and, therefore, the delay or prevention of abnormal cell growth and proliferation, for example, in the formation or growth of tumors. Antagonists can also be employed to prevent hypervascular diseases and prevent proliferation of epithelial lens cells, after extracapsular cataract surgery. The prevention of mitogenic activity of the polypeptides of the present invention may be desired in cases, such as restenosis, after balloon-like or inflated angioplasty.

Antagonists can also be employed to prevent the growth of scar tissue during wound healing. These antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described below. The polypeptides, agonists and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier, to comprise a pharmaceutical composition for parenteral administration. Such compositions comprise a therapeutically effective amount of the polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, saline solutions, regulated salt solutions, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be appropriate to the mode of administration. The invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with the containers may be a notice, in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, which reflects the approval by the manufacturing, use or sale agency for the administration in humans. In addition, the polypeptides, agonists and antagonists of the present invention can be used in conjunction with other therapeutic compounds. The pharmaceutical compositions can be administered in a convenient manner, such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route. The pharmaceutical compositions are administered in an amount that is effective to treat and / or prophylaxis for the specific indication. In general, they are administered in an amount of at least 10 μg / kg of body weight and, in many cases, they are administered in an amount of no more than about 8 mg / kg of body weight per day. In many cases, the dose is approximately 10 μg / kg up to 1 mg / kg of body weight daily, taking into account the routes of administration, symptoms, etc. In the specific case of topical administration, the doses are preferably administered from about 0.1 μg to 9 mg per cm. The polypeptide of the invention and the agonist and antagonist compounds, which are polypeptides, can also be employed, according to the present invention, by the expression of this polypeptide in vivo., which is often referred to as "gene therapy". Thus, for example, cells can be treated with a polynucleotide (DNA or RNA) encoding the polypeptide ex vivo, the treated cells are then delivered to a patient to be treated with this polypeptide. Such methods are well known in the art. For example, cells can be treated by methods known in the art by the use of a retroviral particle containing the RNA encoding the polypeptide of the present invention. Similarly, cells can be treated in vivo for expression of the polypeptide in vivo, for example by methods known in the art. As is known, a producer cell that produces a retroviral particle containing the RNA encoding the polypeptide of the present invention, can be administered to a patient by cells treated in vivo and expression of the polypeptide in vivo. These and other methods of administering a polypeptide of the present invention by these methods will be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for the treated cells can be another in addition to a retroviral particle, for example, an adenovirus, which can be used to treat the cells in vivo after combination with a suitable delivery vehicle.Retroviruses from which the aforementioned retroviral plasmid vectors can be derived include, but are not limited to: Murine Maloney Leukemia Virus, spleen necrosis virus, retrovirus, such as Rous Sarcoma Virus, Harvey Virus of Sarcoma, bird leukosis virus, gibbon simian leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector was derived from the Muroney Maloney Leukemia Virus. The vector includes one or more promoters. Suitable promoters that may be employed include, but are not limited to, the retroviral LTR, the SV40 promoter; and the human cytomegalovirus (CMV) promoter, described in Miller et al. Biotechniques, Vol. 7, No. 9, 980-990 (1989) or any other promoter (for example cellular promoters, such as eukaryotic cell promoters including, but not limited to, histone, pol III and β-actin promoters). ). Other viral promoters that can be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters and parvovirus B19 promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that may be employed include, but are not limited to, adenoviral promoters, such as the late adenoviral major promoter; or heterologous promoters, such as the cytomegalo-virus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; promoters of thermal shocks; the albumin promoter; the ApoAI promoter; promoters of human globin; viral thymidine kinase promoters, such as the herpes simplex thymidine kinase promoter retroviral LTRs (including modified retroviral LTRs, described above); the ß-actin promoter; and the promoters of human growth hormone. The promoter can also be a native promoter that controls the gene encoding the polypeptide. The retroviral plasmid vector is used to transduce packets of cell lines to form producer cell lines. Examples of cellular packets that can be transfected include, but are not limited to, PE501, PA317,? -2,? -AM, PA12, T19-14X, VT-19-17-H2,? -CRE,? -CRIP , GP + E-86, GP + envAml2 and DAN cell lines, as described in Miller, Human Gene Therapy, Vol. 1, pages 5-14 (1990), which are incorporated herein by reference in their entirety. The vector can transduce the packed cells through any means known in the art. Such resources include, but are not limited to, electroporation, the use of liposomes, and precipitation of CaP 4. In an alternative, the retroviral plasmid vector can be encapsulated in a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles, which include one or more nucleic acid sequences encoding the polypeptides. Such retroviral vector particles can then be used to transduce the eukaryotic cells, in vi tro or in vivo. The transduced eukaryotic cells will express one or more nucleic acid sequences encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells and bronchial epithelial cells. This invention also relates to the use of the genes of the present invention as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences encoding the polypeptide of the present invention. .

Individuals carrying mutations in a gene of the present invention can be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis can be obtained from the cells of patients, such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by the use of PCR (Saiki et al., Na ture, 324: 163-166 (1986)) before analysis. The RNA or the cDNA can also be used for the same purpose. As an example, primers of the PCTR complementary to the nucleic acid, which encode a polypeptide of the present invention, can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in the size of the amplified product, compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to the radiolabelled RNA or alternatively, radiolabelled antisense DNA sequences. Sequences that correspond perfectly can be distinguished from the duplexes that do not correspond by the digestion of the RNase A or by differences in the fusion temperatrums. Genetic testing based on DNA sequence differences can be achieved by detecting the alteration in the electrophoretic mobility of the DNA fragments in the gels, with or without denaturing agents. The deletions and small sequence insertions can be visualized by high resolution gel electrophoresis. The DNA fragments of different sequences can be distinguished in denatured formamide gradient gels in which the mobilities of the different DNA fragments are retarded in the gel at different positions, according to their specific melting point or temperatures. partial fusion (see, for example Myers et al., Science, 230: 1242 (1985)). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (eg, Cotton et al., PNMAS, USA, 85: 4397 -4401 (1985)). Thus, the detection of a specific DNA sequence can be achieved by methods, such as hybridization, RNase protection, chemical cleavage, direct DNA sequence formation or the use of restriction enzymes, (for example, polymorphisms of the Length of the Restriction Fragment and Southern blot of genomic DNA In addition to conventional gel electrophoresis and DNA sequence, mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detecting altered levels of FGF-11 proteins in various tissues, since an over-expression of the proteins, compared to normal control tissue samples, can detect the presence of abnormal cell proliferation, for example, a tumor. The assays used to detect protein levels in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding analysis, Western blot analysis, ELISA assays and the assay. of "sandwich". An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1 (2), Chapter 6, (1991)), comprises initially preparing an antigen-specific antibody to the polypeptides of the present invention, preferably an antibody monoclonal In addition, an information antibody is prepared against the monoclonal antibody. To the information antibody is attached a detectable reagent, such as radioactivity, fluorescence or, in this example, a strong horseradish peroxidase enzyme. A sample is removed from the host and incubated on a solid support, for example a polystyrene disk, which binds the proteins in the sample. Any free protein binding site on the disk is then covered by incubation with a non-specific protein, similar to bovine serum albumin. Next, the monoclonal antibody is incubated on the disk during that time the monoclonal antibodies attack any polypeptide of the present invention attached to the polystyrene disk. All unbound monoclonal antibodies are washed and separated with a buffer solution. The information antibody bound to strong horseradish peroxidase is now placed on the disk and results in the binding of the information antibody to any monoclonal antibody bound to the protein of interest. The unbound information antibody is then separated by washing. The peroxidase substrates are then added to the disc and the amount of color developed in a given period of time is the measure of the amount of the polypeptide of the present invention present in a given volume of the patient sample when compared against a standard curve. . A competition assay in which antibodies specific to a polypeptide of the present invention, binds to a solid support and labeled FGF-11 and a sample derived from the host can be used on the solid support and the amount of label detected. , for example by liquid scintillation chromatography, can be correlated to an amount of the polypeptide of the present invention in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay a polypeptide of the present invention is passed over a solid support and ligated to the antibody bound to a solid support. A second anti-body is then ligated to the polypeptide of interest. A third antibody that is labeled and specific to the second antibody, is then passed over the solid support and ligated to the second antibody and an amount can then be quantified. The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is specifically objective and can hybridize to a particular location on an individual human chromosome. Also, there is a current need to identify particular sites on the chromosome. Few reagents that mark the chromosome, based on real sequence data (repeated polymorphisms) are currently available to mark the cormosomal location. The mapping of the DNA to chromosomes, according to the present invention, is a first important step in correlating those sequences with the genes associated with the disease. In brief, the sequences can be mapped to chromosomes by preparing PCR primers (preferably 15 to 25 bp) from the cDNA. Computer analysis of the 3 'untranslated region is used to quickly select primers that do not expand more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for the PCR examination of somatic cell hybrids containing individual human chromosomes. Only those hybrids that contain the human gene that corresponds to the initiator will supply an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be accomplished with panels of fragments from specific chromosomes or sets of large genomic clones in an analogous manner. Other mapping strategies that can be used similarly for mapping to their chromosome include in situ hybridization, prior examination with labeled chromosomes, classified in flow, and prior selection by hybridization to construct a collection of specific chromosomes of the cDNA. Fluorescence in-fluorescence (FISH) hybridization of a cDNA clone to an extended chromosomal metaphase can be used to provide a precise chromosomal location in one step. This technique can be used with the cDNA as short as 50 to 60 bases. For a review of this technique, see Verma et al. , Human Chromosomes: a Manual of Basic Techniques, Pargamon Press, New York (1989).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with the genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library.) Relationships between genes and diseases that have been mapped to the same cormosomal region are Then, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals, if a mutation is observed in some or all of the individuals. affected, but not in any normal individual, then the mutation is probably the causative agent of the disease.With the current resolution of physical mapping and genetic mapping techniques, a cDNA located precisely to a chromosomal region, associated with the disease, can be one of between 50 and 500 potential causative genes. (This assumes 1 resolution of megabase mapping and one gene per 20 kb). peptides, their fragments or other derivatives, or their analogs, or cells expressing them, can be used as an immunogen to produce antibodies. These anti-bodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments. The antibodies generated against the polypeptides, corresponding to a sequence of the present invention, can be obtained by direct injection of the polypeptides into an animal or by administration of the polypeptides to an animal, preferably non-human. The antibody, thus obtained, will then be ligated to the polypeptides themselves. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies that bind to the entire native polypeptides. These antibodies can then be used to isolate the polypeptide from the tissue expressing that polypeptide. For the preparation of monoclonal antibodies, any technique that delivers antibodies produced by continuous cultures of cell lines can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Na ture, 256: 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72 ) and the EBV-hybridoma technique, to produce human monoclonal antibodies (Colé, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). The techniques described for the production of single chain antibodies (patent of U. U. A., No. 4,946,778) can be adapted to produce single chain antibodies to the immunogenic polypeptide products of this invention. Also, transgenic mice can be used to express the humanized antibodies to the immunogenic polypeptide products of this invention. The present invention will be further described with reference to the following examples; however, it will be understood that the present invention is not limited to such examples. All parts or quantities, unless otherwise specified, are by weight. In order to facilitate understanding of the following examples, certain methods and / or terms that occur frequently will be described. The "plasmids" are designated by a lower condition p preceded and / or followed by uppercase letters and / or numbers. The plasmids starting here or are commercially available, publicly available on an unrestricted basis or can be constructed from available plasmids, according to published procedures. In addition, plasmids equivalent to those described are known in the art and will be apparent to one of ordinary skill in the art. The "digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme, which acts only in certain sequences in DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, co-factors and other requirements are used as will be known to one of ordinary skill in the art. For analytical purposes, typically 1 μg of the plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of the buffer. In order to isolate the DNA fragments for the construction of plasmids, typically 5 to 50 μg of the DNA are digested with 20 to 250 units of enzyme in a larger volume. Regulators and appropriate substrate amounts for the particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour, at 37 ° C, are commonly used, but may vary according to the dispenser's instructions. After digestion, the reaction is subjected to electrophoresis directly on a polyacrylamide gel, to isolate the desired fragment. The separation by size of the split fragments is carried out using 8 percent of the polyacrylamide gel described by Goeddel, D. et al. , Nucleic Acids Res. , 8: 4057 (1980).

"Oligonucleotides" refers to a single-stranded polydeoxynucleotide or two strands of complementary polydeoxy-nucleotides, which can be chemically synthesized. These synthetic oligonucleotides do not have 5 'phosphate and thus do not bind to another oligonucleotide without adding a phosphate with an ATP, in the presence of a kinase. A synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. "Ligation" refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (Maniatis, T., et al., Id., P.146). Unless otherwise provided, ligation can be accomplished using known regulators and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated. Unless stated otherwise, the transformation is executed as described by the method of Graham, F and Van der Eb, A., Virology, 52: 456-457 (1973). Example 1 Bacterial Expression and Purification of FGF-11 Proteins The DNA Sequence, Which Encodes FGF-11, ATCC # 97150, was initially amplified using oligonucleotide primers from the PCR, which correspond to the 5 'sequences of the processed protein (minus the signal peptide sequence) and the 3' vector sequences to the gene. Additional nucleotides, corresponding to the gene, were added to the 5 'and 3' sequences, respectively. The 5 'oligonucleotide primer, 51 CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3' (SEQ ID NO: 3) contains a BamHI restriction enzyme site. The 3 'sequence, the 5' CGCGGATCCCGTTGATTCATTGTGGCTCAT 3 '(SEQ ID NO: 4) contains sequences complementary to the BamHI site and is followed by 21 nucleotides of the coding sequence FGF-11. The restriction enzyme sites correspond to the restriction enzyme sites in the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, CA 91311). PQE-60 codes for antibiotic resistance (Ampr), a replication bacterial origin (ori), an IPTG-regulatable promoter operator (P / O), a ribosome binding site (RBS), a 6-His tag, and restriction enzyme sites. The pQE-60 was then digested with Ncol and BamHI. The amplified sequence was ligated into pQE-60 and inserted into the frame with the sequence coding for the histidine tag and the ribosome binding site (RBS). The ligand mixture was then used to transform E. coli strain M15 / rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J et al. , Molecular Cloning: A Labora tory Manual, Cold Spring Laboratory Press (1989). Ml5 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers resistance to kanamycin (Kanr). Transformants were identified by their ability to grow on LB plates and colonies resistant to ampicillin / kanaminin were selected. Plasmid DNA was isolated and confirmed by restriction analysis. The clones containing the desired builders were grown overnight (0 / N) in a liquid LB culterin medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml). The O / N culture was used to inoculate a large culture at a ratio of 1: 100 to 1: 250. The cells grew at an optical density 600 (D.O. 600) between 0.4 and 0.6 m Then the IPTG ("isopropyl-B-D-thiogalacto-pyranoside") was added to a final concentration of 1 mM. The IPTG induces by inactivation the repressor of lacl, clarifies the p / O leading to an increased expression of gene. The cells grew an extra 3 to 4 hours. The cells were then harvested by centrifugation. The cell pellet was solubilized in the 6 molar chaotropic agent of Guanidine HCl. After clarification, the solubilized FGF-11 was purified from this solution by chromatography on a Nickel-NAT resin under conditions that allow firm binding by proteins containing the 5-His label (Hochuli, R. et al., J. Chromatography 411: 177-184 (1984) .The proteins were eluted from the column in 6 molar guanidine HCl, pH 5.0, and for renaturation purposes were adjusted to 3 molar guanidine HCl, 10 mM sodium phosphate, 10 mmolar of glutathione (reduced) and 2 mmolar of glutathione (oxidized) After incubation in this solution for 12 hours, the proteins were dialyzed to 10 mmolar sodium phosphate.

Example 2 Expression of FGF-11 by in vivo transcription and translation. The FGF-11 cDNA, ATCC # 97150, was transcribed and moved in vi tro to determine the size of the translatable polypeptide encoded by the full length FGF-11 cDNA. The inserts of the full length cDNA of FGF-11 in the pBluescript SK vector. The in vitro transcription / translation reaction was performed in a volume of 25 ul, using the T ^ T® Reticulocyte Linked Lysate Systems (promega, CAT # L4950 =. Specifically, the reaction contains 12.5 ul of rabbit reticulocyte lysate. T ^ T, 2 μl of the reaction regulator of T ^, 1 μl of T3 polymerase, 1 μl of 1 mM amino acid mixture (minus methionine), 4 μl of 35s_methionine (> 1000 Ci / mmol, 10 mCi / ml ), 1 μl of 40 U / μl, RBasin ribo-nuclease inhibitor, 0.5 or 1 μg of plasmid pBluescript FGF-11 The H2 = nuclease-free was added to bring the volume to 25 μl. The reaction was incubated 30 ° C for 2 hours Five microliters of the reaction product were analyzed in a gradient of 4-20% of the SDS-PAGE gel After fixing in 25% isopropanol and 10% acetic acid, the gel was dried and dried. exposed to an x-ray film overnight at 70 ° C.

Example 3 Cloning and expression of FGF-11 using the baculovirus expression system The DNA sequence encoding the full-length FGF-11 protein, ATCC # 97150, was amplified using the oligonucleotide primers of the PCR corresponding to the 5 'and 3' sequences of the gene. The primer of FGF-11 51 has the sequence 5 'CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3' (SEQ ID NO: 5) and contains the BamHI restriction enzyme site (highlighted) so that cloning at this site places the baculovirus signal sequence in frame with 21 nucleotides of the FGF-11 gene downstream of the peptide cleavage site of the putative FGF-11 signal. The 3 'initiator has the sequence 5' CGCGGTACCCTACGTTGATTCATTGTGGCT 3 '(SEQ ID NO: 6) and contains the cleavage site for the restriction endonuclease Asp718 and 21 nucleotides complementary to the sequence not translated 3 'of the gene. The amplified sequences were isolated from a 1% agarose gel using commercially available equipment ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment was then digested with the respective endonucleases and purified again on a 1% agarose gel. This fragment was designated F2. The vector pA2 (modification of vector pVL941, discussed below) was used for the expression of proteins using the baculovirus expression system (for review see: Summers, MD and Smith, GE 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by the recognition sites for the restriction endonucleases BamHI and Asp718. The polyadenylation site of simian virus (SV) 40 was used for efficient polyadenylation. For an easy selection of the recombinant virus, the E. coli beta-galactosidase gene was inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences were flanked on both sides by the viral sequences for the cell-mediated homologous recombination of the co-transfected wild-type viral DNA. Many other baculovirus vectors may be used in place of pA2, such as pRGl, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Viro.Zogy, 170: 31-39). The plasmid was digested with the restriction enzymes and dephosphorylated using the calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from 1% agarose gel using commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla, Ca.) This vector DNA was designated V2. The F2 fragment and the dephosphorylated V2 plasmid were ligated with T4 DNA ligase. The DH5a cells of E. coli were then transformed and the identified bacteria containing the plasmid (pBacFGF-11) using the respective restriction enzymes. The sequence of the cloned fragment was confirmed by the DNA sequence. 5 μg of plasmid pBacFGF-11 was co-transfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold® baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al., Proc. Nati, Acad. Sci. USA, 84: 7413-7417 (1987)). 1 μg of BaculoGold® virus DNA or 5 μg of the plasmids, in each case, were mixed in a sterile cavity of microtiter plates containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Next, 10 μl of Lipofectin plus 90 μl of Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added, in drops, to the Sf9 insect cells (ATC CRL 1711) seeded in 35 mm tissue culture plates with 1 ml of Grace's medium, without serum. The plates were vibrated again back and forth to mix the newly added solution. These plates were then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution was removed from the plate and 1 ml of Grace's insect medium, supplemented with 10% fetal calf serum, was added. Plates were placed back in an incubator and culture was continued at 27 ° C for four days. After four days, the supernatant was collected and plaque assays performed in a manner similar to that described by Summers and Smith (supra). As a modification, an agar gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used, which allows easy isolation of the blue-stained plates. (A detailed description of a "plaque assay" can be found in the user guide for the cultivation of insect cells and baculovirology, distributed by Life Technologies Inc., Gaithersburg, page 9-10). Four days after the serial dilution, the virus was added to the cells and the blue stained plates were taken with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect the Sf9 cells seeded on 35 mm discs. Four days later, the supernatants from these culture discs were collected and then stored at 4 ° C. The Sf9 cells grew in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-FGF-11 at a multiplicity of infection (MOI) of 2. Six hours later, the medium was removed and replaced with the SF900 I medium minus methionine and cysteine (Life Technologies Inc. ., Gaithersburg). 42 hours later, 5 μCi of 35s-methionine and 5 μCi of 3E >; s-cysteine (Amersham) were added. The cells were further incubated for 16 hours, before they were collected by centrifugation and the labeled proteins were visualized by SDS-PAGE and autoradiography.

Example 4 Expression of FGF-11 Recombinant in COS Cells The expression of plasmids, FGF-11-HA, derived from vector pcDNA3 / Amp) Invitrogen) containing: 1) replication origin SV40, 2) ampicillin-resistant gene, 3) replication origin E. coli, 4) CMV promoter followed by a poly-linker region, an SV40 intron and polyadenylation site. The DNA fragments encoding the entire precursor of FGF-11 and a HA tag fused in frame to the 3 'end, is cloned in the poly-linker region of the vector, therefore, expression of the recombinant protein is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein, as previously described (I. Wilson, H. Niman, R. Heithten, A. Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37: 767 (1984)). Infusion of the HA tag to the target protein allows easy detection of the re-combining protein with an antibody that recognizes the epitope. The strategy of plasmid construction is described in the following: The DNA sequence encoding FGF-11, ATCC # 97150, was constructed by PCR using two primers: the 5 'primer. that is to say 5 'CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3' (SEQ ID NO: 8) contains sequences complementary to an Xbal site and the last 21 nucleotides of the sequence encoding FGF-11 (not including the stop codon). Therefore, the PCR product contains an Xhol site, which encodes the sequence followed by the HA label fused in the frame, a translational stop arrest codon followed by the HA tag, and an Xhol site.

The DNA fragments amplified by the PCR and the pcDNA3 / Amp vector were digested with the respective restriction enzymes and ligated. The ligand mixture was transformed into the SURE E. coli strain (available from Stratagene Cloning Systems, La Jolla, CA 92037), the transformed culture was plated on ampicillin medium plates and the resistant colonies were selected. The plasmid DNA was isolated from the transformants and examined by restriction analysis in the presence of the correct fragment. For the expression of recombinant FGF-11, cells were transfected with the expression vector by the DEAR-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Labora tory Manual, Cold Spring Laboratory Press ( 1989)). The expression of the FGF-11-HA protein was detected by the radio-labeling and immuno-precipitation method (E. Harlow, D. Lane, Antibodies: A Labora tory Manual, Cold Spring Harbor Laboratory Press (1988)). The cells were labeled for 8 hours with 35g_cysteine two days after transfection. The culture medium was then harvested and the cells were lysed with detergent (RIPA regulator (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 40 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37: 767 (1984)) Both the cell lysate and the culture medium were precipitated with specific monoclonal antibody, the precipitated proteins were analyzed in 15% of the gels SDS-PAGE.

Example 5 Expression by Gene Therapy Fibroblasts were obtained from a subject by skin biopsy. The resulting tissue was placed in a tissue culture medium and separated into small pieces. Small pieces of tissue were placed on a wet surface of a tissue culture flask, approximately ten pieces were placed in each flask. The flask was turned down, closed tightly and left at room temperature overnight. After 24 hours at room temperature, the flask was inverted and the pieces of tissue remained fixed to the bottom of the flask and the fresh medium (for example, Ham's F12 medium, with 10% FBS, penicillin and streptomycin, were added). This was then incubated at 37 ° C for about a week.At this time, the fresh medium was added and subsequently changed every several days.After two additional weeks in the culture, a monolayer of fibroblasts emerged. The monolayer was trypsinized and scaled in larger flasks. pMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988) flanked by long-terminal repeats of Moloney murine sarcoma virus, was digested with EcoRI and HindIII and then treated with calf intestinal phosphatase The linear vector was fractionated on an agarose gel and purified using glass beads The cDNA encoding a polypeptide of the present invention was amplified using the PCT primers corresponding to the 5 'end sequences and 3 ', respectively. The 5' primer containing an EcoRI site and the 3 'primer also include a HindIII site Equal amounts of the linear backbone of the Maloney murine sarcoma virus and the amplified EcoRI and HindIII fragments were added together, in the presence of the T4 DNA ligase The resulting mixture was maintained under conditions appropriate to the ligand of the two fragments.The ligand mixture was used to trans the HB101 bacterium, which was then placed on agar that contains kanamycin in order to confirm that the vector has the gene of interest inserted properly. The amphotropic packed cells pA317 or GP + aml2 were grown in the culture medium at the confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene was then added to the medium and the packed cells were transduced with the vector. The packed cells now produce infectious viral particles that contain the gene (the packed cells are now named as produced cells).edium was added to the transduced produced cells and thereafter, the medium was collected from the 10 cm plate of the confluent produced cells. The spent medium, which contains the infectious viral particles, was filtered through a millipore filter to remove the produced detached cells and this medium was then used to infect the fibroblast cells. The medium was removed from the sub-confluent plate of fibroblasts and rapidly replaced with the medium of the produced cells. This medium was removed and replaced with fresh medium. If the virus titer is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector having a selectable marker, such as neo or his. The treated fibroblasts were then injected into the host, either alone or after growing in confluence in cytodex 3 microcarrier beads. Fibroblasts now produce the protein product. Numerous modifications and variants of the present invention are possible in the light of the foregoing teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than that particularly described.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: HU, ET AL (ii) TITLE OF THE INVENTION: Fibroblast Growth Factor-11 (Üi) SEQUENCE NUMBER: 8 (iv) CORRESPONDENCE ADDRESS (A) RECIPIENT: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN (B) STREET: 6 BECKER FARM ROAD (C) CITY: ROSELAND (D) STATE: NEW JERSEY (E) COUNTRY: E.U.A. (F) POSTAL ZONE: 07068 (V) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: 3.5-INCH (B) COMPUTER DISK: IBM PS / 2 (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT INFORMATION OF THE APPLICATION: (A) NUMBER OF THE APPLICATION: (B) DATE OF SUBMISSION: Concurrently (C) CLASSIFICATION: «(VÜ) DATA FROM THE PREVIOUS APPLICATION (A) NUMBER OF THE APPLICATION: 08 / 207,412 (B) DATE OF SUBMISSION: MARCH 8, 994 (viil) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: FERRARO GREGORY D. (B) REGISTRATION NUMBER: 36,134 (C) REFERENCE NUMBER / FILE: 325800 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION OF SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH : 768 BASIC COUPLES (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l ATGAGTGGAA AGGTGACCAA GCCCAAAGAG GAGAAAGATG CTTCTAAGGT TCTGGATGAC 60 GCCCCCCCTG GCACACAGGA ATACATTATG T ACGACAAG ATTCCATCCA ATC GCGGAA 120 TTAAAGAAAA AAGAGTCCCC CTTTCGTGCT AAGTGTCACG AAATCTTCTG CTGCCCGCTG 180 AAGCAAGTAC ACCACAAAGA GAACACAGAG CCGGAAGAGC CTCAGCTTAA GGGXATAGTT 240 ACCAAGCTAT ACAGCCGACA AGGCTACCAC TTGCAGCTGC AGGCGGATGG AACCATTGAT 300 GGCACCAAAG ATGAGGACAG CACTTACACT CTGTTTAACC tCATCCCTGT GGGTCTGCGA 360 GTGGTGGCTA TCCAAGGAGT TCAAACCAAG CTGTACTTGG CAATGAACAG TGAGGGATAC 420 TTGTACACCT CGGAACTTTT CACACCTGAG TGCAAATTCA AAGAATCAGT GTTTGAAAAT B0 TATTATGTGA CATATTCATC AATGATATAC CGTCAGCAGC AGTCAGGCCG AGGGTGGTAT 540 CTGGGTCTGA ACAAAGAAGG AGAGATCATG AAAGGCAACC ATGTGAAGAA GAACAAGCCT 600 GCAGCTCATT TTCTGCCTAA ACCACTGAAA GTGGCCATGT ACAAGGAGCC ATCACTGCAC 660 GATCTCACGG AGTTCTCCCG ATCTGGAAGC GGGACCCCAA CCAAGAGCAG GTCTCT AAG 720 GGCGTGCTGA ACGGAGGCAA ATCCAXGAGC CACAATGAAT CAACGTAG_768_(2) INFORMATION OF SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 255 AMINO ACIDS (B) TYPE :. AMINO ACID (C) CORD CLASS: (D) TOPOLOGY: LINEAR (Ü) TYPE OF MOLECULE: PROTEIN (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Met Ser Gly Lys Val Thr Lye Pro Lys Glu Glu Lys Asp Wing Ser 5 10 15 Lys Val Leu Asp Asp Ala Pro Pro Gly Thr Gln Glu Tyr He Met 20 25 30 Leu Arg Gln Asp Ser He Gln Ser Ala Glu Lue Lys Lys Lys Glu 35 40 45Ser Pro Phe Arg Ala Lye Cys His Glu He Phe Cye Cys Pro Leu 50 55 60 Lys Gln Val His HIs Lys Glu Asn Thr Glu Pro Glu Glu Pro Gln 65 70 75 Leu Lys Gly He Val Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His 80 85 90 Leu Gln Leu Gln Wing Aep Gly Thr He Asp Gly Thr Lys Asp Glu 95 100 105 Asp Ser Th_r Tyr Thr Leu Phe Asn Leu He Pro Val Gly Leu Arg Val Val Ala He Gln Giy Val Gln Thr Lye Leu Tyr Leu Ala Met 125 130 135 Asn Ser Glu Gly Tyr Leu Tyr Thr Ser Glu Leu Phe Thr Pro Glu 140 145 150 Cys Lye Phe Lye Glu Ser Val Phe Glu Asn Tyr Tyr Val Thr Tyr 155 160 165 Ser Ser Met He Tyr Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr 170 175 180 Leu Gly Leu Aen Lye Glu Gly Glu He Met Lye Gly Asn His Val 185 Lye Lys Asn Lys Pro Wing Ala His Phe Leu Pro Lys Pro Leu Lys Val Wing Met Tyr Lys Glu Pro Ser Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr Lys Ser Arg Ser Val Ser Gly Val Leu Aen Gly Gly Lye Ser Met Ser His Asn Glu Ser Thr 1 245 250 -Í-JO (2) INFORMATION OF SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 33 BASIC FACTS (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SINGLE (D) TOPOLOGY: LINEAR (ii) ) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: CGCGGATCCA TCATGAGTGG AAAGGTGACC 33 (2) INFORMATION OF SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 30 BASIC COUPLES (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SINGLE (D) TOPOLOGY: LINEAR (ii) ). TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4 CGCGGATCCC GTTGATTCAT TGTGGCTCAT 30 (2) INFORMATION OF SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: BASIC COUPLES (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (i) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: (2) INFORMATION OF SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: BASIC COUPLES (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide »(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: (2) INFORMATION OF SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: BASIC COUPLES (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: (2) INFORMATION OF SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: BASIC COUPLES (B) TYPE: NUCLEIC ACID (C) TYPE OF CORD: SIMPLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8;

Claims (20)

1. CLAIMS 1. An isolated polynucleotide, which comprises a member selected from the group consisting of: (a) a polynucleotide encoding the polypeptide, which includes amino acid 1 to amino acid 255, indicated in Sequence Identification No. 2; (b) a polynucleotide, capable of hybridizing and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotides (a) or (b).
2. 2. The polynucleotide of claim 1, which encodes the polypeptide comprising amino acid 1 to amino acid 255 as indicated in Sequence Identification No. 23.
3. The polynucleotide of claim 1, wherein the polynucleotide is DNA.
4. 4. An isolated polynucleotide, which comprises a member selected from the group consisting of: (a) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Repository No. 97150; (b) a polynucleotide encoding the polypeptide expressed by the DNA contained in ATCC Deposit No. 97150; (c) a polynucleotide, capable of hybridizing and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotides of (a), (b) or (c).
5. 5. A vector containing the DNA of claim 2.
6. 6. A host cell genetically treated with the vector of claim 5.
7. 7. A method for producing a polypeptide, which comprises: expressing from the host cell of claim 6, the polypeptide encoded by the DNA.
8. 8. A method for producing cells capable of expressing a polypeptide, which comprises genetically treating the cells with the vector of claim 5.
9. 9. A polypeptide comprising a member selected from the group consisting of (i) a polypeptide having the amino acid sequence deduced from Sequence Identification No. 2, and its fragments, analogs and derivatives; and (ii) a polypeptide encoded by the cDNA of ATCC Repository No. 97150, and fragments, analogs and derivatives of this polypeptide.
10. 10. An antibody against the polypeptide of claim 9.
11. 11. A compound, which inhibits the polypeptide of claim 9.
12. 12. A compound, which activates a receptor for the polypeptide of claim 9.
13. 13. A method for the treatment of a patient in need of a FGF-11 polypeptide, this method comprises: administering to the patient a therapeutically effective amount of the polypeptide of claim 9.
14. 14. A method for the treatment of a patient that has to inhibit the FGF-11 polypeptide, this method comprises: administering to the patient a therapeutically effective amount of the polypeptide of claim 11.
15. 15. The method of claim 13, wherein the therapeutically effective amount of the polypeptide is administered by supplying the patient with the DNA encoding the polypeptide and expressing this polypeptide in vivo.
16. 16. The method of claim 14, wherein the compound is a polypeptide and the therapeutically effective amount of the compound is administered by providing the patient with the DNA encoding the antagonist and expressing this antagonist in vivo.
17. 17. A method for identifying compounds active as agonists to the polypeptide of claim 9, this method comprises: (a) combining a compound to be classified and a reaction mixture containing cells, under conditions where the cells are normally stimulated by the polypeptide, this reaction mixture contains a label incorporated into the cells as they proliferate; Y (b) determining the extent of proliferation of the cells to identify whether the compound is an effective agonist.
18. 18. A method for identifying active compounds as antagonists to the polypeptide of the claim 9, this method comprises: (a) combining a compound to be classified, the polypeptide and the reaction mixture containing cells under conditions where the cells are normally stimulated by the polypeptide, this reaction mixture contains a tag incorporated in the cells as they proliferate; and (b) determining the extent of proliferation of the cells to identify whether the compound is an effective antagonist.
19. 19. A method for diagnosing a disease or susceptibility to a disease related to a sub-expression of the polypeptide of claim 9, this method comprises: determining a mutation in the nucleic acid sequence encoding the polypeptide.
20. 20. A diagnostic method, which comprises: analyzing the presence of the polypeptide of claim 9 in a sample derived from a host.