MXPA96003897A - Factor 10 of fibroblas growth - Google Patents

Abstract

The human FGF-10 polypeptides and the DNA (RNA) encoding such FGF-10 polypeptides are described. A method for producing such polypeptides by recombination techniques is also provided. Methods for using such polypeptides to stimulate revascularization, to treat wounds and to prevent neuronal damage are also described. Antagonists against such polypeptides and their uses as abnormal cellular, hypervascular diseases and proliferation of epithelial crystalline cells are also described. Diagnostic methods to detect mutations in the sequence encoding FGF-10 and alterations in the concentration of the FGF-10 protein in a sample derived from a host are also described.

Description

FACTOR 10 OF GROWTH OF FIBROBLASTS This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are the factor 10 growth of fibroblasts / growth factor 10 bound to heparin, hereinafter referred to as "FGF-10". The invention also relates to the inhibition of the action of such polypeptides. The growth factors of fibroblasts are a family of characteristic proteins that bind to heparin and are, therefore, also called growth factors attached to heparin (HBGF). The expression of different members of these proteins is found in several tissues and is under particular temporal and spatial control. These proteins are potent mitogens for a variety of cells of mesodermal, ectodermal, and endodermal origin, including fibroblasts, corneal and vascular endothelial cells, granulocytes, adrenocortical cells, chondrocytes, myoblasts, vascular smooth muscle cells, crystalline epithelial cells, REF: 23Di * í melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells. Each member has functions that overlap with others and also has its unique spectrum of functions. In addition to the ability to stimulate the proliferation of vascular endothelial cells, both FGF-1 and 2 are chemotactic for endothelial cells and FGF-2 has been shown to enable endothelial cells to penetrate the basement membrane. Consistent with these properties, both FGF-1 and 2 have the ability to stimulate angiogenesis. Another important feature 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 the promotion of angiogenesis and wound healing. Several members of the FGF gene family have been shown to introduce mesoderm formation 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 the promotion of tumorigenesis in carcinomas and sarcomas by promoting tumor vascularization and as transforming proteins when their expression is deregulated. The FGF family currently consists of eight structurally related polypeptides. The genes for each one have been cloned and sequenced. Two of the members, FGF-1 and FGF-2, have been characterized under many names, but most often as a growth factor for the acid and basic fibroblasts, respectively. Normal gene products influence the overall proliferative capacity of most cells derived from mesoderm and neuroectoderm. They are capable of inducing angiogenesis in vivo and can play important roles in primary development (Burgess, W. H. and Maciag, T., Annu, Rev. Biochem. 58: 575-606, (1989)). 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. Expression of FGF-1 induced intimal thickening in the porcine arteries 21 days after the gene transfer (Nabel, E.G., et al., Nature, 362: 844-6 (1993)). In addition, it has been shown that the growth factor of the basic fibroblast can regulate the glioma growth and the independent progress of its role in tumor angiogenesis and that the release or secretion of the growth factor of the basic fibroblasts can be required for these actions ( Morrison RS, et al., J. Neurosci. Res., 34: 502-9 (1993)). Growth factors of fibroblasts, such as basic FGF, have been further implicated in the growth of Kaposi sarcoma cells in vi tro (Huang, YQ, et al., J. Clin. Invest., 91: 1191 -7 (1993)). Also, the sequence of the cDNA encoding the human basic fibroblast growth factor has been cloned downstream of the transcription promoter recognized by the RNA polymerase of bacteriophage T7. The growth factors of the basic fibroblasts thus obtained have been shown to have biological activity indistinguishable from the growth factor of human placental fibroblasts in mitogenicity, synthesis of the plasminogen activator and angiogenesis assays (Squires, CH, et al., J. Biol. Chem., 263: 16297-302 (1988)). U.S. Patent No. 5,155,214 discloses growth factors of basic fibroblasts of substantially pure mammals and their production. It describes the amino acid sequences of the growth factor of the basic bovine and human fibroblasts, as well as the DNA sequence that codes for the polypeptide of the bovine species. The polypeptide of the present invention has been invocatively 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 which are FGF-10 as well as biologically active, useful diagnostic and therapeutically useful fragments, analogs and derivatives thereof were provided. The polypeptides of the present invention are of human origin. In accordance with another aspect of the present invention, isolated nucleic acid molecules encoding human FGF-10 were provided, including mRNAs, DNAs, cDNA, genomic DNA, as well as antisense analogs thereof and fragments thereof. very biologically active and useful in diagnosis or therapeutically useful. According to another aspect of the present invention, a process for producing such polypeptide by recombinant techniques was provided through the use of recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of FGF proteins. -10, as well as recombinant prokaryotic and / or eukaryotic host cells comprising a nucleic acid sequence for human FGF-10. According to a further aspect of the present invention, there is provided a process for using such a polypeptide, or polynucleotide encoding such a polypeptide, for therapeutic purposes, for example, promoting healing due to wounds, burns or ulcers, to prevent damage neuronal due to neuronal diseases, and to prevent skin aging and hair loss. According to a further aspect of the present invention, antibiotics are provided against such polypeptides. According to yet another aspect of the present invention, antagonists are provided against such polypeptides, which can be used to inhibit the action of such a polypeptide, for example, in the treatment of tumors and hypervascular diseases. In accordance with another aspect of the present invention, nucleic acid probes comprising nucleic acid molecules of sufficient length are provided to specifically hybridize to the sequences of human FGF-10.

According to yet another aspect of the present invention, diagnostic assays are provided to detect diseases or susceptibility to diseases related to mutations in the nucleic acid sequences of FGF-10 or overexpression of the polypeptides encoded by such sequences. In accordance with another aspect of the present invention, there is provided a process for using such polypeptides, or polynucleotides encoding such polypeptides, for purposes related to scientific research, DNA synthesis, and DNA vector manufacturing. Those and other aspects of the present invention will be apparent to those skilled in the art from the teachings herein. The following drawings are intended only as illustrations of the specific embodiments of the present invention and are not intended to be limitations in any way. Figure 1 describes the cDNA sequence and the corresponding deduced amino acid sequence of FGF-10. The amino acid sequence shown represents the mature form of the protein. The one-letter standard abbreviation for amino acids was used. Inaccuracies in the sequence are a common problem when trying to determine polynucleotide sequences. Sequencing was performed using an automatic DNA sequencer 373 (Applied Biosystems, Inc.). The accuracy of the sequencing was predicted to be greater than 97%. Figure 2 illustrates the homology of the amino acid sequence between FGF-10 and the other members of the FGF family. The conserved amino acids are indicated in bold. Figure 3 shows a PAGE gel with SDS after transcription / translation in vi tro of the FGF-10 protein. 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 polypeptide mature coding by the clone cDNA deposited in the ATCC deposit No. 75696 on March 4, 1994. The polynucleotide of this invention was initially discovered in a cDNA library derived from human tissue at the primary stage for 8 weeks and subsequently the Full length cDNA was found in a library derived from the human amygdala. This is structurally related to all members of the fibroblast growth factor gene family and contains an open reading frame that encodes a polypeptide of 181 amino acids. Among the main similarities are: 1) 37% identity and 67% similarity in the sequence with the FGF-9 isolated from the brain over an extension of 129 amino acids; 2) 36% identity and 64% similarity to FGF-7 (keratinocyte growth factor) in a region of 121 amino acids; 3) 33% identity and 55% similarity to FGF-1 (acidic FGF) over an extension of 110 amino acids. In addition, the signature of the FGF / HBEF family GXLX (S, T, A, G) X6 (D, E) CXFXE was retained in the polypeptide of the present invention (X means any residual amino acid, (D, E) means a residue either D or E; X6 means any 6 residual amino acid). The polynucleotide of the present invention can be in the form of DNA or in the form of DNA, DNA which includes cDNA, genomic DNA and synthetic DNA. DNA can be double-stranded or single-stranded. The coding sequence coding for the mature polypeptide can be identical to the coding sequence shown in Figure 1 (SEQ ID No. 1) or to that of the deposited clone or it can be a different coding sequence as a result of redundancy or degeneracy of the genetic code, which encodes the same, the mature polypeptide 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 for the mature polypeptide encoded by the deposited cDNA 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 leader or secretory sequence or a protein sequence; the coding sequence for the mature polypeptide (and optionally the additional coding sequence) and the non-coding sequence, such as introns or 5 'and / or 3' non-coding sequences of the coding sequence for the mature polypeptide. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only the coding sequence for the polypeptide as well as a polynucleotide that includes additional coding and / or non-coding sequences. The present invention further relates to the variants of the polynucleotides described hereinbefore, which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNA of the deposited clone. Variants of the polynucleotide can be natural allelic variants of the polynucleotide or an unnatural variant of the polynucleotide. Thus, the present invention includes polynucleotides that encode the same natural polypeptide shown in Figure 1 (SEQ ID NO 2) or the same wild-type polypeptide encoded by the deposited clone cDNA, as well as variants of such polynucleotides , variants which code for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. As indicated hereinbefore the polynucleotide may have a coding sequence which is a natural allelic variant of the coding sequence shown in Figure 1 (SEQ ID No. 1) or of the coding sequence of the deposited clone. 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 formation of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide can be fused in the reading frame thereof to a polynucleotide sequence that aids the expression and secretion of a polypeptide from a host cell, eg, a leader sequence which functions as a secretory sequence to control the transport of the polypeptide from the cell. The polypeptide having a leader sequence is a protein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode a proprotein, which is the mature protein plus additional 5 'residual amino acids. A mature protein that has a prosequence is a protein and is an active form of the protein. Once the prosequence is cleaved, an active mature protein remains. Thus, for example, the polynucleotide of the present invention can code for a mature protein, or for a protein having prosequence or for a protein having both a prosequence and a presequence (leader sequence). The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence, which allows purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag provided by a pQE-9 vector for proportion. The purification of the mature polypeptide fused to the marker in the case of a bacterial host or, for example, the marker sequence can be a mark of haemagglutinin (HA) when a mammalian host is used, eg, COS-7 cells. The HA mark corresponds to the epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37: 767 (1984)): The present invention is further related to the polynucleotides that are hybridized to the sequences described above if there is at least 50% and preferably 70% identity between the sequences. The present invention relates in particular to polynucleotides that hybridize under stringent conditions to the polynucleotides described above. As used herein, the term "strict combinations" means that hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides that hybridize with the polynucleotides described hereinbefore in a preferred embodiment code for polypeptides that retain substantially the same function or biological activity as the mature polypeptide encoded by the cDNA of Figure 1 (SEQ ID No.1) or the deposited cDNA. . The deposits referred to here will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent procedures. These deposits were provided simply for convenience and do not admit that a deposit under 35 U.S.C. § 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded by these, are incorporated herein by reference and are controlled in the case of any conflict with the description of the sequences herein. . A license may be required to make, use or sell the deposited materials, and such a license is not granted hereby. The present invention further relates to a FGF-10 polypeptide, which has the amino acid sequence deduced from Figure 1 (SEQ ID No. 2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide. The terms "fragment", "derivative" and "analogue" when referring to the polypeptide of Figure 1 (SEQ ID No. 2) or to that encoded by the deposited cDNA, mean a polypeptide that retains essentially the same function or biological activity as such polypeptide. In this way, an analog includes a protein that can be activated by cleaving a portion of the protein to produce a mature active polypeptide. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO 2) or of the one encoded by the deposited cDNA can be (i) one in which one or more of the residual amino acids are substituted with the residual amino acid conserved or not conserved (preferably a residual conserved amino acid) and such substituted residual amino acid may or may not be one encoded by the genetic code, or (ii) one in which one or more of the residual amino acids includes a substituent group, or (iii) one wherein the mature polypeptide is fused to another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence that is employed for the purification of the mature polypeptide or a protein sequence. It is considered that such fragments, derivatives and analogs are within the scope of those skilled in the art from the teachings herein. The polypeptides and polynucleotides of the present invention are preferably provided in isolated form and preferably are purified homogeneously. The term "isolated" means that the material was removed from its original environment (for example, the natural environment if found in nature). For example, a natural polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide separated from one or all of the coexisting materials in the natural system is isolated. Such a polynucleotide could be part of a vector and / or such a polynucleotide or polypeptide could be part of a composition, and still be isolated since such a composition vector is not part of its natural environment. The present invention also relates to vectors that include the polynucleotides of the present invention, host cells which are genetically engineered with the vectors of the invention and the production of the polypeptides of the invention by recombinant techniques.

The host cells can be genetically engineered (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 engineered host cells can be cultured in modified conventional nutrient media as appropriate to activate the promoters, select transformants or amplify the FGF-10 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cells selected for expression, and will be apparent to those skilled 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 for the expression of a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV40 derivatives; bacterial plasmids; Phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccina, adenovirus, smallpox virus, and pseodorabies. 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 the appropriate endonuclease restriction sites by methods known in the art. It is considered that such procedures and others are within the reach of those skilled in the art. The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct the synthesis of the mRNA. As representative examples of such promoters, there may be mentioned: the LTR or SD40 promoter, the lac or trp of E. coli, the phage lambda PL promoter and other promoters that are known to control the expression of genes in prokaryotic or eukaryotic cells or your viruses. The expression vector also contains a ribosome binding site to initiate translation and a transcription terminator. The vector may also include the appropriate sequences to amplify the expression. In addition, the expression vectors preferably contain a gene to provide a phenotypic trait or characteristic for the selection of transformed host cells such as resistance to dihydrofolate reductase or neomycin for the culture of eukaryotic cells, or such as resistance to tatracycline or ampicillin in E. coli. The vector containing the appropriate DNA sequence described above, as well as an appropriate promoter or 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: bacterial cells such as E. coli, Salmonella typhi urium, 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. It is considered that the selection of an appropriate host is within the reach of those skilled in the art and forms part of the teaching herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, in which a sequence of the invention has been inserted, in a forward or inverted orientation. A preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, 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. Bacteria: pQE70, pQE60, pQE-9 (Qiagen), pBs, Phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a, (Stratagene); pTRL99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacy) Eukaryotes: pWLneo, pSV2cat, pOG 4, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacy). However, any other plasmid or vector can be used so 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 selectable marker vectors. Two appropriate vectors are pKK232-8 and pCM7. Particularly named bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include the CMV intermediate, HSV thymidine kinase, and initial and final SV40, retrovirus LTR, and mouse metallothionine I. The selection of the appropriate vector and promoter is also within the level of one skilled in the art.

In a further embodiment, the present invention relates to host cells that contain the construct described above. The host cell can 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 with calcium phosphate, transfection mediated by DEAE-Dextran, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, 1986) ). The constructs in the host cells can be used in a conventional manner to produce the genetic product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be produced synthetically or by conventional peptide synthesizers. Mature cells can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using the RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, NY, 1989), the description of which is Incorporates here as a reference. The transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an amplifying sequence into the vector. Amplifiers are cis-acting elements of DNA, usually around 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 amplifier on the end side of the duplication origin (100 to 270 bp), in a cytomegalovirus primary promoter amplifier, a polyoma amplifier on the end side of the duplication origin, and adenovirus amplifiers. Generally, recombinant expression vectors will include duplication origins and selectable markers that allow transformation of the host cell, for example, the E. coli ampicillin resistance gene and the TRP1 gene of S. cerevisiae, and a promoter derived from a highly expressed gene to direct the transcription of a downstream structural sequence. Such promoters can be derived from operons that encode glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), factor a, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in the appropriate phase with the translation, initiation and termination sequences. Optionally, the heterologous sequences can encode a fusion protein that includes an N-terminal identification peptide that 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 a desired protein together with the appropriate translation, initiation and termination signals in the operable reading phase with a functional promoter. The vector should comprise one or more phenotypically selectable markers and a duplication origin to ensure maintenance of the vector and, if desirable, provide amplification within the host. Prokaryotic hosts suitable for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and several species within the genus Pseudomonas, Streptomyces, and Staphylococcus, although others may also be used as the material of choice.

As a representative example but not limiting, expression vectors useful for bacterial use may comprise a selectable marker and bacterial origin of duplication derived from commercially available plasmids comprising genetic elements of the cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, WI, USA). Those sections of the "skeleton" of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is depressed with the appropriate means (e.g., temperature modification or chemical induction) and the cells are cultured for a period of time. additional period. The cells are typically harvested by centrifugation, broken by physical or chemical means, and the resulting crude extract is preserved for further purification. Microbial cells used in the expression of proteins can be disrupted by any conventional method, including a freeze-thaw cycle, sonication, mechanical disruption, or the use of agents for cell lysis. Various mammalian cell culture systems may also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 lines of the monkey kidney fibroblasts, described by Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing a compatible vector, eg, the cell lines, C127, 3T3, CHO, HeLa and BHK. Mammalian expression vectors will comprise a suitable duplication origin, promoter and amplifier, and also any necessary ribosome binding sites, polyadenylation site, donor sites and splice receptors, transcriptional termination sequences, 5 'non-transcribed flanking sequences. . DNA sequences derived from the SV40 viral genome, eg, SV40 origin sites, primary promoter, amplifier, splice, and polyadenylation can be used to provide the required non-transcribed genetic elements. FGF-10 can be recovered and purified from recombinant cell cultures by the above methods, including precipitation with ammonium sulfate or ethanol, acid extraction, anionic or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity, chromatography with hydroxyapatite and chromatography with lecithin. Protein reconstitution steps may be used, as necessary, to complete the configuration of the mature protein. Finally, high-performance liquid chromatography (CLAP) can be used for the final purification steps. The polypeptides of the present invention can be a naturally purified product, or a product of synthetic chemical processes, or produced by recombination techniques from a prokaryotic or eukaryotic host (for example, by bacterial cells, yeasts, from higher plants, insects and mammals in crops). Depending on the host employed in a recombinant production process, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. The polypeptides of the invention may also include a residual amino acid initial methionine. The polypeptide of the present invention, as a result of the ability to stimulate the growth of vascular endothelial cells, can be employed in the treatment to stimulate revascularization in ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and others. cardiovascular conditions. FGF-10 can also be used to treat injuries due to damage, burns, tissue repair after an operation, and ulcers since it has the ability to be a mitogenic agent for several cell types, such as fibroblast cells and skeletal muscle cells. FGF-10 can also be used to treat and prevent neuronal damage that occurs in certain neuronal conditions or neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complexes. FGF-10 has the ability to stimulate the growth of chondrocytes, therefore, it can be used to amplify bone and periodontal regeneration and aid in tissue transplants or bone grafts. FGF-10 can also be used to prevent skin aging due to sunburn stimulating the growth of keratinocytes. FGF-10 can also be used to prevent hair loss, since FGF-10 activates the cells that form the hair and promotes the growth of melanocytes. Following the same lines, FGF-10 stimulates the growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytosines.

FGF-10 can also be used to maintain organs before transplantation or to support primary tissue cell cultures. According to yet another aspect of the present invention, there is provided a process for using such polypeptides, or polynucleotides encoding such polypeptides, for purposes related to scientific research, DNA synthesis, manufacturing of DNA vectors and for the purpose of providing diagnosis and therapeutic treatment for the treatment of human diseases. The full length FGF-10 gene fragments can be used as a hybridization probe for a cDNA library to isolate the full-length FGF-10 gene and to isolate other genes having a sequence highly similar to these genes or having similar biological activity. Probes of this type generally have at least 20 bases. Preferably, however, the probes have at least 30 bases and generally do not exceed 50 bases, although they may have a larger number of bases. The probe can also be used to identify a cDNA clone that corresponds to a full-length transcript and a clone or genomic clones containing the complete FGF-10 gene including the regulatory and promoter regions, exons, and introns. An example of a selection comprises isolating the coding region of the FGF-10 gene using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides have a complementary sequence so that the gene of the present invention is used to select a library of human cDNA, genomic DNA or mRNA to determine which members of the library will hybridize the probe. This invention provides a method for the identification of FGF-10 polypeptide receptors. The gene encoding the receptor can be identified by numerous methods known to those skilled in the art, for example, ligand selection and FACS classification (Coligan, et al., Current Protocols in Immun., 1 (2), Chapter 5 , (1991)). Preferably, expression cloning is employed wherein the polyadenylated RNA was prepared from a cell responsive to the polypeptides, and a cDNA library created from this RNA is divided into groups and used to transfect COS cells or other cells that they do not respond to polypeptides. Transfected cells that are grown on glass plates are exposed to the labeled polypeptides. The polypeptides can be labeled by a variety of means including the iodination or inclusion of a recognition site by a site-specific protein kinase. After fixation and incubation, the plates are subjected to an autoradiographic analysis. Positive groups are identified and groups are prepared and retransfected using an iterative subpool and selection processes, eventually producing a single clone encoding the putative receptor. As an alternative method for identification of the receptor, the tagged polypeptides can be ligated by photoaffinity with the cell membrane or extract preparations expressing the receptor molecule. The crosslinked material is resolved by PAGE analysis and exposed to an X-ray film. The labeled complex containing the polypeptide receptors can be cleaved, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from the microsequencing could be used to design a set of degenerate oligonucleotide probes to select a cDNA library to identify the genes encoding the putative receptors.

This invention provides a method for selecting compounds to identify those that modulate the action of FGF-10. An example of such an assay comprises combining a mammalian fibroblast cell, FGF-10, the compound to be selected and 3 [H] thymidine under cell culture conditions where the fibroblast cells could proliferate normally. A control assay can be performed in the absence of the compound to be selected and compare the amount of fribroblast proliferation in the presence of the compound to determine whether the compound stimulates the proliferation of the keratinocytes by determining the consumption of 3 [H] thymidine in each case. For the selection of antagonists, the same assay can be prepared and the ability of the compound to avoid proliferation of the fibroblasts and a determination of the antagonist capacity can be measured. The amount of proliferation of fibroblast cells is measured by liquid flash chromatography, which measures 3 [H] thymidine incorporation. In another method, a mammalian cell or membrane preparation expressing the FGF-10 receptor with labeled FGF-10 could be incubated in the presence of the compound. The ability of the compound to increase or block this interaction could then be measured. Alternatively, the response of the second known messenger system could be measured by following the interaction of the FGF-10 and the receptor may be compared in the presence or absence of the compound. Such a second messenger system includes but is not limited to, cAMP guanylate cyclase, iron channels or phosphoinositide hydrolysis. Examples of potential antagonists of FGF-10 include an antibody, or in some cases, an oligonucleotide, which binds to the polypeptide. Alternatively, a potential antagonist of FGF-10 may be a mutant form of FGF-10, which binds to the FGF-10 receptors, however, no response of the second messenger is detected and, therefore, the action of FGF-10 was effectively blocked. Another potential antagonist of FGF-10 is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene ession through the formation of a triple helix or DNA or antisense RNA, both methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which codes for the polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. The DNA oligonucleotide is designed to complement 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 avoiding the transcription and production of FGF-10. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation of the mRNA molecule into the FGF-10 polypeptide (Atisentido-Okano, J. Neurochem., 56: 560 (1991); Oligodeoxynucleotides as antisense inhibitors of ession Genetics, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be released into cells so that the antisense RNA or DNA can be essed in vivo to inhibit the production of FGF-10. Potential antagonists of FGF-10 include small molecules, which bind to and occupy the binding site of the FGF-10 receptor, thereby rendering the receptor inaccessible to FGF-10, so that normal biological activity is avoided. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules. FGF-10 antagonists can be used to inhibit cell growth and affect the proliferation of FGF-10 on neoplastic cells and tissues and, therefore, retard or prevent abnormal cell growth and proliferation, e.g., tumor growth. . Antagonists of FGF-10 can also be used to prevent hypervascular diseases, and prevent the proliferation of epithelial crystalline cells after extracapsular cataract surgery. Antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described hereinafter. The polypeptides, agonists and antagonists of the present invention can be used in combination with a pharmaceutically acceptable carrier to comprise a pharmaceutical composition. Such compositions comprise a therapeutically active amount of polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to saline solutions, buffered saline solutions, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must conform to the mode of administration. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such containers may be a note in the form prescribed by a governmental agency that regulates the manufacture, use or sale of pharmaceutical or biological products, that reflects the approval of the agency for the manufacture, use or sale for human administration. 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 conveniently such as by means of the oral, topical intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount that is effective for the treatment and / or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg / kg of body weight and in many cases will be administered in an amount not to exceed about 8 mg / kg of body weight per day. In many cases, the dose is from about 10 μg / kg to about 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 preferably administered are from about 0.1 μg / kg to 9 mg per cm. The polypeptides, agonists and antagonists of FGF-10, which are polypeptides, can also be employed in accordance with the present invention for the expression of such polypeptide in vivo which is often referred to as "gene therapy". Thus, for example, cells can be designed with a polynucleotide (DNA or RNA) that codes for the polypeptide ex vivo, the designed cells are then provided to a patient to be treated with the polypeptide. Such methods are well known in the art. For example, the cells can be designed 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 designed in vivo for expression of the polypeptide in vivo, for example, by methods known in the art. As is known in the art, a producer cell for producing a retroviral particle containing the RNA encoding the polypeptide of the present invention can be administered to a patient to design cells in vivo and for expression of the polypeptide in vivo. Those and other methods for administering a polypeptide of the present invention by such methods will be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for designing cells can be different from a viral particle, for example, an adenovirus, which can be used to design cells in vivo after combination with a suitable delivery vehicle. This invention also relates to the use of the FGF-10 gene as part of a diagnostic assay to detect diseases or the susceptibility of diseases related to the presence of mutations in the nucleic acid sequences of FGF-10. Individuals that carry mutations in the FGF-10 gene can be detected at the DNA level by a variety of techniques. The nucleic acids for diagnosis can be obtained from the cells of a patient, 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., Nature, 324: 163-166 (1986)) before analysis. RNA or cDNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding FGF-10 can be used to identify and analyze mutations of FGF-10. For example, deletions and insertions can be detected by a change in the size of the amplified product compared to that of the normal genotype. Punctual mutations can be identified by hybridizing the amplified DNA to the radiolabelled FGF-10 RNA or alternatively, the radiolabelled FGF-10 antisense DNA sequences. The perfectly matched sequences can be distinguished from the dissimilar duplicates by the RNase A digestion or by the differences in the melting temperatures. Genetic testing based on DNA sequence differences can be achieved by detecting the alteration in the electrophoretic mobility of the DNA fragments in gels with or without denaturing agents. Small deletions and insertions in the sequence can be visualized by high-resolution gel electrophoresis. The DNA fragments of the different sequences can be distinguished on denaturing formaldehyde gradient gels in which the mobilities of the different DNA fragments are delayed in the gel at different positions according to their specific melting or partial melting temperatures ( see, for example, Myers et al., Science, 230: 1242 (1985)).

Changes in the sequence at specific locations can also be revealed by nuclease protection assays, such as protection with RNase and SI or the chemical cleavage method (e.g., Cotton et al. , PNAS, USA, 85: 4397-4401 (1985)). In this way, the determination of a specific DNA sequence can be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (for example, Fragment Length Polymorphisms of Restriction (PLFR)) and Southern blotting of genomic DNA. In addition to conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis. The present invention also relates to a diagnostic assay for detecting altered levels of FGF-10 protein in various tissues since over expression of the proteins compared to normal control tissue samples can detect the presence of a disease or susceptibility to a disease, for example, a tumor. The assays used to detect levels of FGF-10 protein in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, ELISA assays and "sandwich" assays. " An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1 (2), Chapter 6, (1991)) initially comprises preparing an antibody specific for the FGF-10 antigen, preferably a monoal antibody. In addition, a reporter antibody is prepared against the monoal antibody. A detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme is attached to the reporter antibody. A sample of a host is removed and incubated on a solid support for example a polystyrene disk, which binds the proteins in the sample. Any free protein binding sites on the disk are then covered by incubation with a non-specific protein similar to bovine serum albumin. Next, the monoal antibody is incubated on the disk during which time the monoal antibodies bind to any FGF-10 proteins bound to the polystyrene disk. All unbound monoal antibodies are washed with buffer. The reporter antibody bound to horseradish peroxidase is now placed on the disk resulting in the binding of the reporter antibody to any monoal antibody bound to FGF-10. The unbound reporter antibody is then washed. Next, peroxidase substrates are added to the disc and the amount of color developed in a given period of time is a measure of the amount of FGF-10 protein present in a given volume of patient sample, when compared against a standard curve. . A competition assay may be employed, in which antibodies specific for FGF-10 bind to a solid support and are labeled with FGF-10 and a sample derived from the host is passed over the solid support and the amount of label detected. , for example by flash chromatography in the liquid state, can be correlated with an amount of FGF-10 in the sample. A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay FGF-10 is passed over a solid support and binds to the antibody bound to a solid support. Next, a second antibody is bound to FGF-10. A third antibody, which is labeled and specific for the second antibody, is then passed over the solid support and is bound to the second antibody and then an amount can be quantified. The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is specifically directed towards and can hybridize to a particular location on an individual human chromosome. In addition, there is a current need to identify particular sites on the chromosomes. Few chromosome marker reagents based on current sequence data (repeated polymorphism) are currently available for chromosomal localization. The tracing of the DNA for chromosomes according to the present invention is an important first step in the correlation of those sequences with the genes associated with the disease. In summary, the sequences for the chromosomes can be plotted by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3 'untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thereby complicating the amplification process. These primers are then used to select the PCR of the somatic cell hybrids containing the individual human chromosomes. Only those hybrids that contain the human gene that corresponds to the primer will produce an amplified fragment. The PCR tracing of somatic cell hybrids is a rapid procedure to evaluate a particular DNA for a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization with panels of fragments of chromosomes or groups of large genomic clones can be achieved analogously. Other mapping or tracing strategies that can be similarly used to map their chromosomes include in situ hybridization, preselection with labeled, flow-labeled chromosomes, and pre-selection by hybridization to construct chromosome-specific cDNA libraries. Fluorescent in situ hybridization (FISH) of a cDNA clone for a diffused metaphase chromosome can be used to provide a precise chromosomal location in one step. This technique can be used with cDNAs as short as 500 or 600 bases; however, clones larger than 2000 bp are more likely to bind to a single chromosomal location with sufficient signal strength for simple detection. FISH requires the use of the clones from which the label of the expression sequence (a fragment of the gene of the present invention) was derived, and the larger the better. For example, 2000 bp is good, 4,000 is better, and over 4000 is probably not necessary to give good results in a reasonable percentage of time. For a review of this technique, see Verma et al., Human Chromoso is: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been plotted for an accurate chromosomal location, the physical position of the sequence on the chromosome can be correlated with the data on the genetic map. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library.) The relationship between genes and diseases that have been plotted for the same chromosomal region is identified Then, it is necessary to determine the differences in the sequence of the cDNA or genomic between the affected and unaffected individuals, if a mutation is observed in some or all of the individuals affected, but not in a normal individual, then the mutation is probably the causal agent of the disease.With the current resolution of the physical tracing and genetic tracing techniques, a cDNA located precisely for a chromosomal region associated with the disease could be one between 50 and 500 potential causal genes. (It is assumed a resolution of the trace of 1 megabase and one gene per 20 kb) The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies therefor. These antibodies can be, for example, polyclonal 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. The various methods known in the art can be used for the production of such antibodies and fragments. Antibodies raised against 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 a human. The antibody that has been obtained will then bind to the polypeptides by itself. In this way, a sequence encoding only a fragment of the polypeptides can still be used to generate the antibodies that bind to all the native polypeptides. Such polypeptides can then be used to isolate the polypeptide from the tissue expressing that antibody. For the preparation of the monoclonal antibodies, any technique that provides antibodies produced by cultures of continuous cell lines can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256: 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72 ), and the EBV hydridome technique to produce human monoclonal antibodies (Colé, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). The techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single chain antibodies for the immunogenic polypeptide products of this invention. Also, transgenic mice can be used to express 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 should be understood that the present invention is not limited to such examples. All parts or quantities, unless otherwise specified, are by weight. To facilitate the understanding of the following examples, certain methods and / or terms that are frequently encountered will be described. The "Plasmids" are designated by a letter p preceded and / or followed by capital letters and / or numbers.

The starting plasmids here are either commercially available, publicly available on an unrestricted basis, or can be constructed from the available plasmids according to published procedures. further, plasmids equivalent to those described are known in the art and will be apparent to those skilled in the art. "Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme that only acts on certain sequences in DNA. The different restriction enzymes used here are commercially available and their reaction conditions, cofactors, and other factors were used as would a person skilled in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment with approximately 2 units of enzyme in approximately 20 μl of buffer was used. For the purpose of isolating the DNA fragments for the construction of the plasmid, typically from 5 to 50 μg of DNA were digested with 20 to 250 units of enzyme in a larger volume. The buffers and substrate amounts appropriate for the particular restriction enzymes are specified by the manufacturer. Incubation times of approximately 1 hour at 37 ° C were used, but may vary according to the distributor's instructions. After digestion the reaction was electroporated directly onto a polyacrylamide gel to isolate the desired fragment. The size preparation of the excised fragments was carried out using the 8 percent polyacrylamide gel described by Goeddel, D. et al, Nucleic Acids Res., 8: 4057 (1980). "Oligonucleotides" refer to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands, which can be chemically synthesized. Such synthetic oligonucleotides do not have 5 'phosphate and thus will not be ligated 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 stated, ligation can be performed using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0. 5 ng of approximately equimolar amounts of the DNA fragments to be ligated. Unless otherwise stated, the transformation was carried out according to that described in the method of Graham, F. and Van der Eb, A., Virology, 52: 456-457 (1973).

Example 1 Tissue distribution of FGF-10 mRNA in adult human tissues To analyze the expression of the FGF-10 mRNA, a Northern analysis was carried out with two ug of poly A + mRNA from several human adult tissues using radioactively labeled FGF-10 cDNA as a probe. The results indicate that a message of 1.4 kb was expressed more abundantly in the skeletal muscle, at an intermediate level in the heart, brain, placenta, kidney and pancreas, at a lower level in the liver. In the heart, 3 other fragments with sizes of 4.4 kb, 2.4 kb and 0.5 kb were also present. It is likely that that different size of mRNA in the heart is the result of the alternative splicing. In the brain, a 4.4 kb mRNA was also present. Nylon staining with 2 ug of poly A + mRNA from various human adult tissues attached to the membrane was obtained from Clontech Laboratories, Inc. Palo Alto, CA. The stain was hiked with all the cDNA of FGF-10 labeled with radioactive dCTP for marking a random priming. Hybridization was carried out in 7% SDS, 0.5 M. NaP04 pH 7.2, and 1% BSA at 65 ° C overnight. After washing 2 x 30 min in 0.2 x SSC, 0.1% SDS at 65 ° C, the stain was exposed to an X-ray film with an intensifying screen overnight.

Example 2 Expression of FGF-10 by transcription and translation In vi tro The FGF-10 cDNA, ATCC # 75696, was transcribed and translated in vi tro to determine the size of the translatable or translatable polypeptide encoded by the full-length and partial FGF-10 cDNA. The full-length and partial cDNA inserts the FGF-10 into the pBluescript SK vector where it is amplified by PCR with three primer pairs, 1) forward and forward M13 primers; 2) backward primer M13 and primer P20 of FGF; 3) backward primer M13 and primer P22 of FGF. The sequence of these primers is as follows: forward primer M13-2: 5'-ATGCTTCCGGCTCGTATG-3 '(SEQ ID No. 3) This sequence is located upstream of the 5' end of the FGF-10 cDNA insert in the pBluescript vector and is in an antisense orientation with respect to the cDNA. A promoter sequence of T3 is located between this primer and the cDNA of FGF-10. forward primer M13-2: 5'-GGGTTTTCCCAGTCACGAC-3 '(SEQ ID No. 4) This sequence is located downstream of the 3' end of the FGF-10 cDNA insert in the pBluescript vector and is in an antisense orientation with with respect to the cDNA insert. FGF primer P20: 5'-GTGAGATCTGAGGGAAGAAGGGGA-3 '(SEQ ID No. 5) The 15 bp sequence of this primer on the 3' primer is antisense to the FGF-10 cDNA sequence of 780-766 bp, the which is 12 bp downstream of the stop codon. FGF primer P22: 5'-CCACCGATAATCCTCCTT-3 '(SEQ ID No. 6) This sequence is located within the cDNA of the FGF-10 in an antisense orientation and is approximately 213 bp downstream of the stop codon. The PCR reaction with the three primer pairs produces products amplified with the T3 promoter sequence in the front of the cDNA insert.

The three pairs of primers produce PCR products that code for the full-length FGF-10 polypeptide. Approximately 1 μg of PCR product from the first pair of primers, 0.3 μg of the second pair of primers, 0.3 μg of the third pair of primers was used for transcription / translation in vi tro. The transcription / translation reaction in vi tro was carried out in a volume of 25 ul, using the Lisado systems of TNT Coupled Reticulocyte (Promega, CAT # L4950) Specifically, the reaction contains 12.5 ul of rabbit reticulocyte lysate TNT, 2 μl of TNT reaction buffer, 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; inhibitor of the RNase ribonuclease, 0.5 or 1 μg of PCR products. Se) added nuclease-free H20 to bring the volume to 25 ul. The reaction was incubated at 30 ° C for 2 hours. Five microliters of the reaction product was analyzed on a gradient of 4-20% SDS-PAGE gel. After fixing the 25% isopropanol and 10% acetic acid, the gel was dried and exposed to an X-ray film overnight at 70 ° C. As shown in Figure 3, the PCR products contain the full length FGF-10 cDNA and the cDNA lacks approximately 340 bp and approximately 140 bp in the 3 'n region. translated (3'-UTR) produced the same length of the translated or translational products, whose molecular weights were estimated to be around 19 kd (lanes 2-4). Numerous modifications and variations 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 in other circumstances than those particularly described.

SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: HU, ET AL. (ii) TITLE OF THE INVENTION: Factor 10 of Growth of the Fibroblasts (iii) NUMBER OF SEQUENCES: 6 (iv) DOMICILE FOR CORRESPONDENCE: (A) RECIPIENT: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN (B) STREET: 6 BECKER FARM ROAD (C) CITY: ROSELAND (D) STATE: NEW JERSEY (E) COUNTRY: USA (F) C.P .: 07068 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: 3.5 INCH DISK (B) COMPUTER: IBM PS / 2 (C) OPERATING SYSTEM: MS-DOS (D) PROGRAM: WORD PERFECT 5.1 (vi) DATA OF THE CURRENT APPLICATION. (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: Jointly (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION (A) APPLICATION NUMBER: 08 / 207,412 (B) DATE OF SUBMISSION: MARCH 8, 1994 (viii) INFORMATION FROM THE MANDATORY / AGENT (A) NAME: FERRARO, GREGORY D. (B) REGISTRATION NUMBER: 36,134 (C) REFERENCE NUMBER / FILE: 325800-347 (ix) INFORMATION BY TELECOMMUNICATION: (A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO: l (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 1121 PAIRS OF BASES (B) TYPE: NUCLEIC ACID (C) HEBRA: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l GGCAAAGTGG GATGATCTGT CACTACACCT GCAGCACCAC GCTCGGAGGA CAGCTCCTGC 60 CTGCAGC? TC CAGACCCAGG AASCCTGAGG GGAAOQAAOG AAGTACGOGC GAAATCATCA 120 GATTGGCTTC CCAGATTTGG GAATCTGAAG CGGGCCCACA TCITCCQGCC AACTTCCATT 180 GAACTTCCCA GCACTCGAAA GßGACCGAAA TGGAGAGCAA AGAACCCCAG CTCAAAGGGA 240 TGTG? CAAG GTATTCAOC CAGCAGGGAT ACTTCCTGC? GATGCACCCA GATGßTACCA 300 CTGATGGGAC CAAGQACGAA AACAGCGACT ACACTCTCTT CAATCTAATT CCCGTGGGCC 360 TGCGTGTAGT GGCCATCCAA GGAGTGAAGG CTAGCCTCTA TGTGGCCATG AATGGTGAAG 420 GCTATCTCTA CAGTrCAGAT GTT TCACTC CAGAATGCAA ATTCAAGGAA TCTGTGTTG 480 AAAACTACTA TOTOATCTAT TCTTCCACAC TOTACCGCCA GCAAGAATCA GGCCGAGCTT 540 GGTTTCTOGO ACTCAATAAA ßAAOGTCAAA TTATOAAOOs QAACAQAOTO AAOAAAACCA 600 AGCCCTCATC ACATTTTGTA CCGAACCTA TTßAAGTQTG TATOTACAQA GAACCATOGC 660 TACATGAAAT TGOAGAAAAA CAA? GQCSTT CA? GGAAA? G TTCTOOAACA CCAACCATQA 720 ATGGAGGCAA AGTTQTQAAT CAAQATTCAA CATAOCTOA? AACTCTCCCC TTCTTCCCTC 780 TCGCATCCCT TCCCCTTCCC TTCCTTCCCA TTTACCOOT TCCGGCCAOT AAATCCACCC 84O AAG AGA? GA AAATAAAATQ ACAACGCAAG CACCTAOTOO IAASATTCT GCACTCAAAA 900 TCTTCC? TG TOTAGOACAA GAAAATTGAA CCAAAGCGGG CGGGTTGCAA 'GG ?? TAGAA 9β AATTCACGTT CACAAAQATT ATCACACTGA AAAGCAAAQG AAAAAATAAA TCAGAA GCC 1020 ATAAATATGA AACTAAACGG TATGGTGATT AGTAGAAGGC TAATTGTAAT GAAGACATGA íoßc ATAAAGGTGA AATAAACTTA AAAAAAAAAA AAAAAAAAAA A 1121 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 181 AMINO ACIDS (B) TYPE: AMIONACIDES (C) HEBRA: (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: PROTEIN (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Met Glu Ser Lys Glu Pro Gln Leu Lyß Gly lie Val Thr Arg Leu 5 10 15 Phe Ser Gln Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr 20 25 30 lie Asp Gly Thr Lyß Asp Glu Asn Ser Asp Tyr Thr Leu Phe Aßn 35 40 45 Leu He Pro Val Gly Leu Arg Val Val Ala He Gln Gly Val Lys 50 55 60 Wing Ser Leu Tyr Val Wing Met Aßn Gly Glu Gly Tyr Leu Tyr Ser 65 70 75 Be Asp Val Phe Thr Pro Glu Cys Lyß Phe Lyß Glu Ser Val Phe 80 85 90 Glu Aßn Tyr Tyr Val He Tyr Ser Ser Thr Leu Tyr Arg Gln Gln 95 100 105 Glu Ser Gly Arg Wing Trp Phe Leu Gly Leu Aßn Lyß Glu Gly Gln 110 115 120 He Met Lys Gly Aßn Arg Val Lyß Lyß Thr Lyß Pro Ser Ser His 125 130 135 Phe Val Pro Lyß Pro He Glu Val Cyß Met Tyr Arg Glu Pro Ser 140 145 150 Leu Hiß Glu He Gly Glu Lyß Gln Gly Arg Ser Arg Lyß Ser Ser 155 160 165 (2) INFORMATION FOR SEQ ID NO: 3 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 18 PAIRS OF BASES (B) TYPE: NUCLEIC ACID (C) HEBRA: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: ATGCTTCCGG CTCGTATG 18 (2) INFORMATION FOR SEQ ID NO:: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 19 PAIRS OF BASES (B) TYPE: NUCLEIC ACID (C) HEBRA: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GGGTTTTCCC AGTCACGAC 19 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 24 PAIRS OF BASES (B) TYPE: NUCLEIC ACID (C) HEBRA: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GTGAGATCTG AGGGAAGAAG GGGA 24 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 18 PAIRS OF BASES (B) TYPE: NUCLEIC ACID (C) HEBRA: SINGLE (D) TOPOLOGY: LINEAR (ii) TYPE OF MOLECULE: Oligonucleotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CCACCGATAA TCCTCCTT 18 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (24)

1. An isolated polynucleotide, characterized in that it is selected from the group consisting of: (a) a polynucleotide encoding the polypeptide having the deduced amino acid sequence of SEQ ID No. 2 or a fragment, analog or derivative of such polypeptide; (b) a polynucleotide encoding the polypeptide having the amino acid sequence encoded by the cDNA contained in the ATCC Deposit No. 75696 or a fragment, analog or derivative of such polypeptide.

2. The polynucleotide according to claim 1, characterized in that the polynucleotide is DNA.

3. The polynucleotide according to claim 1, characterized in that the polynucleotide is RNA.

4. The polynucleotide according to claim 1, characterized in that the polynucleotide is genomic DNA.

5. The polynucleotide according to claim 2, characterized in that the polynucleotide encodes a polypeptide having the amino acid sequence deduced from SEQ ID No. 2.

6. The polynucleotide according to claim 2, characterized in that the polynucleotide encodes the polypeptide encoded by the cDNA of ATCC Deposit No. 75696.

7. The polynucleotide according to claim 1, characterized in that it has the coding sequence shown in SEQ ID No. 1.

8. The polynucleotide according to claim 2, characterized in that it has the coding sequence of the polypeptide deposited in the deposit ATCC 75696.

9. A vector, characterized in that it contains DNA according to claim 2.

10. A host cell, characterized in that it was designed with the vector according to claim 9.

11. A process for producing a polypeptide, characterized in that it comprises: expressing the host cell according to claim 10 of the polypeptide encoded by the DNA.

12. A process characterized in that it is for producing cells capable of expressing a polypeptide comprising the cells genetically engineered with the vector in accordance with. claim 9.

13. An isolated DNA, characterized in that it is hybridizable to DNA according to claim 2 and codes for a polypeptide having FGF-10 activity.

14. A polypeptide, characterized in that it is selected from the group consisting of: (i) a polypeptide having the deduced amino acid sequence of SEQ ID No. 2 and fragments, analogs and derivatives thereof, and (ii) a polypeptide encoded by the cDNA of ATCC Deposit No. 75696 and fragments, analogs and derivatives of such polypeptide.

15. The polypeptide according to claim 14, characterized in that the polypeptide is FGF-10 having the deduced amino acid sequence of SEQ ID No. 2.

16. An antibody, characterized in that it is against the polypeptide of claim 14.

17. A compound, characterized in that it is effective as an antagonist for the polypeptide according to claim 14.

18. A compound, characterized in that it is effective as an antagonist against the polypeptide according to claim 14.

19. A method for the treatment of a patient in need of FGF-10, characterized in that it comprises: administering to the patient a therapeutically effective amount of the polypeptide according to claim 14.

20. A method for the treatment of a patient having a need to inhibit FGF-10, characterized in that it comprises: administering to the patient a therapeutically effective amount of the compound according to claim 18.

21. The method according to claim 19, characterized in that the therapeutically effective amount of polypeptide is administered by providing the patient with the DNA encoding the polypeptide and expressing the polypeptide in vivo.

22. A process for identifying active compounds as agonists and antagonists for FGF-10, characterized in that it comprises: (a) combining FGF-10, a compound to be selected, and a reaction mixture containing cells under conditions wherein the cells are normally stimulated by FGF-10, the reaction mixture contains a tag incorporated into the cells as they proliferate; and (b) determining the degree of proliferation of the cells to identify whether the compound is an effective agonist or antagonist.

23. A process for diagnosing a disease or susceptibility to a disease related to a low expression of the polypeptide according to claim 14, characterized in that it comprises: determining a mutation in the sequence of nucleic acids encoding the polypeptide.

24. A diagnostic process, characterized in that it comprises: analyzing the presence of the polypeptide according to claim 14, in a sample derived from a host.