GLIAL MITOGENIC FACTORS, THEIR PREPARATION AND USE

Abstract: Disclosed is the characterization and purification of DNA encoding a numerous polypeptides useful for the stimulation of glial cell (particularly, Schwann cell) mitogenesis and treating glial cell tumors. Also disclosed are DNA sequences encoding novel polypeptides which may have use in stimulating glial cell mitogenesis and treating glial cell tumors. Methods for the synthesis, purification and testing of both known and novel polypeptides for their use as both therapeutic and diagnostic aids in the treatment of diseases involving glial cells are also provided. Methods are also provided for the use of these polypeptides for the preparation of antibody probes useful for both diagnostic and therapeutic use in diseases involving glial cells.

Description: GLIAL MITOGENIC FACTORS, THEIR PREPARATION AND USE Cross Reference to Related Applications This application is a continuation-in-part of Serial No. 08/036,555, filed March 24, 1993, Serial No. 07/965,173, filed October 23, 1992, Serial No. 07/940,389, filed September 3, 1992, Serial No. 07/907,138, filed June 30, 1992 and Serial No. 07/863,703, filed April 3, 1992. Background of the Invention This invention relates to polypeptides found in vertebrate species, which polypeptides are mitogenic growth factors for glial cells, including Schwann cells. The .invention is also concerned with processes capable of producing such factors, and the therapeutic application of such factors. The glial cells of vertebrates constitute the specialized connective tissue of the central and peripheral nervous systems. Important glial cells include Schwann cells which provide metabolic support for neurons and which provide myelin sheathing around the axons of certain peripheral neurons, thereby forming individual nerve fibers. Schwann cells support neurons and provide a sheath effect by forming concentric layers of membrane around adjacent neural axons, twisting as they develop around the axons. These myelin sheaths are a susceptible element of many nerve fibers, and damage to Schwann cells, or failure in growth and development, can be associated with significant demyelination or nerve degeneration characteristic of a number of peripheral nervous system diseases and disorders. In the development of the nervous system, it has become apparent that cells require various factors to regulate their division and growth, and various such factors have been identified in recent years, including some found to have an effect on Schwann cell division or development. Thus, Brockes et al., inter alia, in J. Neuroεcience, (1984) 75-83 describe a protein growth 5 factor present in extracts from bovine brain and pituitary tissue, which was named Glial Growth Factor (GGF) . This factor stimulated cultured rat Schwann cells to divide against a background medium containing ten percent fetal calf serum. The factor was also described as having a 10 molecular weight of 31,000 Daltons and as readily di erizing. In Meth. Enz., 147 (1987), 217-225, Brockes describes a Schwann cell-based assay for GGF. Brockes et al., supra, also describes a method of purification of GGF to apparent homogeneity. In brief, one 15 large-scale purification method described involves extraction of the lyophilized bovine anterior lobes and chromatography of material obtained thereby using NaCl gradient elution from CM cellulose. Gel filtration is then carried out with an Ultrogel column, followed by elution 20 from a phosphocellulose column, and finally, small-scale SDS gel electrophoresis. Alternatively, the CM-cellulose material was applied directly to a phosphocellulose column, fractions from the column were pooled and purified by preparative native gel electrophoresis, followed by a final 25 SDS gel electrophoresis. Brockes et al. observed that in previously reported gel filtration experiments (Brockes et al., J. Biol. Chem. • ' ^55 (1980) 8374-8377) , the major peak of growth factor activity was observed to migrate with a molecular weight of 30 56,000 Daltons, whereas in the first of the above-described procedures activity was predominantly observed at molecular SUBSTITUTESHEET(RULE26^ weight 31,000. It is reported that the GGF dimer is largely removed as a result of the gradient elution from CM-cellulose in this procedure. Benveniste et al. (PNAS, SI (1985), 3930-3934) describes a T lymphocyte-derived glial growth promoting factor. This factor, under reducing conditions, exhibits a change in apparent molecular weight on SDS gels. Kimura et al. (Nature, 2Λ3. (1990), 257-260) describes a factor they term Schwannoma-derived growth factor (SDGF) which is obtained from a sciatic nerve sheath tumor. The authors state that SDGF does not stimulate the incorporation of. tritium-labelled TdR into cultured Schwann cells under conditions where, in contrast, partially purified pituitary fraction containing GGF is active. SDGF has an apparent molecular weight of between 31,000 and 35,000. Davis and Stroobant (J. Cell. Biol., 110 (1990), 1353-1360) describe the screening of a number of candidate mitogens. Rat Schwann cells were used, the chosen candidate substances being examined for their ability to stimulate DNA synthesis in the Schwann cells in the presence of 10% FCS (fetal calf serum), with and without forskolin. One of the factors tested was GGF-carboxymethyl cellulose fraction (GGF-CM) , which was itogenic in the presence of FCS, with and without forskolin. The work revealed that in the presence of forskolin, inter alia, platelet derived growth factor (PDGF) was a potent mitogen for Schwann cells, PDGF having previously been thought to have no effect on Schwann cells. Holmes et al. Science (1992) £££: 1205 and Wen et al. Cell (1992) ££: 559 demonstrate that DNA sequences which encode proteins binding to a receptor (plδS"* 82 ) are associated with several human tumors. The pl 5 tΛm protein is a 185 kilodalton membrane spanning protein with tyrosine kinase activity. The protein is encoded by the erbB2 proto-oncogene (Yarden and Ullrich Ann. Rev. Biochem. SI; 443 (1988)). The erbB2 gene, also referred to as HER-2 (in human cells) and neu (in rat cells) , is closely related to the receptor for epidermal growth factor (EGF) . Recent evidence indicates that proteins which interact with (and activate the kinase of) p 185 « __ 2 i n( j uce proliferation in the cells bearing plSS"* 82 (Holmes et al. Science 256; 1205 (1992); Dobashi et al. Proc. Natl. Acad. Sci. 88: 8582 (1991) ; Lupu et al. Proc. Natl. Acad. Sci. ££: 2287 (1992)). Furthermore, it is evident that the gene encoding pies'* 82 binding proteins produces a number of variably-sized, differentially-spliced RNA transcripts that give rise to a series of proteins, which are of different lengths and contain some common peptide sequences and some unique peptide sequences. This is supported by the differentially-spliced RNA transcripts recoverable from human breast cancer (MDA-MB-231) (Holmes et al. Science 256; 1205 (1992)). Further support derives from the wide size range of proteins which act as (as disclosed herein) ligands for the plSS** 82 receptor (see below) . Summary of the Invention In general the invention provides methods for stimulating glial cell (in particular, Schwann cell and glia of the central nervous system) mitogenesis, as well as new proteins exhibiting such glial cell mitogenic activity. In addition, DNA encoding these proteins and antibodies which bind these and related proteins are provided. The novel proteins of the invention include alternative splicing products of sequences encoding known polypeptides. Generally, these known proteins are members of the GGF/pl85 tΛB2 family of proteins. Specifically, the invention provides polypeptides of a specified formula, and DNA sequences encoding those polypeptides. The polypeptides have the formula WYBAZCX wherein WYBAZCX is composed of the a ino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 141-147, 160, 161, 173-178, 42-44, 77); wherein W comprises the polypeptide segment F, or is absent; wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprises the polypeptide segments C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, or C/D C/D' D' HKL; provided that, either a) at least one of F, Y, B, A, Z, C, or X is of bovine origin; or b) Y comprises the polypeptide segment E; or c) X comprises the polypeptide segments C/D HKL, C/D D, C/D' HKL, C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, C/D'H, C/D C/D'H, or C/D C/D' HL. In addition, the invention includes the DNA sequence comprising coding segments 3' FBA 3' as well as the with corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 139, 173-175) ; the DNA sequence comprising the coding segments 5 'FBA' 3 ' as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 140, 173, 174); the DNA sequence comprising the coding segments 'FEBA 3' as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 173-175); the DNA sequence comprising the coding segments 5' FEBA' 3' as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-138, 140, 173, 174); and the DNA sequence comprising the polypeptide coding segments of the GGF2HBS5 cDNA. clone (ATCC Deposit No. 75298, deposited September 2, .1992). The invention further includes peptides of the formula FBA, FEBA, FBA' FEBA' and DNA sequences encoding these peptides wherein the polypeptide segments correspond to amino acid sequences shown in Figure 31, SEQ ID Nos. (136, 138, 139, 173-175), (136-139, 173-175) and (136, 138, 140, 173, 174) and (136-138, 140, 173, 174) respectively. The polypeptide purified GGF-II polypeptide (SEQ ID No. 167) is also included as a part of the invention. Further included as an aspect of the invention are peptides and DNA encoding such peptides which are useful for the treatment of glia and in particular oligodendrocytes, microglia and astrocytes, of the central nervous system and methods for the administration of these peptides. suBStmπES** ^ Also included in this invention is the mature GGF peptide and the DNA encoding said peptide, exclusive of the N-terminal signal sequence, which is also useful for the treatment of conditions of the central nervous system and for the preparation of antibodies specific for said peptides. These antibodies may be useful for purification of peptides described herein and for diagnostic applications. The invention further includes vectors including DNA sequences which encode the amino acid sequences, as defined above. Also included are a host cell containing the isolated DNA encoding the amino acid sequences, as defined above. The invention further includes those compounds which bind the plδδ 0 * 82 receptor and stimulate glial cell mitogenesis in vivo and/or in vitro. Also a part of the invention are antibodies to the novel peptides described herein. In addition, antibodies to any of the peptides described herein may be used for the purification of polypeptides described herein. The antibodies to the polypeptides may also be used for the therapeutic inhibitor of glial cell mitogenesis. The invention further provides a method for stimulating glial cell mitogenesis comprising contacting glial cells with a polypeptide defined by the formula WYBAZCX wherein WYBAZCX is composed of the polypeptide segments shown in Figure 31 (SEQ ID Nos. 136-139, 141-147, 160, 161, 173-178, 42-44, 77); wherein W comprises the polypeptide segment F, or is absent wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprises the polypeptide segment C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, or C/D C/D' D' HKL. The invention also includes a method for the preparation of a glial cell mitogenic factor which consists of culturing modified host cells as defined above under conditions permitting expression of the DNA sequences of the invention. The peptides of the invention can be used to make a pharmaceutical or veterinary formulation for pharmaceutical or veterinary use. Optionally, the formulation may be used together with an acceptable diluent, carrier or excipient and/or in unit dosage form. A method for stimulating mitogenesis of a glial cell by contacting the glial cell with a polypeptide defined above as a glial cell itogen in vivo or in vitro is also an aspect of the invention. A method for producing a glial cell mitogenic effect in a vertebrate (preferably a mammal, more preferably a human) by administering an effective amount of a polypeptide as defined is also a component of the invention. Methods for treatment of diseases and disorders using the polypeptides described are also a part of the invention. For instance, a method of treatment or prophylaxis for a nervous disease or disorder can be effected with the polypeptides described. Also included are a method for the prophylaxis or treatment of a pathophysiological condition of the nervous system in which a cell type is involved which is sensitive or responsive to a polypeptide as defined are a part of the invention. Included in the invention as well, are methods for treatment when the condition involves peripheral nerve damage; nerve damage in the central nervous system; neurodegenerative disorders; demyelination in peripheral or central nervous system; or damage or loss of Schwann cells oligodendrocytes, microglia, or astrocytes. For example a neuropathy of sensory or motor nerve fibers; or the treatment of a neurodegenerative disorder are included. In any of these cases, treatment consists of administering an effective amount of the polypeptide. The invention*also includes a method for inducing neural regeneration and/or repair by administering an effective amount of a polypeptide as defined above. Such a medicament is made by administering the polypeptide with a pharmaceutically effective carrier. The invention includes the use of a polypeptide as defined above in the manufacture of a medicament. The invention further includes the use of a polypeptide as defined above -to immunize a mammal for producing antibodies, which can optionally be used for therapeutic or diagnostic purposes -in a competitive assay to identify or quantify molecules having receptor binding characteristics corresponding to those of the polypeptide; and/or -for contacting a sample with a polypeptide, as mentioned above, along with a receptor capable of binding specifically to the polypeptide for the purpose of detecting competitive inhibition of binding to the polypeptide. SUBSTITUTE SHEET (RULE 2β 10 -in an affinity isolation process, optionally affinity chromatography, for the separation of a corresponding receptor. The invention also includes a method for the prophylaxis or treatment of a glial tumor. This method consists of administering an effective amount of a substance which inhibits the binding of a factor as defined by the peptides above. Furthermore, the invention includes a method of stimulating glial cell mitogenic activity by the application to the glial cell of a -30 kD polypeptide factor isolated from the MDA - MB 231 human breast cell line; or -35 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line to the glial cell or -75 kD polypeptide factor isolated from the SKBR-3 human breast cell line; or -44 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line; or -25kD polypeptide factor isolated from activated mouse peritoneal acrophages; or -45 kD polypeptide factor isolated from the MDA - MB 231 human breast cell; or -7 to 14 kD polypeptide factor isolated from the ATL-2 human T-cell line to the glial cell; or -25 kD polypeptide factor isolated from the bovine kidney cell; or -42 kD polypeptide factor (ARIA) isolated from brains. The invention further includes a method for the use of the EGFL1, EGFL2, EGFL3, EGFL4, EGFL5, and EGFL6 11 polypeptides. Figure 38 to 43 and SEQ ID Nos. 154 to 159, respectively, for the stimulation of glial cell mitogenesis in vivo and in vitro. Also included in the invention is the administration of the GGF-II polypeptide whose sequence is shown in Figure 45 for the stimulation of glial cell mitogenesis. An additional aspect of the invention includes the use of the above-referenced peptides for the purpose of stimulating Schwann cells to produce growth factors which may, in turn, be harvested for scientific or therapeutic use. Furthermore, the peptides described herein may be used to induce central glial proliferation and remyelination for treatment of diseases, e.g., MS, where re-myelination is desired. In an additional aspect of the invention, the novel polypeptides described herein may be used to stimulate the synthesis of acetylcholine receptors. As mentioned above, the invention provides new glial growth factors from mammalian sources, including bovine and human, which are distinguished from known factors. These factors are mitogenic for Schwann cells against a background of fetal calf plasma (FCP) . The invention also provides processes for the preparation of these factors, and an improved method for defining activity of these and other factors. Therapeutic application of the factors is a further significant aspect of the invention. Thus, important aspects of the invention are: (a) a basic polypeptide factor having glial cell mitogenic activity, more specifically, Schwann cell mitogenic activity in the presence of fetal calf plasma, a molecular weight of from about 30 kD to about 36 kD, and including within its amino acid sequence any one or more of the following peptide sequences: F K G D A H T E A S L A D E Y E Y M X K T E T S S S G L X L K A S L A D E Y E Y M R K A G Y F A E X A R T T E M A S E Q G A A K E A L A A L K F V L Q A K K E T Q P D P G Q I L K K V P M V I G A Y T E Y K C L K F K W F K K A T V M E X K F Y V P K L E F L X A K; and (b) a basic polypeptide factor which stimulates glial cell mitogenesis, particularly the division of Schwann cells, in the presence of fetal calf plasma, has a molecular weight of from about 55 kD to about 63 kD, and including within its amino acid sequence any one or more of the following peptide sequences: V H Q V W A A K Y I F F M E P E A X S S G L G A W G P P A F P V X Y W F V V I E G K A S P V S V G S V Q E L Q R V C L L T V A A L P P T K V H Q V W A A K K A S L A D S G E Y M X K D L L L X V E G K V H P Q R R G A L D R K P S C G R L K E D S Y I F F M E E L N R K N K P Q N I K I Q K K The novel peptide sequences set out above, derived from the smaller molecular weight polypeptide factor, and from the larger molecular weight polypeptide factor, are also aspects of this invention in their own right. These sequences are useful as probe sources for polypeptide factors of the invention, for investigating, isolating or preparing such factors (or corresponding gene sequences) from a range of different species, or preparing such factors by recombinant technology, and in the generation of corresponding antibodies, by conventional technologies, preferably monoclonal antibodies, which are themselves useful investigative tools and are possible therapeutics. The invention also includes an isolated glial cell mitogenic activity encoding gene sequence, or fragment thereof, obtainable by the methods set out above for the novel peptide sequences of the invention. The availability of short peptides from the highly purified factors of the invention has enabled additional sequences to be determined (see Examples to follow) . Thus, the invention further embraces a polypeptide factor having glial cell mitogenic activity and including an amino acid sequence encoded by: (a) a DNA sequence shown in any one of Figures 28a, 28b or 28c, SEQ ID Nos. 133-135, respectively; 14 (b) a DNA sequence shown in Figure 22, SEQ ID No, 89; (c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in Figure 28a, SEQ ID No. 133; or (d) a DNA sequence hybridizable to any one of the DNA sequences according to (a) , (b) or (c) . The invention further includes sequences which have greater than 60%, preferably 80%, sequence identity of homology to the sequences indicated above. While the present invention is not limited to a particular set of hybridization conditions, the following protocol gives general guidance which may, if desired, be followed: DNA probes may be labelled to high specific activity (approximately 10 8 to 10 93 Pdmp/μg) by nick-translation or by PCR reactions according to Schowalter and Sommer (Anal. Bioche . , 177:90-94, 1989) and purified by desalting on G-150 Sephadex columns. Probes may be denatured (10 minutes in boiling water followed by immersion into ice water) , then added to hybridization solutions of 80% buffer B (2g polyvinylpyrolidine, 2g Ficoll-400, 2g bovine serum albumin, 50ml 1 M Tris HCL (pH 7.5), 58g NaCl, lg sodium pyrophosphate, lOg sodium dodecyl sulfate, 950ml H 2 0) containing 10% dextran sulfate at 10 6 dpm M p per ml and incubated overnight (approximately 16 hours) at 60 * C. The filters may then be washed at 60"C, first in buffer B for 15 minutes followed by three 20-minute washes in 2X SSC, 0.1% SDS then one for 20 minutes in lx SSC, 0.1% SDS. In other respects, the invention provides: SUBSTITUTESHEET(RULE 2 15 (a) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, whether in reducing conditions or not, of from about 30kD to about 36kD on SDS-po yacrylamide gel electrophoresis using the following molecular weight standards: Lysozyme (hen egg white) 14,400 Soybean trypsin inhibitor 21,500 Carbonic anhydrase (bovine) 31,000 Ovalbumin (hen egg white) 45,000 Bovine serum albumin 66,200 Phosphorylase B (rabbit muscle) 97,400; which factor has glial cell mitogenic activity including stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least 50% of said activity after 10 weeks incubation in 0.1% trifluoroacetic acid at (b) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, under non-reducing conditions, of from about 55 kD to about 63 kD on SDS-polyacrylamide gel electrophoresis using the following molecular weight standards: Lysozyme (hen egg white) 14,400 Soybean trypsin inhibitor 21,500 Carbonic anhydrase (bovine) 31,000 Ovalbumin (hen egg white) 45,000 Bovine serum albumin 66,200 ^ Phosphorylase B (rabbit muscle) 97,400; which factor the human equivalent of which is encoded by DNA clone GGF2HBS5 described herein and which factor has glial cell, mitogenic activity including stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least 50% of the activity after 4 days incubation in 0.1% trifluoroacetic acid at 4 * C. For convenience of description only, the lower molecular weight and higher molecular weight factors of this invention are referred to hereafter as "GGF-I" and "GGF-II", respectively. The "GGF2" designation is used for all clones isolated with peptide sequence data derived from GGF-II protein (i.e., GGF2HBS5, GGF2BPP3) . It will be appreciated that the molecular weight range limits quoted are not exact, but are subject to slight variations depending upon the source of the particular polypeptide factor. A variation of, say, about 10% would not, for example, be impossible for material from another source. Another important aspect of the invention is a DNA sequence encoding a polypeptide having glial cell mitogenic activity and comprising: (a) a DNA sequence shown in any one of Figures 28a, 28b or 28c, SEQ ID Nos. 133-135: (b) a DNA sequence shown in Figure 22, SEQ ID No. 89; (c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in Figure 28a, SEQ ID No. 133; or 17 (d) a DNA sequence hybridizable to any one of the DNA sequences according to (a) , (b) or (c) . Another aspect of the present invention uses the fact that the Glial Growth Factors and plβS"* 82 ligand proteins are encoded by the same gene. A variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show plSS"* 82 binding and activation. Several of the (GGF-II) gene products have been used to show Schwann cell mitogenic activity. This invention provides a use for all of the known products of the GGF/plβS 6 * 82 ligand gene (described in the references listed above) as Schwann cell mitogens. This invention also relates to other, not yet naturally isolated splicing variants of the Glial Growth Factor gene. Figure 30, shows the known patterns of splicing derived from polymerase chain reaction experiments (on reverse transcribed RNA) and analysis of cDNA clones (as presented within) and derived from what has been published as sequences encoding plSδ 0 * 82 ligands (Peles et al., Cell £1:205 (1992) and Wen et al.. Cell ££:559 (1992)). These patterns, as well as additional ones disclosed herein, represent probable splicing variants which exist. Thus another aspect of the present invention relates to the nucleotide sequences encoding novel protein factors derived from this gene. The invention also provides processes for the preparation of these factors. Therapeutic application of these new factors is a further aspect of the invention. Thus other important aspects of the invention are : (a) A series of human and bovine polypeptide factors having glial cell mitogenic activity including SUBST,TUTESHEET(^ stimulating the division of Schwann cells. These peptide sequences are shown in Figures 31, 32, 33 and 34, SEQ ID Nos. 136-137, 173, respectively. (b) A series of polypeptide factors having glial cell mitogenic activity including stimulating the division of Schwann cells and purified and characterized according to the procedures outlined by Lupu et al. Science 249: 1552 (1990) ; Lupu et al. Proc. Natl. Acad. Sci USA ££: 2287 (1992); Holmes et al. Science 256: 1205 (1992); Peles et al. j≥£: 205 (1992) ; Yarden and Peles Biochemistry ££): 3543 (1991); Dobashi et al. Proc. Natl. Acad. Sci. _§_£: 8582 (1991); Davis et al. Biochem. Biophys. Res. Commun. 179: 1536 (1991); Beaumont et al. , patent application PCT/US91/03443 (1990); Greene et al. patent application PCT/US91/02331 (1990); Usdin and Fischbach, J. Cell. Biol. 103:493-507 (1986); Falls et al.. Cold Spring Harbor Symp. Quant. Biol. 55:397-406 (1990); Harris et al., Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991); and Falls et al., Cell 72:801-815 (1993). (c) A polypeptide factor (GGFBPP5) having glial cell mitogenic activity including stimulating the division of Schwann cells. The amino acid sequence is shown in Figure 32, SEQ ID No. 148, and is encoded by the bovine DNA sequence shown in Figure 32, SEQ ID No. 148. The novel human peptide sequences described above and presented in Figures 31, 32, 33 and 34, SEQ ID Nos. 136- 150, 173-176, 178, 42-44, 77, respectively, represent a series of splicing variants which can be isolated as full length complementary DNAs (cDNAs) from natural sources (cDNA libraries prepared from the appropriate tissues) or can be SUBSTITUTE SHEET (RULE 2^ assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art. Other compounds in particular, peptides, which bind specifically to the pies'* 82 receptor can also be used according to the invention as a glial cell itogen. A candidate compound can be routinely screened for plδS** 82 binding, and, if it binds, can then be screened for glial cell mitogenic activity using the methods described herein. The invention includes any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included. By way of illustration, in EP-A 109748 mutations of native proteins are disclosed in which the possibility of unwanted disulfide bonding is avoided by replacing any cyεteine in the native sequence which is not necessary for biological activity with a neutral amino acid. The statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention. The new sequences of the invention open up the benefits of reco binant technology. The invention thus also includes the following aspects: (a) DNA constructs comprising DNA sequences as defined above in operable reading frame position within vectors (positioned relative to control sequences so as to permit expression of the sequences) in chosen host cells after transformation thereof by the constructs (preferably the control sequence includes regulatable promoters, e.g. Trp) . It will be appreciated that the selection of a promoter and regulatory sequences (if any) are matters of choice for those of skill in the art; (b) host cells modified by incorporating constructs as defined in (a) immediately above so that said DNA sequences may be expressed in said host cells - the choice of host is not critical, and chosen cells may be prokaryotic or eukaryotic and may be genetically modified to incorporate said constructs by methods known in the art; and, (c) a process for the preparation of factors as defined above comprising cultivating the modified host cells under conditions permitting expression of the DNA sequences. These conditions can be readily determined, for any particular embodiment, by those of skill in the art of recombinant DNA technology. Glial cell mitogens prepared by this means are included in the present invention. None of the factors described in the art has the combination of characteristics possessed by the present new polypeptide factors. As indicated, the Schwann cell assay used to characterize the present factors employs a background of fetal calf plasma. In all other respects, the assay can be the same as that described by Brockes et al. in Meth. Enz., supra, but with 10% FCP replacing 10% FCS. This difference in assay techniques is significant, since the absence of platelet-derived factors in fetal calf plasma (as opposed to serum) enables a more rigorous definition of activity on Schwann cells by eliminating potentially spurious effects from some other factors. The invention also includes a process for the preparation of a polypeptide as defined above, extracting vertebrate brain material to obtain protein, subjecting the resulting extract to chromatographic purification by hydroxylapatite HPLC and then subjecting these fractions to SDS-polyacrylamide gel electrophoresis. The fraction which has an observed molecular weight of about 30kD to 36 kD and/or the fraction which has an observed molecular weight of about 55kD to 63 kD is collected. In either case, the fraction is subjected to SDS-polyacrylamide gel electrophoresis using the following molecular weight standards: Lysozyme (hen egg white) 14,400 Soybean trypsin inhibitor 21,500 Carbonic anhydrase (bovine) 31,000 Ovalbumin (hen egg white) 45,000 Bovine serum albumin 66,200 Phosphorylase B (rabbit muscle) 97,400 In the case of the smaller molecular weight fraction, the SDS-polyacrylamide gel is run in non-reducing conditions in reducing conditions or, and in the case of the larger molecular weight fraction the gel is run under non-reducing conditions. The fractions are then tested for activity stimulating the division of rat Schwann cells against a background of fetal calf plasma. Preferably, the above process starts by isolating a relevant fraction obtained by carboxymethyl cellulose chro atograph , e.g. from bovine pituitary material. It is also preferred that hydroxylapatite HPLC, cation exchange chromatography, gel filtration, and/or reversed-phase HPLC be employed prior to the SDS-Polyacrylamide gel electrophoresis. At each stage in the process, activity may be determined using Schwann cell incorporation of SUBSTITUTE SHEET (RULE W radioactive iododeoxyuridine as a measure in an assay generally as described by Brockes in Meth. Enz., supra, but modified by substituting 10% FCP for 10% FCS. As already noted, such as assay is an aspect of the invention in its own substance for CNS or PNS cell, e.g. Schwann cell, mitogenic effects. Thus, the invention also includes an assay for glial cell mitogenic activity in which a background of fetal calf plasma is employed against which to assess DNA synthesis in glial cells stimulated (if at all) by a substance under assay. Another aspect of the invention is a pharmaceutical or veterinary formulation comprising any factor as defined above formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form. In using the factors of the invention, conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions. Thus, the formulations of this invention can be applied to parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, opthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration. The formulations of this invention may also be administered by the transplantation into the patient of host cells expressing the DNA of the instant invention or by the use of surgical implants which release the formulations of the invention. Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Methods well known in the art for making formulations are to be found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain as excipientε sterile water or saline, polyalkylene glycols such as . polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, • methoxysalicylate for rectal administration, or citric acid for vaginal administration. The present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors. The concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration. In general terms, the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. General dose ranges are from about l mg/kg to about l g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of. the patient, the make up of the formulation, and the route of administration. As indicated above, Schwann cells (the glial cells of the peripheral nervous system) are stimulated to divide in the presence of the factors of the invention. Schwann cells of the peripheral nervous system are involved in supporting neurons and in creating the myelin sheath around individual nerve fibers. This sheath is important for proper conduction of electrical impulses to muscles and from sensory receptors. There are a variety of peripheral neuropathies in which Schwann cells and nerve fibers are damaged, either primarily or secondarily. There are many neuropathies of both sensory and motor fibers (Adams and Victor, Principles of Neurology) . The most important of those neuropathies are probably the neuropathies associates with diabetes, multiple sclerosis, Landry-Guillain-Barr syndrome, neuropathies caused by carcinomas, and neuropathies caused by toxic agents (some of which are used to treat carcinomas) . The invention, however, envisages treatment or prophylaxis of conditions where nervous system damage has been brought about by any basic cause, e.g. infection or injury. Thus, in addition to use of the present factors in the treatment of disorders or diseases of the nervous system where demyelination or loss of Schwann cells is present, such glial growth factors can be valuable in the treatment of disorders of the nervous system that have been caused by damage to the peripheral nerves. Following damage to peripheral nerves, the regeneration process is led by the growth or the re-establishment of Schwann cells, followed by the advancement of the nerve fibre back to its target. By speeding up the division of Schwann cells one could promote the regenerative process following damage. Similar approaches could be used to treat injuries or neurodegenerative disease of the central nervous system (brain and spinal cord) . Furthermore, there are a variety of tumors of glial cells the most common of which is probably neurofibromatosiε, which is a patchy small tumor created by overgrowth of glial cells. Also, it has been found that an activity very much like GGF can be found in some Schwann cell tumors, and therefore inhibitors of the action of the present factors on their receptors provides a therapy of a glial tumor, which comprises administering an effective amount of a substance which inhibits the binding of a factor, as defined above, to a receptor. SUBSTITUTESHEET(RULE2* In general, the invention includes the use of present polypeptide factors in the prophylaxis or treatment of any pathophysiological condition of the nervous εyεtem in which a factor-sensitive or factor-responεive cell type iε involved. The polypeptide factors of the invention can also be uεed as immunogens for making antibodies, such as monoclonal antibodies, following standard techniques. Such antibodies are included within the present invention. These antibodies can, in turn, be used for therapeutic or diagnostic purposes. Thus, conditions perhaps associated with abnormal levels of the factor may be tracked by using such antibodies. IB vitro techniques can be used, employing assays on isolated samples using standard methods. Imaging methods in which the antibodies are, for example, tagged with radioactive isotopes which can be imaged outside the body using techniques for the art of tumour imaging may also be employed. The invention also includes the general use of the present factors as glial cell mitogens in vivo or in vitro. and the factors for such use. One specific embodiment iε thus a method for producing a glial cell mitogenic effect in a vertebrate by administering an effective amount of a factor of the invention. A preferred embodiment iε such a method in the treatment or prophylaxis of a nervous system disease or disorder. A further general aspect of the invention is the use of a factor of the invention in the manufacture of a medicament, preferably for the treatment of a nervous disease or disorder, or for neural regeneration or repair. SU BSTUUTESHEET(R^26) Alsø included in the invention are the use of the factors of the invention in competitive assays to identify or quantify molecules having receptor binding characteristicε correεponding to thoεe of εaid polypeptideε. The polypeptideε may be labelled, optionally with a radioiεotope. A competitive assay can identify both antagoniεtε and agonists of the relevant receptor. In another aspect, the invention provides the uεe of each one of the factorε of the invention in an affinity isolation process, optionally affinity chromatography, for the separation of a respective corresponding receptor. Such processes for the isolation of receptors corresponding to particular proteins are known in the art, and a number of techniques are available and can be applied to the factors of the present invention. For example, in relation to IL-6 and IFN7 the reader is referred to Novick, D.; et al., J. Chromatogr. (1990) 510: .331-7. With respect to gonadotropin releasing hormone reference iε made to Hazu , E., J. (1990) Chromatogr. 510:233-6. In relation to G-CSF reference iε made to Fukunaga, R. , et al., J. Biol. Chem., 265:13366-90. In relation to IL-2 reference iε made to Smart, J.E., et al., (1990) J. Inveεt. Dermatol., «* :15δS-163S, and in relation to human IFN-gamma reference is made to Stefanos, S, e£ aj.. , (I9δ9) J. Interferon Res., 2:719-30. Brief Description of the Drawings The drawings will first be described. Drawings Figures 1 to δ relate to Example 1, and are briefly described below: SUBSTITUTE SHEET (RULE 26^ Fig. 1 iε the profile for product from carboxymethyl cellulose chromatography; Pig. 2 iε the profile for product from hydroxylapatite HPLC; Fig. 3 is the profile for product from Mono S FPLC; Fig. 4 is the profile for product from Gel filtration FPLC; Figs. 5 and 6 depict the profileε for the two partially purified polypeptide productε from reversed-phase HPLC; and Figs. 7 and 8 depict dose-response curves for the GGF-I and GGF-II fractions from reversed-phase HPLC using either a fetal calf serum or a fetal calf plasma background; Figs. 9 to 12 depict the peptide sequences derived from GGF-I and GGF-II, SEQ ID Nos. 1-20, 22-29, 32-53 and 169, (see Example 2 hereinafter), Figures 10 and 12 specifically depict novel εequenceε: In Fig. 10, Panel A, the εequenceε of GGF-I peptides used to design degenerate oligonucleotide probes and degenerate PCR primers are listed (SEQ ID Nos. 20, 1, 22-29, and 17) . Some of the sequences in Panel A were also used to design synthetic peptides. Panel B is a liεting of the sequences of novel peptides that were too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID Nos. 17 and 52); In Fig. 12, Panel A, is a listing of the εequenceε of GGF-II peptides used to design degenerate oligonucleotide probes and degenerate PCR primers (SEQ ID Nos. 45-52). Some of the sequences in Panel A were used to design synthetic peptides. Panel B is a listing of the novel peptide that SUBSTlUTESHEET(R E26) was too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID No. 53) ; Figures 13 to 20 relate to Example 3, below and depict the mitogenic activity of factors of the invention; Figures 21 to 2δ (a, b and c) relate to Example 4, below and are briefly described below: Fig. 21 is a liεting of the degenerate oligonucleotide probeε (SEQ ID Noε. 54-88) designed from the novel peptide sequences in Figure 10, Panel A and Figure 12, Panel A; Fig. 22 (SEQ ID No. 89) depicts a stretch of the putative bovine GGF-II gene sequence from the recombinant bovine genomic phage GGF2BG1, containing the binding site of degenerate oligonucleotide probeε 609 and 650 (see Figure 21, SEQ ID NOs. 69 and 72, respectively). The figure is the coding strand of the DNA sequence and the deduced amino acid sequence in the third reading frame. The εequence of peptide 12 from factor 2 (bold) iε part of a 66 amino acid open reading frame (nucleotideε 75272) ; Fig. 23 iε the degenerate PCR primerε (Panel A, SEQ IS Noε. 90-108) and unique PCR primerε (Panel B, SEQ ID Noε. 109-119) used in experi entε to iεolate segments of the bovine • GGF-II coding sequences present in RNA from posterior pituitary; Fig. 24 depicts of the nine diεtinct contiguous bovine GGF-II cDNA structures and sequences that were obtained in PCR amplification experiments uεing the liεt of primerε in Figure 7, Panels A and B, and RNA from posterior pituitary. The top line of the Figure is a schematic of the coding sequences which contribute to the cDNA structures that were characterized; Fig. 25 is a physical map of bovine recombinant phage of GGF2BG1. The bovine fragment is roughly 20 kb in length and containε two exonε (bold) of the bovine GGF-II gene. Restriction εites for the enzymes Xbal, Spel, Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map. Shaded portions correspond to fragments which were subcloned for sequencing; Fig. 26 is a schematic of the structure of three alternative gene products of the putative bovine GGF-II gene. Exons are listed A through E in the order of their discovery. The alternative splicing patterns 1, 2 and 3 generate three overlapping deduced protein structures (GGF2BPP1, 2, and 3), which are displayed in the various Figures 28a, b, c (described below) ; Fig. 27 (SEQ ID Noε. 120-132) iε a comparison of the GGF-I and GGF-II sequences identified in the deduced protein sequences shown in Figures 28a, 28b and 28c (described below) with the novel peptide εequenceε liεted in Figures 10 and 12. The Figure εhowε that six of the nine novel GGF-II peptide sequences are accounted for in these deduced protein εequenceε. Two peptide εequences similar to GGF-I sequences are also found; Fig. 28a (SEQ ID No. 133) iε a liεting of the coding εtrand DNA sequence and deduced amino acid sequence of the cDNA obtained from εplicing pattern number 1 in Figure 26. ' ' This partial cDNA of the putative bovine GGF-II gene encodes a protein of 206 amino acids in length. Peptides in bold were thoεe identified from the lists presented in Figures 10 and 12. Potential glycosylation εiteε are underlined (along with polyadenylation εignal AATAAA) ; 5U BST,TUTESHEET (RULE« Fig. 28b (SEQ ID No. 134) iε a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 2 in Figure 26. This partial cDNA of the putative bovine GGF-II gene encodeε a protein of 281 amino acids in length. Peptides in bold are those identified from the liεts presented in Figures 10 and 12. Potential glycosylation εites are underlined (along with polyadenylation signal AATAAA) ; Fig. 28c (SEQ ID No. 135) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 3 in Figure 26. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 257 amino acids in length. Peptides in bold are those identified from the lists in Figures 10 and 12. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) . Fig. 29, which relates to Example 6 hereinafter, iε an autoradiogram of a cross hybridization analysiε of putative bovine GGF-II gene εequences to a variety of mammalian DNAs on a southern blot. The filter contains lanes of EcoRI-digested DNA (5 μg per lane) from the εpecies listed in the Figure. The probe detectε a εingle εtrong band in each DNA sample, including a four kilobase fragment in the bovine DNA as anticipated by the physical map in Figure 25. Bands of relatively minor intensity are observed as well, which could represent related DNA εequenceε. The εtrong hybridizing band from each of the other mammalian DNA samples presumably represents the GGF-II homologue of those εpecies. Fig. 30 is a diagram of representative splicing variants. The coding εegmentε are repreεented by F, E, B, SUB ST1TUTESHEET ( RULE26. A, G, C, C/D, C/D' » D, D', H, K and L. The location of the peptide εequences derived from purified protein are indicated by "o". Fig. 31 (SEQ ID NOS. 136-147, 160, 161, 173-178, 42- 44, 77) is a liεting of the DNA εequences and predicted peptide sequences of the coding segments of GGF. Line 1 iε a liεting of the predicted amino acid εequences of bovine GGF, line 2 iε a liεting of the nucleotide εequences of bovine GGF, line 3 iε a listing of the nucleotide sequences of human GGF (heregulin) (nucleotide base matches are indicated with a vertical line) and line 4 is a listing of the predicted amino acid εequences of human GGF/heregulin where it differs from the predicted bovine sequence. Coding segments E, A' and K represent only the bovine εequences. Coding segment D' represents only the human (heregulin) sequence. Fig. 32 (SEQ ID No. 148) is the predicted GGF2 amino acid sequence and nucleotide εequence of BPP5. The upper line iε the nucleotide εequence and the lower line is the predicted amino acid εequence. Fig. 33 (SEQ ID No. 149) is the predicted amino acid εequence and nucleotide sequence of GGF2BPP2. The upper line is the nucleotide sequence and the lower line is the predicted amino acid sequence. Fig. 34 (SEQ ID No. 150) is the predicted amino acid εequence and nucleotide εequence of GGF2BPP . The upper line iε the nucleotide εequence and the lower line is the predicted amino acid sequence. Fig. 35 (SEQ ID Nos. 151-152) depicts the alignment of two GGF peptide εequences (GGF2bpp4 and GGF2bpp5) with SUTUTESHEET ^ SUB the human EGF (hEGF) . Asterisks indicate positions of conserved cysteineε. Fig. 36 depictε the level of GGF activity (Schwann cell mitogenic aεεay) and tyroεine phosphorylation of a ca. 200kD protein (intensity of a 200 kD band on an autoradiogram of a Western blot developed with an antiphosphotyroεine polyclonal antibody) in reεponεe to increaεing amountε of GGF. Fig. 37 iε a liεt of splicing variants derived from the εequenceε shown in Figure 31. Fig. 38 iε the predicted amino acid εequence, bottom, and nucleic εequence, top, of EGFL1 (SEQ ID No. 154) . Fig. 39 iε the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL2 (SEQ ID No. 155) . Fig. 40 iε the predicted amino acid εequence, bottom, and nucleic εequence, top, of EGFL3 (SEQ ID No.. 156). Fig. 41 iε the predicted amino acid sequence, bottom, and nucleic εequence, top, of EGFL4 (SEQ ID No. 157). Fig. 42 is the predicted amino acid εequence, bottom, and nucleic sequence, top, of EGFL5 (SEQ ID No. 158). Fig. 43 iε the predicted amino acid sequence, bottom, and nucleic εequence, top, of EGFL6 (SEQ ID No. 159). Fig. 44 iε a εcale coding segment map of the clone. T3 refers to the bacteriophage promoter uεed to produce RNA from the clone. R «= flanking EcoRI restriction enzyme SU BST Ϊ TUTESHEET(RULE26) εiteε. 5' UT referε to the 5' untranslated region. E, B, A, C, C/D', and D refer to the coding εegmentε. O ••= the tranεlation εtart site. Λ «= the 5' limit of the region homologous to the bovine E segment (εee example 6) and 3' UT referε to the 3' untranslated region. Fig. 45 is the predicted amino acid εequence (middle) and nucleic εequence (top) of GGF2HBS5 (SEQ ID No. 167) . The bottom (intermittent) εequence representε peptide εequenceε derived from GGF-II preparationε (εee Figureε 11, 12). Fig. 46 iε a graph depicting the Schwann cell mitogenic activity of recombinant human and bovine glial growth factors. Fig. 47 is a dose-response curve depicting Schwann cell proliferation activity data resulting from administration of different εize aliquotε of CHO cell conditioned medium. Fig. 48 iε a dose-response curve depicting Schwann cell mitogenic activity secreted into the extracellular medium by SF9 insect cells infected with baculovirus containing the GGF2HBS5 cDNA clone. Fig. 49 iε a Western blot of recombinant CHO cell conditioned medium using a GGF peptide antibody. Fig. 50 (A) is a graph of Schwann cell proliferation activity of recombinant (COS cell produced) human GGF-II (rhGGF-II) peak eluted from the cation exchange column; (B) is an immunoblot against recombinant GGFII peak using polyclonal antibody made against specific peptide of rhGGFII; Fig. 51 (A) is a graph showing the purification of rhGGF-II (CHO cell produced) on cation exchange column by SUBST,TUTESHEET(RULE« ) fraction; (B) iε a photograph of a Western blot using fractions as depicted in (A) and a rhGGF-II specific antibody. Fig. 52 is a photograph of a gel depicting tyrosine phosphorylation in Schwann cells treated with recombinant glial growth factors. Fig. 53 is the sequences of GGFHBS5, GGFHFB1 and GGFBPP5 polypeptides (SEQ ID NOS: 170, 171, and 172). Fig. 54 iε a map of the CHO cell-expression vector pcDHFRpolyA. Fig. 55 is the amino acid εequence of cDNA encoding mature hGGF2 (SEQ ID NO: 179) . Detailed Description The invention pertains to the isolation and purification of novel Glial Growth factors and the cloning of DNA sequences encoding these factors. Other components of the invention are εeveral gene splicing variants which potentially encode a series of glial growth factors, in particular the GGF2HBS5 in particular a variant which encodes the human equivalent of bovine GGF-II. It iε evident that the gene encoding GGF'ε and pl85 ϊA82 binding proteins produces a number of variably-sized, differentially-spliced RNA transcriptε that give riεe to a εeries of proteins, which are of different lengths and contain some common peptide εequenceε and some unique peptide εequences. This iε supported by the differentially- εpliced εequences which are recoverable from bovine posterior pituitary RNA (as presented herein) , human breast cancer (MDA-MB-231) (Holmes et al. Science £S6_: 1205 (1992) and chicken brain RNA (Falls et al. Cell 72:1-20 (1993)). Further support derives from the wide size range of proteins which act as both mitogens for Schwann cells (as disclosed herein) and as ligands for the plδS** 82 receptor (εee below) . Further evidence to support the fact that the genes encoding GGF and pies'* 82 are homologous comes from nucleotide sequence comparison. Science, 256 (1992) , 1205-1210) Holmes ei si. demonstrate the purification of a 45-kilodalton human protein (Heregulin-β) which specifically interacts with the receptor protein plδS** 82 , which is associated with εeveral human malignancies. Several complementary DNA cloneε encoding Heregulin-α were iεolated. Peles et al. (Cell ££:205 (1992)) and Wen et al (Cell 12:559 (1992)) describe a complementary DNA iεolated from rat cells encoding a protein called "neu differentiation factor" (NDF) . The translation product of the NDF cDNA has pies'* 82 binding activity. Usdin and Fischbach, J. Cell. Biol. 103:493-507 (1986); Falls et al.. Cold Spring Harbor Symp. Quant. Biol. 55:397-406 (1990); Harris et al., Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991); and Fallε et al.. Cell 72:801-815 (1993) demonstrate the purification of a 42 Kd glycoprotein which interacts with a receptor protein pies'* 82 and several complementary cDNA clones were iεolated (Fallε et al. Cell 72:601-815 (1993). Several other groupε have reported the purification of proteins of various molecular weights with P185** 82 binding activity. These groups include Lupu et al. (1992) Proc. Natl. Acad. Sci. USA 89:2287; Yarden and Peles (1991) Biochemistry 30:3543; Lupu et al. (1990) Science 249:1552); Dobashi et al. (1991) Biochem. Biophyε. Res. Com . 179:1536; and Huang et al. (1992) J. Biol. Chem. 257:11508-11512. Other Embodiments The invention includes any protein which iε substantially homologous to the coding segments in Figure 31 (SEQ ID Nos. 136-147, 160, 161, 173-176, 42-44, 77) as well as other naturally occurring GGF polypeptides. Also included are: allelic variations; natural mutantε; induced mutantε; proteinε encoded by DNA that hybridizeε under high or low stringency conditions to a nucleic acid naturally occurring (for definitionε of high and low εtringency εee Current Protocolε in Molecular Biology, John Wiley & Sons, New York, 19B9, 6.3.1 - 6.3.6, hereby incorporated by reference) ; and polypeptides or proteins specifically bound by antisera to GGF polypeptide. The term alεo includeε chi eric polypeptides that include the GGF polypeptides comprising sequences from Figure 31. The following examples are not intended to limit the invention, but are provided to usefully illustrate the same, and provide specific guidance for effective preparative techniques. As will be seen from Example 3, below, the present factors exhibit mitogenic activity on a range of cell types. The activity in relation to fibroblaεtε indicateε a wound repair ability, and the invention enco passeε thiε use. The general statements of invention above in relation to formulations and/or medicaments and their manufacture εhould clearly be construed to include appropriate products and uses. This iε clearly a reaεonable expectation for the preεent invention, given reportε of similar activities for fibroblast growth factors (FGFs) . Reference can be made, for example, to Sporn § fil. , "Peptide Growth Factors and their Receptors I", page 396 (Baird and Bohlen) in the section headed "FGFs in Wound Healing and Tiεεue Repair". EXAMPLE 1 Purification of GGF-I and GGF-TI from bovine Pituitaries I. Preparation of Factor-CM Fraction 4,000 frozen whole bovine pituitaries (ca. 12 kg) were thawed overnight, washed briefly with water and then homogenized in an equal volume of 0.15 M ammonium sulphate in batches in a Waring Blender. The homogenate was taken to pH 4.5 with 1.0 M HC1 and centrifuged at 4,900g for 80 minutes. Any fatty material in the supernatant was removed by paεsing it through glasε wool. After taking the pH of the εupernatant to 6.5 uεing 1.0 M NaOH, εolid ammonium sulphate was added to give a 36% εaturated solution. After several hours stirring, the εuεpenεion waε centrifuged at 4,900 g for 80 minuteε and the precipitate diεcarded. After filtration through glass wool, further solid ammonium sulphate was added to the εupernatant to give a 75% εaturated εolution which waε once again centrifuged at 4,900 g for 80 minuteε after εeveral hourε εtirring. The pellet was resuspended in ca. 2 L of 0.1 M sodium phosphate pH 6.0 and dialyzed againεt 3 x 40 L of the εame buffer. After confirming that the conductivity of the dialyεate waε below 20.0 μSiemenε, it waε loaded onto a Eioproceεε column (120 x 113 mm, Pharmacia) packed with carboxymethyl celluloεe (CM-52, Whatman) at a flow rate of 2 ml mini. The column waε waεhed with 2 volumeε of 0.1 M sodium phosphate pH 6.0, followed by 2 volumes of 50 mM NaCl, and finally 2 volumeε of 0.2 M NaCl both in the εame buffer. During the final εtep, 10 mL (5 minute) fractionε were collected. Fractions 73 to llδ inclusive were pooled, dialyzed against 10 volumes of 10 mM sodium phosphate pH 6.0 twice and clarified by centrifugation at 100,000 g for 60 minutes. XL . Hydroxylapatite HPLC Hydroxylapatite HPLC iε not a technique hitherto uεed in isolating glial growth factors, but proved particularly efficacious in thiε invention. The material obtained from the above CM-celluloεe chromatography was filtered through a 0.22 μm filter (Nalgene) , loaded at room temperature on to a high performance hydroxylapatite column (50 x 50 mm, Biorad) equipped with a guard column (15 x 25 mm, Biorad) and equilibrated with 10 mM potassium phosphate pH 6.0. Elution at room temperature waε carried out at a flow rate of 2 ml. inute" 1 uεing the following programmed linear gradient: 10 mM potaεsium phosphate pH 6.0 1.0 M potassium phoεphate pH 6.0 6.0 L (3 minuteε) fractionε were collected during the gradient elution. Fractionε 39-45 were pooled and dialyzed againεt 10 volumeε of 50 mM sodium phosphate pH 6.0. III. Mono S FPLC . Mono S FPLC enabled a more concentrated material to be prepared for εubεeguent gel filtration. Any particulate material in the pooled material from the hydroxylapatite column waε removed by a clarifying spin at 100,000 g for 60 minutes prior to loading on to a SUBSTVTUTESHEETIRULE26) preparative HRlO/10 Mono S cation exchange column (100 x 10 mm, Pharmacia) which was then re-equilibrated to 50mM sodium phosphate pH 6.0 at room temperature with a flow rate of 1.0 ml/minute' 1 . Under these conditions, bound protein waε eluted using the following programmed linear gradient: time (min) %B Solvent A: 50 mM potassium phosphate pH 6.0 Solvent B: 1.2 M sodium chloride, 50 mm sodium phosphate pH 6.0 1 mL (l minute) fractions were collected throughout this gradient program. Fractions 99 to 115 inclusive were pooled. I3 Gel Filtration FPLC This step commenced the separation of the two factors of the invention prior to final purification, producing enriched fractions. For the purposes of this step, a preparative Superose 12 FPLC column (510 x 20 mm, Pharmacia) was packed according to the manufacturers' instructions. In order to standardize this column, a theoretical plateε meaεurement waε made according to the manufacturers' instructions, giving a value of 9,700 theoretical plates. The pool of Mono S eluted material was applied at room temperature in 2.5 Ml aliquots to this column in 50mM sodium phosphate, 0.75 NaCl pH 6.0 (previously passed through a C18 reversed phase column (Sep-pak, Millipore) at a flow rate of l.o mL/minute' 1 . 1 mL (0.5 minute) fractionε were collected from 35 minuteε after each εample waε applied to the column. Fractionε 27 to 41 (GGF-II) and 42 to 57 (GGF-I) incluεive from each run were pooled. V. Reversed-Phase HPLC The GGF-I and GGF-II poolε from the above Superoεe 12 runε were each divided into three equal aliquotε. Each aliquot was loaded on to a C8 reverεed-phaεe column (Aquapore RP-3007 μ Cδ 220 x 4.6 mm. Applied Biosystems) protected by a guard cartridge (RP-δ, 15 x 3.2 mm. Applied Bioεyεtemε) and equilibrated to 40 * C at 0.5 L. inute. Protein was eluted under these conditions using the following programmed linear gradient: time (min) %B Solvent A: 0.1% trifluoroacetic acid (TFA) 0 Solvent B: 90% acetonitrile, 0.1% TFA 60 66.6 62.0 100 72.0 100 75.0 0 200 μL (0.4 minute) fractions were collected in εiliconized tubeε (Multilube tubes, Bioquote) from 15.2 minutes after the beginning of the programmed gradient. VI. SDS-Polyacrylaπtide Gel Electrophoresis In this step, protein molecular weight standards, low range, catalogue no. 161-0304, from Bio-Rad Laboratories Limited, Watford, England were employed. The actual proteins used, and their molecular weight standards, have been listed herein previouεly. Fractions 47 to 53 (GGF-I) and fractions 61 to 67 (GGFII) inclusive from the reversed-phase runs were individually pooled. 7 μL of the pooled material was boiled in an equal volume of 0.0125 M Tris-Cl, 4% SDS, 20% glycerol, and 10% S-mercaptoethanol for GGF-I, for 5 minutes and loaded on to an 11% polyacrylamide Laemmli gel with a 4% stacking gel and run at a conεtant voltage of 50 V for 16 hourε. This gel waε then fixed and stained uεing a silver staining kit (Amersham) . Under these conditions, the factors are each seen aε a εomewhat diffuse band at relative molecular weights 30,000 to 36,000 Daltons (GGF-I) and 55,000 to 63,000 Daltons (GGFII) as defined by molecular weight markers. From the gel staining, it is apparent that there are a εmall number of other protein εpecies present at equivalent levels to the GGF-I and GGF-II specieε in the material pooled from the reverεed-phaεe runε. VII. Stability in Trifluoroacetic Acid Stability data were obtained for the preεent Factorε in the presence of trifluoroacetic acid, as follows:- GGF-I: Material from the reverεed-phase HPLC, in the presence of 0.1% TFA and acetonitrile, was assayed within 12 hours of the completion of the column run and then after 10 weekε incubation at 40 * C. Following incubation, the GGF-I had at leaεt 50% of the activity of that material assayed directly off the column. GGF-II: Material from the reverεed-phaεe HPLC, in the presence of 0.1% TFA and acetonitrile, and εtored at -20'c, waε assayed after thawing and then after 4 days incubation at 40 * C. Following incubation, the GGF-II had at leaεt 50% of the activity of that material freεhly thawed. It will be appreciated that the trifluoroacetic acid concentration used in the above studies is that most commonly used for reversed-phase chromatography. VIII. Activity Assay Conditions Unless otherwise indicated, all operations were conducted at 37'C, and, with reference to Figures 1 to 6, activity at each stage was determined using the Brockes (Meth. Enz., supra) techniques with the following modifications. Thus, in preparing Schwann cells, 5 μM forskolin was added in addition to DMEM (Dulbecco's modified Eagle's medium) , FCS and GGF. Cells used in the aεεay were fibroblaεt-free Schwann cells at passage number leεε than 10, and theεe cells were removed from flaskε with trypsin and plated into flat-bottomed 96-well plates at 3.3 thousand cells per microwell. [ 12i I]IUdR was added for the final 24 hourε after the test solution addition. The background (unstimulated) incorporation to each asεay waε leεs than 100 cpm, and maximal incorporation was 20 to 200 fold over background depending on Schwann cell batch and passage number. In the case of the GGF-I and GGF-II fractions from reversed-phase HPLC as described above, two dose response curves were also produced for each factor, using exactly the above method for one of the curves for each factor, and the above method modified in the aεεay procedure only by εubstituting foetal calf plasma for fetal calf serum to obtain the other curve for each factor. The reεultε are in Figures 7 and β. EXAMPLE 2 Amino acid seguences of purified GGF-1 and GGF-II Amino acid εequence analyεiε studies were performed using highly purified bovine pituitary GGF-I and GGF-II. The conventional single letter code was uεed to deεcribe the εequences. Peptides were obtained by lysyl endopeptidase and proteaεe Vδ digeεts, carried out.on reduced and carboxy ethylated samples, with the lysyl endopeptidase digest of GGF-II carried out on material eluted from the 55-65 RD region of a 11% SDS-PAGE (MW relative to the above-quoted markers) . A total of 21 peptide εequenceε (see Figure 9, SEQ ID Noε. 1-20, 169) were obtained for GGF-I, of which 12 peptides (εee Figure 10, SEQ ID Nos. 1, 22-29, 17, 19, and 32) are not present in current protein databaseε and therefore repreεent unique εequences. A total of 12 peptide εequenceε (see Figure 11, SEQ ID Nos. 33-39, 51, 52, 164- 166) were obtained for GGF-II, of which 10 peptideε (εee Figure 12, SEQ ID Nos. 45-53) are not present in current protein databaseε and therefore represent unique εequenceε (an exception iε peptide GGF-II 06 which εhows identical εequences in many proteins which are probably of no significance given the small number of residues) . These novel sequences are extremely likely to correspond to portions of the true amino acid εequences of GGFs I and II. Particular attention can be drawn to the εequences of GGF-I 07 and GGF-II 12, which are clearly highly related. The εimilarities indicate that the εequences of theεe peptideε are almost certainly those of the assigned GGF εpecieε, and are oεt unlikely to be derived from contaminant proteinε. In addition, in peptide GGF-II 02, the sequence X S S iε consistent with the presence of an N linked carbohydrate moiety on an asparagine at the poεition denoted by X. In general, in Figures 9 and 11, X represents an unknown residue denoting a sequencing cycle where a single position could not be called with certainty either because there was more than one signal of equal size in the cycle or because no signal was present. As asterisk denotes those peptides where the last amino acid called corresponds to the last amino acid present in that peptide. In the remaining peptides, the signal strength after the last amino acid called was insufficient to continue εequence calling to the end of that peptide. The right hand column indicateε the results of a computer database search using the GCG package FASTA and TFASTA programs to analyze the NBRF and EMBL εequence databases. The name of a protein in thiε column denoteε identity of a portion of its sequence with the peptide amino acid εequence called allowing a maximum of two miεmatcheε. A question mark denotes three mismatches allowed. The abbreviations used are as follows: HMG-1 High Mobility Group protein-1 HMG-2 High Mobility Group protein-2 LH-alpha Luteinizing hormone alpha εubunit LH-beta Luteinizing hormone beta εubunit SUBSTV mπεsHff ϊ ^^ 2 ^ EXAMPLE ? Mitogenic Activity of Purified GGF-I and GGF-II The mitogenic activity of a highly purified sample containing both GGFs I and II waε studied uεing a quantitative method, which allows a single microculture to be examined for DNA synthesis, cell morphology, cell number and expression of cell antigens. This technique has been modified from a method previously reported by Muir et al.. Analytical Biochemistry 165, 377-362, 1990. The main modifications are: 1) the use of uncoated microtiter plates, 2) the cell number per well, 3) the use of 5% Foetal Bovine Plasma (FBP) instead of 10% Foetal Calf Serum (FCS), and 4) the time of incubation in presence of mitogens and bromodeoxyuridine (BrdU) , added simultaneously to the cultures. In addition the cell monolayer was not washed before fixation to avoid loss of cells, and the incubation time of monoclonal mouse anti-BrdU antibody and peroxidase conjugated goat anti-mouse immunoglobulin (IgG) antibody were doubled to increase the sensitivity of the assay. The assay, optimized for rat sciatic nerve Schwann cells, has alεo been used for several cell lines, after appropriate modifications to the cell culture conditions. I. Methods of Mitogenesis Testing On day 1, purified Schwann cells were plated onto uncoated 96 well plates in 5% FBP/Dulbecco's Modified Eagle Medium (DMEM) (5,000 cells/well). On day 2, GGFs or other test factors were added to the cultures, as well as BrdU at a final concentration of 10 μm. After 48 hours (day 4) BrdU incorporation was terminated by aspirating the medium and cells were fixed with 200 μl/well of 70% ethanol for 20 min at room temperature. Next, the cells were waεhed with water and the DNA denatured by incubation with 100 μl 2N HC1 for 10 min at 37 * C. Following aspiration, residual acid waε neutralized by filling the wellε with 0.1 M borate buffer, pH 9.0, and the cells were washed with phosphate buffered εaline (PBS) . Cells were then treated with 50 μl of blocking buffer (PBS containing 0.1% Triton X 100 and 2% normal goat serum) for 15 min at 37 * C. After aspiration, monoclonal mouse anti-BrdU antibody (Dako Corp., Santa Barbara, CA) (50 μl/well, 1.4 μg/ml diluted in blocking buffer) was added and incubated for two hours at 37"C. Unbound antibodies were removed by three washes in PBS containing 0.1% Triton X-100 and peroxidase-conjugated goat anti-mouεe IgG antibody (Dako Corp., Santa Barbara, CA) (50 μl/well, 2 μg/ml diluted in blocking buffer) was added and incubated for one hour at 37 * C. After three washeε in PBS/Triton and a final rinse in PBS, wellε received 100 μl/well of 50 mM phoεphate/citrate buffer, pH 5.0, containing 0.05% of the soluble chromogen o-phenylenediamine (OPD) and 0.02% H 2 0 2 . The reaction was terminated after 5-20 min at room temperature, by pipetting 80 μl from each well to a clean plate containing 40 μl/well of 2N εulfuric acid. The absorbance was recorded at 490nm using a plate reader (Dynatech Labs) . The assay plates containing the cell monolayers were washed twice with PBS and immunocytochemically stained for BrdU-DNA by adding 100 μl/well of the substrate diaminobenzidine (DAB) and 0.02% H 2 0 j to generate an insoluble product. After 10-20 min the staining reaction waε εtopped by waεhing with water, and BrdU4-poεitive nuclei obεerved and counted using an inverted microscope, occasionally, negative nuclei were counterεtained with 0.001% Toluidine blue and counted aε before. II. Cell lines used for Mitogenesis Assays Swiss 3T3 Fibroblasts: Cells, from Flow Labs, were maintained in DMEM supplemented with 10% FCS, penicillin and streptomycin, at 37 * C in a humidified atmoεphere of 10% C0 2 in air. Cellε were fed or εubcultured every two days. For mitogenic assay, cellε were plated at a denεity of 5,000 cells/well in complete medium and incubated for a week until cellε were confluent and quiescent. The serum containing medium was removed and the cell onolayer washed twice with serum free-medium. 100 μl of serum free medium containing mitogens and lOμM of BrdU were added to each well and incubated for 48 hours. Dose responses to GGFs and εerum or PDGF (as a positive control) were performed. BHK (Baby Hamster Kidney) 21 C13 Fibroblasts: Cellε from European Collection of Animal Cell Cultureε (ECACC) , were maintained in Glaεgow Modified Eagle Medium (GMEM) εupple ented with 5% tryptoεe phosphate broth, 5% FCS, penicillin and streptomycin, at 37 * C in a humidified atmosphere of 5% C0 2 in air. Cells were fed or εubcultured every two to three dayε. For mitogenic aεsay, cells were plated at a density of 2,000 cell/well in complete medium for 24 hours. The εerum containing medium was then removed and after washing with εerum free medium, replaced with 100 μl of 0.1% FCS containing GMEM or GMEM alone. GGFs and FCS or bFGF as poεitive controls were added, coincident with lOμM BrdU, and incubated for 48 hourε. Cell cultureε were then proceεεed aε deεcribed for Schwann cellε. C6 Rat Glioαa Cell Line: Cellε, obtained at paεsage 39, were maintained in DMEM containing 5% FCS, 5% Horse εerum (HS) , penicillin and streptomycin, at 37'C in a humidified atmosphere of 10% C0 2 in air. Cells were fed or εubcultured every three days. For mitogenic assay, cellε were plated at a denεity of 2,000 cellε/well in complete medium and incubated for 24 hourε. Then medium waε replaced with a mixture of 1:1 DMEM and F12 medium containing 0.1% FCS, after waεhing in εerum free medium. Dose responεes to GGFs, FCS and αFGF were then performed and cellε were processed through the ELISA as previously described for the other cell types. PC12 (Rat Adrenal Pheochromocytoma Cells) : Cells from ECACC, were maintained in RPMI 1640 supplemented with 10% HS, 5% FCS, penicillin and streptomycin, in collagen coated flaskε, at 37°C in a humidified atmoεphere of 5% C0 2 in air. Cellε were fed every three dayε by replacing 80% of the medium. For mitogenic aεεay, cellε were plated at a denεity of 3,000 cellε/well in complete medium, on collagen coated plateε (50 μl/well collagen, Vitrogen Collagen Corp., diluted 1 : 50, 30 min at 37 * C) and incubated for 24 hourε. The medium waε then placed with freεh RPMI either alone or containing 1 mM insulin or 1% FCS. Dose reεponses to FCS/HS (1:2) as positive control and to GGFs were performed as before. After 48 hourε cellε were fixed and the ELISA performed aε previouεly deεcribed. III. Results of Mitogenesis Assays: All the experiments presented in this Example were performed using a highly purified sample from a Sepharose 12 chromatography purification step (εee Example 1, section D) containing a mixture of GGF-I and GGF-II (GGFε) . First, the results obtained with the BrdU incorporation asεay were compared with the claεsical mitogenic assay for Schwann cellε baεed on [125]I-UdR incorporation into DNA of dividing cells, described by J.P.Brockes (Methods Enzymol. 147:217, 1967). Figure 13 shows the comparison of data obtained with the two assays, performed in the εame cell culture conditions (5,000 cells/well, in 5% FBP/DMEM, incubated in presence of GGFs for 4δhrε) . Aε clearly shown, the results are comparable, but BrdU incorporation asεay appearε to be slightly more sensitive, as suggested by the shift of the curve to the left of the graph, i.e. to lower concentrations Of GGFS. As described under the section "Methods of Mitogenesiε Testing", after the immunoreactive BrdU-DNA has been quantitated by reading the intensity of the soluble product of the OPD peroxidase reaction, the original asεay plateε containing cell monolayerε can undergo the εecond reaction reεulting in the insoluble DAB product, which εtainε the BrdU poεitive nuclei. The microcultureε can then be examined under an inverted microscope, and cell morphology and the numbers of BrdU-poεitive and negative nuclei can be observed. In Figure 14a and Figure 14b the BrdU-DNA immunoreactivity, evaluated by reading absorbance at 490 nm, is compared to the number of BrdU-poεitive nuclei and to the percentage of BrdU-positive nuclei on the total number of cells per well, counted in the εame cultureε. Standard deviations were lesε than 10%. The two evaluation ethodε εhow a very good correlation and the diεcrepancy between the valueε at the higheεt doεe of GGFε can be explained by the different extent of DNA εynthesis in cells detected aε BrdU-poεitive. The BrdU incorporation aεεay can therefore provide additional uεeful information about the biological activity of polypeptideε on Schwann cellε when compared to the (125) I-UdR incorporation aεεay. For example, the data reported in Figure 15 εhow that GGFε can act on Schwann cellε to induce DNA synthesis, but at lower doses to increase the number of negative cellε preεent in the microculture after 48 hourε. The assay has then been used on εeveral cell lines of different origin. In Figure 16 the mitogenic responses of Schwann cellε and Swiss 3T3 fibroblasts to GGFs are compared; deεpite the weak response obtained in 3T3 fibroblasts, some clearly BrdU-poεitive nuclei were detected in these cultures. Control cultureε were run in parallel in presence of εeveral doseε of FCS or human recombinant PDGF, εhowing that the cellε could respond to appropriate stimuli (not shown) . The ability of fibroblasts to respond to GGFε waε - further investigated using the BHK 21 C13 cell line. These fibroblasts, derived from kidney, do not exhibit contact inhibition or reach a quiescent εtate when confluent. Therefore the experimental conditionε were deεigned to have a very low background proliferation without compromising the cell viability. GGFs have a significant mitogenic activity on BHK21 C13 cellε as shown by Figure 17 and Figure 18. Figure 17 shows the Brdu incorporation into DNA by BHK 21 C13 cells stimulated by GGFS in the presence of 0.1% FCS. The good mitogenic response to FCS indicates that cell culture conditionε were not limiting. In Figure 18 the mitogenic effect of GGFε is expressed as the number of BrdU-positive and ErdU-negative cellε and as the total number of cellε counted per well. Data are repreεentative of two experimentε run in duplicates; at least three fields per well were counted. Aε obεerved for Schwann cells in addition to a proliferative effect at low doses, GGFs also increase the numbers of nonresponding cells surviving. The percentage of BrdU positive cellε is proportional to the increasing amounts of GGFs added to the cultures. The total number of cells after 48 hourε in presence of higher doses of GGFs is at least doubled, confirming that GGFs induce DNA synthesis and proliferation in BHK21 C13 cells. Under the εame conditions, cells maintained for 46 hourε in the presence of 2% FCS showed an increase of about six fold (not shown) . C6 glioma cellε have provided a uεeful model to study glial cell properties. The phenotype expressed seems to be dependent on the cell pasεage, the cellε more closely resembling an astrocyte phenotype at an early stage, and an oligodendrocyte phenotype at later stages (beyond pasεage 70) . C6 cellε uεed in these experiments were from paεεage 39 to paεεage 52. C6 cells are a highly proliferating population, therefore the experimental conditions were optimized to have a very low background of BrdU incorporation. The presence of 0.1% εerum waε neceεsary to maintain cell viability without significantly affecting the mitogenic responses, as shown by the dose response to FCS (Figure 19). In Figure 20 the mitogenic responseε to aFGF (acidic Fibroblast growth factor) and GGFε are expressed as the percentages of maximal BrdU incorporation obtained in the presence of FCS (8%) . Values are averages of two experiments, run in duplicates. The effect of GGFε waε comparable to that of a pure preparation of aFGF. aFGF has been described as a εpecific growth factor for C6 cellε (Lim R. et al., Cell Regulation 1:741-746, 1990) and for that reason it was used as a poεitive control. The direct counting of BrdU positive and negative cells was not possible because of the high cell density in the icrocultures. In contrast to the cell lines so far reported, PC12 cells did not show any evident responsiveness to GGFS, when treated under culture conditions in which PC12 could respond to sera (mixture of FCS and HS aε uεed routinely for cell maintenance) . Nevertheless the number of cells plated per well εeemε to affect the behavior of PC12 cells, and therefore further experiments are required. EXAMPLE 4 Isolating and Cloning of Nucleotide Seguences encoding proteins containing GGF-I and GGF-II peptides Isolation and cloning of the GGF-II nucleotide εequenceε waε performed aε outlined herein, using peptide εequence information and library εcreening, and was performed as εet out below. It will be appreciated that the peptides of Figures 4 and 5 can be used aε the εtarting point for iεolation and cloning of GGF-I εequenceε by following the techniqueε described herein. Indeed, Figure 21, SEQ ID Nos. 54-86) shows possible degenerate oligonucleotide probes for this purpose, and Figure 23, SEQ ID Nos. 90-119, lists possible PCR primers. DNA εequence and polypeptide εequence εhould be obtainable by thiε meanε aε with GGF-II, and also DNA constructs and expression vectors incorporating εuch DNA εequence, host cells genetically altered by incorporating such constructs/vectorε, and protein obtainable by cultivating εuch hoεt cellε. The invention enviεages εuch εubject matter. I. Design and Synthesis of oligonucleotide Probes and Primers Degenerate DNA oligomer probes were designed by backtranslating the amino acid sequenceε (derived from the peptides generated from purified GGF protein) into nucleotide εequences. Oligo ers represented either the coding strand or the non-coding strand of the DNA sequence. When εerine., arginine or leucine were included in the oligomer deεign, then two εeparate εyntheεeε were prepared to avoid ambiguitieε. For example, εerine waε encoded by either TCN or AGY aε in 537 and 538 or 609 and 610. Similar codon εplitting was done for arginine or leucine (e.g. 544, 545). DNA oligomers were syntheεized on a Bioεearch 8750 4-column DNA εyntheεizer using /S-cyanoethyl chemistry operated at 0.2 micromole scale synthesis. Oligomers were cleaved off the column (500 angstrom CpG resinε) and deprotected in concentrated ammonium hydroxide for 6-24 hourε at 55-60 β C. Deprotected oligomerε were dried under vacuum (Speedvac) and purified by electrophoreεiε in gelε of 15% acrylamide (20 mono : 1 biε) , 50 mM Triε-borate-EDTA buffer containing 7M urea. Full length oligomerε were detected in the gelε by UV εhadowing, then the bands were excised and DNA oligomers eluted into 1.5 is H20 for 4-16 hourε with εhaking. The eluate was dried, redisεolved in 0.1 ml H 2 0 and absorbance measure entε were taken at 260nm. Concentrations were determined according to the following formula: (A 260 x units/ml) (60.6/length « x μM) All oligomers were adjuεted to 50 μM concentration by addition of H 2 0. Degenerate probeε designed as above are shown in Figure 21, SEQ ID Nos. 54-8δ. PCR primers were prepared by essentially the εame procedures that were used for probes with the following modifications. Linkers of thirteen nucleotides containing restriction sites were included at the 5' ends of the degenerate oligomers for use in cloning into vectorε. DNA synthesiε waε performed at 1 micromole εcale using 1,000 angstrom CpG resins and inosine was used at positionε where all four nucleotideε were incorporated normally into degenerate probes. Purifications of PCR primers included an ethanol precipitation following the gel electrophoresiε purification. II. Library Construction and Screening A bovine genomic DNA library was purchased from Stratagene (Catalogue Number: 945701) . The library contained 2 x 10 6 15-2Okb Sau3Al partial bovine DNA fragments cloned into the vector lambda Dashll. A bovine total brain CDNA library was purchased from Clonetech (Catalogue Number: BL 10139). Complementary DNA libraries SUBSTITUTESHEET(RULE W were constructed (In Vitrogen; Stratagene) from mRNA prepared from bovine total brain, from bovine pituitary and from bovine posterior pituitary. In Vitrogen prepared two cDNA libraries: one library was in the vector lambda glO, the other in vector pcDNAI (a plaεmid library) . The Stratagene libraries were prepared in the vector lambda unizap. Collectively, the cDNA libraries contained 14 million primary recombinant phage. The bovine genomic library was plated on £. coli K12 host strain LE392 on 23 x 23 cm plates (Nunc) at 150,000 to 200,000 phage plaques per plate. Each plate represented approximately one bovine genome equivalent. Following an overnight incubation at 37"c, the plates were chilled and replicate filters were prepared according to procedureε of Maniatis et al. (2:60-δl). Four plaque lifts were prepared from each plate onto uncharged nylon membranes (Pall Biodyne A or MSI Nitropure) . The DNA was immobilized onto the membranes by cross-linking under UV light for 5 minutes or, by baking at 80 * C under vacuum for two hours. DNA probes were labelled uεing T4 polynucleotide kinaεe (New England Eiolabs) with gamma 32P ATP (New England Nuclear; 6500 Ci/mmol) according to the specifications of the εupplierε. Briefly, 50 p ols of degenerate DNA oligomer were incubated in the presence of 600 μCi gamma 32 P-ATP and 5 units T4 polynucleotide kinase for 30 minutes at 37 * C. Reactions were terminated, gel electrophoresiε loading buffer waε added and then radiolabelled probeε were purified by electrophoreεis. 32P labelled probes were excised from gel slices and eluted into water. Alternatively, DNA probes were labelled via PCR amplification by incorporation of c-32P-dATP or α-32P dCTP according to the protocol of Schowalter and Sommer, Anal. Biochem 177:90-94 (1989). Probes labelled in PCR reactions were purified by deεalting on Sephadex G-150 columns. Prehybridization and hybridization were performed in GMC buffer (0.52 M NaPi, 7% SDS, 1% BSA, 1.5 mM EDTA, 0.1 M NaCl 10 mg/ml tRNA) . Washing was performed in oligowash (160 ml 1 M Na 2 HP0 4 , 200 ml 20% SDS, 8.0 ml 0.5 M EDTA, 100 ml 5M NaCl, 3632 ml H20). Typically, 20 filterε (400 εq. centimeters each) representing replicate copies of ten bovine genome equivalents were incubated in 200 ml hybridization solution with 100 pmols of degenerate oligonucleotide probe (128-512 fold degenerate) . Hybridization was allowed to occur overnight at 5 * C below the minimum melting temperature calculated for the degenerate probe. The calculation of minimum melting temperature aεεumeε 2 * C for an AT pair and 4 * C for a GC pair. Filterε were washed in repeated changes of oligowash at the hybridization temperatures four to five hourε and finally, in 3.2M tetramethylammonium chloride, 1% SDS twice for 30 min at a temperature dependent on the DNA probe length. For 20merε, the final wash temperature was 60 * C. Filters were mounted, then exposed to X-ray film (Kodak XAR5) using intensifying εcreenε (Dupont Cronex Lightening Pluε) . Usually, a three to five day film exposure at minuε 80 * C was sufficient to detect duplicate signals in these library εcreenε. Following analyεiε of the reεultε, filterε could be εtripped and reprobed. Filterε were εtripped by incubating through two εuccessive cycles of fifteen minutes in a microwave oven at full power in a solution of 1% SDS containing lOmM EDTA pH8. Filterε were taken through at least three to four cycles of stripping and reprobing with various probes. III. Recombinant Phage Isolation, Growth and DNA Preparation These procedures followed standard protocol as described in Recombinant DNA (Maniatis et al 2:60-2:81). IV. Analysis of Isolated Clones Using DNA Digestion and Southern Blots Recombinant Phage DNA samples (2 micrograms) were digested according to conditions recommended by the restriction endonuclease supplier (New England Biolabs) . Following a four hour incubation at 37 * C, the reactions products were precipitated in the presence of 0.1M εodium acetate and three volumeε of ethanol. Precipitated DNA waε collected by centrifugation, rinsed in 75% ethanol and dried. All resuspended sampleε were loaded onto agaroεe gelε (typically 1% in TAE buffer; 0.04M Triε acetate, 0.002M EDTA) . Gel runε were at 1 volt per centimeter from 4 to 20 hourε. Markers included lambda Hind III DNA fragments and/or 0X174HaeIII DNA fragments (New England Biolabs) . The gelε were εtained with 0.5 microgramε/ml of ethidium bromide and photographed. For εouthern blotting, DNA waε first depurinated in the gel by treatment with 0.125 N HC1, denatured in 0.5 N NaOH and transferred in 20x SSC (3M εodium chloride, 0.03 M εodium citrate) to uncharged nylon membranes. Blotting was done for 6 hours up to 24 hours, then the filters were neutralized in 0.5 Tris HC1 pH 7.5, 0.15 M εodium chloride, then rinεed briefly in 50 mM Tris-borate EDTA. For croεs-linking, the filters were wrapped firεt in transparent plastic wrap, then the DNA εide expoεed for five minuteε to an ultraviolet light. Hybridization and waεhing waε performed as described for library εcreening (εee section 2 of thiε Example) . For hybridization analyεiε to determine whether similar genes exiεt in other species slight modifications were made. The DNA filter waε purchaεed from Clonetech (Catalogue Number 7753-1} and containε 5 icrogramε of EcoRI digested DNA from various εpecies per lane. The probe was labelled by PCR amplification reactions as deεcribed in εection 2 above, and hybridizations were done in 80% buffer B(2 g polyvinylpyrrolidine, 2 g Ficoll-400, 2 g bovine εerum albumin, 50 ml 1M Tris-HCl (pH 7.5) 58 g NaCl, 1 g εodium pyrophosphate, 10 g εodium dodecyl εulfate, 950ml H 2 0) containing 10% dextran εulfate. The probeε were denatured by boiling for ten minutes then rapidly cooling in ice water. The probe was added to the hybridization buffer- at 10 6 dp 32 P per ml and incubated overnight at 60 * C. The filters were waεhed at 60'C firεt in buffer B followed by 2X SSC, 0.1% SDS then in lx SSC, 0.1% SDS. For high εtringency, experimentε, final waεhes were done in 0.1 x SSC, 1% SDS and the temperature raised to 65 * C. Southern blot data were used to prepare a restriction map of the genomic clone and to indicate which subfragments hybridized to the GGF probeε (candidateε for, εubcloning) . V. Subcloning of Segments of DNA Homologous to Hybridization Probes DNA digests (e.g. 5 microgramε) were loaded onto 1% agarose gels then appropriate fragments excised from the gelε following staining. The DNA waε purified by adεorption onto glaεε beadε followed by elution uεing the protocol deεcribed by the supplier (Bio 101) . Recovered DNA fragments (100-200 ng) were ligated into linearized dephosphorylated vectors, e.g. pT3T7 (A bion) , which iε a derivative of pUClδ, uεing T4 ligaεe (New England Biolabs). Thiε vector carrieε the E. coli β lactamase gene, hence, transformantε can be εelected on plateε containing a picillin. The vector alεo supplies jS-galactosidase complementation to the host cell, therefore non-recombinants (blue) can be detected using isopropylthiogalactoεide and Bluogal (Bethesda Research Labs) . A portion of the ligation reactions was used to transform £. coli K12 XL1 blue competent cells (Stratagene Catalogue Number: 200236) and then the tranεfor antε were εelected on LB plates containing 50 micrograms per ml ampicillin. White colonieε were εelected and plaε id mini preps were prepared for DNA digeεtion and for DNA εequence analysiε. Selected cloneε were retested to determine if their insert DNA hybridized with the GGF probes. VI. DNA Seouencing Double stranded plasmid DNA templateε were prepared from 5 ml cultures according to standard protocols. Sequencing was by the dideoxy chain termination method using Sequenase 2.0 and a dideoxynucleotide εequencing kit (US Biochemical) according to the manufacturerε protocol (a modification of Sanger et al. PNAS; USA 21:5463 (1977)]. Alternatively, eeguencing was done in a DNA thermal cycler (Perkin Elmer, model 4800) using a cycle sequencing kit (New England Biolabs; Bethesda Research Laboratories) and was performed according to manufacturers instructions using a 5'-end labelled primer. Sequence primerε were either those supplied with the sequencing kits or were synthesized according to εequence determined from the cloneε. Sequencing reactionε were loaded on and reεolved on 0.4mm thick εequencing gels of 6% polyacrylamide. Gels were dried and exposed to X-Ray film. Typically, 35S waε incorporated when εtandard εequencing kitε were uεed and a 32P end labelled primer was used for cycle sequencing reactions. Sequenceε were read into a DNA εequence editor from the bottom of the gel to the top (5' direction to 3') and data were analyzed using programs supplied by Genetics Computer Group (GCG, University of Wisconsin). VII. RNA Preparation and PCR Amplification Open reading frames detected in the genomic DNA and which contained sequence encoding GGF peptides were extended via PCR amplification of pituitary RNA. RNA was prepared from frozen bovine tiεεue (Pelfreeze) according to the guanidine neutral-CεCl procedure (Chirgwin et. al. Bioche iεtry 16:5294(1979).) Polyadenylated RNA was selected by oligo-dT cellulose column chromatography (Aviv and Leder PNAS (USA) 69:140δ (1972)). Specific DNA target εequenceε were amplified beginning with either total RNA or polyadenylated RNA εamples that had been converted to cDNA using the Perkin Elmer PCR/RNA Kit Number: N608-0017. First strand reverse transcription reactions used 1 μg template RNA and either primerε of oligo dT with reεtriction enzyme recognition site linkerε attached or εpecific antiεenεe primers determined from cloned εequences with restriction sites attached. To produce the second strand, the primers either were plus εtrand unique εequences as used in 3' RACE reactions (Frohman et. al., PNAS (USA) ££:6998 (1988)) or were oligo dT primerε with restriction sites attached if the second target site had been added by terminal transferaεe tailing firεt εtrand reaction productε with dATP (e.g. 5' race reactionε, Frohman et. al., ibid). Alternatively, as in anchored PCR reactions the second εtrand primerε were degenerate, hence, representing particular peptide sequenceε. The amplification profileε followed the following general εcheme: 1) five minutes soak file at 95"C; 2) thermal cycle file of 1 minute, 95 * C; 1 minute ramped down to an annealing temperature of 45 * C, 50'C or 55 * C; maintain the annealing temperature for one minute; ramp up to 72 * C over one minute; extend at 72'C for one minute or for one minute plus a 10 second auto extension; 3) extension cycle at 72 * C, five minutes, and; 4) εoak file 4 * C for infinite time. Thermal cycle files (#2) usually were run for 30 cycles. A sixteen μl sample of each 100 μl amplification reaction was analyzed by electrophoresiε in 2% Nuεieve 1% agarose gelε run in TAE buffer at 4 voltε per centimeter for three hourε. The gelε were εtained, then blotted to uncharged nylon membranes which were probed with labelled DNA probes that were internal to the primers. Specific εetε of DNA amplification productε could be identified in the blotting experiments and their positionε used as a guide to purification and reamplification. When appropriate, the remaining portionε of εelected εamples were loaded onto preparative gelε, then following electrophoreεiε four to five εliceε of 0.5 mm thickness (bracketing the expected position of the specific product) were taken from the gel. The agaroεe waε crushed, then soaked in 0.5 ml of electrophoreεiε buffer from 2-16 hourε at 40 * C. The crushed agarose waε centrifuged for two minutes and the aqueous phaεe waε tranεferred to fresh tubes. Reamplification waε done on five microliters (roughly 1% of the product) of the eluted material using the same εetε of primers and the reaction profiles as in the original reactionε. When the reamplification reactionε were completed, εamples were extracted with chloroform and transferred to fresh tubes. Concentrated restriction enzyme bufferε and enzymes were added to the reactionε in order to cleave at the reεtriction εites present in the linkers. The digested PCR products were purified by gel electrophoresis, then εubcloned into vectorε aε described in the subcloning εection above. DNA εequencing waε done described as above. VIII. DNA Seouence Analysis DNA εequenceε were assembled using a fragment assembly program and the amino acid sequences deduced by the GCG programs GelAssemble, Map and Translate. The deduced protein εequenceε were uεed aε a query εequence to search protein εequence databaεes using WordSearch. Analysis was done on a VAX Station 3100 workstation operating under VMS 5.1. The databaεe εearch was done on SwiεsProt release number 21 using GCG Version 7.0. IX. Results of Cloning and Sequencing of genes encoding GGF-I and GGF-II As indicated above, to identify the DNA sequence encoding bovine GGF-II degenerate oligonucleotide probeε were designed from GGF-II peptide εequenceε. GGF-II 12 (SEQ ID No. 44), a peptide generated via lysyl endopeptidase digestion of a purified GGF-II preparation (εee Figures 11 and 12) showed εtrong amino acid εequence homology with GGF-I 07 (SEQ ID No. 39), a tryptic peptide generated from a purified GGF-I preparation. GGF-II 12 was thus used to create ten degenerate oligonucleotide probes (see oligoε 609, 610 and 649 to 656 in Figure 21, SEQ ID Noε. 69, 70, 71 and 79, respectively). A duplicate set of filterε were probed with two sets (set 1=609, 610; set 2=649-5656) of probes encoding two overlapping portions of GGF-II 12. Hybridization signals were observed, but, only one clone hybridized to both probe sets. The clone (designated GGF2BG1) was purified. Southern blot analysis of DNA from the phage clone GGF2EG1 confirmed that both sets of probes hybridized with that bovine DNA εequence, and showed further that both probes reacted with the same set of DNA fragments within the clone. Based on those experiments a 4 kb Eco RI sub-fragment of the original clone was identified, subcloned and partially εequenced. Figure 22 shows the nucleotide sequence, SEQ ID No. 89) and the deduced amino acid sequence of the initial DNA sequence readings that included the hybridization εites of probes 609 and 650, and confirmed that a portion of this bovine genomic DNA encoded peptide 12 (KASLADSGEYM) . Further εequence analyεis demonstrated that GGF-II 12 resided on a 66 amino acid open reading frame (εee below) which has become the starting point for the isolation of overlapping εequences representing a putative bovine GGF-II gene and a cDNA. Several PCR procedures were uεed to obtain additional coding εequences for the putative bovine GGF-II gene. Total RNA and oligo dT-εelected (poly A containing) RNA samples were prepared from bovine total pituitary, anterior pituitary, posterior pituitary, and hypothalamuε. Using primers from the list shown in Figure 23, SEQ ID Nos. 109-119, one-sided PCR reactions (RACE) were uεed to amplify cDNA ends in both the 3' and 5' directions, and anchored PCR reactions were performed with degenerate oligonucleotide primers representing additional GGF-II peptides. Figure 24 summarizes the contiguous DNA εtructures and sequenceε obtained in those experiments. From the 3' RACE reactions, three alternatively spliced cDNA εequenceε were produced, which have been cloned and εequenced. A 5' RACE reaction led to the diεcovery of an additional exon containing coding εequence for at least 52 amino acids. Analyεiε of that deduced amino acid εequence revealed peptides GGF-II-6 and a εequence εimilar to GGF-I-18 (see below) . The anchored PCR reactions led to the identification of (cDNA) coding εequenceε of peptides GGF-II-1, 2, 3 and 10 contained within an additional cDNA segment of 300 bp. The 5' limit of thiε segment (i.e., segment E, εee Fig. 31) iε defined by the oligonucleotide which encodes peptide GGF-II-1 and which was used in the PCR reaction (additional 5' εequence data exiεtε aε described for the human clone in Example 6) . Thus thiε clone contains nucleotide εequences encoding six out of the existing total of nine novel GGF-II peptide εequenceε. The cloned gene waε characterized firεt by conεtructing a phyεical map of GGF2BG1 that allowed uε to poεition the coding εequenceε aε they were found (see below, Figure 25) . DNA probeε from the coding εequenceε deεcribed above have been used to identify further DNA fragments containing the exons on this phage clone and to identify cloneε that overlap in both directionε. The putative bovine GGF-II gene iε divided into at leaεt 5 coding εegmentε. Coding εegmentε are defined aε diεcrete lengthε of DNA εequence which can be tranεlated into polypeptide εequences uεing the univerεal genetic code. The coding εegmentε deεcribed in Figure 31 and referred to in the present application are: 1) particular exons preεent within the GGF gene (e.g. coding εegment a), or 2) derived from εetε of two or more exonε that appear in specific εub- groupε of mRNAs, where each set can be translated into the specific polypeptide εegmentε aε in the gene productε εhown. The polypeptide εegmentε referred to in the claims are the translation productε of the analogouε DNA coding εegmentε. Only coding εegmentε A and B have been defined aε exonε and εequenced and mapped thus far. The summary of the contiguous coding εequenceε identified is εhown in Figure 26. The exons are listed (alphabetically) in the order of their diεcovery. It iε apparent from the intron/exon boundarieε that exon B may be included in cDNAs that connect coding εegment E and coding εegment A. That iε, exon B cannot be εpliced out without compromiεing the reading frame. Therefore, we suggest that three alternative splicing patternε can produce putative bovine GGF-II cDNA εequenceε 1, 2 and 3. The coding sequences of these, deεignated GGF2EPP1.CDS, GGF2BPP2.CDS and GGF2BPP3.CDS, reεpectively, are given in Figures 28a (SEQ ID No. 133), 28b (SEQ ID No. 134), and 28c (SEQ ID No. 135), respectively. The deduced amino acid εequence of the three cDNAs iε also given in Figures 28a, (SEQ ID No. 133) , 28b (SEQ ID No. 134), and 28c (SEQ ID No. 135). The three deduced εtructureε encode proteinε of lengthε 206, 281 and 257 amino acidε. The firεt 183 reεidueε of the deduced protein sequence are identical in all three gene productε. At poεition 184 the cloneε differ εignificantly. A codon for glycine GGT in GGF2BPP1 alεo εerves as a εplice donor for GGF2BPP2 and GGF2BPP3, which alternatively add on exons C, C/D, C/D' and D or C, C/D and D, respectively, and εhown in figure 33, SEQ ID No. 149). GGFIIBPP1 is a truncated gene product which iε generated by reading paεt the coding segment A εplice junction into the following intervening εeguence (intron) . Thiε repreεentε coding εegment A' in figure 31 (SEQ ID No. 140) . The transcript ends adjacent to a canonical AATAAA polyadenylation εequence, and we εuggest that this truncated gene product represents a bona fide mature transcript. The other two longer gene products share the εame 3' untranslated εequence and polyadenylation site. All three of these molecules contain six of the nine novel GGF-II peptide εequenceε (εee Figure 12) and another peptide iε highly homologouε to GGF-I-18 (εee Figure 27) . This finding gives a high probability that this recombinant molecule encodes at least a portion of bovine GGF-II. Furthermore, the calculated isoelectric pointε for the three peptideε are consistent with the physical properties of GGF-I and II. Since the molecular size of GGF-II iε roughly 60 kD, the longeεt of the three cDNAε should encode a protein with nearly one-half of the predicted number of amino acids. A probe encompassing the B and A exons waε labelled via PCR amplification and used to screen a cDNA library made from RNA isolated from bovine posterior pituitary. One clone (GGF2BPP5) showed the pattern indicated in figure 30 and contained an additional DNA coding segment (G) between coding εegments A and C. The entire nucleic acid εequence iε εhown in figure 32 (SEQ ID No. 146) . The predicted translation product from the longest open reading frame is 241 amino acids. A portion of a second cDNA (GGF2BPP4) was also iεolated from the bovine posterior pituitary library using the probe described above. This clone εhowed the pattern indicated in figure 30. Thiε clone iε incomplete at the 5' end, but iε a εplicing variant in the εenεe that it lackε coding εegmentε G and D. EPP4 alεo displayε a novel 3' end with regionε H, K and L beyond region C/D. The εequence of BPP4 iε εhown in figure 34 (SEQ ID No. 150). EXAMPLE 5 GGF Seguences in Various Species Database searching has not revealed any meaningful similarities between any predicted GGF translation products and known protein εequences. Thiε εuggestε that GGF-II is the first member of a new family or superfamily of proteins. In high εtringency cross hybridization studieε (DNA blotting experimentε) with other mammalian DNAs we have shown, clearly, that DNA probes from this bovine recombinant molecule can readily detect specific sequenceε in a variety of εamples tested. A highly homologous εequence iε alεo detected in human genomic DNA. The autoradiogram iε εhown in figure 29. The εignalε in the lanes containing rat and human DNA represent the rat and human equivalents of the GGF gene, the sequences of εeveral cDNA'ε encoded by thiε gene have been recently reported by Holmeε et al. (Science 256: 1205 (1992)) and Wen et al. (Cell §2.: 559 (1992)). EXAMPLE 6 Isolation of a Human Secuence Encoding Human GGF2 Several human clones containing εequences from the bovine GGFII coding εegment E were iεolated by εcreening a human cDNA library prepared from brain εtem (Stratagene catalog #935206) . Thiε εtrategy waε purεued baεed on the εtrong link between most of the GGF2 peptides (unique to GGF2) and the predicted peptide εequence from cloneε containing the bovine E εegment. This library was screened as described in Example 4, Section II using the oligonucleotide probes 914-919 liεted below. 91 CGGGCTCCATGAAGAAGATGTA 915TCCATGAAGAAGATGTACCTGCT 916ATGTACCTGCTGTCCTCCTTGA 917TTGAAGAAGGACTCGCTGCTCA 918AAAGCCGGGGGCTTGAAGAA 919ATGARGTGTGGGCGGCGAAA Clones detected with these probes were further analyzed by hybridization. A probe derived from coding εegment A (εee Figure 21) , which was produced by labeling a polymerase chain reaction (PCR) product from εegment A, waε alεo uεed to εcreen the primary library. Several cloneε that hybridized with both A and E derived probeε were εelected and one particular clone, GGF2HBS5, was εelected for further analyεiε. Thiε clone iε repreεented by the pattern of coding εegmentε (EBACC/D'D aε shown in Figure 31) . The E segment in this clone iε the human equivalent of the truncated bovine verεion of E εhown in Figure 37. GGF2HBS5 iε the most likely candidate to encode GGF-II of all the "putative" GGF-II candidates described. The length of coding sequence εegment E iε 786 nucleotideε pluε 264 bases of untranεlated εequence. The predicted size of the protein encoded by GGF2HBS5 is approximately 423 amino acids (approximately 45 kilodaltonε, εee Figure 45, SEQ ID NO: 167) , which iε εimilar to the εize of the deglycosylated form of GGF-II (εee Example 16) . Additionally, εeven of the GGF-II peptideε listed in Figure 27 have equivalent εequenceε which fall within the protein εequence predicted from region E. Peptideε II-6 and 11-12 are exceptionε, which fall in coding εegment B and coding εegment A, respectively. RNA encoding the GGF2HBS5 protein was produced in an in vitro tranεcription εyεtem driven by the bacteriophage T7 promoter reεident in the vector (Bluescript SK [Stratagene Inc.] εee Figure 44) containing the GGF2HBS5 insert. This RNA was translated in a cell free (rabbit reticulocyte) translation syεtem and the εize of the protein product waε 45 Kd. Additionally, the cell-free product haε been aεεayed in a Schwann cell mitogenic aεεay to confirm biological activity. Schwann cellε treated with conditioned medium εhow both increased proliferation aε measured by incorporation of ,2 I-Uridine and phosphorylation on tyrosine of a protein in the 185 kilodalton range. Thus the εize of the product encoded by GGF2HBS5 and the preεence of DNA sequences which encode human peptides highly homologous to the bovine peptides εhown in Figure 12 confirm that GGF2HBS5 encodes the human equivalent of bovine GGF2. The fact that conditioned media prepared from cells transformed with thiε clone elicitε Schwann cell mitogenic activity confirmε that the GGFIIHBSS gene produce (unlike the BPP5 gene product) is secreted. Additionally the GGFIIBPP5 gene product seems to mediate the Schwann cell proliferation reεponεe via a receptor tyroεine kinase εuch aε pies'* 82 or a closely related receptor (εee Example 14). EXAMPLE 7 Expression of Human Recombinant GGF2 in Mammalian and Insect Cells The GGF2HBS5 cDNA clone encoding human GGF2 (as described in Example 6 and also referred to herein as HBS5) waε cloned into vector pcDL-SRα296 (Takebe et al. Mol. Cell. Biol. 8:466-472 (1988) and COS-7 cellε were tranεfected in 100 mm diεheε by the DEAE-dextran method (Sa brook et al. Molecular Cloning: A Laboratory Manual 2nd ed. CSH Laboratory NY (1989) . Cell lyεates or conditioned media from transiently expreεεing COS cellε were harvested at 3 or 4 days post-transfection. To prepare lysates, cell monolayers were waεhed with PBS, εcraped from the diεheε lyεed by three freeze/thaw cycleε in 150 μl of 0.25 M Triε- HC1, pH8. Cell debriε waε pelleted and the εupernatant recovered. Conditioned media εamples (7 ml.) were collected, then concentrated and buffer exchanged with 10 mM Tris, pH 7.4 using Centiprep-10 and Centricon-10 units aε described by the manufacturer (Amicon, Beverly, MA) . Rat nerve Schwann cells were assayed for incorporation of DNA synthesis precursors, as described (see Example 3). Conditioned media or cell lyεate εamples were tested in the Schwann cell proliferation assay as described in Example 3. The mitogenic activity data are εhown in Fig. 46. The cDNA, GGF2HBS5, encoding GGF2 directed the εecretion of the protein product to the medium. A εmall proportion of total activity waε detectable inside the cellε aε determined by aεεayε uεing cell lyεates. GGF2HFB1 and GGFBPP5 cDNA'ε failed to direct the εecretion of the product to the extracellular medium. GGF activity from theεe clones was detectable only in cell lyεates (Fig. 46) . Recombinant GGF2 was alεo expreεεed in CHO cellε. The GGF2HBS5 cDNA encoding GGF2 was cloned into the EcoRI εite of vector pcdhfrpolyA (Fig. 54) and transfected into the DHFR negative CHO cell line (DG44) by the calcium phosphate coprecipitation method (Graham and Van Der Eb, Virology 52:456-467 (1973). Clones were selected in nucleotide and nucleoside free a medium (Gibco) in 96-well plates. After 3 weekε, conditioned media εamples from individual clones were screened for expreεsion of GGF by the Schwann cell proliferation assay as described in Example 3. Stable cloneε which secreted significant levels of GGF activity into the medium were identified. Schwann cell proliferation activity data from different volume aliquots of CHO cell conditioned medium were used to produce the dose response curve shown in Fig. 47 (Graham and Van Der Eb, Virology 52:456, 1973). Thiε material was analyzed on a Western blot probed with polyclonal antisera raiεed againεt a GGF2 εpecific peptide. A broad band of approximately 69- 90 Kd (the expected εize of GGF2 extracted from pituitary and higher molecular weight glycoforms) is specifically labeled (Fig. 49, lane 12). Recombinant GGF2 was also expressed in insect cells using Baculovirus expression. Sf9 insect cells were infected with baculovirus containing the GGF2HBS5 cDNA clone at a multiplicity of 3-5 (10 6 cells/ml) and cultured in Sf900-11 medium (Gibco) . Schwann cell mitogenic activity was εecreted into the extracellular medium (Fig. 48) . Different volumes of insect cell conditioned medium were tested in the Schwann cell proliferation aεεay in the absence of forskolin and the data used to produce the dose response curve εhown in Fig. 4δ. Thiε material waε alεo analyzed on a Western blot (Fig. 47) probed with the GGF II specific antibody described above. A band of 45 Kd, the εize of deglycoεylated GGF-II (εee Example 16) was seen. The methods used in thiε example were as follows: Schwann cell mitogenic activity of recombinant human and bovine glial growth factors was determined as follows: Mitogenic responses of cultured Schwann cells were measured in the presence of 5 μM forskolin uεing crude recombinant GGF preparations obtained from transient mammalian expression experiments. Incorporation of [ 1J I]-Uridine was determined following an 18-24 hour exposure to materialε obtained from transfected or mock transfected COS cells aε deεcribed in the Methodε. The mean and εtandard deviation of four εetε of data are εhown. The mitogenic response to partially purified native bovine pituitary GGF (carboxymethyl cellulose fraction; Goodearl et al., submitted) is shown (GGF) as a standard of one hundred percent activity. cDNAε (Fig. 53) were cloned into pcDL-SRc296 (Takebe et al., Mol. Cell Biol. 8:466-472 (1988)), and COS-7 cellε SUBSTiTUTESHEET^Erø were transfected in 100 mm dishes by the DEAE-dextran method (Sambrook et al.. In Molecular Cloning. A Laboratory Manual, 2nd. ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)). Cell lyεates or conditioned media were harvested at 3 or 4 dayε poεt-tranεfection. To prepare lyεates, cell monolayerε were washed with PBS, scraped from the dishes, and lysed by three freeze/than cycles in 150 μl of 0.25 M Tris-HCl, pH 8. Cell debriε waε pelleted and the εupernatant recovered. Conditioned media εamples (7 is) were collected, then concentrated and buffer exchanged with 10 mM Triε, pH 7.4 uεing Centriprep-10 and Centricon-10 unitε as described by the manufacturer (Amicon, Beverly, MA) . Rat εciatic nerve Schwann cellε were aεεayed for incorporation of DNA εynthesis precursorε, aε deεcribed (Davis and Stroobant, J. Cell Biol. 110:1353-1360 (1990); Brockes et al.. Brain Res. 165:105-118 (1979)). Western blots of recombinant CHO cell conditioned medium were performed as follows: A recombinant CHO clone was cultured in 7 ml. of MCDB302 protein-free medium for 3 dayε. 2 ml of conditioned medium waε concentrated, buffered exchanged againεt 10 mM Tris-HCl, pH 7.4 and lyophilized to dryness. The pellet was resuspended in SDS-PAGE εample buffer, εubjected to reducing SDS gel electrophoreεiε and analyzed by Weεtern blotting with a GGF peptide antibody. A CHO control was done by using conditioned medium from untransfected CHO-DG44 host and the CHO HBS5 levels were aεεayed using conditioned medium from a recombinant clone. EXAMPLE 8 Isolation of Other Human Segueπces Related to Bovine GGF The result in Examples 5 and 6 indicate that GGF related εequences from human εources can also be easily iεolated by using DNA probes derived from bovine GGF εequences. Alternatively the procedure described by Holmes et al. (Science 256: 1205 (1992)) can be used. In this example a human protein (heregulin a) , which binds to and activates the plδS** 82 receptor (and is related to GGF) , iε purified from a tumor cell line and the derived peptide εequence iε uεed to produce oligonucleotide probeε which were utilized to clone the cDNA'ε encoding heregulin. The biochemical aεεay for plδS'* 82 receptor activation iε distinguished from Schwann cell proliferation. This is a εimilar approach to that used in examples 1-4 for the cloning of GGF sequences from pituitary cDNAs. The heregulin protein and complementary DNAs were isolated from tumor cell lines according to the following procedures. Heregulin was purified from medium conditioned by MDA-MB-231 breast cancer cells (ATCC #HTB 26) grown on Percell Biolytica microcarrier beads (Hyclone Labs) . The medium (10 literε) waε concentrated -25-fold by filtration through a membrane (10-kD cutoff) (Millipore) and clarified by centrifugation and filtration through a filter (0.22 μm) . The filtrate was applied to a heparin Sepharose column (Pharmacia) and the proteins were eluted with εtepε of 0.3, 0.6, and 0.9 M NaCl in phoεphate-buffered εaline. Activity in the various chromatographic fractions was measured by quantifying the increaεe in tyrosine phosphorylation of p 185 eAB2 in MCF _ 7 breast tumor cellε (ATCC # HTB 22). MCF-7 cellε were plated in 24-well Costar plates in F12 (50%) Dulbecco's minimum essential medium (50%) containing serum (10%) (10 s cells per well) , and allowed to attach for at least 24 hourε. Prior to aεεay, cellε were transferred into medium without εerum for a minimum of 1 hour. Column fractions (10 to 100 μl) were incubated for 30 min. at 37 * . Supernatantε were then aεpirated and the reaction waε stopped by the addition of SDS-PAGE εample buffer 100 μl) . Samples were heated for 5 min. at 100"C, and portions (10 to 15 μl) were applied to a tris-glycine gel (4 to 20%) (Novex) . After electrophoreεiε, proteinε were electroblotted onto a polyvinylidenedifluoride (PVDF) membrane and then blocked with bovine εerum albumin (5%) in triε-buffered εaline containing Tween-20 (0.05%) (TBST) . Blots were probed with a monoclonal antibody (1:1000 dilution) to phosphotyrosine (Upstate Biotechnology) for a minimum of 1 hour at room temperature. Blots were washed with TBST, probed with an antibody to mouse immunoglobulin G conjugated to alkaline phoεphataεe (Promega) (diluted 1:7500) for a minimum of 30 min. at room temperature. Reactive bands were visualized with 5-bromo-4-chloro-3-indoyl-l-phoεphate and nitro-blue tetrazolium. Immunoblots were scanned with a Scan Jet Plus (Hewlett-Packard) densitometer. Signal intensitieε for unεtimulated MCF-7 cells were 20 to 30 units. Fully stimulated plβS"* 82 yielded signals of lδO to 200 units. The 0.6 M NaCl pool, which contained most of the activity, was applied to a polyaspartic acid (PolyLC) column equilibrated in 17 mM εodium phosphate (pH 6.δ) containing ethanol (30%). A linear gradient from 0.3 M to 0.6 M NaCl in the equilibration buffer was used to elute bound proteins. A peak of activity (at -0.45 M NaCl) was further fractionated on a C4 reversed-phaεe column (SynChropak RP-4) equilibrated in buffer containing TFA (0.1%) and acetonitrile (15%). Proteins were eluted from this column with an acetonitrile gradient from 25 to 40% over 60 min. Fractions (1 ml) were collected, assayed for activity, and analyzed by SDS-PAGE on tris-glycine gels (4-20%, Novex) . HPLC-purified HRG-o was digested with lysine C in SDS (0.1%), 10 mM dithiothreitol, 0.1 M NH 4 HC0 3 (pH δ.0) for 20 hours at 37 * C and the reεultant fragmentε were resolved on a Synchrom C4 column (4000A", 0.2 by 10 cm). The column was equilibrated in 0.1% TFA and eluted with a 1-propanol gradient in 0.1% TFA (W. J. Henzel, J. T. Stultε, C. Hεu, D. W. Aswad, J. Biol. Chem . 264. 15905 (1969)). Peaks from the chro atographic run were dried under vacuum and εequenced. One of the peptideε (eluting at -24% 1-propanol) gave the εequence [A]AEKEKTF[C]VNGGEXFMVKDLXNP (SEQ ID No. 162). Residues in brackets were uncertain and an X repreεentε a cycle in which it waε not possible to identify the amino acid. The initial yield was 6.5 pmol and the εequence did not correspond to any known protein. Reεidues 1, 9, 15, and 22 were later identified in the cDNA εequence as cysteine. Direct sequencing of the -45-kD band from a gel that had been overloaded and blotted onto a PVDF membrane revealed a low abundance sequence XEXKE[G] [R]GK[G]K[G]KKKEXGXG[K] (SEQ ID No. 30) with a very low initial yield (0.2 pmol). Thiε correεponded to amino acid reεidues 2 to 22 of heregulin-α (Fig. 31) , εuggeεting that εerine 2 iε the NH 2 -terminuε of proHRG-α. Although the NH 2 terminus was blocked, it waε obεerved that occasionally a small amount of a normally blocked protein may not be pbst-translationally modified. The NH 2 terminal assignment was confirmed by maεε εpectrometry of the protein after digeεtion with cyanogen bromide. The COOH-terminuε of the isolated protein has not been definitely identified; however, by mixture εequencing of proteolytic digests, the mature εequence does not appear to extend past residue 241. Abbreviations for amino reεidueε are: A, Ala; C, Cyε; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lyε; L, Leu; M, Met; N, Aεn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. As a source of cDNA clones, an oligo(dT)-primed λgtlO (T. V. Huynn, R. A. Young, R. W. Davis, λgtlO and λgtll DNA Cloning Techniques: A Practical Approach, D. Glover, Ed. (IRC Press, Oxford, (1964)) cDNA library waε conεtructed (U. Gubler and B. J. Hoffman, Gene ££, 263 (1983)) with mRNA purified (J. M. Chirwin, A. E. Przbyla, R. J. MacDonald, W. J. Rutter, Bioche iεtry 18, 5294 (1979)) from MDA-MB-231 cellε. The following eightfold degenerate antiεense deoxyoligonucleotide encoding the 13-amino acid sequence AEKEKTFCVNGGE (SEQ ID No. 31) (13) was designed on the basis of human codon frequency optima (R. Lathe, J. Mol. Biol. 183. 1 (I9δ5)) and chemically synthesized: 5'-CTCGCC (G OR T) CC (A OR G) TTCAC (A OR G) CAGAAGGTCTTCTCCTTCTCAGC-3' (SEQ ID No. 40). For the purpose of probe design a cyεteine was assigned to an unknown residue in the amino acid εequence . The probe was labeled by phosphorylation and hybridized under low-stringency conditions to the cDNA library. The proHRG-α protein waε identified in thiε library. HRB-S1 cDNA was identified by probing a second oligo(dT)-primed λgtlO library made from MDA-MB-231 cell mRNA with εequenceε derived from both the 5' and 3' ends of proHRG-α. Clone 13 (Fig. 2A) was a product of screening a primed (5'-CCTCGCTCCTTCTTCTTGCCCTTC-3' primer (SEQ ID No. 41) ; proHRG-α antiεenεe nucleotideε 33 to 56) MDA-MB-231 λgtlO library with 5' HRG-α sequence. A εequence corresponding to the 5' end of clone 13 aε the probe waε uεed to identify proHRG02 and proHRG/33 in a third oligo(dT)-primed λgtlO library derived from MDA-MB-231 cell mRNA. Two cDNA cloneε encoding each of the four HRGs were sequenced (F. Sanger, S. Milken, A. R. Coulson, Proc. Natl. Acad. Sci.U.S.A. 2A- 5463 1977]). Another cDNA deεignated clone 64 haε an amino acid εequence identical to proHRG02 through amino acid 420. A stop codon at position 421 is followed by a different 3'-untranslated εequence. EXAMPLE 9 Isolation of a Further Splicing Variant The methods in Example 6 produced four closely related sequences (heregulin , βl , β2 , β3 ) which arise as a reεult of εplicing variation. Peles et al. (Cell ≤£, 205 (1992)), and Wen et al. (Cell ££, 559 (1992)) have isolated another εplicing variant (from rat) uεing a εimilar purification and cloning approach to that described in Examples 1-4 and 6 involving a protein which binds to pies'* 2 . The cDNA clone was obtained as follows (via the purification and εequencing of a pies'* 82 binding protein from a transformed rat fibroblast cell line) . A pies'* 82 binding protein was purified from conditioned medium as follows. Pooled conditioned medium from three harvests of 500 roller bottles (120 liters total) was cleared by filtration through 0.2 μ filters and concentrated 31-fold with a Pelicon ultrafiltration εystem using membraneε with a 20kd molecular εize cutoff. All the purification εtepε were performed by uεing a Pharmacia faεt protein liquid chromatography system. The concentrated material waε directly loaded on a column of heparin-Sepharose (150 ml, preeguilibrated with phosphate-buffered saline (PBS) ) . The column waε waεhed with PBS containing 0.2 M NaCl until no absorbance at 280 nm wavelength could be detected. Bound proteins were then eluted with a continuous gradient (250 ml) of NaCl (from 0.2 M to 1.0 M), and 5 ml fractions were collected. Samples (0.01 ml of the collected fractions were used for the quantitative assay of the kinase stimulatory activity. Active fractions from three column runs (total volume = 360 ml) were pooled, concentrated to 25 ml by using a YM10 ultrafiltration membrane (Amicon, Danvers, MA), and ammonium εulfate was added to reach a concentration of 1.7 M. After clearance by centrifugation (10,000 x g, 15 min.), the pooled material was loaded on a phenyl-Superose column (HRlO/10, Pharmacia). The column waε developed with a 45 ml gradient of (NH 4 ) 2 S0 4 (from 1.7 M to no salt) in 0.1 M Na 2 P0 4 (pH 7.4), and 2 ml fractionε were collected and aεεayed (0.002 ml per sample) for kinase stimulation (aε described in Example 6) . The major peak of activity was pooled and dialyzed against 50 mM sodium phosphate buffer (pH 7.3). A Mono-S cation-exchange column (HR5/5, Pharmacia) was preeguilibrated with 50 mM εodium phosphate. After loading the active material (0.8δ4 mg of protein; 35 ml), the column was waεhed with the εtarting buffer and then developed at a rate of 1 l/min. with a gradient of NaCl. The kinaεe stimulatory activity was recovered at 0.45-0.55 M salt and was εpread over four fractions of 2 ml each. These were pooled and loaded directly on a Cu +2 chelating columnε (1.6 ml, HR2/5 chelating Superose, Pharmacia) . Most of the proteinε adsorbed to the resin, but they gradually eluted with a 30 ml linear gradient of ammonium chloride (0-1 M) . The activity eluted in a single peak of protein at the range of 0.05 to 0.2 M NH 4 C1. Samples from various steps of purification were analyzed by gel electrophoresiε followed by silver staining using a kit from ICN (Costa Mesa, CA) , and their protein contentε were determined with a Coomaεεie blue dye binding asεay uεing a kit from Bio-Rad (Richmond, CA). The p44 protein (10 μg) waε reconstituted in 200 μl of 0.1 M ammonium bicarbonate buffer (pH 7.δ). Digestion was conducted with L-1-toεyl-amide 2-phenylethyl chloromethyl ketone-treated trypεin (Serva) at 37 * C for 18 hr. at an enzyme-to-εubεtrate ratio of 1:10. The reεulting peptide mixture was separated by reverεe-phase HPLC and monitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d. x 15 cm, 300 Λ) and an HP 1090 liquid chromatographic εyεtem equipped with a diode-array detector and a workεtation. The column waε equilibrated with 0.1% trifluoroacetic acid (mobile phaεe A) , and elution waε effected with a linear gradient from 0%-55% mobile phaεe B (90% acetonitrile in 0.1% trifluoroacetic acid) over 70 min. The flow rate waε 0.2 ml/min. and the column temperature waε controlled at 25 * C. One-third aliquotε of the peptide peakε collected manually from the HPLC εyεtem were characterized by N-terminal εequence analyεis by Edman degradation. The fraction eluted after 27.7 min. (T27.7) contained mixed amino acid εequences and was further rechroroatographed after reduction as follows: A 70% aliquot of the peptide fraction was dried in vacuo and reconεtituted in 100 μl of 0.2 M ammonium bicarbonate buffer (pH 7.8). DTT (final concentration 2 mM) was added to the solution, which was then incubated at 37 * C for 30 min. The reduced peptide mixture was then separated by reverse-phase HPLC using a Vydac column (2.1 mm i.d. x 15 cm). Elution conditions and flow rat'were identical to those described above. Amino acid εequence analyεiε of the peptide waε performed with a Model 477 protein sequencer (Applied Biosystemε, Inc., Foεter City, CA) equipped with an on-line phenylthiohydantoin (PTH) amino acid analyzer and a Model 900 data analysis system (Hunkapiller et al. (1966) In Methods of Protein Microcharacterization. J.E. Shively, ed. (Clifton, New Jersey: Humana Press p. 223-247). The protein was loaded onto a trifluoroacetic acid-treated glass fiber disc precycled with polybrene and NaCl. The PTH-amino acid analysis was performed with a micro liquid chromatography εyste (Model 120) using dual syringe pumpε and reverεe-phase (C-18) narrow bore columns (Applied Biosystems, 2.1 mm x 250 mm). RNA was isolated from Ratl-EJ cellε by εtandard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York (1962) and poly (A) + waε εelected using an mRNA Separator kit (Clontech Lab, Inc., Palo Alto, CA) . cDNA was synthesized with the Superscript kit (from BRL Life Technologies, Inc., Betheεda, MD) . Column-fractionated double-strand cDNA was ligated into an Sail- and Notl-digeεted pJT-2 plaεmid vector, a derivative of the pCD-X vector (Okayama and Berg, Mol. Cell Biol. 2: 280 (1983)) and transformed into DH10B £. coli cells by electroporation (Dower et al., Nucl. Acidε Reε. 16: 6127 (1988)). Approximately 5 x 10 5 primary transformants were screened with two oligonucleotide probes that were derived from the protein εequenceε of the N-terminuε of NDF (residues 5-24) and the T40.4 tryptic peptide (residueε 7-12) . Their respective εequenceε were aε follows (N indicates all 4 nt): (1) 5'-ATA GGG AAG GGC GGG GGA AGG GTC NCC CTC NGC A T AGG GCC GGG CTT GCC TCT GGA GCC TCT-3' (2) 5'-TTT ACA CAT ATA TTC NCC-3' C G G C (1: SEQ ID No. 167; 2: SEQ ID No. 168) The εynthetic oligonucleotides were end-labeled with t7- 32 P]ATP with T4 polynucleotide kinase and used to screen replicate sets of nitrocellulose filters. The hybridization solution contained 6 x SSC, 50 mM sodium phoεphate (pH 6.8), 0.1% εodium pyrophoεphate, 2 x Denhardt'ε εolution, 50 μg/ml εalmon sperm DNA, and 20% formamide (for probe 1) or no formamide (for probe 2) . The filters were washed at either 50 * C with 0.5 x SSC, 0.2% SDS, 2 mM EDTA (for probe 1) or at 37'C with 2 x SSC, 0.2% SDS, 2 mM EDTA (for probe 2). Autoradiography of the filterε gave ten clones that hybridized with both probes. These cloneε were purified by replating and probe hybridization as described above. The cDNA clones were εequenced using an Applied Biosystems 373A automated DNA sequencer and Applied Biosystems Taq Dy ' eDeoxy™ Terminator cycle εequencing kitε following the manufacture'ε instructions. In εome instances, sequences were obtained using [ 35 S]dATP (Amerεham) and Seguenaεe™ kitε from U.S. Biochemicals following the manufacturer's instructions. Both strands of the cDNA clone 44 were εequenced by uεing εynthetic oligonucleotides as primerε. The εeguence of the most 5' 350 nt was determined in seven independent cDNA clones. The resultant clone demonstrated the pattern εhown in figure 30 (NDF) . EXAMPLE 10 Strategies for Detecting Other Possible Splicing Variants Alignment of the deduced amino acid εequenceε of the cDNA cloneε and PCR productε of the bovine, and the publiεhed human (Fig. 31) and rat εequences εhow a high level of εimilarity, indicating that theεe εequenceε are derived from homologous genes within the three εpecies. The variable number of messenger RNA transcripts detectable at the cDNA/PCR product level is probably due to extenεive tiεsue-specific splicing. The patterns obtained and shown in Figure 30 suggests that other splicing variants exist. A list of probable εplicing variants iε indicated in Figure 37. Many of these variants can be obtained by coding εegment εpecific probing of cDNA librarieε derived from different tiεεues and by PCR experiments using primer pairs εpecific to particular coding εegmentε. Alternatively, the variantε can be assembled from εpecific cDNA cloneε, PCR productε or genomic DNA regions via cutting and εplicing techniques known to one skilled in the art. For example, a rare restriction enzyme cutting site in a common coding εegment (e.g., A), can be uεed to connect the FBA amino terminus of GGF2BPP5 to carboxy terminal εequenceε of GGF2BPP1, GGFBPP2, GGFBPP3, or GGFBPP4. If the preεence or the absence of coding εegment E and/or G provide benefit for contemplated and stated uses, then these coding segments can be included in expression conεtructε. These variant SUBSTITUTESHEET(fiU tE26) εequences can be expresεed in recombinant systems and the recombinant products can be aεsayed to determine their level of Schwann cell mitogenic activity as well as their ability to bind and activate the pl85** 82 receptor. EXAMPLE 11 Identification of Functional Elements of GGF The deduced structures of the family of GGF εequences indicate that the longest forms (as represented by GGF2BPP4) encode transmembrane proteins where the extracellular part contains a domain which resembles epidermal growth factor (see Carpenter and Wahl in Peptide Growth Factors and Their Receptorε I pp. 69-133, Springer-Verlag, NY 1991) . The poεitionε of the cyεteine residues in coding segmentε C and C/D or C/D' peptide εequence are conεerved with respect to the analogous residueε in the epidermal growth factor (EGF) peptide εequence (εee Figure 35, SEQ ID Noε. 151-153). Thiε εuggests that the extracellular domain functionε aε receptor recognition and biological activation εiteε. Several of the variant forms lack the H, K, and L coding εegmentε and thuε may be expreεsed aε εecreted, diffuεible biologically active proteinε. GGF DNA εequences encoding polypeptides which encompass the EGF-like domain (EGFL) can have full biological activity for stimulating glial cell mitogenic activity. Membrane bound versionε of thiε protein may induce Schwann cell proliferation if expressed on the surface of neurons during embryogenesis or during nerve regeneration (where the εurfaceε of neurons are intimately asεociated with the εurfaces of proliferating Schwann cellε) . Secreted (non membrane bound) GGFε may act aε claεεically diffusible factors which can interact with Schwann cellε at εome diεtance from their point of εecretion. Other forms may be released from intracells by εourceε via tissue injury and cell disruption. An example of a εecreted GGF is the protein encoded by GGF2HBS5 (εee example 6) ; thiε iε the only GGF known which haε been found to be directed to the exterior of the cell (example 7) . Secretion iε probably mediated via an N-terminal hydrophobic sequence found only in region E, which is the N-terminal domain contained within recombinant GGF-II encoded by GGF2HBS5. Other GGF'ε appear to be non-εecreted (εee example 6) . These GGFs may be injury response forms which are released as a consequence of tiεsue damage: Other regions of the predicted protein structure of GGF-II (encoded by GGF2HBS5) and other proteins containing regions B and A exhibit εimilarities to the human basement membrane heparan εulfate proteoglycan core protein (ref.). The peptide ADSGEY, which iε located next to the εecond cysteine of the C2 immunoglobulin fold in these GGF's, occurε in nine of twenty-two C-2 repeats found in that basal lamina protein. This evidence εtrongly εuggeεtε that these proteins may associate with matrix proteins εuch aε thoεe aεsociated with neurons and glia, and may εuggeεt a method for εequestration of glial growth factors at target εiteε. EXAMPLE 12 Purification of GGFs from Recombinant Cells In order to obtain full length or portionε of GGFε to assay for biological activity, the proteins can be overproduced using cloned DNA. Several approaches can be used. A recombinant £. coli cell containing the sequenceε described above can be conεtructed. Expression systems such aε pNHβa or pHHl6a (Stratagene, Inc.) can be uεed for thiε purpoεe by following manufacturerε procedureε. Alternatively, these εequenceε can be inserted in a mammalian expression vector and an overproducing cell line can be constructed. As an example, for thiε purpoεe DNA encoding a GGF, clone GGF2BPP5 has been expresεed in both COS cellε and Chinese hamster ovary cells (see Example 7) (J. Biol. Chem. 2&1, 3521-3527, (19B1)). This vector containing GGF DNA sequences can be transfected into host cells using established procedures. Transient expression can be examined or G418-resistant clones can be grown in the presence of methotrexate to select for cells that amplify the dhfr gene (contained on the pMSXND vector) and, in the process, co-amplify the adjacent GGF protein encoding εequence. Becauεe CHO cells can be maintained in a totally εerum-free, protein-free medium (Hamilton and Ham, In Vitro 12., 537-547 (1977)), the, deεired protein can be purified from the medium. Weεtern analyεis using the antisera produced in Example 9 can be used to detect the presence of the desired protein in the conditioned medium of the overproducing cells. The desired protein (rGGF-II) waε purified from the medium conditioned by tranεiently expreεεing coε cellε as follows. rGGF-II was harvested from the conditioned medium and partially purified using Cation Exchange Chromatography (POROS-HS) . The column was equilibrated with 33.3 mM MES pH 6.0. Conditioned media was loaded at flow rate of 10 ml/miή. The peak containing Schwann cell proliferation activity and immunoreactive (using the polyclonal antiεera was against a GGFII peptide described above) was eluted with 50 mM Tris, 1M NaCl pH 8.0. (Figure 50A and 50B respectively) . rGGF-II is also expressed using a stable Chinese Ovary Hamεter cell line. rGGF-II from the harveεted conditioned media waε partially purified using Cation Exchange Chromatograph (POROS-HS) . The column was equilibrated with PBS pH 7.4. Conditioned media was loaded at 10 ml/min. The peak containing the Schwann Cell Proliferative activity and immunoreactivity (uεing GGFII polyclonal antiεera) was eluted with 50 mM Hepes, 500 mM NaCl pH 8.0. An additional peak was observed at 50 mM Hepes, 1M NaCl pH 8.0 with both proliferation as well as immunoreactivity (Fig. 51) . rGGF-II can be further purified using Hydrophobic Interaction Chromatography as a high resolution εtep; Cation exchange/Reserve phase Chromatography (if needed as εecond high resolution step) ; A viral inactivation step and a DNA removal step such as Anion exchange chromatography. Detailed description of procedures used are as follows: Schwann Cell Proliferation Activity of the recombinant GGF-II peak eluted from the Cation Exchange column was determined as follows: Mitogenic responses of the cultured Schwann cells were measured in the presence of 5 M Forεkolin uεing the peak eluted by 50 mM Triε 1 M NaCl 89 pH ' 8.0. The peak was added at 20 1, 10 1 (1:10) 10 l and (1:100) 10 1. Incorporation of I25 I-Uridine was determined and expresεed aε (CPM) following an 18-24 hour expoεure. An immunoblot using polyclonal antibody raised against a peptide of GGF-II waε carried out aε follows: 10 μl of different fractions were ran on 4-12% gradient gelε. The gels were transferred on to Nitrocellulose paper, and the nitrocellulose blots were blocked with 5% BSA and probed with GGF-II-specific antibody (1:250 dilution). ,2i I protein A (1:500 dilution, Specific Activity = 9.0/Ci/g) was used as the secondary antibody. The immunoblots were exposed to Kodax X-Ray filmε for 6 hourε. The peak fractionε eluted with 1 M NaCl εhowed a broad immunoreactive band at 65-90 Kd which is the expected size range for GGFII and higher molecular weight glycoforms. GGF-II purification on cation exchange columns was performed as follows: CHO cell conditioned media expreεεing rGGFII waε loaded on the cation exchange column at 10 ml/mini. The column waε equilibrated with PBS pH 7.4. The elution waε achieved with 50 mM Hepes 500 mM NaCl pH 8.0 and 50 mM Hepes 1M NaCl pH 8.0 respectively. All fractions were analyzed using the Schwann cell proliferation assay (CPM) described herein. The protein concentration (mg/ml) waε determined by the Bradford aεεay using BSA as the εtandard. A Western blot using 10 μl of each fraction was performed. As indicated in Figure 51A and 5IB, immunoreactivity and the Schwann cell activity co-migrates. The Schwann cell mitogenic assay described herein may be uεed to assay the expressed product of the full length clone or any biologically active portions thereof. The full length clone GGF2BPP5 has been expressed transiently in COS cells. Intracellular extracts of transfected COS cellε εhow biological activity when aεεayed in the Schwann cell proliferation aεsay described in Example 1. In addition, the full length close encoding GGF2HBS5 has been expresεed transiently in CHO and insect (Example 7) cells. In this case both cell extract and conditioned media εhow biological activity in the Schwann cell proliferation aεεay deεcribed in Example 1. Any member of the family of εplicing variant complementary DNA'ε derived from the GGF gene (including the Heregulinε) can be expreεsed in thiε manner and aεsayed in the Schwann cell proliferation assay by one skilled in the art. Alternatively, recombinant material may be iεolated from other variantε according to Wen et al. (Cell £2, 559 (1992)) who expressed the splicing variant Neu differentiation factor (NDF) in COS-7 cells. cDNA cloneε inserted in the pJT-2 eukaryotic plasmid vector are under the control of the SV40 early promoter, and are 3'-flanked with the SV40 termination and polyadenylation signals. COS-7 cells were tranεfected with the pJT-2 plaεmid DNA by electroporation as follows: 6 x 10 6 cells (in 0.8 ml of DMEM and 10% FEBS) were transferred to a 0.4 cm cuvette and mixed with 20 μg of plasmid DNA in 10 μl of TE solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Electroporation was performed at room temperature at 1600 V and 25 μF using a Bio-Rad Gene Pulser apparatus with the pulse controller unit set at 200 ohms. The cells were then diluted into 20 ml of DMEM, 10% FBS and transferred into a T75 flask (Falcon) . After 14 hr. of incubation at 37'C, the medium was replaced with DMEM, 1% FBS, and the incubation continued for an additional 48 hr. Conditioned medium containing recombinant protein which was harvested from the cells demonstrated biological activity in a cell line expresεing the receptor for thiε protein. Thiε cell line (cultured human breaεt carcinoma cell line AU 565) waε treated with recombinant material. The treated cellε exhibited a morphology change which iε characteriεtic of the activation of the erbB2 receptor. Conditioned medium of this type also can be tested in the Schwann cell proliferation assay. EXAMPLE 13 Purification and Assay of Other Proteins which bind plSS** 82 Receptor I. Purification of gp30 and p70 Lupu et al. (Science 249. 1552 (1990)) and Lippman and Lupu (patent application number PCT/US91/03443 (1990)), hereby incorporated by reference, have purified a protein from conditioned media of a human breast cancer cell line MDA-MB-231, as follows. Conditioned media collections were carried using well-known procedures. The media was concentrated 100-fold in an Amicon ultra-filtration cell (YM5 membrane) (Amicon, Danvers, MA) . Once clarified and concentrated, the media were stored at -20°C while consecutive collectionε were made during the following dayε. The concentrated media were dialyzed uεing Spectra/por® 3 tubing (Spectrum Medical Induεtries, Los Angeles, CA) against 100 volumes of 0.1 M acetic acid over a two day period at 4"C. The material that precipitated during dialysiε was removed by centrifugation at 4000 rpm for 30 min. at 4 * C; proteaεe inhibitorε were added. The clarified εample waε then lyophilized. Lyophilized conditioned medium waε diεsolved in 1 M acetic acid to a final concentration of about 25 mg/ml total protein. Insoluble material was removed by centrifugation at 10,000 rpm for 15 minuteε. The εample waε then loaded onto a Sephadex G-100 column (XK 16, Pharmacia, Piscataway, NJ) , was equilibrated and waε εubjected to elution with 1 M acetic acid at 4 * C with an upward flow of 30 ml/hr. 100 ng of protein was processed from 4 ml of 100-fold concentrated medium. Fractions containing 3 ml of eluate were lyophilized and reεuεpended in 300 μl PBS for assay and served as a source for further purification. Sephadex G-100 purified material waε run on reverεed-phaεe high pressure liquid chromatography (HPLC) . The first εtep involved a εteep acetonitrile gradient. Steep acetonitrile gradient and all other HPLC εteps were carried out at room temperature after equilibration of the C3-Reversed phase column with 0.05% TFA (Trifluoroacetic acid) in water (HPLC-grade) . The εamples were loaded and fractions were eluted with a linear gradient (0-45% acetonitrile in 0.05% TFA) at a flow rate of 1 ml/min. over a 30 minute period. Absorbance was monitored at 280 nm. One ml fractions were collected and lyophilized before analysiε for EGF receptor-competing activity. A εecond HPLC εtep involved a εhallow acetonitrile gradient. The pool of active fractions from the previous HPLC step was rechromatographed over the same column. Elution waε performed with a 0-18% acetonitrile gradient in 0.05% TFA over a 5 minute period followed by a linear 18-45% acetonitrile gradient in 0.05% TFA over a 30 minute period. The flow rate was l.o ml/min. and l ml fractionε were collected. Human TGFα-like factor was eluted at a 30-32% acetonitrile concentration as a εingle peak detectable by RRA. Lupu et al. (Proc. Natl. Acad. Sci. J£, 2287 (1992)) purified another protein which binds to the piδδ** 82 receptor. This particular protein, p75, was purified from conditioned medium uεed for the growth of SKBr-3 (a human breaεt cancer cell line) propagated in improved Eagle'ε medium (IMEM: GIBCO) εupplemented with 10% fetal bovine serum (GIBCO) . Protein p75 waε purified from concentrated (100X) conditioned medium using a pies'* 82 affinity column. The 94 Kilodalton extracellular domain of pies'* 82 (which binds p75) was produced via recombinant expression and was coupled to a polyacrylamide hydrazido-Sepharoεe affinity chromatography matrix. Following coupling the matrix waε washed extensively with ice cold 1.0 M HC1 and the beadε were activated with 0.5 M NaN0 2 . The temperature waε maintained at 0'C for 20 minutes and this was followed by filtration and washing with ice cold 0.1 M HC1. 500 ml of concentrated conditioned medium was run through the beads by gravity. The column was washed and eluted εtepwise with 1.0 M citric acid at pH valueε from 4.0 to 2.0 (to allow diεsociation of the erbB2 and p75) . All fractions were desalted on Pharmacia PD10 columns. Purification yielded a homogeneous polypeptide of 75kDa at 3.0-3.5 elution pH (confirmed by analyεis on SDS/PAGE by silver εtaining) . II. Binding of gp30 to lSS^B∑ The purified gp30 protein was tested in an asεay to determine if it bound to pies'* 82 . A competition aεεay with a monoclonal antibody againεt pies'* 82 . The gp30 protein diεplaced antibody binding to pies'* 82 in SK-BR-3 and MDA-MB-453 cellε (human breaεt carcinoma cell lineε expreεεing the pies'* 82 receptor) . Schwann cell proliferation activity of gp30 can alεo be demonεtrated by treating Schwann cell cultureε with purified gp30 uεing the aεsay procedure described in Exampleε 1-3. III. Binding of P75 to PISS** 82 To assess whether the 75-kDa polypeptide (p75) obtained from SKBr-3 conditioned medium waε indeed a ligand for the erbB2 oncoprotein in SKBr-3 cellε, a competition aεεay as described above for gp30 was used. It was found that the p75 exhibited binding activity, whereas material from other chromatography.fractions did not show such activity (data not shown) . The flow-through material εhowed εome binding activity. Thiε might be due to the presence of shed erbB2 ECD. IV. Other Pies'* 82 ligands Peles et al. (Cell 69, 205 (1992)) have also purified a lβS"* 82 stimulating ligand from rat cells, (NDF, εee Example δ for method). Holmes et al. (Science 256. 1205 (1992)) have purified Heregulin c from human cells which binds and εtimulateε IBS'* 82 (εee example 6) . Tarakovεky et al. Oncogene 6:218 (1991) have demonstrated bending of a 25 kD polypeptide isolated from activated macrophageε to the Neu receptor, a pies'* 82 homology, herein incorporated by reference. VI. NDF Isolation Yarden and Peles (Biochemistry 3J), 3543 (1991)) have identified a 35 kilodalton glycoprotein which will stimulate the lβS** 82 receptor. The protein waε identified in conditioned medium according to the following procedure. Rat I-EJ cellε were grown to confluence in 175-cm 2 flaεkε (Falcon) . Monolayerε were washed with PBS and left in serum-free medium for 10-16 h. The medium was discarded and replaced by fresh serum-free medium that was collected after 3 days in culture. The conditioned medium waε cleared by low-speed centrifugation and concentrated 100-fold in an Amicon ultrafiltration cell with a YM2 membrane (molecular weight cutoff of 2000) . Biochemical analyses of the neu stimulatory activity in conditioned medium indicate that the ligand is a 35-kD glycoprotein that it iε heat εtable but εensitive to reduction. The factor is precipitable by either high εalt concentrations or acidic alcohol. Partial purification of the molecule by εelective precipitation, heparin-agaroεe chromatography, and gel filtration in dilute acid resulted in an active ligand, which iε capable of εtimulating the protooncogenic receptor but iε ineffective on the oncogenic neu protein, which iε constitutively active. The purified fraction, however, retained the ability to stimulate also the related receptor for EGF, suggesting that these two receptors are functionally coupled through a bidirectional mechanism. Alternatively, the presumed ligand interactε simultaneously with both receptors. The presented biochemical characteristic of the factor may be used to enable a completely purified factor with which to explore these possibilities. In other publications, Daviε et al. (Biochem. Biophyε. Reε. Commun. 179. 1536 (1991), Proc. Natl. Acad. Sci. 8J, 8582 (1991) and Greene et al., PCT patent application PCT/US91/02331 (1990)) describe the purification of a protein from conditioned medium of a human T-cell (ATL-2) cell line. ATL-2 cell line iε an IL-2-independent HTLV-1 (+) T cell line. Mycoplaεm-free ATL-2 cellε were maintained in RPMI 1640 medium containing 10% FCB as the culture medium (10% FCS-RPMI 1640) at 37'C in a humidified atmosphere with 5% C0 2 . For purification of the proteinaceous substance, ATL-2 cellε were waεhed twice in 1 x PBS and cultured at 3 x 10 s ml in εeru -free RPMI 1640 medium/2 mM L-glutamine for seventy-two hours followed by pelleting of the cellε. The culture εupernatant εo produced iε termed "conditioned medium" (CM.). CM. waε concentrated 100 fold, from 1 liter to 10 ml, uεing a YM-2 Diaflo membrane (Amicon, Boεton, MA) with a lOOOd cutoff. For uεe in εome asεayε, concentrated CM. containing componentε greater than 1000 MW were rediluted to original volume with RPMI medium. Gel electrophoreεiε using a polyacrylamide gradient gel (Integrated Separation Systems, Hyde Park, MD or Phorecast Syεtem by Amerεham, Arlington Heightε, IL) followed by εilver εtaining of εome of thiε two column purified material from the one liter preparation revealed at leaεt four to five bands of which the lOkD and 20kD bands were unique to thiε material. Paεεed CM. containing components less than 1000 NW were used without dilution. Concentrated conditioned medium was filter εteriiized with a .45μ uniflo filter (Schleicher and Schuell, Keene, NH) and then further purified by application S< * BSTITUTESHEET(RULE 2 . to a DEAE-SW anion exchange column (Waterε, Inc., Milford, MA) which had been preeguilibrated with lOmM Triε-Cl, pH 8.1 Concentrated CM. proteinε repreεenting one liter of original ATL-2 conditioned medium per HPLC run were absorbed to the column and then eluted with a linear gradient of OmM to OmM NaCl at a flow rate of 4 ml/min. Fractions were asεayed using an is vitro immune complex kinase asεay with 10% of the appropriate DEAE fraction (l column purified material) or 1% of the appropriate C18 fractions (two column purified material) . The activity which increased the tyrosine kinaεe activity of pl85c-neu in a doεe-dependent manner uεing the jjj vitro immune complex kinaεe aεεay waε eluted as one dominant peak across 4 to 5 fractions (36-40) around 220 to 240 mM of NaCl. After HPLC-DEAE purification, the proteinε in the active fractionε were concentrated and pooled, concentrated and εubjected to Clδ (million matrix) reverεe phaεe chromatography (Waterε, Inc., Milford, MA) (referred to aε the C18+1 step or two column purified material) . Elution waε performed under a linear gradient of 2-propanol againεt 0.1% TFA. All the fractions were dialyzed against RPMI 1640 medium to remove the 2-propanol and asεayed using the in vitro immune complex kinase assay, described below, and a 1% concentration of the appropriate fraction. The activity increasing the tyrosine kinase activity of pl85c-neu was eluted in two peakε. one eluted in fraction 11-13, while a εecond, εlightly leεs active peak of activity eluted in fractions 20-23. These two peaks correspond to around 5 to 7% of iεopropanol and 11 to 14% iεopropanol reεpectively. C18#l generated fractions 11-13 were used in the characterization εtudieε. Active fractions 98 obtained from the second chromatographic εtep were pooled, and designated as the proteinaceous substance εample. A twenty liter preparation employed the εame purification εtrategy. The DEAE active fractionε 35-41 were pooled and subjected to cl8 chromatography as discussed above. C18#l fractions 11-13 and 21-24 both had dose-dependent activity. The pool of fractionε 11-13 waε subjected to an additional Clδ chromatographic εtep (referred to aε C18#2 or three column purified material) . Again, fractionε 11-13 and 21-24 had activity. The doεe reεponse of fraction 23 as determined by i vitro immune complex kinase asεay as described in Example 8 may be obtained upon addition of 0.005% by volume fraction 23 and 0.05% by volume fraction 23. This represents the greatest purity achieved. Molecular weight rangeε were determined baεed on gel filtration chromatography and ultrafiltration membrane analysis. Near equal amounts of tyrosine kinase activity were retained and passed by a 10,000 molecular weight cut off filter. Almost all activity was passed by a 30,000 molecular weight cut off filter. Molecular weight ranges for active chromatographic fractionε were determined by comparing fractionε containing doεe-dependent neu-activating activity to the elution profileε of a εet of protein molecular weight εtandardε (Sigma Chemical Co., St. Louiε, MO) generated uεing the εame running conditionε. A low molecular weight region of activity waε identified between 7,000 and 14,000 daltonε. A εecond range of activity ranged from about 14,000 to about 24,000 daltonε. After gel electrophoreεiε uεing a polyacrylamide gradient gel (Integrated Separation Systems, Hyde Park, MD 99 or Phorecaεe System by Amersham, Arlington Heightε, IL) , silver staining of the three-column purified material (cl8#2) was done with a commercially available silver staining kit (BioRad, Rockville Centre, NY) . Fraction 21, 22, 23, and 24 from clβ#2 purification of the twenty liter preparation were run with markers. Fractions 22 and 23 εhowed the oεt potent doεe response in the 18S** 82 (neu) kinase asεay (εee below) . The fact that selected molecular weight fractions interact with lSS** 82 was demonstrated with an immune complex kinase assay. Huang et al. (1992, J. Biol. Chem. 257:11508-11512), hereby incorporated by reference, have isolated an additional neu/erb B2 ligand growth factor from bovine kidney. The 25 kD polypeptide factor was isolated by a procedure of column fractionation, followed by εequential column chromatography on DEAE/cellulose (DE52) , Sulfadex (sulfated Sephadex G-50) , heparin-Sepharoεe 4B, and Superdex 75 (fast protein liquid chromatography) . The factor, NEL- GF, stimulates tyrosine-εpecific autophosphorylation of the neu/erb B2 gene product. VII. Immune complex assay NDF for ligand binding to Pi£L§f_!_E2: Thiε assay reflects the differences in the autophosphorylation activity of immunoprecipitated pl85 driven by pre-incubation of PN-NR6 cell lysate with varying amounts of ATL-2 conditioned medium (C.H.) or proteinaceous subεtance and iε referred to hereinafter as neu-activating activity. Cell lines used in the immune complex kinase assay were obtained, prepared and cultured according to the methods disclosed in Kokai et al., Cell J55, 267-292 (July 28, 1969) the disclosures of which are hereby incorporated by reference as if fully εet forth herein, and U.S. application εerial number 386,820 filed July 27, 1989 in the name of Mark I. Green entitled "Methods of Treating Cancerous Cells with Anti-Receptor Antibodies", the discloεureε of which are hereby incorporated by reference aε if fully εet forth herein. Cell lines were all maintained in DMEM medium containing 5% FCS aε the culture medium (5% FCS-DMEM) at 37 * C in a humidified atmosphere with 5% C0 2 . Dense cultures of cells in 150 mm dishes were washed twice with cold PBS, scraped into 10 ml of freeze-thaw buffer (150 mM NaCl, l mM MgCl 2 , 20 mM Hepes, pH 7.2, 10% Glycerol, 1 mM EDTA, 1% Aprotinin) , and centrifuged (600 x 6, 10 minutes). Cell pellets were resuspended in l ml Lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 3% Brij 35, 1 mM EDTA, 1.5 mM MgCl 2 , 1% Aprotinin, 1 M EGTA, 20 μM Na 3 V0 4 , 10% Glycerol) and rotated for thirty minutes at 4 * C All chemicalε were from Sigma Chemical Co., St. Louiε, Mo, unless otherwise indicated. The insoluble materials were removed by centrifugation at 40,000 x g for thirty minuteε. The clear εupernatant which was subsequently used is designated as cell lysate. The cell lysates were incubated for fifteen minutes • with 50 μl of 50% (volume/volume) Protein A-εepharose (Sigma Chemical Co., St. Louis, Missouri), and centrifugated for two minutes to preclear the lysates. 50 μl aliquots of precleared cell lysate were incubated on ice for fifteen minutes with conditioned medium, proteinaceous substance, or other factors as specified, in a final volume of 1 ml with lysiε buffer. The sample was then incubated with 5 μg of 7.16.4 monoclonal antibody, which recognizes the extracellular domain of the pl85neu and pl85c-neu, or other appropriate antibodies, for twenty minutes on ice, followed by a twenty minute incubation with 50 μl of 50% (vol/vol) protein A-Sepharoεe with rotation at 4 * C Immune complexeε were collected by centrifugation, waεhed four times with 500 μl of washing buffer (50 mM Hepes, pH 7.5, 0.1%, Brij 35, 150 mM NaCl, 2 mM EDTA, 1% Aprontinin, 30 μm Na 3 V0 4 ) , then twice with reaction buffer (20 mM Hepes (pH 7.4), 3 mM MnCl 2 and 0.1% Brij 35, 30 μm Na 3 V0 4 ) . Pellets were reεuspended in 50 μl of reaction buffer and (Gamma- 32 P]-ATP (Amersham, Arlington Heights, IL) was added giving a final concentration of 0.2 μm. The εamples were incubated at 27'C for twenty minutes or at 4 * C for 25 minutes with purer εamples. The reactions were terminated by addition of 3 x SDS sample buffer containing 2 mM ATP and 2 mM EDTA and then incubating them at 100 * C for five minutes. The εamples were then subjected to SDS-PAGE analysis on 10% acrylamide gels. Gels were stained, dried, and exposed to Kodak XAR or XRP film with intensifying εcreenε. VIII. Purification of acetylcholine receptor inducing activity tA I ^ ARIA, a 42 kD protein which εtimulates acetylcholine receptor εyntheεis, has been iεolated in the laboratory of Gerald Fiεchbach (Fallε et al., Cell 72:801-815 (1993)). ARIA induces tyrosine phosphorylation of a 185 Kda muscle transmembrane protein which resembles plβS** 82 , and stimulates acetylcholine receptor synthesiε in cultured embryonic myotubes. Sequence analysis of cDNA clones which encode ARIA shows that ARIA is a member of the GGF/erbB2 ligand group of proteins, and thiε iε potentially useful in the glial cell mitogenesis stimulation and other applicationε of, e.g., GGF2 deεcribed herein. EXAMPLE 14 Protein tyrosine phosphorylation mediated by GGF in Schwann cells Rat Schwann cells, following treatment with sufficient levels of Glial Growth Factor to induce proliferation, show εtimulation of protein tyrosine phosphorylation (figure 36) . Varying amounts of partially purified GGF were applied to a primary culture of rat Schwann cellε according to the procedure outlined in Example 3. Schwann cells were grown in DMEM/10% fetal calf serum/5 μM forskolin/0.5μg per mL GGF-CM (0.5mL per well) in poly D-lysine coated 24 well plates. When confluent, the cells were fed with DMEM/10% fetal calf serum at 0.5mL per well and left in the incubator overnight to quiesce. The following day, the cellε were fed with 0.2mL of DMEM/10% fetal calf εerum and left in the incubator for 1 hour. Test samples were then added directly to the medium at different concentrations and for different lengthε of time aε required. The cellε were then lyεed in boiling lyεis buffer (εodium phoεphate, 5mM, pH 6.8; SDS, 2%, jS-mercapteothanol, 5%; dithiothreitol, 0.1M; glycerol, 10%; Bromophenol Blue, 0.4%; εodium vanadate, lOmM) , incubated in a boiling water bath for 10 minutes and then either analyzed directly or frozen at -70 * C Samples were analyzed by running on 7.5% SDS-PAGE gelε and then electroblotting onto nitrocelluloεe using standard procedures as described by Towbin et al. (1979) Proc. Natl. Acad. Sci. USA 76:4350-4354. The blotted nitrocelluloεe waε probed with antiphoεphotyrosine antibodies using standard methods as described in Kamps and Selton (198B) Oncogene 2:305-315. The probed blotε were expoεed to autoradiography film overnight and developed uεing a εtandard laboratory processor. Densitometrie measurements were carried out using an Ultrascan XL enhanced laser densitometer. (LKB) . Molecular weight aεεignmentε were made relative to preεtained high molecular weight standards (Sigma) . The dose responεes of protein phosphorylation and Schwann cell proliferation are very similar (figure 36) . The molecular weight of the phosphorylated band is very close to the molecular weight of pies'* 82 . Similar results were obtained when Schwann cells were treated with conditioned media prepared from COS cells translates with the GGF2HBS5 clone. These reεults correlate well with the expected interaction of the GGFs with and activation of IBS'* 82 . This experiment has been repeated with recombinant GGF-II. Conditioned medium derived from a CHO cell line stably transformed with the GGF-II clone (GGF2HBS5) stimulates protein tyrosine phoεphorylation using the assay described above. Mock transfected CHO cells fail to stimulate this activity (Fig. 52) . EXAMPLE 15 Assay for Schwann cell Proliferation by Protein Factor from the MDA-MB-231 cell line. Schwann cell proliferation is mediated by conditioned medium derived from the human breast cancer cell line MDA-MB-231. On day 1 of the assay, 10 4 primary rat Schwann cells were plated in 100 μl of Dulbecco's Modified Eagle's medium supplemented with 5% fetal bovine plasma per well in a 96 well microtiter plate. On day 2 of the aεsay, 10 μl of conditioned medium (from the human breast cancer cell line MDA-MB-231, cultured as described in Example 6) was added to each well of the microtiter plate. One day 6, the number of Schwann cells per plate was determined using an acid phosphataεe assay (according to the procedure of Connolly et al. Anal. Biochem. 152: 136 (1986)). The plate was waεhed with 100 μl of phoεphate buffered εaline (PBS) and 100 μl of reaction buffer (0.1M sodium acetate, (pH 5.5)), 0.1% Triton X-100, and 10 mM p-nitrophenyl phosphate) waε added per well. The plate was incubated at 37 * C for two hours and the reaction was stopped by the addition of 10 μl of IN NaOH. The optical density of each sample was read in a εpectrophotometer at 410 nm. A 38% stimulation of cell number over Schwann cells treated with conditioned medium from a control cell line (HS-294T, a non-producer of erbB-2 ligand) was observed. This result shows that a protein secreted by the MDA-MB-231 cell line (which secreteε a pies'* 82 binding activity) εtimulates Schwann cell proliferation. EXAMPLE 16 N-glvcOsylation of GGF The protein sequence predicted from the cDNA sequence of GGF-II candidate clones GGF2BPP1,2 and 3 contains a number of consenεuε N-glycosylation motifs. A gap in the GGFII02 peptide εequence coincides with the asparagine residue in one of these motifs, indicating that carbohydrate is probably bound at this site. N-glycosylation of the GGFs was studied by observing mobility changes on SDS-PAGE after incubation with N- glycanaεe, an enzyme that cleaves the covalent linkages between carbohydrate and aspargine reεidueε in proteinε. N-Glycanaεe treatment of GGF-II yielded a major band of MW 40-42 kDa and a minor band at 45-48 kDa. Activity elution experiments under non-reducing conditions εhowed a eingle active deglycoεylated εpecieε at ca 45-50 kDa. Activity elution experimentε with GGF-I alεo demonεtrate an increaεe in electrophoretic mobility when treated with N-Glycanase, giving an active specieε of MW 26- 2β kDa. Silver εtaining confirmed that there is a mobility shift, although no N-deglycosylated band could be assigned because of background staining in the sample used. 106 EXAMPLE 17 Further tests were carried out to determine the mature GGF2 protein once the protein is expresεed and εecreted from tranεfected cellε. The cDNA encoding human GGF2 was cloned into an amplified vector pcdhfrpolyA and transfected into CHO-DG44 cellε for stable expression. rhGGF2 is εecreted into the conditioned media. The ability of the recombinant GGF2 to be εecreted iε mediated through the N-terminal hydrophobic εtretch, i.e., εignal εequence. According to the εignal hypotheses, a signal εequence, once having initiated the export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. N-terminal analysis of the expresεed and purified rhGGF2 indicates that the site of cleavage iε between A i0 and G 51 . The first 50 amino acid residues are cleaved from the mature protein, thus rhGGF2 consistε of 373 amino acids. The amino acid εequence of the cDNA encoding hGGF2 can be found in Figure 55. The firεt fifteen amino acid residues at the N- terminal of the protein is confirmed by N-terminal εequence analysis aε follows in Table 1. Table 1 - N-terminal sequence analysiε of rhGGF2 Cycle # Primary pMoleε Sequence 1 Gly(G) 210.6 2 Asn(N) 163 3 Glu(E 149 4 Ala(A 220 5 Ala(A 180 6 Pro(P 173 7 Ala(A 177 8 Gly(G 154.9 9 Ala(A 162.4 10 Ser(S 65.4 11 Val(V 132.7 12 Val(V (Cys) 11.7 13 Tyr(Y 112.7 14 Ser(S 47.6 15 Ser(S 27.1 The N-terminal sequence analysis is performed by Edman Degradation Process. The *Cys residues are destroyed by the Edman Degradation Process and cannot be detected. Deposit Nucleic acid encoding GGF-II (cDNA, GGF2HBS5) protein (Example 6) in a plasmid pBluescript 5k, under the control of the T7 promoter, was deposited in the American Type Culture Collection, Rockville, Maryland, on September 2, 1992, and given ATCC Accession No. 75298. Applicant • acknowledges itε reεponεibility to replace thiε plaεmid εhould it become non-viable before the end of the term of a patent issued hereon, and its responsibility to notify the ATCC of the issuance of such a patent, at which time the deposit will be made available to the public. Prior to that time the deposit will be made available to the Commissioner of Patents under the termε of 37 CFR §1.14 and 35 USC §112. 108/1 unuiaul * ~ U mύomHα _Ξ2f tΛffl ' LUD 5250.5 PCT • PCT/US95/06846 INMΓΛTIONS RELATING TO A DEPOSITED MICROORGANISM (?CτKu!el3Mr)

Claims: 109 What is claimed iε: 1. A basic polypeptide factor having Schwann cell mitogenic activity, wherein said polypeptide factor lacks an N-terminal εignal εequence. 2. The basic polypeptide factor of claim 1, wherein said polypeptide factor has an amino acid sequence defined as amino acid position 51 to amino acid position 422 in Figure 55 (SEQ. ID No. 179). 3. Iεolated DNA sequence encoding a polypeptide factor having Schwann cell mitogenic activity, wherein said DNA εequence lacks an N-terminal signal sequence. 4. An isolated DNA sequence encoding the polypeptide factor of claim 2. 5. A method for stimulating mitogenesis of a glial cell, said method comprising contacting said glial cell with an effective amount of said polypeptide of claim 1. 6. A method for the prophylaxis or treatment of a pathophysiological condition of the nervous system in a 110 mammal in which εaid condition involves a cell type which is senεitive or responsive to the polypeptide of claim 1, εaid method compriεing adminiεtering to εaid mammal an effective amount of εaid polypeptide. 7. A polypeptide having a glial cell mitogenic activity wherein εaid polypeptide encoded by the DNA εequence of claim 3, εaid polypeptide obtained by a method compriεing cultivating modified host cells under conditions permitting expression of said DNA sequence. 8. Method for identifying the presence of a receptor for the polypeptide of claim 1, in a sample comprising contacting said sample to said polypeptide and determining binding therebetween, wherein εaid binding is indicative of the presence of said receptor. 9. A method for the prophylaxis or treatment of a glial tumor in a patient, said method comprising administering to said patient an effective amount of a εubstance which inhibits the binding of a polypeptide of claim 1 to a receptor therefor. 111 10. A pharmaceutical or veterinary formulation comprising a polypeptide of claim 1, formulated for pharmaceutical or veterinary uεe, respectively, together with an acceptable diluent, carrier or excipient and/or in unit dosage form. 11. A method for the treatment of a condition which involves peripheral nerve damage in a mammal, said method comprising contacting said peripheral nerves with an effective amount of a polypeptide of claim 1. 12. A method for the prophylaxis or treatment of a condition in a mammal wherein said condition involves demyelination or damage or loss of Schwann cells, said method comprising contacting εaid Schwann an effective amount of a polypeptide of claim 1. 13. The method of claim 12 wherein said condition is a neuropathy of sensory or motor nerve fibers. 1 . A method for the prophylaxis or treatment of a neurodegenerative disorder in a mammal, εaid method comprising contacting glial cells in a mammal with an effective amount of a polypeptide of claim 1. 112 15. A method for inducing neural regeneration and/or repair in a mammal, said method comprising contacting glial cells in a mammal with an effective amount of a polypeptide of claim 1. 16. A method of inducing fibroblast proliferation, said method comprising contacting said fibroblasts with a polypeptide, of claim 1. 17. A method of wound repair in mammals, said method comprising contacting said wound with a polypeptide of claim 1. 18. A method of making a medicament comprising admixing a polypeptide of claim 1, with a pharmaceutically acceptable carrier. 19. A method for producing an antibody, said method comprising immunizing a mammal with a polypeptide of claim 1. 20. A method for detecting a receptor which is capable of binding to a polypeptide of claim 1, said method 113 comprising carrying out affinity isolation on said εample using a said peptide as the affinity ligand. 21. A method for the prophylaxis or treatment of a glial tumor in a patient, said method comprising administering to εaid patient an effective amount of a εubstance which inhibits the binding of a polypeptide of claim 1, to a receptor therefor. 22. A method of investigating, isolating or preparing a glial cell mitogen or gene sequence encoding said glial cell mitogen, said method comprising contacting tiεsue preparations or samples with an antibody, said antibody prepared as defined in claim 19. 23. A method for isolating a nucleic acid sequence coding for a molecule having glial cell mitogenic activity, said method comprising contacting a cell containing sample with a glial cell mitogen specific antibody to determine expression of said mitogen in said sample and isolating εaid nucleic acid sequence from the cells exhibiting said expression. 114 24. A method for inducing myelination of a neural cell by a Schwann cell, εaid method compriεing contacting εaid Schwann cell with a polypeptide of claim 1. 25. A method for inducing acetylcholine receptor εyntheεiε in a cell, εaid method comprising contacting of εaid cell with a polypeptide of claim 1. 26. An antibody to a polypeptide as defined in claim 1. 27. A method of purifying a protein with glial cell mitogenic activity, said method comprising contacting a cell extract with an antibody of claim 26. 28. A method of treating a mammal suffering from a diseaεe of glial cell proliferation, εaid method compriaing adminiεtering to εaid mammal an antibody of claim 26. 29. A vector comprising the DNA sequence of claim 3. 30. A host cell containing the iεolated DNA of claim 3. 115 31. A method for the preparation of a glial cell mitogenic factor, said method comprising cultivating the host cell of claim 30, under conditions permitting expresεion of εaid DNA sequence. 32. A polypeptide of claim 1 for use as a glial cell mitogen. 33. A method for the prophylaxis or treatment of multiple sclerosis in a patient, said method comprising administering to said patient an effective amount of a substance which inhibits the binding of a polypeptide of claim 1 to a receptor therefor. 34. A polypeptide which is a glial cell mitogen, said polypeptide being encoded by a DNA sequence as defined in claim 3, said polypeptide obtained by a method comprising for the preparation of a glial cell mitogenic factor, said method cultivating modified host cells under conditions permitting expression of said DNA sequence.

Inventors: CHEN MAIO SU, GOODEARL ANDREW DAVID, HILES IAN, MARCHIONNI MARK, MINGHETTI LUISA, STROOBANT PAUL, WATERFIELD MICHAEL, CHEN, MAIO, SU, GOODEARL, ANDREW, DAVID, HILES, IAN, MARCHIONNI, MARK, MINGHETTI, LUISA, STROOBANT, PAUL, WATERFIELD, MICHAEL
Applicants: CAMBRIDGE NEUROSCIENCE RES, LUDWIG INST CANCER RES, CAMBRIDGE NEUROSCIENCE, LUDWIG INSTITUTE FOR CANCER RESEARCH
Application year: 1995
Main classification: A61K38
IPC codes: A61K38, A61K39, A61P21, A61P25, A61P35, A61P37, C07K14, C07K16, C12N5, C12N15, C12P21, C12R1, G01N33