EP0607247A1 - Production of gpa neurotrophic factor - Google Patents

Abstract

The invention relates to nucleic acids encoding the GPA protein, and also to the GPA protein produced by methods involving recombinant DNA. This GPA protein is useful for preparing antibodies and for diagnosing and treating various neural disorders.

Description

PRODUCTION OF GPA NEUROTROPHIC FACTOR

Field of the Invention

This application relates to the production of polypeptides involved in neuronal survival and/or growth, in particular the production of purified forms thereof by means of recombinant DNA technology. Background of the Invention

A number of protein neurotrophic factors have been identified which influence growth and development of the vertebrate nervous system. It is believed that these factors may play an important role in sustaining the survival of neurons in both the mature and immature nervous system.

The belief that neuronal survival and function is dependent upon neurotrophic factors is based upon the precedent established by work with nerve growth factor (NGF). NGF has been shown, both jn vitro and m vivo, to support the survival of sympathetic, sensory, and basal forebrain neurons. Administration of exogenous NGF rescues neurons from cell death during development. Conversely, removal or sequestration of endogenous NGF by administration of anti-NGF antibodies promotes such cell death. Heumann, J. Exp. Biol.

122:133-150 (1987); Hefti, J. Neurosci. 6_:2155-2162 (1986); Thoenen and Barde, Annu.

Rev. Physiol. 60:284-335 (1980). The role of NGF as a physiologically important neurotrophic factor is limited, however, insofar as the effects of NGF seem to be specific for only certain types of neurons. For example, NGF does not appear to be a survival factor for parasympathetic neurons, placode- and neural crest-derived sensory neurons, or motorneurons. Accordingly, there is significant interest in identifying additional neurotrophic factors that may act as growth or survival factors for other types of neurons.

Examples of such additional neurotrophic factors that have been identified thusfar include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF). Leibrock, Nature 3_ L: 149-152 (1989); Hohn, et al., Nature

244:339-341 (1990); Rosenthal, et al., Neuron 4:767-773 (1990); Manthorpe, et al., in Nerve Growth Factors, pp.31 -56 (John Wiley & Sons Ltd. 1989).

Ciliary neurotrophic factors (CNTFs) are proteins capable of supporting the survival and growth of chick embryo ciliary ganglion neurons jn vitro. CNTFs have been purified from various tissue sources, including chicken eye and rat sciatic nerve. Manthorpe, et al. J. Neurosci. Res. 38:233-239 (1982); Manthorpe, et al., Brain Res. 3_57:282-286 (1986). The nucleotide sequences encoding rabbit, rat, and human CNTF, as well as the expression of rabbit and rat CNTF in recombinant host cells, has been reported. Lin, et al., Science 246:1023-1025 (1989); Stockli, et al., Nature 342:920-923 (1989); Collins, et al., U.S. Patent No. 4,997,929. Oπe of the co-inventors of the present invention has previously reported that an extract of chick eyes contains two independently acting neurotrophic factors, referred to as growth- promoting activity (GPA) and a cholϊne acetyltransferase-stimulating activity (CSA). Nishi, et al., J. Neurosci. 1:505-513 (1981 ). Recently, two of the co-inventors of the present invention reported the partial purification and preliminary characterization of GPA from chicken sciatic nerves. Eckenstein, et al.. Neuron 4:623-631 (1989). GPA was found to support the survival of ciliary ganglion, dorsal root ganglion, and sympathetic neurons in vitro, jd. Comparison of a partial amino acid sequence obtained for such purified GPA with the known amino acid sequences of rabbit and rat CNTF showed approximately 57% identity between GPA and those CNTFs within the portion of the GPA amino acid sequence that was analyzed. _d. However, the difficulty of obtaining substantial amounts of GPA from chicken sciatic nerves has interfered with efforts to further characterize GPA, and of course has precluded any possible clinical use of GPA.

Accordingly, it is an object of the present invention to provide nucleic acid encoding GPA protein and to use this nucleic acid to produce GPA protein in recombinant host cells for diagnostic use or for therapeutic use with neurological disorders.

It is another object to use such nucleic acids encoding GPA, and portions thereof, to identify related nucleic acids in the cells or tissues of various animal species.

It is yet another object to provide derivatives and modified forms of GPA protein, including amino acid sequence variants and covalent derivatives thereof.

It is an additional object to prepare immunogens for raising antibodies, as well as to obtain antibodies, capable of binding GPA protein, or derivatives or modified forms thereof. These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole. Summary of the Invention

These objects are accomplished by first providing isolated DNA comprising the nucleotide coding sequence for GPA, an expression vector comprising the nucleotide coding sequence for GPA, host cells transformed with the vector, including mammalian and bacterial host cells, and a method of using a nucleic acid molecule encoding GPA to effect the production of GPA, comprising culturing a host cell transfected to express such nucleic acid molecule and recovering GPA from the host cell culture. In this method, preferably the host cell is transfected with an expression vector comprising the nucleotide coding sequence for GPA.

By providing the full nucleotide coding sequence for GPA, the invention enables the production of GPA by means of recombinant DNA technology, thereby making available for the first time sufficient quantities of substantially pure GPA protein for diagnostic and therapeutic uses with a variety of neurological disorders. In a preferred embodiment, the invention provides GPA that is free of contaminating polypeptides of the animal species in which GPA naturally occurs, and compositions comprising GPA that are free of such contaminating polypeptides.

Modified and variant forms of GPA are produced in vitro by means of chemical or enzymatic treatment or in vivo bv means of recombinant DNA technology. Such polypeptides differ from native GPA, for example, by virtue of one or more amino acid substitutions, deletions or insertions, or in the extent or pattern of glycosγlation, but substantially retain a biological activity of native GPA.

Antibodies to GPA are produced by immunizing an animal with GPA or a fragment thereof, optionally in conjunction with an immunogenic polypeptide, and thereafter recovering antibodies from the serum of the immunized animals. Alternatively, monoclonal antibodies are prepared from cells of the immunized animal in conventional fashion. Antibodies obtained by routine screening will bind to GPA but will not substantially bind to (i.e., cross react with) NGF, BDNF, NT-3, CNTF, or other neurotrophic factors. Immobilized anti-GPA antibodies are particularly useful in the detection of GPA in clinical samples for diagnostic purposes, and in the purification of GPA.

GPA, its derivatives, or its antibodies are formulated with physiologically acceptable carriers, especially for therapeutic use. Such carriers are used, for example, to provide sustained-release formulations of GPA. in further aspects, the invention provides a method for determining the presence of a nucleic acid molecule encoding GPA in test samples prepared from cells, tissues, or biological fluids, comprising contacting the test sample with isolated DNA comprising the coding sequence for GPA and determining whether the isolated DNA hybridizes to a nucleic acid molecule in the test sample. DNA comprising the coding sequence for GPA is also used in hybridization assays to identify and to isolate nucleic acids sharing substantial sequence identity to the coding sequence for GPA.

Also provided is a method for amplifying a nucleic acid molecule encoding GPA that is present in a test sample, comprising the use of an oligonucleotide having a portion of the nucleotide coding sequence for GPA as a primer in a polymerase chain reaction.

Brief Description of the Drawings Figure 1 shows the nucleotide sequence of the cDNA insert in the ΛCE15 #19 clone

[SEQ ID NO:1 ], including the complete nucleotide coding sequence and deduced amino acid sequence of GPA [SEQ ID NO:2].

Figure 2 shows the homologies among the amino acid sequences of rabbit, rat, and human CNTF [SEQ ID NOS:3,4,5, respectively] and GPA. Figure 3 shows the effect of conditioned media and cell extracts prepared from cultures of recombinant host cells expressing either CNTF or GPA on ciliary ganglion neuron growth.

Figure 4 shows the nucleotide sequence of pHEB030 [SEQ ID NO:6). The location of certain restriction endonuclease cleavage sites and control elements is indicated in brackets. In the sequence, "N" is used to designate the nucleotides that comprise the arbitrary 350 base pair cDNA insert in pHEBO30.

Detailed Description of the Preferred Embodiments "GPA" or "GPA protein" refers to a polypeptide or protein encoded by the GPA nucleotide sequence set forth in Figure 1 ; a polypeptide that is the translated amino acid sequence set forth in Figure 1 ; fragments thereof having greater than about 5 amino acid residues and comprising an immune epitope or other biologically active site of GPA; amino acid sequence variants of the amino acid sequence set forth in Figure 1 wherein one or more amino acid residues are added at the N- or C-terminus of, or within, said Figure 1 sequence or its fragments as defined above; amino acid sequence variants of said Figure 1 sequence or its fragments as defined above wherein one or more amino acid residues of said Figure 1 sequence or fragment thereof are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above proteins, polypeptides, or fragments thereof, wherein an amino acid residue has been covalently modified so that the resulting product is a non-naturally occurring amino acid. GPA amino acid sequence variants may be made synthetically, for example, by site-directed or PCR mutagenesis, or may exist naturally, as in the case of allelic forms and other naturally occurring variants of the translated amino acid sequence set forth in Figure 1 that may occur in human and other animal species. In any event, such fragments, variants, and derivatives exclude any polypeptide heretofore identified, including any known neurotrophic factor, such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), as well as statutorily obvious variants thereof.

A GPA amino acid sequence variant is included within the scope of the invention provided that it is functionally active. As used herein, "functionally active" and "functional activity" in reference to GPA means that the GPA is able to promote the growth, survival, and/or differentiation of neurons, especially ciliary ganglion neurons, dorsal root ganglion neurons, or sympathetic neurons, in vivo or in vitro, and/or that the GPA is immunologically cross-reactive with an antibody directed against an epitope of naturally occurring GPA.

Therefore, GPA amino acid sequence variants generally will share at least about 75% (preferably greater than 80% and more preferably greater than 90%) sequence identity with the translated amino acid sequence set forth in Figure 1 , after aligning the sequences to provide for maximum homology, as determined, for example, by the Fitch, et al., Proc. Nat.

Acad. Sci. USA £0:1382-1386 (1983), version of the algorithm described by Needleman, et al., J. Mol. Biol. 48:443-453 (1970). Amino acid sequence variants of GPA are prepared by introducing appropriate nucleotide changes into GPA DNA and thereafter expressing the resulting modified DNA in a host cell, or by jn vitro synthesis. Such variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the GPA amino acid sequence set forth in Figure 1 . Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of GPA, provided that such variant possesses the desired characteristics described herein. Changes that are made in the amino acid sequence set forth in Figure 1 to arrive at an amino acid sequence variant of GPA also may result in further modifications of GPA upon its expression in host cells, for example, by virtue of such changes introducing or moving sites of glycosyiation, or introducing membrane anchor sequences as described, for example, in PCT Pat. Pub. No. WO 89/01041 (published February 9, 1989).

There are two principal variables in the construction of amino acid sequence variants of GPA: the location of the mutation site and the nature of the mutation. These are variants from the amino acid sequence set forth in Figure 1 , and may represent naturally occurring allelic forms of GPA, or predetermined mutant forms of GPA made by mutating GPA DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the GPA characteristic to be modified. For example, due to the degeneracy of nucleotide coding sequences, mutations can be made in the GPA nucleotide sequence set forth in Figure 1 without affecting the amino acid sequence of the GPA encoded thereby. Other mutations can be made that will result in a GPA that has an amino acid sequence different from that set forth in Figure 1 , but which is functionally active. Such functionally active amino acid sequence variants of GPA are selected, for example, by substituting one or more amino acid residues in the amino acid sequence set forth in Figure 1 with other amino acid residues of a similar or different polarity or charge.

One useful approach is called "alanine scanning mutagenesis." Here, a an amino acid residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and, by means of recombinant DNA technology, replaced by a neutral or negatively charged amino acid (most preferably alanine or polyaianine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Cunningham, et al., Science 244: 1081 -1085 (1989). Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the sites of substitution. Obviously, such variations that, for example, convert the amino acid sequence set forth in Figure 1 to the amino acid sequence of a known neurotrophic factor, such as NGF, BDNF, NT-3, and CNTF, or another known polypeptide or protein are not included within the scope of this invention, nor are any other fragments, variants, and derivatives of the amino acid GPA that are not novel and unobvious over the prior art. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed GPA variants are screened for functional activity.

Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues, and typically are contiguous. Deletions from regions of substantial homology with CNTF, for example, are more likely to affect the functional activity of GPA. Generally, the number of consecutive deletions will be selected so as to preserve the tertiary structure of GPA in the affected domain, e.g., beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one amino acid residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Iπtrasequence insertions (i.e., insertions made within the amino acid sequence set forth in Figure 1 ) may range generally from about 1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3. Examples of terminal insertions include GPA with an N-terminal methionyl residue (such as may result from the direct expression of GPA in recombinant cell culture), and GPA with a heteroiogous N-termiπal signal sequence to improve the secretion of GPA from recombinant host cells. Such signal sequences generally will be homologous to the host cell used for expression of GPA, and include STII or ipp for E. coli. alpha factor for yeast, and viral signals such as herpes gD for mammalian cells. Other insertions include the fusion to the N- or C- terminus of GPA of immunogenic polypeptides, e.g., bacterial polypeptides such as beta- lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin constant regions, albumin, or ferritin, as described in PCT Pat. Pub. No. WO 89/02922 (published April 6, 1989). The third group of variants are those in which at least one amino acid residue in the amino acid sequence set forth in Figure 1 , and preferably only one, has been removed and a different residue inserted in its place. The sites of greatest interest for making such substitutions are in the regions of the amino acid sequence set forth in Figure 1 that have the greatest homology with CNTF. Those sites are likely to be important to the functional activity of the neurotrophic factors. Accordingly, to retain functional activity, those sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of preferred substitutions. If such substitutions do not result in a change in functional activity, then more substantial changes, denominated exemplary substitutions in Table 1 , or as further described below in reference to amino acid classes, may be introduced and the resulting variant GPA analyzed for functional activity. Original Residue Ala (A) Arg (R) Asn (N) Asp (D) Cys (C) Gin (Q) Glu (E) Gly (G) His (H) lie (I)

Leu (L)

Lys (K) Met (M) Phe (F) Pro (P) Ser (S) Thr (T) Trp (W) Tyr (Y) Val (V)

Insertional, deletional, and substitutional changes in the amino acid sequence set forth in Figure 1 may be made to improve the stability of GPA. For example, trypsin or other protease cleavage sites are identified by inspection of the encoded amino acid sequence for an arginyl or lysinyl residue. These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue. Also, any cysteine residues not involved in maintaining the proper conformation of GPA for functional activity may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

Covalent modifications of GPA molecules also are included within the scope of this invention. For example, covalent modifications are introduced into GPA by reacting targeted amino acid residues of the GPA with an organic derivatizing agent that is capable of reacting with selected amino acid side chains or the N- or C-terminal residues. Cysteinyl residues most commonly are reacted with σ-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, σ-bromo-£-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-riitrobenzo-2-oxa-1 ,3- dϊazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatiziπg σ-amino-containing residues include imidoesters such as methyl picolinimidate; pγridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenyiglγoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK, of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using ^{1 6}l or ¹³¹l to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.

Carboxyl side groups (aspartyl or giutamyl) are selectively modified by reaction with carbodiimϊdes (R'-N = C = N-R'), where R and R' are different alkyl groups, such as 1 - cycIohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and giutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking GPA to a water- insoluble support matrix or surface for use in the method for purifying anti-GPA antibodies, or for therapeutic use. Commonly used crosslinking agents include, e.g., 1 ,1-bis(diazoacetyD-

2-phenyIethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidyipropionate), and bifunctional maleimides such as bis-N-maleimido-1 ,8- octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691 ,01 6; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding giutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the σ-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Creighton, Proteins: Structure and Molecular Properties, pp.79- 86 (W.H. Freeman & Co., 1983). GPA also is covalently linked to nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4, 179,337; 4,301 , 144; 4,496,689; 4,640,835; 4,670,417; or 4,791 ,192.

"Cell," "host cell," "cell line," and "cell culture" are used interchangeably and all such terms should be understood to include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of times the cultures have been passaged. It should also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. "Plasmids" are DNA molecules that are capable of replicating within a host cell, either extrachromosomaliγ or as part of the host cell chromosome(s), and are designated by a lower case "p" preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids as disclosed herein and/or in accordance with published procedures. In certain instances, as will be apparent to the ordinarily skilled artisan, other plasmids known in the art may be used interchangeably with plasmids described herein.

"Control sequences" refers to DNA sequences necessary for the expression of an operably linked nucleotide coding sequence in a particular host cell. The control sequences that are suitable for expression in prokarγotes, for example, include origins of replication, promoters, ribosome binding sites, and transcription termination sites. The control sequences that are suitable for expression in eukaryotes, for example, include origins of replication, promoters, ribosome binding sites, polyadenylation signals, and enhancers. An "exogenous" element is one that is foreign to the host cell, or homologous to the host cell but in a position within the host cell in which the element is ordinarily not found.

"Digestion" of DNA refers to the catalytic cleavage of DNA with an enzyme that acts only at certain Iocations in the DNA. Such enzymes are called restriction enzymes or restriction endonucleases, and the sites within DNA where such enzymes cleave are called restriction sites. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements as established by the enzyme manufacturers are used. Restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters representing the microorganism from which each restriction enzyme originally was obtained and then a number designating the particular enzyme. In general, about 1 μg of DNA is digested with about 1 -2 units of enzyme in about 20 μ\ of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer, and/or are well known in the art.

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest typically is accomplished by separating the digestion products, which are referred to as "restriction fragments," on a polyacrylamide or agarose gel by electrophoresis, identifying the fragment of interest on the basis of its mobility relative to that of marker DNA fragments of known molecular weight, excising the portion of the gel that contains the desired fragment, and separating the DNA from the gel, for example by electroelution. "Ligation" refers to the process of forming phosphodiester bonds between two double- stranded DNA fragments. Unless otherwise specified, ligation is accomplished using_. known buffers and conditions with 10 units of T4 DNA lϊgase per 0.5 / g of approximately equimolar amounts of the DNA fragments to be ligated.

"Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (involving, for example, triester, phosphoramidite, or phosphonate chemistry), such as described by Engels, et al., Agnew.

Chem. Int. Ed. Engl. 2^:716-734 (1989). They are then purified, for example, by polyacrylamide gel electrophoresis.

"Polymerase chain reaction," or "PCR," as used herein generally refers to a method for amplification of a desired nucleotide sequence _n vitro, as described in U.S. Pat. No. 4,683,195. In general, the PCR method involves repeated cycles of primer extension synthesis, using two oligonucleotide primers capable of hybridizing preferentially to a template nucleic acid. Typically, the primers used in the PCR method will be complementary to nucleotide sequences within the template at both ends of or flanking the nucleotide sequence to be amplified, although primers complementary to the nucleotide sequence to be amplified also may be used. Wang, et al., in PCR Protocols, pp.70-75 (Academic Press, 1990); Och aπ, et al., in PCR Protocols, pp. 219-227; Trigtia, et al., Nuc. Acids Res. 16:8186 (1988). "PCR cloning" refers to the use of the PCR method to amplify a specific desired nucleotide sequence that is present amongst the nucleic acids from a suitable cell or tissue source, including total genomic DNA and cDNA transcribed from total cellular RNA. Frohman, et al., Proc. Nat. Acad. Sci. USA ££:8998-9002 (1988); Saiki, et al., Science 2_ϋ_:487-492 (1988); Mullis, et al., Meth. Enzymol. 15^:335-350 (1 987).

"GPA nucleic acid" is RNA or DNA that encodes GPA. "GPA DNA" is DNA that encodes GPA. GPA DNA is obtained from cDNA or genomic DNA libraries, or by jn vitro synthesis. Identification of GPA DNA within a cDNA or a genomic DNA library, or in some other mixture of various DNAs, is conveniently accomplished by the use of an oligonucleotide hybridization probe that is labeled with a detectable moiety, such as a radioisotope. Keller, et al.. DNA Probes, PP.149-213 (Stockton Press, 1989). To identify DNA encoding GPA, the nucleotide sequence of the hybridization probe preferably is selected so that the hybridization probe is capable of hybridizing preferentially to DNA encoding the GPA amino acid sequence set forth in Figure 1 , or a variant or derivative thereof as described herein, under the hybridization conditions chosen. Another method for obtaining GPA nucleic acid is to chemically synthesize it using one of the methods described, for example, by Engels, et al., Agnew. Chem. Int. Ed. Engl. 28:716-734 (1989).

If the entire nucleotide coding sequence for GPA is not obtained in a single cDNA, genomic DNA, or other DNA, as determined, for example, by DNA sequencing or restriction endonuclease analysis, then appropriate DNA fragments (e.g., restriction fragments) may be recovered from several DNAs and covalently joined to one another to construct the entire coding sequence. The preferred means of covalently joining DNA fragments is by ligation using a DNA ligase enzyme, such as T4 DNA ligase.

"Isolated" GPA nucleic acid is GPA nucleic acid that is identified and separated from (or otherwise free from), contaminant nucleic acid encoding other polypeptides. The isolated GPA nucleic acid may be labeled for diagnostic and probe purposes, using a label as described and defined further below in the discussion of diagnostic assays and nucleic acid hybridization methods.

For example, isolated GPA DNA, or a fragment thereof comprising at least about 1 5 nucleotides, is used as a hybridization probe to detect, diagnose, or monitor disorders or diseases that involve changes in GPA expression, such as may result from sensory neuron damage. In one embodiment of the invention, total RNA in a tissue sample from a patient (that is, a human or other mammal) can be assayed for the presence of GPA messenger RNA, wherein the decrease in the amount of GPA messenger RNA is indicative of neuronal degeneration. isolated GPA nucleic acid also is used to produce GPA by recombinant DNA and recombinant cell culture methods. In various embodiments of the invention, host cells are transformed or transfected with recombinant DNA molecules comprising an isolated GPA DNA, to obtain expression of the GPA DNA and thus the production of GPA in large quantities. DNA encoding amino acid sequence variants of GPA is prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants of GPA) or preparation by site-directed (or oligonucieotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding a variant or a non-variant form of GPA.

Site-directed mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of GPA DNA. This technique is well known in the art, Zoller, et al., Meth. Enz. 1Q0.4668-500 (1983); Zoller, et al., Meth. Enz. 15.4:329-350 (1987); Carter,

Meth. Enz. 154:382-403 (1987); Horwitz, et al., Meth. Enz. 185:599-61 1 (1990), and has been used, for example, to produce amino acid sequence variants of trypsin and T4 lysozyme, which variants have certain desired functional properties. Perry, et al.. Science 226:555-557

(1984); Craik, et al.. Science 228:291-297 (1985). Briefly, in carrying out site-directed mutagenesis of GPA DNA, the GPA DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such

GPA DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of GPA

DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

Oligonucleotides for use as hybridization probes or primers may be prepared by any suitable method, such as by purification of a naturally occurring DNA or by jn vitro synthesis. For example, oligonucleotides are readily synthesized using various techniques in organic chemistry, such as described by Narang, et al., Meth. Eπzymol. 6_8:90-98 (1979); Brown, et al., Meth. Enzymol. g£J:109-151 (1979); Caruther, et al., Meth. Enzymol. 1j>4:287-313 (1985). The general approach to selecting a suitable hybridization probe or primer is well known. Keller, et al., DNA Probes, pp.1 1-18 (Stockton Press, 1989). Typically, the hybridization probe or primer will contain 10-25 or more nucleotides, and will include at least 5 nucleotides on either side of the sequence encoding the desired mutation so as to ensure that the oligonucleotide will hybridize preferentially to the single-stranded DNA template molecule.

Multiple mutations are introduced into GPA DNA to produce amino acid sequence variants of GPA comprising several or a combination of insertions, deletions, or substitutions of amino acid residues as compared to the amino acid sequence set forth in Figure 1. If the sites to be mutated are located close together, the mutations may be introduced simultaneously using a single oligonucleotide that encodes all of the desired mutations. If, however, the sites to be mutated are located some distance from each other (separated by more than about ten nucleotides), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.

In the first method, a separate oligonucleotide is generated for each desired mutation. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for introducing a single mutation: a single strand of a previously prepared GPA DNA is used as a template, an oligonucleotide encoding the first desired mutation is annealed to this template, and a heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.

PCR mutagenesis is also suitable for making amino acid sequence variants of GPA. Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990); Vallette, et al., Nuc. Acids Res. 17:723-733 (1989). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, for example, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a nucleotide sequence within the opposite strand of the plasmid DNA, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone. Wagner, et al., in PCR Topics, pp.69-71 (Springer-Verlag, 1991 ).

If the ratio of template to product amplified DNA is extremely low, the majority of product DNA fragments incorporate the desired mutation(s). This product DNA is used to replace the corresponding region in the plasmid that served as PCR template using standard recombinant DNA methods. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer, or performing a second PCR with different mutant primers and ligatϊng the two resulting PCR fragments simultaneously to the plasmid fragment in a three (or more)-part ligation.

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al.. Gene, 2 315-323 (1985). The starting material is the plasmid (or other vector) comprising the GPA DNA to be mutated. The codon(s) in the GPA

DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate Iocations in the GPA DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated GPA DNA sequence.

GPA DNA, whether cDNA or genomic DNA or a product of in vitro synthesis, is ligated into a replicable vector for further cloning or for expression. "Vectors" are plasmids and other DNAs that are capable of replicating autonomously within a host cell, and as such, are useful for performing two functions in conjunction with compatible host cells (a vector-host system) .

One function is to facilitate the cloning of the nucleic acid that encodes the GPA, i.e., to produce usable quantities of the nucleic acid. The other function is to direct the expression of GPA. One or both of these functions are performed by the vector-host system. The vectors will contain different components depending upon the function they are to perform as well as the host cell with which they are to be used for cloning or expression.

To produce GPA, an expression vector will contain nucleic acid that encodes GPA as described above. The GPAs of this invention are expressed directly in recombinant cell culture, or as a fusion with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the junction between the heterologous polypeptide and the GPA.

In one example of recombinant host cell expression, mammalian cells are transfected with an expression vector comprising GPA DNA and the GPA encoded thereby is recovered from the culture medium in which the recombinant host cells are grown. But the expression vectors and methods disclosed herein are suitable for use over a wide range of prokaryotic and eukaryotic organisms.

Prokaryotes may be used for the initial cloning of DNAs and the construction of the vectors useful in the invention. However, prokaryotes may also be used for expression of DNA encoding GPA. Polypeptides that are produced in prokaryotic host cells typically will be non-glycosylated.

Plasmid or viral vectors containing replication origins and other control sequences that are derived from species compatible with the host cell are used in connection with prokaryotic host cells, for cloning or expression of an isolated DNA. For example, E. £θJi typically is transformed using pBR322, a plasmid derived from an E. £oji species. Bolivar, et al., Gene 2:95-1 13 (1987). PBR322 contains genes for ampicillin and tetracycline resistance so that cells transformed by the plasmid can easily be identified or selected. For it to serve as an expression vector, the pBR322 plasmid, or other plasmid or viral vector, must also contain, or be modified to contain, a promoter that functions in the host cell to provide messenger RNA (mRNA) transcripts of a DNA inserted downstream of the promoter. Rangagwala, et al., Bio/Technology 9:477-479 (1991 ).

In addition to prokaryotes, eukaryotic microbes, such as yeast, may also be used as hosts for the cloning or expression of DNAs useful in the invention. Saccharomvces cerevisiae. or common baker's yeast, is the most commonly used eukaryotic microorganism. Plasmids useful for cloning or expression in yeast cells of a desired DNA are well known, as are various promoters that function in yeast cells to produce mRNA transcripts.

Furthermore, cells derived from multiceliular organisms also may be used as hosts for the cloning or expression of DNAs useful in the invention. Mammalian cells are most commonly used, and the procedures for maintaining or propagating such cells jn vitro, which procedures are commonly referred to as tissue culture, are well known. Kruse & Patterson, eds., Tissue Culture (Academic Press, 1977). Examples of useful mammalian cells aretiuman cell lines such as 293, HeLa, and WI-38, monkey cell lines such as COS-7 and VERO, and hamster cell lines such as BHK-21 and CHO, all of which are publicly available from the American Type Culture Collection (ATCC), Rockville, Maryland 20852 USA.

Expression vectors, unlike cloning vectors, should contain a promoter that is recognized by the host organism and is operably linked to the GPA nucleic acid. Promoters are untranslated sequences that are located upstream from the start codon of a gene and that control transcription of the gene (that is, the synthesis of mRNA). Promoters typically fall into two classes, inducibie and constitutive. Inducible promoters are promoters that initiate high level transcription of the DNA under their control in response to some change in culture conditions, for example, the presence or absence of a nutrient or a change in temperature.

A large number of promoters are known, that may be operably linked to GPA DNA to achieve expression of GPA in a host cell. This is not to say that the promoter associated with naturally occurring GPA DNA is not usable. However, heterologous promoters generally will result in greater transcription and higher yields of expressed GPA.

Promoters suitable for use with prokaryotic hosts include the Mactamase and lactose promoters, Goeddel, et al., Nature 281 :544-548 (1 979), tryptophan (trp) promoter, Goeddel, et al., Nuc. Acids Res.1:4057-4074 (1980), and hybrid promoters such as the tac promoter, deBoer, et al., Proc. Natl. Acad. Sci. USA 8_Q_:21 -25 (1983). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, Siebenlist, et al.. Cell 2Q:269-281 (1980), thereby enabling a skilled worker operably to ligate them to DNA encoding GPA using linkers or adaptors to supply any required restriction sites. Wu, et al., Meth. Enz. 152:343-349 (1987).

Suitable promoters for use with yeast hosts include the promoters for 3- phosphoglycerate kinase, Hitzeman, et al., J. Biol. Chem. 255:12073-12080 (1980); Kingsman, et al., Meth. Enz. 185:329-341 (1990), or other glycolytic enzymes such as enofase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxγlase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and giucokinase. Dodson, et al., Nuc. Acids res. 10:2625-2637 (1982); Emr, Meth. Enz. 185:231 -279 (1990).

Expression vectors useful in mammalian cells typically include a promoter derived from a virus. For example, promoters derived from polyoma virus, adenovirus, cytomegalovirus (CMV), and simian virus 40 (SV40) are commonly used. Further, it is also possible, and often desirable, to utilize promoter or other control sequences associated with a naturally occurring DNA that encodes GPA, provided that such control sequences are functional in the particular host cell used for recombinant DNA expression. Other control sequences that are desirable in an expression vector in addition to a promoter are a ribosome binding site, and in the case of an expression vector used with eukaryotic host cells, an enhancer. Enhancers are cis-acτing elements of DNA, usually about from 10-300 bp, that act on a promoter to increase the level of transcription. Many enhancer sequences are now known from mammalian genes (for example, the genes for globiπ, elastase, albumin, σ-fetoprotein and insulin). Typically, however, the enhancer used will be one from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Kriegler, Meth. Enz. J_85_:512-527 (1990). Expression vectors may also contain sequences necessary for the termination of transcription and for stabilizing the messenger RNA (mRNA). Balbas, et al., Meth. Enz. 185:14-37 (1990); Levinson, Meth. Enz. 185:485-51 1 (1990). In the case of expression vectors used with eukaryotic host cells, such transcription termination sequences may be obtained from the untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain polyadenγlation sites as well as transcription termination sites. Birnsteil, et al.. Cell 41:349-359 (1985).

In general, control sequences are DNA sequences necessary for the expression of an operably liked coding sequence in a particular host cell. "Expression" refers to transcription and/or translation. "Operably linked" refers to the covalent joining of two or more DNA sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.

Expression and cloning vectors also will contain a sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosome(s), and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (for example, from SV40, polyoma, or adenovirus) are useful for cloning vectors in mammalian cells. Most expression vectors are "shuttle" vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector may be cloned in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome. The expression vector may also include an ampiifiabie gene, such as that comprising the coding sequence for dihydrofolate reductase (DHFR). Cells containing an expression vector that includes a DHFR gene may be cultured in the presence of methotrexate, a competitive antagonist of DHFR. This leads to the synthesis of multiple copies of the DHFR gene and, concomitantly, multiple copies of other DNA sequences comprising the expression vector, Ringold, et al., J. Mol. Apl. Genet. 1:165-175 (1981 ), such as a DNA sequence encoding GPA. In that manner, the level of GPA produced by the cells may be increased.

DHFR protein encoded by the expression vector also may be used as a selectable marker of successful transfection. For example, if the host cell prior to transformation is lacking in DHFR activity, successful transformation by an expression vector comprising DNA sequences encoding GPA and DHFR protein can be determined by cell growth in medium containing methotrexate. Also, mammalian cells transformed by an expression vector comprising DNA sequences encoding GPA, DHFR protein, and aminoglycoside 3' phosphotransferase (APH) can be determined by cell growth in medium containing an aminoglycoside antibiotic such as kanamycin or neomyciπ. Because eukaryotic cells do not normally express an endogenous APH activity, genes encoding APH protein, commonly referred to as neo^r genes, may be used as dominant selectable markers in a wide range of eukaryotic host cells, by which cells transfected by the vector can easily be identified or selected. Jiminez, et al.. Nature, 287:869-871 (1980); Colbere-Garapin, et al., J. Mol. Biol. 150:1-14 (1981 ); Okayama & Berg, Mol. Cell. Biol., 2=280-289 (1983).

Many other selectable markers are known that may be used for identifying and isolating recombinant host cells that express GPA. For example, a suitable selection marker for use in yeast is the trpl gene present in the yeast plasmid YRp7. Stinchcomb, et al., Nature 2§2:39-43 (1979); Kingsman, et al., Gene 2:141-152 (1979); Tschemper, et al.. Gene 1jQ:157-166 (1980). The trp 1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (available from the American Type Culture Collection, Rockville, Maryland 20852 USA). Jones, Genetics 8_5:12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC Nos. 20622 or 38626) are complemented by known plasmids bearing the Leu2 gene.

Particularly useful in the invention are expression vectors that provide for the transient expression in mammalian cells of DNA encoding GPA. in general, transient expression involves the use of an expression vector that is able to efficiently replicate in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient expression systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties. Yang, et al., Cell 42:3-10 (1986); Wong, et al., Science 228:810-815 (1985); Lee, et al., Proc. Nat Acad. Sci. USA 82:4360-4364 (1985). Thus, transient expression systems are particularly useful in the invention for expressing DNAs encoding amino acid sequence variants of GPA, to identify those variants which are functionally active. Since it is often difficult to predict in advance the characteristics of an amino acid sequence variant of GPA, it will be appreciated that some screening of such variants will be needed to identify those that are functionally active. Such screening may be performed vitro, using routine assays for neuronal survival, Eckenstein, et al.. Neuron 4:623-631 (1990), or using immunoassays with monoclonal antibodies that selectively bind to GPA that is functionally active GPA, such as a monoclonal antibody that selectively binds to the active site or receptor binding site of GPA.

In a preferred embodiment of the invention, GPA expression in mammalian host cells is accomplished by the use of an expression vector comprising the oriP origin of replication from Epstein-Barr virus (EBV) and a host cell that is transformed with and constitutively expresses the EBNA-1 gene of EBV.

Plasmids containing the oriP sequence of EBV are able to replicate in EBV-transformed host cells that express the EBV nuclear antigen (EBNA-1 ). Sugden, et al., J. Mol. cell. Biol. 5:410-413 (1985); Reisman, et al., Mol. Cell Biol.5:1822-1832 (1985); Yates, et al., Proc. Nat. Acad. Sci. 81 :3806-3810 (1984). The expression of GPA that is described in the Examples below involves the use of a plasmid expression vector (pHEBO30) containing the oriP region from EBV, and a cell line (CEN4) that constitutively expresses EBNA-1 . Specifically, pHEBO30 comprises the strong CMV promoter, a multiple cloning region for insertion of foreign (exogenous) genes downstream of the CMV promoter, the oriP region of

EBV for plasmid replication in host cells expressing EBNA-1 (for example, CEN4), a hygromycin resistance gene for selection in eukaryotes, the origin of replication from pBR322 for replication in prokaryotes, and an ampicillin resistance gene for selection in prokaryotes.

Upon transfection, pHEBO30 (including recombinant derivatives thereof) is stably maintained as an episome in the nuclei of host cells expressing EBNA-1 . Unexpectedly, the efficiency of stable transfection of such host cells with pHEBO30 is several fold greater than obtained with a plasmid containing both the oriP region and the EBNA-1 gene from EBV. In general, the efficiency of stable transfection of CEN4 cells with pHEBO30 is from about 5% to 25% or more. Further advantages of pHEBO30 and CEN4 include: (1 ) The level of transient expression in CEN4 cells of foreign genes cloned in pHEBO30 (for example, the genes encoding human tissue-type plasminogen activator (tPA) and human soluble alkaline phosphatase) is several fold higher than obtained with expression vectors lacking the oriP from EBV; (2) Stable expression in CEN4 cells of foreign genes cloned in pHEBO30 can be maintained for four months or more with appropriate selection (for example, hygromycin selection); and (3) pHEB030 and recombinant derivatives thereof are readily recovered from transfected cells, for analysis or modification.

As used herein, the terms "transformation" and "transfection" refer to the process of introducing a desired nucleic acid, such a plasmid or an expression vector, into a host cell. Various methods of transformation and transfection are available, depending on the nature of the host cell. In the case of E. c_2lj cells, the most common methods involve treating the cells with aqueous solutions of calcium chloride and other salts. In the case of mammalian cells, the most common methods are transfection mediated by either calcium phosphate or DEAE-dextran, or electroporation. Sambrook, et al., eds., Molecular Cloning, pp. 1 .74-1 .84 and 16.30-16.55 (Cold Spring Harbor Laboratory Press, 1989). Following transformation or transfection, the desired nucleic acid may integrate into the host cell genome, or may exist as an extrachromosomal element.

Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype. The culture conditions, such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent those skilled in the art. Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, vertebrate, and mammalian host cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli. Bacillus species such as j|. subtilis, Pseudomonas species such as P. aeruoinosa. Salmonella vohimurium. or Serratia marcescans. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for GPA-encoding vectors. Saccharomvces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomvces pombe. Beach and Nurse, Nature 290:140-142 (1981 ), Pichia pastoris. Cregg, et al., Bio Technology 5:479-485 (1987); Sreekrishna, et al., Biochemistry 28:41 17-4125 (1989), Neurospora crassa. Case, et al., Proc. Natl. Acad. Sci. USA 76:5259- 5263 (1979), and Aspergillus hosts such as A. nidulans. Ballance, et al., Biochem. Biophys. Res. Commun. 112:284-289 (1983); Tilburn, et al., Gene 2g:205-221 (1983); Yelton, et al., Proc. Natl. Acad. Sci. USA 51:1470-1474 (1984), and A. niger, Kelly, et al., EMBO J. 4:475-479 (1985).

Suitable host cells for the expression of GPA also are derived from ππulticellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture. It will be appreciated, however, that because of the species-, tissue-, and cell-specificity of glycosylation, Rademacher, et al., Ann. Rev. Biochem. 57:785-838 (1988), the extent or pattern of glycosylation of GPA in a foreign host cell typically will differ from that of GPA obtained from a cell in which it is naturally expressed.

Examples of invertebrate cells include insect and plant cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera fruoiperda (caterpillar), Aedes aeovpti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombvx mori host cells have been identified. Luckow, et al., Bio Technology 5:47-55 (1988); Miller, et al., in Genetic Engineering, vol. 8, pp.277- 279 (Plenum Publishing, 1986); Maeda, et al.. Nature 315:592-594 (1985).

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens. which has been previously altered to contain

GPA DNA. During incubation of the plant cells with A. tumefaciens. the DNA encoding the

GPA is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the GPA DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences, and the ribulose biphosphate carboxylase promoter. Depicker, et al., J.

Mol. Appl. Gen. 1:561 -573 (1982). Herrera-Estrella, et al., Nature 21 :1 15-120 (1984). In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. European Pat. Pub. No. EP 321 ,196 (published

June 21 , 1989).

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Kruse &

Patterson, eds., Tissue Culture (Academic Press, 1973). Examples of useful mammalian host cells are the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line 293 (or 293 cells subcioned for growth in suspension culture),

Graham, et al., J. Gen Virol. 25:59-72 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (including DHFR-deficient CHO cells, Urlaub, et al., Proc.

Natl. Acad. Sci. USA 77:4216-4220 (1980); mouse sertoli cells (TM4, Mather, Biol. Reprod.

23:243-251 (1980); monkey kidney cells (CV1 , ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL

2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung ce'-. (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor ( MT 060562, ATCC CCL51 ); TRI cells (Mather, et al., Annals N.Y.

Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep

G2).

Construction of suitable vectors containing the nucleotide sequence encoding GPA and appropriate control sequences employs standard recombinant DNA methods. DNA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required.

For analysis to confirm correct sequences in the vectors constructed, the vectors are analyzed by restriction digestion (to confirm the presence in the vector of predicted restriction endonuclease) and/or by sequencing by the dideoxy chain termination method of Sanger, et al., Proc. Nat. Acad. Sci. USA 72:3918-3921 (1979).

The mammalian host cells used to produce the GPA of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal

Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham, et al., Meth. Enz. 52:44-93 (1979); Barnes, et al., Anal. Biochem.

102:255-270 (1980); Bottenstein, et al., Meth. Enz. 58:94-109 (1 979); U.S. Pat. Nos.

4,560,655; 4,657,866; 4,767,704; or 4,927,762; or in PCT Pat. Pub. Nos. WO 90/03430 (published April 5, 1990), may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromoiar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in culture jn vitro as well as cells that are within a host animal, for example, as a result of transplantation or implantation.

It is further envisioned that the GPA of this invention may be produced by homologous recombination, for example, as described in PCT Pat. Pub. No. WO 91 /06667 (published May

16, 1991 ). Briefly, this method involves transforming cells containing an endogenous gene encoding GPA with a homologous DNA, which homologous DNA comprises (1 ) an amplifiable gene, such as DHFR, and (2) at least one flanking sequence, having a length of at least about

150 base pairs, which is homologous with a nucleotide sequence in the cell genome that is within or in proximity to the gene encoding GPA. The transformation is carried out under conditions such that the homologous DNA integrates into the cell genome by recombination.

Cells having integrated the homologous DNA then are subjected to conditions which select for amplification of the amplifiable gene, whereby the GPA gene amplified concomitantly.

The resulting cells then are screened for production of desired amounts of GPA. Flanking sequences that are in proximity to a gene encoding GPA are readily identified, for example, by the method of genomic walking, using as a starting point the GPA nucleotide sequence set forth in Figure 1. Spoerel, et al., Meth. Enz. 152:598-603 (1987).

Gene amplification and/or gene expression may be measured in a sample directly, for example, by conventional Southern blotting to quantitate DNA, or Northern blotting to quantitate mRNA, using an appropriately labeled oligonucleotide hybridization probe, based on the sequences provided herein. Various labels may be employed, most commonly radioisotopes, particularly ³²P. However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polγnucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radioisotopes, fluorophores, chromophores, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of the gene product, GPA. With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like. A particularly sensitive staining technique suitable for use in the present invention is described by Hsu, et al., Am. J. Clin. Path., 75:734-738 (1980). Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or poiyclonal. Conveniently, the antibodies may be prepared against a synthetic peptide based on the DNA sequences provided herein.

GPA preferably is recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates. To obtain GPA that is substantially free of contaminating proteins or polypeptides of the host cell in which it is produced it is necessary to purify the GPA, based on the differential physical properties of GPA as compared to the contaminants with which it may be associated. For example, as a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. GPA thereafter is purified from contaminant soluble proteins and polypeptides, for example, by ammonium sulfate or ethanol precipitation, gel filtration (molecular exclusion chromatography), ion- exchange chromatography, immunoaffinity chromatography, reverse phase HPLC, and/or gel electrophoresis.

Amino acid sequence variants and derivatives of GPA are recovered in the same fashion, taking account of any distinguishing features or physical properties of the particular GPA. For example, in the case of a fusion protein comprising GPA and another protein or polypeptide, such as a bacterial or viral antigen, a significant degree of purification may be obtained by using an immunoaffinity column containing antibody to the antigen. In any event, the ordinarily skilled artisan will appreciate that purification methods suitable for naturally occurring GPA may require modification to account for changes in the character of GPA or its variants or derivatives produced in recombinant host cells.

The purity of GPA produced according to the present invention is determined according to methods well known in the art, such as by analytical sodium dodecyl sulfate (SDS) gel electrophoresis, immunoassaγ, or amino acid composition or sequence analysis electrophoresis. Preferably, the GPA is purified to such an extent that it is substantially free of other proteins. For therapeutic uses, the purified GPA will be greater than 99% GPA and, accordingly, non-GPA proteins will comprise less than 1 % of the total protein in the purified GPA composition. GPA may be used as an immunogen to generate aπti-GPA antibodies. Such antibodies, which specifically bind to GPA, are useful as standards in assays for GPA, such as by labeling purified GPA for use as a standard in a radioimmunoassay, enzyme-linked immunoassay, or competitive-type receptor binding assays radioreceptor assay, as well as in affinity purification techniques.

Poiyclonal antibodies directed toward GPA generally are raised in animals by multiple subcutaneous or intraperitoneal injections of GPA and an adjuvant. It may be useful to conjugate GPA or a peptide fragment thereof to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, for example, maleimidobenzoγl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimϊde (conjugation through lysine residues), glutaraldehyde, succinic anhydride, S0CI₂, or R'N = C = NR, where R and R¹ are different alkyl groups.

Animals are immunized with such GPA-carrier protein conjugates combining 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1 /5th to 1 /10th the original amount of conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later animals are bled and the serum is assayed for anti-GPA antibody titer. Animals are boosted until the antibody titer plateaus. Preferably, the animal is boosted by injection with a conjugate of the same GPA with a different carrier protein and/or through a different cross-linking agent. Conjugates of GPA and a suitable carrier protein also can be made in recombinant cell -culture as fusion proteins. Also, aggregating agents such as alum are used to enhance the immune response. Monoclonal antibodies directed toward GPA are produced using any method which provides for the production of antibody molecules by continuous cell lines in culture. Examples of such methods include the original hybridoma method of Kohler, et al.. Nature 256:495-497 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133:3001 (1984); Brodeur, et al.. Monoclonal Antibodv Production Technioues and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987).

For diagnostic applications, anti-GPA antibodies typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radϊoisotope, such as ³H, ¹⁴C, ³²P, ^3BS, or ¹²⁵l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocγanate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., ^t26l, ³²P, ^WC, or ³H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by David, et al., Biochemistry 12:1014-1021 (1974); Pain, et al., J. Immunol. Meth. 4Q:219-231 (1981 ); and Bayer, et al., Meth. Enz. 1_8_4:138-163 (1990). The anti-GPA antibodies may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Technioues. pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard (e.g., GPA or an immunologically reactive portion thereof) to compete with the test sample analyte (GPA) for binding with a limited amount of antibody. The amount of GPA in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex. David, et al., U.S. Pat No. 4,376, 1 10. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunogiobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

The anti-GPA antibodies of the invention also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety is administered to a host, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of various neurological disorders. The antibody may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

GPA is believed to be useful in promoting the development, maintenance, or regeneration of neurons jn vivo, including ciliary, sensory, and sympathetic neurons. Accordingly, GPA may be utilized in methods for the diagnosis and/or treatment of a variety of neurologic diseases and disorders.

In various embodiments of the invention, purified GPA can be administered to patients in whom the nervous system has been damaged by trauma, surgery, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, or toxic agents, to promote the survival or growth of neurons. For example, GPA can be used to promote the survival or growth of motorneurons that are damaged by trauma or surgery. Also, GPA can be used to treat motorneuron disorders, such as amyotrophic lateral sclerosis (Lou Gehrig's disease), Bell's palsy, and various conditions involving spinal muscular atrophy, or paralysis. Further, GPA can be used to treat human neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, and Down's Syndrome.

In still further embodiments of the invention, antibodies directed toward GPA can be administered to patients suffering from neurologic diseases and disorders characterized by excessive production of GPA. Anti-GPA antibodies can be used in the prevention of aberrant regeneration of sensory neurons such as may occur post-operatively, or in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.

Therapeutic formulations of GPA and anti-GPA antibodies for treating neurologic diseases and disorders are prepared by mixing GPA or anti-GPA antibody, having the desired degree of purity, with optional physiologically acceptable carriers, excipients, or stabilizers which are well known. Acceptable carriers, excipients or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpγrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; -and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

It may be desirable to adsorb GPA onto a membrane, such as a silastic membrane, which can be implanted in proximity to damaged neural tissue, or to complex GPA with liposomes. PCT Pat. Pub. No. WO 91 /04014 (published April 4, 1991 ).

GPA optionally is combined with or administered in concert with other neurotrophic factors to achieve a desired therapeutic effect. For example, GPA may be used together with NGF or BDNF or another neurotrophic factor to achieve a synergistic stimulatory effect on the growth of sensory neurons, wherein the term "synergistic" means that the effect of the combination of GPA with a second neurotrophic factor is greater than that achieved with either substance used individually.

GPA and anti-GPA antibodies to be used for in vivo administration must be sterile. This is readily accomplished by filtration of a solution of GPA or anti-GPA antibody through sterile filtration membranes. Thereafter, the filtered solution may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The filtered solution also may be lyophiiized to produce sterile GPA or anti-GPA antibody in a powder form. Methods for administering GPA and anti-GPA antibodies _n vivo include injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, and by means of sustained-release formulations.

Sustained-release formulations generally consist of GPA or anti-GPA antibodies and a matrix from which the GPA or anti-GPA antibodies are released over some period of time. Suitable matrices include semipermeable polymer matrices in the form of shaped articles, for example, membranes, fibers, or microcapsules. Sustained release matrices may comprise polyesters, hydrogeis, polylactides, U.S. Pat. No. 3,773,919, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, Sidman, et al., Biopolymers, __2: 547-556 (1983), poly (2- hydroxyethyl-methacrylate), or ethylene vinyl acetate, Langer, et al., J. Biomed. Mater. Res. 1 : 167-277 (1981 ); Langer, Chem. Tech. 12:98-105 (1982).

In one embodiment of the invention, the sustained release formulation comprises GPA or anti-GPA antibodies entrapped within or complexed with liposomes. In a further embodiment, the sustained release formulation comprises cells actively producing GPA or anti-GPA antibodies. Such cells may be directly introduced into the tissue of a patient, or may be encapsulated within porous membranes which are then implanted in a patient, in either case providing for the delivery of GPA or anti-GPA antibody into areas within the body of the patient in need of increased or decreased concentrations of GPA. An effective amount of GPA or anti-GPA antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 1 g/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Where possible, it is desirable to determine appropriate dosage ranges first in vitro, for example, using assays for neuronal cell survival or growth which are known in the art, and then in suitable animal models, from which dosage ranges for human patients may be extrapolated. Based on the use of GPA in such in vitro assays, Eckenstein, et al., Neuron 4:623-631 (1990), in a specific embodiment of the invention, a pharmaceutical composition effective in promoting the survival or growth of neurons will provide a local GPA concentration in vivo of between about 0.1 and 10 ng/ml.

In summary, by providing nucleic acid molecules encoding GPA, the present invention enables for the first time the production of GPA by recombinant DNA methods, thus providing a reliable source of sufficient quantities of GPA for use in various diagnostic and therapeutic applications. In view of its distinct biological properties, purified recombinant GPA will be especially useful in a variety of circumstances where it is necessary or desirable to assure neuronal growth and survival, but where other neurotrophic factors either cannot be used or are ineffective. The following examples are offered by way of illustration only and are not intended to limit the invention in any manner.

Examples

1. Construction of Λ-HEBO vector The mammalian expression plasmid pRK.CXRHN, Leung, et al.. Science 45:1306-

1309 (1989), was used as a starting material for construction of the Λ-HEBO vector. pRK.CXRHN contains an Sfii restriction endonuclease site downstream of its multiple cloning region. An additional Sfil site was introduced into the multiple cloning region by inserting between the Clal and NotI restriction endonuclease sites a synthetic DNA having the sequence:

5' CGATTGGCCTTGGTGGCCTAAGCTTGC 3' [SEQ ID NO:7]

3' TAACCGGAACCACCGGATTCGAACGCCGG 5' [SEQ ID NO:8].

The resulting plasmid then was cleaved with Spel and HinPI restriction endonucleases, and the 1 1 10 base pair restriction fragment was isolated. That restriction fragment, which comprises the entire expression unit from pRK.CXRHN, including the CMV promoter, two Sfil sites for efficient cDNA cloning, and the SV40 polγ-adenylation sequence for transcription termination, was inserted at the unique Xbal restriction endonuclease site of the plasmid

P220.2.

Plasmid p220.2 was obtained from Dr. William Sugden, McCardle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin USA. p220.2 is a derivative of the plasmid p201 , Yates, et al., Nature 313:812-815 (1985), constructed by inserting the multiple cloning region from pUC12 (containing BamHi, Xbal, Sail, and Hindlll restriction endonuclease sites) at the unique Narl site within the herpes simplex virus (HSV) thymidine kinase (tk) terminator sequence of p201. Although p220.2 should contain a Narl restriction endonuclease site, it was determined both by restriction analysis and DNA sequence analysis that the particular p220.2 cloned used in this instance did not. Therefore, the above 1 1 10 base pair Spel-HϊnPI restriction fragment was joined to Xbal digested p220.2 by way of legitimate ligation of Spel end of the restriction fragment to one Xbal end of the linearized p220.2 plasmid, and forced ligation of the HinPI end of the restriction fragment to the other Xbal end of that plasmid. The resulting plasmid, referred to as pHEB02, contains the CMV promoter, the EB A1 gene and oriP region of Epstein-Barr virus, and a hygromycin resistance gene, and the pML sequence for replication and selection in E. coli.

Finally, the Λ-HEBO vector was constructed by inserting BamHI linearized pHEB02 DNA at the BamHI site of the Λ-DASHII vector (Stratagene, Inc., La Jolla, California USA).

2. Preparation of cDNA library

Because of the rarity of naturally occurring GPA, an important first step toward isolating nucleic acid encoding GPA was to determine which tissue source, if any, contained a relatively greater amount of GPA protein as compared to other tissue sources. It was hoped that a tissue source enriched in GPA also would be enriched in nucleic acid encoding GPA.

Based on the finding that the eyes of embryonic day 15-17 chickens contain relatively greater amounts of GPA than other embryonic chicken tissues, eyes from embryonic day 15 chicken were used as a source for preparing a cDNA library.

To obtain messenger RNA, eyes from embryonic day 15 (E15) chicken embryos were dissected and the vitreous humor was removed, then the eyes were stored at -70° C. before further processing. Total RNA was prepared from 3 gms. of eye tissue (approximately 30 eyes) essentially as described by Cathala, et al., DNA 2:329-335 (1983). The yield of total RNA was approximately 2.8 mgs. The size distribution of the total RNA was analyzed by electrophoresis of an aliquot of the total RNA on a formaldehyde-agarose gel. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., pp.7.43-7.45 (Cold Spring Harbor Laboratory Press, 1989).

Poly(A) ⁺ RNA (messenger RNA) was isolated cDNA was selected by affinity chromatography of total RNA on a column of oligo(dT)-cellulose essentially as described, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., pp.7.26-7.29 (Cold Spring Harbor Laboratory Press, 1989), except that poly(A)^* RNA that was eluted from the column was passed over the column a second time, for a total of two cycles of selection. Approximately 5% of the total RNA was recovered as poly(A)⁺ RNA. cDNA was prepared by reverse transcription of the poly(A) ⁺ RNA using an oiigo-dT primer and a reverse transcriptase that lacks RNase H activity (Bethesda Research Laboratories, Gaithersburg, Maryland). The reaction conditions for the first and second strand synthesis were as specified by the manufacturer.

The resulting double-stranded cDNAs were ligated with a molar excess of 5' phosphorylated Sfil restriction endonuclease adaptors having the sequence:

5' TGGCCAGCTGAGCTCACCTGC 3' [SEQ ID NO:9] 3' ACCACCGGTCGACTCGAGTGGACG 5' [SEQ ID NO:10].

The cDNAs with cohesive Sfil ends were fractionated by polyacrylamide gel electrophoresis, and those cDNAs greater than 600 base pairs in length ends were eluted from the gel and ligated to Sfil digested Λ-HEBO vector.

3. Screening of cDNA library

Repeated attempts to identify and isolate GPA cDNA by using the polymerase chain reaction (PCR) nucleic acid amplification method were unsuccessful.

Instead, to identify and isolate GPA cDNA, it was necessary to screen the CE15 library by low-stringency hybridization, using multiple oligonucleotide probes. The oligonucleotide probes were designed on the basis of partial amino acid sequences of GPA obtained by microsequencing of three different peptide fragments of purified GPA protein. Three oiigonucleotides, referred to as o-GPA-1 , o-GPA-2, and o-GPA-3, having the following sequences:

5' GACAACCTGGCTGCCTACCGCGCCTTCCGCACCCTGTT 3'

[SEQ ID N0:11 ]

5' ATGCTGCTGCAGGTGTCTGCCTTCGTGTACCACCTGGA 3'

[SEQ ID NO:12]

5' AGGGTGCTGCGAGAGCTGGCACAGTGGGCTGTGAGGTCTGT 3' [SEQ ID NO:13] were radiolabeled at their 5' ends using -³²P-ATP and T4 polynucleotide kinase for use as probes in screening the chick El 5 cDNA library.

Screening of the library was accomplished by filter hybridization, essentially as described by Wahl, et al., Meth. Enzymol. 152:415-423 (1987). Triplicate filters containing DNA derived from Λ phage plaques were prehybridized at 42° C. for 5 hours in 20% formamide, 6X SSC (1 X SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5X

Denhardt's solution (1X Denhardt's solution is 0.02% Ficoll, 0.02% bovine serum albumin, and 0.02% polyvinyl-pyrrolidone), 0.1 % SDS (sodium dodecyl sulfate), and 50 //g/ml salmon sperm DNA. Each of the radiolabeled probes, o-GPA-1 , o-GPA-2, and o-GPA-3, then was separately hybridized to the filters. Hybridization was carried out in the same buffer as used for prehybridization. After hybridization, filters were washed in 1 X SSC at 42° C, and autoradiographed.

Of the approximately 2 x 10" lambda-phage clones from the chick E15 cDNA library that were screened, six clones were identified that hybridized with at least one of the three probes. When those six clones were re-screened, only one, referred to as ΛCE15 #19 hybridized with all three probes.

4. Nucleotide Seouencino of GPA cDNA

Nucleotide sequencing of the cDNA insert in the ΛCE15 #19 clone was accomplished by the dideoxy chain termination method of Sanger, et al., Proc. Nat. Acad. Sci. USA 72:3918-3921 (1979), but with several modifications. Specifically, various anomalies in the electrophoretic mobility of the DNA sequencing fragments that were initially encountered were overcome by the use of the single-stranded binding protein gp32, Schwarz, et al., Nuc.

Acids Res. 18:1079 (1990), and the nucleotide analogs 7-deaza-dGTP, Mizusawa, etal., Nuc.

Acids Res.14 1319-1324 (1986), and 7-deaza-dATP, Jensen, et al., J. DNA Sequencing and Mapping 1:233-239 (1991 ), in the sequencing reactions.

5. Construction of pHEB02-GPA

ΛCE15 #19 DNA was digested with the restriction endonuclease NotI, thereby generating a restriction fragment comprising the complete nucleotide coding sequence for

GPA flanked by nucleotide sequences of the pHEB02 plasmid. That fragment was circularized by ligation with T4 DNA ligase to generate the plasmid pHEB02-GPA. 6. Cloning and Expression of GPA DNA in E. coli

The complete nucleotide coding sequence for GPA was excised from pHEB02-GPA by complete digestion of that plasmid DNA with restriction endonuclease Ncol and partial digestion with restriction endonuclease Espl. The Ncol-Espl DNA fragment comprising the complete coding sequence for GPA was modified by iigating to the Ncol cohesive-end of the fragment a Xbal-Ncol adaptor having the sequence:

5' CTAGGAGCTTTTG 3' [SEQ ID NO: 14] 3' CTCGAAAACGTAC 5' [SEQ ID NO: 15], which Xbal-Ncol adaptor includes a ribosome binding site (Shine-Dalgarno sequence), and by Iigating to the Espl cohesive-end of the fragment a Espl-Bglll adaptor having the sequence:

5' TTAGAATTCGCGATT 3' [SEQ ID NO:16]

3' CTTAAGCGCTAACTAG 5' [SEQ ID NO: 17], thereby producing a Xbal-Bglll DNA fragment encoding GPA.

Next, plasmid pHGH207-1 (described in U.S. Patent No. 4,551 ,433), which contains the human growth hormone gene under the control of a trp promoter-operator, was digested with restriction endonucleases Xbal and Bglll to release the human growth hormone gene.

The Xbal site in pHGH207-1 is immediately downstream of the trp promoter-operator. The above Xbal-Bglll DNA fragment encoding GPA then was joined to the remaining restriction DNA fragment of pHGH207-1 by ligation, thereby effectively substituting the GPA nucleotide coding sequence for the human growth hormone gene.

The resulting plasmid, referred to as ptrp-GPA, thus contains the complete GPA nucleotide coding sequence downstream of and in the correct orientation for expression under the control of the trp promoter of pHGH207-1 and the ribosome binding site provided by the above Xbal-Ncol adaptor. E. coli DH1 OB cells (BRL/Gibco, Gaithersburg, Maryland 20877 USA) were transformed with ptrp-GPA according to the method of Chung, et al., Proc. Nat. Acad. Sci. 86:2172-2175 (1989). Expression of the cloned GPA DNA in the transformed E. coli cells was confirmed by polyacrylamide gel electrophoresis of total cellular protein. Transformed E. coli cells, but not untransformed E. coli cells, contained a protein of the size expected for GPA, approximately 21 , 500 Daltons .

7. Construction of pHEB030 As indicated above, the plasmid pHEB02 contains the EBNA1 gene and the oriP region of Epstein-Barr virus. A plasmid containing these two viral elements can replicate autosomally in the nucleus of a host cell. Because the function of the EBNA1 gene can be provided by a cell that expresses EBNA1 constitutively, it was possible to reduce the size of the plasmid pHEB02 by removing an 1840 base pair EcoNI-SgrAI restriction fragment from pHEB02 that spans most of the EBNA1 gene. Specifically, pHEB02 DNA was digested with the restriction endonucleases EcoNI and SgrAI and the cohesive ends of the larger resulting restriction fragment were filled-in with Klenow DNA polymerase I in the presence of all four deoxyribonucleotides. The resulting blunt-end fragment then was circularized by ligation with

T4 DNA ligase to generate the plasmid pHEBO20.

To establish that cDNAs can be efficiently cloned in pHEB020, cDNA fragments prepared from rat atrial mRNA were joined to Sfil adaptors having the sequence:

5' TGGCCAGCTGAGCTCACCTGC 3' [SEQID NO:18] 3' ACCACCGGTCGACTCGAGTGGACG 5' [SEQ ID NO:19], and the resulting cDNAs having Sfil cohesive ends were joined to Sfil linearized pHEBO20 DNA by ligation with T4 DNA ligase. One of the resulting clones, consisting of an arbitrary

350 base pair cDNA insert at the Sfil site of pHEBO20, is referred to as pHEBO30.

8. Construction of PCEN1

The starting plasmids for the construction of pCEN1 were pRK.CXRHN, pHEB02, pKAN2, and pSW2.RXR. pRK.CXRHN and pHEB02 are described above. pKAN2 is described in Yates, et al., Proc. Nat. Acad. Sci 81 :3806-3810 (1984), and was obtained from Dr.

William Sugden, McCardle Laboratory for Cancer Research, University of Wisconsin, Madison,

Wisconsin USA. pSW2.RXR was derived from pRK.CXRHN by deleting three small regions within the pUCI 18-derived sequence of pRK.CXRHN that are not essential for replication and selection of the plasmid in E. £2il -ⁿd by introducing an EcoRI site upstream of the M13 intergenic region of pRK.CXRHN, using PCR mutagenesis. Higuchi, in PCR Protocols, pp.177-

183 (Academic Press, 1990). pRK.CXRHN DNA was digested with the restriction endonuclease Xhol, and the resulting Xhol cohesive ends were filled-in with Klenow DNA polymerase in the presence of all four deoxyribonucleotides. The blunt end DNA then was digested with the restriction endonuclease Spel, and a 900 base pair Spel-Xhol' restriction fragment, comprising the CMV promoter of pRK.CXRHN, was isolated. pHEB02 DNA was digested with the restriction endonuclease Hinfl, and the resulting

Hinfl cohesive ends were filled-in with Klenow DNA polymerase in the presence of all four deoxyribonucleotides. The blunt end DNA then was digested with the restriction endonuclease Mrol, and a 215 base pair Hinfl'-Mrol restriction fragment, comprising the N- terminal coding sequence of the EBNA1 gene, was isolated.

The 900 base pair Spel-Xhol' restriction fragment (from pRK.CXRHN) and the 215 base pair Hinfl'-Mrol restriction fragment (from pHEB02> then were joined by ligation with T4 DNA ligase, to generate a 1 1 15 base pair Spel-Mrol DNA fragment. The 11 15 base pair Spel-Mrol DNA fragment was combined with (1 ) the 4290 base pair Mrol-BamHI restriction fragment from pHEB02 which comprises the remainder of the

EBNA1 gene (i.e., that portion of the EBNA1 gene not present in the above 215 base pair

Hinfl'-Mrol restriction fragment) and oriP, (2) the 1600 base pair BamHI-EcoRI restriction fragment from pKan2 which comprises the G418 resistance gene, and (3) the 2900 base pair EcoRI-Spel restriction fragment from pSW.RXR which comprises the elements necessary for replication and selection in E. coli. and those four DNA fragments were joined together by ligation with T4 DNA ligase to generate the plasmid pC.EBNA. To delete oriP, pC.EBNA was digested with the restriction endonucleases Accl and

BamHI. The resulting cohesive ends were filled-in with Klenow DNA polymerase in the presence of all four deoxyribonucleotides, then the larger restriction fragment was isolated, and circularized by ligation with T4 DNA ligase, to generate the plasmid p.CENI .

9. Preparation of the cell line CEN4 The cell line CEN4 is a derivative of the human embryonic kidney cell line 293, which was prepared by stably integrating into the genome of 293 cells the plasmid pCEN1 . pCEN1 DNA was linearized by digestion with the restriction endonuclease Seal (the unique Seal site in pCEN1 occurs within the ampicillin resistance gene). The linearized plasmid DNA then was transfected into 293 cells by electroporation. Potter, et al., Proc. Nat. Acad. Sci. 51:7161 -7165 (1984). Linearizing the pCEN1 DNA increased the frequency of stable integration of the plasmid DNA into the cell chromosomal DNA.

To identify transfected cells which had incorporated the pCEN1 DNA, the cells were grown in the presence of neomycin, and six neomycin resistant clones were selected for further study. Specifically, pHEB0.20 was transfected into ceils of each of the six clones by electroporation, and in each case the efficiency of transfection was determined by counting the number of hygromycin resistant colonies obtained. Because pHEBO20 lacks the EBNA1 gene, but requires the EBNA1 gene product for replication from oriP, efficient transfection depends upon the host cell being able to express the EBNA1 gene that is present within the cell by virtue of the integrated pCEN1 DNA. One of the six clones was found to be most efficiently transfected with pHEBO20 DNA. The cells of that clone are referred to as CEN4 cells.

10. Cloning and Expression of GPA DNA in Mammalian Cells The complete nucleotide coding sequence for GPA was excised from pHEB02-GPA by digestion of that plasmid DNA with the restriction endonuclease Sstl. The GPA DNA then was ligated to Sstl linearized plasmid pUC219, to form plasmid pUC219-GPA. pUC219-GPA then was digested with restriction endonucleases Eagl and EcoNI. The cohesive ends of the resulting DNA fragment having the complete nucleotide coding sequence for GPA were filled in with Klenow DNA polymerase and all four deoxynucloside triphosphates, and adaptors having Sfil cohesive ends then were joined to the DNA fragment by ligation. That fragment with Sfil cohesive ends then was ligated to Sfil linearized pHEBO30. The resulting plasmid, referred to as pHEBO30-GPA, thus contains the complete GPA nucleotide coding sequence downstream of, and in the correct orientation for transcription from the CMV promoter of the plasmid. CEN4 cells were transfected with pHEBO30-GPA by electroporation. Potter, et al., Proc. Nat. Acad. Sci. USA 51:7161-7165 (1984). Transfected cells were cultured in high glucose DMEM, 10% fetal bovine serum, 200 //g/ml hygromycin, 800μg/ml neomycin. For assay of the biological activity of the expressed GPA, the transfected cells were transferred to serum free medium, and the serum free conditioned medium then was assayed for growth promoting activity.

1 1 . Biological Activity of Recombinant GPA Conditioned media and cell extracts prepared from cultures of CEN4 cells transfected with p.HEBO30-GPA were added to ciliary ganglion neurons cultured in 24 well tissue culture plates at a density of one ciliary ganglion per well in Eagle's Minimal Essential Medium (MEM, Gibco) supplemented with 25mM potassium, 10% heat inactivated horse serum, 50 units/ml penicillin, and 5 mg/ml streptomycin. Conditioned media and cell extracts from cultures of CEN4 cells transfected with either pHEB030 or pHEB030-tPA (pHEBO30 containing the coding sequence for human tissue-type plasminogen activator) were used as controls.

Ciliary ganglion neurons were cultured for a total of nine days, and were fed with medium containing freshly thawed samples of conditioned media or cell extract every third day. The amount of neuronal cytoplasm present after nine days was quantitated by measuring the amount of lactate dehγdrogenase (LDH) released upon extracting the neurons in each well of the tissue culture plate with detergent. Thus, the level of LDH is a measure of the number of neurons surviving as well as the amount of neuronal process outgrowth and increases in somal volume.

Specifically, the neuron cultures were washed once with balanced salt solution and then extracted with 100 μ\ homogenate buffer (0.05M. Tris, pH 7.2, 1 mM EDTA, 0.5% Triton X-100, and 2mg/ml bovine serum albumin). LDH activity in duplicate 20 - 25 μ\ aliquots of the detergent extracts was determined using a spectrophotometric assay that measures the conversion of lactate to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD) and a tetrazolium dye. Reaction rates were measured on a Molecular Devices kinetic microplate reader.

In the following Tables I and II, one unit of activity is the amount of sample which gives half-maximal levels of LDH in the neuron cultures. N/D indicates that no activity greater than background levels was detectable. TABLE I

Assays of Conditioned Medium from

CEN4 cells transfected with kunits/ml

PHEBO30-GPA (10 g DNA) 333 pHEB030-GPA (10 μg DNA) 233

PHEB030-GPA ( 1 μg DNA) 1334

PHEB030-GPA ( 2 μg DNA) 1250

PHEBO30-GPA (10 μg DNA) 3448

PHEB030-GPA (20 μg DNA) 5207

PHEBO30 (10 μg DNA) N/D

PHEBO30 d O μgDNA) N/D pHEB030-tPA (10 μg DNA) N/D pHEBO30-tPA (10 μg DNA) N/D

TABLE II

Assays of Cell Extracts from

CEN4 cells transfected with kunits/ml pHEB030-GPA (10 μg DNA) 2631 pHEBO30-GPA (10 μg DNA) 2538

PHEB030-GPA ( 1 μg DNA) 20000

PHEB030-GPA ( 2 μg DNA) 20408

PHEB030-GPA (10 μg DNA) 28000

PHEBO30-GPA (20 μg DNA) 25000

PHEBO30 (10 μg DNA) N/D pHEBO30 (10 μgDNA) N/D pHEB030-tPA (10 μg DNA) N/D pHEB030-tPA (10 μg DNA) N/D

To determine whether cell lysis accounted for the presence of GPA activity in the conditioned medium from cultures of CEN4 cells transfected with pHEB030-GPA, 40 - 50 μl samples of conditioned medium were directly assayed for LDH activity. In the following Table

III, LDH activity is indicated in terms of the reaction rate (change in optical density (OD) per minute) in the LDH spectrophotometric assay. N/D indicates that no activity greater than background levels was detectable.

TABLE III

Assay of LDH Activity in Conditioned Medium from

CEN4 cells transfected with LDH (mODs/min) pHEBO30-GPA (10 μg DNA) N/D pHEBO30-GPA d O μg DNA) N/D

PHEB030-GPA ( 1 μg DNA) N/D

PHEB030-GPA ( 2 μg DNA) N/D

PHEBO30-GPA (10 μg DNA) N/D

PHEBO30-GPA (20 μg DNA) N/D pHEB030 (10 μg DNA) N/D

PHEBO30 (10 μg DNA) N/D pHEBO30-tPA (10 μg DNA) 0.032 pHEBO30-tPA (10 μg DNA) N/D

The absence of detectable LDH activity in the samples of conditioned medium indicates that the GPA in the conditioned medium results from secretion of the GPA from the recombinant CEN4 cells rather than from cell lysis.

12. Secretion of GPA from CEN4 Cells Conditioned media and cell extracts prepared from CEN4 cells transfected with either pHEBO30-GPA or pHEBO30-CNTF (pHEB030 containing the coding sequence for rat ciliary neurotrophic factor) were assayed for their ability to promote ciliary ganglion neuron growth as described above. The results of those assays are shown in Figures 3A and 3B. The growth promoting activities of conditioned media and cell extracts from CEN4 cells transfected with pHEBO30-GPA are shown in Figure 3A. The growth promoting activities of conditioned media and cell extracts from CEN4 cells transfected with pHEB030-CNTF are shown in Figure 3B. in each of the figures, various sample dilutions are shown on the horizontal axis and relative levels of LDH activity extracted from the neuron cultures are shown on the vertical axis. Each point is the average of assay results obtained from two cultures of ciliary ganglion neurons.

Conditioned media from CEN4 cells transfected with pHEBO30-GPA contained significant amounts of neuron growth promoting activity, whereas conditioned media from

CEN4 cells transfected with pHEB030-CNTF contained no such activity in excess of that found in the conditioned media of non-transfected CEN4 cells. However, extracts prepared from CEN4 cells transfected with pHEBO30-CNTF did have significant neuron growth promoting activity, indicating that pHEBO30-CNTF DNA was being expressed in those cells.

These results indicate that recombinant GPA expressed in CEN4 cells is secreted from the cells into the culture medium, but recombinant CNTF expressed in the same type cells is not secreted. SEQUENCE LISTING

(1 ) GENERAL INFORMATION: (i) APPLICANT: GENENTECH, INC. and STATE OF OREGON BY AND THROUGH THE

OREGON STATE BOARD OF HIGHER EDUCATION ON BEHALF OF THE OREGON HEALTH SCIENCES UNIVERSITY

(ii) TITLE OF INVENTION: PRODUCTION OF GPA NEUROTROPHIC FACTOR (iii) NUMBER OF SEQUENCES: 19

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genentech, Inc. (B) STREET: 460 Point San Bruno Boulevard

(C) CITY: South San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94080-4990

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 5.25 inch, 360 Kb floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: patin (Genentech)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 29 September 1992 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Johnston, Sean A.

(B) REGISTRATION NUMBER: P-35,910

(C) REFERENCE/DOCKET NUMBER: 731

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415/225-3562

(B) TELEFAX: 415/952-9881 /9882

(C) TELEX: 910/371-7168

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1469 bases (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

GAGCTCCCGG CTGGAATGCA CCAGCTCAGC AAGGGATTGT CCTCCCCGGG 50 CTGCGCGGGCACTGCGCCGTGAGCCTGGGA CGGCCGCCTG GGCC AT 96

Met 1

G GCG GCG GCA GAC ACC CCT TCA GCC ACC CTC CGG CAC CAC 136 Ala Ala Ala Asp Thr Pro Ser Ala Thr Leu Arg His His 5 10 GAC CTG TGC AGC CGC GGC ATC CGC CTG GCC AGG AAG ATG 175

Asp Leu Cys Ser Arg Gly lie Arg Leu Ala Arg Lys Met 15 20 25

CGC TCG GAC GTC ACA GAC CTC CTG GAC ATC TAC GTG GAG 214 Arg Ser Asp Val Thr Asp Leu Leu Asp Ile Tyr Val Glu

30 35 40

CGG CAG GGC CTG GAC GCC TCC ATC AGC GTG GCG GCG GTG 253 Arg Gin Gly Leu Asp Ala Ser Ile Ser Val Ala Ala Val 45 50

GAT GGG GTG CCG ACG GCG GCA GTG GAG CGC TGG GCC GAG 292 Asp Gly Val Pro Thr Ala Ala Val Glu Arg Trp Ala Glu 55 60 65

CAG ACG GGC ACC CAG CGC CTC CTG GAC AAC CTG GCC GCC 331 Gin Thr Gly Thr Gin Arg Leu Leu Asp Asn Leu Ala Ala 70 75 TAC CGG GCC TTC CGC ACG CTG CTG GCG CAG ATG CTG GAG 370

Tyr Arg Ala Phe Arg Thr Leu Leu Ala Gin Met Leu Glu 80 85 90

GAG CAG CGG GAG CTG CTG GGA GAC ACC GAT GCT GAG CTG 409 Glu Gin Arg Glu Leu Leu Gly Asp Thr Asp Ala Glu Leu

95 100 105

GGC CCG GCG CTG GCG GCC ATG CTG CTG CAG GTC TCG GCC 448 Gly Pro Ala Leu Ala Ala Met Leu Leu Gin Val Ser Ala 1 10 1 15

TTT GTC TAC CAC CTG GAG GAG CTG CTG GAG CTG GAG AGC 487 Phe Val Tyr His Leu Glu Glu Leu Leu Glu Leu Glu Ser 120 125 130

CGC GGG GCC CCC GCT GAG GAG GGC TCC GAG CCC CCC GCG 526 Arg Gly Ala Pro Ala Glu Glu Gly Ser Glu Pro Pro Ala 135 140 CCC CCA CGC CTC AGC CTC TTC GAG CAG AAG CTG CGG GGC 565

Pro Pro Arg Leu Ser Leu Phe Glu Gin Lys Leu Arg Gly 145 150 155

CTG CGG GTG CTG CGG GAG CTG GCC CAG TGG GCC GTC AGG 604 Leu Arg Val Leu Arg Glu Leu Ala Gin Trp Ala Val Arg

160 165 170 TCG GTG CGG GAC CTG CGG CAG CTC TCC AAG CAC GGC CCG 643 Ser Val Arg Asp Leu Arg Gin Leu Ser Lys His Gly Pro 175 180 GGC AGC GGC GCG GCA CTG GGT CTG CCT GAG AGC CAG T 680

Gly Ser Gly Ala Ala Leu Gly Leu Pro Glu Ser Gin 185 190 195

GACGGCGGTG TGGGGAGCCC CGGCCCGGAT GGGGCTTAGC TGGGGGGCCC 730

ATCCCTGTGC AGGGCTGCTG ACAGGCACAG GGGCTTCGGG GGGTGGGGGG 780

GGCCATGCTA CTAACCCAGG ACACTTCTGC TTCCTAATGG GCCCACTTCC 830

CTGCGATGGA AACTGAGGCA CGGGGAGGGA ACTCCCGGTG AGGTGGGCCT 880

TGTGGGCGAC CCGCAGCCCC ACCGTGGCCT GGAAGCCGTG ACCTCCGGCG 930

TGCTGTGCAG GGCCGGCCTG TTCTGTGGAC CGGGTTGCTG CCTGTCTCCT 980

GGCAGCCAGG TGATGCTTCT GGAGGAGGGG AGCTGCCTGG GGGCATTGCA 1030

GAGGTGAGAG CGATGTGGAA GCATCACCTG CTGAAAGGCG GCTTAGGAAT 1080

TGTCCCCCCT CCGCAAGGCC TGAGGCTTTG CCTCCTCCAG GTGCTCCCTT 1 130

TTCTCTCGGC TCCTGCACAG CTCCCACCCT GCCTGTGTGT TCTGCGTGCT 1 180

GAATTTCTGC TCTGACCGCT TGGGGCCTTT CTCAGGAGGG GGGGTGGATT 1230

TTGAGCTGGA TGGCATTTCT TAGCTGGTCG GACGGGATAT GGAGAGGGTA 1280

TTTATATAGA AGGCTGACTT GGAAGACCCT CTGCCAGGGT CTTTGTGCCT 1330

CTGGGGGCTG CTTTAAGACT GGAAAGGGAG AAGCGAACAG CCCCAGCCGC 1380

TGTCGGCCACTGTTTTCTCC CCGGGTGGCTGTGGTGGATG CTGAGCAAAT 1430

AAAGCATCTG GAGAGCCAAAAAAAAAAAAAAAAAAAAAA 1469 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 195 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Ala Ala Asp Thr Pro Ser Ala Thr Leu Arg His His Asp

1 5 10 15

Leu Cys Ser Arg Gly lie Arg Leu Ala Arg Lys Met Arg Ser Asp 20 25 30

Val Thr Asp Leu Leu Asp Ile Tyr Val Glu Arg Gin Gly Leu Asp 35 40 45

Ala Ser lie Ser Val Ala Ala Val Asp Gly Val Pro Thr Ala Ala 50 55 60

Val Glu Arg Trp Ala Glu Gin Thr Gly Thr Gin Arg Leu Leu Asp 65 70 75 Asn Leu Ala Ala Tyr Arg Ala Phe Arg Thr Leu Leu Ala Gin Met

80 85 90

Leu Glu Glu Gin Arg Glu Leu Leu Gly Asp Thr Asp Ala Glu Leu 95 100 105

Gly Pro Ala Leu Ala Ala Met Leu Leu Gin Val Ser Ala Phe Val 110 115 120

Tyr His Leu Glu Glu Leu Leu Glu Leu Glu Ser Arg Gly Ala Pro 125 130 135

Ala Glu Glu Gly Ser Glu Pro Pro Ala Pro Pro Arg Leu Ser Leu 140 145 150 Phe Glu Gin Lys Leu Arg Gly Leu Arg Val Leu Arg Glu Leu Ala

155 160 165

Gin Trp Ala Val Arg Ser Val Arg Asp Leu Arg Gin Leu Ser Lys 170 175 180

His Gly Pro Gly Ser Gly Ala Ala Leu Gly Leu Pro Glu Ser Gin 185 190 195

(2) INFORMATION FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 199 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ala Phe Met Glu His Ser Ala Leu Thr Pro His Arg Arg Glu 1 5 10 15

Leu Cys Ser Arg Thr Ile Trp Leu Ala Arg Lys lie Arg Ser Asp 20 25 30

Leu Thr Ala Leu Thr Glu Ser Tyr Val Lys His Gin Gly Leu Asn 35 40 45

Lys Asn lie Asn Leu Asp Ser Val Asp Gly Val Pro Met Ala Ser 50 55 60 Thr Asp Gin Trp Ser Glu Leu Thr Glu Ala Glu Arg Leu Gin Glu

65 70 75

Asn Leu Gin Ala Tyr Arg Thr Phe His lie Met Leu Ala Arg Leu 80 85 90

Leu Glu Asp Gin Gin Val His Phe Thr Pro Ala Glu Gly Asp Phe 95 100 105

His Gin Ala lie His Thr Leu Leu Leu Gin Val Ala Ala Phe Ala 1 10 1 15 120

Tyr Gin lie Glu Glu Leu Met Val Leu Leu Glu Cys Asn lie Pro 125 130 135 Pro Lys Asp Ala Asp Gly Thr Pro Val Ile Gly Gly Asp Gly Leu

140 145 1 50

Phe Glu Lys Lys Leu Trp Gly Leu Lys Val Leu Gin Glu Leu Ser 155 160 165

His Trp Thr Val Arg Ser lie His Asp Leu Arg Val Ile Ser Cys 170 175 180

His Gin Thr Gly lie Pro Ala His Gly Ser His Tyr ile Ala Asn 185 190 195

Asp Lys Glu Met 199 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 200 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ala Phe Ala Glu Gin Thr Pro Leu Thr Leu His Arg Arg Asp 1 5 10 15 Leu Cys Ser Arg Ser lie Trp Leu Ala Arg Lys lie Arg Ser Asp 20 25 30

Leu Thr Ala Leu Met Glu Ser Tyr Val Lys His Gin Gly Leu Asn 35 40 45

Lys Asn lie Asn Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser 50 55 60 Thr Asp Arg Trp Ser Glu Met Thr Glu Ala Glu Arg Leu Gin Glu

65 70 75

Asn Leu Gin Ala Tyr Arg Thr Phe Gin Gly Met Leu Thr Lys Leu 80 85 90

Leu Glu Asp Gin Arg Val His Phe Thr Pro Thr Glu Gly Asp Phe 95 100 105

His Gin Ala Ile His Thr Leu Met Leu Gin Val Ser Ala Phe Ala 1 10 1 15 120

Tyr Gin Leu Glu Glu Leu Met Val Leu Leu Glu Gin Lys lie Pro 125 130 135 Glu Asn Glu Ala Asp Gly Met Pro Ala Thr Val Gly Asp Gly Gly

140 145 150

Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys Val Leu Gin Glu Leu 155 160 165

Ser Gin Trp Thr Val Arg Ser Ile His Asp Leu Arg Val lie Ser 170 175 180

Ser His Gin Met Gly lie Ser Ala Leu Glu Ser His Tyr Gly Ala 185 190 195

Lys Asp Lys Gin Met 200 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 200 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp 1 5 10 15

Leu Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys lie Arg Ser Asp 20 25 30 Leu Thr Ala Leu Thr Glu Ser Tyr Val Lys His Gin Gly Leu Asn

35 40 45 Lys Asn Ile Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser 50 55 60

Thr Asp Gin Trp Ser Glu Leu Thr Glu Ala Glu Arg Leu Gin Glu 65 70 75

Asn Leu Gin Ala Tyr Arg Thr Phe His Val Leu Leu Ala Arg Leu 80 85 90 Leu Glu Asp Gin Gin Val His Phe Thr Pro Thr Glu Gly Asp Phe

95 100 105

His Gin Ala lie His Thr Leu Leu Leu Gin Val Ala Ala Phe Ala 1 10 1 15 120

Tyr Gin Ile Glu Glu Leu Met ile Leu Leu Glu Tyr Lys lie Pro 125 130 135

Arg Asn Glu Ala Asp Gly Met Pro lie Asn Val Gly Asp Gly Gly 140 145 150

Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys Val Leu Gin Glu Leu 155 160 165 Ser Gin Trp Thr Val Arg Ser Ile His Asp Leu Arg Phe Ile Ser

170 175 180

Ser His Gin Thr Gly lie Pro Ala Arg Gly Ser His Tyr Ile Ala 185 190 195

Asn Asn Lys Lys Met 200

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8575 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GATCCTCACA GGCCGCACCC AGCTTTTCTT CCGTTGCCCC AGTAGCATCT 50

CTGTCTGGTG ACCTTGACCG GTGTGTTCGT ATATGGAGGT AGTAAGACCT 100

CCCTTTACAA CCTAAGGCGA GGAACTGCCC TTGCTATTCC ACAATGTCGT 150

CTTACACCAT TGAGTCGTCT CCCCTTTGGA ATGGCCCCTG GACCCGGCCC 200 ACAACCTGGC CCGCTAAGGG AGTCCATTGT CTGTTATTTC ATGGTCTTTT 250

TACAAACTCA TATATTTGCT GAGGTiTTGA AGGATGCGAT TAAGGACCTT 300

GTTATGACAA AGCCCGCTCC TACCTGCAAT ATCAGGGTGA CTGTGTGCAG 350

CTTTGACGAT GGAGTAGATT TGCCTCCCTG GTTTCCACCT ATGGTGGAAG 400

GGGCTGCCGC GGAGGGTGAT GACGGAGATG ACGGAGATGA AGGAGGTGAT 450

GGAGATGAGG GTGAGGAAGG GCAGGAGTGA TGTAACTTGT TAGGAGACGC 500

CCTCAATCGT ATTAAAAGCC GTGTATTCCC CCGCACTAAA GAATAAATCC 550

CCAGTAGACA TCATGCGTGC TGTTGGTGTA TTTCTGGCCA TCTGTCTTGT 600

CACCATTTTC GTCCTCCCAA CATGGGGCAA TTGGGCATAC CCATGTTGTC 650

ACGTCACTCA GCTCCGCGCT CAACACCTTC TCGCGTTGGA AAACATTAGC 700

GACATTTACC TGGTGAGCAA TCAGACATGC GACGGCTTTA GCCTGGCCTC 750

CTTAAATTCA CCTAAGAATG GGAGCAACCA GCAGGAAAAG GACAAGCAGC 800

GAAAATTCAC GCCCCCTTGG GAGGTGGCGG CATATGCAAA GGATAGCACT 850

CCCACTCTAC TACTGGGTAT CATATGCTGA CTGTATATGC ATGAGGATAG 900

CATATGCTAC CCGGATACAG ATTAGGATAG CATATACTAC CCAGATATAG 950

ATTAGGATAG CATATGCTAC CCAGATATAG ATTAGGATAG CCTATGCTAC 1000

CCAGATATAA ATTAGGATAG CATATACTAC CCAGATATAG ATTAGGATAG 1050

CATATGCTAC CCAGATATAG ATTAGGATAG CCTATGCTAC CCAGATATAG 1 100

ATTAGGATAG CATATGCTAC CCAGATATAG ATTAGGATAG CATATGCTAT 1 150 CCAGATATTT GGGTAGTATA TGCTACCCAG ATATAAATTA GGATAGCATA 1200

TACTACCCTA ATCTCTATTA GGATAGCATA TGCTACCCGG ATACAGATTA 1250

GGATAGCATA TACTACCCAG ATATAGATTA GGATAGCATA TGCTACCCAG 1300

ATATAGATTA GGATAGCCTA TGCTACCCAG ATATAAATTA GGATAGCATA 1350

TACTACCCAG ATATAGATTA GGATAGCATA TGCTACCCAG ATATAGATTA 1400

GGATAGCCTA TGCTACCCAG ATATAGATTA GGATAGCATA TGCTATCCAG 1450

ATATTTGGGT AGTATATGCT ACCCATGGCA ACATTAGCCC ACCGTGCTCT 1 500

CAGCGACCTC GTGAATATGA GGACCAACAA CCCTGTGCTT GGCGCTCAGG 1 550

CGCAAGTGTG TGTAATTTGT CCTCCAGATC GCAGCAATCG CGCCCCTATC 1600

TTGGCCCGCC CACCTACTTA TGCAGGTATT CCCCGGGGTG CCATTAGTGG 1650

TTTTGTGGGC AAGTGGTTTG ACCGCAGTGG TTAGCGGGGT TACAATCAGC 1700

CAAGTTATTA CACCCTTATT TTACAGTCCA AAACCGCAGG GCGGCGTGTG 1750

GGGGCTGACG CGTGCCCCCA CTCCACAATT TCAAAAAAAA GAGTGGCCAC 1800

TTGTCTTTGT TTATGGGCCC CATTGGCGTG GAGCCCCGTT TAATTTTCGG 1850

GGGTGTTAGA GACAACCAGT GGAGTCCGCT GCTGTCGGCG TCCACTCTCT 1900

TTCCCCTTGT TACAAATAGA GTGTAACAAC ATGGTTCACC TGTCTTGGTC 1950

CCTGCCTGGG ACACATCTTA ATAACCCCAG TATCATATTG CACTAGGATT 2000

ATGTGTTGCC CATAGCCATA AATTCGTGTG AGATGGACAT CCAGTCTTTA 2050

CGGCTTGTCC CCACCCCATG GATTTCTATT GTTAAAGATA TTCAGAATGT 2100 TTCATTCCTA CACTAGTATT TATTGCCCAA GGGGTTTGTG AGGGTTATAT 2150

TGGTGTCATA GCACAATGCC ACCACTGAAC CCCCCGTCCA AATTTTATTC 2200

TGGGGGCGTC ACCTGAAACC TTGTTTTCGA GCACCTCACA TACACCTTAC 2250

TGTTCACAAC TCAGCAGTTA TTCTATTAGC TAAACGAAGG AGAATGAAGA 2300

AGCAGGCGAA GATTCAGGAG AGTTCACTGC CCGCTCCTTG ATCTTCAGCC 2350

ACTGCCCTTG TGACTAAAAT GGTTCACTAC CCTCGTGGAA TCCTGACCCC 2400

ATGTAAATAA AACCGTGACA GCTCATGGGG TGGGAGATAT CGCTGTTCCT 2450

TAGGACCCTT TTACTAACCC TAATTCGATA GCATATGCTT CCCGTTGGGT 2500

AACATATGCT ATTGAATTAG GGTTAGTCTG GATAGTATAT ACTACTACCC 2550

GGGAAGCATA TGCTACCCGT TTAGGGTTAA CAAGGGGGCC TTATAAACAC 2600

TATTGCTAAT GCCCTCTTGA GGGTCCGCTT ATCGGTAGCT ACACAGGCCC 2650

CTCTGATTGA CGTTGGTGTA GCCTCCCGTA GTCTTCCTGG GCCCCTGGGA 2700

GGTACATGTC CCCCAGCATT GGTGTAAGAG CTTCAGCCAA GAGTTACACA 2750

TAAAGGCAAT GTTGTGTTGC AGTCCACAGA CTGCAAAGTC TGCTCCAGGA 2800

TGAAAGCCAC TCAGTGTTGG CAAATGTGCA CATCCATTTA TAAGGATGTC 2850

AACTACAGTC AGAGAACCCC TTTGTGTTTG GTCCCCCCCC GTGTCACATG 2900

TGGAACAGGG CCCAGTTGGC AAGTTGTACC AACCAACTGA AGGGATTACA 2950

TGCACTGCCC GTGACCAATA CAAAACAAAA GCGCTCCTCG TACCAGCGAA 3000

GAAGGGGCAG AGATGCCGTA GTCAGGTTTA GTTCGTCCGG CGGCGGGGGA 3050 TCCTCTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC 3100

ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT 3150

GACCGCCCAA CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC 3200

ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT 3250

ACGGTAAACT GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA 3300

CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC 3350

CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT 3400

AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC 3450

GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC 3500

GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG 3550

TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT 3600

GGGAGGTCTA TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG 3650

AGACGCCATC CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC 3700

CAGCCTCCGC GGCCGGGAAC GGTGCATTGG AACGCGGATT CCCCGTGCCA 3750

AGAGTGACGT AAGTACCGCC TATAGAGTCT ATAGGCCCAC CCCCTTGGCT 3800

TCGTTAGAAC GCGGCTACAA TTAATACATA ACCTTATGTA TCATACACAT 3850

ACGATTTAGG TGACACTATA GAATAACATC CACTTTGCCT TTCTCTCCAC 3900

AGGTGTCCAC TCCCAGGTCC AACTGCACCT CGGTTCTATC GATTGGCCTT 3950

GGTGGCCAGC TGAGCTCACC TGCNNNNNNN NNNNNNNNNN NNNNNNNNNN 4000 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNN 4050

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4100

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNN 4150

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN4200

NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN4250

NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN4300

NNNNNNNNNN NNNNNNNNNN NNNGCAGGTGAGCTCAGCTG GCCACCACGG 4350

CCGCAGCTTG GCCGCCATGG CCCAACTTGT TTATTGCAGC TTATAATGGT 4400

TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CA I I I I I I I C 4450

ACTGCATTCT AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG 4500

TCTGGATCGG GAATTAATTC GGGCGGCGGA GTCGACCTGC AGCCCAAGCT 4550

TGGCACGCCA GAAATCCGCG CGGTGGTTTT TGGGGGTCGG GGGTGTTTGG 4600

CAGCCACAGA CGCCCGGTGT TCGTGTCGCG CCAGTACATG CGGTCCATGC 4650

CCAGGCCATC CAAAAACCAT GGGTCTGTCT GCTCAGTCCA GTCGTGGACC 4700

TGACCCCACG CAACGCCCAA AAGAATAACC CCCACGAACC ATAAACCATT 4750

CCCCATGGGG GACCCCGTCC CTAACCCACG GGGCCCGTGG CTATGGCGGG 4800

CTTGCCGCCC CGACGTTGGC TGCGAGCCCT GGGCCTTCAC CCGAACTTGG 4850

GGGTTGGGGT GGGGAAAAGG AAGAAACGCG GGCGTATTGG CCCCAATGGG 4900

GTCTCGGTGG GGTATCGACA GAGTGCCAGC CCTGGGACCG AACCCCGCGT 4950 TTATGAACAA ACGACCCAAC ACCCGTGCGT TTTATTCTGT C I I I I I ATTG 5000

CCGTCATAGC GCGGGTTCCT TCCGGTATTG TCTCCTTCCG TGTTTCAGTT 5050

AGCCTCCCCC ATCTCCCGAT CCGGGGCGTC GGTTTCCACT ATCGGCGAGT 5100

ACTTCTACAC AGCCATCGGT CCAGACGGCC GCGCTTCTGC GGGCGATTTG 5150

TGTACGCCCG ACAGTCCCGG CTCCGGATCG GACGATTGCG TCGCATCGAC 5200

CCTGCGCCCA AGCTGCATCA TCGAAATTGC CGTCAACCAA GCTCTGATAG 5250

AGTTGGTCAA GACCAATGCG GAGCATATAC GCCCGGAGCC GCGGCGATCC 5300

TGCAAGCTCC GGATGCCTCC GCTCGAAGTA GCGCGTCTGC TGCTCCATAC 5350

AAGCCAACCA CGGCCTCCAG AAGAAGATGT TGGCGACCTC GTATTGGGAA 5400

TCCCCGAACA TCGCCTCGCT CCAGTCAATG ACCGCTGTTA TGCGGCCATT 5450

GTCCGTCAGG ACATTGTTGG AGCCGAAATC CGCGTGCACG AGGTGCCGGA 5500

CTTCGGGGCA GTCCTCGGCC CAAAGCATCA GCTCATCGAG AGCCTGCGCG 5550

ACGGACGCAC TGACGGTGTC GTCCATCACA GTTTGCCAGT GATACACATG 5600

GGGATCAGCA ATCGCGCATA TGAAATCACG CCATGTAGTG TATTGACCGA 5650

TTCCTTGCGG TCCGAATGGG CCGAACCCGC TCGTCTGGCT AAGATCGGCC 5700

GCAGCGATCG CATCCATGGC CTCCGCGACC GGCTGCAGAA CAGCGGGCAG 5750

TTCGGTTTCA GGCAGGTCTT GCAACGTGAC ACCCTGTGCA CGGCGGGAGA 5800

TGCAATAGGT CAGGCTCTCG CTGAATTCCC CAATGTCAAG CACTTCCGGA 5850

ATCGGGAGCG CGGCCGATGC AAAGTGCCGA TAAACATAAC GATCTTTGTA 5900 GAAACCATCG GCGCAGCTAT TTACCCGCAG GACATATCCA CGCCCTCCTA 5950

CATCGAAGCT GAAAGCACGA GATTCTTCGC CCTCCGAGAG CTGCATCAGG 6000

TCGGAGACGC TGTCGAACTT TTCGATCAGA AACTTCTCGA CAGACGTCGC 6050

GGTGAGTTCA GGCI I I I I CA TATCTCATTG CCCCCGGACG AGGGATCTGC 6100

GGCACGCTGT TGACGCTGTT AAGCGGGTCG CTGCAGGGTC GCTCGGTGTT 6150

CGAGGCCACA CGCGTCACCT TAATATGCGA AGTGGACCTC GGACCGCGCC 6200

GCCCCGACTG CATCTGCGTG TTCGAATTCG CCAATGACAA GACGCTGGGC 6250

GGGGTTTGTG TCATCATAGA ACTAAAGACA TGCAAATATA TTTCTTCCGG 6300

GGACACCGCC AGCAAACGCG AGCAACGGGC CACGGGGATG AAGCAGGGCA 6350

GCGCTCTGGG TCATTTTCGG CGAGGACCGC TTTCGCTGGA GCGCGACGAT 6400

GATCGGCCTG TCGCTTGCGG TATTCGGAAT CTTGCACGCC CTCGCTCAAG 6450

CCTTCGTCAC TGGTCCCGCC ACCAAACGTT TCGGCGAGAA GCAGGCCATT 6500

ATCGCCGGCA TGGCGGCCGA CGCGCTGGGC TACGTCTTGC TGGCGTTCGC 6550

GACGCGAGGC TGGATGGCCT TCCCCATTAT GATTCTTCTC GCTTCCGGCG 6600

GCATCGGGAT GCCCGCGTTG CAGGCCATGC TGTCCAGGCA GGTAGATGAC 6650

GACCATCAGG GACAGCTAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA 6700

GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC 6750

ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA 6800

AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC 6850 GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 6900

TGGCGCTTTC TCAATGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC 6950

GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG 7000

CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG 7050

ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG 7100

TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA 71 50

CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 7200

TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT 7250

AGCGGTGGTT I I I I I GTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG 7300

ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA 7350

ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC 7400

TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG 7450

TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 7500

CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC 7550

CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG 7600

TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG 7650

CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT 7700

TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG 7750

TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTGCAGGCA 7800 TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC 7850

CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT 7900

TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT 7950

TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA 8000

TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG 8050

AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAACACGGG 8100

ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA 8150

CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG 8200

TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT 8250

TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA 8300

AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT 8350

TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA 8400

TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT 8450

CCCCGAAAAG TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT 8500

AACCTATAAA AATAGGCGTA TCACGAGGCC CTTTCGTCTT CAAGAATTCT 8550

CATGTTTGAC AGCTTATCAT CGATA 8575

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGATTGGCCT TGGTGGCCTA AGCTTGC 27

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 bases (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GGCCGCAAGC TTAGGCCACC AAGGCCAAT 29

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 bases

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

TGGCCAGCTG AGCTCACCTG C 21

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GCAGGTGAGC TCAGCTGGCC ACCA 24

(2) INFORMATION FOR SEQ ID NO:1 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 1 GACAACCTGG CTGCCTACCG CGCCTTCCGC ACCCTGTT 38

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 bases

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:

ATGCTGCTGC AGGTGTCTGC CTTCGTGTAC CACCTGGA 38

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

AGGGTGCTGC GAGAGCTGGC ACAGTGGGCT GTGAGGTCTG T 41

(2) INFORMATION FOR SEQ ID N0:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CTAGGAGCTT TTG 13

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CATGCAAAAG CTC 13

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TTAGAATTCG CGATT 15

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GATCAATCGC GAATTC 1 6

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

TGGCCAGCTG AGCTCACCTG C 21

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 bases (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCAGGTGAGC TCAGCTGGCC ACCA 24

Claims

CLAIMS What is claimed is:

1 . An isolated nucleic acid molecule encoding GPA.

2. A nucleic acid molecule of claim 1 which is DNA and which comprises the nucleotide sequence shown in Figure 1 for mature GPA.

3. A nucleic acid molecule of claim 2 further comprising a promoter operably linked to the nucleotide sequence shown in Figure 1 for mature GPA.

4. An expression vector comprising the nucleic acid molecule of claim 2 operably linked to control sequences recognized by a host cell transformed with the vector.

5. A host cell transformed with the vector of claim 4.

6. An isolated DNA comprising a nucleotide sequence which (i) has at least 90% sequence identity with the nucleotide sequence shown in Figure 1 for mature GPA, and (ii) encodes a protein that is capable of promoting the growth, survival, and/or differentiation of ciliary ganglion neurons, dorsal root ganglion neurons, or sympathetic neurons.

7. A method of using a nucleic acid molecule encoding GPA, comprising transforming a host cell with an expression vector comprising the nucleic acid molecule operably linked to control sequences recognized by the host cell, expressing the nucleic acid molecule in the host cell, and thereafter recovering GPA from the host cell.

8. A method of claim 7 wherein the GPA is recovered from the host cell culture medium.

9. A method of diagnosing a disease or disorder of the nervous system comprising:

(a) contacting nucleic acid from a cell or a tissue with a labelled DNA which comprises at least 10 nucleotides of the nucleotide sequence shown in Figure 1 , under conditions which will allow hybridization of complementary nucleotide sequences; and

(b) detecting any hybridization which occurs.

10. A method comprising:

(a) contacting nucleic acid from a cell or a tissue with a DNA which comprises at least 10 nucleotides of the nucleotide sequence shown in Figure 1 , under conditions which will allow hybridization of complementary nucleotide sequences;

(b) amplifying in a polymerase chain reaction the nucleic acid to which the DNA of step (a) hybridizes; and (c) detecting any nucleic acid produced by the polymerase chain reaction of step

(b). . A method for producing GPA comprising:

(a) transforming a cell containing an endogenous GPA gene with a homologous

DNA comprising an amplifiable gene and a flanking sequence of at least about 150 base pairs that is homologous with a DNA sequence within or in proximity to the endogenous GPA gene, whereby the homologous DNA integrates into the cell genome by recombination; (b) culturing the cells under conditions that select for amplification of the amplifiable gene, whereby the GPA gene is also amplified; and thereafter (c) recovering GPA from the cells.

12. GPA that is unaccompanied by associated native glycosylation.

13. A composition comprising GPA that is free of other polypeptides of the animal species in which GPA naturally occurs.

14. A composition of claim 13 further comprising a physiologically acceptable carrier.

15. A composition of claim 13 that is sterile.

16. An antibody that is capable of binding to GPA but not to NGF, BDNF, NT-3, or CNTF.

17. An antibody of claim 16 that is a monoclonal antibody.

18. A method of treating a neurologic disease or disorder in a mammal comprising administering a therapeutically effective amount of the composition of claim 15.

19. A method of claim 18 wherein the neurologic disease or disorder is Alzheimer's disease or amyotrophic lateral sclerosis.

20. A method of claim 18 that further comprises administering a therapeutically effective amount of NGF, BDNF, or NT-3.