Definitions

• the present invention relates to nucleic acid and amino acid sequences of a novel human 10 myeloid terminal differentiation response gene found in cDNA libraries 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.
• Animal cells respond to differentiation signals which turn on or off the appropriate genes resulting in conversion of proliferating, undifferentiated cells into nonproliferating, highly specialized, differentiated cells.
• An example of this process is the differentiation of myeloid precursor cells into mature granulocytes and macrophages. Blocks in the differentiation process
• MyD genes function as positive regulators of terminal hematopoietic cell differentiation, which is associated with inhibition of cell growth and 0 apoptosis.
• Selvakumaran et al. supra provide evidence that MyD family member, murine YD118, described as a terminal differentiation response gene, is expressed in M1D+ myeloid precursor cells following induction of terminal differentiation and growth arrest by IL6 and has been shown to be a positive regulator of apoptosis induced by TGF ⁇ l .
• 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 nude mice.
• MyD genes specifically MyD21 , MyD42 and MyD63
• MyD63 function as positive regulators of hematopoietic cell differentiation
• the cDNA sequence and deduced amino acid sequence of murine MYD1 18 is disclosed in Abdollahi A et al. (1991 Oncogene 6:165-17) who indicate that the cDNA nucleotide sequence of murine MYD1 18 predicts a protein of 160 amino acids, which does not contain protein secretory signals, transmembrane domains or known protein-DNA binding motifs, but does appear to contain a protein kinase phosphorylation site at position 204, two casein kinase II phosphorylation sites at positions 215 and 231 and several AT 3 motifs in its 3' untranslated region.
• MYD1 18 RNA in myeloid precursor enriched murine bone marrow, but not in several other non-myeloid murine tissues, such as liver or brain.
• Abdollahi et al. also observed that MYD1 18 expression was induced in the absence of protein synthesis, following stimulation of M1 D+ cells by IL-1, LPS and leukemia inhibitory factor (LIF).
• IL-1 myeloid precursor enriched murine bone marrow
• LPS leukemia inhibitory factor
• the amino acid sequence for murine MYD1 18 is 75% similar (57% identical) to the amino acid sequence for murine GADD gene, GADD45, which is regulated in part by the tumor suppressor gene p53 (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 growth of hematopoietic cell lineages.
• GenBank 92 cDNA libraries made from tissue from hippocampus (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), human white blood cells (T33963), human brain (T35368), human pancreas (T29941), liver (T40088), prostate gland (T35225) and lung (T35563) .
• the complete nucleotide sequence encoding human MYD1 18 has not been disclosed.
• Myeloproliferative diseases and leukemias are neoplasms of the hematopoietic stem cell 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 (AMM/MF); and essential thrombocytosis (ET).
• ALL acute lymphocytic leukemia
• AML acute myelogenous leukemia
• CLL chronic lymphocytic leukemia
• CML chronic myelogenous leukemia
• PV polycythemia vera
• ALM/MF agnogenic myeloid metaplasia with myelofibrosis
• ET essential thrombocytosis
• leukemic cells proliferate primarily in the bone marrow and lymphoid tissues and are characterized according to the cell type involved (myeloid or lymphoid).
• Acute leukemia is characterized by proliferation of immature myeloid or lymphoid cells.
• CLL is a hematologic neoplasm characterized by the accumulation of mature- appearing lymphocytes in the peripheral blood associated with infiltration of the bone marrow.
• 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 an elevated hemoglobin concentration.
• AMM/MF is characterized by the tendency of the neoplastic stem cells to lodge and grow in multiple sites outside the marrow, progressive splenomegaly, the gradual replacement of marrow elements by 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 from individuals subject to ET, colonies of megakaryocytes from megakaryocyte progenitors form in the absence of added stimulus, whereas such colonies do not occur with marrow cell cultures from normal individuals. (Braunwald et al. (Eds, 1987) Harrison's Principles of Internal Medicine, l lth ed. McGraw-Hill, New York NY).
• the present invention relates to human MYDl 18 whose nucleic acid sequence has been identified among the polynucleotide sequences of cDNA libraries made from human fetal liver- spleen tissue and non-adherent peripheral blood mononuclear cells and to the use of the nucleic acid and amino acid sequences of MYDl 18 in the study, diagnosis and treatment of disease states related to proliferation, specifically myeloproliferative diseases and leukemias.
• the nucleic acid sequence of myd 1 18 (SEQ ID NO: 1 ) and the protein it encodes,
• MYDl 18 (SEQ ID NO:2) is disclosed herein in Figure 1.
• the amino acid homology among human MYDl 18, murine MYDl 18 (SEQ ID NO:3) and human GADD 45 (SEQ ID NO:4) is disclosed herein in Figure 1.
• the present invention is based in part on the amino acid homology that human MYDl 18 shares with murine MYDl 18 and the ability of murine MYDl 18 and other MYD family members to stimulate 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 MYDl 18 in a cDNA library made from fetal liver/spleen tissue and non-adherent peripheral blood mononuclear cells, where hematopoietic cells would be expected to be found.
• Nucleic acid sequences encoding MYDl 18 are not detected in samples of cDNA libraries made from malignant hematopoietic cells sources where expression of genes related to cell growth arrest and apoptosis may be absent, aberrant, deleted or expressed at low levels.
• human MYDl 18 may be altered, absent or at low levels in individuals subject to myeloproliferative disease and leukemias.
• Human MYDl 18, and nucleic acid sequences that encode it and oligonucleotides, peptide nucleic acid (PNA), fragments, portions or antisense molecules thereof, provide the basis for diagnostic methods for the early and accurate detection and/or quantitation of MYDl 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, chronic myelogenous leukemia (CML); polycythemia vera (PV); agnogenic myeloid metaplasia with myelofibrosis (AMM/MF); and essential thrombocytosis (ET).
• ALL acute
• the nucleotide sequence for mydl 18 disclosed herein, or fragments thereof may be used in hybridization assays of biopsied cells or tissues or bodily fluids to diagnose abnormalities in mydl 18 gene expression in individuals subject to or at risk for a myeloproliferative disease or leukemia.
• Mydl 18 gene expression may be present at low levels, entirely absent or altered in such a disease states.
• the nucleotide and amino acid sequences for MYDl 18 may also be used in the diagnosis and treatment of other disease states related to abnormal proliferation of cells associated with severe inflammation, such as rheumatoid arthritis, psoriasis (characterized by proliferation of epidermal cells) and lymphomatoid granulomatosis (characterized as a lymphoproliferative disease) where it would be desirable to terminally differentiate proliferating cells and arrest their growth.
• the present invention provides diagnostic compositions and diagnostic tests for the detection of mydl 18 nucleotide sequences in biological samples.
• a diagnostic test comprises the steps of combining the biological sample with a first nucleotide sequence which comprises an mydl 18 nucleotide sequence, or a non-conserved fragment thereof, 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 mydl 18 nucleotide sequences in said biological sample; and comparing the amount of the second nucleotide sequence in said sample with a standard thereby determining whether the amount of said second nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said second nucleotide sequence correlates positively with a myeloproliferative disease.
• nucleotide sequences encoding MYDl 18 in a biological sample may reflect a chromosomal aberration, such as a nucleic acid deletion or mutation. Accordingly, nucleotide sequences encoding MYDl 18 provide the basis for probes which can be used diagnostically to detect chromosomal aberrations such as deletions, mutations or chromosomal translocations in the gene encoding MYDl 18.
• the present invention also provides a diagnostic test for the detection of mydl 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:l, detecting amplified nucleotide sequences, and comparing the amount of amplified nucleotide sequences in said biological sample with a standard thereby determining whether the amount of said nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said nucleotide sequence correlates positively with a myeloproliferative disease.
• human MYDl 18 and the nucleic acid sequences that encode 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.
• myeloproliferative diseases such as ALL, AML, CLL, hairy cell leukemia, CML, PV, AMM/MF and ET.
• nucleotide sequences that encode MYDl 18 can be administered alone or in combination with nucleotide sequences encoding tumor suppressor genes, such as p53 (known to regulate the expression of related family member GADD45), pi 6 and p21, through gene therapy techniques to individuals with CML to induce terminal differentiation of granulocytes, thereby arresting leukemic cell proliferation.
• tumor suppressor genes such as p53 (known to regulate the expression of related family member GADD45), pi 6 and p21
• mydl 18 nucleic acid antisense molecules or antagonists of MYDl 18 protein may be used to block the activity of human MYDl 18 in conditions where it would be preferable to block positive regulators of cell growth arrest.
• MYDl 18 may be used alone or in combination with other agents in the ex vivo culturing of hematopoietic stem cells intended for autologous transplant to individuals lacking cells of the hematopoietic lineage such as, for example, individuals subject to HIV infection 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 MYD 1 18 for the in vivo or in vitro production of MYD 1 18 protein.
• the present invention relates to the use of MYDl 18 polypeptides, or fragments or variants thereof, to produce anti-MYDl 18 antibodies and to screen for antagonists or inhibitors of MYDl 18 polypeptides which can be used diagnostically to detect and quantitate MYDl 18 protein levels in disease states related to proliferation.
• the present invention further relates to methods of treating individuals subject to myeloproliferative disease comprising administering compositions comprising purified MYDl 18 polypeptides or variants thereof, to subjects at risk for or having myeloproliferative disease or leukemia.
• the present invention also relates to pharmaceutical compositions comprising effective amounts of MYDl 18 protein or nucleic acid encoding MYDl 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 MYDl 18 into individuals having or at risk for myeloproliferative diseases, or leukemias.
• the invention further provides diagnostic assays and kits for the detection of MYDl 18 in cells and tissues comprising purified MYDl 18 which may be used as a positive control, and anti- MYDl 18 antibodies.
• Such antibodies may be used in solution-based, membrane-based, or tissue-based technologies to detect any disease state or condition related to the expression of MYD 1 18 protein or expression of deletions or variants thereof.
• Figure 1 displays the polynucleotide (SEQ ID NO:l)and deduced amino acid (SEQ ID NO:2) sequence for MyDl 18. Sequences shown in this Figure were produced using the multisequence alignment program of DNASTAR software (DNASTAR Ine, Madison WI). SEQ ID NO: l also contains untranslated 5' and 3' regions of mydl 18.
• Figure 2 displays the amino acid alignment of human MYDl 18 (SEQ ID NO:2), murine
• Figure 3 displays an analysis of the hydrophobicity and other characteristics of MYDl 18 based on the predicted amino acid sequence (produced using DNASTAR software).
• the present invention relates to human MYDl 18 and to the use of the nucleic acid and amino acid sequences of MYDl 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 MYD 118 shares with murine MYDl 18 and the ability of murine MYDl 18 and other MYD family members to stimulate terminal differentiation of hematopoietic cells, to arrest 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 MYDl 18 in a cDNA library made from fetal liver/spleen tissue and non-adherent peripheral blood mononuclear cells, where hematopoietic cells would be expected to be found.
• Nucleic acid sequences encoding MYDl 18 were not detected in randomly selected samples of about 500 to 5700 usable sequences in cDNA libraries made from THP-1 cells, the human promonocyte line derived from the peripheral blood of an individual subject to acute monocytic leukemia (ATCC accession TIB 202; INCYTE libraries: THP1NOB01, THPIPEBOI, THPIPLBOI and THP1PLB02); U937 cells, made from malignant cells from the pleural effusion of an individual subject to histiocytic lymphoma (ATCC accession CRL 1593; Sundstrom C and K Nilsson (1976) Int J Cancer 17:565-577; INCYTE library U937NOT01); T/B lymphoblasts from a leukemia source (Stratagene (La Jolla, CA), STR 937214; INCYTE library TBLYNOT01); peripheral blood white blood cells from an individual with myelogenous leukemia (INCYTE
• MYDl 18 were not detected in a cDNA library from peripheral blood granulocytes (INCYTE library NEUTFMT01); pooled bone marrow samples (INCYTE library BMARNOR02); ataxia telangiectasia fibroblast cell line (INCYTE libraries: FIBRAGT01, FIBRAGT02 and FIBRANTOl); or adult spleen (INCYTE library SPLNNOT02).
• M YD 1 18 related family member, GADD45 is known to be absent in individuals subject to ataxia telangiectasia (Zhan et al. (1994) Mol and Cell Biol 14:2361-2371).
• human MYDl 18 may be altered, absent or not detected in individuals subject to myeloproliferative disease and leukemias. Additionally, the gene encoding MYDl 18 may be involved in a chromosomal aberration such as a deletion, mutation ie, a point or internal mutation, translocation or may contain tri-nucleotide repeats known to be present in chromosomal abnormalities.
• Human MYDl 18, nucleic acid sequences that encode MYDl 18 and oligonucleotides, peptide nucleic acid (PNA), fragments, portions or antisense molecules thereof, provide the basis for diagnostic methods for the early and accurate detection and/or quantitation of MYDl 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 (AMM/MF); and essential thrombocytosis (ET).
• ALL acute lymphocytic leukemia
• AML acute myelogenous leukemia
• CLL chronic lymphocytic leukemia
• CML chronic myelogenous leukemia
• PV polyc
• nucleotide sequence for mydl 18 disclosed herein, or fragments thereof may be used in hybridization assays of biopsied cells or tissues to diagnose abnormalities in mydl 18 gene expression in individuals with or at risk for a myeloproliferative disease or leukemias.
• Mydl 18 gene expression may be present at low levels, entirely absent or altered in such disease states.
• nucleic acid sequences disclosed herein may be used in the detection of aberrations, such as mutations and deletions, in the gene encoding MYDl 18.
• the nucleotide sequences disclosed herein may be used to identify and isolate a genomic sequence for mydl 18.
• PCR primers can be designed from various portions of the introns and exons of the genomic mydl 18 that will allow detection of aberrations in the genomic sequence.
• human MYDl 18 and the nucleic acid sequences that encode 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.
• myeloproliferative diseases such as ALL, AML, CLL, hairy cell leukemia, CML, PV, AMM MF and ET.
• nucleic acid sequences that encode MYDl 18 can be administered to individuals subject to CML or AMM to induce terminal differentiation of granulocytes, thereby arresting cell proliferation.
• Administration of MYDl 18 or nucleic acid sequences that encode it may alleviate the symptoms associated with myeloproliferative diseases and leukemias, such as anemia, fatigue, splenomegaly, hypermetabolism and thrombohemorrhagic complications.
• mydl 18 nucleic acid antisense molecules or antagonists of MYDl 18 protein may be used to block the activity of human MYDl 18 in conditions where it would be preferably to block positive regulators of cell growth arrest.
• MYDl 18 may be used alone or in combination with other agents in the ex vivo culturing of hematopoietic stem cells intended for autologous transplant to HIV infected individuals 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 MYDl 18 for the in vivo or in vitro production of MYDl 18 protein.
• Nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide sequence, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be double-stranded or single-stranded whether representing the sense or antisense strand.
• amino acid sequence refers to peptide or protein sequences or portions thereof.
• lower case mydl 18 refers to a nucleic acid sequence whereas upper case MYDl 18 refers to a protein sequence.
• peptide nucleic acid refers to a class of informational molecules that have a neutral "peptide like" backbone combined with nucleotides that allow hybridization to complementary DNA or RNA with higher affinity and specificity than corresponding oligonucleotides (PerSeptive Biosystems, Marlborough MA).
• MYDl 18 refers to MYDl 18 from bovine, ovine, porcine, equine and preferably human, in naturally occurring or in variant form, or from any source, whether natural, synthetic, semi-synthetic or recombinant.
• naturally occurring refers to an MYDl 18 with an amino acid sequence found in nature
• biologically active refers to an MYDl 18 having structural, regulatory or biochemical functions of the naturally occurring MYDl 18.
• immunological activity is defined as the capability of the natural, recombinant or synthetic MYDl 18 or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
• MYDl 18 refers to the chemical modification of MYDl 18. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. An MYDl 18 polypeptide derivative would encode a polypeptide which retains essential biological characteristics of MYDl 18.
• purified refers to molecules, either nucleic 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.
• the nucleotide sequence of mydl 18 (SEQ ID NO:l) is shown in Figure 1.
• the entire coding region for human mydl 18 was initially identified within a cDNA library made from human fetal liver/spleen tissue where it was found 1 time in 2899 usable sequences.
• a BLAST search (Basic Local Alignment Search Tool; Altschul SF (1993) J. Mol. Evol. 36: 290-300; Altschul SF et al (1990) J. Mol. Biol.
• Polynucleotide sequences encoding MYDl 18 were subsequently found in a cDNA library made from non-adherent peripheral blood mononuclear cells (PBMN) where it was found 1 time in 3941 usable sequences.
• PBMN peripheral blood mononuclear cells
• usable sequences refers to the total number of clones in a library after the removal of vector, nucleotide repeats, contamination, and mitochondrial DNA.
• nucleotide sequence encoding MYDl 18 have been found in GenBank from the following human tissue sources: hippocampus, retina, olfactory epithelium, fetal lung, breast, placenta, white blood cells, brain, pancreas, liver, prostate gland and lung. Polynucleotide sequences encoding MYDl 18 have not been detected however, in cDNA libraries made from malignant cells of hematopoietic lineage, including THP-1 cells; U937 cells; T/B lymphoblasts from a leukemia source; peripheral blood white blood cells from an individual with myelogenous leukemia; and human mast cells from an individual with mast cell leukemia.
• the mydl 18 nucleotide sequence 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 DNA sequencing are well known in the art and employ such enzymes as the 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 polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg MD).
• the quality of any particular cDNA library from which polynucleotides encoding MYDl 18 are found may be determined by performing a pilot scale analysis of the cDNAs and checking for percentages of clones containing vector, lambda or J_. coli DNA, mitochondrial or
• the polynucleotide sequence of mydl 18 may be extended utilizing the nucleotide sequences from SEQ ID NO:l 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-
• PCR reverse transcriptase chain reaction
• RNA 25 transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
• Inverse 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 may be designed using Oligo 4.0 (National Biosciences Ine, Plymouth MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal 0 to the target sequence at temperatures about 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 by intramolecular ligation and used as a PCR template.
• Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1 :1 1 1-19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome (YAC) DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before PCR.
• PromoterFinderTM is a new kit available from Clontech (Palo Alto CA) which uses PCR, nested primers and special libraries to "walk in” genomic DNA. This process avoids the need to screen libraries and is useful in finding intron exon junctions.
• Another PCR method employs XL-PCRTM enzymes (Perkin-Elmer, Foster City CA) to amplify and/or extend nucleotide sequences.
• Preferred libraries for screening for full length cDNAs are ones that have been size- selected to include larger cDNAs.
• random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
• a randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
• Genomic libraries are useful for obtaining introns and extending 5' sequence.
• a new method for analyzing either the size or confirming 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 flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
• Output/light intensity is converted to electrical signal using appropriate software (eg. GenotyperTM and Sequence NavigatorTM from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled.
• Capillary electrophoresis is particularly suited to the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
• the reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has been reported (Ruiz- Martinez MC et al (1993) Anal Chem 65:2851-8).
• mydl 18 polynucleotide sequences which encode MYDl 18 polypeptide sequences, fragments, fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of mydl 18 in appropriate host cells.
• DNA 5 sequences other than the polynucleotide sequences of SEQ ID NO: 1 which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express mydl 18.
• RNA transcript 10 17: can be selected, for example, to increase the rate of mydl 18 expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
• polynucleotide sequences that are capable of hybridizing to SEQ ID NO:l under conditions of intermediate to maximal
• Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Wahl GM et al. (1987, Methods Enzymol 152:399-407) incorporated herein by reference, and confer a defined "stringency” as explained below.
• Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); “high stringency” at about 5°C to 10°C below Tm; “intermediate stringency” at about 10°C to 20°C below Tm; and “low stringency” at about 20°C to 25°C below Tm.
• a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency 5 hybridization can be used to identify or detect similar or related polynucleotide sequences.
• hybridization refers to "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology. Stockton Press, New York NY). Amplification as carried out in polymerase chain reaction technologies is described in Dieffenbach CW and GS Dveksler (1995, PCR 0 Primer, a Laboratory Manual. Cold Spring Harbor Press, Plainview NY) and inco ⁇ orated herein by reference.
• deletion is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
• an "insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring molecule.
• substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
• Variant mydl 18 polynucleotide sequences may be used in accordance with the invention and include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent MYDl 18 polypeptide.
• Variant MYDl 18 protein may also be used in accordance with the invention and may include deletions, insertions or substitutions of amino acid residues as long as the result is a functionally equivalent MYDl 18.
• the term functionally equivalent refers to a variant polynucleotide or variant polypeptide sequence that retains at least one of the biological activities of the naturally occurring sequence.
• Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as a biological activity of MYDl 18 is retained.
• negatively charged amino acids include aspartic acid and glutamic acid
• positively charged amino acids include lysine and arginine
• amino acids with uncharged polar head groups having similar hydrophilicity values are grouped as follows: leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and lastly, serine, threonine phenylalanine, and tyrosine.
• alleles of mydl are included within the scope of the present invention.
• an "allele” or “allelic sequence” is an alternative form of mydl 18. Alleles result from a mutation, ie, 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 may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of nucleic acids. Each of these types of changes may occur alone, or in combination with the others, and at the rate of one or more times in a given sequence.
• nucleotide sequences of the present invention may be engineered in order to alter an mydl 18 coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
• mutations may be introduced using techniques which are well known in the art, eg. site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.
• an mydl 18 natural, modified or recombinant sequence may be ligated to a heterologous sequence to encode a fusion protein.
• a heterologous sequence For example, for screening of peptide libraries for inhibitors of MYDl 18 activity, it may be useful to encode a chimeric MYDl 18 protein expressing a heterologous epitope that is recognized by a commercially available antibody.
• a fusion protein may also be engineered to contain a cleavage site located between an MYDl 18 sequence and the heterologous protein sequence, so that the MYDl 18 may be cleaved and purified away from the heterologous moiety.
• the coding sequence of mydl 18 could be synthesized, 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 et al(1980) Nuc Acids Res Symp Ser 225- 32, etc).
• the protein itself could be produced using chemical methods to synthesize an MYDl 18 amino acid sequence, whole or in part.
• peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (eg, Creighton (1983) Proteins Structures And Molecular Principles. WH Freeman and Co, New York NY).
• composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (eg, the Edman degradation procedure; Creighton, supra)
• Direct peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
• the amino acid sequence of MYDl 18, or any part thereof may be altered during direct synthesis and/or combined using chemical methods with other sequences to produce a variant polypeptide.
• the nucleotide sequence coding for MYDl 18, or a functional equivalent thereof is inserted into an appropriate expression vector, ie, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
• an appropriate expression vector ie, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
• Methods which are well known to those skilled in the art can be used to construct expression vectors containing an MYDl 18 coding sequence and appropriate transcriptional or translational controls. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination or genetic recombination. Such techniques are described in Maniatis et al (1989) 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.
• a preferred expression system for the expression of MYDl 18 in host cells is one that allows for expression of proteins toxic to the host cell.
• Zhan et al. describe a mammalian expression system for the expression of MYDl 18 related family members GADD45, which has been shown to be regulated by p53, and murine MYDl 18.
• This expression system employs the cytomegalovirus promoter and cotransfection with a selectable marker, pSV2neo. Expression studies were performed in human tumor lines with a null p53 phenotype (HI 299), in cells with a normal p53 phenotype (RKO), and in cells containing a viral protein, E6, that interferes with p53 function (HeLa).
• marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
• mydl 18 is inserted within a marker gene sequence, recombinant cells containing mydl 18 can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with an mydl 18 sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of mydl 18 as well.
• host cells which contain the coding sequence for mydl 18 and express mydl 18 may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-based, solution-based, or chip- based technologies for the detection and/or quantification of the nucleic acid or protein.
• the presence of the mydl 18 polynucleotide sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of mydl 18 disclosed in SEQ ID NO: 1.
• Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the mydl 18 sequence to detect transformants containing mydl 18 DNA or RNA.
• oligonucleotides or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer.
• oligonucleotides are derived from the 3' region of the mydl 18 nucleotide sequence shown in Figure 1.
• a variety of protocols for detecting and measuring the expression of MYD 1 18 polypeptide, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• FACS fluorescent activated cell sorting
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on MYDl 18 polypeptides is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, a Laboratory Manual. APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 158:121 1).
• Means for producing labeled hybridization or PCR probes for detecting mydl 18 polynucleotide sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
• the mydl 18 sequence, or any portion of it may be cloned into a vector for the production of an mRNA probe.
• Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
• reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
• Patents teaching the use of such labels include US Patents 3,817.837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.
• recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567 and inco ⁇ orated herein by reference. Purification of MYDl 18
• Host cells transformed with a mydl 18 nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
• the protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
• expression vectors containing mydl 18 can be designed with signal sequences which direct secretion of MYDl 18 through a particular prokaryotic or eukaryotic cell membrane.
• MYDl 18 may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
• purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3:263- 281), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ , Seattle WA).
• cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and MYDl 18 is useful to facilitate purification.
• MYDl 18 cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA)
• MYDl 18 appears to induce terminal differentiation of hematopoietic cells thereby arresting cell proliferation and inducing apoptosis.
• Polynucleotide sequences encoding MYDl 18, or portions thereof, have been found in a variety of cDNA libraries from tissues including fetal liver/spleen, PBMN cells, hippocampus, retina, olfactory epithelium, human fetal lung, breast, placenta, human white blood cells, human brain, human pancreas, liver, prostate gland and lung.
• Polynucleotide sequences encoding MYDl 18 are not detected in cDNA libraries made from malignant cells of hematopoietic lineage, where genes related to cell growth arrest and apoptosis would be expected to be expressed at undetectable levels or not expressed at all.
• human MYDl 18 disclosed herein may be used as a diagnostic or therapeutic agent in the detection or treatment of proliferative diseases of hematopoietic cells, specifically in myeloproliferative diseases and leukemias.
• MYDl 18 can be used to treat or ameliorate the symptoms of myeloproliferative diseases or 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 (AMM/MF); and essential thrombocytosis (ET).
• ALL acute lymphocytic leukemia
• AML acute myelogenous leukemia
• CLL chronic lymphocytic leukemia
• CML chronic myelogenous leukemia
• PV polycythemia vera
• ALM/MF agnogenic myeloid metaplasia with myelofibrosis
• ET essential thrombocytosis
• MYDl 18 administration of MYDl 18 to individuals subject to myeloproliferative disease, which is characterized by the production of increased numbers of immature cells in the peripheral blood, may induce terminal differentiation of the immature cells thereby leading to the maturation of the cells and eventual apoptosis.
• Administration of MYDl 18 may be used to augment chemotherapy, such as administration of busulfan, radiation therapy and/or bone marrow transplant.
• MYDl 18 may be used therapeutically to ameliorate the symptoms associated with myeloproliferative diseases and leukemias, such as, anemia, fatigue, splenomegaly hypermetabolism and thrombohemorrhagic complications.
• MYDl 18 may also be used to treat disease states related to abnormal proliferation of cells associated with severe inflammation including, but not limited, to rheumatoid arthritis, psoriasis, and lymphomatoid granulomatosis.
• antibodies or antagonists of MYDl 18 may be used in the ex vivo culturing of cells from a hematopoietic cell lineage intended for autologous transplant where it would be desirable to eliminate a cell growth arrest factor that might interfere with desirable cell proliferation.
• MYDl 18 Antibodies may be used in the ex vivo culturing of cells from a hematopoietic cell lineage intended for autologous transplant where it would be desirable to eliminate a cell growth arrest factor that might interfere with desirable cell proliferation.
• Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
• Neutralizing antibodies, ie, those which inhibit biological activity of MYDl 18 polypeptides, are especially preferred for diagnostics and therapeutics.
• various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with MYDl 18 polypeptide or any portion, fragment or oligopeptide which retains immunogenic properties.
• various adjuvants may be used to increase immunological response.
• adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
• BCG Bacilli Calmette-Guerin
• Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified MYDl 18 polypeptide is administered to immunologically compromised individuals for the pu ⁇ ose of stimulating systemic defense.
• Monoclonal antibodies to MYDl 18 polypeptide may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV- hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy. Alan R Liss Ine, pp 77-96).
• Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi R et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991 ; Nature 349:293-299).
• Antibody fragments which contain specific binding sites for MYDl 18 may also be generated.
• such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab'), fragments.
• Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al ( 1989) Science 256: 1275- 1281).
• MYDl 18-specific antibodies are useful for the diagnosis of conditions and diseases associated with expression of MYDl 18 polypeptide.
• a variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between MYDl 18 polypeptides and its specific antibody (or similar MYDl 18-binding molecule) and the measurement of complex formation.
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific MYDl 18 protein is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox (supra). Diagnostic Assays Using MYDl 18 Specific Antibodies
• Anti-MYDl 18 antibodies are useful for the diagnosis of myeloproliferative disease or leukemias or other conditions, disorders or diseases characterized by abnormal expression of MYDl 18. Diagnostic assays for MYDl 18 include methods utilizing the antibody and a label to detect MYDl 18 polypeptide in human body fluids, cells, tissues or sections or extracts of such tissues.
• the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule.
• a wide variety of reporter molecules are known to those of skill in the art.
• a variety of protocols for measuring MYDl 18 polypeptide, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• FACS fluorescent activated cell sorting
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on MYDl 18 polypeptide is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox (supra).
• normal or standard values for MYDl 18 polypeptide expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with antibody to MYDl 18 polypeptide under conditions suitable for complex formation which are well known in the art. The amount of standard complex formation may be quantified by comparing it with a dilution series of positive controls where a known amount of antibody is combined with known concentrations of purified MYDl 18 polypeptide. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to MYDl 18 polypeptide expression. Deviation between standard and subject values establishes the presence of the disease state. Drug Screening
• MYDl 18 polypeptide, its immunogenic fragments or oligopeptides can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
• the fragment employed in such a test may be free in solution, affixed to a solid support, bome on a cell surface, or located intracellularly.
• the abolition of activity or the formation of binding complexes, between MYDl 18 polypeptide and the agent being tested, may be measured.
• the present invention provides a method for screening a plurality of compounds for specific binding affinity with MYDl 18, or a portion thereof, comprising providing a plurality of compounds; combining MYDl 18, or a portion thereof, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions; and detecting binding of MYDl 18, or portion thereof, to each of the plurality of compounds, thereby identifying the compounds which specifically bind MYDl 18.
• Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the MYDl 18 polypeptides and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference.
• large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
• the peptide test compounds are reacted with MYDl 18 fragments and washed. Bound MYDl 18 is then detected by methods well known in the art.
• Purified MYDl 18 can also be coated directly onto plates for use in the aforementioned drug screening techniques.
• non- neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
• This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding MYDl 18 specifically compete with a test compound for binding MYD 1 18. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with MYDl 18.
• An mydl 18 polynucleotide, or any part thereof, may be used for diagnostic and/or therapeutic purposes.
• mydl 18 polynucleotide sequences may be used to detect and quantitate gene expression in conditions, disorders or diseases in which mydl 18 activity may be implicated, for example in myeloproliferative diseases or leukemias, where mydl 18 gene expression appears to be absent, aberrant or not detected.
• mydl 18 polynucleotide sequences may be administered to individuals with myeloid proliferative disease or leukemia to induce terminal differentiation of proliferating immature hematopoietic cells origin, thereby arresting cell proliferation, ameliorating the symptoms of disease, and reversing the leukemic phenotype.
• myeloid proliferative disease or leukemia to induce terminal differentiation of proliferating immature hematopoietic cells origin, thereby arresting cell proliferation, ameliorating the symptoms of disease, and reversing the leukemic phenotype.
• DNA molecules and ribozymes which function to inhibit translation of an mydl 18.
• Such nucleotide sequences may be used in conditions where is would be preferable to block positive regulators of cell growth arrest, for example, in ex vivo culturing of cells intended for autologous transplant to individuals lacking cells of the hematopoietic lineage, such as individuals who are immunocompromised due to disease, such as HIV infection, or individuals subject to chemotherapy or radiation therapy which depletes certain cells of hematopoietic lineage.
• Another aspect of the subject invention is to provide for nucleic acid hybridization or PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding MYDl 18 or closely related molecules, such as alleles.
• the specificity of the probe ie, whether it is derived from a highly conserved, conserved or non-conserved region or domain, and the stringency of the hybridization or amplification (high, intermediate or low) will determine whether the probe identifies only naturally occurring mydl 18, or related sequences.
• Probes for the detection of related nucleic acid sequences are selected from conserved or highly conserved nucleotide regions of MYD family members and such probes may be used in a pool of degenerate probes.
• nucleic acid probes are selected from the non-conserved nucleotide regions or unique regions of mydl 18 polynucleotides.
• non-conserved nucleotide region refers to a nucleotide region that is unique to mydl 18 and does not occur in related family members, such as the nucleotide sequence encoding GADD45.
• An mydl 18 encoding polynucleotide sequence may be used for the diagnosis of diseases resulting from abnormal expression of mydl 18 or other genes associated with myeloproliferative disease or leukemia.
• polynucleotide sequences encoding MYDl 18 may be used in hybridization or PCR assays of tissues from biopsies or autopsies or biological fluids, such as serum, to detect abnormalities in mydl 18 expression.
• the form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane- based technologies; PCR technologies; dip stick, pin or chip technologies; and ELISA or other multiple sample format technologies. All of these techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits.
• Such assays may be tailored to evaluate the efficacy of a particular therapeutic treatment regime and may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
• a normal or standard profile for myd 1 18 expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with mydl 18 or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained for normal subjects with a dilution series of positive controls run in the same experiment where a known amount of purified mydl 18 is used.
• Standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to mydl 18 expression. Deviation between standard and subject values establishes the presence of the disease state. If disease is established, an existing therapeutic agent is administered, and treatment profile or values may be generated. Finally, the assay may be repeated on a regular basis to evaluate whether the values progress toward or return to the normal or standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
• PCR as described in US Patent Nos. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the myd 1 18 sequence.
• oligomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source.
• Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3') and one with antisense (3' ⁇ -5') employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
• samples may be speeded up by running the assay in an ELISA format where the an oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• An mydl 18 polynucleotide sequence may provide the basis for treatment of various abnormal conditions involving abnormally proliferating cells 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 (AMM/MF); and essential thrombocytosis (ET), where it would be advantageous to induce terminal differentiation of the proliferating cells thereby arresting cell proliferation.
• myeloproliferative diseases and leukemias such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelog
• Stable transformation of appropriate germ line cells, or a zygote, with a vector containing polynucleotide sequences encoding MYDl 18 may produce a transgenic organism (US Patent No. 4,736,866, 12 April 1988) producing enough copies of the polynucleotide sequence to induce terminal differentiation of cells of hematopoietic lineage, thereby leading to cell growth arrest
• a transgenic organism US Patent No. 4,736,866, 12 April 1988
• Such vectors and expression systems intended for in vivo or gene therapy use should be designed for expression of molecules toxic to the host cell as disclosed infra.
• a preferable expression vector would be one which drives expression of mydl 18 at levels comparable to its expression in normal non-proliferating cells.
• an mydl 18 polynucleotide sequence may also provide the basis for design of antisense molecules that are able to inhibit transcription or translation of mydl 18 in conditions where it would be advantageous to block positive regulators of cell growth arrest, such as for example in ex vivo culturing of cells from a hematopoietic lineage intended for autologous transplant, where proliferation of cells of hematopoietic lineage would be desirable.
• positive regulators of cell growth arrest such as for example in ex vivo culturing of cells from a hematopoietic lineage intended for autologous transplant, where proliferation of cells of hematopoietic lineage would be desirable.
• Expression vectors derived from retroviruses, adenovirus, he ⁇ es or vaccinia viruses, or from various bacterial plasmids may be used for delivery of recombinant mydl 18, sense or antisense molecules, to the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors containing mydl 18. See, for example, the techniques described in Maniatis et al (supra) and Ausubel et al(supra).
• recombinant mydl 18 can be delivered to target cells in liposomes.
• cDNA sequence and/or its regulatory elements enable researchers to use mydl 18 as a tool in sense (Youssoufian H and HF Lodish 1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) investigations of gene function.
• Oligonucleotides, designed from the cDNA or control sequences obtained from the genomic DNA can be used in vitro or in vivo to inhibit expression. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions.
• mydl 18 expression can be modulated by transfecting a cell or tissue with expression vectors which express high levels of an mydl 18 fragment in conditions where it would be preferably to block the activity of positive regulator of cell growth arrest, such as in bone marrow transplant therapy and ex vivo culturing of cells intended for autologous transplant.
• Such constructs can flood cells with untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies of the vector are disabled by endogenous nucleases.
• Such transient expression may last for a month or more with a non-replicating vector (Mettler I, personal communication) and even longer if appropriate replication elements are part of the vector system.
• Modifications of gene expression can be obtained by designing antisense sequences to the control regions of the mydl 18 gene, such as the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence, are preferred. Antisense RNA and DNA molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using Hogeboom base-pairing methodology, also known as "triple helix" base pairing. Triple helix pairing compromises 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 ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
• engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of mydl 18 RNA sequences.
• ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide sequence inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
• RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
• antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.
• DNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone 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 the ex vivo therapy.
• mydl 18 polynucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely 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 Mapping of Polynucleotide Sequences Related to mydl 18
• the nucleic acid sequence for mydl 18 can also be used to generate hybridization probes as previously described, for mapping the endogenous genomic sequence.
• the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in ____ hybridization to chromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques. Pergamon Press, New York City), flow- sorted chromosomal preparations, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries.
• telangiectasia AT
• any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc between normal, carrier or affected individuals.
• compositions which may comprise all or portions of mydl 18 polynucleotide sequences, MYDl 18 polypeptides, inhibitors or antagonists of MYDl 18 bioactivity, including antibodies, alone or in combination with at least one other agent, such as stabilizing compound, and may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
• Mydl 18 nucleotide and MYDl 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, pi 6 and p21, drugs or hormones or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers.
• the pharmaceutically acceptable carrier is pharmaceutically inert.
• a preferred route of administration for treatment of myeloproliferative diseases or leukemias would be intravenous delivery whereas a preferred route of administration for treatment of conditions related to inflammation would be local administration at the site of inflammation, such as the joint affected in rheumatoid arthritis.
• Mydl 18 polynucleotide sequences or MYDl 18 amino acid sequences may be administered alone to individuals subject to myeloproliferative diseases or leukemias or in combination with other types of agents or therapy including chemotherapy, radiation therapy or stem cell transplant therapy.
• these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton PA). Suitable routes may, for example, include oral or transmucosal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
• compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
• physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
• penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
• an effective amount of MYDl 18 may be that amount that ameliorates the symptoms of anemia, fatigue, splenomegaly, hypermetabolism and thrombohemorrhagic complications. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided below.
• compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
• compositions of the present invention may be manufactured in a manner that is itself known, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
• Suitable lipophilic 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.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after 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 from corn, wheat, rice, potato, etc; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
• disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
• 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 dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, dosage.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
• compositions comprising a compound of the invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• conditions indicated on the label may include treatment of myeloproliferative diseases or leukemias.
• the pharmaceutical composition may 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. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
• the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
• the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models (particularly murine models) to achieve a desirable circulating concentration range that adjusts MYDl 18 levels.
• a therapeutically effective dose refers to that amount of MYD 1 18 which ameliorates symptoms of the disease state. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
• the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state; age, weight, and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature. See US Patent No.
• the human spleen cell cDNA library (SPLNFET01) was custom constructed by Stratagene.
• the tissue was obtained from fetal spleens pooled from different sources and contained many different types of cells.
• Poly(A+) RNA (mRNA) was purified, and cDNA was synthesized from the mRNA.
• Synthetic adaptor oligonucleotides were ligated onto cDNA ends enabling its insertion into Uni-ZAP TM vector system (Stratagene), allowing high efficiency unidirectional (sense orientation) lambda library construction and the convenience of a plasmid system with blue/white color selection to detect clones with cDNA insertions.
• Alternative unidirectional vectors are pcDNAl (Invitrogen, San Diego CA) and pSHlox-1 (Novagen, Madison WI).
• the custom-constructed library phage particles were transfected into E. coli host strain XL 1 -Blue® (Stratagene), which has a high transformation efficiency, increasing the probability of obtaining rare, under-represented clones in the cDNA library.
• the phagemid forms of individual cDNA clones were obtained by the in vivo excision process, in which the host bacterial strain was coinfected with both the lambda library phage and an fl helper phage. Proteins derived from both the library -containing phage and the helper phage nicked the lambda DNA, initiated new DNA synthesis from defined sequences on the lambda target DNA and created a smaller, single stranded circular phagemid DNA molecule that included all DNA sequences of the pBluescript® plasmid and the cDNA insert. The phagemid DNA was secreted from the cells and purified, then used to re-infect fresh host cells, where the double stranded phagemid DNA was produced. Because the phagemid carries the gene for ⁇ - lactamase, the newly-transformed bacteria were selected on medium containing ampicillin.
• Phagemid DNA was purified using the QIAwell-8 Plasmid, QIAwell PLUS, or QIAwell ULTRA DNA purification system (QIAGEN Ine Chatsworth CA). This product line provides a convenient, rapid and reliable high-throughput method to lyse bacterial cells and isolate highly purified phagemid DNA using QIAGEN anion-exchange resin particles with EMPORE TM membrane technology (3M, Minneapolis MN) in a multiwell 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 94:44 If), using the Catalyst 800 and 373 DNA Sequencing System (Perkin Elmer). II Homology Searching of cDNA Clones and Their Deduced Proteins Each cDNA was compared to sequences in GenBank using a search algorithm d inco ⁇ orated into the ABI INHERITTM 670 Sequence Analysis System (Perkin Elmer). In this algorithm, Pattern Specification Language (TRW Ine, Los Angeles CA) was used to determine regions of homology. The three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance.
• the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith- Waterman alignments were used to display the results of the homology search.
• BLAST which stands for Basic Local Alignment Search Tool (Altschul SF (1993) J Mol
• BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps.
• the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
• An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
• the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
• the parameter E establishes the statistically significant threshold for reporting database sequence matches.
• E is inte ⁇ reted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
• MYDl 18 was identified using the ABI INHERITTM DNA Analysis System (Perkin Elmer) software which identified clone 25214 as being related to human GADD45 and murine MYDl 18. 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 (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO:2) of MYDl 18 are disclosed herein in Figure 1. III Determination of Reading Frame of cDNA Clone
• the reading frame of individual cDNA clones obtained from the SPLNFET01 library was obtained by analyzing the polynucleotide sequences for the presence of start (ATG, GTG, etc.) and stop codons (TGA, TAA, TAG). Typically, one frame will continue throughout the major portion of all of a cDN A sequence and the other two pending frames tend to contain numerous stop codons. Algorithms for determining reading frame have been developed 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 in the third codon position.
• These algorithms have been inco ⁇ orated into widely available software and can be easily used to determine coding potential (and frame) of a given stretch of DNA. This algorithm-derived information, combined with start/stop codon information, was used to determine proper frame of individual clones within the SPLNFET01 library with a high degree of certainty, thus permitting the correct reading frame alignment with appropriate expression vehicles. IV Extension of mydl 18 to Recover Regulatory Elements
• the nucleic acid sequence of mydl 18 may be used to design oligonucleotide primers for obtaining full length sequences from genomic libraries.
• One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF).
• the primers allow the known mydl 18 sequence to be extended "outward" generating amplicons containing new, unknown nucleotide sequence for the control region of interest.
• the initial primers are designed from the cDNA using Oligo 4.0 (National Biosciences Ine, Plymouth MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C. Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations is avoided.
• a human genomic library is used to extend and amplify 5' upstream sequence. If necessary, a second set of primers is designed to further extend the known region.
• a second set of primers is designed to further extend the known region.
• PCR is performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters: Step 1 94 ° C for 1 min (initial denaturation)
• Step 2 65° C for l min
• Step 3 68 ° C for 6 min
• Step 4 94° C for 15 sec
• Step 5 65 ° C for l min
• Step 6 68° C for 7 min
• Step 7 Repeat step 4-6 for 15 additional cycles
• Step 8 94° C for 15 sec
• Step 9 65 ° C for l min
• Step 10 68 ° C for 7:15 min Step 1 1
• Step 8-10 for 12 cycles
• a 5-10 ⁇ l aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. The largest products or bands were selected and cut out of the gel. Further purification involves using a commercial gel extraction method such as QIAQuickTM (QIAGEN Ine). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning. After ethanol precipitation, the products are redissolved in 13 l of ligation buffer, l ⁇ l
• T4-DNA ligase (15 units) and l ⁇ l T4 polynucleotide kinase are added, and the mixture is incubated at room temperature for 2-3 hours or overnight at 16° C.
• Competent __. coH cells (in 40 ⁇ l of appropriate media) are transformed with 3 ⁇ l of ligation mixture and cultured in 80 ⁇ l of SOC medium (Sambrook J et al, supra). After incubation for one hour at 37° C, the whole transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra) containing 2x Carb.
• PCR amplification For PCR amplification, 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers used for the extension reaction are added to each well. Amplification is performed using the following conditions:
• Step l 94° C for 60 sec
• Step 2 94° C for 20 sec
• Step 3 55° C for 30 sec
• Step 5 Repeat steps 2-4 for an additional 29 cycles
• Step 7 4° C (and holding) Aliquots of the PCR reactions are run on agarose gels together with molecular weight markers. The sizes of the PCR products are compared to the original partial cDNAs, and appropriate clones are selected, ligated into plasmid and sequenced. V Labeling of Hybridization Probes
• Hybridization probes derived from SEQ ID NO: 1 may be employed to screen cDNAs, mRNAs or genomic DNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure may be used with larger cDNA fragments. Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 mCi of [g- 32 P] adenosine triphosphate (Amersham, Chicago IL) and T4 polynucleotide kinase (DuPont NEN ® , Boston MA).
• the labeled oligonucleotides are purified with Sephadex G-25 super fine resin column (Pharmacia). A portion containing IO 7 counts per minute of each is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, EcoR I, Pst I, Xba 1, or Pvu II; DuPont NEN ® ).
• the DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale CA) for several hours, hybridization patterns are compared visually. VI Antisense Molecules
• the mydl 18 sequence may be used to inhibit in vivo or in vitro expression of endogenous mydl 18.
• antisense oligonucleotides consisting of about 20 base-pairs
• essentially the same procedure may be used with larger cDNA fragments.
• An oligonucleotide based on the coding sequence of mydl 18 may be used to inhibit expression of endogenous mydl 18.
• Oligo 4.0 the complementary oligonucleotide can be designed from the conserved 5' sequence and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of an mydl 18 transcript by preventing the ribosome from binding to the mRNA. VII Production of MYDl 18 Specific Antibodies
• the deduced amino acid sequence of MYDl 18 is analyzed using DNASTAR software (DNASTAR Ine) to determine regions of high immunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies in rabbits. Analysis to select appropriate epitopes, such as those near the C-terminus or in adjacent hydrophilic regions is described by Ausubel FM et al (supra).
• oligopeptide of about 15 residues in length is synthesized using an ABI Peptide Synthesizer Model 431 A (Perkin Elmer, Norwalk, CN) using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel FM et al, supra).
• MBS M-maleimidobenzoyl-N-hydroxysuccinimide ester
• Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
• the resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated, goat anti-rabbit IgG.
• Endogenous or recombinant MYDl 18 can be purified by immunoaffinity chromatography using antibodies specific for MYDl 18.
• An immunoaffinity column is constructed by covalently coupling MYDl 18 antibody to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
• Media containing MYDl 18 is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of MYDl 18 (eg, high ionic strength buffers in the presence of detergent).
• the column is eluted under conditions that disrupt antibody /MYDl 18 binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and MYDl 18 is collected.

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