MXPA98006628A - Gene of response of differentiation terminal honey honey noved - Google Patents

Abstract

The present invention provides polynucleotide and amino acid sequences that encode a novel human myeloid terminal differentiation response gene designated MYD118. The present invention also provides MYD118 antisense molecules. The invention further provides genetically engineered expression vectors and host cells for the production of purified MYD118 polypeptide, antibodies, antagonists and inhibitors of MYD118 polypeptide, and pharmaceutical compositions and methods of treatment based on polynucleotide sequences encoding MYD118 polypeptide. The invention specifically provides for the use of MYD118 polynucleotide sequences as a diagnostic composition for the detection of myeloproliferative diseases and leukemias. The invention also relates to therapeutic methods and compositions based on nucleotide and amino acid sequences for MYD118. The invention also provides antibodies that specifically bind to MYD1

Description

GENE OF DIFFERENTIATION RESPONSE TERMINAL HONEY HUMAN NOVEDOUS RELATED REQUESTS The present invention is a continuation request in part of the Application of E. U.A. Series No. 08 / 602,208, filed on February 15, 1996, which is incorporated herein by reference.

TECHNICAL FIELD The present invention relates to nucleic acid and amino acid sequences of a novel human myeloid terminal differentiation response gene found in AD Nc collections made from human fetal liver / spleen tissue and non-adherent peripheral blood mononuclear cells and to the use of these sequences in the diagnosis, study and treatment of disease.

BACKGROUND OF THE INVENTION The numbers of cells are regulated through a balance between the proliferation, growth and programmed cell death (apoptosis; Fornace AJ et al., 1992 Ann New York Acad Sci 663: 139-153). The genes induced by various growth arrest and apoptotic stimuli are the tumor suppressor p53 gene, the myeloid differentiation primary response genes (MyD genes), and inducible genes for growth arrest and DNA damage (GADD genes; Selvakumaran M; et al., 1994 Mol Cell Biol 14: 2352-60). The animal cells respond to signals of differentiation, which in turn activate or deactivate genes, resulting in the conversion of non-differentiated, proliferating cells to non-proliferating, highly specialized, differentiated cells. An example of this process is the differentiation of myeloid precursor cells to granulocytes and mature macrophages. Blocks in the differentiation process appear to be a major step in tumor progression, and lesions in genes involved in terminal differentiation contribute to the development of malignancies (Liebermann MA et al., 1994, Stem Cells 12: 352-69). Liebermann and others suggest that MyD genes function as positive regulators of terminal hematopoietic cell differentiation, which is associated with the inhibition of cell growth and apoptosis. Selvakumaran et al., Supra, provide evidence that the member of the MyD family, murine MYD118, described as a terminal differentiation response gene, is expressed in M1D + myeloid precursor cells after the induction of terminal differentiation and growth arrest by IL6y it has been shown to be a positive regulator of apoptosis induced by TGFßl. In addition, leucine zipper transcription factors of the fos / jun family have been identified as MyD genes, specifically MyD21, MyD42 and MyD63, and function as positive regulators of hematopoietic cell differentiation, increasing the differentiation of myeloblastic leukemia cells in vitro and reducing the aggressiveness of the leukemic phenotype in mice discovered. Liebermann and others, supra. suggest that lesions in the MyD genes of the fos / jun family that affect the expression or function of genes contribute to the development of leukemias. The cDNA sequence and deduced amino acid sequence of MYD118 of mupno is described by Abdollahi A et al. (1991, Oncogene 6: 165-17), who indicate that the murine MYD118 cDNA nucleotide sequence predicts a protein of 160 amino acids, which does not contain signals of protein secretion, transmembrane domains or protein-DNA binding motifs, but appears to contain a protein kinase phosphorylation site at position 204, two phosphorylation sites of casein kinase II in positions 215 and 231 and various AT3 motifs, in their 3 'untranslated region. Abdollahi et al. Observed detectable levels of MYD118 RNA in murine spinal cord rich in myeloid precursor, but not in several other non-myeloid murine tissues, such as liver or brain. Abdollahi and others also observed that expression MYD118 was induced in the absence of protein synthesis, after stimulation of M1D + cells through IL-2, LPS and leukemia inhibitory factor (LIF). The amino acid sequence for murine MYD118 is 75% similar (57% identical) to the amino acid sequence for the GADD gene, GADD45, which is regulated in part by the p53 tumor suppressor gene (Zhan Q et al., (1994 ) Cancer Res 54: 2755-60; Carrier F et al. (1994) J Biol Chem 269: 32672-32677). GADD and MYD118 are two separate but closely related genes and act synergistically to suppress the growth of hematoetic cell lineages. Several potions of the nucleotide sequence encoding human MYD118 have been described in GenBank 92 in collections of cDNAs made from hippocampal tissue (M77995), retina (H84533 and H83991), olfactory epithelium (H71592), human fetal lung (D310470 and D31559). ), breast (H44355, R55161 and R82994), placenta (R24009, R63425, R21918 and R22497), white blood cells of human (T33963), human brain (T35368), human pancreas (T29941), liver (T40088), gland of prostate (T35225) and lung (T35563). The complete nucleotide sequence encoding human MYD118 has not been described. Myeloproliferative diseases and leukemias are hematopoietic stem cell neoplasms and include acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myelogenous leukemia (CML); polycythemia vera (PV); Agnogenic myeloid metaplasia with myelofibrosis (MMA / MF); and essential thrombocytosis (ET). The research suggests that proliferative diseases arise as chronic expansions of an individual transformed stem cell and that all myeloid cells in the blood are derived from the neoplastic clone. In leukemia, leukemic cells proliferate mainly in the bone marrow and lymphoid tissues and are characterized according to the type of cell involved (myeloid or lymphoid). Acute leukemia is characterized by the proliferation of immature myeloid or lymphoid cells. CLL is a hematological neoplasm characterized by the accumulation of mature-appearing lymphocytes in the peripheral blood associated with bone marrow infiltration. Hairy cell leukemia is characterized by peripheral blood cytopenias, splenomegaly, and malignant cells in the blood and bone marrow. CML is characterized by marked splenomegaly and the production of increased numbers of granulocytes, particularly neutrophils, in the marrow and blood. PV is characterized by splenomegaly and an increased production of all myeloid elements, but is dominated by a high concentration of hemoglobin. AMM / MF is characterized by the tendency of neoplastic stem cells to lodge and grow in multiple sites outside the marrow, progressive splenomegaly, gradual replacement of marrow elements through fibrosis, and variable changes in the number of granulocytes and platelets. ET is characterized by an elevated platelet count and represents the overproduction of platelets in the absence of a recognizable stimulus. In cultures of bone marrow cells of individuals subjected to ET, megakaryocyte progenitor megakaryocyte colonies are formed in the absence of an aggregated stimulus, whereas such colonies do not occur with bone cell cultures of normal individuals. (Braunwald et al., (Ed. 1987) Harrison's Principles of Internal Medicine, 11th Edition, McGraw-Hill, New York, NY).

DESCRIPTION OF THE INVENTION The present invention relates to human MYD118, whose nucleic acid sequence has been identified between the polynucleotide sequences of cDNA collections made from human fetal liver-spleen tissue and non-adherent peripheral blood mononuclear cells and to the use of the sequences of nucleic acid and amino acid of MYD118 in the study, diagnosis and treatment of disease states related to proliferation, specifically with myeloproliferative diseases and leukemias. The myd118 nucleic acid sequence (SEQ ID NO: 1) and the protein encoding MYD118 (SEQ ID NO: 2) is described in Figure 1 herein. The amino acid homology between human MYD118, murine MYD118 (SEQ ID NO: 3) and human GADD 45 (SEQ ID NO: 4) is shown in Figure 2. The present invention is based in part on the homology of the amino acid that the Human MYD118 shares human MYD118 and the ability of MYD118 of murine and other members of the MYD family to stimulate the terminal differentiation of hematopoietic cells, to arrest cell growth and to modulate the leukemic phenotype in vivo. The present invention is also based in part on the presence of nucleic acid sequences encoding MYD1 18 in a cDNA library made of fetal liver / spleen tissue and non-adherent peripheral blood mononuclear cells, where hematopoietic cells could be found. Nucleic acid sequences encoding MYD1 18 are not detected in samples from cDNA collections made from sources of malignant hematopoietic cells, where the expression of genes related to cell growth arrest and apoptosis may be absent, aberrant, eliminated or expressed at low levels. Therefore, the expression of human MYD1 may be altered, absent or at low levels in individuals subjected to myeloproliferative disease or leukemias. Human MYD 18, and the nucleic acid sequences encoding it and oligonucleotides, peptide nucleic acid (PNA), fragments, potions or antisense molecules thereof, provide the basis for diagnostic methods for early and accurate detection and / or quantitation of MYD 1 18 associated with abnormally proliferating hematopoietic cells such as myeloproliferative diseases and leukemias, including, but not limited to, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia and chronic myelogenous leukemia (CML); polycythemia vera (PV); Agnogenic myeloid metaplasia with myelofibrosis (MMA / MF); and essential thrombocytosis (ET). For example, the nucleotide sequence for myd 1 18 described herein, or its fragments, can be used in hybridization assays of cells or biopsy tissues or body fluids to diagnose abnormalities in the expression of the myd 1 18 gene in individuals subjected to or at risk of a myeloproliferative disease or leukemia. The expression of the myd 1 18 gene may be present at low levels, completely absent or altered in said disease states. In addition, there is a chromosomal aberration, such as a deletion or mutation, present in the region of the gene encoding MYD 1 18 in disease states related to the proliferation of hematopoietic cells. Nucieotide and amino acid sequences for MYD 1 18 can also be used in the diagnosis or treatment of other disease states related to the abnormal proliferation of cells associated with severe inflammation, such as rheumatoid arthritis, psoriasis (characterized by cell proliferation epidermal) and lymphomatoid granulomatosis (characterized by a lymphoproliferative disease), where it is desirable to terminally differentiate the proliferation of cells and stop their growth. Accordingly, the present invention provides diagnostic compositions and diagnostic tests for the detection of myd 1 18 nucleotide sequences in biological samples. Said diagnostic test comprises the steps of combining the biological sample with a first nucleotide sample, which comprises a myd1 18 nucleotide sequence, or its non-conserved fragment, under conditions suitable for the formation of a nucleic acid hybridization complex.; detecting said hybridization complex, wherein the presence of said complex correlates with the presence of a second nucleotide sequence comprising nucleotide sequences myd 1 18 in said biological sample; and comparing the amount of the second nucleotide sequence in said sample with a normal one, to thereby determine whether the amount of said second nucleotide sequence varies from normal to ia, wherein the presence of an abnormal level of said second nucleotide sequence is correlated positively with a myeloproliferative disease. An abnormal DNA of nucleotide sequences encoding MYD 1 1 8 in a biological sample may reflect a chromosomal aberration, such as a deletion or mutation of nucleic acid. Accordingly, the nucleotide sequences encoding MYD 1 18 provide the basis for probes that can be used to detect chromosomal aberrations such as deletions, mutations or chromosomal translocations in the gene encoding MYD1 18. The present invention also provides a diagnostic test for the detection of myd 1 18 nucleotide sequences in a biological sample, comprising the steps of combining the biological sample with polymerase chain reaction primers under conditions suitable for nucleic acid amplification, wherein said primers comprise non-conserved fragments of the nucleotide sequence of SEQ ID NO: 1, detecting amplified nucleotide sequences, and comparing the amount of amplified nucleotide sequences in said biological sample with a normal one to thereby determine whether the amount of said nucleotide sequence varies from the normal, where the presence of a level The abnormality of said nucleotide sequence correlates positively with a myeloproliferative disease. In addition, human MYD118 and the nucleic acid sequences encoding it, will provide the basis for pharmaceutical compositions for the treatment of myeloproliferative diseases, such as ALL, AML, CLL, hairy cell leukemia, CML, PV, AMM / MF and ET. . For example, the nucleotide sequences encoding MYD118 can be administered alone or in combination with nucleotide sequences encoding tumor suppressor genes, such as p53 (known to regulate the expression of the related family member GADD45), p16 and p21, a through gene therapy techniques to individuals with CML to induce the terminal differentiation of granulocytes, thus stopping the proliferation of leukemic cells. Alternatively, myd118 nucleic acid antisense molecules or MYD118 protein antagonists can be used to block the activity of human MYD118 under conditions where it would be preferable to block positive regulators of cell growth arrest. For example, MYD 1 18 can be used alone or in combination with other agents in the ex vivo culture of hematopoietic stem cells intended for autologous transplantation to individuals lacking cells of the hematopoietic lineage such as, for example, individuals subjected to infection of VI H or individuals who have undergone chemotherapy or radiation therapy. The present invention also relates, in part, to expression vectors and host cells comprising polynucleotide sequences encoding MYD1 18 for the in vivo or jjn vitro production of the MYD1 protein 18. In addition, the present invention relates to the use of MYD1 polypeptides 18, or fragments or variants thereof, to produce anti-MYD18 antibodies and to classify MYD118 polypeptide antagonists or inhibitors, which can be used to detect and quantify protein levels diagnostically MYD 1 18 in disease states related to proliferation.

The present invention further relates to methods of treating individuals subjected to myeloproliferative disease comprising administering compositions comprising purified MYD 1 18 polypeptides or variants thereof, to subjects at risk or having a myeloproliferative disease or leukemia. The present invention also relates to pharmaceutical compositions comprising effective amounts of MYD 1 18 protein or nucleic acid encoding MYD 1 18 for the treatment of myeloproliferative diseases or leukemias. The present invention also encompasses the use of gene therapy methods for the introduction of nucleotide sequences encoding MYD118 in individuals who have or are at risk for myeloproliferative diseases or leukemias. The invention further provides diagnostic assays and equipment for the detection of MYD118 in cells and tissue comprising purified MYD118, which can be used as a positive control, and anti-MYD118 antibodies. Said antibodies can be used in solution-based, membrane-based, or tissue-based technologies to detect any disease state or condition related to the expression of the MYD118 protein or the expression of its deletions or variants.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 displays the polynucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO: 2) sequence for MyD118. The sequences shown in this figure were produced using the multiple sequence alignment program of the DNASTAR software (DNASTAR Inc., Madison Wl). SEQ ID NO: 1 also contains the 5 'or 3' untranslated regions of myd118. Figure 2 displays the amino acid alignment of human MYD118 (SEQ ID NO: 2), murine MYD118 (SEQ ID NO: 3) and human GADD45 (SEQ ID NO: 4). The residues inside the boxes coincide with the consensus sequence, exactly. Figure 3 displays an analysis of hydrophobic character and other characteristics of MYD1 18 based on the predicted amino acid sequence (produced using the DNASTAR software).

MODES FOR CARRYING OUT THE INVENTION The present invention relates to human MYD 1 18 and to the use of the nucleic acid and amino acid sequences of MYD1 18 in the study, diagnosis and treatment of disease states, specifically myeloproliferative diseases and leukemias. The present invention is based in part on the amino acid homology that human MYD1 18 shares with murine MYD118 and the ability of MYD 1 18 of murine and other members of the MYD family to stimulate terminal differentiation of hematopoietic cells, to arrest the cell growth and to reduce a leukemic phenotype in vivo. The present invention is also based in part on the presence of nucleic acid sequences encoding MYD1 18 in a cDNA library made of fetal liver / spleen tissue and non-adherent peripheral blood mononuclear cells, where hematopoietic cells could be found. Nucleic acid sequences encoding MYD1 18 were not detected in randomly selected samples of approximately 500 to 5700 sequences usable in cDNA collections made of TH P-1 cells, the human promocito line derived from the peripheral blood of an individual submitted to Acute monocytic leukemia (ATCC access TIB 202, INCYTE collections: THP1NOB01, THP1PEB01, THP1PLB01 and THP1PLB02); U937 cells, made of malignant cells from the pleural effusion of an individual subjected to histiocytic lymphoma (ATTC access CRL 1593, Sundstrom C and K Nilsson (1976) Int J Cancer 17: 565-577, INCYTE collection U9337NOT01); T / B lymphoblasts from a leukemia source (Stratagene (LaJolla, CA), STR 937214; INCYTE TBLYNOT01 collection); white blood cells of peripheral blood of an individual with myelogenous leukemia (INCYTE AMLBNOT01 collection); and a barley cell line from an individual subjected to barley cell leukemia (INCYTE collection HMC1NOT01), wherein the gene expression related to the arrest of cell growth and apoptosis may be absent or aberrant. Nucleic acid sequences encoding MYD118 were not detected in a collection of peripheral blood granulocyte cDNAs (INCYTE NEUTFMT01 collection); Bone marrow samples combined (INCYTE BMARNOR02 collection); ataxia telangestasia fibroblast cell line (INCYTE, FIBRAGT01, FIBRAGT02, and FIBRANT01 collections); or adult spleen (INCYTE SPLNOT02 collection). It is known that the related family member MYD118, GADD45, is absent in individuals subjected to ataxia telangestasia (Zhan et al. (1994) Mol and Cell Biol 14: 2361-2371). Therefore, the expression of human MYD118 may be altered, absent or undetected in individuals subjected to myeloproliferative disease and leukemias. In addition, the gene encoding MYD1 18 may be involved in a chromosomal aberration such as a deletion, mutation, i.e., a point or internal mutation, translocation or may contain tri-nucleotide repeats that are known to be present in chromosomal abnormalities. Human MYD1, nucleic acid sequences encoding MYD1 18 and oligonucleotides, peptide nucleic acid (PNA), fragments, portions or antisense molecules thereof, provide the basis for diagnostic methods for detection and / or early quantification and exact MYD1 18 associated with myeloproliferative diseases and leukemias, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML); polycythemia vera (PV); Agnogenic myeloid metaplasia with myelofibrosis (MMA / MF); and essential thrombocytosis (ET). For example, the nucleotide sequence for myd 1 18 described herein, or its fragments, can be used in hybridization assays of cells or biopsy tissues to diagnose abnormalities in the expression of the myd 1 18 gene in individuals at risk of myeloproliferative disease or leukemia. The expression of the myd 1 18 gene may be present at low levels, completely absent or altered in said disease states. In addition, the nucleic acid sequences described herein can be used in the detection of aberrations, such as mutations and deletions, in the gene encoding MYD118. For example, the nucleotide sequences described herein can be used to identify and isolate a genomic sequence for myd188. PCR primers can be designed from several portions of introns and exons of the genomic myd118 that will allow the detection of aberrations in the genomic sequence. Additionally, human MYD118 and the nucleic acid sequences encoding it will provide the basis for pharmaceutical compositions for the treatment of myeloproliferative diseases, such as ALL, AML, CLL, hairy cell leukemia, CML, PV, AMM / MF and ET. For example, nucleic acid sequences encoding MYD118 can be administered to individuals undergoing CML or AMM to induce terminal granulocyte differentiation, thereby stopping cell proliferation. Administration of MYD118 or nucleic acid sequences encoding it may alleviate symptoms associated with myeloproliferative diseases and leukemias, such as anemia, fatigue, splenomegaly, hypermetabolism and thromboemorrhagic complications. Alternatively, myd118 nucleic acid antisense molecules or protein antagonists MYD118 can be used to block the activity of human MYD118 in conditions where it could preferably block the positive regulators of cell growth arrest. For example, MYD118 can be used alone or in combination with other agents in the ex vivo culture of hematopoietic stem cells intended for autologous transplantation to HIV-infected individuals, who have undergone chemotherapy or radiation therapy. The present invention also relates, in part, to expression vectors and host cells comprising polynucleotide sequences encoding MYD1 18 for the in vivo and m vitro production of MYD1 protein 18. "Nucleic acid sequence", as used herein, refers to a sequence of oligonucleotide, nucleotide or polynucleotide, and fragments and portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be double or single chain structure if represented the structure of sense or antisense. As used herein, "amino acid sequence" refers to peptide or protein sequences or portions thereof. As used herein, myd 1 18 in lowercase refers to a nucleic acid sequence, while MYD1 18 in uppercase refers to a protein sequence. As used herein, "peptide nucleic acid" (PNA) refers to a class of information molecules that have a "peptide type" base structure combined with nucleotides that allow hybridization to complementary DNA or RNA with affinity and specific character greater than the corresponding oligonucleotides (PerSeptive Biosystems, Marlborough MA). As used herein, MYD 1 18 refers to MYD 1 1 8 of bovine, ovine, porcine, equine and preferably human, naturally occurring or in a variable form, or from any source, whether natural, synthetic, semi synthetic or recombinant. As used herein, "natural existence" refers to a MYD118 with an amino acid sequence found in nature, and "biologically active" refers to a MYD118 that has structural, regulatory or biochemical functions of the MYD118 of natural existence. Also, "immunological activity" is defined as the ability of the natural, recombinant or synthetic MYD118 or any oligopeptide thereof to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. The term "derivative", as used herein, refers to the chemical modification of MYD118. Illustrative of such modifications could be the replacement of hydrogen by an alkyl, acyl or amino group. A MYD118 polypeptide derivative could encode a polypeptide that retains essential biological characteristics of MYD118. As used herein, the term "purified" refers to molecules, either nucleic acid or amino acid sequences, that are removed from their natural environment and isolated or separated from at least one other component with which they are naturally associated .

Coding Sequences of myd1 18 The coding sequence of myd118 (SEQ ID NO: 1) is shown in Figure 1. The entire coding region for human myd1 was initially identified within a cDNA library made of liver tissue. human fetal spleen, where it was found 1 time in 2899 usable sequences. A BLAST search (Basic Local Aligment Search Tool; Altschul SF (1993) J. Mol. Evol. 36: 290-300; Altschul SF et al. (1990) J. Mol. Biol. 215: 403-410) comparing the cDNAs of the human fetal liver / spleen collection (SPLN FET01) against the primate database of GenBank 91 identified the clone Incyte 25214 as an inaccurate comparison with murine mRNA for myd 1 18 and the related human member of the family, gadd45 (NCBI number Gl 53291), see Figure 2. The nucleotide sequence for myd 18 was identified within the Incyte 25214 clone through a computer-generated search for nucleotide sequence alignments. The clone was sequenced again, and the coding region was determined. The polynucleotide sequences encoding MYD 1 18 were subsequently found in a cDNA library made from non-adherent peripheral blood mononuclear cells (PBMN) where 1 in 3941 usable sequences were found. As used herein, the term "usable sequences" refers to the total number of clones in a collection after vector removal, nucleotide repeats, contamination and mitochondrial A DN.

Several portions of the nucleotide sequence encoding MYD118 have been found in GenBank from the following sources of human tissue: hippocampus, retina, olfactory epithelium, fetal lung, breast, placenta, white blood cells, brain, pancreas, prostate gland and lung. However, the polynucleotide sequences encoding MYD118 have not been detected in cDNA collections made of malignant cells of hematopoietic lineage, including THP-1 cells; U938 cells; T / B lymphoblasts from a leukemic source; white blood cells of peripheral blood of an individual with myelogenous leukemia; and human mast cells from an individual with barley cell leukemia. The nucleotide sequence myd118 encodes an acidic protein of 160 amino acids having a predicted isoelectric point of 4.4 and discrete hydrophobic and hydrophilic regions as shown in Figure 3. Methods for sequencing DNA are well known in the art and employ enzymes such as Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer, Norwalk CT), thermostable T7 polymerase (Amersham, Chicago IL), or combinations of recombinant polymerase and exonucleases tested of reading such as ELONGASE Amplification System sold by Gibco BRL (Gaithersburg MD). Methods for extending the DNA of an oligonucleotide primer attached to the DNA template of interest have been developed for both single and double chain structure templates. The chain termination reaction products were separated using electrophoresis and detected through their incorporated, tagged precursors. Recent improvements in the preparation of mechanized reaction, sequencing and analysis have allowed the expansion in the number of sequences that can be determined per day. Preferably, the process is automated with machines such as Hamilton Micro Lab 2200 (Hamilton, Reno NV), Peltier Thermal Cycler (PTC200, MJ Research, Watertown MA) and ABI Catalyst 800 and DNA sequencers 377 and 373 (Perkin Elmner). The quality of any particular cDNA library from which the polynucleotides encoding MYD1 18 are found can be determined by performing a pilot scale analysis of cDNAs and verifying the percentages of clones containing a vector, lambda DNA or E. coli , Mitochondrial or repetitive DNA, and clones with exact comparisons or homologous to public databases.

Polynucleotide sequence extension myd 1 18 Polynucleotide sequence of myd 1 18 can be extended using the oligonucleotide sequences of SEQ ID NO: 1 in various methods known in the art to detect upstream sequences such as promoters and regulatory elements . Gobinda et al. (1993; PCR Methods Applic 2: 318-22) describe the "restriction site polymerase chain reaction (PCR)" as a direct method using universal primers to recover the unknown sequence adjacent to a known site.

First, genomic DNA is amplified in the presence of an initiator to an adapter sequence and an initiator specific to the known region. The amplified sequences are subjected to a second round of PCR with the same adapter initiator and another specific primer internal to the first. The products from each round of PCR were transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase. Reverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia T et al. (1988) Nucleic Acids Res 16: 8186). The primers can be designed using Oligo 4.0 (National Biosciences Inc., Plymouth MN), or another suitable program, so that they have a length of 22-30 nucleotides, a GC content of 50% or more, and to be fixed to the target sequence at temperatures of approximately 68-72 ° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized through intramolecular ligation and used as a PCR template. Capture PCR (Lagerstrom M et al. (1991) PCR Methods Applic 1: 111-19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and artificial yeast chromosome DNA (YAC) . Capture PCR also requires digestions and multiple restriction enzyme ligations to place a double-stranded structure sequence engineered into an unknown portion of the DNA molecule prior to PCR. Parker JD et al (1991, Nucleic Acids Res 19: 3055-60), teach step PCR, a method for the passage of the target gene, which allows recovery of the unknown sequence. PromoterFinder ™ in a new team from Clontech (Palo Alto CA) that uses PCR, nested primers and special collections to "pass on" genomic DNA. This procedure avoids the need to classify collections and is useful in the finding of intron / exon junctions. Another PCR method, "Improved Method for Obtaining Full Lenght cDNA Sequences "(Improved Method for Obtaining Full-Length cDNA Sequences) by Guegler et al., U.S. Patent Application Serial No. 08/487, 12, filed June 7, 1995 and incorporated herein by reference, employs XL-PCR ™ enzymes (Perkin-Elmer, Foster City CA) to amplify and / or extend nucleotide sequences.The preferred collections for classifying full-length cDNAs are those that have been selected by size to include Larger cDNAs Also, randomized initiation collections are preferred, as they will contain more sequences containing the 5 'regions and upstream of genes A randomly initiated collection can be particularly useful if an oligo d (t) collection does not produce a Full length cDNA Genomic libraries are useful for obtaining introns and extending the 5 'sequence.

A new method to analyze either the size or confirm the nucleotide sequence of sequencing or PCR products is capillary electrophoresis. Systems for rapid sequencing are available from Perkin Elmer, Beckman Instruments (Fullerton CA), and other companies. Capillary sequencing employs polymers capable of flowing for electrophoretic separation, four different dyes (one for each nucleotide), which are activated by laser, and detection of the wavelengths emitted through a development camera coupled to a charge. The output / light intensity is converted to an electrical signal using appropriate software (for example, Perkin Elmer's Genotyper ™ and Sequence navigator ™) and the entire process of loading the samples for computer analysis and displaying the electronic data is controlled by a computer. Capillary electrophoresis is particularly suitable for the sequencing of small pieces of DNA that may be present in limited quantities in a particular sample. Sequencing that can reproduce up to 350 bp of M 13 phage DNA in 30 minutes has been reported (Ruiz-Martinez MC et al. (1993) Anal, Chem. 65: 2851-8).

Expression of MYD1 1 8 In accordance with the present invention, the myd 1 18 polynucleotide sequences, which encode MYD 1 18 polypeptide sequences, their fragments, fusion proteins or functional equivalents, can be used to generate recombinant DNA molecules. which direct the expression myd118 in appropriate host cells. Due to the inherent degeneracy of the genecode, the DNA sequences other than the polynucleotide sequences of SEQ ID NO: 1, which encode a substantially equal or functionally equivalent amino acid sequence, can be used to clone and express myd 118. As it will be understood by those skilled in the art, it may be advantageous to produce nucleotide sequences encoding MYD1 18 that possess codons of non-natural existence. Preferred codons, through a parlar prokaryoor eukaryohost (Murray E. et al. (1989) N uc Acids Res. 17 :), can be selected, for example, to increase the expression rate of myd 118 or produce transcripts of recombinant RNA having desirable properties, such as a longer life than transcripts produced from a naturally occurring sequence. Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to SEQ ID NO: 1 under conditions of intermediate to maximal severity so long as the polynucleotide sequence capable of hybridization encodes a protein that retains a biological activity. of MYD1 18 of natural existence. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught by Wahl GM et al. (1987, Methods Enzymol 152: 399-407) incorporated herein by reference, and confer an explanation of "severity" as explained later. The "maximum severity" typically occurs at a Tm of approximately -5 ° C (5 ° C below the Tm of the probe); "high severity" at about 5 ° C to 10 ° C below the Tm; "intermediate severity" at approximately 10 ° C to 20 ° C below the Tm; and "low severity" at about 20 ° C to 25 ° C below the Tm. As will be understood by those skilled in the art, a hybridization of maximal severity can be used to identify or detect idenl polynucleotide sequences, while an intermediate (or low) hybridization can be used to identify or detect similar or related polynucleotide sequences. The term "hybridization", as used herein, refers to the "process by which a nucleic acid structure binds with a complementary structure through base pairs" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY). Amplification as performed in polymerase chain reaction technologies is described in Dieffenbach CW and GS Dvksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY) and is incorporated herein by reference. As used herein a "deletion" is defined as a change in either the nucleotide or amino acid sequence, wherein one or more nucleotide or amino acid residues, respectively, are absent. As used herein, an "insertion" or "addition" is that change in a nucleotide or amino acid sequence, which has resulted in the addition of one or more nucleotide or amino acid residues, respectively, as compared to the molecule of natural existence. As used herein, "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. Polynucleotide sequences of variant m and d 18 according to the invention can be used and include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide encoding the same MYD1 polypeptide 18 or equivalent functionality. The protein of variant MYD1 18 can also be used according to the invention and can include elim inations, insertions or substitutions of amino acid residues, provided that the result is a functionally equivalent MYD 1 18. As used herein, the term "functionally equivalent" refers to a variant polynucleotide or variant polynucleotide sequence that retains at least one of the biological activities of the natural sequence. Amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilic character, and / or the amphipathic nature of the residues provided that a biological activity of MYD118 is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include Usin and arginine; and amino acids with uncharged polar head groups having similar hydrophilic character values are grouped as follows: leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and finally, serine, threonine, phenylalanine and tyrosine. Included within the scope of the present invention are the myd118 alleles. As used herein, an "allele" or "allelic sequence" is an alternative form of myd118. The alleles result from a mutation, i.e., a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene can have none, one or many allelic forms. Mutational changes that give rise to alleles that are generally attributed to deletions, additions or substitutions of nucleic acids. Each of these types of changes can occur alone, or in combination with each other, and at the speed of one or more times in a given sequence. The nucleotide sequences of the present invention can be engineered for the purpose of altering a myd118 coding sequence for a variety of reasons, including, but not limited to, alterations that modify the cloning, processing and / or expression of the product. gen. For example, mutations can be introduced using techniques that are well known in the art, for example, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc. In another embodiment of the invention, a natural, modified or recombinant sequence of myd1 18 can be linked to a heterologous sequence to encode a fusion protein. For example, to classify collections of peptides for inhibitors of MYD1 18 activity, it may be useful to encode a chimeric MYD 18 protein by expressing a heterologous epitope that is recognized by a commercially available antibody. Also, a fusion protein can be engineered to contain a cleavage site between a MYD1 sequence 18 and the heterologous protein sequence, so that MYD 1 18 can be cleaved and purified away from the heterologous portion. In an alternative embodiment of the invention, the coding sequence of myd1 18 can be synthesized, in whole or in part, using chemical methods well known in the art (See, Caruthers MH et al. (1980) Nuc. Acids Res. Symp. Ser. 215-23, Horn T and others (1980) Nuc Acids Res. Symp. Ser 225-32, etc.). Alternatively, the same protein can be produced using chemical methods to synthesize an amino acid sequence MYD1 18, in whole or in part. For example, peptides can be synthesized through solid phase techniques, cleaved from the resin, and purified through preparative high performance liquid chromatography (eg, Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co New York, NY). The composition of the synthetic peptides can be confirmed by acid analysis or sequencing (for example, the Edman degradation procedure, Creighton, supra). Direct peptide synthesis can be carried out using the various solid phase techniques (Roberge JY et al. (1995) Science 269: 202-204) and automated synthesis can be achieved, for example, by using an ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. In addition, the amino acid sequence of MYD118, or any part thereof, may be altered during direct synthesis and / or using chemical methods with other sequences to produce a variant polypeptide.

Expression Systems In order to express a biologically active MYD118, the nucleotide sequence encoding MYD118, or a functional equivalent thereof, is inserted into an appropriate expression vector, i.e., a vector containing the necessary elements for the expression. transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing an MYD 1 18 coding sequence and appropriate transcription and translation controls. These methods include recombinant DNA techniques in vitro. synthetic techniques and in vivo recombination or genetic recombination. These techniques are described by Maniatis et al. (1989) in Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Plainview NY and Ausubel FM et al. (1989) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY. The analysis of the functions of growth inhibitory genes, such as myd 18, using expression vectors, can be technically difficult, since even a low level expression can block the growth of the host cell. Stable transfectants that grow usually are inverters that have loss expression or have other compensation changes (Zhan et al., Supra). Therefore, a preferred expression system for the expression of MYD 18 in host cells is one that allows the expression of toxic proteins to the host cell. For expression in mammalian cells, Zhan et al (supra, p 2366), describes a mammalian expression system for the expression of family members related to MYD1 18, GADD45, which has been shown to be regulated by p53, a murine MYD1 18. This expression system employs the cytomegalovirus promoter and cotransfection with a selectable marker, pSV2neo. Expression studies were performed on tumor lines in humans with a null p53 phenotype (H 1299), in cells with a normal p53 phenotype (R KO), and in cells containing a viral protein, E6, that interferes with the p53 function (HeLa). For the expression of MYD118 in E. coli. Brown et al. (Gene 1993, 127: 99-103) describe a method that was used for the expression of the toxic POL3 gene of S. cerevisiae. which involves the use of the pT7SC vector. Brown obtained up to 15 mg of protein from only 3 grams of cells and the expressed protein was in the form of inclusion bodies.

Identification of Transformants Containing myd118 Although the presence / absence of marker gene expression suggests that the gene of interest is also present, its presence and expression must be confirmed. For example, if the myd118 is inserted into a marker gene sequence, recombinant cells containing myd118 can be identified through the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with a myd118 sequence under the control of an individual promoter. Expression of the marker gene in response to induction or selection usually also indicates the expression of myd118. Alternatively, host cells, which contain the coding sequence for myd118 and express myd118, can be identified through a variety of methods known to those skilled in the art. These methods include, but are not limited to, bioassay or immunoassay techniques of hybridization and DNA-DNA or DNA-RNA protein, which include membrane-based, solution-based or wafer-based technologies for detection and / or quantification of the nucleic acid or protein. The presence of the myd 1 18 polynucleotide sequence can be detected through hybridization or amplification of DNA-DNA- or DNA-RNA using probes, portions or fragments of very -18 described in SEQ ID NO: 1. Assays based on nucleic acid amplification involve the use of oligonucleotides or oligomers based on the sequence of myd1 18 to detect transformants containing DNA or RNA of myd1 18. As used herein, "oligonucleotides" or "oligomers" refers to a nucleic acid sequence of at least about 10 nucleotides and as many as 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20-25 nucleotides, which They can be used as a probe or amplifier. Preferably, the oligonucleotides are derived from the 3 'region of the nucleotide sequence myd 1 18 shown in Figure 1. A variety of protocols are known in the art for detecting and measuring MYD1 18 polypeptide expression, using either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay is preferred, using monoclonal antibodies reactive to two epitopes without interference in MYD118 polypeptides, but a competitive binding assay can be employed. These and other assays are described, inter alia, in Hampton R et al. (1990, Seroloaical Methods, a Laboratorv Manual, APS Press, St Paul MN) and Maddox DE et al. (1983, J. Exp. Med. 158: 1211). ) A wide variety of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting myd118 polynucleotide sequences include oligotag, nick translation, final labeling or PCR amplification using a labeled nucleotide. Alternatively, the myd118 sequence, or a portion thereof, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available and can be used to synthesize RNA probes in vitro by the addition of an appropriate RNA polymerase, such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wl), and US Biochemical Corp. (Cleveland OH) supply commercial equipment and protocols for these procedures. Suitable reporter molecules or labels include those radionucleotides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. The patents that teach such marks include the US patents. 3,817, 837; 3,850,752; 3,393,350; 3,996,345; 4,277,437; 4,275, 149 and 4,366,241. Recombinant immunoglobulins can also be produced as shown in the U.A. No. 4,816,567 and are incorporated herein by reference.

Purification of MYD118 The host cells transformed with a myd1 nucleotide sequence 18 can be cultured under conditions suitable for the expression and recovery of the encoded protein from the cell culture. The protein produced through a recombinant cell can be secreted or it can be contained intracellularly depending on the sequence and / or the vector used. As will be understood by those skilled in the art, expression vectors containing myd 1 18 can be designed with signal sequences that direct the secretion of MYD1 18 through a particular prokaryotic or eukaryotic cell membrane. Other recombinant instructions can bind myd 1 18 to the nucleotide sequence encoding a polypeptide domain, which will facilitate the purification of soluble proteins (Kroll DJ et al. (1993) DNA Cell Biol. 12: 441-53; see also the above discussion of vectors containing fusion proteins). MYD 1 18 can also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate purification of the protein. Such domains that facilitate purification include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Exp. Pur. 3: 263-281), protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (Immunex Corp., Seattle WA). The inclusion of separable adapter sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and MYD118 is useful to facilitate purification.

Uses of MYD118 It seems that MYD118 induces the terminal differentiation of hematopoietic cells thus stopping cell proliferation and inducing apoptosis. Polynucleotide sequences encoding MYD118, or portions thereof, have been found in a variety of tissue cDNA collections including fetal liver / spleen, PBMN cells, hippocampus, retina, olfactory epithelium, human fetal lung, breast, placenta, globules human targets, human brain, human pancreas, liver, prostate gland and lung. The polynucleotide sequences encoding MYD118 are not detected in cDNA collections made from malignant cells of hematopoietic lineage, where the genes related to cell growth arrest and apoptosis could be expected to be expressed at undetectable levels or not expressed. Based on 1) its characterization as a member of the MYD family, 2) the ability of members of the MYD family to terminally differentiate cells from a hematopoietic lineage and 3) the ability of related MYD family members to reverse a phenotype In vivo leukemia, human MYD1 18, described herein, can be used as a diagnostic or therapeutic agent in the detection or treatment of hematopoietic cell proliferative diseases, specifically in myeloproliferative diseases and leukemias. Accordingly, MYD1 18 can be used to treat or mitigate the symptoms of myeloproliferative diseases or leukemias, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia and myelogenous leukemia chronic (CML); polycythemia vera (PV); Agnogenic myeloid metaplasia with myelofibrosis (MMA / MF); and essential thrombocytosis (ET). MYD118 can be used alone or in combination with other drugs or agents in the treatment of such diseases. For example, the administration of MYD1 18 to individuals subjected to myeloproliferative disease, which is characterized by the production of increased numbers of cells in peripheral blood, can induce terminal differentiation of immune cells thus leading to maturation of the cells. cells and final apoptosis. The administration of MYD1 18 can be used to increase chemotherapy, such as the administration of busalfan, radiation therapy and / or bone marrow transplantation. MYD 1 18 can be used therapeutically to mitigate the symptoms associated with myeloproliferative diseases and leukemias, such as anemia, fatigue, splenomegaly, hypermetabolism and thromboemorrhagic complications. MYD1 18 can also be used to treat disease states related to the abnormal proliferation of cells associated with severe inflammation including, but not limited to, rheumatoid arthritis, psoriasis, and infomatoid granulomatosis. In another embodiment of the present invention, antibodies or antagonists of MYD1 18 can be used in the ex vivo culture of cells of a hematopoietic cell lineage intended for autologous transplantation, where it might be desirable to eliminate a cell growth arrest factor that could interfere with the desirable proliferation of cells.

MYD1 Antibodies 18 Methods well known in the art can be used for the production of antibodies to MYD1 polypeptides 18. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments. produced through a collection of Fab expression. Neutralizing antibodies, that is to say, those that exhibit biological activity of MYD 1 8 polypeptides, are especially preferred for diagnosis and therapy.

For the production of antibodies, several hosts, including goats, rabbits, rats, mice, etc. , they can be immunized by injection with MYD1 polypeptide 18 or any portion, fragment or oligopeptide that retains the immunogenic properties. Depending on the host species, several auxiliaries can be used to increase the immune response. Such auxiliaries include, but are not limited to, Freund mineral gels, such as aluminum hydroxide and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, key limpet hemocyanin and dinitrophenol. Potentially useful human auxiliaries with BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. which may be employed if the MYD 18 polypeptide is administered to immunologically compromised individuals for the purpose of stimulating a systemic defense. The monoclonal antibodies of MYD 1 18 can be prepared using any technique that provides for the production of antibody molecules through continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehier and Milstein (1975 Nature 256: 495-497), the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol.; Cote et al. (1983) Proc. Nati. Acad. Sci. 80: 2026-2030) and the EBV hybridoma technique (Colé et al. (1985) Monoclonal Antibodies and Cancer Therapy.) Alan R. Liss Inc. P. 77- 96). In addition, techniques developed for the production of "chimeric antibodies", the binding of mouse antibody genes to human antibody genes can be used to obtain a molecule with specific antigen-specific character and biological activity (Morrison et al. (1984) Proc. Nati, Acad. Sci. 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314-452-454). Alternatively, the techniques described for the production of single chain antibodies (Patent of US Pat. No. 4,946,778) can be adapted to produce specific single chain antibodies. Antibodies can also be produced by inducing m. live the production in the lymphocyte population or by sorting collections of recombinant immunoglobulin or panels of highly specific binding reagents as described by Orlandi R et al. (1989, Proc. Nati Acad. Sci. 86: 3833-3837), and Winter G and M ilstein C (1991; Nature 349: 293-299). Antibody fragments containing specific binding sites for MYD 1 18 can also be generated. For example, such fragments include, but are not limited to, fragments of F (ab ') 2, which can be produced through pepsin digestion of the antibody molecule and Fab fragments, which can be generated by reducing the disulfide bridges of the F (ab ') 2 fragments. Alternatively, Fab expression libraries can be constructed to allow a rapid and easy identification of monoclonal Fab fragments with the desired specific character (Huse WD et al. (1989) Science 256: 1275-1281). The MYD118-specific antibodies are useful for the diagnosis of conditions and diseases associated with the expression of the MYD1 polypeptide 18. A variety of protocols for competitive binding or immunoradiometric assays of either polyclonal or monoclonal antibodies with specific characters are well known in the art. established. Such immunoassays typically involve complex formation between MYD1 polypeptides 18 and their specific antibody (or similar binding molecule MYD1 18) and the measurement of complex formation. A monoclonal-based immunoassay is preferred, from two sites, using two epitope monoclonal antibodies without interference in a specific MYD 1 18 protein, but a binding assay can also be employed. These assays are described by Maddox (supra).

Diagnostic Assays Using Specific Antibodies MYD1 18 Anti-MYD 1 18 antibodies are useful for the diagnosis of myeloproliferative disease or leukemias or other conditions, disorders or diseases characterized by the abnormal expression of MYD118. Diagnostic assays for MYD1 18 include methods that use the antibody and a label to detect MYD1 polypeptide 18 in fluids of a human body, cells, tissues or sections or extracts of said tissues. The polypeptides and antibodies of the present invention can be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of report molecules is known to those skilled in the art. A variety of protocols for measuring MYD 1 18 polypeptide, using either polyclonal or monoclonal antibodies specific to the respective protein, is known in the art. Examples include assay with protein-linked immunosorbent (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay is preferred using monoclonal antibodies reactive to two epitopes without interference to MYD1 polypeptide 18, but a competitive binding assay may be employed. These tests are described, inter alia, in Maddox (supra). In order to provide a basis for the diagnosis of a disease, normal or standard values for the expression of MYD 1 18 polypeptide should be established. This is achieved by combining body fluids or cell extracts taken from normal subjects, either animal or human, with antibody to MYD 1 polypeptide 18 under conditions suitable for complex formation, which are well known in the art. The amount of standard complex formation can be quantified by comparing it with a series of dilutions of positive controls wherein a known amount of antibody is combined with known concentrations of the purified MYD118 polypeptide. Then, the standard values obtained from normal samples can be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to MYD 1 polypeptide expression 18. The deviation between the standard and submitted values establishes the presence of the state of disease.

Drug Classification The MYD1 18 polypeptide, its immunogenic fragments or oligopeptides can be used to classify therapeutic compounds in any variety of drug classification techniques. The fragment employed in said test may be in solution, fixed to a solid support, carried on a cell surface, or located intracellularly. The abolition of the activity or the formation of binding complexes can be measured between the MYD1 polypeptide 18 and the agent being tested. Accordingly, the present invention provides a method for classifying a plurality of compounds for specific binding affinity with MYD1 18, or a portion thereof, comprising providing a plurality of compounds; combining MYD 1 18, or a portion thereof, with each of a plurality of compounds for a sufficient time to allow binding under stable conditions; and detecting the binding of MYD 1 18, or a portion thereof, to each of a plurality of compounds, thereby identifying the compounds that specifically bind MYD1 18. Another technique for drug classification provides a classification of high production compounds that have adequate affinity to the MYD 1 polypeptides and is described in detail by Geysen, European Patent Application 84/03564, published September 13, 1984, incorporated herein by reference. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or other surface. The peptide test compounds are reacted with fragments of MYD1 18 and washed. The bound MYD 18 is then detected by methods well known in the art. The purified MYD 1 18 can also be coated directly onto plates for use in the aforementioned drug classification techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. This invention also contemplates the use of drug classification assays, in which neutralizing antibodies capable of binding MYD 1 18 specifically compete with a test compound for the binding of MYD 1 18. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with MYD 1 18.

Uses of Polynucleotide myd 1 18 A polynucleotide myd1 18, or any part thereof, can be used for diagnostic and / or therapeutic purposes. For diagnostic purposes, the myd1 polynucleotide sequences 18 can be used to detect and quantify the expression of the gene in conditions, disorders or diseases where the activity of myd 1 18 may be involved, for example, in myeloproliferative diseases or leukemias, in where the expression of the myd 1 18 gene to be absent, aberrant or not detected. For therapeutic purposes, the myd 1 18 polynucleotide sequences can be administered to individuals with myeloproliferative disease or leukemia to induce terminal differentiation of immature hematopoietic proliferation cells, thus arresting cell proliferation, mitigating the symptoms of the disease and reversing the leukemic phenotype. Included within the scope of the invention are oligonucleotide sequences, antisense DNA and RNA molecules and ribozymes, which function to inhibit the translation of a myd 1 18. Said nucleotide sequences can be used in conditions where it may be preferable to block positive regulators of cell growth arrest, for example, in the ex vivo culture of cells intended for autologous transplantation to individuals lacking cells of the hematopoietic lineage, said individuals who are immunocompromised due to the disease, such as HIV infection. , or individuals undergoing chemotherapy or radiation therapy, who lack certain cells of hematopoietic lineage. Another aspect of the present invention is to provide nucleic acid hybridization or PCR probes, which are capable of detecting polynucleotide sequences, including genomic sequences, encoding MYD118 or closely related molecules, such as alleles. The specific character of the probe, that is, if it is derived from a region or domain highly conserved, conserved or not conserved, and the severity of the hybridization or amplification (high, intermediate or low), will determine if the probe only identifies myd 1 18 of natural existence, or related sequences. Probes for the detection of nucleic acid sequences are selected from conserved or highly conserved nucleotide regions of members of the MYD 1 family 18 and such probes can be used in a combination of degeneration probes. For the detection of identical nucleic acid sequences, or where a specific maximum character is desired, the nucleic acid probes are selected from the non-conserved nucleotide regions or unique regions of the polynucleotides myd 1 18. As used herein, the term "non-conserved nucleotide region" refers to a nucleotide region that is unique to myd 18 and does not occur in related family members, such as the nucleotide sequence encoding GADD45.

Diagnostic Uses of myd1 Polynucleotide 18 A polynucleotide sequence encoding myd118 can be used for the diagnosis of diseases resulting from the abnormal expression of myd1 18 or other genes associated with myeloproliferative disease or leukemia. For example, polynucleotide sequences encoding MYD1 18 can be used in hybridization or PCR assays of biopsy or autopsy tissues or biological fluids, such as serum, to detect abnormalities in the expression of myd 1 18. The form of such qualitative methods or quantitative may include Southern or Northern analysis, spot staining technologies or other membrane-based technologies; PCR technologies; level rod, pin or wafer technologies; and ELISA or other multiple sample format technologies. All these techniques are well known in the field, and, in fact, they are the basis of commercially available diagnostic equipment. Such trials can be designed to evaluate the efficacy of a particular therapeutic treatment regimen and can be used in animal studies., in clinical experiments or in the verification of the treatment of an individual patient. In order to provide a basis for diagnosis of a disease, a normal or standard profile must be established for the expression of myd 1 18. This is achieved by combining the body fluids or cell extracts taken from normal subjects, either animal or human, with myd1 18 or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization can be quantified by comparing the values obtained from normal subjects with a series of dilutions of positive controls that operate in the same experiment, where a known amount of myd118 purifies is used. The standard values obtained from normal samples can be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to the expression of myd118. The deviation between the standard values and subjects establishes the presence of the disease state. If a disease is established, an existing therapeutic agent is administered, and a treatment profile or values can be generated. Finally, the test can be repeated on a regular basis to assess whether the values progress toward or return to the normal or standard pattern. Successive treatment profiles can be used to show the effectiveness of the treatment over a period of several days or several months. PCR, as described in the patents of E.U.A. Nos. 4,683,195; 4,800,195; and 4,965,188, provides additional uses for oligonucleotides based on the sequence of myd118. Said oligomers are generally chemically synthesized, but can be generated enzymatically or produced from a recombinant source. The oligomers generally comprise two nucleotide sequences, one with sense orientation (5'-> 3 ') and one with antisense (3' < -5 '), used under optimal conditions for the identification of a specific gene or condition . The same two oligomers, nested groups of oligomers, or even a combination of degeneration of oligomers, may be employed under less stringent conditions for the detection and / or quantification of closely related DNA or RNA sequences. In addition, methods for quantifying the expression of a particular molecule include radiolabelling nucleotides (Melby PC et al 1993 J. Immunol Methods 159: 235-44) or biotinylation (Duplaa C et al 1993 Anal. Biochem. 229-36), coamplification of a control nucleic acid and standard curves on which the experimental results are interpolated. The quantification of multiple samples can be accelerated by performing the assay in an ELISA format, wherein the oligomer of interest is present in various dilutions and a spectrophotometric or colorimetric response gives a rapid quantification.

Therapeutic Uses of a myd1 Polynucleotide 18 A myd 1 18 polynucleotide sequence may provide the basis for the treatment of various abnormal conditions involving cells of abnormal proliferation of hematopoietic lineage, including myeloproliferative diseases and leukemias, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML); polycythemia vera (PV); agnogenic myeloid metaplasia with myelofibrosis (AM M / MF); and essential thrombocytosis (ET), where it may be advantageous to induce terminal differentiation of the proliferation cells thus stopping cell proliferation. Stable transformation of appropriate germ line cells, or a zygote, with a vector containing polynucleotide sequences encoding MYD118, can produce a transgenic organism (U.S. Patent No. 4,736,866, April 12, 1988) producing sufficient copies of the polynucleotide sequence to induce terminal differentiation of hematopoietic lineage cells, thus leading to the arrest of cell growth. Said vectors and expression systems intended for in vivo use or gene therapy must be designed for the expression of toxic molecules to the host cell as described infra. A preferred expression vector may be one that conducts the expression of myd118 at levels comparable to its expression in normal non-proliferating cells. Alternatively, a myd118 polynucleotide sequence may also provide the basis for the design of antisense molecules that are capable of inhibiting the transcription or translation of myd118 under conditions where it may be advantageous to block positive regulators of cell growth arrest, such as , for example, in the ex vivo culture of cells of a hematopoietic lineage intended for autologous transplantation, where the proliferation of hematopoietic lineage cells may be desirable. The introduction of vectors into stem cells taken from a patient and propagated clonally for autologous transplantation is described in the U.S. Patents. Nos. 5,399,493 and 5,437,994, described herein by reference.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or from several bacterial plasmids, can be used for the delivery of recombinant myd 1 18, sense or antisense molecules, to the target cell population. Methods that are well known to those skilled in the art can be used to construct recombinant vectors containing myd 1 18. See, for example, the techniques described by Maniatis et al. (Supra) and Ausubel et al. (Supra). Alternatively, the recombinant myd1 18 can be delivered to the target cells in liposomes. The full-length cDNA sequence and / or its regulatory elements allow the researchers to use myd1 18 as a tool in sense investigations (Youssoufian H and HF Lodish 1993 Mol.Cell.Biol.13: 98-104) or antisense ( Eguchi et al. (1991) Annu. Rev. Biochem 60: 631-652) of the gene function. Oligonucleotides, designed from the cDNA or control sequences obtained from genomic DNA, can be used in vitro or in vivo to inhibit expression. Such technology is now well known in the art, and sense and antisense oligonucleotides or larger fragments can be designed from various locations throughout the coding or control regions. Additionally, the expression of myd 1 18 can be modulated by transfecting a cell or tissue with expression vectors, which express high levels of a myd 1 18 fragment in conditions where it could preferably be to block the activity of the positive regulator of the arrest. of cell growth, such as in bone marrow transplantation therapy and in the ex vivo culture of cells intended for autologous transplantation. Such constructions can flood cells with sense or antisense sequences without translation. Even in the absence of integration towards DNA, said vectors can continue transcribing RNA molecules until all copies of the vector are incapacitated through endogenous nucleases. Such a passing expression can last a month or more with a vector without replication (Mettier I personal communication) and even more time if the elements of appropriate replication are part of the vector system. Modifications of gene expression can be obtained by designing antisense sequences for the control regions of the myd118 gene, such as promoters, enhancers and introns. Oligonucleotides derived from the transcription initiation site, for example, regions between -10 and +10 of the leader sequence, are preferred. Antisense DNA and RNA molecules can also be designed to block the translation of mRNA by preventing transcription of the ribosome binding. Similarly, inhibition can be achieved using Hogeboom's base pair methodology, also known as "triple helix" base pairs. Triple helix pairs compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of action of ribozyme involves sequence-specific hybridization of the ribozyme molecule to the complementary target RNA, followed by an endonucleolytic cleavage. Within the scope of the invention, ribozyme molecules of hammerhead motif are designed engineered that specifically and efficiently catalyze the endonucleolytic cleavage of myd 1 18 sequences. The specific ribozyme cleavage sites within any potential RNA target are initially identified exploring the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GU U and G UC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural aspects, which can make the oligonucleotide sequence inoperable. The suitability of candidate targets can also be evaluated by testing the ability to access hybridization with complementary oligonucleotides using ribonuclease protection assays. Both the RNA and antisense DNA molecules and the ribozymes of the invention can be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as the chemical synthesis of solid phase phosphoramidite. Alternatively, RNA molecules can be generated through in vitro or in vivo transcription of DNA sequences encoding the antisense RNA molecule. Said DNA sequences can be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, antisense cDNA constructs that constitutively or inducibly synthesize antisense RNA can be introduced into cell lines, cells or tissues. DNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences to 5 'and / or 3' ends of the molecule or the use of phosphorothioate or 2 'O-methyl instead of phosphodiesterase bonds within the structure of the molecule. Methods for introducing vectors into cells or tissue include those methods discussed infra. In addition, several of these transformation or transfection methods are equally suitable for ex vivo therapy. In addition, the myd118 polynucleotide sequences described herein may be used in molecular biology techniques that have not yet been developed, provided that the new techniques are based on properties of nucleotide sequences that are currently known, including but not limited to , such properties as the triplet genetic code and specific base pair interactions.

Detection and Plotting of Polynucleotide Sequences with Relation to myd118 The nucleic acid sequence for myd118 can also be used to generate hybridization probes as previously described, to plot the endogenous genomic sequence. The sequence can be mapped to a particular chromosome or to a specific region of the chromosome using well-known techniques. These include in situ hybridization to chromosomal extensions (Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York), chromosomal preparations classified by flow, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes ( BACs), bacterial P1 constructs or collections of individual chromosome cDNAs. In situ hybridization of chromosomal preparations and physical tracing techniques such as ligature analysis using established chromosomal markers is inevitable in extension genetic maps. Examples of genetic maps can be found in Science (1995; 270: 410f and 1994; 265: 1981f). Typically, placement of the gene on the chromosome of another mammalian species may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be designed for chromosomal arms, or parts thereof, through physical tracing. This provides valuable information to researchers looking for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome, such as ataxia telangestasia (AT), has been crudely localized through genetic ligation to a particular genomic region, for example, AT a 1 1 q22-23 (Gatti et al. (1988) Nature 336 : 577-580), any sequence of tracing to that area may represent associated or regulatory genes for further investigation. The nucleotide sequence of the present invention can also be used to detect differences in chromosomal location due to translocation, inversion, etc. , between normal individuals, carriers or affected.

Pharmaceutical Compositions The present invention relates to pharmaceutical compositions, which may comprise all or portions of myd 1 18 polynucleotide sequences, MYD 1 18 polypeptides or MYD 1 18 bioactivity antagonists, including antibody, alone or in combination with at least another agent, such as a stabilization compound, and can be administered in any sterile, biocompatible pharmaceutical vehicle, including, but not limited to, pH regulated saline, dextrose, and water. The myd 1 18 nucleotide and MYD 1 18 amino acid sequences can be administered to a patient alone or in combination with other nucleotide sequences, such as nucleotide sequences encoding tumor suppressor genes, such as p53, p16 and p21, drugs or hormones or in pharmaceutical compositions where it is mixed with excipients or other pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert. A preferred route of administration for the treatment of myeloproliferative diseases or leukemias could be intravenous delivery, while a preferred route of administration for the treatment of conditions related to inflammation could be local administration at the site of inflammation, such as the affected joint. in rheumatoid arthritis. The myd 1 18 polynucleotide sequences or MYD 1 18 amino acid sequences can be administered alone to individuals subjected to myeloproliferative diseases or leukemias or in combination with other types of agents or therapy including chemotherapy, radiation therapy or cell transplantation therapy. stem. Depending on the condition being treated, these pharmaceutical compositions can be formulated and administered systemically or locally. The techniques for formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton PA). Suitable routes may include, for example, oral or transmucosal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal or intranasal administration. For injection, the pharmaceutical compositions of the invention can be formulated in aqueous solutions, preferably in physiologically compatible pH regulators such as Hank's solution, Ringer's solution, or physiologically regulated salt at their pH. For administration to the tissue or cell, suitable penetration agents are used in the formulation for the particular barrier to be penetrated. Such penetrating agents are generally known in the art. The pharmaceutical compositions can be formulated using pharmaceutically acceptable carriers well known in the art, in doses suitable for oral administration. Said vehicles allow the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. For example, an effective amount of MYD 18 may be that amount that mitigates the symptoms of anemia, fatigue, splenomegaly, hypermetabolism and thromboemorrhagic complications. The determination of effective amounts is within the ability of those skilled in the art, especially in light of the description provided below. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate the processing of the active compounds to preparations that can be used pharmaceutically. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules or solutions. The pharmaceutical compositions of the present invention can be manufactured in a form that is known per se, for example, by conventional methods such as mixing, dissolving, granulating, dragee-making, spraying, emulsifying, encapsulating, entrapping or lyophilizing. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Optionally, the suspension may also contain stabilizers or agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. Pharmaceutical preparations for oral use can be obtained by combining the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, then adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch of corn, wheat, rice, potatoes, etc .; cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabica and tragacanth; and proteins such as gelatin and collagen. If desired, disintegration or solubilization agents can be added, such as the interlaced polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. The dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium oxide, lacquer solutions and suitable organic solvents or mixtures of solvent. Dye stuff or pigments can be added to the tablet or dragee coatings for product identification or to characterize the amount of the active compound, i.e., the dose.

Pharmaceutical preparations, which can be used orally, include soft-fit capsules made of gelatin, as well as soft, sealed gelatin capsules and a coating such as glycerol or sorbitol. The soft-fit capsules may contain the active ingredients blended with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or polyethylene glycol with or without stabilizers. Compositions comprising a compound of the invention formulated in an acceptable pharmaceutical carrier can be prepared, placed in an appropriate container and labeled for the treatment of an indicated condition. For polynucleotide or amino acid sequences of MYD1 18, the indicated conditions on the label may include the treatment of myeloproliferative diseases or leukemias. The pharmaceutical compositions can be provided as a salt and can be formed with many acids, including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. The salts tend to be more soluble in aqueous solvents or other protonic solvents which are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1% -2% sucrose, 2% -7% mannitol at a pH scale of 4.5 to 5.5 which is combined with the pH regulator before use. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from the cell culture assays. Then, preferably, the dose can be formulated in animal models (particularly murine models) to achieve a desirable circulation concentration scale that adjusts the levels of MYD 1 18. A therapeutically effective dose refers to that amount of MYD1 18, which mitigates the symptoms of the disease state. The toxicity and therapeutic efficacy of said compounds can be determined through normal pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (lethal dose at 50% of the population) and ED50 (therapeutically effective dose). in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and can be expressed as the LD50 / ED50 ratio. Preferred are compounds that exhibit high therapeutic indices. The data obtained from these cell culture assays and additional animal studies can be used in the formulation of a dose scale for human use. The dose of said compounds preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dose varies within this scale depending on the dosage form used, patient sensitivity, and the route of administration. The exact dose is chosen by the individual doctor in view of the patient who will be treated. The dose and administration are adjusted to provide sufficient levels of the active portion or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease status; age, weight and gender of the patient; diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to therapy. The long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and the rate of removal of the particular formulation. The normal dose amounts may range from 0.1 to 100,000 micrograms, up to a total dose of approximately 1 g, depending on the route of administration. A guide as to the doses and particular methods of supply is provided in the literature. See the patents of E. U.A. Nos. 4,657,760; 5,206, 344; or 5,225,212. Those skilled in the art will employ different formulations for MYD1 18 than for inhibitors of MYD1 18. Administration to the bone marrow may need delivery in a form other than intravenous injections. These examples are provided by way of illustration and are not included for the purpose of limiting the invention.

INDUSTRIAL APPLICABILITY I Construction of the ADNC Collection SPLNFET01 and Isolation of DNA Clones The human spleen cell cDNA library (SPLNFET01) was constructed as usual by Stratagene. The tissue was obtained from combined fetal spleens from different sources and containing many different types of cells. Poly (A +) RNA (mRNA) was purified, and the cDNA was synthesized from the mRNA. The synthetic adapter oligonucleotides were ligated into the cDNA ends allowing their insertion into the Uni-ZAP ™ vector system (Stratagene), allowing a high-efficiency unidirectional lambda collection construction (sense orientation) and the convenience of a plasmid with blue / white color selection to detect clones with cDNA inserts. Alternative unidirectional vectors are pcDNAl (Invitrogen, San Diego CA) and pSHIox-1 (Novagen, Madison, Wl). The custom-built collection phage particles were transfected into the host strain of E ^ coli XL1-Blue (Stratagene), which has a high transformation efficiency, increasing the probability of obtaining rare clones, of low representation in the collection of CDNA The phagemid forms of individual cDNA clones were obtained through the in vivo excision procedure, in which the host bacterial strain was coinfected with both the lambda collection phage and an auxiliary phage f1. Proteins derived from both the phage containing the collection and the auxiliary phage cut out the lambda DNA, initiated new DNA synthesis from the sequences defined in the lambda target DNA and created a circular chain phagemid DNA molecule. individual, smaller, which included all the DNA sequences of the plasmid pBluescript ™ and the cDNA insert. The phagemid DNA was secreted from the cells and purified, then used to re-infect fresh host cells, where the phagemid DNA of double-stranded structure was produced. Since the phagemid carries the β-lactamase pair gene, newly transformed bacteria were selected in a medium containing ampicillin. The phagemid DNA was purified using the QIAwell-8 Plasm id, QIAwell PLUS or QIAwell ULTRA DNA purification system (QIAGEN Inc. Chatsworth CA). This product line provides a convenient, fast and reliable high production method for lysing bacterial cells and isolating highly isolated phagemid DNA using QIAGEN anion exchange resin particles with EMPORE ™ membrane technology (3M, Minneapolis MN) in a Multiple cavities format. The DNA was eluted from the purification resin already prepared for DNA sequencing and other analytical manipulations. The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975; J. Mol. Biol. 94: 441 f), using the Catalyst 800 and 373 DAN Sequencing System (Catalyst 800 and DNA Sequencing System 373) (Perkin Elmer) II Homology Search of cDNA Clones and their Deducted Proteins Each cDNA was compared to sequences in GenBank using a search algorithm incorporated into the 670 ABI IN HERIT ™ Sequence Analysis System (Perkin Elmer). In this algorithm, Pattern Specification Language (TRW Inc., Los Angeles CA) was used to determine regions of homology. The three parameters that determine how sequence operations operate when there is window size, window deviation and error tolerance. Using a combination of these three parameters, the A DN database was searched for sequences containing regions of homology to the inquiry sequence, and the appropriate sequences were classified with an initial value. Subsequently, these homologous regions were examined using dot matrix homology graphs to distinguish regions of homology from opportunity comparisons. The Smith-Waterman alignments were used to display the results of the homology search. BLAST was used, which means Basic Local To the ignition Search Tool (Basic Local Alignment Search Tool) (Altsch ul SF (1993) J. Mol.Evol.36: 290-300; Altsch ul, SF (1990) J. Mol. Biol. 215: 403-10), to look for local sequence alignments. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of the alignments, BLAST is especially useful for determining exact comparisons or identifying homologs. BLAST is useful for comparing which ones do not contain gaps. The fundamental unit of the BLAST algorithm output is the High Class Segment Par (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment classification satisfies or exceeds a threshold or cutoff rating set by the user. The BLAST aspect is to search for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any comparison found, and to report only those comparisons that meet the threshold of importance selected by the user. Parameter E sets the threshold significantly important for reporting database sequence comparisons. E is interpreted as the superior union of the expected frequency of occurrence of opportunity of an HSP (or group of HSPs) within the context of the entire database search. Any database sequence whose comparison satisfies E is reported in the output of the program. MYD1 18 was identified using the ABI I N H ER IT ™ DNA Analysis System software (Perkin Elmer), which identified clone 25214 as being related to human GADD45 and murine MYD118. PCR extension analysis was performed to determine the 5 'coding region. The nucleotide sequence for clone 25214 was subjected to a confirmatory DNA sequence analysis. The polynucleotide sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of MYD118 are described herein in Figure 1.

III Determination of the Reading Framework of the cDNA Clone The reading frame of individual cDNA clones obtained from the SPLNFET01 collection was obtained by analyzing the polynucleotide sequences for the presence of start codons (ATG, GTG, etc.) and arrest ( TGA, TAA, TAG). Typically, one frame will continue through the main portion of an entire cDNA sequence and two other pending frames contain numerous stop codons. Algorithms have been developed to determine the reading frame, which analyze the occurrence of individual nucleotide bases of each putative codon triplet (eg, Fickett, JW (1982) Nucleic Acids Res. 10: 5303-18). Coding DNA tends to contain predominantly certain nucleotides within certain triplet periodicities, such as a significant preference for pyrimidines at the third codon position. These algorithms have been incorporated into widely available software and can be easily used to determine the coding potential (and frame) of a given stretch of DNA. This information derived from algorithm, combined with start / stop codon information, was used to determine the appropriate frame of individual clones within the SPLNFET01 collection with a high degree of certainty, thus allowing the correct alignment of the reading frame with vehicles of appropriate expression.

IV Extension of myd1 18 to Retrieve Regulatory Elements The nucleic acid sequence of myd 1 18 can be used to design oligonucleotide primers to obtain full length sequences from genomic collections. One initiator is synthesized to initiate the extension in the antisense direction (XLR) and the other is synthesized to extend the sequence in the direction of sense (XLF). The primers allow the known myd 1 18 sequence to be extended "out" by generating amplicons containing a new, unknown nucleotide sequence for the control region of interest. The initial primers are designed from the cDNA using Oligo 4.0 (National Biosciences Inc. Plymouth M N), or another suitable program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to bind to the target sequence at temperatures of approximately 68o-72 ° C. Any stretching of nucleotides that could result in heparin structures and initiator-initiator dimerizations is avoided.

A human genomic library was used to extend and amplify the 5 'upstream sequence. If necessary, design a second group of initiators to further extend the known region. Following the instructions for the XL-PCR equipment (Perkin Elmer) and thoroughly mixing the enzyme and the reaction mixture, a high fidelity amplification is obtained. Starting with 40 pmol of each primer and the recombinant concentrations of all the other components of the kit, PCR was carried out using the Peltier Thermal Cycler (PTC200, MJ Research, Watertown MA) and the following parameters: Step 1 94 ° C during 1 minute (initial denaturation) Step 2 65 ° C for 1 minute Step 3 68 ° C for 6 minutes Step 4 94 ° C for 15 seconds Step 5 65 ° C for 1 minute Step 6 68 ° C for 7 minutes Step 7 Repeat steps 4-6 for 15 additional cycles Step 8 94 ° C for 15 seconds Step 9 65 ° C for 1 minute Step 10 68 ° C for 7:15 minutes Step 11 Repeat steps 8-10 for 12 cycles Step 12 72 ° C for 8 minutes Step 13 4 ° C (and keep) An aliquot of 5-10 μl of the reaction mixture was analyzed through electrophoresis at a low concentration (approximately 0.6-0.8%) of agarose mini-gel to determine which reactions were successful in extending the sequence. Larger products or bands were selected and cut out of the gel. Another purification involves using a commercial gel extraction method such as QIAQuick ™ (QIAGEN Inc). After DNA recovery, the Klenow enzyme was used to cut nucleotide protrusions, of individual chain structure creating shaved ends that facilitate religation and cloning. After precipitation with ethanol, the products were redissolved in 13 μl of ligation pH buffer, 1 μl of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2-3 hours or overnight at 16 ° C. Competent E. coli cells (in 40 μl of appropriate medium) were transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium (Sambrook J and gold, supra). After incubation for one hour at 37 ° C, the whole transformation mixture was plated on Luria Bertani (LB) agar (Sambrook J et al., Supra) containing 2x Carb. The next day, several colonies from each plate were randomly collected and cultured in 150 μl of LB / 2x Carb liquid medium placed in a single well of a sterile, commercially available, appropriate 96-well microtiter plate. On the next day 5 μl of each overnight culture was transferred to a non-sterile 96-well plate and after a 1: 10 dilution with water, 5 μl of each was transferred to a PCR setup. For PCR amplification, 18 μl of the concentrated PCR reaction mixture (3.3x) containing 4 units of rTth DNA polymerase, was added, to each well, a vector primer and one or both gene-specific primers used for the assay. extension reaction. The amplification was carried out using the following conditions: Step 1 94 ° C for 60 seconds Step 2 94 ° C for 20 seconds Step 3 55 ° C for 30 seconds Step 4 72 ° C for 90 seconds Step 5 Repeat steps 2-4 for 29 additional cycles Step 6 72 ° C for 180 seconds Step 7 4 ° C (and maintain) The aliquots of the PCR reactions were operated on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and appropriate clones were selected, ligated to a plasmid and sequenced.

V Labeling of Hybridization Probes Hybridization probes derived from SEC I D NO: 1 can be used to classify cDNAs, A R Nms or genomic DNAs.

Although the labeling of oligonucleotides, consisting of approximately 20 base pairs, is specifically described, essentially the same procedure can be used with larger cDNA fragments. Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 mCi of [g-33P] adenosine triphosphate (Amersham, Chicago IL) and T4 polynucleotide kinase (DuPont NEN®, Boston MA). The labeled oligonucleotides are purified with a superfine resin column of Sephadex G-25 (Pharmacia). A portion containing 107 counts per minute of each was used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bg 1 II, EcoR I, Pst I, Xba I, or Pvu II; DuPont NEN®). The DNA of each digestion is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham NH). Hybridization was carried out for 16 hours at 40 ° C. To remove nonspecific signals, the stains were sequentially washed at room temperature unextremely severe conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After exposing the XOMAT AR ™ film (Kodak, Rochester NY) to stains in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale CA) for several hours, the hybridization patterns were compared visually.

VI Antisense Molecules The myd118 sequence, or a part thereof, can be used to inhibit the expression of endogenous myd118 in vivo or in vitro. Although the use of antisense oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure can be used with larger cDNA fragments. An oligonucleotide based on the coding sequence of myd118 can be used to inhibit the expression of endogenous myd118. Using Oligo 4.0, the complementary oligonucleotide can be designed from the 5 'sequence conserved and used either to inhibit transcription by preventing the binding of the promoter to the upstream untranslated sequence or the translation of a myd118 transcript by avoiding the ribosome of the binding to mRNA.

VII Production of Specific Antibodies MYD118 For the production of polyclonal antibodies, the amino acid sequence deduced from MYD118 is analyzed using the DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity and a corresponding oligopeptide in synthesized and used to increase the antibodies in rabbits. . The analysis for selecting suitable epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, is described by Ausubel FM et al. (Supra). An oligopeptide of approximately 15 residues in length is synthesized using an ABI Model 431A Peptide Synthesizer (Perkin Elmer, Norwalk, CN) using fmoc chemistry, and coupled to boundary limpet hemocyanin (KLH, Sigma) through the reaction with M-maleimidobenzoyl-Nh-idroxysuccinimide ester (MBS; Ausubel FM et al., supra). Rabbits were immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. The resulting antisera were tested for the antipeptide activity, for example, by binding the peptide to the plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with goat anti-rabbit IgG, radioiodinated.

VIII Purification of MYD1 18 Utilizing Specific Antibodies Endogenous or recombinant MYD1 18 can be purified through immunoaffinity chromatography using antibodies specific for MYD 1 1 8. An immunoassay column was constructed by covalently coupling a MYD antibody 1 1 8 to an activated chromatographic resin such as Sepharose activated with Cn-Br (Pharmacia Biotech). After coupling, the resin was blocked and washed according to the manufacturer's instructions. A medium containing the MYD 1 1 8 was passed over the affinity cell, and the column was washed under conditions which allow the preferential absorbance of MYD 1 1 8 (eg p H regulators of high ionic strength in the presence of detergent) . Column was eluted under conditions that break the binding of the antibody / MYD 18 (for example, a pH regulator with a pH of 2-3 or a high concentration of cariotrope such as urea or thiocyanate ion), and was collected the MYD1 18.

IX Identification of Molecules Interacting with MYD1 18 MYD 1 18, or biologically active fragments thereof, are labeled with the Bolton-Hunter 125I reagent (Bolton, AE and Hunter, WM (1973) Biochem J. 133: 529). Small candidate molecules previously disposed in the cavities of a 96-well plate were incubated with the marked MYD 1 18, washed and the wells analyzed with the marked MYD1 18 complex. The data obtained using different concentrations of MYD 1 18 were used to calculate the values for the number, affinity and association of mYD1 18 with the candidate molecules. All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and system of the invention described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described together with the preferred specific embodiments, it should be understood that the invention as claimed should not be unduly limited to said specific modalities. Rather, various modifications of the modes described to carry out the invention, which are obvious to those skilled in molecular biology or related fields, are intended to be within the scope of the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: INCYTE PHARMACEUTICALS, INC. (ii) TITLE OF THE INVENTION: RESPONSE GENE OF HUMAN NOVELTY HONEY TERMINAL DIFFERENTIATION (iii) NUMBER OF SEQUENCES: 4 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Incyte Pharmaceuticals, Inc. (B) STREET: 3174 Porter Drive (C) CITY. Palo Alto (D) STATE: CA (E) COUNTRY: E.U.A. (F) ZONE: 94303 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: TWO (D) SOFTWARE: FastSEC Version 1.5 (vi) DATA CURRENT APPLICATION: (A) PCT APPLICATION NO .: will be assigned (B) SUBMISSION DATE, filed with it (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION SERIAL NUMBER: US 08 / 221,531 (B) DATE OF SUBMISSION: February 2, 1994 (vii) PREVIOUS APPLICATION DATA:; (A) SERIAL NUMBER OF APPLICATION: US 08 / 602,208 (B) DATE OF SUBMISSION: February 15, 1996 (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: Billings, Lucy J. (B) No. REGISTRATION: 36,749 (C) REFERENCE / PENALTY NUMBER: PF-0054 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-845-4166 (C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 745 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) COLLECTION: Myeloid terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CGGACTACCG TTGGTTTCCG CAACTTCTTG GATTATCCTC GCCAAGGACT TTGNAATATA 60 l I l II CCGCC TTTTCTGGAA GGATTTCGCT GCTTCCCGAA GGTCTTGGAC GAGCGCTCTA 120 GCTCTGTGGG AAGGTTTNGG GCTCTCTGGC TCGGATTTTG GAATTTCTCC CTGGGGACTCC 180 CCGTGGAGCC GCATCCACTG TGGATTATAA TTGCAACATG ACGCTGGAAG AGCTCGTGGC 240 GTGCGACAAC GCGGCGCAGA AGATGCAGAC GGTGACCGCC GCGGTGGAGG AGCTTTTGGT 300 GGCCGCTCAG CGCCAGGATC GCCTCACAGT GGGGGTGTAC GAGTCGGCCA AGTTGATGAA 360 TGTGGACCCA GACAGCGTGG TCCTCTGCCT CTTGGCCATT AACGAGGAGG AGGAGGATGA 420 CATCGCCCTG CAAATCCACT TCACGCTCAT CCAGTCCTTC TCCTGTAACA ACGACATCAA 480 CATCGTGCGG GTTTCGGGCA TGCAGCGCCT GGCGCAGCTC CTGGGAGAGC CGGCCGAGAC 540 CCAGGGCACC ACCGAGGCCC GAGACCTGCA TTGTCTCCTG GTCACGAACC CTCACACGGA 600 CGCCCGGAAG AGCCACGGCT TGGTGGAGGT GGCCAGCTAC TGCGAAGAAA GCCGGGGCAA 660 CAACCAGTGG GTCCCCTACA TCTCTCTTCA GGAACGCTGA GGCCTTCCCA GCAGCAGAAT 720 CTGTTTGAGT TGCTGCCACA ACCAA 745 (2) IN FORMATION FOR SEC ID NO: 2: (i) SECU E NCE CHARACTERISTICS: (A) LONGITU D: 160 base pairs (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: peptide (vii) FU ENTE IN MEDIATA: (A) COLLECTION: Myeloid terminal (xi) DESCR ICECTION OF SEQUENCE: SEQ ID NO: 2: Met Thr Leu Glu Glu Leu Val Wing Cys Asp Asn Wing Wing Gln Lys Met 1 5 10 15 Gln Thr Val Thr Ala Wing Val Glu Glu Leu Leu Val Wing Wing Gln Arg 20 25 30 Gln Asp Arg Leu Thr Val Gly Val Tyr Glu Ser Wing Lys Leu Met Asn 35 40 45 Val Asp Pro Asp Ser Val Val Leu Cys Leu Leu Ala lie Asn Glu Glu 50 55 60 Glu Glu Asp Asp lie Ala Leu Gln lie His Phe Thr Leu He Gln Ser 65 70 75 80 Phe Ser Cys Asn Asn Asp He Asn He Val Arg Val Ser Gly Met Gln 85 90 95 Arg Leu Wing Gln Leu Leu Gly Glu Pro Wing Glu Thr Gln Gly Thr Thr 100 105 110 Glu Wing Arg Asp Leu His Cys Leu Leu Val Thr Asn Pro His Thr Asp 115 120 125 Ala Arg Lys Ser His Gly Leu Val Glu Val Ala Ser Tyr Cys Glu Glu 130 135 140 Ser Arg Gly Asn Asn Gln Trp Val Pro Tyr He Ser Leu Gln Glu Arg 145 150 155 160 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 165 base pairs (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vii) IMMEDIATE SOURCE. (A) COLLECTION: Myeloid terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Thr Leu Glu Glu Phe Be Wing Gly Glu Gln Lys Thr Glu Arg Met 1 5 10 15 Asp Lys Val Gly Asp Ala Leu Glu Glu Val Leu Ser Lys Ala Leu Ser 20 25 30 Gln Arg Thr He Thr Val Gly Val Tyr Glu Ala Wing Lys Leu Leu Asn 40 45 Val Asp Pro Asp Asn Val Val Leu Cys Leu Leu Ala Wing Asp Glu Asp 50 55 60 Asp Asp Arg Asp Val Wing Leu Gln He His Phe Thr Leu He Gln Wing 65 70 75 80 Phe Cys Cys Glu Asn Asp He Asn He Leu Arg Val Ser Asn Pro Gly 85 90 95 Arg Leu Wing Glu Leu Leu Leu Glu Thr Asp Wing Gly Pro Wing Wing 100 105 110 Ser Glu Gly Wing Glu Gln Pro Pro Asp Leu His Cys Val Leu Val Thr 115 120 125 Asn Pro His Ser Ser Gln Trp Lys Asp Pro Wing Leu Ser Gln Leu He 130 135 140 Cys Phe Cys Arg Glu Ser Arg Tyr Met Asp Gln Trp Val Pro Val He 145 150 155 160 Asn Leu Pro Glu Arg 165 (2) IN FORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LONGITU D: 160 base pairs (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLÉCU LA: peptide (vii) SOURCE INM EDIATA: (A) COLLECTION: Myeloid terminal (xi) DESCR I PTION OF SEC EU NC IA: SEQ ID NO: 4: Met Thr Leu Glu Glu Leu Val Wing Ser Asp Asn Wing Val Gln Lys Met 1 5 10 15 Gln Ala Val Thr Ala Ala Val Glu Gln Leu Leu Val Ala Ala Gln Arg 20 25 30 Gln Asp Arg Leu Thr Val Gly Val Tyr Glu Ala Wing Lys Leu Met Asn 40 45 Val Asp Pro Asp Ser Val Val Leu Cys Leu Leu Ala He Asp Glu Glu 50 55 60 Glu Glu Asp Asp He Ala Leu Gln He His Phe Thr Leu He Gln Ser 65 70 75 80 Phe Cys Cys Asp Asn Asp He Asp He Val Arg Val Ser Gly Met Gln 85 90 95 Arg Leu Wing Gln Leu Leu Gly Glu Pro Wing Glu Thr Leu Gly Thr Thr 100 105 110 Glu Wing Arg Asp Leu His Cys Leu Leu Val Thr Asn Cys His Thr Asp 115 120 125 Ser Trp Lys Ser Gln Gly Leu Val Glu Val Ala Ser Tyr Cys Glu Glu 130 135 140 Ser Arg Gly Asn Asn Gln Trp Val Pro Tyr He Ser Leu Glu Glu Arg 145 150 155 160

Claims (20)

1. - A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence shown in SEQ ID NO: 2.

2. The purified p-nucleotide according to claim 1, wherein the nucleic acid sequence comprises the sequence shown in SEQ ID NO: 1.

3. An antisense molecule comprising the complement of the polynucleotide of claim 1 or a portion thereof.

4 - An expression vector comprising the polynucleotide of claim 1.

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

6 - A method for producing the polypeptide having the amino acid sequence for MYD118 (SEC ID NO: 2), said method comprising the steps of: a) culturing the host cell of claim 5 under conditions suitable for the expression of said polypeptide, and b) recovering said polypeptide from the host cell culture. 7 -.

7 - A purified polypeptide comprising the amino acid sequence for MYD118 as shown in SEQ ID NO: 2.

8 - A diagnostic composition for the detection of myd118 polynucleotide sequences comprising the polynucleotide of claim 1.

9. - A diagnostic test for the detection of myd118 polynucleotide sequences in a biological sample, comprising the steps of: a) combining the biological sample with a first nucleotide sequence of SEQ ID NO: 1, or a non-conserved fragment thereof , under conditions suitable for the formation of a nucleic acid hybridization complex, b) detecting said hybridization complex, wherein the presence of said complex correlates with the presence of a second nucleotide sequence comprising the polypeptide sequences in the sample biological, and c) comparing the amount of the second nucleotide sequence in said sample with a standard one determined thus if the amount of said second nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said second nucleotide sequence is correlates positively with a myeloproliferative disease.

10. A diagnostic test for the detection of myd 1 18 polypeptide sequences in a biological sample, comprising the steps of: a) combining the biological sample with the polymerase chain reaction primer under conditions suitable for the amplification of nucleic acid, wherein said primers comprise non-conserved fragments of the nucleotide sequence of SEQ ID NO: 1, b) detect amplified nucleotide sequences, and c) compare the amount of amplified nucleotide sequences in said biological sample to a standard thereby determining if the amount of said nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said nucleotide sequence is positively correlated with a myeloproliferative disease.

11. A method for classifying a plurality of compounds for specific binding affinity with the polypeptide of claim 7 or a portion thereof, comprising the steps of: a) providing a plurality of compounds; b) combining the polypeptide of claim 7 with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions; and c) detecting the binding of said polypeptide of claim 7 to each of the plurality of compounds, thereby identifying the compounds that specifically bind said polypeptide of claim 7.

12.- A method for inducing terminal differentiation of proliferating myeloid cells human which comprises administering an effective amount of the polypeptide of claim 1 to said myeloid cells under conditions suitable for the expression of MYD118 (SEQ ID NO: 2).

13 - A method for inducing the terminal differentiation of human proliferating myeloid cells comprising administering an effective amount of the polypeptide of claim 7 to said myeloid cells.

14. A method for inhibiting the expression of MYD118 in human hematopoietic cells which comprises administering a effective amount of the antisense molecule of claim 3 to said cells.

15 - The method according to claim 14, where the hematopoietic cells are in vitro.

16. A pharmaceutical composition for treating an individual having a myeloproliferative disease comprising administering an effective amount of the polynucleotide of claim 1, or a biologically active fragment thereof.

17 - A method for treating an individual having a myeloproliferative disease comprising administering an effective amount of the pharmaceutical composition of claim 16 to said individual.

18. A pharmaceutical composition for treating an individual having a myeloproliferative disease comprising an effective amount of the polypeptide of claim 7, or a biologically active fragment thereof.

19 - A method for treating an individual having a myeloproliferative disease comprising administering an effective amount of a pharmaceutical composition of claim 18 to said individual.

20. - An antibody specific for the polypeptide of claim 7.