WO2000006731A2 - PERSEPHIN ARF, A PROTEIN ENCODED BY UNSPLICED PERSEPHIN mRNA - Google Patents

Abstract

A novel protein, persephinARF is disclosed and the human, mouse and rat amino acid sequences thereof are identified. Nucleotide sequences encoding these persephinARF amino acid sequences are also disclosed.

Description

PERSEPHIN ARF, A PROTEIN ENCODED BY UNSPLICED PERSEPHIN mRNA

Reference to Government Grant

This mvention was made with government support under Grant Numbers AG13729 and AG13730. The government has certain rights in this invention.

Background of the Invention (1) Field of the Invention

This mvention relates generally to novel expression products of the persephm gene and, more particularly to a novel protein encoded by an alternative reading frame in unsphced persephm mRNA. (2) Descπption of the Related Art

Recently, a family of neurotrophic factors that are related to the TGFβ superfamily of growth factors but structurally distinct from the neurotrophms has been identified. This family was identified on the basis of amino acid sequences that are conserved between ghal cell line-derived neurotrophic factor (GDNF) and neurtuπn (NTN) which are not present in other members of the TGFβ superfamily. Kotzbauer et al., Nature 384:461-410, 1996. A PCR based strategy using degenerate primers corresponding to these conserved regions in what is now termed the GDNF family was used to discover an additional family member, persephm (PSP), as described in copendmg U.S. Seπal No. 08/931,858 filed September 16, 1997; Milbrandt et al., Neuron 20:245-253, 1998. GDNF, neurtunn and persephm have similarities and differences in their biological activity profiles While each family member exhibits neurotrophic activity on mesencephahc, dopaminergic and motor neurons and act as a kidney ramogen, persephm, unlike neurtunn and GDNF, does not appear to act on penpheral sympathetic, sensory or enteric neurons Milbrandt et al , supra

Sequence analysis of genomic and cDNA clones for GDNF, neurtunn and persephm revealed that each family member is likely expressed within a cell as a pre-pro-protem, with the pre-region containing an ammo terminal signal sequence followed by a pro domain which contains an RXXR proteolytic processing site immediately before the N-termmal ammo acid of the predicted mature protein The genes encoding GDNF, neurtunn and persephm are also similar in that each contains an intron that interrupts the coding region of the pro-domain While the mtron for neurtunn was reported to be about 500 nt m length, the mtron in the persephm gene was reported to be much shorter: 88 nt in mouse and rat genes and 85 nt in the human persephm gene. See U.S. Patent 5,739,307 and 08/931,858 While the latter reference disclosed the sequence of the mtron in the persephm rat and mouse genes and identified the location of the predicted splice site m human persephm protein, the mtron sequence for the human persephm gene has not been disclosed.

Based on the observation that unsphced and spliced persephm mRNA have been reported to be present m equally low levels m rat heart, kidney, liver and brain, it has been suggested that the regulation of persephm mRNA processing might be involved in regulating the production of persephm protein m tissues. Milbrandt et al., supra This would be consistent with the reported use of alternative splicing of the GDNF mtron to produce two GDNF mRNAs of diffenng lengths The longer form, GDNFα, corresponds to the cDNA ongmally isolated by Lm et al., (GenBank citation) and the shorter form, GDNFβ, contains a 78 bp deletion m the prepro region of the coding sequence probably resulting from use of an alternative donor splice site dunng GDNF mRNA processing Trupp et al., J Cell Biol 730.137-148, 1995, Springer et al , Exper Neurol 131:41-52, 1995 However, this deletion does not include the secretion signal sequence or the consensus proteolytic cleavage sequence and does not alter the reading frame of the coding sequence downstream of the splice site Moreover, the deletion apparently does not interfere with the processing of the polypeptide encoded by GDNFB as identical mature GDNF proteins are believed to be produced from the GDNFα and GDNFβ mRNA isoforms. Trupp et al., supra

Alternative splicing resulting in mRNAs encoding entirely distinct proteins has been reported for other mammalian genes. For example, the human INK4a tumor suppressor gene yields α and β transcnpts of similar length which differ by the presence of alternative first exons joined through the identical splice acceptor site to sequences in exon 2 Mao et al., Cancer Res 55 2995-2997, 1995, Stone et al , Cancer Res 55 2988-2994, 1995 It has been determined that translation of the β transcnpt begins at a 5 AUG diffenng from that used in the α transcript and consequently the exon 2 nucleotide sequence is translated m an alternative reading frame from that used dunng translation of the α transcript Quelle et al , Cell 83 993-1000, 1995 Although the polypeptides encoded by the α and β transcripts, named pl6^INK4a and pl9^ΛRF, respectively, have entirely different ammo acid sequences, they have both been shown to be naturally expressed and surpnsmgly both are reported to induce growth arrest in mammalian fibroblasts Quelle et al , supra Alternative splicing has also been shown to generate two transcnpts of the munne peπaxin gene m approximately equal amounts via a retained intron mechanism Dytnch et al , J Bwl Chem 273:5794-5800, 1998 Because a stop codon is present in the retained mtron, the larger mRNA containing the intron produces a truncated protein, S-penaxm, with an lntron-encoded C terminus of 21 amino acids that is not present the larger protein, L- penaxm, encoded by the shorter mRNA Dytrych et al reported that although both L- peπaxm and S-peπaxin are expressed in Schwann cells, they are targeted to different subcellular localizations, and suggested the two penaxm proteins may have distinct functions

All references cited in this specification are hereby incorporated m their entirety by reference The discussion of the references herein is intended merely to summanze the assertions made by their authors and no admission is made that any reference constitutes prior art Applicants reserve the nght to challenge the accuracy and pertinency of the cited references

Summary of the Invention Briefly, therefore, the present invention is directed to the identification of an alternative reading frame in the persephm genomic sequence that encodes a novel polypeptide This polypeptide, referred to herein as persephm^ΛRF, is predicted to be the translation product of the unsphced transcnpt of the persephm gene Because the unsphced transcript is present in various tissues m amounts comparable to or larger than quantities of the spliced transcnpt and because the unsphced transcnpt encodes a polypeptide with a secretion signal sequence, it is believed that persephm^ARF is synthesized in vivo and has biological activity by virtue of its secreted nature

T e invention thus provides isolated human, mouse, and rat persephιn^ARF polypeptides which compnse the ammo acid sequences set forth m SEQ ID NO:l, 2 and 3 respectively. In addition, the invention includes mature persephιn^ARF polypeptides lacking the secretion signal sequence and comprising SEQ ID NO:4 (human), SEQ ID NO:5 (mouse) or SEQ ID NO:6 (rat).

In another embodiment the present invention provides isolated polynucleotides encoding the human, mouse, and rat persephin^ARF polypeptides of SEQ ID NOS: 1-6. Preferred polynucleotides comprise human nucleotide sequences as set forth in SEQ ID NO: 7 and 8; mouse nucleotide sequences as set forth in SEQ ID NOS:9 and 10; and rat nucleotide sequences as set forth in SEQ ID NO:l 1 and 12.

Expression vectors and stably transformed cells comprising polynucleotides encoding persephm ^ARF are also within the scope of this invention. The transformed cells can be used in a method for producing persephin ^ARF.

In another embodiment, the invention provides isolated polynucleotides comprising nucleotide sequences complementary to SEQ ID NOS-.7-12 as set forth in SEQ ID NOS:13-18 as well as isolated polynucleotides that specifically hybridize to the intronic portion of polynucleotides comprising any one of SEQ ID NOS:7, 8, 13 and 14. Such hybridizing polynucleotides include but are not limited to SEQ ID NO: 19 and its complement as well as fragments thereof. These complementary and hybridizing polynucleotides can be used in methods for detecting the persephin gene and unsphced transcription products thereof.

The present invention also provides antibodies which specifically react with persephin^ARF and methods for purifying persephin^ARF or detecting its expression using such antibodies.

In still further embodiments, the present mvention includes a composition comprising persephin^ARF and a pharmaceutically acceptable carrier as well as a method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of persephin^ARF. Among^' the several advantages found to be achieved by the present mvention, therefore, may be noted the provision of a novel protein, persephin^ARF, the provision of polynucleotides and methods for obtaining persephin^ARF by recombinant techniques; the provision of polynucleotides and antibodies useful in methods for detecting the persephm gene and expression products thereof; and the provision of methods for preventing or treating medical conditions.

Brief Description of the Drawings

Figure 1 illustrates the alignment of the nucleotide sequences of the mouse, rat and human persephin genes (SEQ ID NOS:26, 27 and 7, respectively) with the arrows denoting the beginning and ending of the intron that is not spliced out in the transcript encoding persephin^ARF and the stop codon for each species indicated by an asterisk (*); Figure 2 illustrates the alignment of the amino acid sequences of human, mouse and rat persephin ^ARF with the first amino acid of the predicted mature protein indicated by the asterisk (*) at amino acid position 24;

Figure 3 illustrates relative levels of unsphced and spliced persephin mRNA in various rat embryonic (El 8) and adult tissues determined by semiquantitative RT/PCR using 32, 29 or 26 cycles, with unsphced mRNA indicated by the open arrowhead and spliced mRNA indicated by the closed arrowhead; and

Figure 4 illustrates an agarose gel showing products resulting from PCR analysis of human genomic DNA (lane 1) and reverse transcribed human fetal brain poly A⁺ RNA (lane 3, no RT control, and lane 4) using PCR primers corresponding to the start and stop sites of the coding sequence for human persephin, with size markers present in lane 2 and unsphced and spliced persephin mRNA species indicated by the filled and open arrowheads, respectively.

Description of the Preferred Embodiments

The present invention is based upon the identification of an alternative reading frame in unsphced persephin mRNA that encodes a novel protein, persephin^AR . As used herein, "an alternative reading frame" in connection with a persephin gene or an unsphced transcript thereof is a reading frame which begins at the same initiation codon used in the synthesis of persephin and continues through the intron sequence. As shown in Figure 1, the alternative reading frame in the mouse and rat persephin genes stops at the end of the intron while in the human persephin gene the alternative reading frame continues for another 261 bases. Thus, the alternative reading frame in the human persephin gene encodes a 164 amino acid polypeptide that is twice as long as the 81 amino acid polypeptides encoded by the corresponding alternative reading frames in the mouse and rat persephin genes.

As shown in Fig. 2, alignment of the predicted amino sequences of the human, mouse and rat persephin^ARF polypeptides indicates that these three polypeptides share some homology in the region encoded by the intron. However, there is no homology between the persephin^ARF polypeptide and mature persephin. In addition, a search of the GenBank^® database revealed no homologies between human persephin^ARF and other proteins.

As described more fully below, unsphced human, mouse and rat persephin transcripts are present in amounts comparable to or greater than the corresponding spliced transcripts. Thus, the inventors herein believe that the unsphced transcript is translated in vivo to produce persephin^ARF polypeptides whose predicted amino acid sequences are set forth in SEQ ID NOS: 1-3. These persephin^ARF polypeptides are believed to be precursor forms in that they have a 23 amino acid long signal sequence identical to that present in precursor forms of perseph . The presence of the signal sequence indicates that persephm^ARF is a secreted protein The precursor form of persephm^ΛRF does not appear to contain an RXXR cleavage site; thus, cleavage of the predicted signal sequence is predicted to produce a 141 am o acid long mature human persephm^ARF consisting of SEQ ID NO:4 and 58 amino acid long mouse and rat persephm^ARF polypeptides consisting of SEQ ID NO:5 and SEQ ID NO:6, respectively

As used herein, a "secreted protein" is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its ammo acid sequence. Secreted proteins include without limitation proteins or polypeptides secreted wholly (e.g. soluble proteins) or partially (e.g. receptors) from the cell in which they are expressed. Secreted proteins also include without limitation proteins or polypeptides which are transported across the membrane of the endoplasmic reticulum.

Accordingly, one embodiment of the invention provides an isolated and punfied polypeptide compns g a persephm^ARF ammo acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ LD NO:5 and SEQ ID NO:6. As used herein, "isolated and purified" means that the polypeptide constitutes at least about 50 percent of a composition on a molar basis compared to total proteins or other macromolecular species present m the composition. Preferably, the polypeptide of the invention will constitute at least about 75 to about 80 mole percent of the total protein or other macromolecular species present. More preferably, an isolated and punfied polypeptide will constitute about 85 to about 90 mole percent of a composition and still more preferably, at least about 95 mole percent or greater.

The invention also includes polypeptides compnsmg any naturally occurnng allelic vanants of the disclosed persephi ^ARF amino acid sequences; that is, polypeptides encoded by the alternative reading frame in different alleles of the human, mouse or rat persephin genes Homologs of the disclosed mammalian persephin ^ARF polypeptides are also embraced by the invention. As used herein, a persephιn^ARF homolog is a polypeptide encoded by the alternative reading frame of a persephin gene of any other mammalian or nonmamma an species. A persephin gene m another species can be identified by making suitable probes or primers from the genomic sequences shown m Figure 1 , screening a suitable library from the desired species and aligning the sequence of an identified clone against one or more of the genomic persephin sequences of Figure 1.

The scope of the present invention also includes conservatively substituted vanants of the persephin^ARF amino acid sequences disclosed herein. Conservative amino acid substitutions refer to the lnterchangeabi ty of residues having similar side chains.

Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains. For example, one grouping of amino acids includes those ammo acids that have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M), another grouping is those ammo acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those ammo acids having basic side chains (K, R, and H); another grouping is those ammo acids having acidic side chains (D and E); another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those amino acids having ahphatic-hydroxyl side chains (S and T); another grouping is those amino acids having amine-contaimng side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those ammo acids having sulfur-containmg side chains (C and M). Preferred conservative ammo acid substitutions groups are: R-K; E-D, Y-F, L-M; V-I, and Q-H.

A persephm ^ARF ammo acid sequence as used herein can also include modified sequences in which one or more ammo acids have been inserted, deleted, or replaced with a different amino acid or a modified ammo acid or unusual ammo acid, as well as modifications such as glycosylation or phosphorylation so long as the polypeptide containing the modified sequence retains the biological activity of persephm^ARF. By retaining the biological activity, it is meant that the modified polypeptide has a biological activity profile that is qualitatively similar to, but not necessarily quantitatively equivalent to, that of human, mouse or rat persephm^ARF identified herein Fragments of precursor or mature persephin ^ARF are also encompassed by the present invention. Such fragments may be of any length but should retain the biological activity of precursor or mature persephm ^ARF or should be antigenic. The minimum length of such biologically active or antigenic fragments can readily be determined by those skilled in the art using known techniques. Antigenic fragments are capable of eliciting persephin ^ARF-specιfιc antibodies when administered to a host animal and includes those smaller fragments that require conjugation to a carrier molecule to be immunogenic. Typically, antigenic fragments will be at least 5 or 6 ammo acids in length and may be any length up to the length of precursor persephιn^ARF, preferably 10 to 12 amino acids in length and more preferably, an antigenic fragment will comprise at least 15 to 20 ammo acids, or larger.

Because it is believed that unsphced persephin mRNA is translated, it is likely that a precursor or mature persephιn^ARF polypeptide can be isolated from tissues or cells which naturally express persephin. Alternatively, polypeptides of the present invention can be made by recombinant DNA technology by expressing a nucleotide sequence encoding the desired persephin^ARF amino acid sequence in a suitable transformed host cell. Using methods well known in the art, a polynucleotide encoding persephin^ARF may be operably linked to an expression vector, transformed into a host cell and culture conditions established that are suitable for expression of the polypeptide by the transformed cell.

Any suitable expression vector may be employed to produce recombmant persephm^ARF such as, for example, the mammalian expression vector pCB6 (Brewer, Meth Cell Biol 43.223-245, 1994) or the E coh pET expression vectors, specifically, pET-30a (Studier et al , Methods Enzymol 755:60-89, 1990) Other suitable expression vectors for expression in mammalian and bacterial cells are known m the art as are expression vectors for use in yeast or insect cells Baculovirus expression systems can also be employed.

A number of cell types may be suitable as host cells for expression of recombmant precursor or mature persephιn^ARF Mammalian host cells include, but are not limited to, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo 205 cells, 3T3 cells, CV-1 cells, other transformed pnmate cell lines, normal diploid cells, cell strains denved from in vitro culture of pnmary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells. Yeast strains that may act as suitable host cells include Saccharomyces cerevisiae,

Schizosaccharomyces pombe, Kluyveromyces strains, Candida, and any other yeast strain capable of expressing heterologous proteins. Host bactenal strains include Escherichia coh, Bacillus subtihs, Salmonella typhimurium and any other yeast strain capable of expressing heterologous proteins. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide, for example, by phosphorylation or glycosylation of the appropriate sites using known chemical or enzymatic methods, to obtain a biologically active polypeptide

The polypeptide of the invention may also be expressed in transgenic animals, e.g., cows, goats, pigs, or sheep whose somatic or germ cells contain a nucleotide sequence encoding persephin ^ARF. The expressed persephιn^ARF polypeptide can be purified using known purification procedures, such as gel filtration and ion exchange chromatography. Punfication may also include affinity chromatography using an agent that will specifically bind the persephm^ARF polypeptide, such as a polyclonal or monoclonal antibody raised against persephm^ARF or fragment thereof. Other affinity resms typically used in protein punfication may also be used such as concanava n A-agarose, hepann-toyopearl^® or Cibacrom blue 3GA Sepharose^®. Purification of persephm^ARF can also include one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether

It is also contemplated that a persephm^ARF polypeptide may be expressed as a fusion protein to facilitate purification. Such fusion proteins, for example, include a persephιn^ARF amino acid sequence fused to a histidme tag such as when expressed in the pET bacterial expression system as well as the persephm^ARF amino acid sequence fused to the amino acid sequence of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxm (TRX) Similarly, the polypeptide of the invention can be tagged with a heterologous epitope and subsequently purified by immunoaffmity chromatography using an antibody that specifically binds such epitope. Kits for expression and punfication of such fusion proteins and tagged proteins are commercially available

Precursor or mature persephm^ΛRF and fragments thereof may also be produced by chemical synthesis using methods known to those skilled in the art

In another embodiment, the present invention provides an isolated and punfied polynucleotide compnsmg a nucleotide sequence that encodes a persephm^ΛRF ammo acid sequence As used herein, a polynucleotide includes DNA and/or RNA and thus the nucleotide sequences recited in the Sequence Listing as DNA sequences also include the identical RNA sequences with uracil substituted for thymine residues. Nucleotide sequences included m the invention are those encoding the human, mouse and rat persephm^ARF ammo acid sequences set forth m SEQ ID NOS- 1-6 It is understood by the skilled artisan that degenerate nucleotide sequences can encode the persephm^ARF ammo acid sequences described herein and these are also intended to be included within the present invention. Such nucleotide sequences include modifications of naturally-occurnng sequences in which at least one codon is substituted with a corresponding redundant codon preferred by a given host cell, such as E. coh or insect cells, so as to improve expression of recombmant persephin ^ARF therein. A preferred polynucleotide of the invention encodes precursor human persephιn^ARF and compnses SEQ ID NO:7 Another preferred polynucleotide encodes mature human persephm^ARF and compnses SEQ ID NO:8.

The present invention also encompasses vectors comprising an expression regulatory element operably linked to any of the persephin ^ARF-encodιng nucleotide sequences included within the scope of the invention This mvention also includes host cells, of any vanety, that have been transformed with such vectors

In another embodiment, the present invention also provides a polynucleotide compnsmg a first nucleotide sequence which is complementary to a second nucleotide sequence encoding a persephm^ARF ammo acid sequence As used herein, complementary nucleotide sequences have the same length. Complementary nucleotide sequences include SEQ ID NOS: 13-18. Preferably the first nucleotide sequence is complementary to a human nucleotide sequence encoding precursor or mature persephm^ARF and comprises SEQ ID NO:13 or SEQ ID NO:14.

In yet another embodiment, a polynucleotide which specifically hybndizes to the lntromc portion of a human persephιn^ARF-encodιng polynucleotide or its complement is provided. Specific hybridization is defined herein as the formation of hybrids between a polynucleotide, including oligonucleotides, and a specific reference polynucleotide (e g., a polynucleotide compnsmg a nucleotide sequence complementary to a nucleotide sequence encoding persephm^ARF) wherein the polynucleotide preferentially hybndizes to the specific reference polynucleotide over other polynucleotides Specific hybridization is preferably done under high stringency conditions which, as well understood by those skilled in the art, can readily be determined by adjusting several factors dunng hybridization and during the washing procedure, including temperature, ionic strength, length of hybridization or washing times, and concentration of formamide (see for example, Sambrook et al , 1989, supra) The present invention also provides methods for detecting expression of persephιn^AR mRNA in a sample comprising detecting an unsphced transcript of the persephin gene In one embodiment, the method compnses contacting the unsphced transcript with a polynucleotide probe which specifically hybndizes to the intron of the unsphced transcnpt and detecting a hybnd duplex between the probe and the unsphced transcript The sample may compnse cultured cells of any type or may be a blood or tissue sample from a mammal The sample is treated using techniques known in the art to make its mRNA available for hybridization to the probe or, alternatively, the mRNA may be extracted from the sample and may then be subjected to gel electrophoresis or other size separation techniques

The polynucleotide probe compnses a nucleotide sequence containing a minimum of at least about 6, preferably at least about 8, more preferably at least about 10 to 12, and even more preferably at least about 15 to 20 contiguous nucleotides which are complementary to a sequence of the same length in the mtron of unsphced persephin mRNA Preferably, the polynucleotide probe is complementary to the intron sequence of human, rat or mouse persephin mRNA as set forth in SEQ ID NOS.19-21 or a fragment thereof The polynucleotide probe may be composed of DNA and/or RNA, and/or a synthetic oligonucleotide analog As used herein, "synthetic oligonucleotide analog" includes oligonucleotides containing one or more modified bases, oligonucleotides with a modified phosphate backbone such as methylphosphonate, phosphorothioate and phosphoramidate, and oligonucleotides with a phosphate-free backbone such as polyamide (peptide nucleic acids) The polynucleotide probe may be prepared by any method known in the art such as, for example, chemical synthesis, excision from a DNA fragment, transcnption, or reverse transcription

To enable detection of the hybnd duplex, the probe may be labeled with any detectable label known in the art such as, for example, radioactive or fluorescent labels or enzymatic markers. Labeling of the probe can be accomplished by any method known m the art such as by PCR, random pnming, end labeling, nick translation or the like. One skilled m the art will also recognize that other methods not employing a labeled probe can be used to detect the hybnd duplex. Examples of methods that can be used for detecting hybndization include Southern blotting, fluorescence in situ hybridization, and single-strand conformation polymorphism with PCR amplification

Hybridization conditions for the type of probe used may be readily determined by those skilled in the art High stringency conditions are preferred in order to prevent false positives The stnngency of hybridization is determined by a number of factors in the hybridization and washing steps. Such factors are well known to those skilled in the art and outlined in, for example, Sambrook et al , 1989, supra.

In another embodiment, detection of the unsphced persephin transcript in the sample comprises performing a reverse transcnption reaction on the unsphced transcnpt to produce a cDNA encoding persephιn^ARF, amplifying a target sequence in the cDNA, and detecting the amplified target sequence. The target sequence can be of any length and can mclude the full length of the cDNA. In one embodiment, the target sequence comprises the mtron sequence of unsphced persephm mRNA or a fragment thereof. Amplification of the target sequence can be by any amplification method known m the art, including polymerase chain reaction (PCR) and hgase chain reaction (LCR). Detection of the amplified target sequence can be accomplished using any known method such as separation of reaction products by gel electrophoresis and detecting a fragment of the expected size and/or incubation of the reaction products with a probe which specifically hybndizes to the target sequence. Methods are also provided herein for producing persephιn^ARP. Preparation can be by isolation from conditioned medium from a cell type that produces persephm^ARF. A second and prefened method involves utilization of recombmant methods by isolating a polynucleotide encoding precursor or mature persephm^ARF, cloning the polynucleotide along with appropnate regulatory elements into suitable vectors and cell types, and expressing the polynucleotide to produce precursor or mature persephm^ARF.

Another embodiment of the invention provides an isolated and punfied antibody which specifically reacts with a persephm^ARF polypeptide or an epitope thereof. By epitope reference is made to an antigenic determinant of a polypeptide. An epitope could compnse 3 ammo acids in a spatial conformation which is unique to the epitope. Methods of determining the spatial conformation of amino acid sequences are known m the art, and include, for example, x-ray crystallography and 2 dimensional nuclear magnetic resonance. Typically an epitope consists of at least 5 or 6 contiguous ammo acids of a persephιn^ARF ammo acid sequence.

One approach for prepanng antibodies to a protein is the selection and preparation of an amino acid sequence of all or part of the protein, chemically synthesizing the sequence and injecting it into an appropriate animal, usually a rabbit or a mouse. Peptide candidates for the production of an antibody specific to a persephin ^ARF polypeptide can be based upon amino acid sequences lying in hydrophilic regions which are thus likely to be exposed in the mature protein. Such peptides can be prepared by chemical synthesis using methods known in the art, e.g., the classical Merrifeld method of solid phase peptide synthesis (Merrifeld, J Am Chem Soc <°5:2149, 1963 or the FMOC strategy on a Rapid Automated Multiple Peptide Synthesis system (DuPont Company, Wilmington, DE) (Caprino and Han, J Org Chem 37:3404, 1972). In a preferred embodiment, the anti-persephin^ARF antibody is specific for human persephin^ARF and reacts with an epitope from SEQ ID NO: 1 or SEQ ID NO:4. The invention includes both polyclonal antibodies and monoclonal antibodies. Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate intervals. The animals are bled and sera assayed against purified persephin^ARF, usually by ELISA or by bioassay based upon the ability to block one or more of the biological activities of persephin^ARF. When using avian species, e.g. chicken, turkey and the like, the antibody can be isolated from the yolk of the egg. Monoclonal antibodies can be prepared, for example, by fusing antibody-producing cells from immunized mice with continuously replicating tumor cells such as myeloma or lymphoma cells. (Kohler and Milstein, Nature 256:495-491, 1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone and Banatis eds., Academic Press, 1981). The hybridoma cells so formed are then cloned by limiting dilution methods and supernates thereof are assayed for antibody production by ELISA, RIA or bioassay.

Polyclonal or monoclonal antibodies to persephin^ARF or an epitope thereof may be used in a method for detecting persephin^ARF in a sample. Any antibody-based method known in the art for detecting proteins may be used. Such methods include, but are not limited to immunodiffusion, immunoelectrophoresis, immunochemical methods, binder-ligand immunoassays, immunohistochemical techniques, agglutination and complement assays. (For example, see Basic and Clinical Immunology, Sites and Ten, eds., Appleton & Lange, Norwalk, Conn, pp 217-262, 1991). Preferred are binder-ligand immunoassay methods which involve reacting antibodies with an epitope or epitopes of persephin^ARF or derivative thereof to competitively displace a labeled persephin^ARF polypeptide.

Antibodies useful in detecting persephin^ARF are intended to include complete anti- persephin^ARF antibody molecules and persephin^ARF -binding fragments of such antibody molecules. The anti-persephin^ARF antibody may be unlabeled, for example as used in agglutination tests, or labeled for use in a wide variety of assay methods. Labels that can be used include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates or co- factors, enzyme inhibitors, particles, dyes and the like for use in radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays and the like.

The present invention also includes therapeutic or pharmaceutical compositions comprising a persephιn^ΛRF polypeptide and a method for treating a patient suffenng from a medical condition associated with a lack of, or reduced amount of, at least one of the biological activities of persephιn^ARF, the method comprising administenng to the patient a therapeutically effective amount of a persephm^ΛRF polypeptide. Potential biological activities for persephm^ARF and medical conditions associated therewith are descnbed below. As used herein, "therapeutically effective amount" means the amount of the persephιn^ARF polypeptide sufficient to show a meaningful benefit to the patient, l e , prevention, healing or amelioration of the relevant medical condition, or an increase in rate of healing or amelioration of the condition.

In certain circumstances, it may be desirable to modulate or decrease the effect of endogenously synthesized persephm^ARF. This may be achieved by blocking the activity of endogenously synthesized persephm^ARF or by decreasing expression of persephιn^ARF. Thus, appropriate treatments, for example, may involve administration of antι-persephm^ARF antibodies or other compounds having persephin ^ARF antagonist properties, or the use of antisense polynucleotides to modulate persephm^ARF expression

Specific antibodies, either polyclonal or monoclonal, including persephin ^ARF-bmdmg fragments thereof, may be capable of preventing persephm^ARF from exerting one or more of its biological effects Such antibodies can be produced by any suitable method known m the art. For example, munne or human monoclonal antibodies can be produced by hybndoma technology or by combmatonal antibody library technology, including panning a phage display library The antibody may be engineered using recombmant techniques to produce an antibody with desirable characteristics such as being "humanized" to be better tolerated by the patient. Such antibody engineenng techniques are known in the art. See for example, Hayden et al , Curr Opin Immunol 9(2).201-212, 1997 Alternatively, a persephm^ARF polypeptide, or an immunologically active fragment thereof, or an anti-idiotypic antibody, or fragment thereof can be administered to an animal to elicit the production of antibodies capable of recognizing and binding to persephm^ARF. Such antibodies can be from any class of antibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE or in the case of avian species, IgY and from any subclass of antibodies

In another aspect of the present invention, persephm^ARF antisense oligonucleotides can be made and used in a method for diminishing the level of expression of persephin ^AR protein by a cell which compnses administenng one or more persephιn^ARF antisense oligonucleotides. By persephm^ARF antisense oligonucleotides reference is made to oligonucleotides that have a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of persephin^ARF such that the expression of persephin^ARF is reduced. Preferably, the specific nucleic acid sequence involved in the expression of persephin ^ARF is contained within a genomic DNA molecule or mRNA molecule that encodes persephin^ARF. The antisense oligonucleotide may be directed against a regulatory region of the persephin gene and/or coding sequences for precursor or mature persephin^ARF. By complementary to a nucleotide sequence in the context of antisense oligonucleotides and methods therefor is meant sufficiently complementary to such a sequence as to allow hybridization to that sequence in a cell, i.e., under physiological conditions. The persephin^ARF antisense oligonucleotides preferably comprise about 8 to about 100 nucleotides and more preferably the persephin^ARF antisense oligonucleotides comprise from about 15 to about 30 nucleotides.

The persephin^ARF antisense oligonucleotides can also include derivatives which contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified intemucleoside linkages, modified nucleic acid bases and/or sugars and the like (Uhlmann and Peyman, Chemical Reviews 90:543-584, 1990; Schneider and Banner, Tetrahedron Lett 37:335, 1990; Milligan et al., JMed Chem 36: 1923-1937, 1993; Tseng et al., Cancer Gene Therap 7:65-71, 1994; Miller et al., Parasitology 70:92-97, 1994). Such derivatives include but are not limited to backbone modifications such as phosphotriester, phosphorothioate, methylphosphonate, phosphoramidate, phosphorodithioate and formacetal as well as morpholino, peptide nucleic acid analogue and dithioate repeating units.

The therapeutic or pharmaceutical compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that a persephin^ARF polypeptide or an antibody thereto be administered to cells in the central nervous system, administration can be by intravenous injection with one or more agents capable of promoting penetration of persephin^ARF or the anti- persephin^ARF antibody across the blood-brain barrier such as an antibody to the transferrin receptor. Co-administration may comprise physically coupling any known blood-brain penetrating agent to the persephin^ARF polypeptide. (See for example, Friden et al., Science 259:313-311, 1993). A persephin^ARF polypeptide or antibody thereto can also be linked or conjugated with agents that provide other desirable pharmaceutical or pharmacodynamic properties. For example, a persephin ^ARF polypeptide can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties. (See, e.g., Davis et al Enzyme Eng 4 169-73, 1978, Burnham, Am JHosp Pharm 57:210-218, 1994) The compositions are usually employed in the form of pharmaceutical preparations

Such preparations are made in a manner well known m the pharmaceutical art One prefened preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, stenle water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophilized and stored in a stenle ampoule ready for reconstitution by the addition of stenle water for ready injection. The pnmary solvent can be aqueous or alternatively non- aqueous. A persephm^ARF polypeptide can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requinng treatment. The earner can also contain other pharmaceuticaHy-acceptable excipients for modifying or maintaining the pH, osmolanty, viscosity, clanty, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the earner may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barner. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or penodic infusion

It is also contemplated that certain formulations containing a persephιn^ARF polypeptide are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable earners in solid dosage forms. Some examples of suitable earners, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalhne cellulose, polyvmylpyrrohdone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubncating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavonng agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known m the art. The formulations can also contain substances that dimmish proteolytic degradation and promote absorption such as, for example, surface active agents. The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied The dose will also be calculated dependent upon the particular route of administration selected Further refinement of the calculations necessary to determine the appropnate dosage for treatment is routinely made by those of ordinary skill in the art Exact dosages are determined in conjunction with standard dose-response studies It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the seventy of the patient's symptoms, and the chosen route of administration Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used

It is also contemplated that the expression of persephm^ARF could be increased in tissues defective m such expression by gene therapy Patients may be implanted with vectors or cells capable of producing a biologically-active persephm^ARF polypeptide Cells can be grown ex vivo, for example, for use in transplantation or engraftment into patients (Muench et al , Leuk & Lymph 76-1-11, 1994). In one approach, cells that secrete persephιn^ARF may be encapsulated into semipermeable membranes for implantation into a patient The cells can be those that normally express a persephm^ARF polypeptide or the cells can be transformed to express a persephm^ARF polypeptide When the patient is human, it is prefened that the persephm^ARF be human persephm^ARF. However, the formulations and methods herein can be used for vetennary as well as human applications and the term "patient" as used herein is intended to include human and vetennary patients

The isolated polynucleotides and polypeptides of the present invention are expected to exhibit one or more of the uses or biological activities identified below Research Uses and Utilities

The polynucleotides and polypeptides provided by the present invention can be commercialized as reagent grade research products or in kit format for use by the research community for vanous purposes For example, polynucleotides encoding persephm^ARF can be used to express recombmant persephm^ARF for charactenzmg its biological activity in vitro or in vivo Polynucleotides complementary to persephm^ARF-encodmg polynucleotides and fragments thereof can also be used to identify tissues that express persephιn^ARF and in subtractive hybndization techniques that remove known polynucleotides to facilitate discovery of other novel polynucleotides expressed in those tissues. The polynucleotides of the invention can be used to analyze genomic DNA sequences in vanous populations to identify polymorphisms that may be useful in DNA fingerprinting applications or may be linked to genetic disorders. In addition, persephin^ARF-encoding polynucleotides and fragments thereof can be administered to an animal to raise anti- persephin^ARF antibodies using DNA immunization techniques. Similarly, persephin^ARF polypeptides provided by the invention can be used in various assays to characterize biological activity, including in a panel of multiple proteins for high- throughput screening to identify a candidate drug that interacts with a target receptor or to induce or inhibit a desired cellular response. In addition, persephin^ARF polypeptides can be used to raise antibodies for use in identifying cell types that synthesize and secrete persephin ^ARF' The polypeptides of the invention can also be used to identify proteins that bind to persephin^ARF including potential receptors or ligands.

Methods for performing the research activities described above are well known to those skilled in the art and are described in various references, including without limitation "Molecular Cloning: A Laboratory Manual", 2d ed. Cold Spring Harbor Laboratory Press, Sambrook et al., eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987. Cell Proliferation/Differentiation Activity

Because the persephin^ARF mRNA encodes a polypeptide with a secretion signal sequence and is expressed by a gene that also expresses persephin, a growth factor that promotes the survival of certain types of neurons, persephin^ARF protein may also act as a cytokine, i.e., persephin^ARF may induce or inhibit cell proliferation or cell differentiation or may regulate the expression of other cytokines, including possibly persephm. A persephin^ARF polypeptide of the present invention can be tested for such activities on a number of known cytokine-dependent cell types grown as primary cultures or from established cell lines including, without limitation, sympathetic neurons, sensory neurons, mesencephalic cells 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, Tl 165, HT2, CTLL2, TF-1, Mo7e and CMK, using well-known assays routinely performed in the art.

For example, persephin^ARF may be tested for neurotrophic activity using assays such as, but not limited to, the superior cervical ganglion survival and nodose ganglion survival assays described in U.S. Patent No. 5,747,655, the dopaminergic neuron survival assay described in Lin et al., Science 260: 1130-1132, 1993, the chick embryo ciliary ganglia survival assay described in U.S. 5,011,914, and assays for nerve tissue generation as described in International Patent Publication No. WO95/05846. Demonstration of neurotrophic activity for persephin ^ARF would indicate its utility in treating one or more diseases and medical conditions associated with neuronal degeneration such as peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Shy-Drager syndrome, lschemic stroke, acute brain or spinal chord injury, nervous system tumors, multiple sclerosis, peripheral nerve trauma or injury, exposure to neurotoxms, metabolic diseases such as diabetes or renal dysfunction and damage caused by infectious agents.

Similarly, persephm^ARF may be tested for hematopoietic activity using assays for proliferation and differentiation of vanous cell types in the hematopoietic system, including without limitation (1) assays for stem cell survival and differentiation as described in Johansson et al., Cellular Biology 75:141-151, 1995; Keller et al., Molecular and Cellular Biology 73:473-486, 1993; McClanahan et al., Blood 57:2903-2915, 1993; Hirayama et al, Proc. Natl. Acad. Sci. USA 59:5907-5911, 1992; Neben et al., Exper. Hematol. 22:353-359; and Culture of Hematopoietic Cells, R. I. Freshney et al. eds., Wiley-Liss, Inc., New York, NY 1994, pp. 1-21, 23-39, 139-179, and 265-268; (2) assays for thymocyte or T-cell proliferation as descnbed m Current Protocols in Immunology, J.E. Cohgan et al. eds., John Wiley and Sons, 1998, Chapters 3 and 7; Takai et al., J Immunol. 737:3494-3500, 1986; Bertagnoh et al., J. Immunol. 745:1706-1712, 1990; Bertagnoh et al., Cellular Immunol. 733:327-341, 1991; Bertagnoh et al., J. Immunol. 749:3778-3783, 1992; Bowman et al., J. Immunol. 752: 1756-1761, 1994; (3) assays for proliferation and differentiation of other myeloid- and lymphoid-denved cells as descnbed in Current Protocols in Immunology, infra at pp. 6.3.1-6.3.12, 6.6.1-6.6.5, 6.13.1 and 6.15.1; deVries et al., J. Exp. Med. 773:1205-1211, 1991; Moreau et al., Nature 33(5:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. USA 50:2935-2938, 1983; and Smith et al., Proc Natl Acad. Sci USA 53: 1857-1861, 1986; and (4) assays for stimulation or inhibition of cytokine production such as descnbed in Current Protocols in Immunology, infra at pp..3.12.1-3.12.14 and 6.8.1-6.8.8. Demonstration that persephm^ARF has hematopoietic activity would indicate utility in treating one or more diseases or conditions associated with an insufficient number of blood cells, such as for example, leukopenias including eosinopenia and/or basopema, lymphopema, monocytopema, neutropema, anemias, thrombocytopema or conditions associated with an insufficient number of stem cells including stem cell disorders such as aplastic anemia and paroxysmal nocturnal hemoglobinuna, and stem-cell deficient bone manow following inadiation or chemotherapy.

Persephιn^ARF may also promote or inhibit growth and differentiation of one or more of the following tissues: bone, cartilage, tendon, ligament, muscle (smooth, skeletal or elastic), vascular, and organs including without limitation endothe um, intestine, kidney, liver pancreas, skin, and pancreas. A growth promoting activity may promote the growth and/or differentiation of cells comprising such tissues and/or promote the generation or regeneration of these tissues or alternatively, may inhibit fibrotic scarnng to allow normal tissue to regenerate. Persephm^ΛRF may also exhibit wound healing activity.

A persephin ^ARF polypeptide of the invention can be tested for tissue generation activity using assays known in the art including, without limitation, assays for promoting growth of bone, cartilage and tendon as descnbed in International Patent Publication No WO95/16035 and assays for promoting skin and endothe um growth as descnbed in International Patent Publication No. WO/91/07491. The ability of persephm^ARF to promote wound healing can be tested using assays such as those described in Epidermal Wound Healing, Maibach, H.I. and Rovee, D.T., eds, Year Book Medical Publishers, Inc., Chicago, 1992, pp 71-112, as modified by Eaglstem and Mertz, J. Invest Dermatol 77.382-84, 1978 Demonstration that persephm^ARF promotes cartilage and/or bone growth would indicate its utility m healing bone fractures and cartilage damage, m improving fixation of artificial joints, m repair of congenital or trauma-mduced cramalfacial defects, and in cosmetic plastic surgery A protein that promotes bone and cartilage growth may also be useful m treating osteoporosis, osteoarthntis or penodontal disease. The ability to induce tendon and/or ligament formation would make persephm^ARF a candidate agent for treating tears or deformities in tendon and/or ligament tissues, for improving fixation of tendon or ligament to bone or other tissues and for treating conditions such as tendinitis and carpal tunnel syndrome It is also expected that persephm^ARF may have activity in promoting better or faster healing of wounds. Such activity would make persephm^ARF useful, for example, m treating non-healing wounds such as pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. Immune Stimulating or Suppressing Activity Persephm^ARF may also stimulate or suppress the immune system by, for example, regulating growth and proliferation of T, B, and NK cells, effecting or blocking the cytolytic activity of NK cells and other cytotoxic cell populations, inducing or suppressing T cell responses, regulating ratio of T_hl and T_h2 cells, activating expression of class I and/or class II MHC molecules, and inducing or blocking the production of cytokmes. Exhibition of an immune stimulating activity by persephm^ARF would indicate a utility in treating vanous immune deficiencies such as severe combined immunodeficiency (SCID) and AIDS or in treating infectious diseases caused by bacterial, viral and fungal infections , including infections by mycobactena, Leishmania spp., malana spp., Salmonella typhi, pathogenic strains of E. coli, HIV, hepatitis viruses, herpesviruses, and candidiasis. Alternatively, a determination that persephιn^ARF has immune suppressing activity would indicate this protein would be useful in treating one or more autoimmune disorders and allergic reactions or conditions including without limitation multiple sclerosis, systemic lupus erthematosus, rheumatoid arthritis, insulin dependent diabetes melhtis, connective tissue disease, autoimmune pulmonary inflammation, Guillam-Bane syndrome, autoimmune thyroiditis, Goodpasture's syndrome, myasthema gravis, graft-versus-host disease and autoimmune inflammatory eye disease, asthma, hay fever, glomerulonephntis, and contact dermatitis

Testing persephm^ARF for immune stimulating and/or suppressing activity may be done using assays known in the art, including without limitation the hematopoietic assays described above as well as the following methods-

Assays for modulation of T-cell dependent antibody responses and T_hl/Tι,2 ratios including, e g , those described m Mahszewski, J Immunol 144 3028-3033, 1990,

Assays for B cell function, as descnbed, e.g., in Current Protocols in Immunology, infra at pp. 3.8.1-3.8.16;

Mixed lymphocyte reaction (MLR) assays, such as those descnbed in Current Protocols in Immunology, infra at pp. 3.1-3.19 and Chapter 7, Takai et al , J Immunol 137, supra, Takai et al., J Immunol 740:508-512, 1988, and Bertagnoh et al, J Immunol 149, supra;

Assays for thymocyte or splenocyte cytotoxicity which are descnbed, e.g., in Current Protocols in Immunology, infra at pp. 3.1-1.19 and Chapter 7, Hermann et al , Proc Natl Acad Sci USA 75-2488-2492, 1981, Herrmann et al , J Immunol 725:1968-1974, 1982; Handa et al., J Immunol 735:1564-1572, 1985, Takai et al., J Immunol 137, supra,

Bowman et al., J Virol 67:1992-1998, 1987, Takai et al., J Immunol 740, supra, Bertagnoh et al , Cell Immunol 133, supra, and Brown et al , J Immunol 753:3079-3092, 1994;

Assays for effect on T cell commitment and development include those described in Antica et al., fi/ood 54:111-117, 1994; Fine et al., Cellular Immunol. 755:111-122, 1994; Galy et al., Blood 55:2770-2778, 1995; Toki et al , Proc Nat Acad Sci USA 55:7548-7551, 1991; and

Assays for effect on lymphocyte survival and/or apoptosis such as those described in Darzynkiewicz et al., Cytometry 73:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993, Gorczyca et al, Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991 ; Zacharchuk, J of Immunol 745:4037-4045; 1990; Zamai et al., Cytometry 74:891-897, 1993; Gorczyca et al., Internal J Oncol 7:639-648, 1992 Activm/Inhibm Activity

Persephin ^ARF may also exhibit activity similar to that of activms and inhibins, polypeptide factors which induce or inhibit release of follicle stimulating hormone (FSH) from antenor pituitary cells, respectively. Thus, based on the ability of activins and mhibins to increase or decrease fertility in mammals, a similar activity for persephm^ARF would indicate this protein, alone or in combination with another activin or mhibin, may be useful as a contraceptive or conversely, in treating infertility. Persephιn^ARF may be tested for activm or mhibin activity using assays known in the art, including without limitation those descnbed m Vale et al., Endocrmol 97:562-572, 1972; Ling et al., Nature 327:779-782, 1986; Vale et al, Nature 321-116-119, 1986; Mason et al., Nature 375:659-663, 1985; Forage et al , Proc Natl Acad Sci USA 53:3091-3095, 1986. Chemotactic/Chemokinetic Activity

It is also expected that persephιn^ARF may exhibit chemotactic or chemokinetic activity for one or more mammalian cells, including without limitation monocytes, fibroblasts, neutrophils, T cells, mast cells, eosmophiles, epithelial and/or endothelial cells A protein has chemotactic activity for a particular cell population if it can directly or indirectly stimulate directed orientation or movement of cells in the population. Due to an ability to attract cells involved m the immune response to a desired site of action, proteins having chemotactic or chemokinetic activity may be useful in treating wounds and other tissue damage, as well as in treating localized infections and tumors.

Persephιn^ARF may be tested for chemotactic activity using known assays that measure the ability of a protein to induce migration of cells across a membrane as well as the ability to induce adhesion of one cell population to another cell population. Such assays include, wthout limitation, those descnbed in Current Protocols in Immunology, infra at Chapter 6.12; Taub et al , J Clin Invest. 95:1370-1376, 1995; Lind et al., APMIS 703: 140-146, 1995; Muller et al, Eur J Immunol 25:1744-1748, 1995; Gruber et al., J. Immunol 752:5860- 5867, 1994, Johnston et al., J Immunol 753:17 '62-1 '68, 1994. Anti-inflammatory Activity

A persephin ^ARF polypeptide of the invention may have anti-mflammatory activity which may include one or more of the following: inhibiting vascular permeability, inhibiting production or binding of cell adhesion molecules, inhibiting chemotaxis and release of chemical mediators by cells involved m the inflammatory response, and/or inhibiting activation or proliferation of cells involved in initiating or promoting the inflammatory response. A protein having anti-inflammatory activity may have utility in treating acute or chronic inflammatory conditions, including without limitation conditions associated with infection such as septic shock, lschemia-reperfusion injury, arthritis, nephntis, inflammatory bowel disease, Crohn's disease, anaphylaxis, cytokme or chemokine-mduced lung injury, complement-mediated hyperacute rejection, and overproduction of cytokmes such as TNF or IL-1. Hemostatic or Thrombolytic Activity

Persephιn^ARF may exhibit hemostatic activity, which would indicate its utility m treating vanous coagulation disorders, such as hemophilias, or to enhance coagulation m treating wounds resulting from trauma, surgery and the like Alternatively, persephm^ARF may have thrombolytic activity and would thus be a candidate therapeutic for inhibiting or dissolving formation of thrombosis and for preventing or treating conditions resulting therefrom Persephm^ARF may be tested for hemostatic or thrombolytic activity using known assays such as Linet et al , J Clin Pharmacol 26 131-140, 1986, Burdick et al , Thrombosis Res 45-413-419, 1987, Humphrey et al, Fibnnolysis 5.71-79, 1991, Schaub, Prostaglandins 35 467-474, 1988

Other Biological Activities

It is expected that persephιn^ARF may have any one or more of other activities that would make persephm^ARF useful in treating medical conditions that would benefit from providing the activity. Such activities include without limitation: inhibiting tumor growth directly or indirectly; receptor activity for ligands such as growth factors and other cytokmes; analgesic or other pain reducing activity, activities that inhibit or increase the metabolism, catabohsm, anabohsm, processing, utilization, storage or elimination of dietary fat, hpid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or components; activities that alter behavioral charactenstics, including appetite, libido, stress, cognition, depression, addiction and violent behaviors; activities involved m regulating biorhythms; inhibiting or increasing fertility of male or female mammals, and activities that affect body charactenstics such as height, weight, muscle mass, breast size, tissue pigmentation, hair growth or color

Preferred embodiments of the invention are descnbed m the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spmt of the invention being indicated by the claims which follow the examples Example 1

This example illustrates the relative levels of unsphced and spliced transcnpts of the persephin gene in various rat tissues as determined by semi-quantitative RT-PCR analysis

Poly A⁺ RNA was isolated from heart, kidney, liver and brain tissues of embryonic and adult rat and semiquantitative RT-PCR was performed essentially as descnbed by Freeman et al. Neuron 72.343-355, 1994. In bnef, 1 μg of each RNA sample was used as template in separate reverse transcnption (RT) reactions earned out at 37° C for 1 hr after which the reaction mixtures were diluted (5:1) with H₂O and boiled for 5 mm to kill the reverse transcnptase Control reactions for each RNA sample were performed in the absence of reverse transcnptase to test for genomic DNA contamination

A quots of the diluted RT reaction mixtures were used in PCR reactions with forward (5 ^'-AGCTCAGCCACTGGTAGGGTCAGG-3 ') (SEQ ID N0.22) and reverse

(5 '-AGCTCAGCCACTGGTAGGGTCAGG-3') (SEQ ID NO 23) primers corresponding to the persephin cDNA sequence The amount of each RT reaction used for PCR analysis was normalized by performing preliminary PCR reactions with pnmers to β-actm The PCR parameters were 95°C for 30 s, 65°C for 30 s, then 72°C for 30 s for 26, 29 or 32 cycles. Products of the PCR reactions were separated on a 2.8% agarose gel, blotted onto a nylon membrane and then detected by hybndization to a ³²P-lableled persephin probe. The results, as visualized by a Phosphorlmager® (Molecular Dynamics, Sunnyvale, CA), are shown in Figure 3

Two species of persephin mRNA were amplified by the RT/PCR reaction with the larger, more abundant species corresponding to the unsphced transcnpt of the persephin gene. The levels of both spliced and unsphced mRNAs were constant from embryonic day 10 through embryonic day 18 (El 8) (data not shown) Slightly higher levels of both persephin mRNA species were observed in embryonic tissues other than bram

Example 2 This example illustrates the detection of unsphced and spliced transcripts of the persephin gene in human fetal bram tissue

Human fetal bra poly A⁺ RNA (1 μg) (CLONTECH) was used as the template in a reverse transcnptase (RT) reaction A control reaction was also performed in which no reverse transcnptase enzyme was added. These reactions were incubated at 37° C for 1 hr, diluted (5 1) with H₂0, and boiled for 5 mm to kill the enzyme

PCR reactions were then performed using forward and reverse primers, [5'- GTCACAATGGCCGTAGGGAAGTT-3'] (SEQ ID NO:24) and [5'- CACCCTCAGCCACCACAGCCGCA-3'] (SEQ ID NO:25) conespondmg to the start and stop sites in the human persephin coding sequence, respectively. The expected size of fragments amplified with these pnmers would be 566 nt and 481 nt conespondmg to the unsphced and spliced species of persephin mRNA, respectively. Templates for the PCR reactions included human genomic DNA as a control for the size of the gene itself (including the intron); the human fetal bram RT reaction (5 μl); and the mock human fetal bram RT reaction (5 μl) in which the RT enzyme was omitted as a control for DNA contamination of the RNA sample. The PCR cycling parameters used were an initial denaturation at 98° C for 2 mm, followed by 40 cycles of 68° C for 1 5 mm and 98° C for 10 sec The PCR products were electrophoresed on a 2 5 % agarose gel and detected by ethidium bromide staining

As shown in Figure 4, fragments conespondmg to the expected size for unsphced and spliced human persephin transcnpts were amplified, with the larger unsphced transcript being present in a much larger amount than the smaller spliced form These results indicate that human fetal brain tissue contains a significant amount of unsphced persephin mRNA, at least a portion of which should be available for translation into human precursor persephin ^ARF

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained As vanous changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained m the above descnption and shown in the accompanying drawings shall be interpreted as illustrative and not m a limiting sense

Claims

What is Claimed is:

1. An isolated and purified polypeptide compnsmg a persephm^ARF amino acid sequence

2. The isolated and punfied polypeptide of claim 1 wherein the persephιn^ΛRr amino acid sequence is a human sequence compnsmg SEQ ID NO. l or SEQ ID NO.4

3 The isolated and punfied polypeptide of claim 1 wherein the persephm^ΛRF ammo acid sequence is a mouse sequence comprising SEQ ID NO:2 or SEQ ID NO.5

4 The isolated and punfied polypeptide of claim 1 wherein the persephm^ARF ammo acid sequence is a rat sequence compnsmg SEQ ID NO: 3 or SEQ ID NO:6.

5. A composition comprising the isolated and punfied polypeptide of claim 2 and a pharmaceutically acceptable earner.

6. An isolated and purified polynucleotide compnsmg a nucleotide sequence encoding a perseph╬╣n^ARF ammo acid sequence as set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO.3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

7. The isolated and punfied polynucleotide of claim 6 wherein the nucleotide sequence comprises a human sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 8.

8. The isolated and purified polynucleotide of claim 6 wherein the nucleotide sequence comprises a mouse sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

9 The isolated and punfied polynucleotide of claim 6 wherein the nucleotide sequence comprises a rat sequence as set forth in SEQ ID NO:l 1 or SEQ ID NO: 12

10. A vector compnsmg a recombmant DNA molecule comprising expression regulatory elements operably linked to a nucleotide sequence encoding the human persephin ^ARF ammo acid sequence of claim 2.

11. A host cell transformed with the vector of claim 10.

12. A persephm^ARF polypeptide expressed by the host cell of claim 11.

13. An isolated and punfied polynucleotide compnsmg a nucleotide sequence complementary to the nucleotide sequence of claim 6

14. The isolated and punfied polynucleotide of claim 12 wherein the complementary nucleotide sequence is SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:18.

15. An isolated and punfied polynucleotide which specifically hybridizes to a polynucleotide consisting of SEQ ID NO: 19 or its complement

16. The isolated and purified polynucleotide of claim 15 which is a perseph╬╣n^ARF antisense oligonucleotide.

17. An isolated and purified antibody which specifically reacts with a persephιn^ARF polypeptide or an epitope thereof, wherein the persephm^ΛRF polypeptide comprises SEQ ID NO- 1 , SEQ ID NO:2, or SEQ ID NO:3

18 A method for detecting expression in a sample of a perseph╬╣n^ARF polypeptide comprising reacting an antibody according to claim 17 with the persephm^ARF polypeptide and detecting binding of the antibody to the persephm^ARr polypeptide.

19 A method for detecting expression of perseph╬╣n^ARF mRNA in a sample comprising detecting an unsphced transcript of the persephin gene in the sample

20. The method of claim 19 wherein detecting the unsphced transcript compnses contacting the unsphced transcript with a polynucleotide probe which specifically hybridizes to the intron of the unsphced transcnpt and detecting a hybrid duplex between the probe and the unsphced transcnpt.

21. The method of claim 20 wherein detecting the unsphced persephin transcript comprises performing a reverse transcnption reaction on the unsphced transcnpt to produce a cDNA encoding persephm^ARF, amplifying a target sequence in the cDNA, and detecting the amplified target sequence.