EP1402010A2 - 8843, a human dual specificity phosphatase family member and uses therefor - Google Patents

Abstract

The invention provides isolated nucleic acids molecules, designated 8843 nucleic acid molecules, which encode novel dual specificity phosphatase members. The invention also provides methods of modulating the differentiation and proliferation of erythroid progenitors, and CD34 positive cells. The invention further provides methods of treating, preventing and diagnosing eythroid-associated disorders such as anemias, leukemias, and erythrocytosis.

Description

8843, A NOVEL HUMAN DUAL SPECIFICITY PHOSPHATASE FAMILY MEMBER

AND USES THEREOF

Related Applications This application claims priority to U.S. application number 09/686,673 filed on

October 11, 2000, the contents of which are incorporated herein by reference.

Background ofthe Invention The intracellular phosphorylation of proteins is critical for a plethora of regulatory and signalling mechanisms in eukaryotic cells. Phosphorylation events can govern a wide range of cellular processes, including cell proliferation, differentiation, transcription, and morphology. Serine/threonine protein kinases, also called serine protein kinases, are frequently utilized in signalling cascades as the activity of these enzymes can be finely regulated by stimuli. A common stimulus is phosphorylation ofthe serine protein kinase itself. Hence, signalling pathways, such as the MAP protein kinase cascade, can contain multiple proteins kinases which are sequentially activated. Ultimately, kinase cascades can result in the phosphorylation of cytoskeletal proteins, transcription factors, and biosynthetic enzymes. Another class of kinases includes the receptor tyrosine kinases. Activated receptor tyrosine kinases not only autophosphorylate, but phosphorylate other intracellular signalling molecules, including those specifically bound to autophosphorylated receptors. An essential component ofthe aforementioned signalling pathways is the ability of the cell to desensitize, recycle, and counteract phosphorylation signals. The cell primarily utilizes enzymes, termed phosphatases, which remove the phosphate on tyrosine, serine, and threonine side chains. Dual specificity phosphatases hydrolyze phospho yrosine, phosphothreonine, and phosphoserine residues (for a review, see, e.g., Fauman and Saper (1996) Trends in Biochem. 21:412). This class of proteins is exemplified by the VHl or vaccinia virus late HI gene protein, whose catalytic activity is required for vaccinia virus replication. A human homolog of VHl, VHR, has also been identified. VHl -like dual specificity phosphatase can also include the phosphatases PAC-1 and CL100/MKP-1, hVH-2/MKP-2, hVH-3, MKP-3, MKP-X, MKP-4, hVH-5, and M3/6 proteins. The PAC-1 and CE100 proteins hydrolyze phosphothreonine and phosphotyrosine residues on phosphorylated MAP (mitogen activated protein) kinases. In order to modulate signalling events, the activity and expression of dual specificity phosphatases can be finely regulated. For example, the PAC-1 and CL100 phosphatase can be induced by growth factors (Keyse, S (1995) Biochim. Biophys. Actal265:152-160).

Thus, the function of dual specificity phosphatase proteins can be critical for the regulation of cellular processes such as proliferation and differentiation.

Summary ofthe Invention

The present invention is based, in part, on the discovery of a novel dual specificity phosphatase family member, referred to herein as "8843". The nucleotide sequence of a cDNA encoding 8843 is shown in SEQ ID NO:l, and the amino acid sequence of an 8843 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of he coding region are depicted in SEQ ID NO: 3.

Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes an 8843 protein or polypeptide, e.g., a biologically active portion ofthe 8843 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 8843 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number . In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number .

In other embodiments, the invention provides a nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3, or the sequence of he DNA insert ofthe plasmid deposited with ATCC Accession Number , wherein the nucleic acid encodes a full length 8843 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs which include an 8843 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules ofthe invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 8843 nucleic acid molecules ofthe invention e.g., vectors and host cells suitable for producing 8843 nucleic acid molecules and polypeptides.

In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 8843 -encoding nucleic acids. In still another related aspect, isolated nucleic acid molecules that are antisense to an 8843 encoding nucleic acid molecule are provided. hi another aspect, the invention features, 8843 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 8843-mediated or -related disorders, e.g., erythroid associated disorders. In another embodiment, the invention provides 8843 polypeptides having an 8843 activity. Preferred polypeptides are 8843 proteins including at least one dual specificity phosphatase domain, and, preferably, having an 8843 activity, e.g., an 8843 activity as described herein. In other embodiments, the invention provides 8843 polypeptides, e.g., an 8843 polypeptide having the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with ATCC Accession

Number ; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO.l or SEQ ID NO:3, or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC

Accession Number , wherein the nucleic acid encodes a full length 8843 protein or an active fragment thereof. In a related aspect, the invention further provides nucleic acid constructs which include an 8843 nucleic acid molecule described herein.

In a related aspect, the invention provides 8843 polypeptides or fragments operatively linked to non- 8843 polypeptides to form fusion proteins.

In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 8843 polypeptides.

In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity ofthe 8843 polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating 8843 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression ofthe 8843 polypeptides or nucleic acids, such as conditions involving aberrant (e.g., enhanced or deficient) erythroid cell activity (e.g., aberrant erythroblast proliferation, e.g., an erythroleukemia, and/or aberrant erythroblast differentiation, e.g., an anemia).

In another aspect, the invention features a method for identifying an agent, e.g., a compound, that interacts with, e.g., binds to, or modulates an activity of an 8834 polypeptide or nucleic acid. The method includes contacting the 8834 polypeptide or nucleic acid, or a cell containing, e.g., expressing, the 8834 polypeptide or nucleic acid with a test compound, and determining whether the polypeptide or nucleic acid interacts with, e.g., binds to, the test compound, or whether the test compound modulates an activity of an 8834 polypeptide.

In a preferred embodiment, the interaction, e.g., binding, ofthe test compound to the 8834 polypeptide or nucleic acid is detected by one ofthe following methods: a) detection of binding (e.g., direct binding) by labeling the test compound or the 8843 polypeptide or nucleic acid with a detectable signal, e.g., a radioactive metal ion or a fluorescent signal, and detecting the bound labeled complex ofthe test compound/polypeptide or nucleic acid binding relative to the unbound fraction; b) detection of binding using a competition binding assay; or c) detection of binding using an assay for 8843 polypeptide activity, e.g., protein phosphatase activity.

In a preferred embodiment, the 8834 activity is hematopoiesis, e.g., erythropoiesis.

In a preferred embodiment, the 8834 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 8834 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, or more contiguous amino acids of SEQ ID NO:2.

In a preferred embodiment, the 8834 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:l or 3. In other embodiments, the 8834 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO: 1 or 3.

In preferred embodiments, the compound is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody. In additional preferred embodiments, the compound is an antisense, a ribozyme, or a triple helix molecule.

In another aspect, the invention features a method of identifying a compound which modulates the activity (e.g., hematopoiesis, e.g., erythropoiesis) or expression of an 8834 polypeptide or nucleic acid. The method includes contacting the polypeptide or nucleic acid with a test compound; and determining the effect ofthe test compound on the activity or expression ofthe polypeptide or nucleic acid. In a preferred embodiment, the 8834 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 8834 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, or more contiguous amino acids of SEQ ID NO:2. In a preferred embodiment, the determined activity is protein phosphatase activity or erythropoiesis.

In a preferred embodiment, the 8834 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:l or 3. In other embodiments, the 8834 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO: 1 or 3.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression ofthe 8843 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody. In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule.

In a preferred embodiment, the contacting step occurs in vitro or ex vivo.

In a preferred embodiment, the contacting step occurs in vivo. For example, the contacting step can occur in an animal model for an erythroid-associated disorder.

In yet another aspect, the invention features a method for modulating (e.g., increasing or decreasing) the activity or expression of an 8834 polypeptide or nucleic acid, comprising contacting the 8843 polypeptide or nucleic acid, or a cell containing (e.g., expressing) the 8843 polypeptide or nucleic acid with a agent which interacts with, e.g., binds to, the 8843 polypeptide or nucleic acid in a sufficient concentration to modulate the activity or expression ofthe polypeptide or nucleic acid. In a preferred embodiment, the 8834 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 8834 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, or more contiguous amino acids of SEQ ID NO:2.

In a preferred embodiment, the determined activity is protein phosphatase activity. In a preferred embodiment, the 8834 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:l or 3. In other embodiments, the 8834 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO:l or 3. hi a preferred embodiment, the agent modulates (e.g., increases or decreases) expression ofthe 8843 nucleic acid by, e.g., modulating transcription, nucleic acid stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 8843 nucleic acid, or any combination thereof.

In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) the proliferation, survival, or differentiation of a cell, e.g., an 8834-expressing cell, e.g., a hematopoietic cell (e.g., a bone marrow cell, such as a CD34 positive cell or a cell expressing a glycophorin A protein). The method includes contacting the cell with a agent that modulates the activity or expression of an 8834 polypeptide or nucleic acid, in an amount effective to modulate the proliferation and/or differentiation ofthe cell.

In a preferred embodiment, the 8834 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 8834 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, or more contiguous amino acids of SEQ ID NO:2.

In a preferred embodiment, the 8834 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:l or 3. In other embodiments, the 8834 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO:l or 3.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein phosphatase activity.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression ofthe 8843 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion. In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 8843 nucleic acid, or any combination thereof.

In a preferred embodiment, the agent is administered in combination with a cytotoxic agent. In a preferred embodiment, the cell, e.g., the 8834-expressing cell, is a pluripotent hematopoietic stem cell, e.g., a granulocytic, monocytic, erythroid, megakaryocytic, and lymphoid cell, or a precursor cell thereof. Examples of such cells include myelocytic cells (polymorphoneuclear cells), erythrocytic cells, lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes, as well as stem cells for the different lineages, and precursors for the committed progenitor cells, for example, precursors of red blood cells (erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts).

In a preferred embodiment, the cell, e.g., the 8834-expressing cell, is a bone marrow cell, e.g., a CD34-expressing cell. Examples of CD34-expressing cells include immature haematopoietic precursor cells, haematopoietic colony-forming cells in bone marrow, including unipotent (CFU-GM, BFU-E) and pluripotent progenitors (CFU-GEMM, CFU- Mix and CFU-blast); as well as stromal cell precursors, terminal deoxynucleotidyl transferase (TdT) expressing B- and T-lymphoid precursors, early myeloid cells and early erythroid cells. hi another preferred embodiment, the cell is a progenitor cell, e.g., a CFU-GEMM (colony forming unit - granulocyte, erythrocyte, macrophage, megakaryocyte), a CFU-E (colony forming unit - erythrocyte), a CFU-GM (colony forming unit - granulocyte, monocytes), or a CFU-G (colony forming unit - granulocyte). In one embodiment, the cell is a neutrophil precursor, e.g., a CFU-GM, CFU-G

(colony forming unit - granulocyte), myeloblast, promyelocyte, myelocyte, a metamyelocyte, or a band cell. In another embodiment, the cell is platelet precursor, e.g., a promegakaiyoblast, a megakaryoblast, or a megakaryocyte. In still another embodiment, the cell is a monoblast or other macrophage precursor, a lymphoblast. In a preferred embodiment, the cell is a BFU-E (blast forming unit - erythroid),

CFU-E, normoblast or erythroblast, a basophilic pronormoblast, polychromatophilic pronormoblast, orthochromatophilic pronormoblast, a reticulocyte, or erythrocyte.

In a preferred embodiment, the cell, e.g., the 8834-expressing cell, is an erythroid cell, e.g., an erythroid progenitor or differentiated cell, e.g., an erythrocyte, or a cell expressing a glycophorin A protein.

In a preferred embodiment, the cell, e.g., the 8834-expressing cell, is further contacted with a protein selected from the group consisting of G-CSF, GM-CSF, stem cell factor, Flt-3 ligand, IL-3, IL-4, thrombopoietin, and erythropoietin. Preferably, the protein is erythropoietin. The protein contacting step can occur before, at the same time, or after the agent is contacted. The protein contacting step can be effected in vitro or ex vivo. For example, the cell, e.g., the 8834-expressing cell is obtained from a subject, e.g., a patient, and contacted with the protein ex vivo. The treated cell can be re-introduced into the subject. Alternatively, the protein contacting step can occur in vivo.

In a preferred embodiment, the agent and the 8834-polypeptide or nucleic acid are contacted in vitro or ex vivo.

In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. Preferably, the subject is a human, e.g., a patient with an erythroid-associated disorder. For example, the subject can be a patient with an anemia, e.g., a drug- (e.g., chemotherapy-) induced anemia, hemolytic anemia, aberrant erythropoiesis, secondary anemia in nonhematolic disorders, anemia of chronic disease such as chronic renal failure; endocrine deficiency disease; and/or erythrocytosis (e.g., polycythemia). Preferably, the anemia is a drug- (e.g., chemotherapy-) induced anemia. Alternatively, the subject can be a cancer patient, e.g., a patient with leukemic cancer, e.g., an erythroid leukemia, or a carcinoma, e.g., a renal carcinoma.

The contacting step(s) can be repeated.

In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation ofthe cell, e.g., the 8834-expressing cell, e.g., erythroid cell. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas, e.g., renal carcinomas.

In a preferred embodiment, the agent increases the number of erythroid cells, by e.g., increasing the proliferation, survival, and/or stimulating the differentiation, of eythroid progenitor cells. Such agents can be used to treat or prevent anemias, e.g., a drug- (e.g., chemotherapy-) induced anemia, hemolytic anemias, aberrant erythropoiesis, secondary anemias in nonhematolic disorders, anemias of chronic diseases such as chronic renal failure; endocrine deficiency diseases; and/or erythrocytosis (e.g., polycythemias). Preferably, the anemia is a drug- (e.g., chemotherapy-) induced anemia

In a preferred embodiment, the agent increases the number of erythroid cells, by e.g., increasing the proliferation, survival, and/or stimulating the differentiation, of granulocytic and monocytic progenitor cells, e.g., CFU-GM, CFU-G (colony forming unit - granulocyte), myeloblast, promyelocyte, myelocyte, a metamyelocyte, or a band cell. Such agents can be used to treat or prevent neutroponeia and granulocytopenia, e.g., conditions caused by cytotoxic chemotherapy, AIDS, congenital and cyclic neutropenia, myelodysplastic syndromes, or aplastic anemia.

In another aspect, the invention features a method of modulating hematopoiesis, e.g., erythropoiesis, comprising contacting a hematopoietic cell, e.g., an erythroid cell (e.g., an 8834-expressing cell), with a agent that increases or decreases the activity or expression of an 8834 polypeptide or nucleic acid, thereby modulating the differentiation ofthe hematopoietic cell, e.g., erythroid cell.

In a preferred embodiment, the 8834 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 8834 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, or more contiguous amino acids of SEQ ID NO:2.

In a preferred embodiment, the 8834 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:l or 3. In other embodiments, the 8834 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO: 1 or 3.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein phosphatase activity.

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion. hi additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 8843 nucleic acid, or any combination thereof.

In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.

In a preferred embodiment, the hematopoietic cell is is a pluripotent hematopoietic stem cell, e.g., a cell that can give rise to progenitors ofthe granulocytic, monocytic, erythroid, megakaiyocytic, and lymphoid lineages.

In a preferred embodiment, the erythroid cell is an erythroid progenitor or differentiated cell, e.g., an erythrocyte, or a cell expressing a glycophorin A protein.

In a preferred embodiment, the agent and the 8834-polypeptide or nucleic acid are contacted in vitro or ex vivo. In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. Preferably, the subject is a human, e.g., a patient with an erythroid-associated disorder. For example, the subject can be a patient with an anemia, e.g., hemolytic anemia, aberrant erythropoiesis, secondary anemia in nonhematolic disorders, anemia of chronic disease such as chronic renal failure; endocrine deficiency disease; and/or erythrocytosis (e.g., polycythemia). Alternatively, the subject can be a cancer patient, e.g., a patient with leukemic cancer, e.g., an erythroid leukemia.

In a preferred embodiment, the method further includes contacting ofthe erythroid cell with a protein selected from the group consisting of G-CSF, GM-CSF, stem cell factor, Flt-3 ligand, IL-3, IL-4, thrombopoietin, and erythropoietin. Preferably, the protein is erythropoietin. The protein contacting step can occur before, at the same time, or after the agent is contacted. The protein contacting step can be effected in vitro or ex vivo. For example, the cell, e.g., the erythroid cell can be obtained from a subject, e.g., a patient, and contacted with the protein ex vivo. The treated cell can be re-introduced into the subject. Alternatively, the protein contacting step can occur in vivo.

The contacting step(s) can be repeated.

In a preferred embodiment, the agent increases the number of erythroid cells, by e.g., increasing the proliferation, survival, and/or stimulating the differentiation, of eythroid progenitor cells, in the subject Such agents can be used to treat anemias, e.g., hemolytic anemias, aberrant erythropoiesis, secondary anemias in nonhematolic disorders, anemias of chronic diseases such as chronic renal failure; endocrine deficiency diseases; and/or erythrocytosis (e.g., polycythemias).

In yet another aspect, the invention features a method of treating or preventing a hematopoietic disorder, e.g., an erythroid-associated disorder, in a subject. The method includes administering to the subject an effective amount of a agent that modulates the activity or expression of an 8834 polypeptide or nucleic acid such that the hematopoietic disorder is ameliorated or prevented.

In a preferred embodiment, the 8834 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 8834 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, or more contiguous amino acids of SEQ ID NO:2.

In a preferred embodiment, the 8834 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:l or 3. In other embodiments, the 8834 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NOT or 3. hi a preferred embodiment, the agent modulates (e.g., increases or decreases) protein phosphatase activity. In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression ofthe 8843 nucleic acid by, e.g., modulating transcription, mRNA stability, etc. hi preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion. i additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 8843 nucleic acid, or any combination thereof.

In a preferred embodiment, the agent is administered in combination with a cytotoxic agent. In a preferred embodiment, the subject is a human, e.g., a patient with a hematopoietic disorder, e.g., an erythroid-associated disorder. For example, the subject can be a patient with an anemia, e.g., a drug- (e.g., chemotherapy-) induced anemia, hemolytic anemia, aberrant erythropoiesis, secondary anemia in nonhematolic disorders, anemia of chronic disease such as chronic renal failure; endocrine deficiency disease; and/or erythrocytosis (e.g., polycythemia). Preferably, the anemia is a drug- (e.g., chemotherapy-) induced anemia. Alternatively, the subject can be a cancer patient, e.g., a patient with leukemic cancer, e.g., an erythroid leukemia, or a patient with a carcinoma, e.g., a renal carcinoma.

In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of a cell, e.g., an 8834-expressing cell, e.g., an erythroid cell, in the subject. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas, e.g., renal carcinomas.

In a preferred embodiment, the agent increases the number of erythroid cells, by e.g., increasing the proliferation, and/or stimulating the differentiation, of eythroid progenitor cells, in the subject. Such agents can be used to treat anemias, e.g., a drug- (e.g., chemotherapy-) induced anemia, hemolytic anemias, aberrant erythropoiesis, secondary anemias in nonhematolic disorders, anemias of chronic diseases such as chronic renal failure; endocrine deficiency diseases; and/or erythrocytosis (e.g., polycythemias). In a preferred embodiment, the hematopoietic disorder is a differentiative disorder, e.g., a disorder of granulocyte, monocytes or macrophage, megakaryocyte, or lymphocyte differentiation. For example, the disorder is a disorder of granulopoiesis, lymphopiesis, or megakaryopoiesis. In another embodiment, the hematopoietic disorder is a proliferative disorder, e.g., a disorder of granulocyte, monocytes or marcophage, megakaryocyte, or lymphocyte progenitor cell proliferation.

In a preferred embodiment, the disorder is an erythroid-associated disorder. Examples of erythroid-associated disorder include a drug- (e.g., chemotherapy-) induced anemia, hemolytic anemia, aberrant erythropoiesis, secondary anemia in nonhematolic disorders, anemia of chronic disease such as chronic renal failure; endocrine deficiency disease; and/or erythrocytosis (e.g., polycythemia).

In a preferred embodiment, the disorder is a cancer, e.g., a leukemic cancer, e.g., an erythroid leukemia, or a carcinoma, e.g., a renal carcinoma

In a preferred embodiment, the method further includes administering an effective amount of a protein selected from the group consisting of G-CSF, GM-CSF, stem cell factor, Flt-3 ligand, IL-3, IL-4, thrombopoietin, and erythropoietin to the subject. Preferably, the protein is erythropoietin. The protein can be administered before, at the same time or after, administration ofthe agent.

The administration ofthe agent and/or protein can be repeated. In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder, in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of an 8834 nucleic acid or 8834 polypeptide, such that a change in the level of 8834 nucleic acid or 8834 polypeptide after treatment, relative to the level before treatment, is indicative ofthe efficacy ofthe treatment ofthe disorder. In a preferred embodiment, the disorder is an erythroid-associated disorder.

Examples of erythroid-associated disorders include anemias, e.g., a drug- (e.g., chemotherapy-) induced anemia, hemolytic anemias, aberrant erythropoiesis, secondary anemia in nonhematolic disorders, anemias of chronic disease such as chronic renal failure; endocrine deficiency diseases; and/or erythrocytosis (e.g., polycythemia). In a preferred embodiment, the disorder is a cancer, e.g., leukemic cancer, e.g., an erythroid leukemia, or a carcinoma, e.g., a renal carcinoma

In a preferred embodiment, the subject is a human. In a preferred embodiment, the subject is an experimental animal, e.g., an animal model for an erythroid-associated disorder.

In a preferred embodiment, the method can further include treating the subject with a protein selected from the group consisting of G-CSF, GM-CSF, stem cell factor, Flt-3 ligand, IL-3, IL-4, thrombopoietin, and erythropoietin prior to assessing expression levels. Preferably, the protein is erythropoietin.

The invention also features a method of diagnosing a hematopoietic disorder, e.g., an erythroid-associated disorder, in a subject. The method includes evaluating the expression or activity of a 8834 nucleic acid or a 8834 polypeptide, such that, a difference in the level of 8834 nucleic acid or 8834 polypeptide relative to a normal subject or a cohort of normal subjects is indicative ofthe hematopoietic disorder.

In a preferred embodiment, the subject is a human. i a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, is obtained from the subject. In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 8834 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression ofthe 8834 nucleic acid or polypeptide.

In a preferred embodiment, the hematopoeitic disorder is an erythroid-associated disorder, e.g., an erythroid-associated disorder as described herein.

In yet another aspect, the invention features a method for identifying an agent, e.g., a agent, which modulates the activity of a 8834 polypeptide, e.g., a 8834 polypeptide as described herein, or the expression of a 8834 nucleic acid, e.g., a 8834 nucleic acid as described herein, including contacting the 8834 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect ofthe test agent on the activity ofthe polypeptide or nucleic acid to thereby identify a agent which modulates the activity ofthe polypeptide or nucleic acid.

In a preferred embodiment, the activity ofthe 8834 polypeptide is protein phosphatase activity. In a preferred embodiment, the activity ofthe 8834 polypeptide is hematopoiesis, e.g., erythropoiesis.

In a preferred embodiment, the activity ofthe 8834 polypeptide is proliferation, differentiation, and/or survival of a cell, e.g., a 8834-expressing cell, e.g., a hematopoietic cell (e.g., a bone marrow cell such as a CD34 positive cell, an erythroid cell, a megakaryocyte).

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.

In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 8834 nucleic acid, or any combination thereof.

The invention also provides assays for determining the activity of or the presence or absence of 8843 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

In further aspect the invention provides assays for determining the presence or absence of a genetic alteration in an 8843 polypeptide or nucleic acid molecule, including for disease diagnosis.

In another aspect, the invention features a two dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address ofthe plurality has a capture probe that recognizes a 8843 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 8843 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 8843 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding ofthe sample to the array.

The terms "agent" and "compound" are used interchangeably herein.

Other features and advantages ofthe invention will be apparent from the following detailed description, and from the claims.

Brief Description ofthe Drawings Figure 1 depicts a cDNA sequence (SEQ ID NO:l) and predicted amino acid sequence (SEQ ID NO:2) of human 8843. The methionine-initiated open reading frame of human 8843 (withoutthe 5' and 3' untranslated regions) until the end of SEQ ID NO.T is shown also as coding sequence SEQ ID NO:3.

Figure 2 depicts a hydropathy plot of human 8843. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 8843 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 9 to 25, from about 45 to 52, and from about 142 to 150, of SEQ ID NO:2; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 27 to 36, from about 70 to 78, and from about 151 to 160, of SEQ ID NO:2; a sequence which includes a Cys, or a glycosylation site.

Figure 3 depicts an alignment ofthe dual specificity phosphatase domain of human 8843 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:4), while the lower amino acid sequence corresponds to amino acids 37 to 185 of SEQ ID NO:2.

Figure 4 depicts an alignment ofthe dual specificity phosphatase domain of human 8843 with a consensus amino acid sequence derived from a hidden Markov model (HMM) in the SMART domain library. The upper sequence is the consensus amino acid sequence (SEQ ID NO:5), while the lower amino acid sequence corresponds to amino acids 37 to 185 of SEQ IDNO:2.

Figure 5 is a bar graph depicting relative 8843 mRNA expression as determined by the TaqMan assay on mRNA derived from a hematological human samples, e.g. , erythroid cells (ery), megakaryocytes (meg), neutrophils, platelets, mast cells, and erythroid burst forming units.

Figure 6 is a bar graph depicting relative 8843 mRNA expression as determined by the TaqMan assay on mRNA derived from tissues including cord blood, fetal liver, and bone marrow, e.g., glycophorin A positive (low) bone marrow, and CD34+ bone marrow.

Figure 7 is a bar graph depicting relative 8843 mRNA expression as determined by the TaqMan assay on mRNA on from tissues including K562 cells, an erythroid- megakaryocyte line, and hepatic tissues.

Figure 8 is a bar graph depicting relative 8843 mRNA expression as determined by the TaqMan assay on mRNA derived from lung, fetal liver, and other tissues. Detailed Description The human 8843 sequence (Figure 1; SEQ ID NOT), which is approximately 839 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 606 nucleotides, including the termination codon (nucleotides indicated as coding of SEQ ID NO:l in Fig. 1; SEQ ID NO:3). The coding sequence encodes a 201 amino acid protein (SEQ ID NO:2).

Human 8843 contains the following regions or other structural features: a dual specificity phosphatase domain (PFAM Accession Number PF00782) located at about amino acid residues 37 to 185 of SEQ ID NO:2; a tyrosine specific protein phosphatase active site signature (Prosite PS00383), also termed "C-X₅-R" motif, located at about amino acid residues 130 to 142 of SEQ ID NO:2, including an active site cysteine at about amino acid 132 of SEQ ID NO:2, and an active site arginine at about amino acid 138 of SEQ ID NO:2; a dual specificity phosphatase extended active site signature

(VXVHCXXGXSRSXTXXXAY[LI]M; SEQ IDNO:8; Mudaet /. (1996) J Biol Chem 271:27205) locate at about amino acid residues 128 to 158 of SEQ ID NO:2; a VHl -like dual specificity phosphatase loop located at about amino acid residues 106 to 110 of SEQ ID NO:2, include a conserved general acid, aspartic acid at about residue 109 of SEQ ID NO:2; one predicted N-glycosylation site (PS00001) at about amino acids 82 to 85 of SEQ ID NO.2; one predicted protein kinase C phosphorylation sites (PS00005) at about amino acids 187 to l89 of SEQ ID O:2; four predicted casein kinase II phosphorylation sites (PS00006) located at about amino acids 70 to 73, 83 to 86, 98 to 101, and 154 to 157 of SEQ ID NO:2; and one predicted N-myristylation sites (PS00008) from about amino acid 114 to 119 of SEQ ID NO-2.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc. edu general/ software/packages/pfam/pfam.html.

A plasmid containing the nucleotide sequence encoding human 8843 was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

The 8843 protein contains a significant number of structural characteristics in common with members ofthe dual specificity phosphatase family. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non- human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

Dual specificity phosphatases are exemplified by the VHl or vaccinia virus late HI gene protein, which hydrolyzes both phosphotyrosine, phosphothreonine, and phosphoserine. VHl catalytic activity is required for viral replication. A human homolog of VHl, VHR, has been identified. The three dimensional structure of this family is based on models from x-ray crystallographic data of protein tyrosine phosphatases, and human VHR The VHR structure includes a core domain consisting of a five-stranded mixed β- sheet and six α-helices. This structure closely superimposes on the structure of phosphotyrosine protein phosphatases. However, dual specificity phosphatases lack the KNRY motif, and the N-terminal structures of tyrosine protein phosphatases which endow these enzymes with a deep active site specific for aryl phosphates. Thus, dual specificity phosphatases have a shallower active site relative to tyrosine protein phosphatases and can accommodate phosphoserine and phosphothreonine substrates. Even so dual specificity phosphatases can have a greater than 50-fold faster rate of phosphatase activity for phosphotyrosine substrates than phosphothreonine or phosphoserine substrates. Similar to the broader class of phosphatases, dual specificity phosphatases have a highly conserved active site including three catalytic residues, a cysteine, an arginine, and an aspartic acid. The active site cysteine and arginine are found in the "C-X5-R" motif ofthe tyrosine phosphatase signature (Prosite PS00383). This motif forms a binding pocket for three ofthe phosphate oxyanions. The cysteine acts as a nucleophile to accept the PO₃ group. The reaction transiently generates a phospho-cysteine intermediate before the phosphate is transferred to water. The active site arginine stabilizes the transition-state by hydrogen bonding to phosphate oxygens. In addition the histidine preceding the active site cysteine and the serine or Ihreonine following the active site arginine are responsible for lowering the pKa ofthe cysteine to stabilize a negative charge on the cysteine. The active site aspartic acid accelerates the reaction by donating a protein to generate an uncharged hydroxyl (for a review, see Fauman and Saper (1996) Trends in Biochem. 21:412).

An 8843 polypeptide can include a "dual specificity phosphatase catalytic domain" or regions homologous with a "dual specificity phosphatase domain".

As used herein, the term "dual specificity phosphatase domain" includes an amino acid sequence of about 80 to 220 amino acids, more preferably about 100 to 180 amino acid residues, or about 130 to 160 amino acid residues in length and having a bit score for the alignment ofthe sequence to the dual specificity phosphatase domain (HMM) of at least 10, preferably 15, and more preferably 20. The dual specificity phosphatase catalytic domain (HMM) has been assigned the PFAM Accession Number PF00782 (http;//genome.wustl.edu/Pfam .html). An alignment ofthe dual specificity phosphatase domain (amino acids 37 to 185 of SEQ ID NO:2) of human 8843 with a consensus amino acid sequence (SEQ ID NO:4) derived from a hidden Markov model is depicted in Figure 3, and a similar alignment with a consensus amino acid sequence (SEQ ID NO:5) derived from the SMART domain HMM model is depicted in Figure 4.

A dual specificity phosphatase domain preferably includes a perfect match to the Prosite tyrosine specific protein phosphatase active site signature (PS00383; [LIVMF]-H-C- x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein X is any amino acid and a number in parenthesis indicates the amino acid pattern is repeated that number of times; SEQ ID

NO:6). Even more preferably, a dual specificity phosphatase includes the extended active site signature (VXVHCXXGXSRSXTXXXAY[LI]M; SEQ ID NO:8; Mudaet /. (1996)J Biol Chem 271 :27205). A dual specificity phosphatase domain also includes the conserved active site residues cysteine, arginine, and aspartic acid. The aspartic acid is preferably located in a loop region N-terminal to the active site signature.

In a preferred embodiment 8843 polypeptide or protein has a "dual specificity phosphatase domain" or a region which includes at least about 80 to 220 more preferably about 100 to 180 or 130 to 160, or about 148 amino acid residues in length and has at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a "dual specificity phosphatase domain," e.g., the dual specificity phosphatase domain of human 8843 (e.g., residues 37 to 185 of SEQ ID NO:2). In a preferred embodiment, the 8834 polypeptide has a tyrosine specific protein phosphatase active site signature located at about amino acids 130 to 142 of SEQ ID NO:2. The 8834 polypeptide also preferable has a conserved active site serine at about amino acid residue 132 of SEQ ID NO:2, a conserved active site arginine at about amino acid residue 138 of SEQ ID NO:2, and a conserved active site aspartic acid at about amino acid residue 109 of SEQ ID NO:2. Preferably, the active site aspartic acid is in an mobile loop, approximately 20 to 30, or preferably, 20 to 25 amino acids N-terminal to the active site cysteine.

To identify the presence of a "dual specificity phosphatase" domain in an 8843 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http ://www. sanger. ac. uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description ofthe Pfam database can be found in Sonhammer etal. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183 : 146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh etα .(1994)J. Mol. Biol. 235:1501-1531; and Stultz etal.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a "dual specificity phosphatase" domain in the amino acid sequence of human 8843 at about residues 37 to 185 of SEQ ID NO:2 (see Figure 1 ).

An 8843 molecule can further include: preferably at least one N-glycosylation site; preferably at least one protein kinase C phosphorylation sites; at least one, two, three, and preferably four casein kinase II phosphorylation sites; and preferably at least one N- myristylation site.

As used herein, a "8843 activity", "biological activity of 8843" or "functional activity of 8843", refers to an activity exerted by an 8843 protein, polypeptide or nucleic acid molecule on e.g., an 8843-responsive cell or on an 8843 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, an 8843 activity is a direct activity, such as an association with an 8843 target molecule. A "target molecule" or "binding partner" is a molecule with which an 8843 protein binds or interacts in nature, an 8843 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction ofthe 8843 protein with an 8843 receptor. Based on the above-described sequence similarities, the 8843 molecules ofthe present invention are predicted to have similar biological activities as dual specificity phosphatase family members. For example, the 8843 proteins ofthe present invention can have one or more ofthe following activities: (1) catalyzing the removal of a phosphate group attached to a tyrosine residue in a protein; (2) catalyzing the removal of a phosphate group attached to a serine or threonine residue in a protein; (3) modulating an intracellular signaling pathway, e.g., a MAP kinase or ERK kinase pathway; (4) modulating cell differentiation, e.g., differentiation of erythroid progenitor cells, such as, CD34+ progenitors; (5) modulating cell proliferation, e.g., proliferation erythroid progenitor cells; (6) inactivating cell surface growth factor receptors, e.g., tyrosine kinase receptors; or (7) modulating apoptosis, of a cell, e.g., a leukemic cell, (e.g., an erythroleukemia cell).

As the 8843 mRNA is found in hematopoietic cells, and in particular, in erythroid cell lineages (Figures 5-8), the molecules ofthe invention can be used to develop novel agents or compounds to treat and/or diagnose disorders involving aberrant activities of those cells e.g., hematopoietic and, in particular, erythroid disorders, as described below. For example, an 8843 polypeptide is expressed in CD34 positive cells, e.g., mobilized peripheral blood CD34+ cells, normal adult bone maπow CD34+ cells, cord blood CD34+ cells, normal adult bone marrow CD34+ cells, G-CSF-treated bone marrow CD34+ cells, and fetal liver CD34+ cells; and erythroid progenitor cells, e.g., bone marrow glycophorin A positive cells and erythropoietin treated erythroid burst forming units (BFUs) (Figures 5-8). 8843 mRNA is also expressed in hepatic cells, kidney, lung, and dermal cells, and thus diagnostic and therapeutic methods of using the molecules ofthe invention to treat/diagnose hepatic, kidney, lung, and dermal disorders are also contemplated by the present invention.

As used herein, the term " pluripotent hematopoietic stem cell" includes a cell that can give rise to a spleen colony forming unit (day 12 CFU-S), which, in turn, can give rise to progenitors ofthe granulocytic, monocytic, erythroid, megakaryocytic, and lymphoid lineages. As used herein, a "CD34-positive cell" refers to a cell that expresses detectable levels ofthe CD34 antigen, preferably human CD34 antigen. The sequence for human CD34 is provided in SwissProt Accession Number P28906. The CD34 antigen is typically present on immature hematopoietic precursor cells and hematopoietic colony-forming cells in the bone marrow, including unipotent (CFU-GM, BFU-E) and pluripotent progenitors (CFU- GEMM, CFU-Mix and CFU-blast). The CD34 is also expressed on stromal cell precursors. Terminal deoxynucleotidyl transferase (TdT)-positive B- and T-lymphoid precursors in normal bone also are CD34+. The CD34 antigen is typically present on early myeloid cells that express the CD33 antigen, but lack the CD14 and CD15 antigens and on early erythroid cells that express the CD71 antigen and dimly express the CD45 antigen. The CD34 antigen is also found on capillary endothelial cells and approximately 1% of human thymocytes. Normal peripheral blood lymphocytes, monocytes, granulocytes and platelets do not express the CD34 antigen. CD34 antigen density is highest on early haematopoietic progenitor cells and decreases as the cells mature. The antigen is undetectably on fully differentiated haematopoietic cells. Approximately 60% of acute B-lymphoid leukemia's and acute myeloid leukemia express the CD34 antigen. The antigen is not expressed on chronic lymphoid leukemia (B or T lineage) or lymphomas.

As the 8843 polypeptides ofthe invention may modulate 8843-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 8843- mediated or related disorders, e.g., erythroid-associated disorders.

As used herein, the term "erythroid associated disorders" include disorders involving aberrant (increased or deficient) erythroblast proliferation, e.g., an erythroleukemia, and aberrant (increased or deficient) erythroblast differentiation, e.g., an anemia. Erythrocyte- associated disorders include anemias such as, for example, hemolytic anemias due to hereditary cell membrane abnormalities, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis; hemolytic anemias due to acquired cell membrane defects, such as paroxysmal nocturnal hemoglobinuria and spur cell anemia; hemolytic anemias caused by antibody reactions, for example to the RBC antigens, or antigens ofthe ABO system, Lewis system, Ii system, Rh system, Kidd system, Duffy system, and Kell system; methemoglobinemia; a failure of erythropoiesis, for example, as a result of aplastic anemia, pure red cell aplasia, myelodysplastic syndromes, sideroblastic anemias, and congenital dyserythropoietic anemia; secondary anemia in nonhematolic disorders, for example, as a result of chemotherapy (e.g., a drug- (e.g., chemotherapy-) induced anemia), alcoholism, or liver disease; anemia of chronic disease, such as chronic renal failure; and endocrine deficiency diseases. Preferably, the anemia is a drug- (e.g., chemotherapy-) induced anemia.

Compounds that modulate 8843 polypeptide or nucleic acid activity or expression can be used to treat anemias, in particular, anemias associated with cancer chemotherapy, chronic renal failure, malignancies, adult and juvenile rheumatoid arthritis, disorders of haemoglobin synthesis, prematurity, and zidovudine treatment of HIV infection. A subject receiving the treatment can be additionally treated with a second agent, e.g., erythropoietin, to futher ameliorate the condition. As used herein, the term "erythropoietin" or "EPO" refers to a glycoprotein produced in the kidney, which is the principal hormone responsible for stimulating red blood cell production (erythrogenesis). EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow. Normal plasma erythropoietin levels range from 0.01 to 0.03 Units/mL, and can increase up to 100 to 1,000- fold during hypoxia or anemia. Graber and Krantz,--4rø. Rev. Med. 29:51 (1978); Eschbach and Adamson, Kidney Intl. 28:1 (1985). Recombinant human erythropoietin (rHuEpo or epoetin alfa) is commercially available as EPOGEN.RTM. (epoetin alfa, recombinant human erythropoietin) (Amgen Inc., Thousand Oaks, Calif.) and as PROCRIT.RTM.

(epoetin alfa, recombinant human erythropoietin) (Ortho Biotech Inc., Raritan, N. I).

Another example of an erythroid-associated disorder is erythrocytosis. Erythrocytosis, a disorder of red blood cell overproduction caused by excessive and/or ectopic erythropoietin production, can be caused by cancers, e.g., a renal cell cancer, a hepatocarcinoma, and a central nervous system cancer. Diseases associated with erythrocytosis include polycythemias, e.g., polycythemia vera, secondary polycythemia, and relative polycythemia. Treatment, prevention and diagnosis of cancer or neoplastic disorders related to the erythroid lineage are also included in the present invention. Such neoplastic disorders are exemplified by erythroid leukemias, or leukemias of erythroid precursor cells, e.g., poorly differentiated acute leukemias such as erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) CritRev. in Oncol./Hemotol. 11:267-97). In particular, AML can include the uncontrolled proliferation of CD34+ cells such as AML subtypes Ml and M2, myeloblastic leukemias with and without maturation, and AML subtype M6, erythroleukemia (Di Guglielmo's disease). Additional neoplastic disorders include a myelodysplastic syndrome or preleukemic disorder, e.g., oligoblastic leukemia, smoldering leukemia. Additional cancers ofthe erythroid lineage include erythroblastosis, and other relevant diseases ofthe bone marrow. As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.

The term "leukemia" or "leukemic cancer" is intended to have its clinical meaning, namely, a neoplastic disease in which white corpuscle maturation is arrested at a primitive stage of cell development. The disease is characterized by an increased number of leukemic blast cells in the bone marrow, and by varying degrees of failure to produce normal hematopoietic cells. The condition may be either acute or chronic. Leukemias are further typically categorized as being either lymphocytic i.e., being characterized by cells which have properties in common with normal lymphocytes, or myelocytic (or myelogenous), i.e., characterized by cells having some characteristics of normal granulocytic cells. Acute lymphocytic leukemia ("ALL") arises in lymphoid tissue, and ordinarily first manifests its presence in bone marrow. Acute myelocytic leukemia ("AML") arises from bone marrow hematopoietic stem cells or their progeny. The term acute myelocytic leukemia subsumes several subtypes of leukemia: myeloblastic leukemia, promyelocytic leukemia, and myelomonocytic leukemia. In addition, leukemias with erythroid or megakaryocytic properties are considered myelogenous leukemias as well.

The 8843 protein, fragments thereof, and derivatives and other variants ofthe sequence in SEQ ID NO: 2 thereof are collectively referred to as "polypeptides or proteins of the invention" or "8843 polypeptides or proteins". Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as "nucleic acids ofthe invention" or "8843 nucleic acids." 8843 molecules refer to 8843 nucleic acids, polypeptides, and antibodies.

As used herein, the term "nucleic acid molecule" includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs ofthe DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

The term "isolated or purified nucleic acid molecule" includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules, which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50°C (the temperature ofthe washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding an 8843 protein, preferably a mammalian 8843 protein, and can further include non-coding regulatory sequences, and introns.

An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language "substantially free" means preparation of 8843 protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-8843 protein (also referred to herein as a "contaminating protein"), or of chemical precursors or non-8843 chemicals. When the 8843 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume ofthe protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight. A "non-essential" amino acid residue is a residue that can be altered from the wild- type sequence of 8843 (e.g., the sequence of SEQ ID NO:l or 3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number ) without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides ofthe present invention, e.g., those present in the dual specificity phosphatase domain, are predicted to be particularly unamenable to alteration.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an 8843 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an 8843 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 8843 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOT or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.

As used herein, a "biologically active portion" of an 8843 protein includes a fragment of an 8843 protein which participates in an interaction between an 8843 molecule and a non-8843 molecule. Biologically active portions of an 8843 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe 8843 protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length 8843 proteins, and exhibit at least one activity of an 8843 protein. Typically, biologically active portions comprise a domain or motif with at least one activity ofthe 8843 protein, e.g., protein phosphatase activity. A biologically active portion of an 8843 protein can be a polypeptide which is, for example, 10, 25, 50,

100, 200 or more amino acids in length. Biologically active portions of an 8843 protein can be used as targets for developing agents, which modulate an 8843 mediated activity, e.g., protein phosphatase activity.

Particularly preferred 8843 polypeptides ofthe present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:2. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%o, 98% or 99% identity to SEQ ID NO:2 are termed substantially identical.

In the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOT or 3 are termed substantially identical. Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% ofthe length ofthe reference sequence (e.g., when aligning a second sequence to the 8843 amino acid sequence of SEQ ID NO:2 having 149 amino acid residues, at least 45, preferably at least 60 , more preferably at least 75, even more preferably at least 90, and even more preferably at least 104, 120, or 134 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. hi yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdn CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation ofthe invention) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a P AMI 20 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990)J Mol. Biol. 215:403-10.

BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to 8843 nucleic acid molecules ofthe invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to 8843 protein molecules ofthe invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. See http ://www. ncbi. nlm.nih. go v. "Misexpression or aberrant expression", as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non- wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms ofthe time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms ofthe splicing size, amino acid sequence, post- transitional modification, or biological activity ofthe expressed polypeptide; a pattern of expression that differs from wild type in terms ofthe effect of an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength ofthe stimulus. "Subject", as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.

A "purified preparation of cells", as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% ofthe subject cells.

Various aspects ofthe invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes an 8843 polypeptide described herein, e.g., a full length 8843 protein or a fragment thereof, e.g., a biologically active portion of 8843 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to a identify nucleic acid molecule encoding a polypeptide ofthe invention, 8843 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule ofthe invention includes the nucleotide sequence shown in SEQ ID NOT, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 8843 protein (i.e., "the coding region" of SEQ ID NOT, as shown in SEQ ID NO:3), as well as 5' untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 1 (e.g., SEQ ID NO:3) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment ofthe protein from about amino acid 25 to 190, or about 37 to 185 of SEQ ID NO:2.

In another embodiment, an isolated nucleic acid molecule ofthe invention includes a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:l_or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule ofthe invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOT or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession

Number such that it can hybridize to the nucleotide sequence shown in SEQ ID NOT or 3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as

Accession Number , thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule ofthe present invention includes a nucleotide sequence which is at least about 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%o, 97%), 98%, 99%, or more homologous to the entire length ofthe nucleotide sequence shown in SEQ ID NOT or SEQ ID NO:3, or the entire length ofthe nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number or a portion, preferably ofthe same length, of any of these nucleotide sequences.

8843 Nucleic Acid Fragments

A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NOT or 3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number . For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of an 8843 protein, e.g., an immunogenic or biologically active portion of an 8843 protein. A fragment can comprise those nucleotides of SEQ ID NO: 1 , which encode a dual specificity phosphatase domain of human 8843. The nucleotide sequence determined from the cloning ofthe 8843 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 8843 family members, or fragments thereof, as well as 8843 homologues, or fragments thereof, from other species. In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100, 150, or 200 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, an 8843 nucleic acid fragment can include a sequence corresponding to a dual specificity phosphatase domain.

8843 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NOT or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number , or of a naturally occurring allelic variant or mutant of SEQ ID NO or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .

In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a dual specificity phosphatase domain, e.g., about amino acids 25 to 201, or about 37 to 185 of SEQ ID NO:2. hi another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of an 8843 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a dual specificity phosphatase domain from about amino acid 25 to 201, or from 37 to 185 of SEQ ID NO:2. A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

A nucleic acid fragment encoding a "biologically active portion of an 8843 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NOT or 3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with

ATCC as Accession Number , which encodes a polypeptide having an 8843 biological activity (e.g., the biological activities ofthe 8843 proteins are described herein), expressing the encoded portion ofthe 8843 protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 8843 protein. For example, a nucleic acid fragment encoding a biologically active portion of 8843 includes a dual specificity phosphatase domain, e.g., amino acid residues about 37 to 185 of SEQ ID NO:2. A nucleic acid fragment encoding a biologically active portion of an 8843 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NOT, or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number .

8843 Nucleic Acid Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NOT or SEQ ID NO:3, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number . Such differences can be due to degeneracy ofthe genetic code (and result in a nucleic acid which encodes the same 8843 proteins as those encoded by the nucleotide sequence disclosed herein, another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:2. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

Nucleic acids ofthe invention can be chosen for having codons, which are preferred, or non preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one colon, at preferably at least 10%, or 20% ofthe codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non- conservative amino acid substitutions (as compared in the encoded product). hi a preferred embodiment, the nucleic acid differs from that of SΕQ ID NO: 1 or 3, or the sequence in ATCC Accession Number , e.g., as follows: by at least one but less than

10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% ofthe in the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SΕQ ID NO:2 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SΕQ ID NO 2 or a fragment ofthe sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants ofthe 8843 cDNAs ofthe invention can further be isolated by mapping to the same chromosome or locus as the 8843 gene.

Preferred variants include those that are correlated with protein phosphatase activity. Allelic variants of 8843, e.g., human 8843, include both functional and nonfunctional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 8843 protein within a population that maintain the ability to hydrolyze the phosphate of phosphorylated proteins. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SΕQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Nonfunctional allelic variants are naturally-occurring amino acid sequence variants ofthe 8843, e.g., human 8843, protein within a population that do not have the ability to to hydrolyze the phosphate of phosphorylated proteins. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO: 2, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.

Moreover, nucleic acid molecules encoding other 8843 family members and, thus, which have a nucleotide sequence which differs from the 8843 sequences of SEQ ID NOT or SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC as Accession Number are intended to be within the scope ofthe invention.

Antisense Nucleic Acid Molecules. Ribozymes and Modified 8843 Nucleic Acid Molecules

In another aspect, the invention features, an isolated nucleic acid molecule that is antisense to 8843. An "antisense" nucleic acid can include a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 8843 coding strand, or to only a portion thereof (e.g., the coding region of human 8843 corresponding to SEQ ID NO:3). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding 8843 (e.g., the 5' and 3* untranslated regions).

An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 8843 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of 8843 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 8843 mRNA, e.g., between the -10 and +10 regions ofthe target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules ofthe invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an 8843 protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation.

Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an -anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625- 6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid ofthe invention is a ribozyme. A ribozyme having specificity for an 8843-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of an 8843 cDNA disclosed herein (i.e., SEQ ID NOT or SEQ ID NO:3), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an 8843-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech etal. U.S. Patent No. 5,116,742. Alternatively, 8843 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 :1411- 1418.

8843 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe 8843 (e.g., the 8843 promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe 8843 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807- 15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

An 8843 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule. For example, the deoxyribose phosphate backbone ofthe nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of 8843 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 8843 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaή e et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g. , PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross- linking agent, transport agent, or hybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to an 8843 nucleic acid ofthe invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence ofthe 8843 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Patent No. 5,854,033; Nazarenko et al., U.S. Patent No. 5,866,336, and Livak etal., U.S. Patent 5,876,930.

Isolated 8843 Polypeptides In another aspect, the invention features, an isolated 8843 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-8843 antibodies. 8843 protein can be isolated from cells or tissue sources using standard protein purification techniques. 8843 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically. Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell. In a preferred embodiment, an 8843 polypeptide has one or more ofthe following characteristics:

(i) it has the ability to hydrolyze the phosphate of phosphorylated proteins, i.e., it has protein phosphatase activity, e.g., for phosphotyrosine, phosphoserine, and phosphothreonine substrates; (ii) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of an 8843 polypeptide, e.g., a polypeptide of SEQ ID NO:2;

(iii) it has an overall sequence similarity of at least 90, 95, 96, 97, 98, 99, 100%, with a polypeptide of SEQ ID NO:2; (iv) it can be found in erythroid cells, especially erythroid progenitor cells, e.g.,

CD34+ progenitor cells, bone marrow cells (CD34+ and glycophorin A positive), erythroblasts, erythropoietin treated erythroid cells;

(v) it can be found in a CD34+ cell;

(v) it has a dual specificity phosphatase domain which is preferably about 70%, 80%, 90% or 95% with amino acid residues about 37 to 185 of SEQ ID NO:2;

(vii) it has an active site cysteine at about residue 132 of SEQ ID NO:2, an active site arginine at about residue 138 of SEQ ID NO: 2, and an active site aspartic acid at about residue 109 of SEQ ID NO:2;

(viii)it has a dual specificity phosphatase extended active site signature (VXVHCXXGXSRSXTXXXAY[LI]M; SEQ ID NO:8; Muda et al. (1996) JBiol Chem

271:27205) locate at about amino acid residues 128 to 158 of SEQ ID NO:2; in addition to a tyrosine specific protein phosphatase active site signature (Prosite PS00383) located at about amino acid residues 130 to 142 of SEQ ID NO:2;

(ix) it has a VHl -like dual specificity phosphatase loop located at about amino acid residues 106 to 110 of SEQ ID NO:2, include a conserved general acid, aspartic acid at about residue 109 of SEQ ID NO:2;

(x) it has one predicted N-glycosylation site (PS00001) at about amino acids 82 to 85 ofSEQ ID NO:2; (xi) it has one predicted protein kinase C phosphorylation sites (PS00005) at about amino acids 187 to 189 of SEQ ID NO:2;

(xii) it has four predicted casein kinase II phosphorylation sites (PS00006) located at about amino acids 70 to 73, 83 to 86, 98 to 101, and 154 to 157 of SEQ ID NO:2; or (xiii)it has one predicted N-myristylation sites (PS00008) from about amino acid 114 to 119 of SEQ ED NO:2.

In a preferred embodiment the 8843 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO:2 by at least one residue but less than 20%), 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO:2. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution, hi a preferred embodiment the differences are not in the dual specificity phosphatase domain. In another preferred embodiment one or more differences are in the dual specificity phosphatase domain.

Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 8843 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at least about 80%, 85%, 90%), 95%., 98%), 99%, or more homologous to SEQ ID NO:2.

A 8843 protein or fragment is provided which varies from the sequence of SEQ ID NO.2 in regions defined by amino acids about 37 to 185 of SEQ ID NO.2 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO.2 in regions defined by amino acids about 37 to 185 of SEQ ID NO.2. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) h some embodiments the difference is at a non essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non conservative substitution. In one embodiment, a biologically active portion of an 8843 protein includes a dual specificity phosphatase domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 8843 protein.

In a preferred embodiment, the 8843 protein has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the 8843 protein is substantially identical to SEQ ID NO:2. In yet another embodiment, the 8843 protein is substantially identical to SEQ ID NO:2 and retains the functional activity ofthe protein of SEQ ID NO:2, as described in detail in the subsections above.

8843 Chimeric or Fusion Proteins

In another aspect, the invention provides 8843 chimeric or fusion proteins. As used herein, an 8843 "chimeric protein" or "fusion protein" includes an 8843 polypeptide linked to a non-8843 polypeptide. A "non-8843 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 8843 protein, e.g., a protein which is different from the 8843 protein and which is derived from the same or a different organism. The 8843 polypeptide ofthe fusion protein can correspond to all or a portion e.g., a fragment described herein of an 8843 amino acid sequence, hi a preferred embodiment, an 8843 fusion protein includes at least one (or two) biologically active portion of an 8843 protein. The non-8843 polypeptide can be fiised to the N-terminus or C-terminus ofthe 8843 polypeptide.

The fusion protein can include a moiety, which has a high affinity for a ligand. For example, the fusion protein can be a GST-8843 fusion protein in which the 8843 sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purification of recombinant 8843. Alternatively, the fusion protein can be an 8843 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 8843 can be increased through use of a heterologous signal sequence.

Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

The 8843 fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 8843 fusion proteins can be used to affect the bioavailability of an 8843 substrate. 8843 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding an 8843 protein; (ii) mis-regulation ofthe 8843 gene; and (iii) aberrant post-translational modification of an 8843 protein. Moreover, the 8843-fusion proteins ofthe invention can be used as immunogens to produce anti-8843 antibodies in a subject, to purify 8843 ligands and in screening assays to identify molecules that inhibit the interaction of 8843 with an 8843 substrate.

Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An 8843 -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 8843 protein.

Variants of 8843 Proteins

In another aspect, the invention also features a variant of an 8843 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants ofthe 8843 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of an 8843 protein. An agonist ofthe 8843 proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occurring form of an 8843 protein. An antagonist of an 8843 protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe 8843 protein by, for example, competitively modulating an 8843-mediated activity of an 8843 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe 8843 protein. Variants of an 8843 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an 8843 protein for agonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of an 8843 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of an 8843 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred. Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 8843 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Cell based assays can be exploited to analyze a variegated 8843 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 8843 in a substrate-dependent manner. The transfected cells are then contacted with 8843 and the effect ofthe expression ofthe mutant on signaling by the 8843 substrate can be detected, e.g., by measuring protein phosphatase activity. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 8843 substrate, and the individual clones further characterized.

In another aspect, the invention features a method of making an 8843 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 8843 polypeptide, e.g., a naturally occurring 8843 polypeptide. The method includes: altering the sequence of an 8843 polypeptide, e.g., altering the sequence, e.g. , by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragment or analog of an 8843 polypeptide a biological activity of a naturally occurring 8843 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of an 8843 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

Anti-8843 Antibodies In another aspect, the invention provides an anti-8843 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent ofthe framework region and CDR's has been precisely defined (see, Kabat, E.A., etal. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NM Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incoφorated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from ammo-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The anti-8843 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts with an antigen. The constant regions ofthe antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq) ofthe classical complement system.

As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH— terminus. Full-length immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one ofthe other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 8843 polypeptide or fragment thereof. Examples of antigen-binding fragments ofthe anti-8843 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CHI domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).

Furthermore, although the two domains ofthe Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. The anti-8843 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

Phage display and combinatorial methods for generating anti-8843 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. Intemational Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. Intemational Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) HumAntibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBOJ 12:725-734; Hawkins et al. (1992) JMolBiol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incoφorated by reference herein).

In one embodiment, the anti-8843 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art. Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. Intemational Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. Intemational Application WO 92/03918; Kay et al.

International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 EurJ Immunol 21:1323-1326). An anti-8843 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention. Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent

Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., Intemational Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041- 1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559). A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some ofthe CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding ofthe humanized antibody to an 8843 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the "donor" and the immunoglobulin providing the framework is called the "acceptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

As used herein, the term "consensus sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A "consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences ofthe Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incoφorated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 8843 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Patent 5,225,539; Jones et al. 1986 Nature 321 :552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141 :4053-4060; Winter US 5,225,539, the contents of all of which are hereby expressly incoφorated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies ofthe present invention (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), the contents of which is expressly incoφorated by reference.

Also within the scope ofthe invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues ofthe humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations ofthe substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., US 5,585,089). Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, the e.g., columns 12-16 of US 5,585,089, the contents of which are hereby incoφorated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al, published on December 23, 1992. In preferred embodiments, an antibody can be made by immunizing with purified

8843 antigen, or a fragment thereof, e.g., a fragment described herein, tissue, e.g., crude tissue preparations, whole cells, lysed cells, or cell fractions.

A full-length 8843 protein or, antigenic peptide fragment of 8843 can be used as an immunogen or can be used to identify anti-8843 antibodies made with other immunogens, e.g., cells, and the like. The antigenic peptide of 8843 should include at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of 8843. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

Fragments of 8843 which include residues about 9 to 25, about 45 to 52, or about 142 to 150 of SEQ ID NO.2 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophobic regions ofthe 8843 protein. Similarly, a fragment of 8843 which include residues about 27 to 36, about 70 to 78, or about 151 to 160 of SEQ ID NO.2 can be used to make an antibody against a hydrophilic region ofthe 8843 protein; a fragment of 8843 which include residues about 25 to 201 , or about 37 to 185 of SEQ ID NO.2 can be used to make an antibody against the dual specificity phosphatase region ofthe 8843 protein.

Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

Antibodies which bind only native 8843 protein, only denatured or otherwise non- native 8843 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies that bind to native but not denatured 8843 protein.

Preferred epitopes encompassed by the antigenic peptide are regions of 8843 are located on the surface ofthe protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis ofthe human 8843 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface ofthe 8843 protein and are thus likely to constitute surface residues useful for targeting antibody production.

The anti-8843 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N YAcad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes ofthe same target 8843 protein.

In a prefeπed embodiment the antibody has effector function and/or can fix complement, hi other embodiments the antibody does not recruit effector cells; or fix complement.

In a prefeπed embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has amutagenized or deleted Fc receptor binding region.

In a preferred embodiment, an anti-8843 antibody alters (e.g., increases or decreases) the phosphatase activity of a 8843 polypeptide. For example, the antibody can bind at or in proximity to the active site, e.g., to an epitope that includes a residue located from about 37 to 185 of SEQ ID NO:2 (e.g., 130 to 142, 128 to 158, and 106 to 110 of SEQ ID NO:2).

The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e.g., ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels that produce detectable radioactive emissions or fluorescence are preferred.

An anti-8843 antibody (e.g., monoclonal antibody) can be used to isolate 8843 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-8843 antibody can be used to detect 8843 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe protein. Anti-8843 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵1, ¹³¹I, ³⁵S or ³H

The invention also includes a nucleic acid that encodes an anti-8843 antibody, e.g., an anti-8843 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells. The invention also includes cell lines, e.g., hybridomas, which make an anti-8843 antibody, e.g., an antibody described herein, and method of using said cells to make an 8843 antibody.

Recombinant Expression Vectors. Host Cells and Genetically Engineered Cells i another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses. A vector can include an 8843 nucleic acid in a form suitable for expression ofthe nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 8843 proteins, mutant forms of 8843 proteins, fusion proteins, and the like).

The recombinant expression vectors ofthe invention can be designed for expression of 8843 proteins in prokaryotic or eukaryotic cells. For example, polypeptides ofthe invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve three puφoses: 1) to increase expression of recombinant protein; 2) to increase the solubility ofthe recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Purified fusion proteins can be used in 8843 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 8843 proteins. In a prefeπed embodiment, a fusion protein expressed in a retroviral expression vector ofthe present invention can be used to infect bone maπow cells which are subsequently transplanted into iπadiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g., six weeks).

To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

The 8843 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Non-hmiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) roα Natl. Acad. Sci. USA 86:5473- 5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion ofthe regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986. Another aspect the invention provides a host cell which includes a nucleic acid

A host cell can be any prokaryotic or eukaryotic cell. For example, an 8843 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DΕAΕ-dextran-mediated transfection, lipofection, or electroporation. A host cell ofthe invention can be used to produce (i.e., express) an 8843 protein.

Accordingly, the invention further provides methods for producing an 8843 protein using the host cells ofthe invention. In one embodiment, the method includes culturing the host cell ofthe invention (into which a recombinant expression vector encoding an 8843 protein has been introduced) in a suitable medium such that an 8843 protein is produced, h another embodiment, the method further includes isolating an 8843 protein from the medium or the host cell. i another aspect, the invention features, a cell or purified preparation of cells which include an 8843 transgene, or which otherwise misexpress 8843. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In prefeπed embodiments, the cell or cells include an 8843 transgene, e.g., a heterologous form of an 8843, e.g., a gene derived from humans (in the case of a non-human cell). The 8843 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous 8843, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis- expressed 8843 alleles or for use in drug screening.

In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject 8843 polypeptide.

Also provided are cells, preferably human cells, e.g., human hematopoietic or fibroblast cells, in which an endogenous 8843 is under the control of a regulatory sequence that does not normally control the expression ofthe endogenous 8843 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome ofthe cell such that the inserted regulatory element is operably linked to the endogenous 8843 gene. For example, an endogenous 8843 gene which is "transcriptionally silent," e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071; WO 91/06667, published in May 16, 1991.

Transgenic Animals The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of an 8843 protein and for identifying and/or evaluating modulators of 8843 activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a reaπangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome ofthe cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 8843 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell of the animal, prior to development ofthe animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene ofthe invention to direct expression of an 8843 protein to particular cells. A transgenic founder animal can be identified based upon the presence of an 8843 transgene in its genome and/or expression of 8843 mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals caπying a transgene encoding an 8843 protein can further be bred to other transgenic animals carrying other transgenes. 8843 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protem or polypeptide can be introduced into the genome of an animal. In prefeπed embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

8834 Molecules In Erythropoietin-Responsive Cells

As 8834 molecules are abundantly expressed in eiythroid progenitor cells, 8834 molecules can modulate the differentiative process undergone by progenitors to produce erythrocytes. The final stages of erythroid differentiation are regulated by erythropoietin, which is an extremely potent soluble polypeptide regulator. Erythropoietin can be administered at approximately 50 units per kilogram of body weight every other day in order to increase red blood cell mass. Erythropoietin can be used to treat anemias, especially anemia due to chronic renal failure. Alternatively, it can be used to increase patient hemocrit prior to operations.

In normal subjects, the peritubular intestinal cells ofthe kidney produce erythropoietin in response to lowered oxygen levels. However, in pathological states, such as cancers, especially renal cancers, erythropoietin can be oveφroduced resulting in erythrocytosis due to excess red blood cell production.

Contemplated by the cuπent invention are methods of modulating the erythropoietin- induce differentiative response by agonizing or antagonizing 8834 polypeptides and nucleic acids. In one embodiment, 8834 dual specificity phosphatase can increase cellular signalling in response to erythropoietin. In another embodiment, it can antagonize erythropoietin-induce phosphorylation signals. It will be readily apparent to one skilled in the art, that 8834 activity can be modulated by altering 8834 expression levels, or by small molecules, or proteins which bind to 8834 polypeptides in order alter a cell's response to erythropoietin.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more ofthe following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). The isolated nucleic acid molecules ofthe invention can be used, for example, to express an 8843 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect an 8843 mRNA (e.g., in a biological sample) or a genetic alteration in an 8843 gene, and to modulate 8843 activity, as described further below. The 8843 proteins can be used to treat disorders characterized by insufficient or excessive production of an 8843 substrate or production of 8843 inhibitors. In addition, the 8843 proteins can be used to screen for naturally occurring 8843 substrates, to screen for drugs or compounds which modulate 8843 activity, as well as to treat disorders characterized by insufficient or excessive production of 8843 protein or production of 8843 protein forms which have decreased, abeπant or unwanted activity compared to 8843 wild type protein (e.g., abeπant, increased or deficient, erythroid proliferation, e.g., an erythroleukemia and, aberrant, increased or deficient erythroid differentiation, e.g., an anemia). Moreover, the anti-8843 antibodies ofthe invention can be used to detect and isolate 8843 proteins, regulate the bioavailability of 8843 proteins, and modulate 8843 activity.

A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 8843 polypeptide is provided. The method includes: contacting the compound with the subject 8843 polypeptide; and evaluating ability ofthe compound to interact with, e.g., to bind or form a complex with the subject 8843 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with subject 8843 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 8843 polypeptide. Screening methods are discussed in more detail below.

Screening Assays:

The invention provides methods (also refeπed to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 8843 proteins, have a stimulatory or inhibitory effect on, for example, 8843 expression or 8843 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of an 8843 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 8843 genes) in a therapeutic protocol, to elaborate the biological fiinction of the target gene product, or to identify compounds that disrupt normal target gene interactions. hi one embodiment, the invention provides assays for screening candidate or test compounds, which are substrates of an 8843 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds, which bind to or modulate the activity of an 8843 protein or polypeptide or a biologically active portion thereof.

The test compounds ofthe present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. et al. J. Med. Chem. 1994, 37: 2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, KS. (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Caπell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell etal. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwiria et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J Mol. Biol. 222:301-310); (Ladner supra.). In one embodiment, an assay is a cell-based assay in which a cell which expresses an 8843 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 8843 activity is determined. Determining the ability ofthe test compound to modulate 8843 activity can be accomplished by monitoring, for example, protein phosphatase activity. The cell, for example, can be of mammalian origin, e.g., human.

The ability ofthe test compound to modulate 8843 binding to a compound, e.g., an 8843 substrate, or to bind to 8843 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g., the substrate, to 8843 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 8843 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 8843 binding to an 8843 substrate in a complex. For example, compounds (e.g.,

8843 substrates) can be labeled with H^I, ³^S₅ 1 c, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

The ability of a compound (e.g., an 8843 substrate) to interact with 8843 with or without the labeling of any ofthe interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 8843 without the labeling of either the compound or the 8843. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a Iight- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator ofthe interaction between a compound and 8843. i yet another embodiment, a cell-free assay is provided in which an 8843 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to bind to the 8843 protein or biologically active portion thereof is evaluated. Preferred biologically active portions ofthe 8843 proteins to be used in assays ofthe present invention include fragments, which participate in interactions with non-8843 molecules, e.g., fragments with high surface probability scores. Soluble and/or membrane-bound forms of isolated proteins (e.g., 8843 proteins or biologically active portions thereof) can be used in the cell-free assays ofthe invention. When membrane-bound forms ofthe protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-l 14, Thesit®, Isotridecypoly(ethylene glycol ether)_n, 3-[(3-cholamidopropyl)dimethylamminio]-l-propane sulfonate (CHAPS), 3 -[(3 -chola-midopropyl)dimethylamminio] -2-hydroxy- 1 -propane sulfonate (CHAPSO), orN-dodecyl=N,N-dimethyl-3-ammonio-l-propane sulfonate. Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz etal, U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that ofthe 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission ofthe 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the ability ofthe 8843 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product test compound complexes anchored on the solid phase can be detected at the end ofthe reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize either 8843, an anti-8843 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to an 8843 protein, or interaction of an 8843 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, glutathione-S-transferase/8843 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 8843 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 8843 binding or activity determined using standard techniques.

Other techniques for immobilizing either an 8843 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 8843 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). i order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non- immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with 8843 protein or target molecules but which do not interfere with binding ofthe 8843 protein to its target molecule. Such antibodies can be derivatized to the wells ofthe plate, and unbound target or 8843 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 8843 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 8843 protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minion, A.P., Trends Biochem Sci 1993 Aug;18(8):284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Cuπent Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N.H., J MolRecognit 1998 Winter;ll(l-6):141-8; Hage, D.S., and Tweed, S.A. JChromatogr B Biomed Sci Appl 1997 Oct 10;699(l-2):499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification ofthe complex from solution.

In a prefeπed embodiment, the assay includes contacting the 8843 protein or biologically active portion thereof with a known compound which binds 8843 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an 8843 protein, wherein determining the ability ofthe test compound to interact with an 8843 protein includes determining the ability ofthe test compound to preferentially bind to 8843 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

The target gene products ofthe invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the puφoses of this discussion, such cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity ofthe target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 8843 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability ofthe test compound to modulate the activity of an 8843 protein through modulation ofthe activity of a downstream effector of an 8843 target molecule. For example, the activity ofthe effector molecule on an appropriate target can be deteπnined, or the binding ofthe effector to an appropriate target can be determined, as previously described. To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence ofthe test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition ofthe target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction ofthe target gene product and the interactive binding partner.

Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end ofthe reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence ofthe test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. h order to conduct the assay, the partner ofthe immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence ofthe test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one ofthe binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment ofthe invention, a homogeneous assay can be used. For example, a preformed complex ofthe target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

In yet another aspect, the 8843 proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent

WO94/10300), to identify other proteins, which bind to or interact with 8843 ("8843 -binding proteins" or "8843-bp") and are involved in 8843 activity. Such 8843-bps can be activators or inhibitors of signals by the 8843 proteins or 8843 targets as, for example, downstream elements of an 8843-mediated signaling pathway. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an 8843 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 8843 protein can be the fused to the activator domain.) If the "bait" and the "prey" proteins are able to interact, in vivo, forming an 8843-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 8843 protein. In another embodiment, modulators of 8843 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 8843 mRNA or protein evaluated relative to the level of expression of 8843 mRNA or protein in the absence ofthe candidate compound. When expression of 8843 mRNA or protein is greater in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of 8843 mRNA or protein expression. Alternatively, when expression of 8843 mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of 8843 mRNA or protein expression. The level of 8843 mRNA or protein expression can be determined by methods described herein for detecting 8843 mRNA or protein.

In another aspect, the invention pertains to a combination of two or more ofthe assays described herein. For example, a modulating agent can be identified using a cell- based or a cell free assay, and the ability ofthe agent to modulate the activity of an 8843 protein can be confirmed in vivo, e.g., in an animal such as an animal model for abeπant (increased or deficient) erythroid proliferation and differentiation.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., an 8843 modulating agent, an antisense 8843 nucleic acid molecule, an 8843-specific antibody, or an 8843-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

Detection Assays

Portions or fragments ofthe nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 8843 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below. Chromosome Mapping

The 8843 nucleotide sequences or portions thereof can be used to map the location of the 8843 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 8843 sequences with genes associated with disease.

Briefly, 8843 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 8843 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene coπesponding to the 8843 sequences will yield an amplified fragment.

A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990)

Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 8843 to a chromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents coπesponding to noncoding regions ofthe genes actually are prefeπed for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping. Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be coπelated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available online through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 8843 gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymoφhisms.

Tissue Typing

8843 sequences can be used to identify individuals from biological samples using, e.g. , restriction fragment length polymoφhism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057). Furthermore, the sequences ofthe present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 8843 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NOT can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. If a panel of reagents from 8843 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification ofthe individual, living or dead, can be made from extremely small tissue samples.

Use of Partial 8843 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification ofthe origin ofthe biological sample.

The sequences ofthe present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 1 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 1 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

The 8843 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., a blood sample, a bone maπow sample, or a spleen. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 8843 probes can be used to identify tissue by species and/or by organ type. i a similar fashion, these reagents, e.g., 8843 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

Predictive Medicine The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) puφoses to thereby treat an individual.

Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 8843. Such disorders include, e.g., a disorder associated with the misexpression of 8843 gene; a disorder of erythroid cells; a disorder of CD34+ cells. The method includes one or more ofthe following: detecting, in a tissue ofthe subject, the presence or absence of a mutation which affects the expression ofthe 8843 gene, or detecting the presence or absence of a mutation in a region which controls the expression ofthe gene, e.g., a mutation in the 5' control region; detecting, in a tissue ofthe subject, the presence or absence of a mutation which alters the structure ofthe 8843 gene; detecting, in a tissue ofthe subject, the misexpression ofthe 8843 gene, at the mRNA level, e.g., detecting a non- wild type level of a mRNA ; detecting, in a tissue ofthe subject, the misexpression ofthe gene, at the protein level, e.g., detecting a non-wild type level of an 8843 polypeptide.

In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 8843 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides ofthe gene, a gross chromosomal rearrangement ofthe gene, e.g., a translocation, inversion, or deletion. For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NOT, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the 8843 gene; (ii) exposing the probe/primer to nucleic acid ofthe tissue; and detecting, by hybridization, e.g., in situ hybridization, ofthe probe/primer to the nucleic acid, the presence or absence ofthe genetic lesion.

In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript ofthe 8843 gene; the presence of a non- wild type splicing pattern of a messenger RNA transcript ofthe gene; or a non-wild type level of 8843.

Methods ofthe invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structure of an 8843 gene, an abnormal structure being indicative of risk for the disorder.

In preferred embodiments the method includes contacting a sample form the subject with an antibody to the 8843 protein or a nucleic acid, which hybridizes specifically with the gene. There and other embodiments are discussed below.

Diagnostic and Prognostic Assays

Diagnostic and prognostic assays ofthe invention include method for assessing the expression level of 8843 molecules and for identifying variations and mutations in the sequence of 8843 molecules.

Expression Monitoring and Profiling. The presence, level, or absence of 8843 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 8843 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 8843 protein such that the presence of 8843 protein or nucleic acid is detected in the biological sample. The term "biological sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A prefeπed biological sample is serum. The level of expression ofthe 8843 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 8843 genes; measuring the amount of protein encoded by the 8843 genes; or measuring the activity ofthe protein encoded by the 8843 genes.

The level of mRNA coπesponding to the 8843 gene in a cell can be determined both by in situ and by in vitro formats. The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe aπays. One prefeπed diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 8843 nucleic acid, such as the nucleic acid of SEQ ID NO: 1 , or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 8843 mRNA or genomic DNA. The probe can be disposed on an address of an aπay, e.g., an aπay described below. Other suitable probes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip aπay described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 8843 genes.

The level of mRNA in a sample that is encoded by one of 8843 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli etal, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh etal, (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal, (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi etal, U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 8843 gene being analyzed.

In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 8843 mRNA, or genomic DNA, and comparing the presence of 8843 mRNA or genomic DNA in the control sample with the presence of 8843 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Patent No. 5,695,937, is used to detect 8843 transcript levels.

A variety of methods can be used to determine the level of protein encoded by 8843. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a prefeπed embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

The detection methods can be used to detect 8843 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 8843 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 8843 protein include introducing into a subject a labeled anti- 8843 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-8843 antibody positioned on an antibody aπay (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label. In another embodiment, the methods ftirther include contacting the control sample with a compound or agent capable of detecting 8843 protein, and comparing the presence of 8843 protein in the control sample with the presence of 8843 protein in the test sample.

The invention also includes kits for detecting the presence of 8843 in a biological sample. For example, the kit can include a compound or agent capable of detecting 8843 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 8843 protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide coπesponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule coπesponding to a marker ofthe invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component ofthe kit can be enclosed within an individual container and all ofthe various containers can be within a single package, along with instructions for inteφreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or abeπant or unwanted 8843 expression or activity. As used herein, the term "unwanted" includes an unwanted phenomenon involved in a biological response such as hematopoietic disorder.

In one embodiment, a disease or disorder associated with abeπant or unwanted 8843 expression or activity is identified. A test sample is obtained from a subject and 8843 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 8843 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abeπant or unwanted 8843 expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue. The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abeπant or unwanted 8843 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a hematopoietic disorder.

In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 8843 in a sample, and a descriptor ofthe sample. The descriptor ofthe sample can be an identifier ofthe sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g. , a prefeπed treatment), hi a prefeπed embodiment, the data record further includes values representing the level of expression of genes other than 8843 (e.g., other genes associated with a 8843-disorder, or other genes on an aπay). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database ofthe Oracle or Sybase database environments).

Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile ofthe sample, wherein the profile includes a value representing the level of 8843 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile ofthe sample can be obtained by any ofthe methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an aπay). The method can be used to diagnose a hematopoietic disorder in a subject wherein a change, e.g., an increase or decrease, in 8843 expression is an indication that the subject has or is disposed to having a hematopoietic disorder. The method can be used to monitor a treatment for hematopoietic disorder in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset ofthe disorder (see, e.g., Golub etal. (1999) Science 286:531). hi yet another aspect, the invention features a method of evaluating a test compound (see also, "Screening Assays", above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 8843 expression. In a prefeπed embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated , favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from anuncontacted cell. In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 8843 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length ofthe distance vector that is the difference between the two profiles. Each ofthe subject and reference profile is represented as a multi- dimensional vector, wherein each dimension is a value in the profile.

The method can further include tiansmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison ofthe subject expression profile with another profile, a most similar reference profile, or a descriptor of any ofthe aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 8843 expression. Arrays and Uses Thereof hi another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address ofthe plurality includes a capture probe that binds specifically to a 8843 molecule (e.g., a 8843 nucleic acid or a 8843 polypeptide). The aπay can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm², and ranges between. In a prefeπed embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a prefeπed embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three- dimensional substrate such as a gel pad. Addresses in addition to address ofthe plurality can be disposed on the aπay.

In a preferred embodiment, at least one address ofthe plurality includes a nucleic acid capture probe that hybridizes specifically to a 8843 nucleic acid, e.g., the sense or anti- sense strand. In one prefeπed embodiment, a subset of addresses ofthe plurality of addresses has a nucleic acid capture probe for 8843. Each address ofthe subset can include a capture probe that hybridizes to a different region of a 8843 nucleic acid. In another prefeπed embodiment, addresses ofthe subset include a capture probe for a 8843 nucleic acid. Each address ofthe subset is unique, overlapping, and complementary to a different variant of 8843 (e.g., an allelic variant, or all possible hypothetical variants). The aπay can be used to sequence 8843 by hybridization (see, e.g., U.S. Patent No. 5,695,940).

An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

In another preferred embodiment, at least one address ofthe plurality includes a polypeptide capture probe that binds specifically to a 8843 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 8843 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see "Anti- 8843 Antibodies," above), such as a monoclonal antibody or a single-chain antibody.

In another aspect, the invention features a method of analyzing the expression of 8843. The method includes providing an array as described above; contacting the aπay with a sample and detecting binding of a 8843-molecule (e.g., nucleic acid or polypeptide) to the aπay. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the aπay. In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 8843. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k- means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 8843. For example, the aπay can be used for the quantitation ofthe expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

For example, aπay analysis of gene expression can be used to assess the effect of cell-cell interactions on 8843 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression. In another embodiment, cells are contacted with a therapeutic agent. The expression profile ofthe cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect ofthe therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis ofthe undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the aπay. Such analysis can identify and/or characterize the development of a 8843-associated disease or disorder; and processes, such as a cellular transformation associated with a 8843-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 8843-associated disease or disorder

The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g. , including 8843) that could serve as a molecular target for diagnosis or therapeutic intervention.

In another aspect, the invention features an aπay having a plurality of addresses. Each address ofthe plurality includes a unique polypeptide. At least one address ofthe plurality has disposed thereon a 8843 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking etal. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, 1-VII; MacBeath, G, and Schreiber, S.L. (2000). Science 289, 1760-1763; and WO 99/51773 Al. In a prefeπed embodiment, each addresses ofthe plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99 % identical to a 8843 polypeptide or fragment thereof. For example, multiple variants of a 8843 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses ofthe plurality. Addresses in addition to the address ofthe plurality can be disposed on the array.

The polypeptide aπay can be used to detect a 8843 binding compound, e.g., an antibody in a sample from a subject with specificity for a 8843 polypeptide or the presence of a 8843-binding protein or ligand.

The array is also useful for ascertaining the effect ofthe expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 8843 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

each address ofthe plurality being positionally distinguishable from each other address of the plurality, and each address ofthe plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 8843 (or does not express as highly as in the case ofthe 8843 positive plurality of capture probes) or from a cell or subject which in which a 8843 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the aπay with one or more inquiry probes (which is preferably other than a 8843 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. i another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional aπay having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, contacting the aπay with a first sample from a cell or subject which express or mis-express 8843 or from a cell or subject in which a 8843-mediated response has been elicited, e.g., by contact ofthe cell with 8843 nucleic acid or protein, or administration to the cell or subject 8843 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 8843 (or does not express as highly as in the case of the 8843 positive plurality of capture probes) or from a cell or subject which in which a 8843 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding ofthe first sample with the binding ofthe second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same aπay can be used for both samples or different aπays can be used. If different aπays are used the plurality of addresses with capture probes should be present on both aπays.

In another aspect, the invention features a method of analyzing 8843, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 8843 nucleic acid or amino acid sequence; comparing the 8843 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 8843.

Detection of Variations or Mutations

The methods ofthe invention can also be used to detect genetic alterations in a 8843 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 8843 protein activity or nucleic acid expression, such as a hematopoietic disorder, hi prefeπed embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 8843 -protein, or the mis-expression ofthe 8843 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 8843 gene; 2) an addition of one or more nucleotides to a 8843 gene; 3) a substitution of one or more nucleotides of a 8843 gene, 4) a chromosomal rearrangement of a 8843 gene; 5) an alteration in the level of a messenger RNA transcript of a 8843 gene, 6) abeπant modification of a 8843 gene, such as ofthe methylation pattern ofthe genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a 8843 gene, 8) a non-wild type level of a 8843-protein, 9) allelic loss of a 8843 gene, and 10) inappropriate post-translational modification of a 8843 -protein.

An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 8843 -gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 8843 gene under conditions such that hybridization and amplification ofthe 8843 -gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amphfication step in conjunction with any ofthe techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used. In another embodiment, mutations in a 8843 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in 8843 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional aπays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address ofthe plurality. A probe can be complementary to a region of a 8843 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 8843 nucleic acid (e.g., a destabilizing mismatch). The aπays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M.T. etal. (1996) Human Mutation 7: 244-255; Kozal, M.J. etal. (1996) Nature Medicine 2: 753- 759). For example, genetic mutations in 8843 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. etal. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller; specialized probe aπays complementary to all variants or mutations detected. Each mutation aπay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 8843 gene and detect mutations by comparing the sequence ofthe sample 8843 with the coπesponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

Other methods for detecting mutations in the 8843 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242; Cotton etal. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et /. (1992) Methods Enzymol. 217:286-295). In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in 8843 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu etal. (1994) Carcinogenesis 15:1657-1662; U.S. Patent No. 5,459,039). In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 8843 genes. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita etal. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single- stranded DNA fragments of sample and control 8843 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a prefeπed embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet :5).

In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki etα/. (1986) Nature 324:163); Saiki eta/. (1989) roc. NatlAcad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in X etal. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site ofthe sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization) (Gibbs etal. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end ofthe 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 8843 nucleic acid. In a prefeπed embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 1 or the complement of SEQ ID NO: 1. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands. The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of

8843. In a prefeπed embodiment, each oligonucleotide ofthe set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymoφhic locus. i another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The inteπogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides ofthe plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one ofthe oligonucleotides ofthe set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the T_m of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles. hi a prefeπed embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 8843 nucleic acid.

The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 8843 gene.

Use of 8843 Molecules as Surrogate Markers

The 8843 molecules ofthe invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers ofthe pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity ofthe 8843 molecules ofthe invention may be detected, and may be coπelated with one or more biological states in vivo. For example, the 8843 molecules ofthe invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a "surrogate marker" is an objective biochemical marker which coπelates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent ofthe disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Suπogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a suπogate marker, and an analysis of HIV infection may be made using HTV RNA levels as a surrogate marker, well in advance ofthe undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples ofthe use of suπogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209. The 8843 molecules of the invention are also useful as pharmacodynamic markers.

As used herein, a "pharmacodynamic marker" is an objective biochemical marker which coπelates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drag is being administered; therefore, the presence or quantity ofthe marker is indicative ofthe presence or activity ofthe drag in a subject. For example, a pharmacodynamic marker may be indicative ofthe concentration ofthe drag in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level ofthe drag. In this fashion, the distribution or uptake ofthe drag may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity ofthe metabolic product of a drag, such that the presence or quantity ofthe marker is indicative ofthe relative breakdown rate ofthe drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drag is administered in low doses. Since even a small amount of a drag may be sufficient to activate multiple rounds of marker (e.g., a 8843 marker) transcription or expression, the amplified marker may be in a quantity that is more readily detectable than the drag itself. Also, the marker may be more easily detected due to the nature ofthe marker itself; for example, using the methods described herein, anti- 8843 antibodies may be employed in an immune-based detection system for a 8843 protein marker, or 8843-specific radiolabeled probes may be used to detect a 8843 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. US 6,033,862; Haτtis etal. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999)^, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

The 8843 molecules ofthe invention are also useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is an objective biochemical marker which coπelates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod etal. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity ofthe pharmacogenomic marker is related to the predicted response ofthe subject to a specific drag or class of drags prior to administration ofthe drag. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 8843 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment ofthe specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 8843 DNA may coπelate 8843 drug response. The use of pharmacogenomic markers therefore permits the application ofthe most appropriate treatment for each subject without having to administer the therapy.

Pharmaceutical Compositions The nucleic acid and polypeptides, fragments thereof, as well as anti-8843 antibodies

(also referred to herein as "active compounds") ofthe invention can be incoφorated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incoφorated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The caπier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absoφtion ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incoφorating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity ofthe disease or disorder, previous treatments, the general health and/or age ofthe subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible.

Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193). The present invention encompasses agents or compounds which modulate expression or activity. An agent or compound may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts ofthe small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency ofthe small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid ofthe invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health, gender, and diet ofthe subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabohtes (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates ofthe invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drag moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, .alpha. - interferon, .beta. -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Methods of Treatment: The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abeπant or unwanted 8843 expression or activity. As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the puφose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of phaimacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drags in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drag response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 8843 molecules ofthe present invention or 8843 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drag-related side effects. hi one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an abeπant or unwanted 8843 expression or activity, by administering to the subject an 8843 or an agent which modulates 8843 expression or at least one 8843 activity. Subjects at risk for a disease which is caused or contributed to by abeπant or unwanted 8843 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe 8843 abeπance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 8843 aberrance, for example, an 8843, 8843 agonist or 8843 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

It is possible that some 8843 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms. Relevant disorders can include erythroid-associated disorders, e.g., anemias, neoplasias ofthe erythroid lineage or of CD34+ cells, e.g., leukemias, and erythrocytosis, i.e. excessive erythroid differentiation.

As the 8834 molecules are expressed in hepatic cells, kidney, lung, and dermal cells, these molecules can be used diagnostically and therapeutically to treat diagnose hepatic, kidney, lung, and dermal disorders. Examples of disorders of the lung include, but are not limited to, congenital anomalies; atelectasis; diseases of vascular origin, such as pulmonary congestion and edema, including hemodynamic pulmonary edema and edema caused by microvascular injury, adult respiratory distress syndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, and infarction, and pulmonary hypertension and vascular sclerosis; chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis; diffuse interstitial (infiltrative, restrictive) diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia (pulmonary infiltration with eosinophilia), Bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemoπhage syndromes, including Goodpasture syndrome, idiopathic pulmonary hemosiderosis and other hemoπhagic syndromes, pulmonary involvement in collagen vascular disorders, and pulmonary alveolar proteinosis; complications of therapies, such as drag-induced lung disease, radiation-induced lung disease, and lung transplantation; tumors, such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies ofthe pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

Disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and ciπhosis, such as cirrhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn errors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, α/-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as secondary biliary ciπhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and anomalies ofthe biliary tree; circulatory disorders, such as impaired blood flow into the liver, including hepatic artery compromise and portal vein obstruction and thrombosis, impaired blood flow through the liver, including passive congestion and centrilobular necrosis and peliosis hepatis, hepatic vein outflow obstruction, including hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease associated with pregnancy, such as preeclampsia and eclampsia, acute fatty liver of pregnancy, and intrehepatic cholestasis of pregnancy; hepatic complications of organ or bone marrow transplantation, such as drag toxicity after bone maπow transplantation, graft-versus-host disease and liver rejection, and nonimmunologic damage to liver allografts; tumors and tumorous conditions, such as nodular hypeφlasias, adenomas, and malignant tumors, including primary carcinoma ofthe liver and metastatic tumors.

Disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) polycystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis-associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heymann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, including but not limited to, poststreptococcal glomerulonephritis and nonstreptococcal acute glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis), hereditary nephritis, including but not limited to, Alport syndrome and thin membrane disease (benign familial hematuria), chronic glomerulonephritis, glomerular lesions associated with systemic disease, including but not limited to, systemic lupus erythematosus, Henoch-Schόnlein puφura, bacterial endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary and immunotactoid glomerulonephritis, and other systemic disorders; diseases affecting tubules and interstitium, including acute tubular necrosis and tubulointerstitial nephritis, including but not limited to, pyelonephritis and urinary tract infection, acute pyelonephritis, chronic pyelonephritis and reflux nephropathy, and tubulointerstitial nephritis induced by drags and toxins, including but not limited to, acute drug- induced interstitial nephritis, analgesic abuse nephropathy, nephropathy associated with nonsteroidal anti-inflammatory drags, and other tubulointerstitial diseases including, but not limited to, urate nephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases of blood vessels including benign nephrosclerosis, malignant hypertension and accelerated nephrosclerosis, renal artery stenosis, and thrombotic microangiopathies including, but not limited to, classic (childhood) hemolytic-uremic syndrome, adult hemolytic-uremic syndrome thrombotic thrombocytopenic puφura, idiopathic HUS/TTP, and other vascular disorders including, but not limited to, atherosclerotic ischemic renal disease, atheroembolic renal disease, sickle cell disease nephropathy, diffuse cortical necrosis, and renal infarcts; urinary tract obstruction (obstructive uropathy); urolithiasis (renal calculi, stones); and tumors ofthe kidney including, but not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomeduUary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypernephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.

Examples of disorders ofthe skin include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, seboπheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors ofthe dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular immigrants to the skin, including but not limited to, histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis; disorders of epidermal maturation, including but not limited to, ichthyosis; acute inflammatory dermatoses, including but not limited to, urticaria, acute eczematous dermatitis, and erythema multiforme; chronic inflammatory dermatoses, including but not limited to, psoriasis, lichen planus, and lupus erythematosus; blistering (bullous) diseases, including but not limited to, pemphigus, bullous pemphigoid, dermatitis heφetiformis, and noninflammatory blistering diseases: epidermolysis bullosa and poφhyria; disorders of epidermal appendages, including but not limited to, acne vulgaris; panniculitis, including but not limited to, erythema nodosum and erythema induratum; and infection and infestation, such as verracae, moUuscum contagiosum, impetigo, superficial fungal infections, and arthropod bites, stings, and infestations.

As discussed, successful treatment of 8843 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 8843 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')₂ and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression ofthe target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 8843 expression is through the use of aptamer molecules specific for 8843 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osbome, et al. Curr. Opin. Chem Biol. 1997, 1(1): 5-9; and Patel, D.J. Curr Opin Chem Biol 1997 Jun;l(l):32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 8843 protein activity may be specifically decreased without the introduction of drags or other molecules which may have pluripotent effects.

Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 8843 disorders. For a description of antibodies, see the Antibody section above.

In circumstances wherein injection of an animal or a human subject with an 8843 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 8843 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. AnnMed 1999;31(l):66-78; and Bhattacharya-Chatterjee, M., and Foon, KA. Cancer Treat Res 1998;94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti- idiotypic antibodies, which should be specific to the 8843 protein. Vaccines directed to a disease characterized by 8843 expression may also be generated in this fashion.

In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be prefeπed. Lipofectin or liposomes can be used to deliver the antibody or a fragment ofthe Fab region that binds to the target antigen into cells. Where fragments ofthe antibody are used, the smallest inhibitory fragment that binds to the target antigen is prefeπed. For example, peptides having an amino acid sequence coπesponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesis and or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 8843 disorders. A therapeutically effective dose refers to that amount ofthe compound sufficient to result in amelioration of symptoms ofthe disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅0 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are prefeπed. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method ofthe invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum ofthe test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate 8843 activity is used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal ofthe imprinted molecule leaves a polymer matrix that contains a repeated "negative image" ofthe compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, KJ. (1994) Trends in Polymer Science 2:166-173. Such "imprinted" affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example ofthe use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361 :645-647. Through the use of isotope-labeling, the "free" concentration of compound which modulates the expression or activity of 8843 can be readily monitored and used in calculations of IC50.

Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. An rudimentary example of such a "biosensor" is discussed in Kriz, D. etal (1995) Analytical Chemistry 67:2142-2144. Another aspect ofthe invention pertains to methods of modulating 8843 expression or activity for therapeutic puφoses. Accordingly, in an exemplary embodiment, the modulatory method ofthe invention involves contacting a cell with an 8843, agent or compound that modulates one or more ofthe activities of 8843 protein activity associated with the cell. An agent that modulates 8843 protein activity can be an agent or a compound as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an 8843 protein (e.g., an 8843 substrate or receptor), an 8843 antibody, an 8843 agonist or antagonist, a peptidomimetic of an 8843 agonist or antagonist, or other small molecule. The terms "agent" and "compound" are used interchangeably herein. In one embodiment, the agent stimulates one or 8843 activities. Examples of such stimulatory agents include active 8843 protein and a nucleic acid molecule encoding 8843. In another embodiment, the agent inhibits one or more 8843 activities. Examples of such inhibitory agents include antisense 8843 nucleic acid molecules, anti8843 antibodies, and 8843 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by abeπant or unwanted expression or activity of an 8843 protein or nucleic acid molecule, hi one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 8843 expression or activity. In another embodiment, the method involves administering an 8843 protein or nucleic acid molecule as therapy to compensate for reduced, abeπant, or unwanted 8843 expression or activity,

Stimulation of 8843 activity is desirable in situations in which 8843 is abnormally downregulated and/or in which increased 8843 activity is likely to have a beneficial effect. For example, stimulation of 8843 activity is desirable in situations in which an 8843 is downregulated and/or in which increased 8843 activity is likely to have a beneficial effect. Likewise, inhibition of 8843 activity is desirable in situations in which 8843 is abnormally upregulated and/or in which decreased 8843 activity is likely to have a beneficial effect.

Pharmacogenomics

The 8843 molecules ofthe present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 8843 activity (e.g., 8843 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 8843 associated disorders (e.g., abeπant or deficient erythroid proliferation and differentiation) associated with abeπant or unwanted 8843 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drag) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an 8843 molecule or 8843 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an 8843 molecule or 8843 modulator.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymoφhisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drags (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome- wide association", relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymoφhic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymoφhisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occuπence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drag's target is known (e.g., an 8843 protein ofthe present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version ofthe gene versus another is associated with a particular drug response.

Alternatively, a method termed the "gene expression profiling", can be utilized to identify genes that predict drag response. For example, the gene expression of an animal dosed with a drug (e.g., an 8843 molecule or 8843 modulator ofthe present invention) can give an indication whether gene pathways related to toxicity have been turned on.

Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an 8843 molecule or 8843 modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.

The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more ofthe gene products encoded by one or more ofthe 8843 genes ofthe present invention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent. Specifically, the activity ofthe proteins encoded by the 8843 genes ofthe present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more ofthe resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to. Monitoring the influence of agents (e.g., drags) on the expression or activity of an 8843 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 8843 gene expression, protein levels, or upregulate 8843 activity, can be monitored in clinical trials of subjects exhibiting decreased 8843 gene expression, protein levels, or downregulated 8843 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 8843 gene expression, protein levels, or downregulate 8843 activity, can be monitored in clinical trials of subjects exhibiting increased 8843 gene expression, protein levels, or upregulated 8843 activity. In such clinical trials, the expression or activity of an 8843 gene, and preferably, other genes that have been implicated in, for example, an 8843-associated disorder can be used as a "read out" or markers ofthe phenotype of a particular cell.

8843 Informatics

The sequence of a 8843 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 8843. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 8843 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymoφhic sequences including single nucleotide polymoφhisms (SNPs), epitope sequence, and the like. In a prefeπed embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device. As used herein, "machine-readable media" refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g. , an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine- readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD- ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice ofthe data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information ofthe present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g. , text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information ofthe present invention. In a prefeπed embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g. , a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality ofthe sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymoφhisms (e.g., SNP's) translational regulatory sites and splice junctions. Non- limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

By providing the nucleotide or amino acid sequences ofthe invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of puφoses. For example, one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions ofthe sequences ofthe invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

Thus, in one aspect, the invention features a method of analyzing 8843, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 8843 nucleic acid or amino acid sequence; comparing the 8843 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 8843. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

The method can include evaluating the sequence identity between a 8843 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

As used herein, a "target sequence" can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems ofthe present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

Thus, the invention features a method of making a computer readable record of a sequence of a 8843 sequence which includes recording the sequence on a computer readable matrix. In a prefeπed embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain, region, or site; identification ofthe start of transcription; identification ofthe transcription terminator; the full length amino acid sequence ofthe protein, or a mature form thereof; the 5' end ofthe translated region. In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 8843 sequence, or record, in machine-readable form; comparing a second sequence to the 8843 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 8843 sequence includes a sequence being compared. In a prefeπed embodiment the 8843 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 8843 or second sequence can be stored in a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer, hi a prefeπed embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain, region, or site; identification ofthe start of transcription; identification ofthe transcription terminator; the full length amino acid sequence ofthe protein, or a mature form thereof; the 5' end ofthe translated region. In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 8843- associated disease or disorder or a pre-disposition to a 8843-associated disease or disorder, wherein the method comprises the steps of determining 8843 sequence information associated with the subject and based on the 8843 sequence information, determining whether the subject has a 8843-associated disease or disorder or a pre-disposition to a 8843- associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 8843-associated disease or disorder or a pre- disposition to a disease associated with a 8843 wherein the method comprises the steps of determining 8843 sequence information associated with the subject, and based on the 8843 sequence information, determining whether the subject has a 8843-associated disease or disorder or a pre-disposition to a 8843-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a prefeπed embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 8843 sequence ofthe subject to the 8843 sequences in the database to thereby determine whether the subject as a 8843-associated disease or disorder, or a pre-disposition for such.

The present invention also provides in a network, a method for determining whether a subject has a 8843 associated disease or disorder or a pre-disposition to a 8843-associated disease or disorder associated with 8843, said method comprising the steps of receiving 8843 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network coπesponding to 8843 and/or coπesponding to a 8843-associated disease or disorder (e.g., a hematopoietic disorder), and based on one or more ofthe phenotypic information, the 8843 information (e.g. , sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 8843-associated disease or disorder or a pre-disposition to a 8843-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

The present invention also provides a method for determining whether a subject has a 8843 -associated disease or disorder or a pre-disposition to a 8843-associated disease or disorder, said method comprising the steps of receiving information related to 8843 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 8843 and/or related to a 8843-associated disease or disorder, and based on one or more ofthe phenotypic information, the 8843 information, and the acquired information, determining whether the subject has a 8843-associated disease or disorder or a pre-disposition to a 8843-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incoφorated herein by reference. EXAMPLES

Example 1: Identification and Characterization of Human 8843 cDNA

The human 8843 sequence (Fig. 1; SEQ ID NOT), which is approximately 839 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 606 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NOT in Fig. 1; SEQ ID NO:3). The coding sequence encodes a 201 amino acid protein (SEQ ID NO:2).

Example 2: Tissue Distribution of 8843 mRNA

Endogenous human 8834 gene expression was determined using the Perkin- Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (refeπed to as a probe) that has a fluorescent dye coupled to its 5' end (typically 6-F AM) and a quenching dye at the 3' end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5' dye is quenched. As PCR proceeds, the 5* to 3' nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-F AM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount ofthe gene of interest in the test sample, thus providing a way of quantitating the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5' end (typically VIC). To determine the level of 8834 in various human tissues a primer/probe set was designed using Primer Express (Perkin-Elmer) software and primary cDNA sequence information. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction.

8834 mRNA levels were analyzed in a variety of samples of isolated and/or treated blood cells. High relative expression levels of 8834 mRNA, e.g., greater than 40 units, were observed for megakaryocytes, mast cells, blast forming units (BFU), especially BFUs treated with erythropoietin (Fig. 5). Moderate relative expression levels of 8834 mRNA, between 10 and 40 units, were observed for multiple erythroid samples, and a subset of neutrophil samples (Fig. 5)

High relative expression levels of 8834 mRNA, e.g., greater than 40 units, were observed for mobilized CD34+ peripheral blood cells (mBM), normal bone marrow CD34+ cells, as well as for glycophorin A (low levels) bone maπow cells (>60 units), which are erythroid progenitors (Fig. 6). Moderate relative expression levels of 8834 mRNA, between

10 and 40 units, were observed for CD34+ cord blood cells, CD34+ fetal liver cells, and mobilized CD34+ bone maπow cells (Fig. 6). 8834 mRNA expression levels were also monitored in other hematopoietic lineages and tissues. K582 cells, an erythroid/megakaryocyte cell line, and Hep3b cells had high

8834 expression levels relative to controls (Fig. 7).

Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2xSSC at 65°C. A DNA probe corresponding to all or a portion ofthe 8843 cDNA (SEQ ID NOT) can be used. The

DNA was radioactively labeled with 32p_dCTP using the Prime-It Kit (Stratagene, La Jolla, CA) according to the instructions ofthe supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, CA) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 3: Recombinant Expression of 8843 in Bacterial Cells

In this example, 8843 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 8843 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression ofthe GST-8843 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates ofthe induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined. Example 4: Expression of Recombinant 8843 Protein in COS Cells

To express the 8843 gene in COS cells, the pcDNA Amp vector by Invitrogen Coφoration (San Diego, CA) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 8843 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end ofthe fragment is cloned into the polylinker region ofthe vector, thereby placing the expression ofthe recombinant protein under the control ofthe CMV promoter. To construct the plasmid, the 8843 DNA sequence is amplified by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides ofthe 8843 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides ofthe 8843 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA). Preferably the two restriction sites chosen are different so that the 8843_gene is inserted in the coπect orientation. The ligation mixture is transformed intoE. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jόlla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence ofthe correct fragment.

COS cells are subsequently transfected with the 8843-pcDNA Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran- mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The expression ofthe

8843 polypeptide is detected by radiolabelling (³^S-methionine or ³⁵S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIP A buffer, 150 mM NaCl, 1% NP-40, 0.1 % SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 8843 coding sequence is cloned directly into the polylinker ofthe pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 8843 polypeptide is detected by radiolabelling and immunoprecipitation using an 8843 specific monoclonal antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to be encompassed by the following claims.

in

Claims

What is claimed is:

1. A method for identifying an agent which modulates the activity of an 8834 polypeptide, comprising: a) providing an 8843 polypeptide which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO:3, the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number , or a complement thereof; b) contacting the 8843 polypeptide, or a cell expressing the polypeptide with a test compound; and c) determining the effect ofthe test agent on the activity ofthe 8843 polypeptide.

2. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.

3. The method of claim 1, wherein the polypeptide is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3.

4. The method of claim 1 , wherein the polypeptide further comprises a heterologous amino acid sequence.

5. The method of claim 1, wherein the activity ofthe 8834 polypeptide is proliferation, differentiation, or survival of a CD34 positive cell.

6. A method of modulating erythropoiesis, comprising contacting an erythroid cell with an agent that modulates the activity or expression of an 8834 polypeptide, thereby modulating the differentiation ofthe erythroid cell, wherein the 8834 polypeptide is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO:3, the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number , or a complement thereof.

7. The method of claim 6, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.

8. The method of claim 6, wherein the erythroid cell is an erythroid progenitor or differentiated cell.

9. The method of claim 6, wherein the agent and the 8834 polypeptide are contacted in vitro oτ ex vivo.

10. The method of claim 6, wherein the contacting step is effected in vivo in a subject.

11. The method of claim 10, wherein the subject is a human.

12. The method of claim 11 , wherein the subject is a patient having an erythroid- associated disorder.

13. The method of claim 6, further comprising the step of contacting ofthe erythroid cell with a protein selected from the group consisting of G-CSF, GM-CSF, stem cell factor, Flt-3 ligand, IL-3, IL-4, thrombopoietin, and erythropoietin.

14. The method of claim 13 , wherein the protein is erythropoietin.

15. A method of modulating erythropoiesis, comprising contacting an erythroid cell with an agent that modulates the activity or expression of an 8834 nucleic acid, thereby modulating the proliferation, differentiation or survival ofthe erythroid cell, wherein the 8834 nucleic acid is selected from the group consisting of: a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number ; and b) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, or a complement thereof, or the sequence ofthe insert ofthe plasmid deposited with the ATCC as Accession Number .

16. A method of treating or preventing an erythroid-associated disorder, in a subject, comprising administering to the subject an effective amount of an agent that modulates the activity or expression of an 8834 polypeptide such that the erythroid-associated disorder is ameliorated or prevented, wherein the 8834 polypeptide is selected from the group consisting of:

(i) a polypeptide which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO:3, the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number , or a complement thereof; and

(ii) a polypeptide which comprises the amino acid sequence of SEQ ID NO:2.

17. A method of treating or preventing an erythroid-associated disorder, in a subject, comprising administering to the subject an effective amount of an agent that modulates the activity or expression of an 8834 polypeptide such that the erythroid-associated disorder is ameliorated or prevented, wherein the 8834 polypeptide is selected from the group consisting of: a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number ; and b) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, or a complement thereof, or the sequence ofthe insert ofthe plasmid deposited with the ATCC as Accession Number .

18. A method of modulating the proliferation, differentiation or survival of a CD34 positive cell, comprising contacting the cell with an agent that modulates the activity or expression of an 8834 polypeptide, in an amount effective to modulate the proliferation, differentiation or survival ofthe cell, wherein the 8834 polypeptide is selected from the group consisting of:

(i) a polypeptide which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO:3, the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number , or a complement thereof; and

(ii) a polypeptide which comprises the amino acid sequence of SEQ ID NO:2.

19. The method of claim 18, wherein the CD34 positive cell is an erythroid cell.

20. The method of claim 18, wherein the CD34 positive cell is a bone marrow cell.

21. The method of claim 18, wherein the CD34 positive cell is positive for glycophorin A protein.

22. The method of claim 18, wherein the agent is selected from the group consisting of a peptide, a phosphopeptide, a small molecule, and an antibody.

23. The method of claim 18, wherein the agent is administered in combination with a cytotoxic agent.

24. A method of modulating the proliferation, differentiation or survival of a CD34 positive cell, comprising contacting the cell with an agent that modulates the activity or expression of an 8834 nucleic acid, in an amount effective to modulate the proliferation, differentiation or survival ofthe cell, wherein the 8834 nucleic acid is selected from the group consisting of: a) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number ; and b) a nucleic acid molecule comprising the nucleotide sequence of SEQ

ID NO: 1, 3, or a complement thereof, or the sequence ofthe insert ofthe plasmid deposited with the ATCC as Accession Number .

25. A method for evaluating the efficacy of a treatment of an erythroid-associated disorder, in a subject, comprising: treating a subject with a protocol under evaluation; assessing the expression level of a 8834 nucleic acid or 8834 polypeptide, wherein a change in the expression level of 8834 nucleic acid or 8834 polypeptide after the treatment, relative to the level before the treatment, is indicative ofthe efficacy ofthe treatment ofthe erythroid-associated disorder.

26. The method of claim 25 further comprising treating the subject with erythropoietin prior to assessing expression levels.

27. The method of claim 25, wherein the subject is a patient having an erythroid- associated disorder.

28. The method of claim 27, wherein the erythroid disorder is an anemia.

29. The method of claim 27, wherein the erythroid disorder is a leukemia.

30. A method of diagnosing an erythroid-associated disorder, in a subject, comprising: evaluating the expression or activity of a 8834 nucleic acid or a 8834 polypeptide, such that, a difference in the level of 8834 nucleic acid or 8834 polypeptide relative to a normal subject or a cohort of normal subjects is indicative ofthe erythroid- associated disorder.

31. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID

NO: 1 , SEQ ID NO:3 or the cDNA insert ofthe plasmid deposited with the ATCC as

Accession Number ; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number .

32. An isolated polypeptide selected from the group consisting of: a) a polypeptide which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number , or a complement thereof; and b) a polypeptide comprising the amino acid sequence of SEQ ID NO:2.