WO1997012978A1 - NOVEL c-mpl RECEPTOR AGONISTS - Google Patents

NOVEL c-mpl RECEPTOR AGONISTS Download PDF

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WO1997012978A1
WO1997012978A1 PCT/US1996/015938 US9615938W WO9712978A1 WO 1997012978 A1 WO1997012978 A1 WO 1997012978A1 US 9615938 W US9615938 W US 9615938W WO 9712978 A1 WO9712978 A1 WO 9712978A1
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Prior art keywords
leu
gly
ser
pro
val
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PCT/US1996/015938
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French (fr)
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Nicholas R. Staten
Lyle E. Pegg
Charles A. Mcwherter
Yiqing Feng
John P. Mckearn
Neena L. Summers
Judith G. Giri
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G.D. Searle & Co.
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Priority to JP09514462A priority Critical patent/JP2000513924A/en
Priority to EP96936192A priority patent/EP0870027A1/en
Priority to AU73901/96A priority patent/AU722759B2/en
Publication of WO1997012978A1 publication Critical patent/WO1997012978A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to human c-mpl receptor agonists (thrombopoietin) with activity on hematopoietic differentiation and expansion.
  • MK Megakaryocyte maturation and platelet production has been long thought to be regulated by lineage specific humoral growth factors in a manner similar to cytokines that induce erythrocyte (erythropoietin) and granulocyte (G-CSF) expansion and maturation. Platelets are responsible for the prevention of bleeding in response to vascular injury. Therefore, platelet production is a vital component of hematopoietic regulation. Patients undergoing chemotherapy or bone marrow transplantation usually experience severely depressed platelet levels (thrombocytopenia) which may result in life threatening bleeding episodes.
  • IL-3 IL-6, IL-11, SCF
  • Plasma, serum and urine from thrombocytopenic dogs and humans have been found to contain growth factors that have specific megakarypoietic and thrombopoietic activities distinct from all known cytokines. These factors have been termed Meg-CSF, MK-CSF, megakaryocyte growth and development factor (MGDF) , megakaryopoietin, and thrombopoietin but the molecular structure has not been identified until recently.
  • Meg-CSF Meg-CSF
  • MK-CSF megakaryocyte growth and development factor
  • MGDF megakaryopoietin
  • thrombopoietin thrombopoietin
  • the identification of the thrombopoietic cytokine, c-mpl ligand, originated with the identification of a myeloproliferative leukemia virus (MPLV, Wending et al., Virology 149:242-246, 1986) . Mice infected with this virus gave rise to multi-lineage myeloproliferation. Subsequent studies (Souyri et al. , Cell 63:1137-1147, 1990) demonstrated that the retrovirus encoded an oncogene (v-mpl) that when fused with viral envelope gene gave rise to a membrane anchored protein that resembles the cytoplasmic domain of the hematopoietic growth factor receptor family.
  • v-mpl oncogene
  • V-mpl was used to probe both human and murine RNA libraries for homologous genes. Clones were identified in both species and termed c- mpl (Vigon et al. , PNAS USA 89:5640-5644, 1992; Vigon et al . , Oncogene 8:2607-2615, 1993).
  • C-mpl is a member of the cytokine receptor super-family with regions of homology to mIL-5rc, II_3rc, IL4rc, mEPOrc and mGCSFrc. A chimera of the intracellular domain of c-mpl and the extracellular domain of hIL4rc was transfected into a growth factor dependent cell line (BaF3).
  • RT PCR reverse transcriptase polymerase chain reaction
  • c-mpl ligand or thrombopoietin is a peptide with a predicted molecular mass of 35,000 kDa.
  • the protein has a two domain structure with an amino-terminal domain (153 amino acids) with homology to erythropoietin and a carboxy-terminus rich in serine, threonine and proline residues which also contains several glycosylation sites. There are two potential arginine cleavage sites resulting in two shorter peptides of 25 kDa and 31 kDA forms both of which are biologically active. There is high inter species homology between human, murine, porcine, canine, rat and rabbit c-mpl ligand and most forms are active on all species tested.
  • C-mpl ligand has been shown to stimulate the differentiation of CD34+ cells into cells megakaryocyte characteristics.
  • CD34+ cells in the presence of c-mpl ligand underwent endomitosis (Kaushansky et al . , Nature 369:568-571, 1994), expressed the megakaryocyte lineage specific cell surface antigen CD41a and had morphology characteristic of megakaryocytes.
  • endomitosis Korean et al. , Nature 369:568-571, 1994
  • CD41a the megakaryocyte lineage specific cell surface antigen CD41a
  • morphology characteristic of megakaryocytes had morphology characteristic of megakaryocytes.
  • In vivo administration of c-mpl ligand have given rise to increased circulating platelets in normal mice (Lok et al., Nature 369:565-568, 1994).
  • C-mpl deficient mice generated by gene targeting demonstrated a 85% decrease in circulating platelets and megakaryocytes but had normal amounts of other hematopoietic lineages (Gurney et al. , Science 265:1445-1447, 1994). Absolute thrombocytopenia was not observed in these animals indicating that other cytokines may have some activity in expansion of the MK lineage.
  • c-mpl ligand is a cytokine with specific activity on the maturation of megakaryocytes and in platelet production.
  • Other cytokines have been shown to have activity on megakaryocyte expansion and differentiation, including IL-3, IL-6, IL-11 and c-kit ligand.
  • IL-3 IL-3
  • IL-6 IL-6
  • IL-11 IL-11
  • c-mpl ligand may have therapeutic use in restoring circulating platelets to normal amounts in those cases where the number of platelets have been reduced due to diseases or therapeutic treatments such as radiation and/or chemotherapy.
  • EP 675,201 Al relates to the c-mpl ligand (Megakaryocyte growth and development factor or MGDF) , allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
  • MGDF Megakaryocyte growth and development factor
  • WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof.
  • the proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.
  • WO 95/27732 discloses circularly permuted GM-CSF, G- CSF, IL-2 and IL-4 and fusions with Pseudomonas exotoxin.
  • the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
  • linker an additional portion of sequence
  • proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) .
  • the proteins examined have represented a broad range of structural classes, including proteins that contain predominantly ⁇ -helix (interleukin-4; Kreitman et al. , Cytokine 7:311-318, 1995), ⁇ -sheet (interleukin-1; Horlick et al. , Protein Eng.
  • the positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N- terminus to the breakpoint is ca. 10 to 80% of the total sequence length) .
  • the linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc. Natl . Acad. Sci . U. S.A . 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus.
  • the present invention relates to novel c-mpl receptor agonists of the following formulas:
  • Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met;
  • Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met;
  • Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
  • Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or
  • N-termmus is ⁇ omed to the C-termmus directly or through a linker capable of joining the N-termmus to the C-termmus and having new C- and N- termini at ammo acids;
  • said c-mpl receptor agonist can be immediately preceded by (methionine -1 ), (alanine -1 ) or (methionine -2 , alanine -1 ) .
  • Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro,
  • Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
  • Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn;
  • N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
  • said c-mpl receptor agonist can be immediately preceded by (methionine -1 ), (alanine -1 ) or (methionine -2 , alanine- ) .
  • the more preferred breakpoints at which new C-termmus and N-termmus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-
  • the most preferred breakpoints at which new C-termmus and N-termmus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 81-82, 108-109, 115-116, 119-120, 122-123 and 125-126.
  • human c-mpl receptor agonists may contain am o acid substitutions, deletions and/or insertions and may also have amino acid deletions at either/or both the N- and C- termini.
  • the modified human c-mpl receptor agonists of the present mvention can be represented by the Formula: X 1 - ( L ) a -X 2
  • X 1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+l through J;
  • X 2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n; n is an integer ranging from 1 to J-1; and
  • L is a linker
  • residues of human c-mpl ligand are numbered sequentially 1 through J from the amino to the carboxy terminus.
  • a pair of adjacent amino acids within this protein may be numbered n and n+l respectively where n is an integer ranging from 1 to J-1.
  • the residue n+l becomes the new N-terminus of the new c- pl receptor agonist and the residue n becomes the new C- terminus of the the new c-mpl receptor agonist.
  • the linker (L) joining the N-terminus to the C-terminus is a polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:73); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74);
  • GlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:75); SerGlyGlySerGlyGlySer (SEQ ID NO:76); GluPheGlyAsnMetAla (SEQ ID NO:77); GluPheGlyGlyAsnMetAla (SEQ ID NO:78); GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and GlyGlySerAspMetAlaGly (SEQ ID NO:80) .
  • the present invention also encompasses recombinant human c-mpl receptor agonists co-administrated with one or more additional colony stimulating factors (CSF) including, cytokines, lymphokines, interleukins, hematopoietic growth factors (herein collectively referred to as "colony stimulating factors”) each of which may act through a different and specific cell receptor to initiate complementary biological activities.
  • CSF colony stimulating factors
  • co-administrated molecules may be characterized by having the usual activity of both of the peptides or it may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of human c-mpl receptor agonist or the second colony stimulating factor alone.
  • the co ⁇ administration may also unexpectedly provide an enhanced effect on the activity or an activity different from that expected by the presence of human c-mpl receptor agonist or the second colony stimulating factor or human c-mpl ligand variant.
  • the co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human c-mpl ligand or native cytokine.
  • in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.
  • Figure 1 schematically illustrates the sequence rearrangement of a protein.
  • the N-termmus (N) and the C- term us (C) of the native protem are joined through a lmker, or joined directly.
  • the protem is opened at a breakpoint creating a new N-termmus (new N) and a new C- term us (new-C) resultmg in a protein with a new linear am o acid sequence.
  • a rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C-termmus and opening of the protem at the breakpoint.
  • Figure 2 shows a schematic of Method I, for creating new protems in which the original N-terminus and C-termmus of the native protem are joined with a linker and different N- termmus and C-termmus of the protem are created.
  • the sequence rearrangement results in a new gene encoding a protein with a new N-termmus created at ammo acid 97 of the original protein, the original C- termmus (a.a. 174) joined to the ammo acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-termmus created at amino acid 96 of the original sequence.
  • Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-termmus and C-terminus of the native protem are joined without a lmker and different N-terminus and C-termmus of the protein are created.
  • the sequence rearrangement results in a new gene encoding a protem with a new N- terminus created at ammo acid 97 of the original protem, the original C-terminus (a.a. 174) joined to the original N- termmus and a new C-termmus created at am o acid 96 of the original sequence.
  • Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created.
  • the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
  • Receptor agonists of the present invention may be useful in the treatment of diseases characterized by a decreased levels of megakaryocyte cells of the hematopoietic system.
  • a c-mpl receptor agonist may be useful in the treatment or prevention of thrombocytopenia.
  • thrombocytopenia Currently the only therapy for thrombocytopenia is platelet transfusions which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization.
  • a c-mpl receptor agonist may alleviate or dimmish the need for platelet transfusions. Severe thrombocytopenia may result from genetic defects such as Fanconi ' s Anemia, Wiscott-Aldrich, or May-Hegglm syndromes.
  • thrombocytopenia may result from auto- or allo-antibodies as Immune Thrombocytopenida Purpura, Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility.
  • splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection or prosthetic heart valves may result in thrombocytopenia.
  • Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer.
  • Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia or fibrosis.
  • the c-mpl receptor agonists of the present mvention may be useful in the mobilization of hematopoietic progenitors and stem cells into peripheral blood.
  • Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation.
  • Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of plasmaphereses required.
  • the c-mpl receptor agonists may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
  • drugs may cause bone marrow suppression or hematopoietic deficiencies.
  • examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics.
  • the c-mpl receptor agonists may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.
  • Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis, c-mpl ligand may be useful in treating such hematopoietic deficiency.
  • a novel method for producing the novel family of human c-mpl receptor agonists involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel c-mpl receptor agonist polypeptide.
  • suitable cells or cell lines may include various strains of E. coli , yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.
  • Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel c-mpl receptor agonists.
  • These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention.
  • Appropriate vectors which can transform microorganisms capable of expressing the c-mpl receptor agonists include expression vectors comprising nucleotide sequences coding for the c-mpl receptor agonists joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.
  • Vectors incorporating modified sequences as described above are mcluded in the present invention and are useful in the production of the c-mpl receptor agonist polypeptides.
  • the vector employed in the method also contams selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.
  • Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above.
  • Such compositions comprise a therapeutically effective amount of one or more of the c-mpl receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously.
  • the therapeutic composition for use m this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • a parenterally acceptable protein solution having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
  • the c-mpl receptor agonists of the present invention may be useful in the mobilization of multipotential hematopoietic progenitors m peripheral blood.
  • Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation.
  • Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem ceils in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required.
  • the c-mpl receptor agonists may be useful in mobilization of multipotential hematopoietic cells and further enhance the efficacy of peripheral blood cell transplantation.
  • the c-mpl receptor agonists of the present invention may also be useful in the ex vivo expansion of multipotential hematopoietic cells.
  • Colony stimulating factors such as hIL-3
  • CSFs colony stimulating factors
  • hIL-3 colony stimulating factors
  • myeloid lineage which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening.
  • Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.
  • Bone marrow transplants have been used to treat this patient population.
  • several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells.
  • Stem cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exits such that a greater number of stem cells will enhance hematopoietic recovery.
  • Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant.
  • Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors.
  • An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back mto the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection.
  • stem cells can be specifically isolated selected based on the presence of specific surface antigen such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
  • 5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separatmg the stem cells from dedicated cells.
  • 5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
  • a growth factor selected from the group consisting of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3 fusion protein and combinations thereof.
  • 5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.
  • WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells.
  • WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.
  • WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokines, for use in transplants.
  • WO 93/08268 describes a method for selectively expanding ste cells, comprising the steps of (a) separating CD34+ stem cells from other cells and (b) incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.
  • WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.
  • WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.
  • WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
  • WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.
  • WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.
  • WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.
  • stem cell refers to the multipotential hematopoietic cells as well as early progenitor and precursors cells which can be isolated from bone marrow, spleen or peripheral blood.
  • expansion refers to the differentiation and proliferation of the cells.
  • the present mvention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells, (b) culturing said separated stem cells with a selected medium which contains a c-mpl receptor agonist and optionally a colony stimulating factor; and (c) harvesting said stems cells.
  • Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics.
  • progenitor marker antigens such as CD34
  • the phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population.
  • the CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affmity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+.
  • CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules .
  • the most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90(thy-l).
  • CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors.
  • the separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers.
  • Various colony stimulating factors may be utilized in order to selectively expand cells.
  • Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3 G-CSF, GM-CSF, IL-l, IL-6, IL-11, flt-3 ligand or combinations thereof.
  • the proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g.
  • hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al . , Blood 73:1836-1841, 1989) .
  • no single factor has been shown to be as effective as the combination of multiple factors.
  • the present invention provides methods for ex vivo expansion that utilize c-mpl receptor agonists that are more effective.
  • Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a c-mpl receptor agonist of the present invention.
  • a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a c-mpl receptor agonist of the present invention.
  • hematopoietic progenitor cells are good candidates for ex vivo gene transfection.
  • Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy.
  • Gene therapy Potential applications include; 1) the treatment of many congenital metabolic disorders and immunodifiencies (Kay and Woo, Trends Genet . 10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet . 10:210-214, 1994) , 3) cancer (Culver and Blaese, Trends Genet . 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet . 10:139-144, 1994) .
  • Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opin . Genet . Dev. 3:102-109, 1993; Boris-Lawrie and Temin, Annal . New York Acad. Sci . 716:59-71, 1994; Miller, Current Top. Microbiol . Immunol .
  • Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA . 90:2122-2126, 1993; Curiel et al. , PNAS USA 88:8850- 8854, 1991; Curiel, Annal. Ne York Acad. Sci . 716:36-58, 1994), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al. , Annal. New York Acad. Sci . 716:23-35, 1994) .
  • the present invention provides an improvement to the existing methods of expanding hematopoietic cells, which new genetic material has been introduced, in that it provides methods utilizing c-mpl receptor agonists that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties.
  • a daily regimen may be in the range of 0.2 - 150 ⁇ g/kg of non- glycosylated c-mpl receptor agonists protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given receptor agonist and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day.
  • c-mpl receptor agonist there may exist specific circumstances where dosages of c-mpl receptor agonist would be adjusted higher or lower than the range of 0.2 - 150 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated c-mpl receptor agonists; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors.
  • a non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, G-CSF, M-CSF, erythropoietin
  • EPO IL-l, IL-4, IL-2, IL-3 , IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 , IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand, (herein collectively referred to as "colony stimulating factors”), or combinations thereof.
  • SCF stem cell factor
  • colony stimulating factors also known as steel factor or c-kit ligand
  • IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention.
  • the c-mpl receptor agonists of the present invention can be co-administered as with another "colony stimulating factor" as discussed above in a fashion taught in WO 95/20976 and WO 95/20977.
  • the dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition.
  • Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.
  • the length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol . Immunol . 20: 483-489, 1983; Kyte & Doolittle, J. Mol . Biol . 157:105- 132, 1992; solvent exposed surface area, Lee & Richards, J. Mol . Biol .
  • linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:3) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
  • Sequences of c-mpl ligand of foldmg to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from withm the original polypeptide chain while using the linker sequence as described above.
  • Ammo and carboxyl termini are selected from withm a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below.
  • a novel ammo acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus.
  • those skilled in the art will recognize that selections of termini anywhere withm the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
  • Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch & Sander, Biopolymers 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann. .Rev. Biochem . 53:537-572, 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber & Mathews, Method ⁇ Enzymol . 154: 511-533, 1987).
  • solvent exposure of residues is a site of post- translational attachment of carbohydrate which is necessarily on the surface of the protein.
  • methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops.
  • Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile & Salvatore, Eur. J. Biochem .
  • regions of ammo acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protem and should also be avoided for selection of ammo and carboxyl termini.
  • those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.
  • Methods for creation of ⁇ enes w th new N-term nus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region.
  • the first primer set (“new start” and “Linker start”) is used to create and amplify, from the gene sequence, the DNA fragment (“Fragment Start”) that contains the sequence encoding the new N-terminal portion of the new protein followed by the linker that connects the C-terminal and N-terminal ends of the original protein.
  • the second primer set (“new stop” and “linker stop”) is used to create and amplify, from the gene sequence, the DNA fragment (“Fragment Stop”) that encodes the same linker as used above, followed by the new C-terminal portion of the new protein.
  • the “new start” and “new stop” primers are designed to include the appropriate restriction sites which allow cloning of the new gene into expression plasmids.
  • Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes.
  • a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
  • a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 UM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12.
  • PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
  • primers "new start” and “new stop” are added to the annealed fragments to create and amplify the full-length new N- terminus/C-terminus gene.
  • Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycle? 94°C denaturation for one minute, 60°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension fov seven minutes.
  • a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
  • a 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2.
  • PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
  • New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation.
  • the steps are illustrated in Figure 3.
  • the first primer set (“new start” and "P-bl start”) is used to create and amplify, from the original gene sequence, the DNA fragment (“Fragment Start”) that contains the sequence encoding the new N-terminal portion of the new protein.
  • the second primer set (“new stop” and "P-bl stop”) is used to create and amplify, from gene sequence, the DNA fragment (“Fragment Stop”) that contains the sequence encoding the new C-terminal portion of the new protein.
  • the “new start” and “new stop” primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for 45 seconds and 72°C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manufacturer.
  • the "P-bl start” and “P-bl stop” primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start” and “Fragment Stop” to each other.
  • a 100 ul reaction contained 150 pmole of each primer and one ug of template DNA; and lx Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit
  • PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
  • PCR reaction products are purified using a Wizard PCR Preps kit (Promega) .
  • the primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors.
  • "Fragment Start” is designed to create Ncol restriction site
  • “Fragment Stop” is designed to create a Hindlll restriction site.
  • Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega) .
  • Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined with and annealed to the ends of the - 3800 base pair Ncol/Hindlll vector fragment of pMON3934 by heating at 50°C for ten minutes and allowed to slow cool.
  • the three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim) .
  • the result is a plasmid containing the full-length new N-terminus/C-terminus gene.
  • a portion of the ligation reaction is used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD) . Plasmid DNA is purified and sequence confirmed as below.
  • New terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng. 5:427- 431, 1992) . Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 4.
  • PCR Polymerase chain reaction
  • the tandemly-duplicated template DNA is created by cloning and contains two similar, but not necessarily identical, copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N-terminal ends of the two copies of the gene.
  • Specific primer sets are used to create and amplify a full-length new N terminus/C- terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72 n extension for one minute; plus one cycle 72°C extension for seven minutes.
  • a Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used.
  • a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 .
  • PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
  • the construction of the PCR templates involves the steps outlined below;
  • Step 1 Reverse transc.rn-r.ase/polvmerase chain reaction
  • c-mpl ligand The two forms of c-mpl ligand, one with a deletion of am o acids 112-115 and one without the deletion, can be isolated RT/PCR technology.
  • Synthetic primers are designed so that they would anneal to either c-mpl ligand DNA or mRNA (c- mpl ligand sequence based on Genebank accession #L33410 or de Sauvage et al., Nature 369, 1994, pp.533 538) for priming first-strand complementary DNA (cDNA) synthesis.
  • the resulting cDNA is used as a template in PCR (Saik , 1985) to generate double-stranded DNA (dsDNA or DNA) which can be used in additional PCR or digested with appropriate restriction enzymes for transfer to E. coli or mammalian expression plasmids.
  • RNA for the reveise transcriptase (RT) reaction, human fetal (lot #38130) and adult liver (lot #46018) A+ RNA can be obtained from Clontech (Palo Alto, CA) .
  • the RT reactions are carried out using a cDNA CycleTM Kit obtamed from Invitrogen (San Diego, CA) .
  • One microgram (ug) of each RNA sample is combmed and denatured at 65°C for 10 mm. in the presence of either random primers, oligo dT primer or a specific 3' anti- sense primer.
  • the samples are cooled for 2 min. on ice and spun down for 10 sec. at 10,000 x g.
  • RNAse inhibitor, reverse transcriptase buffer, deoxynucleotides, sodium pyrophosphate and reverse transcriptase are added as described by manufacturer, and the 20 microliter reaction is cubated at 42°C tor 1 hr.
  • PCR For PCR a specific 5' sense primer and 3' anti-sense primers are added to the RT reactions and the PCR is carried out using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkm-Elmer (Norwalk, CT) as described by the manufacturers using Taq polymerase. The PCR reactions are subjected to 30 cycles of the following; 1 min. @ 94°C, 1 min. @ 58°C, 90 sec. @ 72°C.
  • PCR reactions are purified using a WizardTM PCR Preps kit from Promega (Madison, WI) . Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 mmute incubation at 24°C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resm via vacuum filtration. The column containing the resm is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol.
  • WizardTM PCR Preps kit from Promega (Madison, WI) . Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 mmute incubation
  • the PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7. , via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.
  • Step 2 Subcloning the PCR products mto a mammalian expression vector
  • the c-mpl ligand PCR products are digested with the appropriate restriction enzymes for ligation to a mammalian expression vector.
  • the mammalian expression vector is a derivative of pMON3359 which is a pUCl ⁇ -based vector contammg a mammalian expression cassette.
  • the cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 secretion signal sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned mto the pUCl ⁇ polylinker (Hippenmeyer et al., Bio /Technology, 1037-1041, 1993) .
  • Restriction enzyme digestions are mcubated for 1 hour at 37°C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel. Fragments are first visualized by long-wave UV and gel-purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA) . The DNA fragments are purified from the resin by agarose solublization, addition of a DNA- bindmg resm, and extensive washing of the resm prior to elution with water. The purified DNA products are combined at a molar excess of PCR product to vector fragment and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase.
  • Qiaex DNA Extraction kit Qiagen, Chatsworth, CA
  • An E. coli strain is then transformed with the ligation, plated out onto LB-agar plus ampicillin (100 ug/ml) .
  • the colonies are screened for presence of a c-mpl ligand gene and DNA is isolated for DNA sequencing to identify both forms c-mpl ligand, one with amino acids 112-115 deleted and one with them present.
  • the linkers that join c-mpl (1-153) ligand genes are created by annealing 200 picomoles each of a pair of complementary synthetic oligonucleotides in 5 microliters of ligation buffer (Boehringer-Mannheim #1243 292) .
  • Each linker which has flanking EcoRI and Afllll sites, is ligated overnight with a 3.7 Kbp EcoRI/BstXI fragment from a form of c-mpl ligand with amino acids 1-153 (step 2), and a 1 Kbp NcoI/BstXI fragment from the either of the two types of clones in step 2 above, one with and one without a deletion of amino acids 112-115.
  • the resulting DNA is used to transform E.
  • coli DH5 ⁇ TM cells Transformed cells are selected on LB agar plates containing ampicillin (100 ug/ml). Plasmid DNA is obtained from single colonies of several clones and sequenced to verify correct assembly of the dimer via the linker. The resulting plasmid DNA template can be used in making novel c-mpl ligand molecules via the Horlick method (Prot. Eng. 5:427-433, 1992).
  • 5' sense ana 3' anti-sense primers are combined with a dimer template for PCR using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkin-Elmer (Norwalk, CT) as described by the manufacturersusing Taq polymerase.
  • the PCR reactions are subjected to 30 cycles of the following; 1 min. G. 94°C, 1 min. @ 58°C, 90 sec. @ 72°C.
  • the product (about 480 base pairs) is visualized using a short-wave UV light source.
  • the reactions are purified using a WizardTM PCR Preps kit from Promega (Madison, WI) .
  • PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 minute incubation at 24°C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resin via vacuum filtration. The column containing the resin is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol.
  • the PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7.4, via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.
  • novel c-mpl receptor agonists PCR products are digested with the appropriate restriction enzymes for ligation to either mammalian or E. coli expression vectors.
  • the mammalian expression vectors are derivatives of pMON3359 which is a pUCl ⁇ -based vector containing a mammalian expression cassette.
  • the cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker [See Hippenmeyer et al. , Bio/Technology, 1993, pp.1037-1041] . Restriction enzyme digestions are incubated for 1 hour at 37°C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel.
  • Fragments are first visualized by long-wave UV and gel- purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA) .
  • the DNA fragments are purified from the resin by agarose solublization, addition of a DNA-binding resin, and extensive washing of the resin prior to elution with water.
  • the purified DNA products are combined at a molar excess of PCR product to vector and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase.
  • the £. coli expression vectors that direct high-level production of heterologous proteins in the cytoplasm are derivatives of that described elsewhere (Olins et al., Methods Enzym. , 185:115-119, 1988 and Rangwala et al., Gene, 122: 263-269, 1992).
  • the expression cassette consists of the recA promoter and T7 gene 10 ribosome binding site (RBS) as well as an M13 origin of replication or a tandem inverted repeat of a phage P22 gene which acts as a transcription terminator. These cassettes are on a plasmid with the pBR327 origin of replication and encode a gene either for spectinomycin or ampicillin resistance.
  • E. coli strains DH5 ⁇ TM (Life Technologies, Gaithersburg,
  • E. coli strain MON105 can be obtained from the American Type Culture Collection (ATCC, Rockville, MD) and is the host for expressing alternate forms of c-mpl ligand in the cytoplasm or periplasmic space, respectively, of E. coli .
  • MON105 ATCC#55204 F-, lamda-, IN(rrnD, rrE)l, rpoD+, rpoH358 DH5 ⁇ TM: F-, phi80dlacZdeltaMl5, delta (lacZYA-argF)Ul69 , deoR, recAl, endAl, hsdRl7 (rk-,mk+) , phoA, supE441amda-, thi-1, gyrA96, relAl
  • TGI delta(lac-pro) , supE, thi-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaMl5)
  • DH5 ⁇ TM Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA using a CaCl2 method.
  • 20 to 50 mLs of cells are grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 150 millimolar NaCl) to a density of approximately 1.0 optical density units at 600 nanometers (OD600) as measured by a Baush & Lomb Spectronic spectrophotometer (Rochester, NY) .
  • the cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 millimolar CaCl2, 10 millimolar Tris-Cl, pH7.4) and are held at 4"C for 30 minutes.
  • the cells are again collected by centrifugation and resuspended in one tenth culture volume of CaCl2 solution.
  • Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4'C for 1 hour.
  • the samples are shifted to 42"C for two minute ⁇ and 1.0 mL of LB is added prior to shaking the samples at 37"C for one hour.
  • Cells from these samples are spread on plates (LB medium plus 1.5% bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin- resistant transformants.
  • the plates are incubated overnight at 37'C. Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37'C with shaking.
  • Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking. Before harvesting the cultures, 1 ul of cells are analyzed by PCR for the presence of a c-mpl ligand gene. The PCR is carried out using a combination of primers that anneal to the c-mpl ligand gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. A gene has been ligated to the vector when a PCR product of the expected size is observed.
  • appropriate antibiotic 100 ug/mL ampicillin or 75 ug/mL spectinomycin
  • Plasmid DNA is isolated using the Promega WizardTM Miniprep kit (Madison, WI) or the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) . Both kits follow the same general procedure for plasmid DNA isolation. Briefly, cells are pelleted by centrifugation (5000 x g) , plasmid DNA released with sequential NaOH & acid treatment, and cellular debris is removed by centrifugation (10000 x g) . The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 100 mis of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking. Plasmid DNA is isolated using the Qiagen Plasmid Midi kit
  • Purified recombinant double-stranded DNA is sequenced using the Applied Biosystems Inc. (ABI, Foster City, CA) PRISMTM Ready Reaction DyeDeoxyTM Terminator Sequencing system.
  • the ABI system relies on incorporation of four fluorescence labelled dideoxy nucleotides into single- stranded DNA during multiple rounds of amplification. Plasmid DNA and a sequencing primer are added to the reaction mixture (including Taq DNA polmerase, buffer and nucleotides) , which is subjected to 25 cycles of amplification (30 seconds at 96°C, 15 seconds at 50°C, 4 minutes at 60°C) .
  • the BHK-21 cell line can be obtained from the ATCC (Rockville, MD) .
  • the cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose) , supplemented to 2 millimolar (mM) L-glutamine and 10% fetal bovine serum (FBS).
  • DMEM/high-glucose Dulbecco's modified Eagle media
  • FBS fetal bovine serum
  • This formulation is designated BHK growth media.
  • Selective media is BHK growth media supplemented with 453 units/ml hygromycin B (Caibiochem, San Diego, CA) .
  • the BHK-21 cell line was previously stably transfected with the HSV transactivating protein VPI6, which transactivates the IE110 promoter found on the plasmid pMON3359 (Hippenmeyer et al., Bio/Technology, 1037-1041, 1993).
  • the VP16 protein drives expression of genes inserted behind the IE110 promoter.
  • BHK-21 cells expressing the transactivating protein VP16 is designated BHK-VP16.
  • the plasmid pMONlll ⁇ (Highkin et al.. Poul try Sci . , 70:970-981, 1991) expresses the hygromycin resistance gene from the SV40 promoter.
  • a similar plasmid is available from ATCC, pSV2- hph.
  • BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X IO 5 cells per dish 24 hours prior to transfection.
  • Cells are transfected for 16 hours in 3 mL of "OPTIMEM”TM (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMONlll ⁇ , and 80 ug of Gibco- BRL "LIPOFECTAMINE”TM per dish.
  • the media is subsequently aspirated and replaced with 3 mL of growth media.
  • At 48 hours post-transfection media from each dish is collected and assayed for activity (transient conditioned media) .
  • the cells are removed from the dish by trypsin-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in trypsin/EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluency, the conditioned media is reassayed, and positive clones are expanded into growth media.
  • E. coli strain MON105 harboring the plasmid of interest are grown at 37°C in M9 plus casamino acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey) . Growth is monitored at OD600 until it reaches a value of 1.0 at which time Nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final concentration of 50 ⁇ g/mL. The cultures are then shaken at 37°C for thr.- ⁇ to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microscope for the presence of inclusion bodies (IB) .
  • IB inclusion bodies
  • One mL aliquots of the culture are removed for analysis of protem content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (Maniatis et al. Molecular Cloning: A Laboratory Manual, 1982) .
  • the first step in purification of the protem is either sonication or homogenization of the cells.
  • the cells are resuspended in one-tenth volume (based on culture size) sonication buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA).
  • resuspended cells are subjected to several repeated sonication t, irsts using the microtip from a Sonicator cell disrupter, Model W-375 obtamed from Heat Systems-Ultrasonics Inc. (Farmingdale, New York) .
  • the extent of sonication is monitored by examining the homogenates under a light microscope. After all of the cells are disrupted, the homogenates are fractionated by centrifugation at 10000 x g for 20 mmutes at 4°C in a JA-20 rotor and J2-21 centrifuge (Beckman, Fullerton, CA) .
  • the IBs are released from the cells by lysing the cells in sonication buffer with a Manton-Gaulm homogenizer (Holland) followed by centrifugation as above.
  • the IB pellets which are highly enriched for the recombinant protein, are then subjected to another round of sonication and centrifugation as described above.
  • the recombinant protein is purified by a variety of standard methods.
  • the protein is purified from £. coli contaminants using ion-exhange chomotography, such as Q-sepharose (anion) and S-sepharose (cation) , gel filtration, hydrophobic chromatography or reversed phase HPLC. After dialysis against a low ionic strength buffer, the purified protein is stored frozen or lyophilized.
  • RT reaction random primers and oligo dT primer are used to generate cDNA from a combination of human and fetal liver mRNA.
  • the RT product serves as the template for PCR with a combination of the primers, Forward primer: c-mplNcol (SEQ ID NO:4) and Reverse primer: Ecompl (SEQ ID NO-.5) .
  • the c-mplNcol (SEQ ID NO:4) primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand sequence from Genebank accession #L33410 or de Sauvage et al., Nature 369:533-538, 1994) and encodes a ⁇ col restriction enzyme site just 5' to the first mature codon (Ser ⁇ ) .
  • the ⁇ col restriction enzyme site codes for methionine and alanine codons prior to ser-1 and includes codon degeneracy for the Ala codon and the first four c-mpl ligand codons (Ser 1 , Pro 2 , Ala 3 , & Pro 4 ) .
  • the Ecompl (SEQ ID NO:5) primer anneals to bases #720-737 of c-mpl ligand and encodes a EcoRI recognition site in-frame with the c-mpl ligand gene immediately following Arg 1 ⁇ _ r ⁇ g EcoRI site creates Glu- ⁇ 4 and Phe-*-55 codons following Argl53 _
  • the ca. 480 bp PCR product is purified, digested with Ncol and EcoRI and ligated to the NcoI-EcoRl vector fragment of pMON3993 (ca. 4550 bp.) .
  • pMON3993 is a derivative of pMON3359.
  • the expression cassette in pMON3359 includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See Hippen eyer et al., Bio/Technology, 1037-1041, 1993).
  • the human IL-3 signal peptide sequence which had been subloned as a BamHI fragment into the unique BamHI site between the IE110 promoter and poly-A signal, contains an Ncol site at its 3' end and is then followed by a unique EcoRI site.
  • the DNA sequence of the signal peptide is shown below (restriction enzyme sites are indicated above) .
  • the ATG (methionine) codon within the Ncol site is in-frame with the initiator ATG of the signal peptide (underlined) ; BamHI
  • pMON26458 coding for c-mpl ligand amino acids 1-153, is a result of this cloning.
  • the RT reaction from Example 1 serves as the template for PCR with a combination of the following primers; c-mplNcol (SEQ ID NO:4) (forward primer) and c-mplHindlll (SEQ ID NO: 6) (reverse primer) .
  • the c- mplNcoI (SEQ ID NO:4) primer is described in Example 1.
  • PCR products Two types are generated from the RT cDNA samples, one with a deletion of the codons for amino acids 112-115 and one without the deletion of these codons.
  • the c- mpl ligand PCR products (ca. 480 bp) are digested with Ncol and Hindlll restriction enzymes for transfer to a mammalian expression vector, pMON3934, which is a derivative of pMON3359 (see Example 1) .
  • pMON3934 is digested with Ncol and Hindlll (ca. 3800 bp) and will accept the PCR products.
  • the plasmid, pMON32132 contians the DNA sequence of (SEQ ID NO:82) which encodes for amino acids 1-153 of c-mpl ligand (SEQ ID NO: .4) was a result of this cloning.
  • the plasmid, pMON32133 contains the DNA sequence of (SEQ ID NO:83) which encodes amino acids 1-153 of c-mpl ligand with a deletion of codons 112-115 ( ⁇ 112-115) (SEQ ID NO:45) was also a result of this cloning.
  • a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of PMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32133 (containing a deletion of amino acids 112-115) along with the EcoRI/AflHI 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3 ' (SEQ ID NO:10) .
  • the EcoRI end of the linker will ligate to the EcoRI end of PMON26458.
  • the Afllll end of the linker will ligate to the Ncol site of pMON32133, and neither restriction site will be retained upon ligation.
  • the BstXl sites of pMON26458 and pMON32133 will ligate as well.
  • Plasmid, pMON28548, is a result of the cloning and contains the DNA sequence of (SEQ ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:43).
  • SEQ ID NO:38 the DNA sequence of (SEQ ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:43).
  • a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflHI 4L synthetic oligonucleotide linker 4L-5' (SEQ ID NO:7) and 4L-3 ' (SEQ ID NO:8) .
  • Plasmid, pMON28500 is a result of the cloning and contains the DNA sequence of (SEQ ID NO:39) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:77) linker (4L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:46) .
  • a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of PMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflHI 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3 ' (SEQ ID NO:10).
  • the EcoRI end of the linker will ligate to the EcoRI end of PMON26458.
  • the Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation.
  • PLasmid, pMON28501 is a result of the cloning and contains the DNA sequence of (SEQ ID NO: 40) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker (5L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:47) .
  • a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from PMON32132 along with the EcoRI/AflHI 8L synthetic oligonucleotide linker 8L-5' (SEQ ID NO:ll) and 8L-3 ' (SEQ ID NO:12) .
  • the EcoRI end of the linker will ligate to the EcoRI end of PMON26458.
  • the Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation.
  • Plasmid, pMON32136 is a result of the cloning which contains the DNA sequence of (SEQ ID NO:41) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79) linker (8L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:48) .
  • Novel c-mpl ligand genes are generated using the Horlick method.
  • the PCR reaction was carried out using dimer template pMON28501 and one of the sets of synthetic primer sets below (number refers to first amino acid of new molecule) .
  • the products that are generated are about 480 bp and are purified via Magic PCR Clean up kits (Promega) .
  • Table 4 shows the template, the primer set used in the PCR reaction and the breakpoint for each Example.
  • the expression vector, pMON3934 is digested with Ncol and
  • Hindlll (ca. 3800 bp) and accepts the PCR products as Ncol- Hindlll or Afllll-Hindlll fragments.
  • Table 4 shows the restriction digest of the PCR product and the resulting expression plasmid pMON designation.
  • dimer templates pMON28500, pMON32136 and pMON28548 could be used in the PCR reaction as described in Examples 7-18.
  • Transfected cell lines Cell lines such as Baf/3 cell line can be transfected with a colony stimulating factor receptor, such as the human IL-3 receptor or human c-mpl receptor, which the cell line does not normally have. These transfected cell lines can be used to determine the activity of the ligand for which the receptor has been transfected into the cell line.
  • a colony stimulating factor receptor such as the human IL-3 receptor or human c-mpl receptor
  • One such transfected Baf/3 cell line was made by cloning the cDNA encoding c-mpl from a library made from a c- mpl responsive cell line and cloned into the multiple cloning site of the plasmid pcDNA3 (Invitrogen, San Diego Ca. ) .
  • Baf/3 cells were transfected with the plasmid via electroporation. The cells were grown under G418 selection in the presence of mouse IL-3 in Wehi conditioned media. Clones were established through limited dilution.
  • the BHK expression levels and bioactivity data of some of the c-mpl receptor agonists of the present invention are shown in Table 5.
  • the supernatant from the transfected BHK cells was evaluated for expression of the c-mpl receptor agonists by western analysis using an antobody raised against c-mpl ligand. Constructs that expressed at a "++++" level were assayed in the Baf-3/c-mpl cell proliferation assay.
  • Bone marrow aspirates are obtained from normal allogeneic marrow donors after informed consent.
  • Cells are diluted 1:3 in phosphate buffered saline (PBS, Gibco- BRL) , 30 mL are layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer is collected and washed in PBS.
  • CD34+ cells are enriched from the mononuclear cell preparation using an affinity column per manufacturers instructions (CellPro, Inc, Bothell WA) .
  • CD34+ cells After enrichment, the purity of CD34+ cells is 70% on average as determined by using flow cytometric analysis using anti CD34 monoclonal antibody conjugated to fluorescein and anti-CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose CA) .
  • Cells are resuspended at 40,000 cells/mL in X-Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1 mL is plated in 12-well tissue culture plates (Costar). Human IL-3 variant, PMON13288, is used at 10 ng/mL or 100 ng/mL.
  • Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand are tested by addition of 100 ⁇ l of supernatant added to 1 mL cultures (approximately a 10% dilution) . Cells are incubated at 37°C for 8-14 days at 5% C02 in a 37°C humidified incubator, b. Cell Harvest and Analysis:
  • MK buffer 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 ⁇ m PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,
  • MK buffer 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 ⁇ m PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,
  • DNA analysis cells are made permeable in MK buffer containing 0.5% Tween 20 (Fisher, Fair Lawn NJ) for 20 minutes on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in Propidium Iodide (Caibiochem , La Jolla Ca) (50 ⁇ g/mL) with RNAase (400 U/mL) in 55% v/v MK buffer (200 mOsm) for 1-2 hours on ice. Cells are analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA) .
  • CD41a-FlTC Green fluorescence
  • PI red fluorescence
  • All cells are collected to determine the percent of cells that are CD41+.
  • Data analysis is performed using LYSIS software (Becton Dickinson, San Jose, CA) .
  • Percent of cells expressing the CD41 antigen is obtained from flow cytometry analysis (Percent) .
  • CD34+ enriched population are isolated as described above.
  • Cells are suspended at 25,000 cells/mL with/without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4 mg/mL apo-transferrin, 6.67 ⁇ M FeCl2, 25 ⁇ g/mL CaCl2, 25 ⁇ g/mL L asparagine, 500 ⁇ g/mL E-amino-n-caproic acid and Penicillin/Streptomycin.
  • thrombin Prior to plating into 35 mm plates, thrombin is added (0.25 units/mL) to initiate clot formation.
  • Cells are incubated at 37°C for 13 days at 5% C02 in a 37°C humidified incubator. At the end of the culture period plates are fixed with methanol:acetone (1:3), air dried and stored at -200C until staining.
  • a peroxidase immunocytochemistry staining procedure is used
  • CFU-MK small colonies, 1-2 foci and less that approx. 25 cells
  • BFU MK large, multi-foci colonies with > 25 cells
  • mixed colonies mixture of both positive and negative cells
  • GTCCTTCACA GCAGACTGAG CCAGTGCCCA
  • GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
  • GTCCTTCACA GCAGACTGAG CCAGTGCCCA
  • GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120 CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
  • GTCCTTCACA GCAGACTGAG CCAGTGCCCA
  • GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 600
  • GTCCTTCACA GCAGACTGAG CCAGTGCCCA
  • GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
  • GTCCTTCACA GCAGACTGAG CCAGTGCCCA
  • GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120

Abstract

Disclosed are c-mpl ligand receptor agonists proteins, DNAs which encode the c-mpl ligand receptor agonists proteins, methods of making the c-mpl receptor agonists proteins and methods of using the c-mpl receptor agonists proteins.

Description

NOVEL c-mpl RECEPTOR AGONISTS
The present application claims priority under 35 USC §119 (e) of United States provisional application Serial No. 60/004,384 filed October 05, 1995.
Field of the Invention The present invention relates to human c-mpl receptor agonists (thrombopoietin) with activity on hematopoietic differentiation and expansion.
Background of the Invention Megakaryocyte (MK) maturation and platelet production has been long thought to be regulated by lineage specific humoral growth factors in a manner similar to cytokines that induce erythrocyte (erythropoietin) and granulocyte (G-CSF) expansion and maturation. Platelets are responsible for the prevention of bleeding in response to vascular injury. Therefore, platelet production is a vital component of hematopoietic regulation. Patients undergoing chemotherapy or bone marrow transplantation usually experience severely depressed platelet levels (thrombocytopenia) which may result in life threatening bleeding episodes. Several known growth factors and cytokines have been found to stimulate megakaryocytes and platelet production but most are pleiotropic both in vitro and in vivo (IL-3, IL-6, IL-11, SCF) . Plasma, serum and urine from thrombocytopenic dogs and humans have been found to contain growth factors that have specific megakarypoietic and thrombopoietic activities distinct from all known cytokines. These factors have been termed Meg-CSF, MK-CSF, megakaryocyte growth and development factor (MGDF) , megakaryopoietin, and thrombopoietin but the molecular structure has not been identified until recently. The identification of the thrombopoietic cytokine, c-mpl ligand, originated with the identification of a myeloproliferative leukemia virus (MPLV, Wending et al., Virology 149:242-246, 1986) . Mice infected with this virus gave rise to multi-lineage myeloproliferation. Subsequent studies (Souyri et al. , Cell 63:1137-1147, 1990) demonstrated that the retrovirus encoded an oncogene (v-mpl) that when fused with viral envelope gene gave rise to a membrane anchored protein that resembles the cytoplasmic domain of the hematopoietic growth factor receptor family. V-mpl was used to probe both human and murine RNA libraries for homologous genes. Clones were identified in both species and termed c- mpl (Vigon et al. , PNAS USA 89:5640-5644, 1992; Vigon et al . , Oncogene 8:2607-2615, 1993). C-mpl is a member of the cytokine receptor super-family with regions of homology to mIL-5rc, II_3rc, IL4rc, mEPOrc and mGCSFrc. A chimera of the intracellular domain of c-mpl and the extracellular domain of hIL4rc was transfected into a growth factor dependent cell line (BaF3). Once transfected, the cells proliferated in response to hIL4 indicating that the c-mpl cytoplasmic domain was fully sufficient to transduce a proliferative signal (Skoda et al., EMBO J. 12 (7) :2645-2653, 1993).
Message for c-mpl was found in a number of hematopoietic cell lines using reverse transcriptase polymerase chain reaction (RT PCR) including the pluripotential cell lines TF- 1, Mo-7E, UT-7 and KU812; and erythro/megakaryocytic cell lines HEL, DAMI and K153. Transcripts were also identified in bone marrow, fetal liver, megakaryocytes, platelets and CD34+ enriched cells (Methia et al. , Blood 82(51 :1395-1401, 1993) .
The identification of a putative receptor triggered several investigative teams to search for a naturally occurring ligand for c-mpl. In June of 1994 several simultaneous publications reported on a ligand that bound to c-mpl and had megakaryocytopoietic properties (de Sauvage et al., Nature 369:533-539, 1994; Lok et al., Nature 369:565- 568, 1994; Wendling et al., Nature 369:571 574, 1994) and Bartley et al., Cell 77:1117-1124, 1994) . The ligand named c-mpl ligand or thrombopoietin is a peptide with a predicted molecular mass of 35,000 kDa. The protein has a two domain structure with an amino-terminal domain (153 amino acids) with homology to erythropoietin and a carboxy-terminus rich in serine, threonine and proline residues which also contains several glycosylation sites. There are two potential arginine cleavage sites resulting in two shorter peptides of 25 kDa and 31 kDA forms both of which are biologically active. There is high inter species homology between human, murine, porcine, canine, rat and rabbit c-mpl ligand and most forms are active on all species tested.
C-mpl ligand has been shown to stimulate the differentiation of CD34+ cells into cells megakaryocyte characteristics. CD34+ cells in the presence of c-mpl ligand underwent endomitosis (Kaushansky et al . , Nature 369:568-571, 1994), expressed the megakaryocyte lineage specific cell surface antigen CD41a and had morphology characteristic of megakaryocytes. In vivo administration of c-mpl ligand have given rise to increased circulating platelets in normal mice (Lok et al., Nature 369:565-568, 1994). C-mpl deficient mice generated by gene targeting demonstrated a 85% decrease in circulating platelets and megakaryocytes but had normal amounts of other hematopoietic lineages (Gurney et al. , Science 265:1445-1447, 1994). Absolute thrombocytopenia was not observed in these animals indicating that other cytokines may have some activity in expansion of the MK lineage.
Studies to date show that c-mpl ligand is a cytokine with specific activity on the maturation of megakaryocytes and in platelet production. Other cytokines have been shown to have activity on megakaryocyte expansion and differentiation, including IL-3, IL-6, IL-11 and c-kit ligand. Recent studies have demonstrated that these cytokines (with the exception of IL-3) act by stimulating the production of c-mpl ligand and do not have megakaryocyte stimulating activity by themselves (Kaushansky et al. , PNAS USA 92:3234-3236, 1995) .
The ability of c-mpl ligand to stimulate the proliferation and maturation of megakaryocytes and production of platelets indicates that c-mpl ligand may have therapeutic use in restoring circulating platelets to normal amounts in those cases where the number of platelets have been reduced due to diseases or therapeutic treatments such as radiation and/or chemotherapy.
EP 675,201 Al relates to the c-mpl ligand (Megakaryocyte growth and development factor or MGDF) , allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof. The proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.
WO 95/27732 discloses circularly permuted GM-CSF, G- CSF, IL-2 and IL-4 and fusions with Pseudomonas exotoxin.
A previously published abstract (Eaton et al. , Blood 84(10) Suppl. abstract 948, 1994) reported c-DNA for an alternative splice form of c-mpl ligand identified in man, dog and mouse. The encoded protein has 4 amino deletion at position aa!12-115. Although this molecule showed no activity in their bioassays, mRNA for this variant was found to be abundant in all three species indicating that it may be a naturally occurring alternative form of c-mpl ligand.
Rearrangement of Protein Sequences
In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle, Protein Science 1:191-200, 1992) .
The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., Proc. Natl . Acad . Sci . U. S. A . 76:3218-3222, 1979; Teather & Erfle, J. Bacteriol . 172: 3837-3841, 1990; Schimming et al., Eur. J. Biochem . 204: 13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260:127-130, 1991, MacGregor et al., FEBS Lett . 378:263-266, 1996) . The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton ( J. Mol . Biol . 165:407- 413, 1983) . A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) . The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly α -helix (interleukin-4; Kreitman et al. , Cytokine 7:311-318, 1995), β -sheet (interleukin-1; Horlick et al. , Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate isomerase; Luger et al. , Science 243:206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies:
Enzymes
T4 lysozyme Zhang et al., Biochemistry 32:12311-12318 (1993); Zhang et al., Nature Struct. Biol . 1:434-438 (1995)
dihydrofolate Buchwalder et al., Biochemistry reductase 31:1621-1630 (1994); Protasova et al., Prot . Eng. 7:1373-1377 (1995)
ribonuclease Tl Mullins et al., J. Am . Chem . Soc. 116:5529-5533 (1994); Garrett et al.. Protein Science 5:204-211 (1996)
Bacillus β-glucanse Hahn et al., Proc. Natl . Acad. Sci
U. S. A . 91:10417-10421 (1994) aspartate Yang & Schachman, Proc . Nat l . Acad . transcarbamoylase Sci . U. S. A . 90:11980-11984 (1993)
phosphoribosyl Luger et al. , Science 243:206-210 anthranilate (1989); Luger et al. , Prot. Eng. isomerase 3:249-258 (1990)
pepsin/pepsinogen Lin et al., Protein Science 4:159- 166 (1995)
glyceraldehyde-3- Vignais et al. , Protein Sci ence phosphate dehydro¬ 4:994-1000 (1995) genase
ornithine Li & Coffino, Mol . Cell . Biol decarboxylase 13:2377-2383 (1993)
yeast Ritco-Vonsovici et al., Biochemistry phosphoglycerate 34:16543-16551 (1995) dehydrogenase
Enzyme Inhibitor
baεic pancreatic Goldenberg & Creighton, J. Mol trypsin inhibitor Biol . 165:407-413 (1983)
Cytokines
interleukin-lβ Horlick et al., Protein Eng. 5:427- 431 (1992)
interleukin-4 Kreitman et al. , Cytokine 7:311- 318 (1995)
Tyrosine Kinase Recognit ion Domain
α-spectrin SH3 Viguera , et al . , J. domain Mol . Biol . 247 : 670 - 681 ( 1995 )
Transmembrane Prot ein
omp A Koebnik & Kramer, ". Mol . Biol , 250:617-626 (1995)
Chimeric Protein interleukin-4- Kreitman et al., Proc. Natl . Acad.
Pseudomonas Sci . U. S. A . 91:6889-6893 (1994) . exotoxin fusion molecule
The results of these studies have been highly variable. In many cases substantially lower activity, solubility or thermodynamic stability were observed {£. coJi dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast phosphoglycerate dehydrogenase). In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus β
-glucanase, interleukin-lβ, α-spectrin SH3 domain, pepsinogen, interleukin-4) . In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged α-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4— Pseudomonas exotoxin fusion molecule (Kreitman et al., Proc. Natl . Acad. Sci . U. S . A . 91:6889-6893, 1994; Kreitman et al. , Cancer Res . 55:3357-3363, 1995) .
The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range in the original sequence into short-range interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple folding pathways (Viguera, et al. , J. Mol . Biol . 247:670-681, 1995). In the case of the SH3 domain of α -spectrin, choosing new termini at locations that corresponded to β -hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.
The positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N- terminus to the breakpoint is ca. 10 to 80% of the total sequence length) . The linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc. Natl . Acad. Sci . U. S.A . 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus. Flexible hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al . i J. Mol . Biol . 247:670-681, 1995) compared joining the original N- and C- termini with 3- or 4-residue linkers; the 3-residue linker was less thermodynamically stable. Protasova et al. { Protein Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in connecting the original N-termini of £. coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield. More systematic studies of linker length and composition have not been reported.
SUMMARY OF THE INVENTION
The present invention relates to novel c-mpl receptor agonists of the following formulas:
1. A c-mpl receptor agonist of the Formula:
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer 1 5 10 15
HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro 20 25 30 35
ValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu 40 45 50 55
ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla 60 65 70 75
AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly 80 85 90 95
GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa 100 105 110
XaaGlyArgThrThrAlaHisLysAspProAsnAlallePheLeuSerPheGlnHis 115 120 125 130
LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal 135 140 145 150
ArgArgAlaProProThrThrAlaValProSerArgThrSerLeuValLeuThrLeu 155 160 165 170 AsnGluLeuProAsnArgThrSerGlyLeuLeuGluThrAsnPheThrAlaSerAla 175 180 185 190
ArgThrThrGlySerGlyLeuLeuLysTrpGlnGlnGlyPheArgAlaLysIlePro 195 200 205
GlyLeuLeuAsnGlnThrSerArgSerLeuAspGlnlleProGlyTyrLeuAsnArg 210 215 220 225
IleHisGluLeuLeuAsnGlyThrArgGlyLeuPheProGlyProSerArgArgThr 230 235 240 245
LeuGlyAlaProAspIleSerSerGlyThrSerAspThrGlySerLeuProProAsn 250 255 260 265
LeuGlnProGlyTyrSerProSerProThrHisProProThrGlyGlnTyrThrLeu 270 275 280 285
PheProLeuProProThrLeuProThrProValValGlnLeuHisProLeuLeuPro 290 295 300
AspProSerAlaProThrProThrProThrSerProLeuLeuAsnThrSerTyrThr 305 310 315 320
HisSerGlnAsnLeuSerGlnGluGly (SEQ ID NO:l) 325 330 332
wherein;
Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met;
Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met;
Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or
Asn;
wherein from 1 to 179 amino acids can be deleted from the C- terminus;
wherein the N-termmus is ηomed to the C-termmus directly or through a linker capable of joining the N-termmus to the C-termmus and having new C- and N- termini at ammo acids;
26-27 51-52 108-109
27-28 52-53 109-110
28-29 53-54 110-111
29-30 54-55 111-112
30-31 55-56 112-113
32-33 56-57 113-114
33-34 57-58 114-115
34-35 58-59 115-116
36-37 59-60 116-117
37-38 78-79 117-118
38-39 79-80 118-119
40-41 80-81 119-120
41-42 81-82 120-121
42-43 82-83 121-122
43-44 83-84 122-123
44-45 84-85 123-124
46-47 85-86 124-125
47-48 86-87 125-126
48-49 87-88 126-127
50-51 88-89 or 127-128; and
additionally said c-mpl receptor agonist can be immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine-1) .
2. A c-mpl receptor agonist of the Formula:
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer 1 5 10 15 HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro 20 25 30 35
ValLeuLeuProAlaValAspSerLeuGlyGluTrpLysThrGlnMetGluGlu 40 45 50 55
ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla 60 65 70 75 AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly 80 85 90 95
GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa 100 105 110
XaaGlyArgThrThrAlaHisLysAspProAsnAlallePheLeuSerPheGlnHis 115 120 125 130
LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal 135 140 145 150
Arg (SEQ ID NO:2) 153
wherein;
Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro,
Phe, Trp, or Met; Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met;
Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met; Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn;
wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
26-27 51-52 108-109
27-28 52-53 109-110
28-29 53-54 110-111
29-30 54-55 111-112 30-31 55-56 112-113
32-33 56-57 113-114
33-34 57-58 114-115
34-35 58-59 115-116
36-37 59-60 116-117
37-38 78-79 117-118
38-39 79-80 118-119
40-41 80-81 119-120
41-42 81-82 120-121
42-43 82-83 121-122
43-44 83-84 122-123
44-45 84-85 123-124
46-47 85-86 124-125
47-48 86-87 125-126
48-49 87-88 126-127
50-51 88-89 or 127-128; and
additionally said c-mpl receptor agonist can be immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine- ) .
The more preferred breakpoints at which new C-termmus and N-termmus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-
121, 121-122, 122-123, 123-124, 124-125, 125-126 and 126-127.
The most preferred breakpoints at which new C-termmus and N-termmus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 81-82, 108-109, 115-116, 119-120, 122-123 and 125-126.
These human c-mpl receptor agonists may contain am o acid substitutions, deletions and/or insertions and may also have amino acid deletions at either/or both the N- and C- termini.
The modified human c-mpl receptor agonists of the present mvention can be represented by the Formula: X1-(L) a-X2
wherein; a is 0 or 1;
X1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+l through J;
X2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n; n is an integer ranging from 1 to J-1; and
L is a linker.
In the forumla above the constituent amino acids residues of human c-mpl ligand are numbered sequentially 1 through J from the amino to the carboxy terminus. A pair of adjacent amino acids within this protein may be numbered n and n+l respectively where n is an integer ranging from 1 to J-1. The residue n+l becomes the new N-terminus of the new c- pl receptor agonist and the residue n becomes the new C- terminus of the the new c-mpl receptor agonist.
In a preferred embodiment of the present invention the linker (L) joining the N-terminus to the C-terminus is a polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:73); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74);
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:75); SerGlyGlySerGlyGlySer (SEQ ID NO:76); GluPheGlyAsnMetAla (SEQ ID NO:77); GluPheGlyGlyAsnMetAla (SEQ ID NO:78); GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and GlyGlySerAspMetAlaGly (SEQ ID NO:80) .
The present invention also encompasses recombinant human c-mpl receptor agonists co-administrated with one or more additional colony stimulating factors (CSF) including, cytokines, lymphokines, interleukins, hematopoietic growth factors (herein collectively referred to as "colony stimulating factors") each of which may act through a different and specific cell receptor to initiate complementary biological activities.
These co-administrated molecules may be characterized by having the usual activity of both of the peptides or it may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of human c-mpl receptor agonist or the second colony stimulating factor alone. The co¬ administration may also unexpectedly provide an enhanced effect on the activity or an activity different from that expected by the presence of human c-mpl receptor agonist or the second colony stimulating factor or human c-mpl ligand variant. The co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human c-mpl ligand or native cytokine.
In addition to the use of co-administration of the present invention in vivo, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.
BRIEF DESCRIPTION OF THF. FTGURK.ς
Figure 1 schematically illustrates the sequence rearrangement of a protein. The N-termmus (N) and the C- term us (C) of the native protem are joined through a lmker, or joined directly. The protem is opened at a breakpoint creating a new N-termmus (new N) and a new C- term us (new-C) resultmg in a protein with a new linear am o acid sequence. A rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C-termmus and opening of the protem at the breakpoint.
Figure 2 shows a schematic of Method I, for creating new protems in which the original N-terminus and C-termmus of the native protem are joined with a linker and different N- termmus and C-termmus of the protem are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-termmus created at ammo acid 97 of the original protein, the original C- termmus (a.a. 174) joined to the ammo acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-termmus created at amino acid 96 of the original sequence.
Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-termmus and C-terminus of the native protem are joined without a lmker and different N-terminus and C-termmus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protem with a new N- terminus created at ammo acid 97 of the original protem, the original C-terminus (a.a. 174) joined to the original N- termmus and a new C-termmus created at am o acid 96 of the original sequence. Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
DETAILED DESCRIPTION OF THR INVENTION
Receptor agonists of the present invention may be useful in the treatment of diseases characterized by a decreased levels of megakaryocyte cells of the hematopoietic system.
A c-mpl receptor agonist may be useful in the treatment or prevention of thrombocytopenia. Currently the only therapy for thrombocytopenia is platelet transfusions which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization. A c-mpl receptor agonist may alleviate or dimmish the need for platelet transfusions. Severe thrombocytopenia may result from genetic defects such as Fanconi ' s Anemia, Wiscott-Aldrich, or May-Hegglm syndromes. Acquired thrombocytopenia may result from auto- or allo-antibodies as Immune Thrombocytopenida Purpura, Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility. In addition, splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection or prosthetic heart valves may result in thrombocytopenia. Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer. Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia or fibrosis. The c-mpl receptor agonists of the present mvention may be useful in the mobilization of hematopoietic progenitors and stem cells into peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of plasmaphereses required. The c-mpl receptor agonists may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. The c-mpl receptor agonists may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.
Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis, c-mpl ligand may be useful in treating such hematopoietic deficiency.
As another aspect of the present invention, there is provided a novel method for producing the novel family of human c-mpl receptor agonists. The method of the present invention involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel c-mpl receptor agonist polypeptide. Suitable cells or cell lines may include various strains of E. coli , yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.
Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel c-mpl receptor agonists. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform microorganisms capable of expressing the c-mpl receptor agonists include expression vectors comprising nucleotide sequences coding for the c-mpl receptor agonists joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.
Vectors incorporating modified sequences as described above are mcluded in the present invention and are useful in the production of the c-mpl receptor agonist polypeptides. The vector employed in the method also contams selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells. Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the c-mpl receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously. When administered, the therapeutic composition for use m this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
The c-mpl receptor agonists of the present invention may be useful in the mobilization of multipotential hematopoietic progenitors m peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem ceils in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required. The c-mpl receptor agonists may be useful in mobilization of multipotential hematopoietic cells and further enhance the efficacy of peripheral blood cell transplantation.
The c-mpl receptor agonists of the present invention may also be useful in the ex vivo expansion of multipotential hematopoietic cells. Colony stimulating factors (CSFs), such as hIL-3, have been administered alone, co-administered with other colony stimulating factors, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result of such treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated. The myeloid lineage, which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening. Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy. Bone marrow transplants have been used to treat this patient population. However, several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells. Stem cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exits such that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival. Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant. However, Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors. An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back mto the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells. The limited number of stem cells may be overcome by ex-vivo expansion of the stem cells. In addition, stem cells can be specifically isolated selected based on the presence of specific surface antigen such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
The following patents contain further details on separating stem cellε, CD34+ cells, culturmg the cells with hematopoietic factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.
5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separatmg the stem cells from dedicated cells.
5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.
WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells.
WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.
WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokines, for use in transplants.
WO 93/08268 describes a method for selectively expanding ste cells, comprising the steps of (a) separating CD34+ stem cells from other cells and (b) incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.
WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.
WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia. WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.
WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.
WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.
The long term cultur g of highly enriched CD34+ precursors of hematopoietic progenitor cells from human bone marrow in cultures containing IL-la, IL-3, IL-6 or GM-CSF is discussed in Brandt et al J. Clm . Invest . 86:932-941, 1990).
One aspect of the present invention provides a method for selective ex-vivo expansion of stem cells. The term "stem cell" refers to the multipotential hematopoietic cells as well as early progenitor and precursors cells which can be isolated from bone marrow, spleen or peripheral blood. The term "expansion" refers to the differentiation and proliferation of the cells. The present mvention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells, (b) culturing said separated stem cells with a selected medium which contains a c-mpl receptor agonist and optionally a colony stimulating factor; and (c) harvesting said stems cells. Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics. The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affmity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules . The most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90(thy-l).
Other surface antigens such as CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors. The separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers. Various colony stimulating factors may be utilized in order to selectively expand cells. Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3 G-CSF, GM-CSF, IL-l, IL-6, IL-11, flt-3 ligand or combinations thereof. The proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to cubation. Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various colony stimulating factors including c-kit liganα (Brandt et al., Blood 83:1507-1514, 1994; McKenna et al., Blood 86:3413-3420, 1995); IL-3 (Brandt et al., Blood 83:1507-1514, 1994; Sato et al., Blood 82:3600- 3609, 1993), G-CSF (Sato et al., Blood 82:3600-3609, 1993), GM-CSF (Sato et al., Blood 82:3600-3609, 1993), IL-l (Muench et al., Blood 81:3463-3473, 1993), IL-6 (Sato et al. , Blood 82:3600-3609, 1993) , IL-11 (Lemoli et al., Exp . Hem. 21:1668- 1672, 1993; Sato et al., Blood 82:3600-3609, 1993), flt-3 ligand (McKenna et al., Blood 86:3413 3420, 1995) and/or combinations thereof (Brandt et al., Blood 83:1507 1514, 1994; Haylock et al. , Blood 80:1405-1412, 1992; Roller et al., Biotechnology 11:358-363, 1993; (Lemoli et al. , Exp.
Hem. 21:1668-1672, 1993), McKenna et al., Bl ood 86:3413-3420, 1995; Muench et al. , Blood 81:3463-3473, 1993; Patchen et al., Biotherapy 7:13-26, 1994; Sato et al., Blood 82:3600- 3609, 1993; Smith et al., Exp. Hem. 21:870-877, 1993; Steen et al., Stem Cells 12:214-224, 1994; Tsujino et al. , Exp. Hem. 21:1379-1386, 1993). Among the individual colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al . , Blood 73:1836-1841, 1989) . However, no single factor has been shown to be as effective as the combination of multiple factors. The present invention provides methods for ex vivo expansion that utilize c-mpl receptor agonists that are more effective.
Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a c-mpl receptor agonist of the present invention.
Another projected clinical use of growth factors has been in the in vitro activation of hematopoietic progenitors and stem cells for gene therapy. Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are good candidates for ex vivo gene transfection. In order to have the gene of interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy. Potential applications of gene therapy (review Crystal, Science 270:404-410, 1995) include; 1) the treatment of many congenital metabolic disorders and immunodifiencies (Kay and Woo, Trends Genet . 10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet . 10:210-214, 1994) , 3) cancer (Culver and Blaese, Trends Genet . 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet . 10:139-144, 1994) .
There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells. Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opin . Genet . Dev. 3:102-109, 1993; Boris-Lawrie and Temin, Annal . New York Acad. Sci . 716:59-71, 1994; Miller, Current Top. Microbiol . Immunol . 158:1-24, 1992) and replication-de icient recombinant adenovirus (Berkner, BioTechniques 6:616-629, 1988; Berkner, Current Top. Microbiol . Immunol . 158:39-66, 1992; Brody and Crystal, Annal . New York Acad. Sci . 716:90-103, 1994) . Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA . 90:2122-2126, 1993; Curiel et al. , PNAS USA 88:8850- 8854, 1991; Curiel, Annal. Ne York Acad. Sci . 716:36-58, 1994), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al. , Annal. New York Acad. Sci . 716:23-35, 1994) .
The present invention provides an improvement to the existing methods of expanding hematopoietic cells, which new genetic material has been introduced, in that it provides methods utilizing c-mpl receptor agonists that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties.
The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action cf drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.2 - 150 μg/kg of non- glycosylated c-mpl receptor agonists protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given receptor agonist and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day. In addition, there may exist specific circumstances where dosages of c-mpl receptor agonist would be adjusted higher or lower than the range of 0.2 - 150 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated c-mpl receptor agonists; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, G-CSF, M-CSF, erythropoietin
(EPO), IL-l, IL-4, IL-2, IL-3 , IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 , IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand, (herein collectively referred to as "colony stimulating factors"), or combinations thereof. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention. The c-mpl receptor agonists of the present invention can be co-administered as with another "colony stimulating factor" as discussed above in a fashion taught in WO 95/20976 and WO 95/20977. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.
Determination of the Linker.
The length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol . Immunol . 20: 483-489, 1983; Kyte & Doolittle, J. Mol . Biol . 157:105- 132, 1992; solvent exposed surface area, Lee & Richards, J. Mol . Biol . 55:379-400, 1971) and the ability to adopt the necessary conformation without deranging the conformation of the c-mpl receptor agonist (conformationally flexible; Karplus & Schulz, Naturwissenschaften 72:212-213, 1985). Assuming an average of translation of 2.0 to 3.8 A per residue, this would mean the length to test would be between 0 to 30 residues, with 0 to 15 residues being the preferred range. Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly- Gly-Ser (SEQ ID NO:3) repeated n times, where n is 1, 2, 3 or 4. Those skilled in the art will recognize that there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short (cf., Sandhu, Cri tical Rev. Biotech . 12: 437-462, 1992); if they are too long, entropy effects will likely destabilize the three- dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain.
Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that must be tested in an empirical selection of linkers. They will also recognize that it is sometimes the case that the positions of the ends of the polypeptide chain are ill- defined in structural models derived from x-ray diffraction or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their c- alpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8A per residue) are then selected. These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:3) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
Determination of the Amino and Carboxyl T rmim of c-mpl liαand
Sequences of c-mpl ligand of foldmg to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from withm the original polypeptide chain while using the linker sequence as described above. Ammo and carboxyl termini are selected from withm a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel ammo acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, those skilled in the art will recognize that selections of termini anywhere withm the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch & Sander, Biopolymers 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann. .Rev. Biochem . 53:537-572, 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber & Mathews, Methodε Enzymol . 154: 511-533, 1987). In some cases additional information is known about solvent exposure of residues; one example is a site of post- translational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available, or is not feasible to obtain, methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops. Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile & Salvatore, Eur. J. Biochem . 218:603-621, 1993) Thus using either the experimentally derived structural mformation or predictive methods (e.g., Srmivisan & Rose Proteins : Struct . , Funct . & Genetics, 22: 81-99, 1995) the parental am o acid sequence is inspected to classify regions according to whether or not they are mtegral to the maintenance of secondary and tertiary structure. The occurrence of sequences withm regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of ammo acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so- called hydrophobic core of the protem and should also be avoided for selection of ammo and carboxyl termini. In contrast, those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.
All references, patents or applications cited herein are incorporated by reference in their entirety.
Materials and Methods
Unless noted otherwise, all specialty chemicals were obtained from Sigma Co., (St. Louis, MO). Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA) .
Methods for creation of σenes w th new N-term nus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region.
Genes with new N-terminus/C-terminus which contain a linker region separating the original C-terminus and N- terminus can be made essentially following the method described in L. S. Mullins, et al (J. Am. Chem . Soc . 116, 5529-5533, 1994) . Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. The steps are illustrated in Figure 2.
In the first step, the first primer set ("new start" and "Linker start") is used to create and amplify, from the gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N-terminal portion of the new protein followed by the linker that connects the C-terminal and N-terminal ends of the original protein. In the second step, the second primer set ("new stop" and "linker stop") is used to create and amplify, from the gene sequence, the DNA fragment ("Fragment Stop") that encodes the same linker as used above, followed by the new C-terminal portion of the new protein. The "new start" and "new stop" primers are designed to include the appropriate restriction sites which allow cloning of the new gene into expression plasmids. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 UM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12. PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . "Fragment Start" and "Fragment Stop", which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the full- length gene encoding the new protein. The DNA fragments "Fragment Start" and "Fragment Stop" are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined in equimolar quantities, heated at 70°C for ten minutes and slow cooled to allow annealing through their shared sequence in "Linker start" and "linker stop". In the third PCR step, primers "new start" and "new stop" are added to the annealed fragments to create and amplify the full-length new N- terminus/C-terminus gene. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycle? 94°C denaturation for one minute, 60°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension fov seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2. PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
Method II. Creation of genes with new N-terminus/C-terminus without a linker region.
New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation. The steps are illustrated in Figure 3. In the first step, the first primer set ("new start" and "P-bl start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N-terminal portion of the new protein. In the second step, the second primer set ("new stop" and "P-bl stop") is used to create and amplify, from gene sequence, the DNA fragment ("Fragment Stop") that contains the sequence encoding the new C-terminal portion of the new protein. The "new start" and "new stop" primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for 45 seconds and 72°C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manufacturer. The "P-bl start" and "P-bl stop" primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start" and "Fragment Stop" to each other. A 100 ul reaction contained 150 pmole of each primer and one ug of template DNA; and lx Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit
Deep Vent polymerase. PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . PCR reaction products are purified using a Wizard PCR Preps kit (Promega) .
The primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typically "Fragment Start" is designed to create Ncol restriction site , and "Fragment Stop" is designed to create a Hindlll restriction site. Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega) . Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined with and annealed to the ends of the - 3800 base pair Ncol/Hindlll vector fragment of pMON3934 by heating at 50°C for ten minutes and allowed to slow cool. The three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim) . The result is a plasmid containing the full-length new N-terminus/C-terminus gene. A portion of the ligation reaction is used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD) . Plasmid DNA is purified and sequence confirmed as below.
Method III. Creation of new N-terminus/C-terminus genes by tandem-duplication method
New terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng. 5:427- 431, 1992) . Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 4.
The tandemly-duplicated template DNA is created by cloning and contains two similar, but not necessarily identical, copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N-terminal ends of the two copies of the gene. Specific primer sets are used to create and amplify a full-length new N terminus/C- terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72n extension for one minute; plus one cycle 72°C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2.
PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
The construction of the PCR templates involves the steps outlined below;
1. Generation of the c-mpl ligand gene with and without ammo acid codons 112-115 via reverse transcriptase/polymerase chain reaction (RT/PCR) . Human liver contains c-mpl ligand mRNA with and without a deletion of ammo acids 112-115. 2. Subcloning the PCR products into an mammalian expression vector.
3. Assembly of dimer templates as genel/linker/genell from; l) a gene encoding c-mpl ligand amino acids 1-153 for the genel position; n) unique synthetic linkers for separation of the two genes; and m) a gene encoding c-mpl ligand ammo acids 1-153, with or without ammo acids 112-115 for the genell position.
Step 1: Reverse transc.rn-r.ase/polvmerase chain reaction
The two forms of c-mpl ligand, one with a deletion of am o acids 112-115 and one without the deletion, can be isolated RT/PCR technology. Synthetic primers are designed so that they would anneal to either c-mpl ligand DNA or mRNA (c- mpl ligand sequence based on Genebank accession #L33410 or de Sauvage et al., Nature 369, 1994, pp.533 538) for priming first-strand complementary DNA (cDNA) synthesis. The resulting cDNA is used as a template in PCR (Saik , 1985) to generate double-stranded DNA (dsDNA or DNA) which can be used in additional PCR or digested with appropriate restriction enzymes for transfer to E. coli or mammalian expression plasmids.
For the reveise transcriptase (RT) reaction, human fetal (lot #38130) and adult liver (lot #46018) A+ RNA can be obtained from Clontech (Palo Alto, CA) . The RT reactions are carried out using a cDNA Cycle™ Kit obtamed from Invitrogen (San Diego, CA) . One microgram (ug) of each RNA sample is combmed and denatured at 65°C for 10 mm. in the presence of either random primers, oligo dT primer or a specific 3' anti- sense primer. Followmg denaturation, the samples are cooled for 2 min. on ice and spun down for 10 sec. at 10,000 x g. RNAse inhibitor, reverse transcriptase buffer, deoxynucleotides, sodium pyrophosphate and reverse transcriptase are added as described by manufacturer, and the 20 microliter reaction is cubated at 42°C tor 1 hr.
For PCR a specific 5' sense primer and 3' anti-sense primers are added to the RT reactions and the PCR is carried out using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkm-Elmer (Norwalk, CT) as described by the manufacturers using Taq polymerase. The PCR reactions are subjected to 30 cycles of the following; 1 min. @ 94°C, 1 min. @ 58°C, 90 sec. @ 72°C. An equal volume of loading dye (0.01% each bromophenol blue and xylene cyanole blue) is added to 10 microliters of the final product for electrophoresis through a 1% SeaKemR LE agarose (FMC, Rockland, ME) gel in the presence of lx TBE/EtBr (Tris-borate-EDTA plus ethidium bromide;Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Horbor, NY, 1989) . For molecular weight standards, 1 microgram of phιXl74 phage DNA digested with Haelll restriction enzyme (New England Biolabs, Beverly, MA) is loaded onto the gel. The product (about 1090 base pairs) is visualized using a short-wave UV light source. The reactions 97/12978 PC17US96/15938
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are purified using a Wizard™ PCR Preps kit from Promega (Madison, WI) . Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 mmute incubation at 24°C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resm via vacuum filtration. The column containing the resm is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol. The PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7. , via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.
Step 2 : Subcloning the PCR products mto a mammalian expression vector
The c-mpl ligand PCR products are digested with the appropriate restriction enzymes for ligation to a mammalian expression vector. The mammalian expression vector is a derivative of pMON3359 which is a pUClδ-based vector contammg a mammalian expression cassette. The cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 secretion signal sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned mto the pUClδ polylinker (Hippenmeyer et al., Bio /Technology, 1037-1041, 1993) . Restriction enzyme digestions are mcubated for 1 hour at 37°C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel. Fragments are first visualized by long-wave UV and gel-purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA) . The DNA fragments are purified from the resin by agarose solublization, addition of a DNA- bindmg resm, and extensive washing of the resm prior to elution with water. The purified DNA products are combined at a molar excess of PCR product to vector fragment and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase. An E. coli strain is then transformed with the ligation, plated out onto LB-agar plus ampicillin (100 ug/ml) . The colonies are screened for presence of a c-mpl ligand gene and DNA is isolated for DNA sequencing to identify both forms c-mpl ligand, one with amino acids 112-115 deleted and one with them present.
3. Assembly of dimer PCR templates
The linkers that join c-mpl (1-153) ligand genes are created by annealing 200 picomoles each of a pair of complementary synthetic oligonucleotides in 5 microliters of ligation buffer (Boehringer-Mannheim #1243 292) . Each linker, which has flanking EcoRI and Afllll sites, is ligated overnight with a 3.7 Kbp EcoRI/BstXI fragment from a form of c-mpl ligand with amino acids 1-153 (step 2), and a 1 Kbp NcoI/BstXI fragment from the either of the two types of clones in step 2 above, one with and one without a deletion of amino acids 112-115. The resulting DNA is used to transform E. coli DH5α™ cells. Transformed cells are selected on LB agar plates containing ampicillin (100 ug/ml). Plasmid DNA is obtained from single colonies of several clones and sequenced to verify correct assembly of the dimer via the linker. The resulting plasmid DNA template can be used in making novel c-mpl ligand molecules via the Horlick method (Prot. Eng. 5:427-433, 1992).
B. Horlick Method
5' sense ana 3' anti-sense primers are combined with a dimer template for PCR using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkin-Elmer (Norwalk, CT) as described by the manufacturersusing Taq polymerase. The PCR reactions are subjected to 30 cycles of the following; 1 min. G. 94°C, 1 min. @ 58°C, 90 sec. @ 72°C. The product (about 480 base pairs) is visualized using a short-wave UV light source. The reactions are purified using a Wizard™ PCR Preps kit from Promega (Madison, WI) . Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 minute incubation at 24°C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resin via vacuum filtration. The column containing the resin is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol. The PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7.4, via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.
Subcloning novel c-mpl receptor agonists into expression vectors
The novel c-mpl receptor agonists PCR products are digested with the appropriate restriction enzymes for ligation to either mammalian or E. coli expression vectors.
Mammalian Expression Vectors
The mammalian expression vectors are derivatives of pMON3359 which is a pUClβ-based vector containing a mammalian expression cassette. The cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker [See Hippenmeyer et al. , Bio/Technology, 1993, pp.1037-1041] . Restriction enzyme digestions are incubated for 1 hour at 37°C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel. Fragments are first visualized by long-wave UV and gel- purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA) . The DNA fragments are purified from the resin by agarose solublization, addition of a DNA-binding resin, and extensive washing of the resin prior to elution with water. The purified DNA products are combined at a molar excess of PCR product to vector and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase.
E. coli Expression Vectors
The £. coli expression vectors that direct high-level production of heterologous proteins in the cytoplasm are derivatives of that described elsewhere (Olins et al., Methods Enzym. , 185:115-119, 1988 and Rangwala et al., Gene, 122: 263-269, 1992). The expression cassette consists of the recA promoter and T7 gene 10 ribosome binding site (RBS) as well as an M13 origin of replication or a tandem inverted repeat of a phage P22 gene which acts as a transcription terminator. These cassettes are on a plasmid with the pBR327 origin of replication and encode a gene either for spectinomycin or ampicillin resistance.
Transformation of E. coli strains E. coli strains DH5α™ (Life Technologies, Gaithersburg,
MD) and TGI (Amersham Corp., Arlington Heights, IL) are used for all transformation of ligation reactions and are the source of plasmid DNA for transfecting mammalian cells. E. coli strain MON105 can be obtained from the American Type Culture Collection (ATCC, Rockville, MD) and is the host for expressing alternate forms of c-mpl ligand in the cytoplasm or periplasmic space, respectively, of E. coli .
MON105 ATCC#55204: F-, lamda-, IN(rrnD, rrE)l, rpoD+, rpoH358 DH5α™: F-, phi80dlacZdeltaMl5, delta (lacZYA-argF)Ul69 , deoR, recAl, endAl, hsdRl7 (rk-,mk+) , phoA, supE441amda-, thi-1, gyrA96, relAl
TGI: delta(lac-pro) , supE, thi-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaMl5)
DH5α™ Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA using a CaCl2 method. Typically, 20 to 50 mLs of cells are grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 150 millimolar NaCl) to a density of approximately 1.0 optical density units at 600 nanometers (OD600) as measured by a Baush & Lomb Spectronic spectrophotometer (Rochester, NY) . The cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 millimolar CaCl2, 10 millimolar Tris-Cl, pH7.4) and are held at 4"C for 30 minutes. The cells are again collected by centrifugation and resuspended in one tenth culture volume of CaCl2 solution. Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4'C for 1 hour. The samples are shifted to 42"C for two minuteε and 1.0 mL of LB is added prior to shaking the samples at 37"C for one hour. Cells from these samples are spread on plates (LB medium plus 1.5% bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin- resistant transformants. The plates are incubated overnight at 37'C. Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37'C with shaking.
Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking. Before harvesting the cultures, 1 ul of cells are analyzed by PCR for the presence of a c-mpl ligand gene. The PCR is carried out using a combination of primers that anneal to the c-mpl ligand gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. A gene has been ligated to the vector when a PCR product of the expected size is observed.
DNA isolation and characterization
Plasmid DNA is isolated using the Promega Wizard™ Miniprep kit (Madison, WI) or the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) . Both kits follow the same general procedure for plasmid DNA isolation. Briefly, cells are pelleted by centrifugation (5000 x g) , plasmid DNA released with sequential NaOH & acid treatment, and cellular debris is removed by centrifugation (10000 x g) . The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 100 mis of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking. Plasmid DNA is isolated using the Qiagen Plasmid Midi kit
(Chatsworth, CA) which is a scaled-up version of the Qiagen QIAwell Plasmid isolation kit described earlier. The DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian or E. coli cells.
Purified recombinant double-stranded DNA is sequenced using the Applied Biosystems Inc. (ABI, Foster City, CA) PRISM™ Ready Reaction DyeDeoxy™ Terminator Sequencing system. The ABI system relies on incorporation of four fluorescence labelled dideoxy nucleotides into single- stranded DNA during multiple rounds of amplification. Plasmid DNA and a sequencing primer are added to the reaction mixture (including Taq DNA polmerase, buffer and nucleotides) , which is subjected to 25 cycles of amplification (30 seconds at 96°C, 15 seconds at 50°C, 4 minutes at 60°C) . Following amplification, unincorporated nucleotides are removed using Centri-Sep spin columns (equilibrated in water) as described by Princeton Separations, Inc. (Adelphia, NJ) . Briefly, the samples are loaded onto a column that has excess water removed by centrifugation (700 x g) for 2 minutes, and the purified sequencing product is eluted by centrifugation (700 x g) for 4 minutes. The samples are then dried down in a Speed Vac (Savant, Hicksville, NY) prior to addition of loading solution. The samples are electrophoresed through a 4.75% polyacrylamide sequencing gel containing 7M urea in IX TBE at 70 watts constant power. The ABI system uses a detector that recognizes each differntially labelled PCR product as they are being subjected to electrophoresis.
Production of novel c-mpl receptor agonists
Mammalian Cell Transfection/Production of Conditioned Media The BHK-21 cell line can be obtained from the ATCC (Rockville, MD) . The cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose) , supplemented to 2 millimolar (mM) L-glutamine and 10% fetal bovine serum (FBS). This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/ml hygromycin B (Caibiochem, San Diego, CA) . The BHK-21 cell line was previously stably transfected with the HSV transactivating protein VPI6, which transactivates the IE110 promoter found on the plasmid pMON3359 (Hippenmeyer et al., Bio/Technology, 1037-1041, 1993). The VP16 protein drives expression of genes inserted behind the IE110 promoter. BHK-21 cells expressing the transactivating protein VP16 is designated BHK-VP16. The plasmid pMONlllδ (Highkin et al.. Poul try Sci . , 70:970-981, 1991) expresses the hygromycin resistance gene from the SV40 promoter. A similar plasmid is available from ATCC, pSV2- hph.
BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X IO5 cells per dish 24 hours prior to transfection. Cells are transfected for 16 hours in 3 mL of "OPTIMEM"™ (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMONlllδ, and 80 ug of Gibco- BRL "LIPOFECTAMINE"™ per dish. The media is subsequently aspirated and replaced with 3 mL of growth media. At 48 hours post-transfection, media from each dish is collected and assayed for activity (transient conditioned media) . The cells are removed from the dish by trypsin-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in trypsin/EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluency, the conditioned media is reassayed, and positive clones are expanded into growth media.
Expression and purification of recombinant protein from E. col i
E. coli strain MON105 harboring the plasmid of interest are grown at 37°C in M9 plus casamino acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey) . Growth is monitored at OD600 until it reaches a value of 1.0 at which time Nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final concentration of 50 μg/mL. The cultures are then shaken at 37°C for thr.-β to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microscope for the presence of inclusion bodies (IB) . One mL aliquots of the culture are removed for analysis of protem content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (Maniatis et al. Molecular Cloning: A Laboratory Manual, 1982) . After centrifugation (5000 x g) to pellet the cells, the first step in purification of the protem is either sonication or homogenization of the cells. For sonication, the cells are resuspended in one-tenth volume (based on culture size) sonication buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA). These resuspended cells are subjected to several repeated sonication t, irsts using the microtip from a Sonicator cell disrupter, Model W-375 obtamed from Heat Systems-Ultrasonics Inc. (Farmingdale, New York) . The extent of sonication is monitored by examining the homogenates under a light microscope. After all of the cells are disrupted, the homogenates are fractionated by centrifugation at 10000 x g for 20 mmutes at 4°C in a JA-20 rotor and J2-21 centrifuge (Beckman, Fullerton, CA) . Alternatively, the IBs are released from the cells by lysing the cells in sonication buffer with a Manton-Gaulm homogenizer (Holland) followed by centrifugation as above. The IB pellets, which are highly enriched for the recombinant protein, are then subjected to another round of sonication and centrifugation as described above. The recombinant protein is purified by a variety of standard methods. The most common methods mvolve solublization of the IBs with 4-6 molar urea or guanidme-HCl buffers at pH 9-12, and air oxidation/foldmg in the presence of cataytic concentrations of cysteine, beta-mercaptoethanol or dithiothreitol for 24 to 72 hours. The protein is purified from £. coli contaminants using ion-exhange chomotography, such as Q-sepharose (anion) and S-sepharose (cation) , gel filtration, hydrophobic chromatography or reversed phase HPLC. After dialysis against a low ionic strength buffer, the purified protein is stored frozen or lyophilized.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Additional details about recombinant DNA methods which may be used to create the variants, express them in bacteria, mammalian cells or insect cells, purification and refold of the desired proteins and assays for determining the bioactvity of the proteins may be found in co-filed United States Patent Applications WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO 95/21254, and US Serial No. 08/383,035 which are hereby incorporated by reference in their entirety.
Further details known to those skilled in the art may be found in T. Maniatis, et al. , Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference; and in J.
Sambrook, et al. , Molecular Cloning. A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference. All references, patents or applications cited herein are incorporated by reference in their entirety.
TABLE 1 OLIGONUCLEOTIDES c-mplNcol ACGTCCATGGCNTCNCCNGCNCCNCCTGCTTGTGCACTCCGAGTC (SEQ ID NO:4) N=A,C,G or T
Ecompl ATGCACGAATTCCCTGACGCAGAGGGTGGA (SEQ ID NO: 5) c-mplHindlll TGACAAGCTTACCTGACGCAGAGGGTGGACCCT (SEQ ID NO: 6)
4L-5' AATTCGGCAA (SEQ ID NO:7)
4L-3' CATGTTGCCG (SEQ ID NO: 8)
5L-5' AATTCGGCGGCAA (SEQ ID NO: 9)
5L-3' CATGTTGCCGCCG (SEQ ID NO: 10)
8L-5' AATTCGGCGGCAACGGCGGCAA (SEQ ID NO: 11)
8L-3' CATGTTGCCGCCGTTGCCGCCG (SEQ ID NO: 12)
31-5' CGATCCATGGAGGTTCACCCTTTGCCT (SEQ ID NO: 13)
31-3' GATCAAGCTTATGGGCACTGGCTCAGTCT (SEQ ID NO: 14)
35-5' CGATACATGTTGCCTACACCTGTCCTG (SEQ ID NO: 15)
35-3' GATCAAGCTTAAGGGTGAACCTCTGGGCA (SEQ ID NO: 16)
39-5' CGATCCATGGTCCTGCTGCCTGCTGTG (SEQ ID NO: 17)
39-3' GATCAAGCTTAAGGTGTAGGCAAAGGGTG (SEQ ID NO: 18)
43-5' CGATCCATGGCTGTGGACTTTAGCTTGGGA (SEQ ID NO: 19)
43-3' GATCAAGCTTAAGGCAGCAGGACAGGTGT (SEQ ID NO: 20)
45-5' CGATCCATGGACTTTAGCTTGGGAGAA (SEQ ID NO: 21)
45-3' GATCAAGCTTACACAGCAGGCAGCAGGAC (SEQ ID NO: 22)
49-5' CGATCCATGGGAGAATGGAAAACCCAG (SEQ ID NO: 23)
49-3' GATCAAGCTTACAAGCTAAAGTCCACAGC (SEQ ID NO: 24)
82-5' CGATCCATGGGACCCACTTGCCTCTCA (SEQ ID NO:25)
82-3' GATCAAGCTTACAGTTGTCCCCGTGCTGC (SEQ ID NO: 26) 109-5' CAGTCCATGGGAACCCAGCTTCCTCCA (SEQ ID NO:27)
109-3' GATCAAGCTTAAAGGAGGCTCTGCAGGGC (SEQ ID NO-.28) 116-5' CGATCCATGGGCAGGACCACAGCTCAC (SEQ ID NO:29)
116-3' GATCAAGCTTACTGTGGAGGAAGCTGGGTT (SEQ ID NO:30)
120-5' CGATCCATGGCTCACAAGGATCCCAATGCC (SEQ ID NO:31)
120-3' GATCAAGCTTATGTGGTCCTGCCCTGTGG (SEQ ID NO:32)
123-5' CGATCCATGGATCCCAATGCCATCTTCCTG (SEQ ID NO:33) 123-3' GATCAAGCTTACTTGTGAGCTGTGGTCCT (SEQ ID NO:34)
126-5' CGATCCATGGCCATCTTCCTGAGCTTCCAA (SEQ ID NO:35)
126-3' GATCAAGCTTAATTGGGATCCTTGTGAGCTGT (SEQ ID NO:36)
TABLE 2 GENE SEQUENCES
PMON26458
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGA'- AGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC (SEQ ID NO: 37) ;
PMON28548
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC
TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC AGAGCCTCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCT GAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACC CTCTGCGTCAGG (SEQ ID NO:38) ; PMON32132
TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO: 82)
PMON32133
TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGC TCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCC ATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAG GGTCCACCCTCTGCGTCAGG (SEQ ID NO:83)
PMON28500
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCAACATGGCGTCTCCCGCTCC GCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGG ACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGG AGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGA GCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGC CATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGA GGGTCCACCCTCTGCGTCAGG (SEQ ID NO:39); PMON28501
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC
AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC AGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAA
TGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA GGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:40);
PMON32136
TCCCCAGCGCCgCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG TCCTTCACAGCA"-ACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACGGCGGCAACATGGC GTCCCCAGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCAT GTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGC TGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACA GGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTG GGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAA GGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:41);
PMON30373
GAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTG CTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTG GGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACC CAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC TTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTC TGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGA GTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCA (SEQ ID NO:61) ;
PMON30374
TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAG ATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTG ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCT GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAA TTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAA CTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT (SEQ ID NO: 62) ;
PMON30375
GTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACC AAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGG GGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACC ACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAG GTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAAC ATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCT (SEQ ID NO:63) ;
PMON30376
GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT (SEQ ID NO:64) ;
PMON30377
GACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTG GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG (SEQ ID NO:65) ;
PMON30378
GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCCGGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG (SEQ ID NO: 66) ;
PMON30379 GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCAC AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTC CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG (SEQ ID NO:67) ; PMON30380
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT (SEQ ID NO: 68) ;
PMON30381
GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC GGCGGCAACATG-JCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG (SEQ ID NO: 69)
PMON30382 GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG
CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC CTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA (SEQ ID NO:70) ; PMON30383
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG (SEQ ID NO:71) ;
PMON30384
GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT
GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGAC TTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAAT (SEQ ID NO:72) . TABLE 3 PROTEIN SEQUENCES
PMON26458pep
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPhe (SEQ ID NO:42);
PMON28548pep
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnMetAla SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro AsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeu ValGlyGlySerThrLeuCysValArg (SEQ ID NO:43);
PMON32132
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSeiArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:44);
PMON32133 SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro AsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeu ValGlyGlySerThrLeuCysValArg (SEQ ID NO:45);
PMON28500
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyAsnMetAlaSer ProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHisVal LeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeuLeu ProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAlaGln AsplleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGlnLeu GlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeuLeu GlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAlaHis LysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPhe LeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:46);
PMON28501
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnMetAla SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:47);
pMON32136
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla HisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnGlyGly AsnMetAlaSerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArg AspSerHisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThr ProValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAla ArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnVal ArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArg ThrThrAlaHisLysAspProAsnAlallePheLeuSerPheGlnHisLeuLeuArgGly LysValArgPheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:48) ;
PMON30 73
GluValHisProLeuProThrProValLeuLeuProAlaValAspPheSerLeuGlyGlu TrpLysThrGlnMetGluGluThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeu LeuGluGlyValMetAlaAlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeu GlyGlnLeuSerGlyGlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThr GlnLeuProProGlnGlyArgThrThrAlaHisLysAspProAsnAlallePheLeuSer PheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeu CysValArgGluPheGlyGlyAsnMetAlaSerProAlaProProAlaCysAspLeuArg ValLeuSerLysLeuLeuArgAspSerHisValLeuHisSerArgLeuSerGlnCysPro (SEQ ID NO: 49) ;
pMON31074
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly
Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly
Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser
Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly
Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro
Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val
Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu
Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser
Arg Leu Ser Gin Cys Pro Glu Val His Pro (SEQ ID NO:! 50) ;
PMON30375
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr
Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro
Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro
Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met
Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn
Met Ala Ser ro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser
Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin
Cys Pro Glu Val His Pro Leu Pro Thr Pro (SEQ ID NO:51) ;
pMON30376
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro (SEQ ID NO: 52);
pMON30377
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys
Ala Gin Asp ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val
Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu
Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr
Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser
His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro
Leu Pro Thr Pro Val Leu Leu Pro Ala Val (SEQ ID NO: 53);
pMON30378
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile
Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg
Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu
Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp
Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val -ιeu Ser Lys Leu Leu Arg Asp Ser His Val Leu His
Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro
Val Leu Leu Pro Ala Val Asp Phe Ser Leu (SEQ ID NO:54); PMON30379 Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu (SEQ ID NO:55);
PMON30380 Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu (SEQ ID NO:56);
PMON30381 Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin (SEQ ID NO:57);
PMON30382 Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr
Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr (SEQ ID NO:58);
PMON30383
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys (SEQ ID NO:59);
PMON30384
Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID NO:60);
The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.
EXAMPLE 1
Construction of the parental plasmid containing the first gene of the dimer template In order to generate a plasmid DNA with the coding sequence of c-mpl (1-153) ligand followed by a unique EcoRI restriction site, the gene is isolated via reverse transcriptase/polymerase chain reaction (RT/PCR) . Human fetal (lot #38130) and adult liver (lot #46018) A+ RNA are obtained from Clontech (Palo Alto, CA) for source of c-mpl ligand messager RNA (mRNA) . The first strand cDNA reactions are carried out using a cDNA Cycle™ Kit obtained from Invitrogen (San Diego, CA) . In the RT reaction, random primers and oligo dT primer are used to generate cDNA from a combination of human and fetal liver mRNA. For amplification of c-mpl ligand gene fragment encoding amino acids 1-153, the RT product serves as the template for PCR with a combination of the primers, Forward primer: c-mplNcol (SEQ ID NO:4) and Reverse primer: Ecompl (SEQ ID NO-.5) . The c-mplNcol (SEQ ID NO:4) primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand sequence from Genebank accession #L33410 or de Sauvage et al., Nature 369:533-538, 1994) and encodes a Νcol restriction enzyme site just 5' to the first mature codon (Ser^) . The Νcol restriction enzyme site codes for methionine and alanine codons prior to ser-1 and includes codon degeneracy for the Ala codon and the first four c-mpl ligand codons (Ser1, Pro2, Ala3, & Pro4) . The Ecompl (SEQ ID NO:5) primer anneals to bases #720-737 of c-mpl ligand and encodes a EcoRI recognition site in-frame with the c-mpl ligand gene immediately following Arg1^ _ r^g EcoRI site creates Glu-^4 and Phe-*-55 codons following Argl53 _ The ca. 480 bp PCR product is purified, digested with Ncol and EcoRI and ligated to the NcoI-EcoRl vector fragment of pMON3993 (ca. 4550 bp.) . pMON3993 is a derivative of pMON3359. The expression cassette in pMON3359 includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See Hippen eyer et al., Bio/Technology, 1037-1041, 1993). The human IL-3 signal peptide sequence, which had been subloned as a BamHI fragment into the unique BamHI site between the IE110 promoter and poly-A signal, contains an Ncol site at its 3' end and is then followed by a unique EcoRI site. The DNA sequence of the signal peptide is shown below (restriction enzyme sites are indicated above) . The ATG (methionine) codon within the Ncol site is in-frame with the initiator ATG of the signal peptide (underlined) ; BamHI
GGATCCACCA_I£AGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCCGC
Ncol
CATGG (SEQ ID NO:81)
pMON26458, coding for c-mpl ligand amino acids 1-153, is a result of this cloning.
EXAMPLE 2 Construction of the parental plasmids containing the second genes of the dimer templates
For amplification of c-mpl ligand gene fragments starting at amino acid 1 (Ser) with a termination codon following amino acid 153 (Arg) , the RT reaction from Example 1 serves as the template for PCR with a combination of the following primers; c-mplNcol (SEQ ID NO:4) (forward primer) and c-mplHindlll (SEQ ID NO: 6) (reverse primer) . The c- mplNcoI (SEQ ID NO:4) primer is described in Example 1. The c-mplHindlll (SEQ ID NO: 6) primer, which ann?als to bases
#716-737 of c-mpl ligand, adds both a termination codon and a Hindlll restriction enzyme site immediately following the final codon, Arg1^ _
Two types of PCR products are generated from the RT cDNA samples, one with a deletion of the codons for amino acids 112-115 and one without the deletion of these codons. The c- mpl ligand PCR products (ca. 480 bp) are digested with Ncol and Hindlll restriction enzymes for transfer to a mammalian expression vector, pMON3934, which is a derivative of pMON3359 (see Example 1) . pMON3934 is digested with Ncol and Hindlll (ca. 3800 bp) and will accept the PCR products. The plasmid, pMON32132, contians the DNA sequence of (SEQ ID NO:82) which encodes for amino acids 1-153 of c-mpl ligand (SEQ ID NO: .4) was a result of this cloning. The plasmid, pMON32133, contains the DNA sequence of (SEQ ID NO:83) which encodes amino acids 1-153 of c-mpl ligand with a deletion of codons 112-115 (Δ112-115) (SEQ ID NO:45) was also a result of this cloning.
EXAMPLE
Generation of PCR 5L dimer template containing Δ112-115 in the second gene
A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of PMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32133 (containing a deletion of amino acids 112-115) along with the EcoRI/AflHI 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3 ' (SEQ ID NO:10) .
The EcoRI end of the linker will ligate to the EcoRI end of PMON26458. The Afllll end of the linker will ligate to the Ncol site of pMON32133, and neither restriction site will be retained upon ligation. The BstXl sites of pMON26458 and pMON32133 will ligate as well. Plasmid, pMON28548, is a result of the cloning and contains the DNA sequence of (SEQ ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:43). EXAMPLE 4 Generation of PCR 4L dimer template pMON28500
A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflHI 4L synthetic oligonucleotide linker 4L-5' (SEQ ID NO:7) and 4L-3 ' (SEQ ID NO:8) .
The EcoRI end of the linker will ligate to the EcoRI end of PMON26458. The Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation. The BstXl sites of■pMON26458 and pMON32132 will ligate as well. Plasmid, pMON28500 is a result of the cloning and contains the DNA sequence of (SEQ ID NO:39) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:77) linker (4L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:46) .
EXAMPLE 5 Generation of PCR 5L dimer template pMON28501
A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of PMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflHI 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3 ' (SEQ ID NO:10). The EcoRI end of the linker will ligate to the EcoRI end of PMON26458. The Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation. The BstXl sites of pMON26458 and PMON32132 will ligate as well. PLasmid, pMON28501 is a result of the cloning and contains the DNA sequence of (SEQ ID NO: 40) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker (5L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:47) .
EXAMPLE 6 Generation of PCR 8L dimer template pMON32136
A PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from PMON32132 along with the EcoRI/AflHI 8L synthetic oligonucleotide linker 8L-5' (SEQ ID NO:ll) and 8L-3 ' (SEQ ID NO:12) . The EcoRI end of the linker will ligate to the EcoRI end of PMON26458. The Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation. The BstXl sites of pMON26458 and PMON32132 will ligate as well. Plasmid, pMON32136 is a result of the cloning which contains the DNA sequence of (SEQ ID NO:41) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79) linker (8L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:48) .
EXAMPLES 7-18
Generation of novel c-mpl receptor agonists
A. PCR generation of novel c-mnl receptor agonists. Novel c-mpl ligand genes are generated using the Horlick method. The PCR reaction was carried out using dimer template pMON28501 and one of the sets of synthetic primer sets below (number refers to first amino acid of new molecule) . 31-5' (SEQ ID NO:13) and 31-3' (SEQ ID NO:14), 35-5' (SEQ ID NO:15) and 35-3' (SEQ ID NO:16), 39-5' (SEQ ID NO:17) and 39- 3' (SEQ ID NO:18), 43-5' (SEQ ID NO:19) and 43-3' (SEQ ID NO:20), 45-5' (SEQ ID NO:21) and 45-3' (SEQ ID NO:22), 49-5' (SEQ ID NO:23) and 49-3' (SEQ ID NO:24), 82-5' (SEQ ID NO:25) and 82-3' (SEQ ID NO:26), 109-5' (SEQ ID NO:27) and 109-3' (SEQ ID NO:28), 116-5' (SEQ ID NO:29) and 116-3' (SEQ ID NO:30) ,120-5' (SEQ ID NO:31) and 120-3' (SEQ ID NO:32), 123- 5' (SEQ ID NO:33) and 123-3' (SEQ ID NO:34), 126-5' (SEQ ID NO:35) and 126-3' (SEQ ID NO:36)
The products that are generated are about 480 bp and are purified via Magic PCR Clean up kits (Promega) . Table 4 shows the template, the primer set used in the PCR reaction and the breakpoint for each Example.
B. Subcloning of novel c-mnl receptor agonists into mammalian expression vector The c-mpl receptor agonist PCR products are digested with
Ncol and Hindlll or Afllll and Hindlll restriction enzymes
(ca. 470 bp) for transfer to a mammalian expression vector.
The expression vector, pMON3934, is digested with Ncol and
Hindlll (ca. 3800 bp) and accepts the PCR products as Ncol- Hindlll or Afllll-Hindlll fragments. Table 4 shows the restriction digest of the PCR product and the resulting expression plasmid pMON designation.
TABLE 4
Example PCR PCR PCR Product Resulting
# template Primer Breakpoint Linker Restriction Plasmid set Digest pMON
7 pMON28501 31 30-31 5L Ncol/Hindlll PMON30373
8 PMON28501 35 34-35 5L Afllll/Hindlll PMON30374
9 PMON28501 39 38-39 5L Ncol/Hindlll pMON30375
10 PMON2.501 43 42-43 5L Ncol/Hindlll PMON30376
11 PMON28501 45 44-45 5L Ncol/Hindlll PMON30377
12 PMON28501 49 48-49 5 Ncol/Hindlll PMON30378
13 PMON28501 82 81-82 5L Ncol/Hindlll PMON30379
14 PMON28501 109 108-109 5L Ncol/Hindlll PMON30380
15 PMON28501 116 115-116 5L Ncol/Hindlll pMON30381
16 PMON28501 120 119-120 5L Ncol/Hindlll PMON30382
17 PMON28501 123 122-123 5L Ncol/Hindlll PMON30383
18 pMON28501 126 125-126 5L Ncol/Hindlll pMON30384
In a similar manner the dimer templates pMON28500, pMON32136 and pMON28548 could be used in the PCR reaction as described in Examples 7-18.
Bioactivity determination of c-mpl receptor agonits
Transfected cell lines: Cell lines such as Baf/3 cell line can be transfected with a colony stimulating factor receptor, such as the human IL-3 receptor or human c-mpl receptor, which the cell line does not normally have. These transfected cell lines can be used to determine the activity of the ligand for which the receptor has been transfected into the cell line.
One such transfected Baf/3 cell line was made by cloning the cDNA encoding c-mpl from a library made from a c- mpl responsive cell line and cloned into the multiple cloning site of the plasmid pcDNA3 (Invitrogen, San Diego Ca. ) . Baf/3 cells were transfected with the plasmid via electroporation. The cells were grown under G418 selection in the presence of mouse IL-3 in Wehi conditioned media. Clones were established through limited dilution.
The BHK expression levels and bioactivity data of some of the c-mpl receptor agonists of the present invention are shown in Table 5. The supernatant from the transfected BHK cells was evaluated for expression of the c-mpl receptor agonists by western analysis using an antobody raised against c-mpl ligand. Constructs that expressed at a "++++" level were assayed in the Baf-3/c-mpl cell proliferation assay.
TABLE 5
Expression and Bioactivity of c-mpl receiotor aqonist pMON# c-mpl receptor BHK expression Baf-3/c-mpl agonist levels cell proliferation
PMON30373 31-153/5L/1-30 + nd
PMON30374 35-153/5L/1-34 + nd
PMON30375 39-153/5L/1-38 - nd
PMON30376 43-153/5L/1-42 + nd
PMON30377 45-153/5L/1-44 ++ nd
PMON30378 49-153/5L/1-48 ++ nd
PMON30379 82-153/5L/1-81 + nd
PMON30380 109-153/5L/1-108 ++++ +
PMON30381 116-153/5L/1-115 ++++ +
PMON30382 120-153/5L/1-119 + nd
PMON30383 123-153/5L/1-122 + nd
2. Bone marrow proliferation assay a. CD34+ Cell Purification:
Between 15-20 mL bone marrow aspirates are obtained from normal allogeneic marrow donors after informed consent. Cells are diluted 1:3 in phosphate buffered saline (PBS, Gibco- BRL) , 30 mL are layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer is collected and washed in PBS. CD34+ cells are enriched from the mononuclear cell preparation using an affinity column per manufacturers instructions (CellPro, Inc, Bothell WA) . After enrichment, the purity of CD34+ cells is 70% on average as determined by using flow cytometric analysis using anti CD34 monoclonal antibody conjugated to fluorescein and anti-CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose CA) .
Cells are resuspended at 40,000 cells/mL in X-Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1 mL is plated in 12-well tissue culture plates (Costar). Human IL-3 variant, PMON13288, is used at 10 ng/mL or 100 ng/mL. Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand are tested by addition of 100 μl of supernatant added to 1 mL cultures (approximately a 10% dilution) . Cells are incubated at 37°C for 8-14 days at 5% C02 in a 37°C humidified incubator, b. Cell Harvest and Analysis:
At the end of the culture period a total cell count is obtained for each condition. For fluorescence analysis and ploidy determination cells, are washed in megakaryocyte buffer (MK buffer, 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 μm PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,) (Tomer et al., Blood 70(6): 1735-42 [1987]) resuspended in 500 μl of MK buffer containing anti-CD41a FITC antibody (1:200, AMAC, Westbrook, ME) and washed in MK buffer. For
DNA analysis cells are made permeable in MK buffer containing 0.5% Tween 20 (Fisher, Fair Lawn NJ) for 20 minutes on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in Propidium Iodide (Caibiochem , La Jolla Ca) (50 μg/mL) with RNAase (400 U/mL) in 55% v/v MK buffer (200 mOsm) for 1-2 hours on ice. Cells are analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA) . Green fluorescence (CD41a-FlTC) is collected along with linear and log signals for red fluorescence (PI) to determine DNA ploidy. All cells are collected to determine the percent of cells that are CD41+. Data analysis is performed using LYSIS software (Becton Dickinson, San Jose, CA) . Percent of cells expressing the CD41 antigen is obtained from flow cytometry analysis (Percent) . Absolute (Abs) number of CD41+ cells/mL is calculated by: (Abs) = (Cell Count)* (Percent) /IOO.
3. Megakaryocyte fibrin clot assay.
CD34+ enriched population are isolated as described above. Cells are suspended at 25,000 cells/mL with/without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4 mg/mL apo-transferrin, 6.67μM FeCl2, 25μg/mL CaCl2, 25 μg/mL L asparagine, 500 μg/mL E-amino-n-caproic acid and Penicillin/Streptomycin. Prior to plating into 35 mm plates, thrombin is added (0.25 units/mL) to initiate clot formation. Cells are incubated at 37°C for 13 days at 5% C02 in a 37°C humidified incubator. At the end of the culture period plates are fixed with methanol:acetone (1:3), air dried and stored at -200C until staining. A peroxidase immunocytochemistry staining procedure is used
(Zymed, Histostain-SP. San Francisco, CA) using a cocktail of primary monoclonal antibodies consisting of anti CD41a, CD42 and CD61. Colonies are counted after staining and classified as negative, CFU-MK (small colonies, 1-2 foci and less that approx. 25 cells), BFU MK (large, multi-foci colonies with > 25 cells) or mixed colonies (mixture of both positive and negative cells) .
Various other examples will be apparent to the person skilled in t-ia art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: G. D. Searle
(B) STREET: P.O. Box 5110
(C) CITY: Chicago
(D) STATE: Illinois
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP) : 60680
(G) TELEPHONE: (708) 470-6501 (H) TELEFAX: (708) 470-6881
(A) NAME: Monsanto Company
(B) STREET: 88 North Lindbergh Boulevard
(C) CITY: St. Louis
(D) STATE: Missouri
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP) : 63167
(G) TELEPHONE: (314) 694-3131 (H) TELEFAX: (314) 694-5435
(ii) TITLE OF INVENTION: c-mpl ligand Receptor Agonists
(iii) NUMBER OF SEQUENCES: 83
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: US C-2908 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004,824
(B) FILING DATE: 05-OCT-1995
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:112
(D) OTHER INFORMATION: /note= "position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or M... "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:113
(D) OTHER INFORMATION: /note= "position 113 is deleted or Pro, Phe, Ala, Leu, Ile, Trp, or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:114
(D) OTHER INFORMATION: /note= "position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:115
(D) OTHER INFORMATION: /note= "position 115 is deleted or Gin, Gly, Ser, Thr, Tyr or Asn"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Xaa 100 105 110
Xaa Xaa Xaa Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala 145 150 155 160 Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn 165 170 175
Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr 180 185 190
Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly Phe Arg Ala Lys Ile 195 200 205
Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu Asp Gin Ile Pro Gly 210 215 220
Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe 225 230 235 240
Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly 245 250 255
Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gin Pro Gly Tyr Ser 260 265 270
Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr Thr Leu Phe Pro Leu 275 280 285
Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu His Pro Leu Leu Pro 290 295 300
Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr 305 310 315 320
Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu Gly 325 330
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:112
(D) OTHER INFORMATION:/note= "position 112 is deleted or Leu, Ala,VAI, Ile, Pro, Phe, Trp or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:113 (D) OTHER INFORMATION: /note= "positoin 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:114
(D) OTHER INFORMATION: /note= "position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 115
(D) OTHER INFORMATION: /note= "positon 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Xaa 100 105 110
Xaa Xaa Xaa Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg 145 150
(2) INFORMATION FOR SEQ ID NO: 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Gly Gly Gly Ser
1
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ACGTCCATGG CNTCNCCNGC NCCNCCTGCT TGTGCACTCC GAGTC 45
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATGCACGAAT TCCCTGACGC AGAGGGTGGA 30
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TGACAAGCTT ACCTGACGCA GAGGGTGGAC CCT 33
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
AATTCGGCAA 10
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CATGTTGCCG 10
(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
[ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
AATTCGGCGG CAA 13
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CATGTTGCCG CCG 13
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AATTCGGCGG CAACGGCGGC AA 22
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CATGTTGCCG CCGTTGCCGC CG 22
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CGATCCATGG AGGTTCACCC TTTGCCT 27
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GATCAAGCTT ATGGGCACTG GCTCAGTCT 29
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CGATACATGT TGCCTACACC TGTCCTG 27
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CGATCCATGG TCCTGCTGCC TGCTGTG 27
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
CGATCCATGG TCCTGCTGCC TGCTGTG 27
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GATCAAGCTT AAGGTGTAGG CAAAGGGTG 29
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CGATCCATGG CTGTGGACTT TAGCTTGGGA 30
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GATCAAGCTT AAGGCAGCAG GACAGGTGT 29
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CGATCCATGG ACTTTAGCTT GGGAGAA 27
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GATCAAGCTT ACACAGCAGG CAGCAGGAC 29
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CGATCCATGG GAGAATGGAA AACCCAG 27
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GATCAAGCTT ACAAGCTAAA GTCCACAGC 29
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
CGATCCATGG GACCCACTTG CCTCTCA 27
(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GATCAAGCTT ACAGTTGTCC CCGTGCTGC 29
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GATCAAGCTT AAAGGAGGCT CTGCAGGGC 29
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GATCAAGCTT AAAGGAGGCT CTGCAGGGC 29
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CGATCCATGG GCAGGACCAC AGCTCAC 27
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GATCAAGCTT ACTGTGGAGG AAGCTGGGTT 30
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetis) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GATCAAGCTT ACTGTGGAGG AAGCTGGGTT 30
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
GATCAAGCTT ATGTGGTCCT GCCCTGTGG 29
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (syntheitc)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CGATCCATGG ATCCCAATGC CATCTTCCTG 30
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
GATCAAGCTT ACTTGTGAGC TGTGGTCCT 29
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CGATCCATGG CCATCTTCCT GAGCTTCCAA 30
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GATCAAGCTT AATTGGGATC CTTGTGAGCT GT 32
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTC 465
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 927 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120 CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACATGGCG 480
TCTCCCGCTC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 540
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 600
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 660
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 720
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 780
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGGGCAGGA CCACAGCTCA CAAGGATCCC 840
AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AGGTGCGTTT CCTGATGCTT 900
GTAGGAGGGT CCACCCTCTG CGTCAGG 927
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 936 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCAA CATGGCGTCT 480
CCCGCTCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC TGCTTCGTGA CTCCCATGTC 540
CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC TGTCCTGCTG 600
CCTGCTGTGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG 660
GACATTCTGG GAGCAGTGAC CCTTCTGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG 720
GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG TCTCCTCCTT 780
GGGGCCCTGC AGAGCCTCCT TGGAACCCAG CTTCCTCCAC AGGGCAGGAC CACAGCTCAC 840
AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC 900
CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGG 936
(2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 939 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACATGGCG 480
TCTCCCGCTC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 540
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 600
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 660
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 720
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 780 CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 840
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 900
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG 939
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 948 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
TCCCCAGCGC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACGGCGGC 480
AACATGGCGT CCCCAGCGCC GCCTGCTTGT GACCTCCGAG TCCTCAGTAA ACTGCTTCGT 540
GACTCCCATG TCCTTCACAG CAGACTGAGC CAGTGCCCAG AGGTTCACCC TTTGCCTACA 600 CCTGTCCTGC TGCCTGCTGT GGACTTTAGC TTGGGAGAAT GGAAAACCCA GATGGAGGAG 660
ACCAAGGCAC AGGACATTCT GGGAGCAGTG ACCCTTCTGC TGGAGGGAGT GATGGCAGCA 720
CGGGGACAAC TGGGACCCAC TTGCCTCTCA TCCCTCCTGG GGCAGCTTTC TGGACAGGTC 780
CGTCTCCTCC TTGGGGCCCT GCAGAGCCTC CTTGGAACCC AGCTTCCTCC ACAGGGCAGG 840
ACCACAGCTC ACAAGGATCC CAATGCCATC TTCCTGAGCT TCCAACACCT GCTCCGAGGA 900
AAGGTGCGTT TCCTGATGCT TGTAGGAGGG TCCACCCTCT GCGTCAGG 948
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110 Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe 145 150 155
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
145 150 155 160 Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 165 170 175
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 180 185 190
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 195 200 205
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 210 215 220
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 225 230 235 240
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 245 250 255
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Gly 260 265 270
Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin 275 280 285
His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser 290 295 300
Thr Leu Cys Val Arg 305
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val
20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45 Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg 145 150
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 149 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95 Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Gly 100 105 110
Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin 115 120 125
His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser 130 135 140
Thr Leu Cys Val Arg 145
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 312 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140 Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Asn Met Ala Ser 145 150 155 160
Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg 165 170 175
Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His 180 185 190
Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly 195 200 205
Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly 210 215 220
Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu 225 230 235 240
Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val 245 250 255
Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro 260 265 270
Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu 275 280 285
Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val 290 295 300
Gly Gly Ser Thr Leu Cys Val Arg 305 310
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala 145 150 155 160
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 165 170 175
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 180 185 190
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 195 200 205
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 210 215 220
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 225 230 235 240
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 245 250 255
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 260 265 270
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 275 280 285
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 290 295 300
Val Gly Gly Ser Thr Leu Cys Val Arg
305 310 (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 50 55 60
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Gly Gly 145 150 155 160
Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser 165 170 175
Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys 180 185 190
Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp 195 200 205 Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin 210 215 220
Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala 225 230 235 240
Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu 245 250 255
Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly 260 265 270
Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn 275 280 285
Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe 290 295 300
Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg 305 310 315
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe 1 5 10 15
Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp 20 25 30
Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg 35 40 45
Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser 50 55 60
Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr 65 70 75 80
Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala 85 90 95 Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu 100 105 110
Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn 115 120 125
Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys 130 135 140
Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu 1 5 10 15
Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala 20 25 30
Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly 35 40 45
Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg 50 55 60
Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro 65 70 75 80
Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser 85 90 95
Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly 100 105 110
Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro 115 120 125
Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp 130 135 140
Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin 1 5 10 15
Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu 20 25 30
Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu 35 40 45
Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly 50 55 60
Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr 65 70 75 80
Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu 85 90 95
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu 100 105 110
Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala 115 120 125
Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu 130 135 140
His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr 1 5 10 15
Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val 20 25 30
Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu 35 40 45
Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser 50 55 60
Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys 65 70 75 80
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys 85 90 95
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu 100 105 110
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg 115 120 125
Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu 130 135 140
Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala 1 5 10 15
Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala 20 25 30
Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin 35 40 45
Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu 50 55 60
Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro 65 70 75 80
Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg 85 90 95
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly 100 105 110
Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu 115 120 125
Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin 130 135 140
Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu 1 5 10 15 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 20 25 30
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin 35 40 45
Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 50 55 60
Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 65 70 75 80
Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 85 90 95
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
100 105 110
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 115 120 125
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 130 135 140
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val
1 5 10 15
Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro
20 25 30
Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu
35 40 45
Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val
50 55 60 U l
Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser 65 70 75 80
Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg 85 90 95
Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His 100 105 110
Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly 115 120 125
Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly 130 135 140
Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 -imino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro 1 5 10 15
Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg 20 25 30
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly 35 40 45
Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu 50 55 60
Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin 65 70 75 80
Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val 85 90 95
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala 100 105 110
Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala 115 120 125
Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin 130 135 140
Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe 1 5 10 15
Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly 20 25 30
Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala 35 40 45
Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser 50 55 60
His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu 65 70 75 80
Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp 85 90 95
Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val 100 105 110
Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro 115 120 125
Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu 130 135 140 Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu 1 5 10 15
Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys 20 25 30
Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys 35 40 45
Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His 50 55 60
Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val 65 70 75 80
Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met 85 90 95
Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu 100 105 110
Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser 115 120 125
Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala 130 135 140
Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys 1 5 10 15
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu 20 25 30
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg 35 40 45
Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu 50 55 60
Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro 65 70 75 80
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr 85 90 95
Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val 100 105 110
Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
115 120 125
Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser 130 135 140
Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe
1 5 10 15
Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly 20 25 30
Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser 35 40 45
Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys 50 55 60
Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp 65 70 75 80
Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin 85 90 95
Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala 100 105 110
Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu 115 120 125
Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly 130 135 140
Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
GAGGTTCACC CTTTGCCTAC ACCTGTCCTG CTGCCTGCTG TGGACTTTAG CTTGGGAGAA 60 TGGAAAACCC AGATGGAGGA GACCAAGGCA CAGGACATTC TGGGAGCAGT GACCCTTCTG 120
CTGGAGGGAG TGATGGCAGC ACGGGGACAA CTGGGACCCA CTTGCCTCTC ATCCCTCCTG 180
GGGCAGCTTT CTGGACAGGT CCGTCTCCTC CTTGGGGCCC TGCAGAGCCT CCTTGGAACC 240
CAGCTTCCTC CACAGGGCAG GACCACAGCT CACAAGGATC CCAATGCCAT CTTCCTGAGC 300
TTCCAACACC TGCTCCGAGG AAAGGTGCGT TTCCTGATGC TTGTAGGAGG GTCCACCCTC 360
TGCGTCAGGG AATTCGGCGG CAACATGGCG TCTCCGGCGC CGCCTGCTTG TGACCTCCGA 420
GTCCTCAGTA AACTGCTTCG TGACTCCCAT GTCCTTCACA GCAGACTGAG CCAGTGCCCA 480
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (Synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
TTGCCTACAC CTGTCCTGCT GCCTGCTGTG GACTTTAGCT TGGGAGAATG GAAAACCCAG 60
ATGGAGGAGA CCAAGGCACA GGACATTCTG GGAGCAGTGA CCCTTCTGCT GGAGGGAGTG 120
ATGGCAGCAC GGGGACAACT GGGACCCACT TGCCTCTCAT CCCTCCTGGG GCAGCTTTCT 180
GGACAGGTCC GTCTCCTCCT TGGGGCCCTG CAGAGCCTCC TTGGAACCCA GCTTCCTCCA 240
CAGGGCAGGA CCACAGCTCA CAAGGATCCC AATGCCATCT TCCTGAGCTT CCAACACCTG 300 CTCCGAGGAA AGGTGCGTTT CCTGATGCTT GTAGGAGGGT CCACCCTCTG CGTCAGGGAA 360
TTCGGCGGCA ACATGGCGTC TCCGGCGCCG CCTGCTTGTG ACCTCCGAGT CCTCAGTAAA 420
CTGCTTCGTG ACTCCCATGT CCTTCACAGC AGACTGAGCC AGTGCCCAGA GGTTCACCCT 480
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
GTCCTGCTGC CTGCTGTGGA CTTTAGCTTG GGAGAATGGA AAACCCAGAT GGAGGAGACC 60
AAGGCACAGG ACATTCTGGG AGCAGTGACC CTTCTGCTGG AGGGAGTGAT GGCAGCACGG 120
GGACAACTGG GACCCACTTG CCTCTCATCC CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT 180
CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT GGAACCCAGC TTCCTCCACA GGGCAGGACC 240
ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC AACACCTGCT CCGAGGAAAG 300
GTGCGTTTCC TGATGCTTGT AGGAGGGTCC ACCCTCTGCG TCAGGGAATT CGGCGGCAAC 360
ATGGCGTCTC CGGCGCCGCC TGCTTGTGAC CTCCGAGTCC TCAGTAAACT GCTTCGTGAC 420
TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT GCCTACACCT 480
(2) INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
GCTGTGGACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA GGCACAGGAC 60
ATTCTGGGAG CAGTGACCCT TCTGCTGGAG GGAGTGATGG CAGCACGGGG ACAACTGGGA 120
CCCACTTGCC TCTCATCCCT CCTGGGGCAG CTTTCTGGAC AGGTCCGTCT CCTCCTTGGG 180
GCCCTGCAGA GCCTCCTTGG AACCCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG 240
GATCCCAATG CCATCTTCCT GAGCTTCCAA CACCTGCTCC GAGGAAAGGT GCGTTTCCTG 300
ATGCTTGTAG GAGGGTCCAC CCTCTGCGTC AGGGAATTCG GCGGCAACAT GGCGTCTCCG 360
GCGCCGCCTG CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC CCATGTCCTT 420
CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACCCTTTGC CTACACCTGT CCTGCTGCCT 480
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: GACTTTAGCT TGGGAGAATG GAAAACCCAG ATGGAGGAGA CCAAGGCACA GGACATTCTG 60
GGAGCAGTGA CCCTTCTGCT GGAGGGAGTG ATGGCAGCAC GGGGACAACT GGGACCCACT 120
TGCCTCTCAT CCCTCCTGGG GCAGCTTTCT GGACAGGTCC GTCTCCTCCT TGGGGCCCTG 180
CAGAGCCTCC TTGGAACCCA GCTTCCTCCA CAGGGCAGGA CCACAGCTCA CAAGGATCCC 240
AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AGGTGCGTTT CCTGATGCTT 300
GTAGGAGGGT CCACCCTCTG CGTCAGGGAA TTCGGCGGCA ACATGGCGTC TCCGGCGCCG 360
CCTGCTTGTG ACCTCCGAGT CCTCAGTAAA CTGCTTCGTG ACTCCCATGT CCTTCACAGC 420
AGACTGAGCC AGTGCCCAGA GGTTCACCCT TTGCCTACAC CTGTCCTGCT GCCTGCTGTG 480
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 479 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACATTCTGGG AGCAGTGACC 60
CTTCTGCTGG AGGGAGTGAT GGCAGCACGG GGACAACTGG GACCCACTTG CCTCTCATCC 120
CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT 180
GGAACCCAGC TTCCTCCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC 240 CTGAGCTTCC AACACCTGCT CCGAGGAAAG GTGCGTTTCC TGATGCTTGT AGGAGGGTCC 300
ACCCTCTGCG TCAGGGAATT CGGCGGCAAC ATGGCTCTCC GGCGCCGCCT GCTTGTGACC 360
TCCGAGTCCT CAGTAAACTG CTTCGTGACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT 420
GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTGCTGCC TGCTGTGGAC TTTAGCTTG 479
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG TCTCCTCCTT 60
GGGGCCCTGC AGAGCCTCCT TGGAACCCAG CTTCCTCCAC AGGGCAGGAC CACAGCTCAC 120
AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC 180
CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGGGAAT TCGGCGGCAA CATGGCGTCT 240
CCGGCGCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC TGCTTCGTGA CTCCCATGTC 300
CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC TGTCCTGCTG 360
CCTGCTGTGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG 420
GACATTCTGG GAGCAGTGAC CCTTCTGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG 480
(2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
GGAACCCAGC TTCCTCCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC 60
CTGAGCTTCC AACACCTGCT CCGAGGAAAG GTGCGTTTCC TGATGCTTGT AGGAGGGTCC 120
ACCCTCTGCG TCAGGGAATT CGGCGGCAAC ATGGCGTCTC CGGCGCCGCC TGCTTGTGAC 180
CTCCGAGTCC TCAGTAAACT GCTTCGTGAC TCCCATGTCC TTCACAGCAG ACTGAGCCAG 240
TGCCCAGAGG TTCACCCTTT GCCTACACCT GTCCTGCTGC CTGCTGTGGA CTTTAGCTTG 300
GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACATTCTGGG AGCAGTGACC 360
CTTCTGCTGG AGGGAGTGAT GGCAGCACGG GGACAACTGG GACCCACTTG CCTCTCATCC 420
CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT 480
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
GGCAGGACCA CAGCTCACAA GGATCCCAAT GCCATCTTCC TGAGCTTCCA ACACCTGCTC 60
CGAGGAAAGG TGCGTTTCCT GATGCTTGTA GGAGGGTCCA CCCTCTGCGT CAGGGAATTC 120
GGCGGCAACA TGGCGTCTCC GGCGCCGCCT GCTTGTGACC TCCGAGTCCT CAGTAAACTG 180
CTTCGTGACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT GCCCAGAGGT TCACCCTTTG 240
CCTACACCTG TCCTGCTGCC TGCTGTGGAC TTTAGCTTGG GAGAATGGAA AACCCAGATG 300
GAGGAGACCA AGGCACAGGA CATTCTGGGA GCAGTGACCC TTCTGCTGGA GGGAGTGATG 360
GCAGCACGGG GACAACTGGG ACCCACTTGC CTCTCATCCC TCCTGGGGCA GCTTTCTGGA 420
CAGGTCCGTC TCCTCCTTGG GGCCCTGCAG AGCCTCCTTG GAACCCAGCT TCCTCCACAG 480
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
GCTCACAAGG ATCCCAATGC CATCTTCCTG AGCTTCCAAC ACCTGCTCCG AGGAAAGGTG 60
CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA GGGAATTCGG CGGCAACATG 120
GCGTCTCCGG CGCCGCCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC 180
CATGTCCTTC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACCCTTTGCC TACACCTGTC 240 CTGCTGCCTG CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG 300
GCACAGGACA TTCTGGGAGC AGTGACCCTT CTGCTGGAGG GAGTGATGGC AGCACGGGGA 360
CAACTGGGAC CCACTTGCCT CTCATCCCTC CTGGGGCAGC TTTCTGGACA GGTCCGTCTC 420
CTCCTTGGGG CCCTGCAGAG CCTCCTTGGA ACCCAGCTTC CTCCACAGGG CAGGACCACA 480
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
GATCCCAATG CCATCTTCCT GAGCTTCCAA CACCTGCTCC GAGGAAAGGT GCGTTTCCTG 60
ATGCTTGTAG GAGGGTCCAC CCTCTGCGTC AGGGAATTCG GCGGCAACAT GGCGTCTCCG 120
GCGCCGCCTG CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC CCATGTCCTT 180
CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACCCTTTGC CTACACCTGT CCTGCTGCCT 240
GCTGTGGACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA GGCACAGGAC 300
ATTCTGGGAG CAGTGACCCT TCTGCTGGAG GGAGTGATGG CAGCACGGGG ACAACTGGGA 360
CCCACTTGCC TCTCATCCCT CCTGGGGCAG CTTTCTGGAC AGGTCCGTCT CCTCCTTGGG 420
GCCCTGCAGA GCCTCCTTGG AACCCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG 480 (2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
GCCATCTTCC TGAGCTTCCA ACACCTGCTC CGAGGAAAGG TGCGTTTCCT GATGCTTGTA 60
GGAGGGTCCA CCCTCTGCGT CAGGGAATTC GGCGGCAACA TGGCGTCTCC CGCTCCGCCT 120
GCTTGTGACC TCCGAGTCCT CAGTAAACTG CTTCGTGACT CCCATGTCCT TCACAGCAGA 180
CTGAGCCAGT GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTGCTGCC TGCTGTGGAC 240
TTTAGCTTGG GAGAATGGAA AACCCAGATG GAGGAGACCA AGGCACAGGA CATTCTGGGA 300
GCAGTGACCC TTCTGCTGGA GGGAGTGATG GCAGCACGGG GACAACTGGG ACCCACTTGC 360
CTCTCATCCC TCCTGGGGCA GCTTTCTGGA CAGGTCCGTC TCCTCCTTGG GGCCCTGCAG 420
AGCCTCCTTG GAACCCAGCT TCCTCCACAG GGCAGGACCA CAGCTCACAA GGATCCCAAT 480
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73;
Gly Gly Gly Ser
1
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Gly Gly Gly Ser Gly Gly Gly Ser 1 5
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 -imino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Ser Gly Gly Ser Gly Gly Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 -imino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Glu Phe Gly Asn Met Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Glu Phe Gly Gly Asn Met Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
Glu Phe Gly Gly Asn Gly Gly Asn Met Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 -imino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Gly Gly Ser Asp Met Ala Gly 1 5
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
GGATCCACCA TGAGCCGCCT GCCCGTCCTG CTCCTGCTCC AACTCCTGGT CCGCCCCGC 59
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 459 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
TCTCCCGCTC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG 459
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 936 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 60
GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180 CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 240
CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 360
CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCAA CATGGCGTCT 480
CCCGCTCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC TGCTTCGTGA CTCCCATGTC 540
CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC TGTCCTGCTG 600
CCTGCTGTGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG 660
GACATTCTGG GAGCAGTGAC CCTTCTGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG 720
GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG TCTCCTCCTT 780
GGGGCCCTGC AGAGCCTCCT TGGAACCCAG CTTCCTCCAC AGGGCAGGAC CACAGCTCAC 840
AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC 900
CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGG 936

Claims

WHAT IS CLAIMED IS:
1. A c-mpl receptor agonist polypeptide comprising, a modified c-mpl ligand amino acid sequence of the Formula:
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer 1 5 10 15
HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro 20 25 30 35
ValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu 40 45 50 55 ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla 60 65 70 75
AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly 80 85 90 95
GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa 100 105 110
XaaGlyArgThrThrAlaHisLysAspProAsnAlallePheLeuSerPheGlnHis 115 120 125 130
LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal 135 140 145 150
Arg (SEQ ID NO:2) 153
wherein;
Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met; Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met; Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, He, Trp, or Met;
Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn; wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
26-27 51-52 108-109
27-28 52-53 109-110
28-29 53-54 110-111
29-30 54-55 111-112
30-31 55-56 112-113
32-33 56-57 113-114
33-34 57-58 114-115
34-35 58-59 115-116
36-37 59-60 116-117
37-38 78-79 117-118
38-39 79-80 118-119
40-41 80-81 119-120
41-42 81-82 120-121
42-43 82-83 121-122
43-44 83-84 122-123
44-45 84-85 123-124
46-47 85-86 124-125
47-48 86-87 125-126
48-49 87-88 126-127
50-51 88-89 or 127-128; and
additionally said polypeptide can be immediately preceded by (methionine"^) , (alanine-^) or (methionine-2, alanine-1).
2. The c-mpl receptor agonist polypeptide, as recited in claim 1, wherein said linker is selected from the group consisting of;
GlyGlyGlySer (SEQ ID NO:73);
GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74)
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:75);; SerGlyGlySerGlyGlySer (SEQ ID NO:76) ;
GluPheGlyAsnMetAla (SEQ ID NO:77);
GluPheGlyGlyAsnMetAla (SEQ ID NO:78);
GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and
GlyGlySerAspMetAlaGly (SEQ ID NO:80).
3. The c-mpl receptor agonist polypeptide as recited in claim 1 wherein said polypeptide is selected from the group consisting of:
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala
Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin
Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala
His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu
Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu
Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro
Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His
Val Leu His Ser Arg Leu Ser Gin Cys Pro (SEQ ID NO:' 19);
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly
Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser
Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly
Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val
Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu
Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser
Arg Leu Ser Gin Cys Pro Glu Val His Pro (SEQ ID NO:50);
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro (SEQ ID NO-.51);
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro (SEQ ID NO:52);
ASp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys
Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val
Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu
Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu
Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr
Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro
Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser
His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro
Leu Pro Thr Pro Val Leu Leu Pro Ala Val (SEQ ID NO:53) ;
Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu (SEQ ID NO:54);
Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu (SEQ ID NO:55);
Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu (SEQ ID NO: 56) ;
Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin (SEQ ID NO: 57) ;
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr
Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro
Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser
His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly
Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly
Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser
Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly
Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr (SEQ ID NO:! 58) ;
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly
Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys
Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu
His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr
Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys
Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly
Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val
Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu
Pro Pro Gin Gly Arg Thr Thr Ala His Lys (SEQ ID NO: 59) ; and
Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe
Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg
Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu
Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys
Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu
Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin
Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID NO: 60) .
4. The c-mpl receptor agonist polypeptide as recited in claim 3 wherein said polypeptide is selected from the group consisting of:
Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp
Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp
Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His
Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr
Gin Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val Thr
Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro
Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg
Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu (SEQ ID NO; 56) ; and
Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser
Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val
Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala
Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin
Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin (SEQ ID NO:! 57) .
5. A nucleic acid molecule, encoding the c-mpl receptor agonist polypeptide of claim 1.
6. A nucleic acid molecule encoding the c-mpl receptor agonist polypeptide of claim 2.
7. A nucleic acid molecule encoding the c-mpl receptor agonist polypeptide of claim 3.
8. A nucleic acid molecule encoding the c-mpl receptor agonist polypeptide of claim 4.
9. A nucleic acid molecule according to claim 7 selected from the group consisting of:
GAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTG CTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTG GGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACC CAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC TTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTC TGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGA GTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCA (SEQ ID NO: 61) ;
TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAG ATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTG
ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCT
GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA
CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG
CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAA TTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAA
CTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT
(SEQ ID NO:62) ;
GTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACC AAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGG GGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACC ACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAG
GTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAAC
ATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC
TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCT
(SEQ ID NO:63) ;
GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT (SEQ ID NO:64) ;
GACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTG GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG (SEQ ID NO:65) ;
GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCCGGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG (SEQ ID NO:66) ;
GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCAC AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT
CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTC CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG (SEQ ID NO:67) ;
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT (SEQ ID NO:68) ;
GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC GGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG (SEQ ID NO:69) ;
GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC CTGCTGCCTGC1GTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA (SEQ ID NO:70) ;
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG (SEQ ID NO:71) ; and
GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT
GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGAC TTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAAT (SEQ ID NO:72) .
10. A nucleic acid molecule according to claim 8 selected from the group consisting of:
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT (SEQ ID NO:68) ; and GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC GGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG (SEQ ID NO:69) .
11. A method of producing a human c-mpl receptor agonist polypeptide comprising, growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising a nucleic acid molecule of claim 5, 6, 7 or 8 in a manner allowing expression of said human c-mpl receptor agonist polypeptide and recovering said human c-mpl receptor agonist polypeptide.
12. A composition comprising a polypeptide of claim 1, 2, 3 or 4 and a pharmaceutically acceptable carrier.
13. A composition comprising; a polypeptide of claim 1, 2, 3 or 4; a colony stimulating factor; and a pharmaceutically acceptable carrier.
14. A composition comprising a polypeptide of claim 1, 2, 3 or 4; a colony stimulating factor selected from the group consisting of; GM-CSF, G-CSF, G-CSF Ser17, M-CSF, erythropoietin (EPO), IL-l, IL-4, IL-2, IL-3, IL-5, IL 6, IL- 7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor; and
a pharmaceutically acceptable carrier.
15. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering said polypeptide of claim 1, 2, 3 or 4 to said patient.
16. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering the composition of claim 13 to said patient.
17. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering the composition of claim 14 to said patient.
18. A method for selective ex vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2 , 3 or 4; and (c) harvesting said cultured cells.
19. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells; (b) separating stem cells from other cells; (c) culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, 3 or 4; (d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said patient.
20. A method of human gene therapy, comprising the steps of; (a) removing stem cells from a patient;
(b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected culture medium comprising the hematopoietic protein of claim 1, 2, 3 or 4; (d) introducing DNA into said cultured cells; (e) harvesting said transduced cells; and
(f) transplanting said transduced cells into said patient.
PCT/US1996/015938 1995-10-05 1996-10-04 NOVEL c-mpl RECEPTOR AGONISTS WO1997012978A1 (en)

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AU73901/96A AU722759B2 (en) 1995-10-05 1996-10-04 Novel C-MPL receptor agonists

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JP2000513924A (en) 2000-10-24
EP0870027A1 (en) 1998-10-14

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