WO2001029179A2

WO2001029179A2 - Regulation of gene expression by neuromodulators

Info

Publication number: WO2001029179A2
Application number: PCT/US2000/029131
Authority: WO
Inventors: Simon Halegoua; Brian Hilbush; Karl W. Hasel
Original assignee: Digital Gene Technologies, Inc.
Priority date: 1999-10-20
Filing date: 2000-10-20
Publication date: 2001-04-26
Also published as: WO2001029179A3; AU1222601A

Abstract

Polynucleotides, polypeptides, kits and methods are provided related to genes regulated by neuromodulators.

Description

REGULATION OF GENE EXPRESSION BY NEUROMODULATORS

REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application S.N. 60/160,562, filed October 20, 1999, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Nerve growth factor (NGF), the first and prototypic neurotrophin, has been shown to mediate control over neuronal phenotype in vivo (Halegoua et al., Curr. Top. Microbiol. Immunol. 165: 119-70 (1991)). NGF actions which are seen during embryogenesis and in the adult include control of neuronal precursor cell proliferation, differentiation and modulation of neuronal morphology, regulation of neuro transmitter synthesis and secretion, the cessation of cell division and prevention of cell death, the expression of neuronal-specific proteins, synaptogenesis and control of synaptic efficacy (synaptic modulation), and the expression of electrical excitability. The latter includes the specific expression of voltage dependent sodium, calcium and potassium channels, as well as modulation of their activities.

The PC 12 cell line has provided a valuable model for the analysis of the molecular events associated with and which mediate the actions of NGF (Halegoua et al., Curr. Top. Microbiol. Immunol. 165:119-70 (1991)). NGF treatment of PC12 cells results in the promotion and establishment of a neuronal phenotype resembling that of sympathetic and sensory neurons. These neuronal characteristics include the cessation of cell division, the morphological elaboration of neuritic processes, the prevention of cell death in serum-free medium, the synthesis and secretion of catecholaminergic and cholinergic neurotransmitters and their receptors, the expression of neuronal-specific proteins, increased expression of genes encoding various voltage-dependent ion channels and the establishment of a sodium- based action potential mechanism.

Studies on the mechanism of NGF actions in PC 12 cells have revealed a complex signal transduction pathway involving various proto-oncogene products (Halegoua et al., Curr.Top. Microbiol. Immunol. 165:119-70 (1991)). The major functional receptor for NGF is the proto-oncoprotein Trk, a receptor tyrosine kinase. The activation of Trk by NGF has been shown to lead to a sequence of proto-oncoprotein activations involving the Src-Ras-Raf- MAP kinase pathway, which mediates the elaboration of phenotypes including neurite growth, inductions of a variety of genes including those for neuronal specific proteins and ion channels such as voltage-dependent calcium channels, the cessation of cell division and regulation of neurotransmitter synthesis. Branch points along the signaling pathway have been shown to differentially modulate the promotion of specific phenotypes (D'Arcangelo and Halegoua, Mol. Cell. Biol. 13(6):3146-55 (1993)). For example, Ras, but not Raf, leads to the induction of the neuronal gene SCG-10, and the induction of the thy-1 gene is stimulated by Src independently of Ras-Raf-MAP kinase activations.

The establishment of electrical excitability in response to NGF-treatment of PC 12 cells occurs through the induction of the type II and PNl voltage-dependent sodium channel genes (Mandel et al., Proc. Natl. Acad. Sci. USA 85(3):924-28 (1988); D'Arcangelo et al., J. Cell. Biol. 122(4):915-21 (1993)). The induction of PNl, the peripheral neuron-specific sodium channel, is mediated by Trk through a signaling pathway which is independent of Src-Ras-Raf-MAP kinase signaling (D'Arcangelo and Halegoua, Mol. Cell. Biol. 13(6):3146- 55 (1993)). The induction of the PNl gene after 5-24 hours has been found to occur in a triggered manner, in response to a brief (one minute) pulse of NGF, an event which can be mimicked by pulsatile treatment with interferon-gamma (IFN-γ; Toledo-Aral et al., Neuron 14(3):607-11 (1995)). Thus, three aspects of signaling can be used to classify the unique mechanism of PNl gene induction, Ras-independence, triggered induction by NGF, and triggered induction by IFN-γ. Using these criteria, a second gene induction event, for the neuronal intermediate filament protein peripherin, has been similarly classified in PC 12 cells.

The demonstration that two funtionally diverse proteins are similarly classified according to the new induction criteria has led to the hypothesis that a wider subset of neuronal phenotypes induced by NGF and IFN-γ are mediated in a Ras-independent and triggered manner (Toledo-Aral et al., Neuron 14(3):607-11 (1995)). This unique mode of signaling could allow for the long-term manipulation of specific neuronal phenotypes by transient stimulation (or inhibition) of the signaling components. Given the importance of PNl and peripherin to neuronal physiology, additional important phenotypic markers might be expected to be similarly classified. SUMMARY OF THE INVENTION

The PC 12 cell line has served as a useful model in studies examining the mechanisms by which NGF regulates the neuronal phenotype. Such studies have identified some proteins and genes that are associated with and/or which mediate the actions of NGF. Such molecules are useful in therapeutic and diagnostic applications in the treatment of disorders or diseases affecting the control of neuronal precursor cell proliferation, differentiation and modulation of neuronal morphology, regulation of neurotransmitter synthesis and secretion, the cessation of cell division and prevention of cell death, the expression of neuronal-specific proteins, synaptogenesis and control of synaptic efficacy (synaptic modulation), and the expression of electrical excitability. In one embodiment, the invention is useful for adjusting the number of NGF receptors on a cell surface, for modulating the sensitivity of cells to NGF and for modulating the cell's resonse to NGF.

Particularly, polynucleotides and polypeptides of the present invention that effectively modulate NGF metabolism can be used therapeutically and diagnostically in diseases and conditions involving altered target cell metabolism of NGF. Such diseases and conditions include those involving reduced or elevated number of trkA NGF receptors. Also included would be diseases or conditions involving reduced or elevated sensitivity of target cells to NGF due to alterations in internalization of NGF, trkA, or NGF-trkA complex or alternation in intracellular messenger metabolism. Such diseases include forms of Alzheimer's disease characterized by altered trkA numbers and expression (Dubus, P., et al., Expression of trk iso forms in brain regions and in the striatum of patients with Alzheimer's disease, Exp Neurol 2000 Oct;165(2):285-294; Hock, C.H., et al., Alterations in neurotrophins and neurotrophin receptors in Alzheimer's disease, J. Neural Transm. Suppl. 2000;59:171-174; Hock, C.H., et al., Decreased trkA neurotrophin receptor expression in the parietal cortex of patients with Alzheimer's disease, Neurosci Lett 1998 Jan 30;241(2-3):151-154.). Other diseases include diabetic neuropathy (Freeman, R., Human studies of recombinant human nerve growth factor and diabetic peripheral neuropathy, Eur. Neurol. 1999;41 Suppl 1:20-26) and congenital insensitivity to pain with anhidrosis (CIPA) (Miura, Y., et al., Mutation and polymorphism analysis of the trkA (NTRK1) gene encoding a high-affinity receptor for nerve growth factor in congenital insensitivity to pain with anhidrosis (CIPA) families, Hum Genet 2000 Jan; 106(1): 116-24; Shatzky, S., et al., Congenital insensitivity to pain with anhidrosis (CIPA) in Israeli-Bedouins: genetic heterogeneity, novel mutations in the trkA/NGF receptor gene, clinical findings, and results of nerve conduction studies, Am. J. Med. Genet. 2000 Jun 19;92(5):353-60.). The subcutaneous and system administration of nerve growth factor is known to produce painful side effects such as myalgia and hyperalgesia (Rogers, B.C., Development of recombinant human nerve growth factor (rhNGF) as a treatment for peripheral neuropathic disease, Neurotoxicology 1996 Fall-Winter; 17(3-4):865-70; Petty, B.G., The effect of systemically administered recombinant human nerve growth factor in healthy human subjects, Ann. Neurol. 1994 Aug; 36(2):244-246.). Native and modified polynucleotides and polypeptides of the present invention that effectively modulate NGF metabolism can be used therapeutically as an adjunct to NGF therapy to ameliorate such side effects.

The present invention provides novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. Also provided are diagnostic methods for detecting disorders related to the polypeptides and the polynucleotides encoding them, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of the polypeptides.

The PCR-based Total Gene Expression Analysis (TOGA) differential display system has been used in studies to examine how the gene expression is regulated by various agents such neuromodulators. Such studies have examined the mechanism of neuronal differentiation in response to such agents and have examined proteins and genes. Molecules have been identified that correspond to genes that are regulated by neuromodulators in the control of neuronal precursor cell proliferation, differentiation and modulation of neuronal moφhology. Such molecules are useful in therapeutic and diagnostic applications.

The present invention provides novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ED NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29. Also provided is an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to any one of these isolated nucleic acid molecules and an isolated nucleic acid molecule at least ten bases in length that is hybridizable to any one of these isolated nucleic acid molecules under stringent conditions. Any one of these isolated nucleic acid molecules can comprise sequential nucleotide deletions from either the 5 '-terminus or the 3 '-terminus. Further provided is a recombinant vector comprising any one of these isolated nucleic acid molecules and a recombinant host cell comprising any one of these isolated nucleic acid molecules. Also provided is the gene corresponding to the cDNA sequence of any one of these isolated nucleic acids.

Another embodiment of the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ JJD NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29. Also provided is an isolated nucleic acid molecule encoding any of these polypeptides, an isolated nucleic acid molecule encoding a fragment of any of these polypeptides, an isolated nucleic acid molecule encoding a polypeptide epitope of any of these polypeptides, and an isolated nucleic acid encoding a species homologue of any of these polypeptides. Preferably, any one of these polypeptides has biological activity. Optionally, any one of the isolated polypeptides comprises sequential amino acid deletions from either the C-terminus or the N-terminus. Further provided is a recombinant host cell that expresses any one of these isolated polypeptides.

Yet another embodiment of the invention comprises an isolated antibody that binds specifically to an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ DD NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29. The isolated antibody can be a monoclonal antibody or a polyclonal antibody.

Another embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as disorders of altered target cell metabolism of NGF, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention. In other embodiments, the disorder can be Alzheimer's Disease, diabetic neuropathy. congenital insensitivity to pain with anhidrosis, or a side effect of NGF therapy, such as myalgia or hyperalgesia.

A further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide of the invention. A preferred embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as disorders of neuronal differentiation comprising administering to a mammalian subject a therapeutically effective amount of the antibody.

An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject. The method comprises determining the presence or absence of a mutation in a polynucleotide of the invention. A pathological condition or a susceptibility to a pathological condition, such as diseases and conditions involving altered target cell metabolism of NGF, is diagnosed based on the presence or absence of the mutation.

Even another embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject, such as diseases and conditions involving altered target cell metabolism of NGF. The method comprises detecting an alteration in expression of a polypeptide encoded by the polynucleotide of the invention, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition. The alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression. In a preferred embodiment a first biological sample is obtained from a patient suspected of having a disease or condition involving altered target cell metabolism of NGF and a second sample from a suitable comparable control source is obtained. The amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample. The amount of the polypeptide in the first and second samples is determined. A patient is diagnosed as having a disorder of altered target cell metabolism of NGF if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.

Another embodiment of the invention provides a method for identifying a binding partner to a polypeptide of the invention. A polypeptide of the invention is contacted with a binding partner and it is determined whether the binding partner effects an activity of the polypeptide.

Yet another embodiment of the invention is a method of identifying an activity of an expressed polypeptide in a biological assay. A polypeptide of the invention is expressed in a cell and isolated. The expressed polypeptide is tested for an activity in a biological assay and the activity of the expressed polypeptide is identified based on the test results.

Still another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in a disorder of altered target cell metabolism of NGF, chosen from the group consisting of the DNA molecules shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.

Even another embodiment of the invention provides a kit for detecting the presence of a polypeptide of the invention in a mammalian tissue sample. The kit comprises a first antibody which immunoreacts with a mammalian protein encoded by a gene corresponding to the polynucleotide of the invention or with a polypeptide encoded by the polynucleotide in an amount sufficient for at least one assay and suitable packaging material. The kit can further comprise a second antibody that binds to the first antibody. The second antibody can be labeled with enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.

Another embodiment of the invention provides a kit for detecting the presence of genes encoding a protein comprising a polynucleotide of the invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.

Yet another embodiment of the invention provides a method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample. A polynucleotide of the invention or fragment thereof having at least 10 contiguous bases is hybridized with the nucleic acid of the sample. The presence of the hybridization product is detected.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

Figure 1 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases GCAC, showing PCR products produced from mRNA extracted from PC 12 cells treated as follows: A: control wash (CW), B: 1 hour with NGF (lhrNGF), C: 1 hour with IFN-γ (lhrlFN), D: 5 hours with NGF (5hrNGF), E: 5 hours with IFN-γ (5hrIFN), and F: 24 hours with NGF (24hrNGF), where the vertical index line indicates a PCR product of about 349 b. p. that is present in the lhrNGF and lhrlFN samples and enriched in 5hrNGF, 5hrIFN, and 24hrNGF samples;

Figure 2 is a graphical representations of the results of Northern Blot analysis of clone

HAL 18 (GCAC 349), where an agarose gel containing poly A enriched mRNA from PC 12 cells treated with NGF as well as size standards was blotted after electrophoresis and probed with either radiolabelled HAL l 8/rPAST or cyclophilin and imaged using a phosphorimager.. PC 12 cells were treated as follows: 1 hour NGF pulse followed by 2-6 hour chase (samples 1 '2hr, 1 '3hr, 1 '4hr, 1 '5hr, and 1 '6hr), continuous NGF treatment for 2-6 hours (samples 2hr, 3hr, 4hr, 5hr, and 6hr), and no NGF treatment (control). The quantified results for both the 3 kb and 4kb HAL_18/rPast bands normalized to the cyclophilin band in the same lane are presented graphically in Figure 2B.

Figures 3A and 3B are graphical representations of the results of Northern Blot analysis of clone HAL 18 (GCAC 349), where an agarose gel containing poly A enriched mRNA from PC 12 cells treated with NGF as well as size standards was blotted after electrophoresis and probed with either radiolabelled HAL_18/rPAST (Fig. 3 A) or cyclophilin, imaged using a phosphorimager and quantified (Fig. 3B). PC 12 cells were treated as follows: 1 hour NGF pulse followed by 1, 5, or 24 hour chase (samples lhr-NGF-p, 5hr-NGF-p, and 24hr-NGF-p), continuous NGF treament for 1, 5, or 24 hours (samples lhr- NGF-c, 5hr-NGF-c, and 24hr-NGF-c), and no NGF treatment for 1, 5, or 24 hours (lhr-wash, 5hr-wash, 24hr-wash). The quantified results for both the 3 kb and 4kb HAL_18/rPast bands normalized to the cyclophilin band in the same lane are presented graphically in Figure 3B.

Figures 4 A and 4B are graphical representations of the results of Northern Blot analysis of clone HAL l 8, where an agarose gel containing poly A enriched mRNA from rasN17 cells (a PC 12 transfectant expressing a ras dominant negative mutant) treated with NGF as well as size standards was blotted after electrophoresis and probed with either radiolabelled HAL_18/rPAST (Fig. 4A) or cyclophilin, imaged using a phosphorimager and quantified (Fig. 4b). rasN17 cells were treated as follows: 1 hour NGF pulse followed by 1, 5, or 24 hour chase (samples lhr-NGF-p, 5hr-NGF-p, and 24hr-NGF-p), continuous NGF treament for 1, 5, or 24 hours (samples lhr-NGF-c, 5hr-NGF-c, and 24hr-NGF-c), and no NGF treatment for 1, 5, or 24 hours (lhr-wash, 5hr-wash, 24hr-wash). The quantified results for both the 3 kb and 4kb HAL_18/rPast bands normalized to the cyclophilin band in the same lane are presented graphically in Figure 4B.

Figure 5 is a graphical representation of the results of Northern Blot analysis of clone HAL 18, where an agarose gel containing poly A enriched mRNA from rat heart, brain, spleen, lung, liver, skeletal muscle, and kidney as well as size standards was blotted after electrophoresis and probed with radiolabelled HAL_18/rPAST and imaged using a phosphorimager.

Figure 6 shows the results of an experiment demonstrating the tissue specific expression of the rPast gene, (left) Rat tissue polyA+mRNA (Clontech) and (right) total cellular RNA (10 μg) isolated from rat dorsal root ganglia (DRG), lung and heart was hybridized with an antisense RNA probe generated from Hall 8 template. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated.

Figure 7 shows the results of an experiment demonstrating the time course oϊrPast gene induction by NGF in PC 12 cells. Total cellular RNA (10 μg) was prepared from PC 12 cells incubated with NGF (100 μg/ml) for the indicated time (hours). The RNA was hybridized with a DNA probe generated from rPast cDNA (nt.1-454) fragment. The indicated two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) confirmed results using an antisense RNA probe from Hal 18 template. Re-hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane. Expression reaches a peak of 4-fold induction at 4 hours after NGF treatment and then returns to the basal level after 24 hours of NGF treatment.

Figure 8 shows the results of an experiment demonstrating that the induction oϊrPast gene expression does not require de novo synthesis of protein. PC 12 cells treated with NGF (100 μg/ml) were cultured in the absence or presence of the translational inhibitor cycloheximide (CHX, 10 μg/ml). CHX was applied at the indicated times (minutes) after NGF addition. Total RNA was isolated from the cells at 4 hours after the addition of NGF to the medium and analyzed by Northern blotting. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. The blot was re-hybridized with an antisense RNA probe for PN1(1 lkb) as a positive control for CHX. The cyclophilin was re-probed as an internal control for RNA loading.

Figure 9 shows the results of an experiment demonstrating the rPast gene induction by NGF by wild type and mutant Trks. Total RNA (10 μg) was isolated from the PC 12 mutant nn5 (lacking TrkA), and the following nnr5 stable transfectants expressing wild type or mutant TrkAs, T14 (wild type TrkA), Y490 (Y490F mutant), Y785 (Y785F mutant), Y490/785 (double mutant) after a four hour treatment with NGF (100 μg/ml). RNA was blotted and hybridized with an antisense RNA probe generated from a Hall 8 template. The two alternatively spliced forms o rPast transcripts (4kb and 3kb) are indicated. Re- hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane.

Figure 10 shows the results of an experiment demonstrating the ras-independent induction of rPast by NGF. (left) Total cellular RNA (10 μg) was prepared from Rasl7N2 cells, which constitutively express a dominant-negative form of Ras, 5 hours after either 1 minute or continuous treatment of NGF (100 μg/ml). The RNA was blotted and hybridized with an antisense RNA probe generated from Hal 18 template. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. Re-hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane. Two-fold induction by NGF oϊrPast gene expression is detected with both 1 minute treatment and continuous treatment, (right) The parallel experiment was done in PC 12 cells 4 hours after either 1 minute or continuous treatment of NGF. Four-fold induction by NGF oϊrPast gene expression is detected with both brief treatment and continuous treatment.

Figure 11 shows the results of an experiment demonstrating that ras is necessary for NGF induction oϊrPast gene expression. GsRasΔN6 cells, which inducibly express a dominant-negative form of ras (in response to dexamethasone), were treated for four hours with NGF (100 μg/mg) alone or after pre-incubation in DEX (0.5 μM) for sixteen hours, then treated with NGF for four hours. Total RNA (10 μg) was isolated from GsRasDN6 cells, blotted and hybridized with Hal 18 probes and cyclophilin probes as described above; mRNA positions are indicated.

Figure 12 shows the results of an experiment demonstrating that the expression of activated forms of Ras and b-Raf is sufficient to induce sustained rPast gene expression. Total RNA (10 μg) was isolated from GsRasl and GsbΔraf cells treated with dexamethasone (DEX: 0.5 μM) at the indicated times (hours). These cell lines are stable PC12 transfectants which inducibly express (in response to dexamethasone) the ras or braf oncogenes, respectively. The RNA was blotted and hybridized with an antisense RNA probe generated from Hall 8 template. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. Re-hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane.

Figure 13 shows the results of an experiment using confocal microscopy showing the effects of NGF treatment on the co-localization and internalization of HA-rPAST and the TrkA receptor. The trkPC12 cells were transiently transfected with HA-rPAST and treated with NGF for the indicated times. After fixation and permeabilization, cells were stained using antibodies to trkA (α-trk, rabbit polyclonal) and HA (α-HA, mouse monoclonal Ig2a). TrkA labeling is shown on the left, HA labeling is shown in the center and both labels are shown on the right. The photographic images are negatives of black and white images; in the original photographs, trk a antibodies were labeled with a green fluorophore and HA antibodies were labeled with a red fluorophore. In the absence of NGF ("control"), trkA and HA-rPAST staining is present diffusely at the surface membrane (A) with some trkA in the juxtanuclear region (B). After NGF treatment for 5 minutes at 37 degrees Celsius ("NGF 5 min"), trkA and HA-rPAST are both internalized into the cytosol (B) and co-localized together with excess HA-rPAST protein remaining on the plasma membrane (A). After 1 hour of NGF treatment at 37 degrees Celsius ("NGF 1 hour"), HA-rPAST only appears on the plasma membrane (A) while trkA remains only in the cytosol (B).

Figure 14 shows the results of an experiment using confocal microscopy showing that an EH domain deletion mutant of rPast localizes to novel intracellular structures and is unresponsive to NGF. The trkPC12 cells were transiently transfected with HA-rPASTΔEH and treated with NGF for the indicated times. After fixation and permeabilization, cells were stained using antibodies to TrkA (α-trk, rabbit polyclonal) and HA (α-HA, mouse monoclonal Ig2a). HA-rPASTΔEH labeling is shown on the left, trkA labeling is shown in the center and both labels are shown on the right. The photographic images are negatives of black and white images; in the original photographs, trk a antibodies were labeled with a green fluorophore and HA antibodies were labeled with a red fluorophore. In the absence of NGF ("control"), TrkA staining is on the plasma membrane (A) and with some staining in the juxtanuclear region (B), while HA-rPASTΔEH labeling is only shown clustering in large structures, not appearing on the plasma membrane (B). After NGF treatment for 5 minutes at 37 degrees Celsius ("NGF 5 min"), trkA labeling is seen both on the plasma membrane (A) and in the cytosol (B), but no change in the localization of HA-rPASTΔEH labeling is observed. After 1 hour of NGF treatment at 37 degrees Celsius ("NGF 1 hour"), trkA labeling is present on the plasma membrane (A) and in the cytosol. HA-rPASTΔEH labeling remains clustered in large structures, and shows no localization change caused by NGF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.

In the present invention, a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence contained in SEQ ID NOs:l-29. For example, the polynucleotide can contain all or part of the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NOs:l-29, or the complement thereof, or the cDNA. "Stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5X SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50°C with IX SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any

3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

A polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, a polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as postfranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, e.g., T. E. Creighton, Ed., Proteins - Structure And Molecular Properties, 2nd Ed., W. H. Freeman and Company, New York (1993); B. C. Johnson, Ed., Postfranslational Covalent Modification Of Proteins, Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol, 182:626-646 (1990); Rattan et al, Ann. N Y. Acαd. Sci., 663:48-62 (1992)).

"A polypeptide having biological activity" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose- dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about ten-fold less activity and, most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).

The translated amino acid sequence, beginning with the methionine, is identified although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by the translation of these alternative open reading frames are specifically contemplated by the present invention.

SEQ ID NOs:l-29 and the translations of SEQ ID NOs:l-29 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. These nucleic acid molecules will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention.

Similarly, polypeptides identified from the translations of SEQ ID NOs:l-29 may be used to generate antibodies which bind specifically to the secreted proteins encoded by the cDNA clones identified.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1,000 bases).

The present invention also relates to the genes corresponding to SEQ ID NOs:l-29, and translations of SEQ ID NOs.T-29. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are species homologues. Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification (such as multiple histidine residues), or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith & Johnson, Gene, 67:31-40 (1988). Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.

Signal Sequences Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein (Virus Res., 3:271-286 (1985)). The method of von Heiηje uses the information from the residues surrounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino terminus of the secreted protein (Nucleic Acids Res.,

14:4683-4690 (1986)). Therefore, from a deduced amino acid sequence, a signal sequence and mature sequence can be identified.

In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called Signal P (Nielsen et al., Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence corresponding to the translations of SEQ ID NOs:l-29 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants "Variant" refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.

"Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin and Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991)). While there exists a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo et al., SI AM J Applied Math., 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers," Martin J.

Bishop, Ed., Academic Press, San Diego, (1994) and Carillo et al., (1988), Supra. Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al, J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) which uses the local homology algorithm of Smith and Waterman (Adv. in App.Math., 2:482-489 (1981)).

When using any of the sequence alignment programs to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)). The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity. Preferred parameters used in a FASTDB search of a DNA sequence to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, and Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Preferred parameters employed to calculate percent identity and similarity of an amino acid alignment are: Matrix=PAM 150, k-tuple=2, Mismatch Penalty= 1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in amino acid residues, whichever is shorter.

As an illustration, a polynucleotide having a nucleotide sequence of at least 95% "identity" to a sequence contained in SEQ ID NOs: 1-29 means that the polynucleotide is identical to a sequence contained in SEQ ID NOs: 1-29 or the cDNA except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the total length (not just within a given 100 nucleotide stretch). In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to SEQ ID NOs: 1-29, up to 5% of the nucleotides in the sequence contained in SEQ ID NOs: 1-29 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.

Further embodiments of the present invention include polynucleotides having at least 80% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to a sequence contained in SEQ ID NOs: 1 -29. Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity will encode a polypeptide identical to an amino acid sequence contained in the translations of SEQ ID NOs: 1-29.

Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide, is intended that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Further embodiments of the present invention include polypeptides having at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence contained in translations of SEQ ID NOs: 1-29. Preferably, the above polypeptides should exhibit at least one biological activity of the protein.

In a preferred embodiment, polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ ID NOs: 1-29.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to optimize codon expression for a particular host (i.e., codons in the human mRNA may be changed to those preferred by a bacterial host, such as E. coli).

Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Lewin, Εd., Genes II, John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al. reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues (J. Biol. Chem. 268: 2984-2988 (1993)). Similarly, interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology, 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle et al. conducted extensive mutational analysis of human cytokine IL-la (J. Biol. Chem., 268:22105-22111

(1993)). These investigators used random mutagenesis to generate over 3,500 individual IL- 1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators concluded that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." (See, Gayle et al. (1993), Abstract). In fact, only 23 unique amino acid sequences, out of more than 3,500 amino acid sequences examined, produced a protein that differed significantly in activity from the wild-type sequence. Furthermore, even if deleting one or more amino acids from the N-terminus or C- terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N- terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions which have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution may be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine mutations at every residue in the molecule) can be used (Cunningham et al., Science, 244:1081-1085 (1989)). The resulting mutant molecules can then be tested for biological activity. According to Bowie et al., these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, the most buried or interior (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface or exterior side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin; replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Tip; and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Besides conservative amino acid substitution, variants of the present invention include: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as decreased aggregation. As known, aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes, 36: 838-845 (1987); Cleland et al., Crit. Rev. Therap. Drug Carrier Sys., 10:307-377 (1993)).

Polynucleotide and Polypeptide Fragments

In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ ID NOs: 1-29. The short nucleotide fragments are preferably at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. A fragment "at least 20 nt in length," for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ID NOs: 1-29. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, and greater than 150 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments having a sequence from about nucleotide number 1-50, 51- 100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, to the end of SEQ ID NOs: 1-29. In this context "about" includes the particularly recited ranges, larger or smaller by several nucleotides (i.e., 5, 4, 3, 2, or 1 nt) at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity.

In the present invention, a "polypeptide fragment" refers to a short amino acid sequence contained in the translations of SEQ ID NOs: 1-29. Protein fragments may be "freestanding," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, or 60 amino acids in length. In this context "about" includes the particularly recited ranges, larger or smaller by several amino acids (5, 4, 3, 2, or 1) at either extreme or at both extremes.

Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha- helix-forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of the translations of SEQ ID NOs: 1-29 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotide fragments encoding these domains are also contemplated.

Other preferred fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Epitopes & Antibodies In the present invention, "epitopes" refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human. A preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment. A region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." In contrast, an "immunogenic epitope" is defined as a part of a protein that elicits an antibody response. (See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1983)).

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA, 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).

In the present invention, antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, e.g., Wilson et al., Cell, 37:767-778 (1984); Sutcliffe et al., Science, 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, e.g., Sutcliffe et al., (1983) Supra; Wilson et al., (1984) Supra; Chow et al., Proc. Natl. Acad. Sci., USA, 82:910-914; and Bittle et al., J. Gen. Virol, 66:2347-2354 (1985)). A preferred immunogenic epitope includes the secreted protein. The immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse). Alternatively, the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids). However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')₂ fragments) which are capable of specifically binding to protein. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med.,

24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a Fab or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and human and humanized antibodies.

Additional embodiments include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature, 332:323, 1988), Liu et al. (PNAS, 84:3439, 1987), Larrick et al. (Bio/Technology, 7:934, 1989), and Winter and Harris (TIPS, 14:139, May, 1993).

One method for producing a human antibody comprises immunizing a non-human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-29, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-29 are generated in said animal. Procedures have been developed for generating human antibodies in non-human animals. The antibodies may be partially human, or preferably completely human. Non- human animals (such as transgenic mice) into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such transgenic mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization. Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-29 are provided herein.

Mice in which one or more endogenous immunoglobulin genes are inactivated by various means have been prepared. Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos.5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.

Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells may be fused with myeloma cells to produce hybridomas by conventional procedures.

A method for producing a hybridoma cell line comprises immunizing such a transgenic animal with an immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs:l- 29; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a polypeptide translated from a nucleotide sequence chosen from SEQ ED NOs: 1-29. Such hybridoma cell lines, and monoclonal antibodies produced therefrom, are encompassed by the present invention. Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques.

Antibodies may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-29. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-29.

Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-29. Examples of such agents are well known, and include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs. The conjugates find use in in vitro or in vivo procedures.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

In addition, polypeptides of the present invention, including fragments and, specifically, epitopes, can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker et al., Nature, 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone (Fountoulakis et al., J. Biochem., 270:3958-3964 (1995)).

Similarly, EP A 0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (see, e.g., EP A 0 232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (See, Bennett et al., J. Mol. Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem., 270:9459-9471 (1995)).

Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. As described in Gentz et al., for instance, hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl. Acad. Sci. USA 86:821- 824 (1989)). Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984)). Other fusion proteins may use the ability of the polypeptides of the present inention to target the delivery of a biologically active peptide. This might include focused delivery of ataxia to tumor cells, or a growth factor to stem cells.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors. Host Cells, and Protein Production The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the S V40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQΕ70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16A, PNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPN, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may, in fact, be expressed by a host cell lacking a recombinant vector. A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.

Polypeptides of the present invention, and preferably the secreted form, can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N- terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available. Each polynucleotide of the present invention can be used as a chromosome marker. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NOs: 1-29. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NOs: 1-29 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene-mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides of 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross-hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Assuming one megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50- 500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. The polynucleotides of SEQ ID NOs: 1-29 can be used for this analysis of individuals.

First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymoφhism. If a new polymoφhism is identified, this polymoφhic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (see, Lee et al., Nuc. Acids Res., 6:3073 (1979); Cooney et al., Science, 241 :456 (1988); and Dervan et al., Science, 251:1360 (1991) for discussion of triple helix formation) or to the mRNA itself (see, Okano, J. Neurochem, 56:560 (1991); and Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988) for a discussion of antisense technique). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymoφhism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to

RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.

Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymoφhic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, Ed., PCR Technology, M. Stockton Press (1989)). Once these specific polymoφhic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class H HLA gene. Similarly, polynucleotides of the present invention can be used as polymoφhic markers for forensic puφoses.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels; as diagnostic probes for the presence of a specific mRNA in a particular cell type; as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support; to raise anti-DNA antibodies using DNA immunization techniques; and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, et al., J. Cell. Biol, 101:976-985 (1985); Jalkanen, et al., Cell. Biol, 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; and radioisotopes, such as iodine (¹²⁵1, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^99mTc); fluorescent labels, such as fluorescein and rhodamine; and biotin.

In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR), or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incoφorated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (e.g., ¹³¹1, ¹¹²In, ^99mTc), a radio-opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, the quantity of radioactivity necessary for a human subject will normally range from about 5 to 20 millicuries of ^99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing Inc. (1982)).

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.

Moreover, polypeptides of the present invention can be used to treat disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin); to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (e.g., an oncogene); to activate the activity of a polypeptide (e.g., by binding to a receptor); to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation); or to bring about a desired response (e.g., blood vessel growth).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce oveφroduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor). Polypeptides can also be used as antigens to trigger immune responses.

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well-known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Biological Activities

The polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.

Nervous System Activity

A polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of neuroblasts, stem cells, or glial cells. Also, a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the mechanisms of synaptic transmission, synthesis, metabolism and inactivation of neural transmitters, neuromodulators and trophic factors, and by activating or inhibiting the expression and incoφoration of enzymes, structural proteins, membrane channels, and receptors in neurons and glial cells.

The etiology of these deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorder), acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular nervous system disease or disorder. The disorder or disease can be any of Alzheimer's disease, Pick's disease, Binswanger's disease, other senile dementia, Parkinson's disease, parkinsonism, obsessive compulsive disorders, epilepsy, encephaolopathy, ischemia, alcohol addiction, drug addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder. Alternatively, the polypeptide or polynucleotide of the present invention can be used to study circadian variation, aging, or long-term potentiation, the latter affecting the hippocampus. Additionally, particularly with reference to mRNA species occurring in particular structures within the central nervous system, the polypeptide or polynucleotide of the present invention can be used to study brain regions that are known to be involved in complex behaviors, such as learning and memory, emotion, drug addiction, glutamate neurotoxicity, feeding behavior, olfaction, viral infection, vision, and movement disorders.

Immune Activity

A polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorders) acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular immune system disease or disorder. A polynucleotide or polypeptide of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells. A polypeptide or polynucleotide of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Di George's Syndrome, HIN infection, HTLN-BLN infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (bleeding cessation) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotide or polypeptide of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotide or polypeptide of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart attacks (infarction), strokes, or scarring.

A polynucleotide or polypeptide of the present invention may also be useful in the treatment or detection of autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells or in some ways resulting in the induction of tolerance, may be an effective therapy in preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Puφura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,

Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

A polynucleotide or polypeptide of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, a polypeptide or polynucleotide of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

Hyperproliferative Disorders A polypeptide or polynucleotide can be used to treat or detect hypeφroliferative disorders, including neoplasms. A polypeptide or polynucleotide of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polypeptide or polynucleotide of the present invention may proliferate other cells which can inhibit the hypeφroliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hypeφroliferative disorder or by inducing the proliferation, differentiation, or mobilization of T-cells, hypeφroliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hypeφroliferative disorders, such as by administering the polypeptide or polynucleotide as a chemotherapeutic agent.

Examples of hypeφroliferative disorders that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic region, skin, soft tissue, spleen, thoracic region, and urogenital system.

Similarly, other hypeφroliferative disorders can also be treated or detected by a polynucleotide or polypeptide of the present invention. Examples of such hypeφroliferative disorders include, but are not limited to hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, puφura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hypeφroliferative disease, besides neoplasia, located in an organ system listed above.

Infectious Disease A polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, the polypeptide or polynucleotide of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention. Examples of viruses include, but are not limited to, the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Heφesviridae (such as Cytomegalovirus, Heφes Simplex, Heφes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to, arthritis, bronchioUitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to, the following Gram-Negative and Gram-positive bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsielia, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,

Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea infections (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcus. These bacterial or fungal families can cause numerous diseases or symptoms, including, but not limited to, bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections (such as whooping Cough or empyema), sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually-transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, and wound infections. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,

Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but not limited to, Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

Preferably, treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues (see, Science, 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery (including cosmetic plastic surgery), fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, ligament) tissue. Preferably, regeneration occurs without scarring or with minimal scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, caφal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stroke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g.,

Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotide or polypeptide of the present invention.

Chemotaxis

A polynucleotide or polypeptide of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hypeφroliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hypeφroliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that a polynucleotide or polypeptide of the present invention may inhibit chemotactic activity. Such molecules could also be used to treat a variety of disorders. Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (i.e., an agonist), increase, inhibit (i.e., an antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic (see, Coligan et al., Current Protocols in Immunology 1(2), Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, related to a fragment of the receptor capable of being bound by the polypeptide (e.g., an active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ΕLISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.

Other Activities A polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells from a lineage other than the above-described hemopoietic lineage.

A polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

A polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, the response to opiates and opioids, tolerance to opiates and opioids, withdrawal from opiates and opioids, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

A polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors, or other nutritional components.

Other Preferred Embodiments

Where a polynucleotide of the invention is down-regulated and exacerbates a pathological condition, such as diseases and conditions involving altered target cell metabolism of NGF, the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention to the mammalian subject. A polynucleotide of the invention can be administered to a mammalian subject by a recombinant expression vector comprising the polynucleotide. A mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, horse, dog, cat, rabbit, guinea pig, rat or mouse. Preferably, the recombinant vector comprises a polynucleotide shown in SEQ ID NOs: 1-29 or a polynucleotide which is at least 98% identical to a nucleic acid sequence shown in SEQ ID NOs: 1-29. Also, preferably, the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ ED NOs: 1-29.

The administration of a polynucleotide or recombinant expression vector of the invention to a mammalian subject can be used to express a polynucleotide in said subject for the treatment of, for example, a condition involving altered target cell metabolism of NGF. Expression of a polynucleotide in target cells, including but not limited to nerve cells, would effect greater production of the encoded polypeptide. In some cases where the encoded polypeptide is a nuclear protein, the regulation of other genes may be secondarily up- or down- regulated.

There are available to one skilled in the art multiple viral and non-viral methods suitable for introduction of a nucleic acid molecule into a target cell, as described above. In addition, a naked polynucleotide can be administered to target cells. Polynucleotides and recombinant expression vectors of the invention can be administered as a pharmaceutical composition. Such a composition comprises an effective amount of a polynucleotide or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration. Suitable formulation materials preferably are non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsoφtion, or penetration of the composition. See Remington 's Pharmaceutical Sciences (18^th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990).

The pharmaceutically active compounds (i.e., a polynucleotide or a vector) can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. Thus, the pharmaceutical composition comprising a polynucleotide or a recombinant expression vector may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).

The dosage regimen for treating a disease with a composition comprising a polynucleotide or expression vector is based on a variety of factors, including the type or severity of the condition involving altered target cell metabolism of NGF, the age, weight, sex, medical condition of the patient, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above.

The frequency of dosing will depend upon the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.

The cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro. Administration of a polynucleotide or a recombinant vector containing a polynucleotide to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art. For example, U.S. Patent No. 5,672,344 describes an in vivo viral- mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector. The above-described compositions of polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray. The term "parenteral" as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally. While the nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

Another delivery system for polynucleotides of the invention is a "non- viral" delivery system. Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄ precipitation, gene gun techniques, electroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110):926-32). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success (Mulligan, R., (1993) Science, 260 (5110):926-32).

Where a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as a disease involving altered target cell metabolism of NGF, the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, the use of antisense oligonucleotides or ribozymes. Alternatively, drugs or antibodies that bind to and inactivate the polypeptide product can be used.

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell. Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, (1994) Meth. Mol. Biol, 20:1-8; Sonveaux, (1994) Meth. Mol. Biol, 26:1-72; Uhlmann et al., (1990) Chem. Rev., 90:543-583.

Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a gene of the invention. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence. Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide of the invention. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., (1992) Trends Biotechnol, 10:152-158; Uhlmann et al., (1990) CTzem. Rev., 90:543-584; Uhlmann et al, (1987) Tetrahedron. Lett., 215:3539-3542.

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987) Science, 236:1532-1539; Cech, (1990) Ann. Rev. Biochem., 59:543-568; Cech, (1992) Curr. Opin. Struct. Biol, 2:605-609; Couture & Stinchcomb, (1996) Trends Genet, 12:510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a polynucleotide of the invention can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide. Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al., EP 321,201).

Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides conesponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NOs: 1-29 and their complements provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polynucleotide expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al., U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Production of Diagnostic Test Pathological conditions or susceptibility to pathological conditions, such as diseases and conditions involving altered target cell metabolism of NGF, can be diagnosed using methods of the invention. Testing for expression of a polynucleotide of the invention or for the presence of the polynucleotide product can conelate with the severity of the condition and can also indicate appropriate treatment. For example, the presence or absence of a mutation in a polynucleotide of the invention can be determined and a pathological condition or a susceptibility to a pathological condition is diagnosed based on the presence or absence of the mutation. Further, an alteration in expression of a polypeptide encoded by a polynucleotide of the invention can be detected, where the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition. The alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.

As an additional method of diagnosis, a first biological sample from a patient suspected of having a pathological condition, such as diseases and conditions involving altered target cell metabolism of NGF, is obtained along with a second sample from a suitable comparable control source. A biological sample can comprise saliva, blood, cerebrospinal fluid, amniotic fluid, urine, feces, or tissue, such as gastrointestinal tissue. A suitable control source can be obtained from one or more mammalian subjects that do not have the pathological condition. For example, the average concentrations and distribution of a polynucleotide or polypeptide of the invention can be determined from biological samples taken from a representative population of mammalian subjects, wherein the mammalian subjects are the same species as the subject from which the test sample was obtained. The amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample. The amounts of the polypeptide in the first and second samples are compared. A patient is diagnosed as having a pathological condition if the amount of the polypeptide in the first sample falls in the range of samples taken from a representative group of patients with the pathological condition.

Other prefened embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NOs: 1-29.

Also prefened is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ED NOs: 1-29 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the clone sequence and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence.

Also prefened is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-29 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the start codon and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ED NOs:l-29.

Similarly prefened is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-29 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NOs: 1-29.

Also prefened is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NOs: 1-29.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NOs: 1-29.

A further prefened embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 1-29 beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NOs: 1-29.

A further prefened embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NOs: 1-29. Also prefened is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

A further prefened embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 1-29, which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.

Also prefened is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also prefened is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further prefened embodiment is a method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 1-29.

Also prefened is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 1-29. The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also prefened is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of a nucleotide sequence of SEQ ED NOs: 1-29. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also prefened is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in an amino acid sequence translated from SEQ ED NOs: 1-29.

Also prefened is a polypeptide, wherein said sequence of contiguous amino acids is included in amino acids in an amino acid sequence translated from SEQ ED NOs: 1-29, in the range of positions beginning with the residue at about the position of the first amino acid of the secreted portion and ending with the residue at about the last amino acid of the open reading frame.

Also prefened is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in an amino acid sequence translated from SEQ ED NOs: 1-29.

Further prefened is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in an amino acid sequence translated from SEQ ID NOs: 1-29.

Further prefened is an isolated polypeptide comprising an amino acid sequence at least 95% identical to amino acids in an amino acid sequence translated from SEQ ID NOs:l- - βl -

29.

Further prefened is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ED NOs: 1-29, which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.

Also prefened is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-29.

Also prefened is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also prefened is a method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-29.

Also prefened is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group. Also prefened is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also prefened is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-29.

Also prefened is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.

Also prefened is an isolated nucleic acid molecule, wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-29.

Further prefened is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also prefened is the recombinant vector produced by this method. Also prefened is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method. Also prefened is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also prefened is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-29. The isolated polypeptide produced by this method is also prefened.

Also prefened is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

The present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such as brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.

In a related embodiment, a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene conesponding to a polynucleotide of the present invention, or a mRNA in a cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell. The nucleic acid molecule probes can be of a variety of lengths from at least about 10, suitably about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length. Hybridization methods are extremely well known in the art and will not be described further here.

In a related embodiment, detection of genes corresponding to the polynucleotides of the present invention can be conducted by primer extension reactions such as the polymerase chain reaction (PCR). To that end, PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected. Preferably, the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the present invention. Particularly prefened PCR primers can be derived from any portion of a DNA sequence encoding a polypeptide of the present invention, but are preferentially from regions which are not conserved in other cellular proteins.

Prefened PCR primer pairs useful for detecting the genes conesponding to the polynucleotides of the present invention and expression of these genes are described in the Examples, including the conesponding Tables. Nucleotide primers from the corresponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the conesponding gene in any of a variety of tissues.

The diagnostic system includes, in an amount sufficient to perform at least one assay, a subject polypeptide of the present invention, a subject antibody or monoclonal antibody, and/or a subject nucleic acid molecule probe of the present invention, as a separately packaged reagent.

In another embodiment, a diagnostic system, preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample. Such diagnostic kit would be useful for monitoring the fate of a therapeutically administered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention. The system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent.

Instructions for use of the packaged reagent(s) are also typically included.

As used herein, the term "package" refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene, or polycarbonate), paper, foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody, or monoclonal antibody of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or antibody or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed (i.e., linked) so as to be capable of being immunologically bound by an antibody or antigen, respectively.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/ sample admixtures, temperature, buffer conditions, and the like.

A diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.

The word "complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incoφorated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRETC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189- 231 (1982), which is incoφorated herein by reference. Other suitable labeling agents are known to those skilled in the art.

In prefened embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor- ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3- ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used illustratively herein.

An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴1, ¹²⁵1, ¹²⁸1, ¹³²I and ⁵¹Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly prefened is ¹²⁵I. Another group of useful labeling means are those elements such as ^HC, ¹⁸F, ¹⁵O and ¹³N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such ^{π ι} indium or ³H.

The linking of labels or labeling of polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incoφoration of radioisotope-containing amino acids provided as a component in the culture medium (see, e.g., Galfre et al., Meth. Enzymol, 73:3-46 (1981)). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable (see, e.g., Aurameas, et al., Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978); Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Patent No. 4,493,795).

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species is present as part of a complex.

In prefened embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample. A description of the ELISA technique is found in Sites et al., Basic and Clinical Immunology, 4^th Ed., Chap. 22, Lange Medical Publications, Los Altos, CA (1982) and in U.S. Patent No. 3,654,090; U.S. Patent No. 3,850,752; and U.S. Patent No. 4,016,043, which are all incoφorated herein by reference.

Thus, in some embodiments, a polypeptide of the present invention, an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsoφtion from an aqueous medium, although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsoφtion methods are described herein.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ), agarose, polystyrene beads of about 1 micron (μm) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs (sheets, strips or paddles) or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent, or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLE 1 Identification and Characterization of Polynucleotides

Regulated by Neuromodulators

PolyA-mRNA was isolated from PC 12 cells before and at different times after triggered induction by NGF or EFN-γ. PC 12 cells were pulse treated with either NGF (100 ng/ml) for two minutes, EFN-γ (1000 units/ml) for three minutes, or with control medium (Dulbecco's modified Eagles (DME) medium; CW) containing fresh serum (5% fetal calf serum, 10% horse serum) for two minutes. The cells were then washed two times with DME and incubated at 37 degrees Celsius in an atmosphere of 10% CO₂ / 90% air in DME containing serum for varying lengths of time. After incubations for one hour (for NGF, EFN- γ, and CW), 5 hours (for NGF and IFN-γ) and 24 hours (for NGF), the cells were washed with phosphate buffered saline (PBS) and harvested. Cells were lysed in lysis solution containing 0.14 M NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl (pH 8.6), 1 mM DTT, 1000 U/ml RNAsin, and 0.5% NP-40. The nuclei were removed by centrifugation and the cytoplasmic RNA was isolated by the LiCl method. Poly-A containing RNA was isolated by oligo dT chromatography.

RNA isolated from PC 12 cells was analyzed using a method of simultaneous sequence-specific identification of mRNAs known as TOGA (TOtal Gene expression Analysis) described in Sutcliffe et al. Proc. Natl. Acad. Sci. USA, 97(5):1976-1981 (2000); International published application WO 026406; U.S. Patent No. 5,459,037; U.S. Patent No. 5,807,680; U.S. Patent No. 6,030,784; U.S. Patent No. 6,096,503 and U.S. Patent 6,110,680, hereby incoφorated herein by reference. Preferably, prior to the application of the TOGA technique, the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art. In a prefened embodiment, the TOGA method further comprised an additional PCR step performed using four 5' PCR primers in four separate reactions and cDNA templates prepared from a population of antisense cRNAs. A final PCR step that used 256 5' PCR primers in separate reactions produced PCR products that were cDNA fragments that conesponded to the 3 '-region of the starting mRNA population. The produced PCR products were then identified by: a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and isolate the 3' region plus the sequence of the preferably four parsing bases immediately 3' to the remainder of the recognition site, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences. Since the length of the obtained PCR products includes known vector sequences at the 5' and 3' ends of the insert, the sequence of the insert provided in the sequence listing is shorter than the fragment length that forms part of the digital address.

The method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags of the 3' end of mRNAs. DSTs that showed changes in relative levels following exposure to NGF or IFN-γ were selected for further study. The intensities of the laser-induced fluorescence of the labeled PCR products were compared across samples isolated from PC 12 cells treated with NGF for 1, 5, or 24 hours or with IFN-γ for 1 or 5 hours.

In general, double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture of all 48 5'- biotinylated anchor primers of a set to initiate reverse transcription. One such suitable set is G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T- T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 30), where V is A, C or G and N is A, C, G or T. One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double stranded cDNA.

Each biotinylated double stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG. The resulting fragments of cDNA corresponding to the 3' region of the starting mRNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate. Suitable streptavidin- coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles. A prefened streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Lake Success, NY).

After washing the streptavidin-coated substrate and captured biotinylated cDNA fragments, the cDNA fragment product was released by digestion with Notl, which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs. The Mspl-Notl fragments of cDNA conesponding to the 3' region of the starting mRNA, which are of uniform length for each mRNA species, were directionally ligated into Clal-, Notl-cleaved plasmid pBC SK⁺ (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene). The ligation regenerates the Notl site, but not the Mspl site, leaving CGG as the first 3 bases of the 5' end of all PCR products obtained. Each library contained in excess of 5 x 10⁵ recombinants to ensure a high likelihood that the 3' ends of all mRNAs with concentrations of 0.001% or greater were multiply represented. Plasmid preps (Qiagen) were made from the cDNA library of each sample under study. An aliquot of each library was digested with Mspl, which effects linearization by cleavage at several sites within the parent vector while leaving the 3' cDNA inserts and their flanking sequences, including the T3 promoter, intact. The product was incubated with T3 RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts of the cloned inserts containing known vector sequences abutting the Mspl and Notl sites from the original cDNAs.

At this stage, each of the cRNA preparations was processed in a three-step fashion. In step one, 250ng of cRNA was converted to first-strand cDNA using the 5' RT primer (A-G- G-T-C-G-A-C-G-G-T-A-T-C-G-G, (SEQ ID NO: 31). In step two, 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 32), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ED NO: 33).

En step three, the product of each subpool was further divided into 64 subsubpools

(2ng in 20 μl) for the second PCR reaction, with 100 ng each of the fluoresceinated "universal" 3' PCR primer, the oligonucleotide (SEQ ED NO: 33) conjugated to 6-FAM and the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ED NO: 34), using a program that included an annealing step at a temperature X slightly above the T_m of each 5' PCR primer to minimize artifactual misprinting and promote high fidelity copying. Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).

The products from the final polymerase chain reaction step for each of the tissue samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were collected using the GeneScan software package (ABI) and normalized for amplitude and migration. Complete execution of this series of reactions generated 64 product subpools for each of the four pools established by the 5' PCR primers of the first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set of the second PCR reaction.

The mRNA samples from each timepoint as described above were analyzed. Table 1 is a summary of the expression levels of 102 mRNAs determined from cDNA. These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence comprising the remainder of the Mspl site and the four parsing bases for each subsubpool coupled with the length of the molecule, as well as the relative amount of the molecule produced at different time intervals after treatment. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The length of the fragment was determined by inteφolation on a standard curve and as such, may vary ±12 b.p. from the actual length as determined by sequencing.

For example, the entry in Table 1 that describes a DNA molecule identified by the digital address Mspl GCAC 349, is further characterized as having a 5' terminus partial nucleotide sequence of CGGGCAC and a digital address length of 349 b.p. The DNA molecule identified as Mspl GCAC 349 is further described as being expressed at increasing levels after treatment with NGF. However, treatment with NGF results in a marked decrease of the expression of Mspl GACT 280 (HAL 17).

Similarly, the other DNA molecules identified in Table 1 by their Mspl digital addresses are further characterized by 1) the level of gene expression in PC 12 cells following wash with control solution, 2) the level of gene expression in PC 12 cells following treatment with NGF for 1 hour, 3) the level of gene expression in PC 12 cells following treatment with EFN-γ for 1 hour, 4) the level of gene expression in PC 12 cells following treatment with NGF for 5 hours, 5) the level of gene expression in PC 12 cells following treatment with EFN- γ for 5 hours, and 6) the level of gene expression in PC 12 cells following treatment with NGF for 24 hours. Several of the isolated clones were further characterized in Table 2 and their nucleotide sequences are provided as SEQ ID NO: 1-29 in the Sequence Listing below. The isolated clones chosen showed between about two-fold and about 27-fold changes in expression, and were induced by both NFG and IFNγ, EFNγ alone, or NGF alone.

The data shown in Figure 1 were generated with a 5' -PCR primer (C-G-A-C-G-G-T- A-T-C-G-G-G-C-A-C, SEQ ID NO: 35) paired with the "universal" 3 ' primer (SEQ ID NO: 33) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel electrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer).

The results of TOGA analysis using a 5' PCR primer with parsing bases (C-G-A-C-G- G-T- A-T-C-G-G-G-C-A-C; SEQ ID NO: 35) are shown in Figure 1 , which presents the results of TOGA analysis using a 5' PCR primer with parsing bases G-C-A-C, showing PCR products produced from mRNA isolated from PC 12 cells that were treated as described above. The vertical index line indicates a PCR product of about 349 b.p. that is present in cells that are treated with NGF or IGNγ for 1 hour, and whose expression increases when PC 12 cells are treated with NGF or IFN-γ for 5 hours or with NGF for 24 hours.

Some products, which were also differentially represented, appeared to migrate in positions that suggests that the products were novel based on comparison to data extracted from GenBank. In these cases, the PCR product was isolated, cloned into a TOPO vector (Invifrogen) and sequenced on both strands. En order to verify that the clones isolated are from the same peak, PCR primers were designed based on the determined sequence and PCR was performed using the cDNA produced in the first PCR reaction as substrate. For example, for the 349 b.p. product disclosed above, an oligonucleotide was synthesized with the sequence G-A-T-C-G-A-A-T-C extended at the 3' end with a partial Mspl site (C-G-G) and an additional 18 nucleotides from the sequence of the cloned PCR product. This oligonucleotide (SEQ ED NO: 47) was paired with the "universal" 3' primer (SEQ ID NO: 33) using cDNA produced in the 1^st PCR reaction as a substrate. The length of the PCR product generated with this extended primer is compared to that of the original PCR product produced in the TOGA reaction. Primers used in such studies are listed in Table 3.

Using a HAL 18 clone as a probe, a PC 12 cDNA library was screened in order to obtain a full-length cDNA clone for the gene containing the HAL 18 sequence. Sequence analysis of HAL 18 sequence revealed that it encoded a 3' untranslated region. A fiill-length clone was recovered, sequenced, and the sequence compared with known nucleotide sequences. The full-length sequence was found to have a high degree of homology with genes identified in C. elegans, Drosophilia, and Plasmodium, and to human, mouse, and rat EST clones. Although the genes have no known function, recent evidence suggests that the protein encoded by the Drosophila gene, termed PAST-1, plays a role in ligand-induced endocytosis. The full-length cDNA conesponding to the HAL 18 clone encodes the rat homolog of PAST-1, rPAST (SEQ ID NO:29). The sequences of HAL 18 (SEQ ED NO: 12) and rPAST were aligned using MacVector. Base 1 of HAL_18 aligns with base 2360 of rPAST, with discrepancies between HAL_18 and rPAST, respectively, at base 138 of HAL_18 (g v. A), base 155 (a v. G), base 181(gap v. C), base 200(g v. A) and base 289 (c v. T).

A full-length cDNA of the HAL_18/rPAST encoding a HA-tagged rPAST protein has been constructed and expressed in PC 12 cells. The rPAST protein has been found to be expressed in vesicular structures. A massive translocation of PAST protein to what may be the endocytic vesicles in PC 12 cells is observed following NGF-treatment of PC 12 cells transfected with the rPAST construct. It is believed that NGF activation of Trk triggers the induction of PAST, to continued process of NGF/Trk endocytosis, an event required for signaling to the neuronal soma and nucleus.

The current model is that brief activation of Trk by NGF triggers the induction of PAST, to ensure the continued process of NGF/Trk endocytosis, an event required for signaling to the neuronal soma/nucleus. In this framework, the signaling pathway to PAST induction, and the role of PAST in Trk endocytosis and signaling has been determined, using Northern blot analyses to study induction by PAST mRNA using various of the PC 12 cell lines we had created which inducibly express various mutant oncogene forms. We have determined that PAST induction is mediated through both Ras-Raf dependent and independent pathways. The induction by NGF occurs within three hours and does not require the prior expression of any immediate-early genes. The Northern blot data are shown below.

The role of PAST in Trk endocytosis stimulated by NGF has been studied. PC 12 cells are transfected with a HA-tagged PAST construct. Using this approach, we have found that PAST is first localized in the plasma membrane and then co-internalized with Trk after NGF stimulation of PC 12 cells. We have found that the first stage of endocytosis is in clathrin-coated pits, by double staining with anti-clathrin and anti-HA antibodies. The co- endocytosis continues to multivesicular bodies. Deletion of the EH domain of PAST results in a complete relocalization to novel intracellular vesicular structures, a loss of PAST from the plasma membrane and no relocalization in response to NGF. We have found that overexpression of PAST in PC12 cells results in a stimulation of NGF-induced Trk endocytosis. Figures 13 and 14 show PAST and Trk endocytosis.

The results show that PAST is a novel protein involved in Trk endocytosis, stimulated by NGF treatment. Its production is also stimulated by NGF through multiple signaling pathways including the Ras-Raf proto-oncoprotein pathway. Figure 2 illustrates the results of Northern Blot analysis of the clone (HAL 18) conesponding to the 349 b.p. product disclosed above. An agarose gel containing poly A enriched mRNA from PC 12 cells treated with NGF at various time points (either continuously or using a pulse-chase) was blotted after electrophoresis and probed with either radiolabelled HAL_18/rPAST (Fig. 2A) or cyclophilin (Fig. 2B) and imaged using a phosphorimager. Two RNA bands of aproximately 3.8 and 2.2 kb were identified, each of which was inducible by NGF and IFN-γ, verifying the results obtained using TOGA.

Figures 3A-3C further illustrate that two RNA species in PC 12 cells, which hybridize to the HAL 18 probe, are inducible by NGF and EFN-γ treatment. The peak of this induction in PC 12 cells occurs at about 4 hours following NGF or EFN-γ exposure. Figures 4A-4C, in which similar results were obtained using a PC 12 transfectant containing a ras dominant negative mutant (rasN17 cells), illustrate that this induction can occur in a ras-independent manner. It has been further established that these HAL_18 mRNAs are mediated by the gpl30 family of cytokine receptors, specifically IL-6.

Figure 5 illustrates the results of Northern Blot analysis of clone HAL_18, where an agarose gel containing poly A enriched mRNA from rat heart, brain, spleen, lung, liver, skeletal muscle, and kidney as well as size standards was blotted after electrophoresis and probed with radiolabelled HAL_18/rPAST and imaged using a phosphorimager. HAL 18 RNA was found to be primarily expressed in the heart, with moderate expression in the lung and detectable expression in the kidney and brain.

Figure 6 shows the results of an experiment demonstrating the tissue specific expression of the rPast gene, (left) Rat tissue polyA+mRNA (Clontech) and (right) total cellular RNA (10 μg) isolated from rat dorsal root ganglia (DRG), lung and heart was hybridized with an antisense RNA probe generated from Hal 18 template. The two alternatively spliced forms o rPast transcripts (4kb and 3kb) are indicated.

Figure 7 shows the results of an experiment demonstrating the time course oϊrPast gene induction by NGF in PC 12 cells. Total cellular RNA (10 μg) was prepared from PC 12 cells incubated with NGF (100 μg/ml) for the indicated time (hours). The RNA was hybridized with a DNA probe generated from rPast cDNA (nt.1-454) fragment. The indicated two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) confirmed results using an antisense RNA probe from Hall 8 template. Re-hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane.

Expression reaches a peak of 4-fold induction at 4 hours after NGF treatment and then returns to the basal level after 24 hours of NGF treatment.

Figure 8 shows the results of an experiment demonstrating that the induction oϊrPast gene expression does not require de novo synthesis of protein. PC12 cells treated with NGF (100 μg/ml) were cultured in the absence or presence of the translational inhibitor cycloheximide (CHX, 10 μg/ml). CHX was applied at the indicated times (minutes) after NGF addition. Total RNA was isolated from the cells at 4 hours after the addition of NGF to the medium and analyzed by Northern blotting. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. The blot was re-hybridized with an antisense RNA probe for PN1(1 lkb) as a positive control for CHX. The cyclophilin was re-probed as an internal control for RNA loading.

Figure 9 shows the results of an experiment demonstrating the rPast gene induction by NGF by wild type and mutant Trks. Total RNA ( 10 μg) was isolated from the PC 12 mutant nn5 (lacking TrkA), and the following nnr5 stable transfectants expressing wild type or mutant TrkAs, T14 (wild type TrkA), Y490 (Y490F mutant), Y785 (Y785F mutant), Y490/785 (double mutant) after a four hour treatment with NGF (100 μg/ml). RNA was blotted and hybridized with an antisense RNA probe generated from a Hal 18 template. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. Re- hybridization with a cyclophilin pEB15probe (lkb) provided an internal control for the amount of RNA in each lane. Figure 10 shows the results of an experiment demonstrating the rαs-independent induction of rPast by NGF. (left) Total cellular RNA (10 μg) was prepared from Rasl7N2 cells, which constitutively express a dominant-negative form of Ras, 5 hours after either 1 minute or continuous treatment of NGF (100 μg/ml). The RNA was blotted and hybridized with an antisense RNA probe generated from Hal 18 template. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. Re-hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane. Two-fold induction by NGF oϊrPast gene expression is detected with both 1 minute treatment and continuous treatment, (right) The parallel experiment was done in PC 12 cells 4 hours after either 1 minute or continuous treatment of NGF. Four- fold induction by NGF oϊrPast gene expression is detected with both brief treatment and continuous treatment.

Figure 11 shows the results of an experiment demonstrating that ras is necessary for NGF induction oϊrPast gene expression. GsRasΔNό cells, which inducibly express a dominant-negative form of ras (in response to dexamethasone), were treated for four hours with NGF (100 μg/mg) alone or after pre-incubation in DEX (0.5 μM) for sixteen hours, then treated with NGF for four hours. Total RNA (10 μg) was isolated from GsRasDNό cells, blotted and hybridized with Hal_18 probes and cyclophilin probes as described above; mRNA positions are indicated.

Figure 12 shows the results of an experiment demonstrating that the expression of activated forms of Ras and b-Raf is sufficient to induce sustained rPast gene expression. Total RNA (10 μg) was isolated from GsRasl and GsbΔraf cells treated with dexamethasone (DEX: 0.5 μM) at the indicated times (hours). These cell lines are stable PC 12 transfectants which inducibly express (in response to dexamethasone) the ras or braf oncogenes, respectively. The RNA was blotted and hybridized with an antisense RNA probe generated from Hal 18 template. The two alternatively spliced forms oϊrPast transcripts (4kb and 3kb) are indicated. Re-hybridization with a cyclophilin pIB15probe (lkb) provided an internal control for the amount of RNA in each lane.

Figure 13 shows the results of an experiment using confocal microscopy showing the effects of NGF treatment on the co-localization and internalization of HA-rPAST and the TrkA receptor. The trkPC12 cells were transiently transfected with HA-rPAST and treated with NGF for the indicated times. After fixation and permeabilization, cells were stained using antibodies to trkA (α-trk, rabbit polyclonal) and HA (α-HA, mouse monoclonal Ig2a). TrkA labeling is shown on the left, HA labeling is shown in the center and both labels are shown on the right. The photographic images are negatives of black and white images; in the original photographs, trk a antibodies were labeled with a green fluorophore and HA antibodies were labeled with a red fluorophore. En the absence of NGF ("control"), trkA and HA-rPAST staining is present diffusely at the surface membrane (A) with some trkA in the juxtanuclear region (B). After NGF treatment for 5 minutes at 37 degrees Celsius ("NGF 5 min"), trkA and HA-rPAST are both internalized into the cytosol (B) and co-localized together with excess HA-rPAST protein remaining on the plasma membrane (A). After 1 hour of NGF treatment at 37 degrees Celsius ("NGF 1 hour"), HA-rPAST only appears on the plasma membrane (A) while trkA remains only in the cytosol (B).

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Claims

We claim:

1. An isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ED NO:l, SEQ ID NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:5, SEQ ED NO:6, SEQ ID NO:7, SEQ ED NO:8, SEQ ED NO:9, SEQ ID NO: 10, SEQ ED NO:l l, SEQ ED NO:12, SEQ ED NO:13, SEQ ID NO:14, SEQ ED NO:15, SEQ ED NO:16, SEQ ED NO: 17, SEQ ID NO: 18, SEQ ED NO: 19, SEQ ED NO:20, SEQ ED NO:21, SEQ ED NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ED NO:28 and SEQ ID NO:29.

2. An isolated polypeptide encoded by a polynucleotide chosen from the group consisting SEQ ED NO:l, SEQ ED NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:5, SEQ

ID NO:6, SEQ ED NO:7, SEQ ED NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ED NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ED NO:16, SEQ ID NO:17, SEQ ED NO:18, SEQ ED NO:19, SEQ ID NO:20, SEQ ED NO:21, SEQ ID NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ED NO:27, SEQ ED NO:28 and SEQ ED NO:29.

3. An isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to the isolated nucleic acid molecule of claim 1.

4. An isolated nucleic acid molecule at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of claim 1 under stringent conditions.

5. An isolated nucleic acid molecule encoding the polypeptide of claim 2.

6. An isolated nucleic acid molecule encoding a fragment of the polypeptide of claim 2.

7. An isolated nucleic acid molecule encoding a polypeptide epitope of the polypeptide of claim 2.

8. The polypeptide of claim 2 wherein the polypeptide has biological activity.

9. An isolated nucleic acid encoding a species homologue of the polypeptide of claim 2.

10. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the 5' end or the 3 'end.

11. A recombinant vector comprising the isolated nucleic acid molecule of claim

1.

12. A recombinant host cell comprising the isolated nucleic acid molecule of claim 1.

13. A method of making the recombinant host cell of claim 12.

14. The recombinant host cell of claim 12 comprising vector sequences.

15. The isolated polypeptide of claim 2, wherein the isolated polypeptide comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

16. An isolated antibody that binds specifically to the isolated polypeptide of claim 2.

17. The isolated antibody of claim 16 wherein the antibody is a monoclonal antibody.

18. The isolated antibody of claim 16 wherein the antibody is a polyclonal antibody.

19. A recombinant host cell that expresses the isolated polypeptide of claim 2.

20. An isolated polypeptide produced by the steps of: (a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and

(b) isolating the polypeptide.

21. A method for preventing, treating, modulating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 2 or the polynucleotide of claim 1.

22. The method of claim 21 wherein the medical condition is a disorder of altered target cell metabolism of NGF.

23. The method of claim 21 wherein the medical condition is Alzheimer's Disease.

24. The method of claim 21 wherein the medical condition is diabetic neuropathy.

25. The method of claim 21 wherein the medical condition is congenital insensitivity to pain with anhidrosis.

26. The method of claim 21 wherein the medical condition is a side effect of NGF therapy.

27. The method of claim 21 wherein the medical condition is hyperalgesia associated with of NGF therapy.

28. A method for preventing, treating, modulating, or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of the antibody of claim 16.

29. The method of claim 28 wherein the medical condition is a disorder of altered target cell metabolism of NGF.

30. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:

(a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

31. The method of claim 30 wherein the pathological condition is a disorder of altered target cell metabolism of NGF.

32. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising detecting an alteration in expression of a polypeptide encoded by the polynucleotide of claim 1, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.

33. The method of claim 32 wherein the alteration in expression is an increase in the amount of expression or a decrease in the amount of expression.

34. The method of claim 32 wherein the pathological condition is a disorder of altered target cell metabolism of NGF.

35. The method of claim 32 wherein the method further comprises the steps of: obtaining a first biological sample from a patient suspected of suffering a disorder of altered target cell metabolism of NGF and obtaining a second sample from a suitable comparable control source;

(a) determining the amount of at least one polypeptide encoded by a polynucleotide of claim 1 in the first and second sample; and

(b) comparing the amount of the polypeptide in the first and second samples; wherein a patient is diagnosed as having a disorder of neuronal differentiation. if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.

36. The use of the polynucleotide of claim 1 or polypeptide of claim 2 for the manufacture of a medicament for the treatment of a disorder of altered target cell metabolism of NGF.

37. The use of the antibody of claim 16 for the manufacture of a medicament for the treatment of a disorder of altered target cell metabolism of NGF.

38. A method for identifying a binding partner to the polypeptide of claim 2 comprising:

(a) contacting the polypeptide of claim 2 with a binding partner; and

(b) determining whether the binding partner effects an activity of the polypeptide.

39. The gene conesponding to the cDNA sequence of the isolated nucleic acid of claim 1.

40. A method of identifying an activity of an expressed polypeptide in a biological assay, wherein the method comprises: (a) expressing the polypeptide of claim 2 in a cell;

(b) isolating the expressed polypeptide;

(c) testing the expressed polypeptide for an activity in a biological assay; and

(d) identifying the activity of the expressed polypeptide based on the test results.

41. A substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in a disorder of neuronal differentiation, chosen from the group consisting of the DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in response of PC 12 cells to NGF.

42. A kit for detecting the presence of the polypeptide of the claim 2 in a mammalian tissue sample comprising a first antibody which immunoreacts with a mammalian protein encoded by a gene conesponding to the polynucleotide of claim 1 or with a polypeptide encoded by the polynucleotide of claim 2 in an amount sufficient for at least one assay and suitable packaging material.

43. A kit of claim 42 further comprising a second antibody that binds to the first antibody.

44. The kit of claim 43 wherein the second antibody is labeled.

45. The kit of claim 44 wherein the label comprises enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.

46. A kit for detecting the presence of a genes encoding an protein comprising a polynucleotide of claim 1, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.

47. A method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample, comprising the steps of:

(a) hybridizing a polynucleotide of claim 1 or fragment thereof having at least 10 contiguous bases, with the nucleic acid of the sample; and

(b) detecting the presence of the hybridization product.