JP2003525025A - Human transmembrane protein - Google Patents

Abstract

  (57) [Summary] The present invention provides human transmembrane proteins (HTMPs) and polynucleotides that identify and encode HTMPs. The invention also provides expression vectors and host cells, antibodies, agonists, antagonists. Further, the present invention provides a method for diagnosing or treating or preventing a disease associated with HTMP expression.

Description

Detailed Description of the Invention

TECHNICAL FIELD OF THE INVENTION The present invention relates to a nucleic acid sequence and an amino acid sequence of a human transmembrane protein, and cell proliferative disorders and immunological disorders, reproductive disorders, smooth muscle disorders, and neurological disorders using these sequences. It is related to diagnosis, treatment, and prevention of.

BACKGROUND OF THE INVENTION Eukaryotes differ from prokaryotes in that they have multiple membrane-bound compartments, such as intracellular organelles and vesicles. Many of the metabolic reactions that biochemically distinguish eukaryotes from prokaryotes occur within these compartments. In particular, many cell functions require extremely stringent reaction conditions, and intracellular organelles and vesicles compartmentalize and sequester reactions that disrupt the metabolic processes of the cytosol. Intracellular organelles include mitochondria and smooth endoplasmic reticulum, rough endoplasmic reticulum, sarcoplasmic reticulum, and Golgi apparatus. Vesicles include phagosomes and lysosomes, endosomes, peroxisomes, secretory vesicles.
The intracellular organelles and vesicles are surrounded by a single or double membrane.

A biological membrane covers the organelles, vesicles, and the cell itself. Biological membranes are highly selective permeability barriers. This lipid bilayer sheet consists of phosphoglycerides and fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins. Membranes include ion pumps and gates that transduce biochemical signals through the membrane in response to external stimuli, and specific receptors. These membranes also include second messengers that interact with pumps and channels, receptors to amplify and regulate these signals.

Cell Membrane Proteins Cell membrane proteins (MPs) depend on the way proteins are removed from the membrane.
It is divided into two groups. External or surrounding membrane proteins may be disruptors of extreme ionic strength, pH, urea, or other protein interactions.
) Can be used to release it. The only way to isolate internal or integral membrane proteins is to dissolve the lipid bilayer of the membrane with a detergent.

Most of the known integral membrane proteins are transmembrane proteins (TM) characterized by an extracellular domain, a transmembrane domain and an intracellular domain. The TM domain usually consists of 15 to 20 hydrophobic amino acids that are presumed to have an α-helical conformation. TM proteins are classified into bitopic (types I and II) and polytopic (types III and IV). (Singe
r, SJ (1990) Annu. Rev. Cell Biol. 6: 247-96.). Bitopic proteins span the membrane once and polytopic proteins contain multiple transmembrane segments.
TM proteins act as cell surface receptors involved in signal transduction and perform a variety of important cell functions. These functions are represented by growth factor and differentiation factor receptors, receptor-interacting proteins such as Drosophila pecanex and frizzled proteins, LIV-1 proteins, NF2 proteins, GNS1 / SUR4 eukaryotic integral membrane proteins. It TM proteins also act as ion or metabolite transporters such as gap junction channels (connexins) and ion channels, and adhesion proteins such as lectins, integrins, and fibronectin. TM proteins are also found in vesicle organelle-forming molecules such as calveolin, or differentiation antigens (CDs) and glycoproteins, cell recognition molecules such as mucins.

Many membrane proteins (MPs) contain amino acid sequence motifs that localize the protein to specific subcellular sites. Examples of such motifs include, for example, PDZ domains, KDEL, RGD, NGR and GSL sequence motifs, von Willebrand factor A (vWFA) domains, EGF-like domains. Peptides containing RGD and NGR, GSL motifs have been used as drug delivery agents in the targeted cancer therapy of tumor vasculature (Arap, W. et al.
1998) Science, 279: 377-380.). Membrane proteins also have a carbohydrate recognition domain (CRD)
Amino acid sequence motifs that interact with extracellular or intracellular molecules such as

Chemical modification of the side chains of amino acid residues alters the way MP interacts with other molecules such as phospholipid membranes. Examples of chemical modifications of amino acid residue side chains include covalent linkages with glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl moieties, ADP-ribose, phosphate and sulfate groups.

RNAs that encode membrane proteins may also have alternative splicing sites that are capable of producing proteins of different amino acid sequences that are encoded by the same gene but have different mRNAs. Splice variant membrane proteins can interact with other ligands and protein isoforms.

G-protein coupled receptors G-protein coupled receptors (GPCRs) are a superfamily of integral membrane proteins that transduce extracellular signals. GPCRs include receptors for biogenic amines, lipid mediators of inflammation, peptide hormones, and mediators of sensory signals. The highly conserved structure of these receptors is the seven hydrophobic transmembrane (snake skin)
A region and a cysteine disulfide bridge between the second and third extracellular loops,
It consists of an extracellular N-terminus and a cytoplasmic C-terminus. Three extracellular loops and three intracellular loops are alternated to connect the seven transmembrane regions. The most conserved parts of these proteins are the transmembrane region and the first two intracellular loops. A conserved acidic Arg aromatic residue triplet in the second cytoplasmic loop may interact with G proteins. The consensus pattern of GPCRs is characteristic of most proteins in this superfamily. (ExPASy PROSITE document PS00237, Wa
tson, S. and S. Arkinstall (1994) The G-protein Linked. Receptor Facts Book , Academic Press, San Diego, CA, pp 2-6.). Mutations and alterations in the transcriptional activity of the gene encoding the GPCR are associated with neurological disorders such as schizophrenia and Parkinson's disease, Alzheimer's disease, drug addiction, eating disorders.

Scavenger Receptor Macrophage scavenger receptors with broad ligand specificity may be involved in the binding of low density lipoprotein (LDL) to foreign antigens. Type I and type II scavenger receptors are trimeric membrane proteins in which each subunit has a small N-terminal intracellular and transmembrane domain, a large extracellular domain, and a C-terminal high cysteine domain. The extracellular domain includes a short spacer domain and an α-helix coiled-coil domain, a triple helix collagenous domain. These receptors include chemically modified lipoproteins and albumin, polyribonucleotides (polyr
It is known to bind to a range of ligands including ibonucleotides), polysaccharides, phospholipids and asbestos. (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci.
87: 9133-9137; and Elomaa, O. et al. (1995) Cell 80: 603-609.). This scavenger receptor is believed to play a major role in the development of atheroma by mediating the uptake of denatured LDL into the arterial wall. It is also considered to play a major role in host defense by binding to endotoxin, bacteria, and protozoa.

The four-transmembrane family protein (tetraspan) is a four-transmembrane superfamily (TM4SF) or tetraspan family, which is III
It is a multigene family encoding type integral membrane proteins. (Wright, MD
And Tomlinson. MG (1994) Immunol. Today 15: 588.). This TM4SF consists of a membrane protein that penetrates the cell membrane four times. Members of TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colon cancer antigens,
Tumor-associated antigens include schistosoma surface proteins (Jankowski, SA (1994)
Oncogene 9: 1205-1211.). The amino acid sequence identity between each member of TM4SF is about 25-30%. Many TMS4F members are involved in signal transduction, regulation of cell adhesion, regulation of cell growth and proliferation including development and carcinogenesis, and cell motility including tumor cell metastasis. Expression of the TMS4F protein is associated with various tumors and its expression level may change when cells are proliferated or activated.

Tumor Antigens Tumor antigens are surface molecules that are differentially expressed on tumor cells compared to normal cells. Since tumor cells can immunologically distinguish normal cells from tumor cells, they are used for diagnosis and treatment of human cancer. (Takagi, S. et al. (1995) Int. J. Cancer 61: 706.
-715: Liu. E. et al. (1992) Oncogene 7: 1027-I032.).

Ion Channels Ion channels are present in the cell membrane of virtually all cells of the body. For example, chloride channels mediate a variety of cellular functions including regulation of membrane potential and absorption and secretion of ions across epithelial membranes. Further, when the chloride ion channel is present in the intracellular membranes of the Golgi apparatus and the phagocytic cells, it regulates the pH of organelles (for example, Greger, R. (1988) Annu. Rev. Physiol. 50: 111-122). See). From the electrophysiological and pharmacological properties of chloride ion channels, including sensitivity to modulators, ionic conductance, and current-voltage relationship, various chloride ion channels are found in muscle and nerves, fibroblasts, epidermal cells, and lymphocytes. It turned out to exist. Many channels have sites that are phosphorylated by one or more protein kinases. The protein kinases include protein kinase A, protein kinase C, and tyrosine kinase II, each of which regulates the activity of cellular ion channels. Inappropriate phosphorylation of proteins in cells leads to changes in cell cycle progression and differentiation. Changes in the cell cycle lead to the induction of apoptosis and cancer. Also, changes in cell differentiation lead to diseases and abnormalities in the reproductive system, immune system, and skeletal muscle.

Proton Pump Proton ATPases are a large class of membrane proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient on either side of the membrane. The resulting gradient can be used to transport ions (Na ⁺ , K ⁺ , or CI ⁻ ) across the membrane and / or the pH of intracellular organelles.
To maintain. Furthermore, proton ATPase is subdivided into mitochondrial F-ATPase, cell membrane ATPase, and vacuolar ATPase. Vacuolar ATPase establishes and maintains an acidic pH within various vesicles involved in the processes of endocytosis and exocytosis (Mellman, I. et al. (1986) Ann. Rev. Biochem. 55: 663-
700.).

Proton-bound 12-transmembrane domain transporters such as PEPT1 and PEPT2 are involved in gastrointestinal peptide absorption and renal reabsorption using an electrochemical H ⁺ gradient as a driving force. Another type of peptidic transporter, a heterodimer consisting of TAP1 and TAP2, is involved in antigen processing. Peptide antigens can be transported by the TAP across the membrane of the endoplasmic reticulum and bound to MHC molecules and presented on the surface of cells. Each TAP
The protein consists of a number of hydrophobic transmembrane segments and a highly conserved ATP-binding cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 284-289.
). Pathogenic microorganisms such as herpes simplex virus may encode inhibitors of TAP-mediated peptide transporters to evade immune surveillance. (Marusina, K. and Manaco, JJ
(1996) Curt. Opin. Hematol. 3: 19-26.).

ABC Transporters ATP-binding cassette (ABC) transporters, also called “transporting ATPases”, constitute the superfamily of membrane proteins that mediate transport and channel function in prokaryotic and eukaryotic cells. (Higgins, CF (1992) Annu. Rev. Cell Biol. 8: 671-7
9.). The ABC proteins are similar in overall structure to each other and have extremely high sequence identity. All ABC proteins contain a conserved domain of approximately 200 amino acid residues with one or more nucleotide binding domains. Mutations in the ABC transporter gene are associated with type II hyperbilirubinemia / Dubin-Johnson syndrome, recessive Stargardt disease, and X-linked adrenal leukodystrophy (X-linked adrenoluekod
ystrophy), multidrug resistance, childhood steatorrhea, cystic fibrosis and other diseases.

Membrane Proteins Involved in Intracellular Transmission Intracellular transmission is essential for the development and survival of multicellular organisms. Cells communicate with each other by the secretion and uptake of signaling molecule proteins. Uptake of proteins into cells is due to endocytosis. Endocytosis is the formation of vesicles from the cell membrane, surrounding the molecules and transporting them into the cytosol by the interaction of signal molecules with the surface of the cell membrane (usually binding with specific receptors). Secretion of proteins from cells is by exocytosis. In exocytosis, intracellular molecules are trans-Golgi network (TGN)
To be packaged in the membrane-bound transport vesicle of origin. These vesicles fuse with the cell membrane and release their contents out of the cell. Endocytosis and exocytosis result in the removal and addition of cell membrane components, but the recycling of these components is essential for maintaining integrity, identity, and function in the cell membrane and inner membrane-binding compartments, respectively. is there.

[0018] Lysosomes are the site of degradation of intracellular substances during autophagy and also the site of degradation of extracellular molecules after endocytosis. Lysosomal enzymes are packaged in vesicles that arise from the trans-Golgi network. These vesicles fuse with endosomes to form mature lysosomes, which are hydrolyzed substances that have been phagocytosed. Lysosomes can fuse with autophagosomes to form unique compartments where intracellular phase organs and other intracellular components are degraded.

By sorting proteins by transport vesicles such as endosomes, various physiological processes including cell surface growth and development of different intracellular organelles, endocytosis, and regulation of secretion of hormones and neurotransmitters are possible. Lead to significant results (Rothman, J.
E. and Wieland, FT (1996) Science 272: 227-234.). In particular, neurodegenerative and other neurological disorders are associated with biochemical defects during endosome protein sorting or endosome biogenesis (Mayer RJ et al. (1996) Ad.
v. Exp. Med. Biol. 389: 261-269.).

Peroxisomes are intracellular organelles independent of the secretory pathway. The peroxisome is the site of many peroxide-producing oxidation reactions within the cell. It is unique among the eukaryotic organelles in that the size, number, and amount of enzymes vary depending on the type of organism and cells and the need for metabolism. The majority of peroxisome-related proteins are membrane bound and are found proximal to the cytosolic or luminal side of the peroxisome membrane (Waterham, HR and Cregg, JM (1997) BioEssays 19: 57-66). Genetic defects in peroxisomes that cause peroxisome deficiency are associated with many human diseases. Among the diseases are Zellweger's syndrome, tarsal punctate epiphyseal dysplasia, X-linked adrenoleukodystrophy,
Includes acyl-CoA oxidase deficiency, bifunctional enzyme deficiency, classic Refsum disease, DHAP alkyltransferase deficiency, and acatalaseemia. (Moser, HW and Moser,
AB (1996) Ann. NY Acad. Sci. 804: 427-441.). Furthermore, Gartner, J. et al. (1991;
Pediatr. Res. 29: 141-146) discovered a 22 kDa integral membrane protein associated with low-density peroxisome-like intracellular compartments in patients with Zellweger syndrome.

Regulation of normal embryonic development and germ cell maturation is carried out by a number of secreted proteins that interact with their corresponding membrane-bound receptors. The cell fate during embryonic development is
For example, it is determined by the activin / TGF-β superfamily and members of cadherin, IGF-2, and other morphogens. Furthermore, proliferation, maturation, and redifferentiation of germ cells and reproductive tissues are regulated by, for example, IGF-2 and inhibin, activin, follistatin (Petraglia, F. (1997) Placenta 18: 3-8.
and Mather, JP et al. (1997) Proc. Soc. Exp. Biol. Med. 215: 209-222).

Endoplasmic reticulum membrane proteins In order for eukaryotic cells to function normally, all newly synthesized proteins must be correctly folded and modified so that they can be transported to specific intracellular or extracellular sites. . Newly synthesized membrane and secretory proteins enter the intracellular sorting and distribution network stage during synthesis (co-translation) or shortly thereafter (post-translation) and are transported to specific locations inside or outside the cell. . The first compartment of this process is the endoplasmic reticulum (ER), where proteins undergo modifications such as glycosylation, disulfide bond formation, and oligomer assembly. The modified protein is then transported through a series of membrane-bound compartments, including the various Golgi apparatus Cisterna, which undergo further carbohydrate modifications. Transport between compartments is performed by vesicles that are generated and fused depending on the type of protein transported. Once in the secretory pathway, proteins do not need to cross the membrane to reach the cell surface.

The majority of proteins processed by the endoplasmic reticulum are transported outside the endoplasmic reticulum, but some remain inside. In mammalian cells, the signal to remain in the endoplasmic reticulum consists of the tetrapeptide sequence KDEL located at the C-terminus of the protein (Munro,
S. (1986) Cell 46: 291-300.). A protein containing this sequence leaves the endoplasmic reticulum, but is immediately recovered from the initial Golgi apparatus Cisterna and returned to the endoplasmic reticulum. On the other hand, proteins that do not contain this sequence continue to flow in the distribution pathway.

Disruption of the cell's secretory pathway is associated with several human diseases. In familial hypercholesterolemia, the low-density lipoprotein receptor is not transported to the cell surface and remains in the endoplasmic reticulum (Pathak, RK (1988) J. Cell Biol. 106: I831-1841.). Altered transport and processing of β-amyloid precursor protein (βAPP), along with the putative vesicular transport protein presenilin, may play a role in early-onset Alzheimer's disease (Levy-Lahad. E. et al. 1995) Science 269: 973-977). Changes in endoplasmic reticulum-derived calcium homeostasis are associated with cardiomyopathy, cardiac hypertrophy, myotonic dystrophy, Brody's disease
(Brody disease), Smith-McCort dyspasia, and diabetes.

Mitochondrial Membrane Protein The mitochondrial electron transport chain (respiratory chain) is a series of three enzyme complexes of the mitochondrial membrane. These enzyme complexes transport electrons generated in the oxidation process of NADH and link this oxidation action to the synthesis of adenosine triphosphate (ATP) (oxidative phosphorylation). This ATP is the main source of energy for many cellular reactions that require energy.

Most protein components of the mitochondrial respiratory chain are products of nuclear genes, some of which have been imported into mitochondria, and the rest of the proteins are products of mitochondrial genes. Defects and altered expression of enzymes in the respiratory chain are associated with various human pathologies including, for example, neurodegenerative diseases and myopathy, cancer and the like.

The response of B cells to antigens regulated through lymphocyte membrane protein and leukocyte membrane protein receptors is an essential element of the normal immune system. Mature B cells recognize foreign antigens via the membrane-bound B cell receptor (BCR) and produce specific antibodies that bind to the foreign antigen. This complex of antigen and receptor is taken up and the antigen undergoes proteolytic processing.
BCR and BCR-related proteins, T cell responses are required to generate efficient responses to complex antigens. A proteolytic fragment of the antigen forms a complex with a class II MHC (major histocompatibility complex II) molecule on the surface of B cells, which complex can be recognized by T cells. In contrast, macrophages and other lymphocytes are class I
It binds to MHC molecules and presents antigens to T cells. T cells recognize the antigen and interact with the T cell surface multimeric protein, the T cell receptor / CD3 complex, located on the cell membrane and activated by the class I MHC antigen complex. T cells activated by antigen presentation secrete various lymphokines that induce B cell maturation and T cell proliferation,
Activates macrophages that kill target cells.

White blood cells play a fundamental role in inflammatory and immune responses,
Includes monocytes / macrophages, mast cells, polymorphonuclear leukocytes, natural killer cells, neutrophils, eosinophils, basophils, bone marrow precursors. Leukocyte membrane proteins include CD antigen and N-CAM, I-CAM, human leukocyte antigen (HLA) class I and HLA class II gene products, immunoglobulin, immunoglobulin receptor, complement, complement receptor, interferon, It includes members of the interferon receptor, interleukin receptor, and chemokine receptor.

Abnormal lymphocyte and leukocyte activity is associated with acute diseases such as AIDS and immune hypersensitivity, leukemia, leukopenia, systemic lupus, granulomatous disease, eosinophilia.

Apoptosis Various ligands and receptors, enzymes, tumor suppressors, viral gene products, drugs,
Inorganic ions play important positive and negative roles in the regulation and execution of apoptotic destruction of cells. Although several specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are unclear and the main points of the pathway remain unclear.

The requirement for calcium for apoptosis has been previously reported by studies showing that calcium levels are involved in DNA cleavage and Fas-mediated cell death (Hewish, DR and LA Burgoyne (1973) Biochem. .Biophys. Res. Comm.
52: 504-510: Vignaux, F. et al. (1995) J. Exp. Med. 181: 781-786; Oshimi, Y.
And S. Miyazaki (1995) J. Immunol. 154: 599-609.). In another study, when apoptosis in thymocytes was triggered by either T-cell receptor cross-linking or glucocorticoids, intracellular calcium levels increased, and cells were blocked by blocking this increase in calcium levels. (McConkey, DJ et al. (1989) J. Immunol. 143: 1801-1806: and McConkey, D.
.J et al. (1989) Arch. Biochem. Biophys. 269: 365-370.). Therefore, membrane proteins such as calcium channels are important in the apoptotic response.

With the discovery of novel human transmembrane proteins and polynucleotides encoding them, a novel human transmembrane protein useful for diagnosis, treatment, and prevention of abnormal cell proliferation and immune disorders, reproductive disorders, smooth muscle disorders, and neurological diseases Providing a composition can meet the needs of the art.

(Summary of the Invention) The present invention is generically referred to as “HTMP”, individually as “HTMP-1” and “HTMP-2”, respectively.
"HTMP-3", "HTMP-4", "HTMP-5", "HTMP-6", "HTMP-7", "HTMP-8",
"HTMP-9", "HTMP-10", "HTMP-11", "HTMP-12", "HTMP-13", "HTMP-1"
"4", "HTMP-15", "HTMP-16", "HTMP-17", "HTMP-18", "HTMP-19", "
"HTMP-20", "HTMP-21", "HTMP-22", "HTMP-23", "HTMP-24", "HTMP-25"
, "HTMP-26", "HTMP-27", "HTMP-28", "HTMP-29", which are human transmembrane proteins and purified polypeptides. In one embodiment of the invention,
a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) SEQ ID NO: 1
A naturally occurring amino acid sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of −29, c) SEQ ID NO: Biologically of an amino acid sequence selected from the group consisting of 1-29 There is provided an isolated polypeptide comprising an active fragment, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29. Alternatively, provided is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-29.

The present invention further provides: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29 and 90% or more of A naturally occurring amino acid sequence having sequence identity, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, or d) SEQ ID NO: 1-
An isolated polynucleotide encoding a polypeptide that is an immunogenic fragment of an amino acid sequence selected from the group consisting of 29 is provided. Alternatively, the polynucleotide is selected from the group consisting of SEQ ID NO: 30-58.

Furthermore, the present invention relates to: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, and 90% or more thereof. A naturally occurring amino acid sequence having the sequence identity of c), c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, or d) SEQ ID NO: 1
Recombinant polynucleotides comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -29. Alternatively, the invention provides a cell transformed with this recombinant polynucleotide. In a further alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

Further, the present invention comprises: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, and 90% or more thereof. A naturally occurring amino acid sequence having the sequence identity of c), c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, or d) SEQ ID NO: 1
Provided is a method for producing a polypeptide containing an immunogenic fragment of an amino acid sequence selected from the group consisting of -29. This method comprises: a) culturing cells transformed with a recombinant polynucleotide containing a promoter sequence operably linked to a polynucleotide encoding the polypeptide under conditions suitable for expression of the polypeptide. And b) recovering the thus expressed polypeptide.

Furthermore, the present invention relates to: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, and 90% or more thereof. A naturally occurring amino acid sequence having the sequence identity of c), a biologically active fragment of an amino acid sequence selected from the group consisting of c) SEQ ID NO: 1-29, or d) SEQ ID NO: 1
There is provided an isolated antibody that specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -29.

Further, the invention comprises: a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 30-58; b) a polynucleotide acid sequence selected from the group consisting of SEQ ID NO: 30-58. An isolated polynucleotide comprising a naturally occurring polynucleotide sequence having 90% or greater sequence identity, c) a polynucleotide sequence complementary to a) above, or d) a polynucleotide sequence complementary to b) above. I will provide a. Alternatively,
The polynucleotide comprises at least 60 contiguous nucleotides.

Further, the present invention provides: a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 30-58, b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 30-58 and 90% Naturally occurring polynucleotide sequences having the above sequence identities, c)
A method for detecting a target polynucleotide in a sample having a polynucleotide sequence complementary to a) or d) a polynucleotide sequence comprising a polynucleotide sequence complementary to b) is provided. The method comprises the steps of: a) hybridizing the sample with a probe comprising at least 16 contiguous nucleotides containing a sequence complementary to the target polynucleotide in the sample, wherein the probe and the target polynucleotide The probe specifically hybridizes to the target polynucleotide under the condition that a hybridization complex is formed in step b), the presence or absence of the hybridization complex is detected and detected. Optionally measuring the amount.
Alternatively, the probe comprises at least 30 contiguous nucleotides.
In a further alternative, the probe comprises at least 60 contiguous nucleotides.

Furthermore, the present invention relates to: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, and 90% or more thereof. A naturally occurring amino acid sequence having the sequence identity of c), c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, or d) SEQ ID NO: 1
There is provided a pharmaceutical composition comprising an effective amount of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -29 and a suitable pharmaceutical excipient. Furthermore, the present invention provides a method for treating a disease associated with reduced expression of functional HTMP or a symptom thereof, which comprises administering the pharmaceutical composition to a patient.

Furthermore, the present invention provides: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, and 90% or more A naturally occurring amino acid sequence having the sequence identity of c), a biologically active fragment of an amino acid sequence selected from the group consisting of c) SEQ ID NO: 1-29, or d) SEQ ID NO: 1
Provided is a method for screening a compound effective as an agonist of a polypeptide containing an immunogenic fragment of an amino acid sequence selected from the group consisting of -29. The method comprises the steps of a) exposing a sample containing the polypeptide to a compound, and b) detecting agonist activity of the sample. Alternatively,
The present invention provides a pharmaceutical composition comprising an agonist compound identified by the above method and a suitable pharmaceutical excipient. In a further alternative, the present invention provides a method of treating a disease associated with reduced expression of functional HTMP or a symptom thereof, comprising the administration of this pharmaceutical composition to a patient.

Furthermore, the present invention relates to: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, and 90% or more. A naturally occurring amino acid sequence having the sequence identity of c), a biologically active fragment of an amino acid sequence selected from the group consisting of c) SEQ ID NO: 1-29, or d) SEQ ID NO: 1
Provided is a method of screening a compound effective as an antagonist of a polypeptide containing an immunogenic fragment of an amino acid sequence selected from the group consisting of -29.
The method comprises a) exposing a sample containing the polypeptide to a compound, and b) detecting antagonist activity in the sample. Alternatively, the present invention provides a pharmaceutical composition comprising an antagonist compound identified by the above method and a suitable pharmaceutical excipient. In a further alternative, the present invention provides a method of treating a disease or condition associated with overexpression of functional HTMP, comprising administering to a patient this pharmaceutical composition.

The invention further provides a method of screening for compounds effective in altering the expression of a target polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 30-58, which comprises: a) the target Provided is a screening method comprising exposing a sample containing a polynucleotide to a compound, and b) detecting a change in the expression of the target polynucleotide.

(Description of the Present Invention) Before describing the protein and nucleic acid sequences and methods of the present invention, the present invention is not limited to the specific devices and materials and methods disclosed herein, and its embodiments are modified. Please understand what you can do. Also, the terminology used herein is for the purpose of describing particular embodiments only and is limited only by the scope of the following claims,
It should also be understood that it is not intended to limit the scope of the invention.

In the present specification and claims, “a”, “the (the same),” etc. represent the singular form.
Note that includes plural forms unless the context clearly indicates otherwise. Thus, for example, “a host cell” includes a plurality of host cells, “antibody” includes a plurality of antibodies, and equivalents well known to those skilled in the art.

All technical terms used herein, unless defined otherwise,
It has the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.
Although any apparatus and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, suitable apparatus and materials and methods are described herein.
All references cited herein are cited to describe and disclose the cell lines, protocols, reagents, vectors described in the references that may be used in connection with the present invention. The mere fact that the conventional invention is cited does not mean that the novelty of the present invention is impaired.

Definitions The term “HTMP” is obtained substantially from all species including natural, synthetic, semi-synthetic or recombinant (especially mammals including bovine, ovine, porcine, murine, equine and human). Refers to the purified amino acid sequence of HTMP.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of HTMP. This agonist either interacts directly with HTMP or interacts with components of the biological pathways involving HTMP to regulate the activity of HTMP such as proteins, nucleic acids, carbohydrates, small molecules, any other compound or The composition may be included.

The term “allelic variant” refers to another form of the gene encoding HTMP. The allelic variant sequence results from at least one mutation in the nucleic acid sequence resulting in a variant mRNA or variant polypeptide, the structure or function of which may or may not change. Some genes include those in which no naturally-occurring allelic variant is present, or one gene or many genes are present. Generally, mutations that give rise to allelic variant sequences are due to natural deletions, additions, or substitutions of nucleotides. Each of these mutations, either alone or concurrently with other mutations, occurs one or more times within a given sequence.

A “mutant” nucleic acid sequence that encodes HTMP refers to a polypeptide that has the same polypeptide as HTMP or at least one of the functional properties of HTMP, even though various nucleotide deletions, insertions, or substitutions have occurred. . This definition includes improper or unexpected hybridization of an HTMP-encoding polynucleotide sequence with an allelic variant at a position other than the normal chromosomal locus, as well as specific oligonucleotide probes of the HTMP-encoding polynucleotide. Includes polymorphisms that are easily or difficult to detect using. The encoded protein may also be mutated and may contain deletions, insertions, or substitutions of amino acid residues which result in silent changes and are functionally equivalent to HTMP. Intentional amino acid substitutions are similar to each other in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues within the range where the activity of HTMP is retained biologically or immunologically. Can be based on. For example, negatively charged amino acids can include aspartic acid and glutamic acid, and positively charged amino acids can include lysine and arginine. Amino acids with similar hydrophilicity values and having polar uncharged side chains can include asparagine, glutamine, serine, threonine. Amino acids with similar hydrophilicity values and having uncharged side chains can include leucine, isoleucine, valine, glycine, alanine, phenylalanine and tyrosine.

The terms “amino acid” and “amino acid sequence” refer to oligopeptide, peptide, polypeptide, protein sequences, or any fragment thereof, including naturally occurring and synthetic molecules. When the "amino acid sequence" is a naturally occurring protein molecule, "
Amino acid sequence "and like terms are not intended to limit the amino acid sequence to the complete, unaltered amino acid sequence associated with the protein molecules described.

The term “amplification” refers to making copies of nucleic acid sequences. Amplification is generally performed by the polymerase chain reaction (PCR) technique well known in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of HTMP. Antagonists are antibodies, nucleic acids, carbohydrates, small molecules, any other compound or composition that directly interacts with HTMP or interacts with components of the biological pathways in which HTMP is involved to regulate HTMP activity. May include proteins such as things.

The term “antibody” refers to intact molecules such as Fab and F (ab ′) ₂ capable of binding antigenic determinants and fragments thereof, Fv fragments. Antibodies that bind HTMP polypeptides can be produced using intact molecules, or fragments thereof, containing small peptides that immunize the antibody.
The polypeptide or oligopeptide used to immunize an animal (eg, mouse, rat, or rabbit) can be synthesized from the translation of RNA or chemically and is optionally conjugated with a carrier protein. It is also possible. Commonly used carriers that are chemically linked to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemonean (KLH). The conjugated peptide is then used to immunize the animal.

The term “antigenic determinant” refers to the region (ie, epitope) of a molecule that makes contact with a particular antibody.
Refers to. When a protein or a fragment of a protein is used to immunize a host animal, various regions of this protein are antibodies that specifically bind to an antigenic determinant (a specific region on the protein or a three-dimensional structure). Can induce the production of The antigenic determinant may compete with the intact antigen (ie, the immunogen used to elicit an immune response) to bind the antibody.

As used herein, “antisense” refers to any composition capable of base pairing with the sense strand of a particular nucleic acid sequence. Antisense components include DNA, RNA, peptide nucleic acid (PNA), and modified backbone linkages such as phosphorothionate, methylphosphonate, and benzylphosphonate.
And an oligonucleotide having a modified sugar such as 2'-methoxyethyl sugar or 2'-methoxyethoxy sugar, and 5-methylcytosine or 2'-deoxyuracil, 7-deaza-2'-deoxyguanosine, etc. Of the modified bases may be included. Antisense molecules can be produced by any method, including chemical synthesis and transcription. Once introduced into a cell, the complementary antisense molecule base pairs with the naturally occurring nucleic acid sequence produced by the cell to form a duplex and inhibit transcription or translation. The expression "negative" or "minus" is the antisense strand, and the expression "positive" or "plus" is the sense strand.

The term “biologically active” refers to a protein that has the structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, the term "immunologically active" means that natural or recombinant HTMP, synthetic HTMP or any oligopeptide thereof induces a specific immune response in a suitable animal or cell to induce a specific antibody. Refers to the ability to combine with.

The terms “complementary” and “complementarity” refer to the natural binding of polynucleotides to form base pairs. For example, the sequence "5'AG-T3 '" binds to the complementary sequence "3'TC-A5'". The complementarity between two single-stranded molecules may be partial, where only some of the nucleic acids bind, or there may be complete complementarity between the single strands resulting in perfect complementarity. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on binding between nucleic acid strands, as well as in the design or use of peptide nucleic acid (PNA) molecules.

“Composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” refers broadly to any composition comprising a given nucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution.
A composition comprising a polynucleotide sequence encoding HTMP or a fragment of HTMP can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be bound to a stabilizer such as sugar. In hybridization, the probe is developed in an aqueous solution containing salts (eg, NaCl) and detergents (eg, SDS: sodium dodecyl sulfate) and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). obtain.

A “consensus sequence” is a nucleic acid sequence that has been sequenced to separate unwanted bases and is 5'and / or 3'using XL-PCR ^™ (Perkin Elmer, Norwalk, CT). Sequenced nucleic acid sequence or GELVI
EW fragment construction system (GCG, Madison, WI), etc. One or more Insight clones using computer programs for fragment construction,
And, in some cases, refers to nucleic acid sequences constructed by duplication of one or more public domain ESTs. Some consensus sequences are constructed by both extension and overlap.

The term “conservative amino acid substitution” refers to a substitution that does not significantly alter the properties of the original protein. That is, the substitution does not significantly change the structure or function of the protein,
The structure of the protein, in particular its function, is preserved. The following shows conservative amino acid substitutions in which the original amino acid of one protein is replaced with another amino acid. Original residue Conservative substitution Ala Gly, Set Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln , Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile . Leu, Thr In general, in the case of conservative amino acid substitutions, a) the backbone structure of the polypeptide in the substituted region, eg β sheet or α helix conformation, b) the charge of the molecule at the substituted site or Hydrophobic and / or c) most of the side chains are retained.

The term “deletion” refers to a change in an amino acid sequence that lacks one or more amino acid residues, or a change in a nucleic acid sequence that lacks one or more nucleotides.

The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of polynucleotide sequences include, for example, replacement of hydrogen with an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide has at least one biological or immunological function of a natural molecule (unmodified molecule).
Encodes a polypeptide that maintains one. A derivative polypeptide is a polypeptide modified by glycosylation, polyethylene glycolation, or any similar process that maintains at least one of the biological or immunological functions of the original polypeptide. That is.

The term “fragment” refers to a unique portion of HTMP or a polynucleotide encoding HTMP that is identical to its parent sequence but shorter than that sequence. The maximum length of a "fragment" is the parent sequence minus one nucleotide / amino acid residue. For example, a fragment contains 5 to 1000 consecutive nucleotides or amino acid residues. Probe, primer, antigen,
The therapeutic molecule, or fragment used for other purposes, comprises at least 5, 10, 15,
It is the length of 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or 500 consecutive nucleotides or amino acid residues. Fragments may also be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment may comprise a predetermined length of contiguous amino acids selected from the first 250 or 500 amino acids shown in the given sequence (or the first 25% or 50% of the polypeptide). . These lengths are examples, and any length described in the specification including the sequence listing and the tables and drawings can be included in the embodiments of the present invention.

Certain fragments of SEQ ID NO: 30-58 include, for example, a region of unique polynucleotide sequence that uniquely identifies SEQ ID NO: 30-58, which differs from other sequences in the same genome. Certain fragments of SEQ ID NO: 30-58 are useful, for example, in hybridization and amplification techniques, or similar methods of distinguishing SEQ ID NO: 30-58 from related polynucleotide sequences. The exact fragment length and region of SEQ ID NO: 30-58 that matches a fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

Certain fragments of SEQ ID NO: 1-29 are encoded by certain fragments of SEQ ID NO: 30-58. Certain fragments of SEQ ID NO: 1-29 contain regions of unique amino acid sequence that uniquely identify SEQ ID NO: 1-29. For example, certain fragments of SEQ ID NO: 1-29 are useful as immunogenic peptides for the production of antibodies that specifically recognize SEQ ID NO: 1-29. The exact fragment length and region of SEQ ID NO: 1-29 that matches a given fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. This includes partial similarity and complete similarity. The term “identity” can also be called “similarity”. Partially complementary sequences in which hybridization of the same sequence to the target nucleic acid is at least partially blocked are said to be "substantially similar." Inhibition of hybridization between the perfectly complementary sequence and the target sequence is tested using a hybridization assay (Southern or Northern blotting, solution hybridization, etc.) under mildly stringent conditions. Substantially similar sequences or hybridization probes competitively suppress binding of the perfectly similar (identical) sequence to the target sequence under mildly stringent conditions. This does not mean that non-specific binding is allowed under mildly stringent conditions, but under mildly stringent conditions, the binding of two sequences to each other is specific (ie, selective). You have to interact. A second target sequence, which may not be said to be partially complementary (eg, less than 30% similarity or identity), can be used to test for the absence of non-specific binding. In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

The term “percent identity” or “% identity” with respect to polynucleotide sequences refers to matching residues between two or more polynucleotide sequences that are aligned using a standardized algorithm. It is a percentage. Such algorithms can insert gaps in the sequences to make a more meaningful comparison between the two sequences in a standardized and reproducible manner to optimize the alignment between the two sequences.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison W
I). This CLUSTAL V is for Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153, Higgins, DG et al. (1992) CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameter is Kt.
Set uple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. A "weighted" residue weight table was selected as the default. Percentage identity is reported by CLUSTAL V as the "percentage similarity" of aligned polynucleotide sequence pairs.

Alternatively, a set of commonly used and freely available sequence comparison algorithms is
NCBI, Bethesda, MD, and Internet (http://WWW.ncbi.nlm.nih.gov/BLAS
National Center for Biotechnology Information (NCB)
I) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J
Mol. Biol. 215: 403-410). The suite of BLAST software includes various sequence analysis programs, including "blastn" used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool called "BLAST 2 Sequences" is available,
Used to directly compare pairs of two nucleotide sequences. "BLAST 2 Sequen
"ces" can be used interactively by accessing http://WWW.ncbi.nlm.nih.gov/gorf/b12.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). The BLAST program is commonly used with gaps and other parameters that set defaults. For example,
When comparing two nucleotide sequences, one may blast with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set to default parameters.
will use n. Such default parameters are, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on May be measured for the entire length of a given sequence, as determined by the sequence number of, or for shorter lengths, for example, fragments obtained from a given given sequence, for example at least contiguous , 20 or 30
, 40, 50, 70, 100, 200 nucleotide fragments. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

Nucleic acid sequences that do not show high identity may still encode similar amino acid sequences due to the degeneracy of the genetic code. It is to be understood that degeneracy can be utilized to alter nucleic acid sequences to produce different nucleic acid sequences each encoding substantially the same protein.

The term “percent identity” or “% identity” as used in polypeptide sequences refers to matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. It is a percentage. Methods of polypeptide sequence alignment are well known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, described in detail above, generally conserve charge and hydrophobicity at the site of substitution, and result in polypeptide structure (and thus function) as well.
Is saved.

The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program (supra). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are Ktuple = 1, gap
Set penalty = 3, window = 5, and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. Similar to polynucleotide alignments, the percent identity of paired aligned polypeptide sequences is reported by CLUSTAL V as the "percentage of similarity."

Alternatively, the NCBI BLAST software suite is used. For example, when making a pairwise comparison of two polypeptide sequences, one would use blastp with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set with default parameters. . Such default parameters are, for example: Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on It may be measured over the entire length of the peptide sequence, or for shorter lengths, eg fragments derived from one large given polypeptide sequence, eg at least 15, 20, or 30, 40 contiguous. , 50, 70, 150 residues may be measured against the length of the fragment. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

“Human Artificial Chromosome (HAC)” is a DNA of about 6 kb (kilobase) to 10 Mb in size.
It is a linear small chromosome that may contain sequences and contains all the elements necessary for the segregation and maintenance of stable mitotic chromosomes.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been altered in order to resemble a more human antibody while retaining its original binding ability.

“Hybridization” is the process of annealing in which a single-stranded polynucleotide forms a base pair with a complementary single strand under defined hybridization conditions. Specific hybridization means that two nucleic acid sequences have high identity. Under conditions that allow annealing, specific hybridization complexes are formed and remain hybridized after the washing process. The washing process is particularly important in determining the stringency or stringency of the hybridization process, and under more stringent conditions, non-specific binding, ie binding of pairs of nucleic acid strands that are not perfectly matched. Is reduced. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those of skill in the art and are constant during hybridization, but the washing process involves changing the conditions during which to achieve the desired stringency. Is possible and hybridization specificity is obtained. Annealing conditions are, for example, at a temperature of 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and about 100.
Includes μg / ml shear denatured salmon sperm DNA.

In general, the stringency of hybridization also depends on the temperature at which the washing process is performed. The wash temperature is usually selected to be about 5-20 ° C below the thermal melting point (Tm) of the particular sequence at a given ionic strength and pH. This Tm is (
The temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under a given ionic strength and pH). The formula for calculating Tm and the conditions for nucleic acid hybridization are well known, Sambrook, J. et al., 1989, Molecular Cloning : A Laboratory Manual , Volume 2 1-3, Cold Spring Harbor Press, Plainview.
NY; Especially described in Chapter 2 of Volume 2.

High stringency hybridization between the polynucleotides of the present invention involves a wash step at about 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is carried out at temperatures of 65 ° C, 60 ° C, 55 ° C, 42 ° C. SSC concentrations range from about 0.1 to 2x SSC in the presence of about 0.1% SDS. Normally,
Blocking agents are used to prevent non-specific hybridization. Such blocking agents include, for example, about 100-200 μg / ml denatured salmon sperm DNA. Organic solvents such as formamide at a concentration of about 35-50% v / v, for example RNA and
It can be used in specific cases such as hybridization of DNA. Useful modifications of these wash conditions are well known to those of skill in the art. Hybridization, particularly under highly stringent conditions, may suggest evolutionary similarities between the nucleotides. Such similarities strongly suggest that the nucleotides and encoded polypeptides play a similar role.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by hydrogen bonding between complementary bases. Hybridization complexes may be formed in solution (eg, C ₀ t or R ₀ t analysis), or one nucleic acid sequence in solution and a solid support (eg, paper, membrane, filter, chip, pin). ,
Or a slide glass, or any suitable substrate for immobilizing cells and their nucleic acids)
Can be formed with another nucleic acid sequence fixed to.

The term “insertion” or “addition” refers to a change in an amino acid sequence or nucleic acid sequence in which one or more amino acid residues or nucleotides have been added, respectively.

“Immune response” refers to conditions associated with inflammatory diseases and trauma, immune disorders, infectious diseases, genetic diseases and the like. These conditions are characterized by the expression of various factors such as cytokines and chemokines, other signaling molecules that affect the cell and systemic defense systems.

The term “microarray” refers to an array of different polynucleotides arranged on a substrate.

The term “element” or “array element” as used in the context of microarray refers to a hybridizable polynucleotide arrayed on the surface of a substrate.

The term “modulation” refers to a change in the activity of HTMP. For example, modulation results in a change in HTMP protein activity, or binding properties, or other biological, functional, or immunological properties.

The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides, or fragments thereof. In addition, it refers to DNA or RNA of genomic or synthetic origin, which is a single-stranded or double-stranded, sense or antisense strand, peptide nucleic acid (PNA), any DNA-like substance, and RNA-like substance.

“Operably linked” refers to the condition in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are very contiguous or contiguous if the regions encoding the two proteins must be joined in the same reading frame.

“Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent that comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. This terminal lysine renders the composition soluble. PNAs can bind preferentially to complementary single-stranded DNA or RNA to stop elongation of transcripts and polyethylene glycol to prolong life in cells.

The “probe” is a nucleic acid sequence encoding HTMP, a complementary sequence thereof, or a fragment thereof used for detecting the same or allelic nucleic acid sequence or a related nucleic acid sequence. A probe is an oligonucleotide or polynucleotide that has been isolated with a detectable label or reporter molecule attached. Typical labels include radioisotopes and ligands, chemiluminescent reagents, enzymes. A "primer" is capable of forming complementary base pairs and annealing to a target polynucleotide,
A short nucleic acid, usually a DNA oligonucleotide. After the primer anneals to the polynucleotide, it is extended along the target DNA single strand by a DNA polymerase enzyme. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence in a PCR method.

The probes and primers used in the present invention include at least 15 of known sequences.
Of contiguous nucleotides. Longer probes and primers can be used to increase specificity. For example, at least 20 or 25, 30, 30, 40, 50, 60, 70, 80, 90, 100 contiguous of the disclosed nucleic acid sequences.
, 150 nucleotides. It should be understood that probes and primers that are considerably longer than the above examples can be used, and nucleotides of any length shown in the tables and drawings of the present specification and the sequence listing can also be used.

For the preparation and use of probes and primers, see, for example, Sambrook,
J. et al., 1989, Name "Molecular Cloning: A Laboratory Manual", 2nd
Editions 1-3 (Cold Spring Harbor Press, Plainview NY), or Ausubel, FM
In 1987, the name "Current Protocols in Molecular Biology" (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY), and Innis et al.,
In 1990, the name `` PCR Protocols, A Guide to Methods and Applications '' (Acade
mic Press, San Diego CA.). The set of primers for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research
, Cambridge MA), and the like, and can be derived from one known sequence using a computer program for such purpose.

Oligonucleotides used as primers are selected with computer programs for primer selection well known in the art. For example, OLIGO 4.06 software selects primer pairs for PCR, each up to 100 nucleotides,
And is useful for the analysis of large polynucleotides and oligonucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32,000 bases. Similar primer selection programs include additional features that expand capacity. For example, the PrimOU primer selection program (Genome Center at Uni
versity of Texas South West Medical Center, available from Dallas TX)
Since a specific primer can be selected from the megabase sequence, it is useful for designing a primer in a genome-wide scope. Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research,
(Available from Cambridge MA1) allows users to specify sequences that they would like to avoid as primer binding sites.
) ”Can be entered. In addition, Primer3 is especially useful for the selection of oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from each source and modified to meet the needs of users). Can also). PrimerGen Program (UK Human Genome Mapping Pro
(available from the ject Resource Center, Cambridge UK), which designs primers based on multiple sequence alignments, so that they will hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences. Can be selected. Therefore, this program is useful for identifying unique and conserved oligonucleotide or polynucleotide fragments. The fragment of the oligonucleotide or polynucleotide identified by any of the selection methods described above is, for example, a PCR method or a sequencing primer, a microarray element, or a specific identifying a polynucleotide that is completely or partially complementary to a nucleic acid of a sample. It is useful for the hybridization technique such as the probe. The method for selecting the oligonucleotide is not limited to the above method.

As used herein, a “recombinant nucleic acid” is a sequence that is an artificial combination of two or more distant segments of a sequence, rather than the native sequence. Often, this artificial combination is made by chemical synthesis, but it is more common to artificially manipulate distant segments of nucleic acid using genetic engineering techniques such as those described in Sambrook, supra. is there. The "recombinant nucleic acid" also includes a nucleic acid modified by simply adding or substituting a part of the nucleic acid. The recombinant nucleic acid may also include a nucleic acid sequence operably linked to a promoter sequence. Such recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such recombinant nucleic acid is a vaccinia virus-based virus that, when used to vaccinate a mammal expressing the recombinant nucleic acid, elicits a protective immune response in the mammal. It can be part of the vector.

As used herein, an “RNA equivalent” to a DNA sequence is composed of the same linear nucleic acid sequence as the reference DNA sequence, except that the nitrogenous base thymine is replaced with uracil to form a sugar chain. Its spine consists of ribose rather than deoxyribose.

The term “sample” is used in its broadest sense. Samples suspected of containing HTMP-encoding nucleic acid or a fragment thereof, and HTMP itself include body fluids, extracts from cells, chromosomes or intracellular organelles isolated from cells, membranes, cells, and solutions. Genomic DNA, RNA, cDNA present in or immobilized on a substrate, tissues or tissue prints and the like may also be included.

The terms “specific binding” and “specifically bind” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. Point to. This interaction depends on the presence of a particular structure of the protein recognized by the binding molecule, eg, an antigenic determinant or epitope. For example, when the antibody is specific to the epitope "A", the reaction solution containing the unbound labeled "A" and the antibody is bound to the polypeptide containing the epitope A or the unbound unlabeled "A". Is present, the amount of labeled A bound to the antibody is reduced.

The term “substantially purified” refers to a nucleic acid sequence or amino acid sequence that has been removed from the natural environment and then isolated or separated so that the composition to which it is naturally associated has at least about 60 amino acids. % Or more, preferably about 75% or more, and most preferably 90% or more.

“Substitution” is the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microspheres. Includes particles and capillaries. The substrate has various surface morphologies such as walls or trenches, pins, channels, pores, to which polynucleotides and polypeptides bind.

“Transformation” is the process by which exogenous DNA enters and changes the recipient cell. Transformation can occur under natural or artificial conditions by a variety of methods well known in the art and is performed by any well known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. be able to. This transformation method is
The choice depends on the type of host cell to be transformed. This method includes, but is not limited to, viral infection, electroporation, lipofection, and particulate irradiation. "Transformed" cells include stably transformed cells in which the introduced DNA is capable of replicating autonomously, either as a plasmid or as part of a host chromosome. Further, a cell that transiently expresses the introduced DNA or the introduced RNA within a limited time is also included.

As used herein, the term “genetically modified organism” refers to any organism in which a human has introduced a heterologous nucleic acid into one or more cells of the organism using, for example, gene recombination techniques well known in the art. Yes, and includes, but is not limited to animals and plants. By careful genetic manipulation such as microinjection or infection with recombinant virus, the heterologous nucleic acid is introduced into the cell directly or indirectly into the precursor of the cell. “Genetic manipulation” refers to the introduction of recombinant DNA molecules, rather than the typical cross breeding or in vitro fertilization. Genetically modified organisms according to the invention include bacteria and cyanobacteria, fungi, plants, animals. The isolated DNA of the present invention can be introduced into a host by methods well known in the art, such as infection, transfection, transformation, transconjugation and the like. Techniques for introducing the DNA of the present invention into such organisms are well known and described in Sambrook et al. (1989) supra.

A “variant sequence” of a particular nucleic acid sequence refers to the default parameter setting “BLAST
2 Sequences "tool Version 2.0.9 (May-07-1999) shows that the specific nucleic acid sequence identity for a given length of a given nucleic acid sequence is at least 40%.
The nucleic acid sequence determined to be. Such nucleic acid pairs may, for example, be at least 50% or 60%, 70%, 80%, 85%, 90%, over a length.
It may exhibit 95%, 98%, or more identity. A variant sequence can be represented, for example, as an "allelic" variant sequence (described above) or a "splice" variant sequence, a "species" variant sequence, a "polymorphism" variant sequence. Splice variant sequences may have very high identities to the reference molecule, but alternative splicing of exons during mRNA processing may result in more or less polynucleotides. The corresponding polypeptide may have additional functional domains present in the reference molecule or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have a high degree of amino acid identity with each other. Polymorphic variant sequences differ in the polynucleotide sequence of a given gene from a given species and the species. The polymorphic variant sequence also includes one of the polynucleotide sequences
A "single nucleotide polymorphism" (SNP) that differs by two nucleotides may also be included. The presence of SNPs may be indicative of, for example, a population, a pathological condition, a characteristic of the pathological condition.

A “variant” of a particular polypeptide sequence refers to the “parameter” of the default parameter setting.
Blast 2 Sequences "tool version 2.0.9 (May-07-1999) by blastp, the identity of the particular polypeptide sequence to a certain length of a nucleic acid sequence is determined to be at least 40% polypeptide sequence That is. Such polypeptide pairs may, for example, be at least 50% or 60%, 7
It may exhibit 0%, 80%, 85%, 90%, 95%, 98%, or more identity.

(Invention) The present invention is based on the discovery of a novel human transmembrane protein (HTMP) and a polynucleotide encoding HTMP. It relates to the use of these compositions in the diagnosis, treatment and prevention of diseases.

Table 1 shows the Incyte clones used to construct the full-length nucleotide sequence encoding HTMP. Columns 1 and 2 show the SEQ ID NOs for the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone ID of the Incyte clone in which the nucleic acid encoding each HTMP was identified, and column 4 shows the cDNA library in which those clones were isolated. Row 5: Incyte clones and their corresponding
A cDNA library is shown. The Incyte clones for which the cDNA library is not shown are derived from the pooled cDNA library. The Insight clone in column 5
It is used to construct a consensus nucleotide sequence for each HTMP and is useful as a fragment in hybridization techniques.

Each column of Table 2 shows various properties of each polypeptide of the invention. Column 1 is the SEQ ID NO, column 2 is the number of amino acid residues in each polypeptide, column 3 is the potential phosphorylation site, column 4 is the potential glycosylation site, and column 5 is the signature (sign).
ature) amino acid residues with sequence and motif, column 6 shows the identification and homologous sequences of each polypeptide identified by BLAST analysis. Column 7 shows the method of analysis and possibly the searchable database to which the method of analysis can be applied. The analysis method in column 7 was used to characterize each polypeptide by sequence homology and protein motifs.

The columns of Table 3 show the tissue specificity and diseases, disorders, symptoms associated with the nucleotide sequences encoding HTMP. Column 1 of Table 3 shows the nucleotide sequence numbers (SEQ
ID NO) is shown. Column 2 shows a fragment of the nucleotide sequence of column 1.
These fragments are useful, for example, in hybridization or amplification techniques that identify SEQ ID NO: 30-58 and distinguish SEQ ID NO: 30-58 from related polynucleotide sequences. The polynucleotide encoded by the particular fragment of SEQ ID NO: 30-58 is useful, for example, as an immunogenic peptide. Column 3 is
The name of the tissue that expresses HTMP and its proportion in all the tissues that express HTMP are shown. Column 4 shows diseases or disorders associated with tissues expressing HTMP, symptoms, and their proportion in total tissues expressing HTMP. Column 5 shows the vector used for subcloning each cDNA library. In particular, 21 cds expressing SEQ ID NO: 41
Note that 15 (71%) of the NA libraries are associated with neural tissue.

Each column of Table 4 is a description of the tissue used to create the cDNA library in which the clone of the cDNA encoding HTMP was isolated. Column 1 is the SEQ ID of the nucleotide
No, column 2 shows the cDNA library from which those clones were isolated, column 3
Shows the origin and details of the tissues associated with the cDNA library in column 2.

SEQ ID NO: 45 maps to chromosome 3 within the interval 49.50 to 55.40 centimorgans. Genes associated with abnormal cell proliferation are also included in this section. SEQ
ID NO: 47 maps to chromosome 7 within the 74.30 to 76.40 centimorgan interval. Also included in this interval is an EST associated with cell proliferation. SEQ ID NO: 50 maps to chromosome 2 within the interval 111.5 to 115.3 centimorgans. Also included within this interval are genes associated with the immune response. SEQ ID NO: 51 is of chromosome 11
Mapped within the 84.2 to 87.1 cm Morgan section. Also included within this interval are genes associated with the immune response. SEQ ID NO: 53 is from Chromosome 13 77.10 to 86.
Mapped within the 90 centimorgan section. Also included within this interval are genes associated with the immune response. SEQ ID NO: 55 maps within the region of chromosomes 74.80 to 78.30 centimorgans. Also included within this interval are genes associated with the immune response. SEQ ID NO: 58 maps within the 25.30 centimorgan interval from the p-terminus of chromosome 15. Genes associated with cell proliferation are also included in this section.

The present invention also includes variants of HTMP. Preferred variants of HTMP have at least one of the functional or structural characteristics of HTMP and have at least about 80% amino acid sequence identity to the HTMP amino acid sequence, or at least about 90% amino acid sequence. There is identity, and even at least about 95% amino acid sequence identity.

The present invention also provides a polynucleotide encoding HTMP. In a particular embodiment, the invention provides a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 30-58 encoding HTMP. SEQ ID NO: shown in the sequence listing
The polynucleotide sequence of 30-58, thymine of the nitrogenous base is replaced by uracil,
The backbone of the sugar chain contains RNA sequence equivalents consisting of ribose rather than deoxyribose.

The present invention also includes variant sequences of the polynucleotide sequence encoding HTMP. In particular, such variant sequences of the polynucleotide sequence have at least 70% polynucleotide sequence identity with the polynucleotide sequence encoding HTMP, or at least 85% polynucleotide sequence identity, and even at least 95%. Has polynucleotide sequence identity. A particular embodiment of the invention is SEQ ID NO: 30
SEQ ID NO: having at least 70% polynucleotide sequence identity, or at least 85% polynucleotide sequence identity, and even at least 95% polynucleotide sequence identity with a nucleic acid sequence selected from the group consisting of -58. Provided are mutant sequences of a polynucleotide sequence comprising a sequence selected from the group consisting of 30-58. Each of the polynucleotide variants described above encodes an amino acid sequence having at least one functional or structural characteristic of HTMP.

It is noted that various polynucleotide sequences encoding HTMP that may be produced by the degeneracy of the genetic code include those that have minimal similarity to polynucleotide sequences of any known naturally occurring gene. Those of ordinary skill in the art will understand. Thus, the present invention may include all of the possible polynucleotide sequence variations that may be created by the selection of combinations based on possible codon choices. These combinations are based on the standard triplet genetic code applied to naturally-occurring HTMP polynucleotide sequences, and are considered to be the clear disclosure of all mutations.

Nucleotide sequences that encode HTMP and variants thereof are generally hybridizable to the naturally occurring HTMP nucleotide sequence under suitable selected stringent conditions, but with non-naturally occurring codons. It may be advantageous to make nucleotide sequences that encode HTMP or its derivatives with substantially different usage codons, such as inclusion. Codons can be selected based on the frequency with which a particular codon is utilized by the host to increase the rate at which peptide expression occurs in a particular eukaryotic or prokaryotic host. HT without changing the encoded amino acid sequence
Another reason to substantially alter the nucleotide sequences encoding MP and its derivatives is to produce RNA transcripts with favorable properties, such as longer half-life, than transcripts made from naturally occurring sequences.

The invention also includes the production of DNA sequences encoding HTMP and its derivatives, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can be inserted into any of the various available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry may be used to introduce mutations into the sequence encoding HTMP or any fragment thereof.

The present invention further provides a polynucleotide sequence capable of hybridizing to the claimed polynucleotide sequences, in particular SEQ ID NO: 30-58 and fragments thereof, under various stringent conditions. Included (eg Wahl, GM and SL Berger
(1987) Methods Enzymol. 152: 399-407; and Kimmel. AR (1987) Methods En
zymol. 152: 507-511.). Hybridization conditions, including annealing and wash conditions, are described in the "Definitions".

Any of the embodiments of the present invention can be carried out using DNA sequencing methods well known in the art. This method includes, for example, the Klenow fragment of DNA polymerase I, SE
QUENASE (US Biochemical, Cleveland OH), Taq Polymerase (Perkin Elmer)
, Thermostable T7 polymerase (Amersham, Pharmacia Biotech Piscataway NJ),
Alternatively, enzymes such as a combination of proofreading exonuclease and polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD) are used. Preferably, MICROLAB 2200 Liquid Transfer System (Hamilton, Reno, NV),
PTC200 Thermal Cycler200 (MJ Research, Watertown MA) and ABI CATALYST 80
Automate sequence preparation using a device such as 0 (Perkin-Elmer). Then ABI 373
Alternatively, sequencing is performed using the 377 DNA Sequencing System (Perkin-Elmer), MEGABACE 1000 DNA Sequencing System (Molecular Dynamics. Sunnyvale CA) or other methods known in the art. The resulting sequences are analyzed using various algorithms well known in the art (e.g., Ausubel, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7;
Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New
York NY, pp. 856-853.).

A variety of PCR-based methods well known in the art can be used to extend the nucleic acid sequence encoding HTMP using a partial nucleotide sequence to extract upstream sequences such as promoters and regulatory elements. To detect. For example, one method utilizing restriction site PCR is to amplify an unknown sequence from genomic DNA in a cloning vector using common and nested primers (eg, Sarkar, G. (1993) PCR Met.
See hods Applic 2: 318-322). An alternative method using the inverse PCR method uses primers that extend in a wide range of directions and amplify unknown sequences from a circularized template. This template is derived from a restriction fragment containing known genomic loci and sequences surrounding it (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186). Capture PCR
A third method using the method involves PCR amplification of DNA fragments flanking known sequences of human and yeast artificial chromosome DNA (eg, Lagerstrom, M. et al. (1991) PCR Methods Ap.
See plic 1: 111-119). This method allows digestion and ligation with multiple restriction enzymes to insert the recombinant double-stranded sequence into a region of unknown sequence prior to PCR. It is also possible to obtain the unknown sequence using other methods well known in the art. (For example, Parker, JD et al. (1991) Nucleic Acids Res
. 19: 3055-3060). Furthermore, by using PCR, nested primers, and PROMOTER FINDER library (Clontech, Palo Alto CA), walking within genomic DNA is possible. This method does not require screening the library and is useful for looking for intron / exon junctions. In all PCR-based methods, the primers are commercially available OLIGO 4.06 Primer Analysis software (National Bioscien
ces, Plymouth MN) or another suitable program etc.
It is designed to anneal to the template at a temperature of about 68 ° C to 72 ° C with 0 nucleotides, a GC content of 50% or more.

When screening for full-length cDNAs, it is preferable to use libraries that are size-selected to contain large cDNAs. Furthermore, if the oligo d (T) library cannot produce a full-length cDNA, a randomly primed library, which often contains a sequence having the 5'region of the gene, is useful. Genomic libraries will be useful for extension of sequences into the 5'non-transcribed regulatory regions.

Sequencing or PCR using a commercially available capillary electrophoresis system
It is possible to analyze or confirm the size of the nucleotide sequence of the product. For more information,
Capillary sequencing uses a flowable polymer for electrophoretic separation, and a laser-activated fluorochrome specific for four different nucleotides and a CCD camera used to detect emitted wavelengths. It is possible.
The output / light intensity is determined by the appropriate software (eg GENOTYPER and SEQUENCE NAVIGA
TOR, Perkin-Elmer) is used to convert to an electric signal, and the process from sample loading to computer analysis and electronic data display can be controlled by computer. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA, which may be present in small amounts in a particular sample.

In another embodiment of the invention, the polynucleotide sequence encoding HTMP or a fragment thereof is cloned into a recombinant DNA molecule to express HTMP, a fragment thereof or a functional equivalent in a suitable host cell. It is possible. Due to the degeneracy of the genetic code, alternative DNA sequences can be made which encode substantially the same or functionally equivalent amino acid sequences, which sequences are available for cloning and expression of HTMP.

The nucleotide sequences of the invention can be recombined using methods generally known in the art to alter the HTMP coding sequence for a variety of purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Nucleotide sequences can be recombined using DNA mixing with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, create splice variants, and the like.

The nucleotides of the present invention were labeled with MOLECULAR BREEDING (Maxygen Inc., Santa Clara C
A; U.S. Pat.No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793.
-797; Christians, FC et al. (1999) Nat. Biotechnol. 17: 259-264; Crameri, A.
Others (1996) Nat. Biotechnol. 14: 315-319) and the ability to shuffle using DNA shuffling techniques to bind the biological or enzymatic activity of HTMP, or to other molecules or compounds. The biological properties of HTMP such as can be changed or improved. DNA shuffling is the process of creating a library of gene variants by recombination of gene fragments by the PCR method. The library is then selected or screened to identify genetic variants with the desired properties. These preferred variants are pooled and the DNA shuffling and selection / screening repeated. Therefore, a variety of genes are created by artificial breeding and rapid molecular evolution. For example, one gene fragment having a mutation at a random position can be subjected to recombination, screening, and shuffling until the desired property is optimized. Alternatively, a fragment of a given gene may be recombined with a fragment of a homologous gene of the same gene family of the same or a different species, thereby regulating gene polymorphism of a large number of naturally occurring genes in a regulatable manner. Sex can be maximized.

According to another embodiment, the sequences encoding HTMP can be wholly or partially synthesized using chemical methods well known in the art (eg, Caruthers. MH et al. (1980).
) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acid
s Res. Symp. Ser. 225-232). Alternatively, chemical methods can be used to synthesize HTMP itself or fragments thereof. For example, peptide synthesis can be performed using a variety of solid phase techniques (eg, Roberge, JY et al. (1995) Science 269:
See 202-204). Alternatively, automation of synthesis may be achieved using, for example, the ABI 431A Peptide Synthesizer (Perkin Elmer). Furthermore, the amino acid sequence of HTMP, or any part thereof, may be altered by direct synthetic alterations and / or combinations with sequences from other proteins or any part thereof using chemical methods. It is possible to make a polypeptide.

This peptide is used for preparative high performance liquid chromatography (eg, Chiez, RM).
And FZ Regnier (1990) Methods Enzymol. 182: 392-421)). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (see, for example, Creighton. T. (1983) Proteins , Structures and Molecular Properties , WH Freeman, New York, NY).
.

To express biologically active HTMP, the nucleotide sequence encoding HTMP or a derivative thereof is inserted into a suitable expression vector. This expression vector contains the elements necessary for the regulation of transcription and translation of the coding sequence inserted into a suitable host. These elements include regulatory sequences such as enhancers, constitutive and expression-inducible promoters, 5'and 3'untranslated regions in the vector and polynucleotide sequences encoding HTMP. Such elements vary in length and specificity. With specific initiation signals, it is possible to achieve more efficient translation of sequences encoding HTMP. Such signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. If the sequence encoding HTMP and its initiation codon, upstream regulatory sequences were inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals would be required. However,
In-frame AT if only the coding sequence or a fragment thereof is inserted
Exogenous translational control signals, including the G start codon, must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of natural and synthetic sources. The efficiency of expression can be increased by the inclusion of enhancers suitable for the particular host cell system used. (For example, Scharf, D. et al. (19
94) Results Probl. Cell Differ. 201-18-162.).

Methods which are well known to those skilled in the art can be used to generate expression vectors containing sequences encoding HTMP and suitable transcriptional and translational regulatory elements. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination technology. (
For example, Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual ,
Cold Spring Harbor Press, Plainview NY, chapters 4 & 8, and 16-17; and
Ausubel, FM et al. (1995) Current Protocols in Molecular Biology , John Wi
ley & Sons, New York NY, ch. 9 and 13-1-4).

A variety of expression vector / host systems may be utilized to retain and express sequences encoding HTMP. These include, but are not limited to, microorganisms such as recombinant bacteriophage, plasmids, or bacteria transformed with cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors. Insect cell lines infected with (eg, baculovirus), or plants transformed with viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg, Ti or pBR322 plasmids) Cell lines and animal cell lines are included. The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors may be chosen depending upon the use intended for the polynucleotide sequence encoding HTMP. For example, routine cloning, subcloning of a polynucleotide sequence encoding HTMP,
PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (G
A multifunctional E. coli vector such as IBCO BRL) can be used. Ligation of the HTMP-encoding sequence at multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method for identification of transformed bacteria containing recombinant molecules. In addition, these vectors can be used to generate in vitro transcription of cloned sequences, dideoxin screening, single chain rescue by helper phage, and nested deletions. (See, for example, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.). For example, when a large amount of HTMP is required for antibody production, a vector which induces high level expression of HTMP can be used. For example, a vector containing a strongly inducible T5 or T7 bacteriophage promoter can be used.

It is possible to use yeast expression systems for the expression of HTMP. Various vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH promoter can be used for yeast Saccharomyces cerevisiae or Pichia pastoris . Furthermore, such vectors induce either the secretion of the expressed protein or its retention within the cell, integrating foreign sequences into the host genome for stable growth. (For example, Ausubel .; and Bitter, GA et al. (1987) Methods above.
Enzymol. 153: 51-794: Scorer. CA et al. (1994) Bio / Technology 121-181-184.
Plant systems can also be used to express HTMP. Transcription of the sequence encoding HTMP, for example, a viral promoter such as CaMV-derived 35S and 19S promoter alone,
Alternatively, it is promoted in combination with an omega leader sequence derived from TMV (Takamatsu, N. et al. (1987) EMBO J 6: 307-311). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example, The McGraw Hill Yearbook of Science and Technology (1992) Mc
See Graw Hill NY, pp.191-196).

In mammalian cells, a variety of virus-based expression systems may be utilized. When adenovirus is used as an expression vector, the sequence encoding HTMP can be linked to the adenovirus transcript / translation complex consisting of the late promoter and the triple leader sequence. Insertion into the nonessential E1 or E3 regions of the viral genome makes it possible to obtain live virus expressing HTMP in infected host cells (Logan, J.
And Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers, such as the Rous Sarcoma Virus (RSV) enhancer, can be used to increase expression in mammalian host cells. For high level expression of proteins, SV40 or EBV based vectors can be used.

Human artificial chromosomes (HACs) can be used to supply fragments of DNA that are larger than those expressed in and contained in a plasmid. HACs of about 6kb-10Mb for treatment
Are prepared and delivered by conventional delivery methods (liposomes, polycationic amino polymers, or vesicles). (For example, Harrington.JJ et al. (1997) Nat Genet. 15: 3.
45-355.).

For long-term production of recombinant proteins in mammalian systems, stable expression of HTMP in cell lines is desirable. For example, the expression vector can be used to transform a cell line with a sequence encoding HTMP. Such expression vectors contain replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are grown in enriched medium for about 1-2 days before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selective mediators, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells can be propagated using tissue culture techniques suitable for that cell type.

Any number of selection systems can be used to recover transformed cell lines. The selection systems include, but are not limited to, include herpes simplex virus thymidine kinase gene and adenine phosphoribosyltransferase genes, respectively tk ^- or apr ^- used in the cell. (For example, Wigler, M. et al. (1
977) Cell 11: 223-232; and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, and als or pat confers resistance to chlorsulfuron (cHTMPsulfuron) and phosphinotricin acetyltransferase (eg, phosphinotricin acetyltransferase). , Wigl
er, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin,
F. et al. (1981) J. Mol. Biol. 150: 1-14). Further genes available for selection have been described in the literature, eg trpB and hisD which alter what the cell needs for metabolism (eg Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad.
. Sci. 85: 8047-51). Anitocyanin, green fluorescent protein (GFP; Clon
tech), β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin and other visible markers are used. Green fluorescent protein (GFP) (Clon
tech, Palo Alto, CA) can also be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression due to a particular vector system (eg, Rhodes, CA et al. (19
95) Methods Mol. Biol. 55: 121-131).

Even if the presence / absence of expression of a marker gene indicates the presence of a gene of interest, it may be necessary to confirm the presence and expression of that gene. For example, if the sequence encoding HTMP is inserted within a marker gene sequence, transformed cells containing the sequence encoding HTMP can be identified by the lack of marker gene function.
Alternatively, the marker gene can be placed in tandem with the sequence encoding HTMP under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

In general, host cells which contain a nucleic acid sequence encoding HTMP and express HTMP can be identified using a variety of methods well known to those of skill in the art. These methods include
Protein biological tests or immunological methods including DNA-DNA or DNA-RNA hybridization, PCR methods, membrane-based, solution-based, or chip-based techniques for detection and / or quantification of nucleic acids or proteins. Assay included,
It is not limited to these.

HT with either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring the expression of MP are well known in the art. Such techniques include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence activated cell sorters (FACS) and the like. Two-site, monoclonal-based immunoassay using a monoclonal antibody that reacts with two non-interfering epitopes on HTMP
Is preferred, but competitive binding assays can also be used. These and other assays are well known in the art. (For example, Hampton. R
Et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Paul
.MN, Sect. IV; Coligan, JE et al Current Protocols in Immunology , Greene
Pub. Associates and Wiley-Interscience, New York. NY; and Pound, JD (
1990) Immunochemical Protocols , Humans Press, Totowa NJ).

Various labeling and conjugation techniques are well known to those of skill in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to HTMP-encoding polynucleotides include oligo-labeling, nick translation, end-labeling, or labeled nucleotides. Includes the PCR amplification used. Alternatively, the HTMP coding sequence, or any fragment thereof, can be cloned into a vector to generate an mRNA probe. Such vectors, which are well known in the art and are commercially available, can be used for the synthesis of RNA probes in vitro by the addition of a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. . These methods are described, for example, in Amersham Pharmac
ia Biotech and Promega (Madison WI), US Biochemical Corp (Cleveland OH
) Can be used with various kits commercially available. Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents and the like.

Host cells transformed with a nucleotide sequence encoding HTMP are cultured under conditions suitable for the expression and recovery of this protein in cell culture. Whether the protein produced by a transformed cell is secreted or remains intracellular depends on the sequence and / or the vector used. It will be understood by those skilled in the art that the expression vector containing the polynucleotide encoding HTMP can be designed to contain a signal sequence that induces the secretion of HTMP that penetrates the prokaryotic cell membrane and the eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability to regulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Modifications of such polypeptides include acetylation, carboxylation, glycosylation, phosphorylation, lipidation (
lipidation), and acylation, but is not limited thereto.
Post-translational processing which cleaves a "prepro" or "pro" form of the protein can be used to identify target proteins, folds and / or activities. A variety of host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular machinery and characteristic mechanisms for post-translational activity are available in the American Type Culture C
Ollection (ATCC; Bethesda, MD) and is selected to ensure correct modification and processing of foreign proteins.

In another embodiment of the invention, the natural or altered or recombinant nucleic acid sequence encoding HTMP is linked to a heterologous sequence which results in translation of the fusion protein of any of the host systems described above. For example, a chimeric HT containing a heterologous moiety that can be recognized by a commercially available antibody
MP proteins can facilitate the screening of peptide libraries for inhibitors of the activity of HTMP. In addition, the heterologous protein portion and the heterologous peptide portion are
Commercially available affinity substrates can be used to facilitate purification of the fusion protein. Such parts include glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-Hi.
s, FLAG, c-mc, hemagglutinin (HA), but are not limited thereto. GST and MBP, Trx, CBP, 6-His immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
Each of the metal chelate resins allows purification of the cognate fusion protein. FLAG
, C-mc, and hemagglutinin (HA) allow purification of the immunoaffinity of the fusion protein using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Alternatively, HTMP can be cleaved from the heterologous moiety after purification by genetically engineering the fusion protein to include a proteolytic cleavage site between the HTMP coding sequence and the heterologous protein sequence. Methods for expressing and purifying fusion proteins are described in Ausubel. (1995, supra ch 10). A variety of commercially available kits can be used to facilitate expression and purification of the fusion protein.

In another embodiment of the present invention, it is possible to synthesize radiolabeled HTMP in vitro using the TNT rabbit reticulocyte lysate or wheat germ extraction system (Promega). These systems connect transcription and translation of sequences encoding proteins that are operably linked to the T7 or T3, SP6 promoters. Translation occurs in the presence of a radiolabeled amino acid precursor, which is, for example, ³⁵ S methionine.

Fragments of HTMP can be produced by direct peptide synthesis using solid phase techniques as well as recombinant products (see, eg, Creighton, pp. 55-60. Supra). Protein synthesis can be performed manually or automatically. Automated synthesis can be performed, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer). Various fragments of HTMP are synthesized separately and then ligated to produce the full length molecule.

Treatment There are chemical and structural similarities between a region of HTMP and a region of human transmembrane protein, eg in the context of sequences and motifs. In addition, HTMP expression is closely associated with cell proliferation and immune response, as well as with cardiovascular and gastrointestinal tissues, reproductive tissue neural tissue. Therefore, HTMP is thought to play a role in cell proliferative disorders and immune disorders, reproductive disorders, smooth muscle disorders, and neurological disorders.
In the treatment of diseases associated with increased HTMP expression or activity, it is desirable to reduce HTMP expression or activity. In addition, in the treatment of diseases associated with decreased HTMP expression or activity, it is desirable to increase HTMP expression or activity.

Thus, in one embodiment, HTMP or a fragment or derivative thereof may be administered to a patient for the treatment or prevention of disorders associated with decreased HTMP expression or activity. Examples of such disorders include, but are not limited to, cell proliferative disorders, including actinic keratoses and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed Type connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and malformations Cancer, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
Includes parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, uterine cancer, and immune disorders, including acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency. , Adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candid ectoderm Dystrophy (APECED),
Bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphotoxin-induced temporary lymphopenia, erythroblastosis, erythema nodosum,
Atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
Myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, Thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, and viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminthic infections, trauma , And also includes reproductive disorders, including abnormal prolactin production, fallopian tube disease,
Abnormal ovulation and endometriosis, abnormal estrus, abnormal menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune disorders, ectopic pregnancy, and malformation Including infertility, breast cancer, fibrocystic mastopathy, and milk leakage, spermatogenesis, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, Including male breast cancer and gynecomastia,
Also included are smooth muscle abnormalities, among which, stomatitis, anaphylaxis, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and pheochromocytoma, Includes myopathy including cardiomyopathy, encephalopathy, epilepsy, Kerns-Saiya syndrome, lactic acidosis, muscular clonic disorders, and ophthalmoplegia,
Also included are neurological disorders, including epilepsy, ischemic cerebrovascular accident, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, muscle atrophy. Sexual lateral sclerosis and other motor neuron disorders,
Progressive neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess ,
Purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease and Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-
Prion diseases including Straussler-Scheinker syndrome, fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma (cerebell)
oretinal hemangioblastomatosis), trigeminal neurovascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, peripheral nerve diseases, dermatomyositis and polymyositis, and hereditary , Metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic quadriplegia, mood and anxiety disorders, psychosis including schizophrenia, seasonal disorders (SAD), and quiz Disability, amnesia, catatonic disease, diabetic neuropathy, extrapyramidal terminal syndrome, dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease, and progressive supranuclear palsy, corticobasal. degeneration) and familial frontotemporal amnesia.

In another embodiment, HTMP or a fragment or derivative thereof is expressed for the treatment or prevention of diseases associated with decreased expression or activity of HTMP, including but not limited to the diseases listed above. It is also possible to administer the resulting vector to a patient.

In yet another embodiment, substantially purified HTMP is used for the treatment or prevention of diseases associated with decreased expression or activity of HTMP, including but not limited to the diseases listed above. It is also possible to administer the pharmaceutical composition containing it to a patient together with a suitable pharmaceutical carrier.

In yet another embodiment, an agonist that modulates the activity of HTMP for the treatment or prevention of diseases associated with decreased expression or activity of HTMP, including but not limited to the diseases listed above. It is also possible to administer to a patient.

In a further embodiment, a patient may be administered an antagonist of HTMP for the treatment or prevention of a disorder associated with increased HTMP expression or activity. Examples of such diseases include, but are not limited to, the above-mentioned abnormal cell proliferation and immune disorders, reproductive disorders, smooth muscle disorders, and neurological disorders. In one embodiment, HT
Antibodies that specifically bind MP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism to deliver the drug to cells or tissues expressing HTMP.

In another embodiment, a complementary sequence of a polynucleotide encoding HTMP for the treatment or prevention of diseases associated with increased expression or activity of HTMP, including but not limited to the diseases listed above. It is also possible to administer a vector that expresses

In another example, any of the proteins, antagonists, antibodies, agonists, complementary sequences, vectors of the invention can be administered in combination with another suitable therapeutic agent. One of ordinary skill in the art can select suitable therapeutic agents for use in the combination therapy according to conventional pharmaceutical principles. The combination with therapeutic agents may bring about a synergistic effect in the treatment or prevention of the various diseases listed above. Using this method, it is possible to obtain a medicinal effect with a small amount of each drug, and it is possible to reduce the possibility of a wide range of side effects.

Antagonists of HTMP can be prepared using methods common in the art. More specifically, purified HTMP can be used to produce antibodies, or a library of therapeutic agents can be screened to identify those that specifically bind to HTMP. HTMP
Antibodies can also be produced using a method generally used in this field. Such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chains,
Fab fragments and fragments produced by Fab expression libraries are included. However, it is not limited to these. For treatment, neutralizing antibodies (ie,
Those which inhibit the formation of dimers) are particularly preferred.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans and others, can be injected with HTMP or any fragment or oligopeptide thereof with immunogenic properties. Can be immunized with. Depending on the host species, various adjuvants may be used to enhance the immune response. Such adjuvants include Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and surfactants such as dinitrophenol. However, it is not limited thereto. Among the adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly preferable.

Oligopeptides, peptides or fragments used to elicit antibodies to HTMP are preferably composed of at least about 5 amino acids, generally about 10 or more amino acids. These oligopeptides or peptides, or fragments thereof, are preferably identical to a portion of the amino acid sequence of the native protein and also include the entire amino acid sequence of small naturally occurring molecules. Short stretches of HTMP amino acids can be fused with sequences of another protein, such as KLH, to produce antibodies to the chimeric molecule.

Monoclonal antibodies against HTMP can be produced by successive cell lines in culture, using any technique that produces antibody molecules. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler,
G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) .J. Immunol. Met.
hods 81-8-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030.
Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques such as splicing the mouse antibody gene onto the human antibody gene developed to produce “chimeric antibodies” are used to obtain molecules with suitable antigen specificity and biological activity ( For example, Morrison, SL et al. (1984) Proc. N
atl. Acad. Sci. 81-4851-4855; Neuberger, MS et al. (1984) Nature 312: 604.
-608; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, applying the described techniques for the production of single chain antibodies using methods well known in the art,
Generates HTMP-specific single chain antibody. Antibodies with related specificities but of different idiotypic composition can also be generated by chain mixing from random combinatorial immunoglobulin libraries (eg, Burton DR (1991) Proc. Natl. A).
cad. Sci. 88: 11120-3).

Antibodies can be induced by in vivo production in a population of lymphocytes, or by screening immunoglobulin libraries or a panel of highly specific binding reagents, as shown in the literature. (See, for example, Orlandoi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833.
-3837; Winter, G. et al. (1991) Nature 349: 293-299).

Antibodies containing specific binding sites for HTMP can also be produced. For example,
Such fragments include F (ab ') fragments produced by pepsin digestion of antibody molecules and Fabs produced by reducing the disulfide bridges of the fragments to F (ab').
Fragments are included, but are not limited to. Alternatively, generating a Fab expression library allows for rapid and easy identification of monoclonal Fab fragments with the desired specificity (eg, Huse, WD et al. (1989) Science 254: 12.
See 75-1281).

Various immunoassays are used to screen to identify antibodies with the desired specificity. Numerous protocols for competitive binding, or immunoradioactivity, using either polyclonal or monoclonal antibodies with isolated specificity are well known in the art. Usually for such immunoassays, HTMP
Included is the measurement of complex regulation between the antibody and its specific antibody. Two-site, monoclonal-based immunoassays are generally used that use monoclonal antibodies reactive to two non-interfering HTMP epitopes, but competitive binding assays can also be used (Pound, supra).

Using various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques, H 2
Assess the affinity of the antibody for TMP. The affinity is represented by a binding constant Ka, which is a value obtained by dividing the molar concentration of the HTMP antibody complex under the equilibrium state by the molar concentration of the free antibody and the free antigen. The Ka of a polyclonal antibody drug having a heterogeneous affinity for many HTMP epitopes represents the average affinity or binding activity of the antibody for HTMP. Ka of a monoclonal antibody drug specific for a specific HTMP epitope is
Represents a true measure of affinity. The high affinity antibody drug having a Ka value of 10 ⁹ to 10 ¹² L / mol is preferably used in an immunoassay in which the HTMP antibody complex must withstand violent manipulation. The low affinity antibody drug having a Ka value of 10 ⁶ to 10 ⁷ L / mol is
Immunopurification in which HTMP must finally dissociate from the antibody in an activated state (immunopuri
fication) and similar processes. (Catty, D. (1988) Antibodies , Volume I: A Practical Approach .IRL Press, Washington, DC; Liddell,
JE and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies , J
ohn Wiley & Sons, New York NY).

To examine the quality and suitability of such pharmaceuticals in certain downstream applications,
The antibody titer and binding activity of the polyclonal antibody drug are further evaluated. For example, a polyclonal antibody drug containing at least 1-2 mg / ml of the specific antibody, preferably 5-10 mg / ml of the specific antibody, is generally used in the treatment in which the HTMP antibody complex must be precipitated. Specificity of antibodies and antibody titer, binding activity, quality of antibodies and guidelines for usage in various applications are publicly available. (For example, Cat
ty, supra, and Coligan et al., supra).

In another embodiment of the invention, the polynucleotide encoding HTMP, or any fragment or complementary sequence thereof, can be used for therapeutic purposes. In certain embodiments, if the complementary sequence of a polynucleotide encoding HTMP is suitable for blocking the transcription of mRNA, it can be used. In particular, cells can also be transformed with sequences complementary to the polynucleotide encoding HTMP. Thus, complementary molecules or fragments can be used to regulate the activity of HTMP, or the regulation of gene function. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the HTMP coding sequence, or from various positions along the coding region.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia, or various bacterial plasmids can also be used to deliver the nucleotide sequence to the target organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to generate vectors expressing nucleic acid sequences complementary to HTMP-encoding polynucleotides (see, eg, Sambrook et al., Supra, and Ausubel et al., Supra). .

The gene encoding HTMP can be stopped by transforming cells or tissues with an expression vector that expresses high levels of a polynucleotide encoding HTMP or a fragment thereof. Such constructs can be used to introduce non-translatable sense or antisense sequences into cells. Even when not integrated into DNA, such vectors continue to transcribe mRNA molecules until impaired by endogenous nucleases. Non-replicating vectors also have transient expression that lasts for a month or more, and may last even longer if suitable replication elements are part of the vector system.

As described above, the expression of a gene can be regulated by designing a sequence complementary to the regulatory 5 ′ or regulatory region of the gene encoding HTMP or an antisense molecule (DNA or RNA, PNA). . For example, it is possible to use an oligonucleotide derived from the transcription start site from about -10 to about +10 from the start site. Similarly, it can be blocked using "triple helix" and base pairing methods.
The triple helix structure is useful because it prevents the double helix from expanding sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances with triplex DNA have been described in the literature (eg Gee, JE et al. (1994) In: Hub.
er, BE and BI Carr, Molecular and ImmunologiHTMPproaches , Futura Pu
See blishing Co., Mt. Kisco, NY). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by blocking the binding of transcripts to ribosomes.

Ribozymes, enzymatic RNA molecules, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by nucleotide strand scission.
For example, recombinant hammerhead ribozyme molecules that specifically and effectively catalyze the nucleotide scission of sequences encoding HTMP are included.

A specific ribozyme cleavage site within any potential RNA target is followed by the sequence GUA.
, GUU, and GUC, are first identified by scanning the target molecule against ribozyme cleavage sites. Once identified, a short RNA sequence of 15 to 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is evaluated for secondary structural features that impair the function of the oligonucleotide. Is possible. The suitability of candidate targets can also be assessed by testing their ease of hybridization with complementary oligonucleotides using a ribonuclease protection assay.

Complementary ribonucleic acid molecules and ribozymes of the invention can be made for the synthesis of nucleic acid molecules using methods well known in the art. These methods include methods of chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, RNA molecules can be generated in vitro and in vivo by transcription of a DNA sequence encoding HTMP, such DNA sequences using various RNA polymerase promoters, such as T7 or SP6, in various vectors. Can be incorporated into. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.

Modification of RNA molecules can increase intracellular stability and prolong half-life. Possible modifications include the addition of flanking sequences at the 5'and / or 3'ends of the molecule, or the use of phosphorothionate or 2'O methyl rather than phosphodiesterase linkages within the backbone of the molecule. , Are not limited to these. Unique to PNA production, this concept is that adenine, cytidine, guanine, thymine, which are not readily recognized by endogenous endonucleases,
And the acetyl-, methyl-, thio-, and similar modified forms of uridine, as well as the inclusion of non-conventional bases such as inosine, queosine, and wybutosine, in whole of these molecules. Can be expanded to.

[0171] vectors available are a number of ways of introducing into a cell or tissue, in vivo, are equally suitable for use in in vitr o, and ex vivo. For ex vivo treatment, the vector is introduced into hepatocytes taken from a patient and cloned and propagated for autologous transplant back into the same patient. Transfection, ribosome injection or delivery by polycationic aminopolymers can be performed using methods well known in the art (eg Goldman, CK et al. (1997) Nature Biotechnology 15: 462.
-66 :).

Any of the above methods of treatment can be applied to any subject in need of treatment including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits and monkeys.

Another embodiment of the present invention relates to the administration of a pharmaceutical or sterile composition in association with a pharmaceutically acceptable carrier for all of the above mentioned therapeutic effects. Such a pharmaceutical composition comprises HTMP, an antibody of HTMP, a mimetic, an agonist, an antagonist, an inhibitor of HTMP, and the like. The composition can be administered alone or with one or more additional agents such as stabilizers to a sterile biocompatible pharmaceutical carrier. Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water and the like. This composition can be administered alone or with another agent such as a drug or hormone.

The pharmaceutical composition used in the present invention can be administered by various routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, subarachnoid, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Are not limited to these.

In addition to the active ingredients, these pharmaceutical compositions also contain suitable pharmaceutically acceptable excipients and adjuvants that facilitate the formulation of the active compound into pharmaceutically usable agents. The carrier may be included. Detailed techniques for formulation and administration are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, PA).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers allow tablets, pills, dragees, capsules, and pharmaceutical compositions to be ingested by patients.
It is formulated as a liquid, gel, syrup, mud, or suspension.

Medicaments for oral use include tablets or dragee cores (after grinding if desired) by mixing the active compound with solid drug additives and treating the resulting granule mixture. cores). Suitable excipients include lactose, sucrose, mannitol, or sugars including sorbitol, starch from corn, wheat, rice, potato, or other plants, methylcellulose, hydroxypropylmethylcellulose, or cellulose such as sodium carboxymethylcellulose, Gum, including gum arabic and gum tragacanth, carbohydrates or protein excipients such as proteins such as gelatin and collagen.
If desired, a disintegrant or solubilizer such as cross-linked polyvinylpyrrolidone, kanten, alginic acid, or its salt sodium alginate is added.

Dragee cores are used with a suitable coating such as a concentrated sugar solvent. Such concentrated sugar solvents may include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solvents, and suitable organic or mixed solvents. Dyestuffs or pigments are added to the tablets or dragee coatings for identification of the product or to indicate the quantity of active compound, ie the dose.

Pharmaceutical formulations for oral use include push-fit capsules made of gelatin, encapsulated capsules made of gelatin with a coating such as glycerol or sorbitol. Push-fit capsules contain the active ingredient in admixture with excipients or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, if desired, stabilizers. In soft capsules, the active compound, with or without stabilizer,
It may be dissolved or suspended in a suitable solution such as a fatty oil, solution, or polyethylene glycol solution.

Pharmaceutical agents suitable for parenteral administration are formulated in aqueous solution, but physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological buffered saline are preferred. Aqueous suspension injections may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be formulated as suitable oily injection suspensions. Suitable hydrophilic solutions or vehicles include fatty oils such as sesame oil, ethyl oleate, triglycerides or synthetic fatty acids such as ribosomes. Non-lipid polycationic amino polymers are used for delivery purposes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for concentrated solutions.

For local or nasal administration, suitable penetrants that penetrate certain barriers are used in the formulation. Such penetrants are well known to those of ordinary skill in the art.

The pharmaceutical compositions of the present invention may be prepared using methods well known in the art, such as conventional mixing, dissolving, granulating, sugar coating, elevating, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Can be manufactured.

Pharmaceutical compositions are formulated as salts and can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid. Salts are more soluble in water or other protic solvents than are the corresponding free base systems. Other drug forms include 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2%.
Freeze-dried powders containing some or all of ~ 7% mannitol and having a pH range of 4.5-5.5 and combined with a buffer before use can be used.

After the pharmaceutical compositions have been formulated, they are packaged in suitable boxes and labeled as medication for the indicated symptoms. For administration of HTMP, such labels will include amount, frequency and method of administration.

Suitable pharmaceutical compositions for use in the present invention include compositions which contain an effective amount of active ingredients to achieve the objectives. One of ordinary skill in the art can determine a sufficiently effective dose on his or her own ability.

For any composition, the therapeutically effective dose can be estimated initially either in tumor cell assays, eg, of tumor cells, or in animal models. Usually, a mouse, a rabbit, a dog, a pig, or the like is used as the animal model. Animal models can also be used to determine the appropriate concentration range and route of administration. Based on such treatment, a beneficial dose and administration route for human can be determined.

A medically effective dose is related to the amount of active ingredients, such as HTMP or fragments thereof, antibodies of HTMP, agonists or antagonists of HTMP, inhibitors, etc. that ameliorate symptoms or conditions. Medicinal efficacy and toxicity can be calculated, for example, by calculating the ED ₅₀ (50% of the population has a medicinal effect on dose) or LD ₅₀ (50% of the population is fatal on dose) statistics. , Can be determined by standard drug techniques in cell culture or animal studies. Dosage ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD _{50 /} ED _50. Pharmaceutical compositions that exhibit high therapeutic indices are desirable. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human application. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the patient sensitivity, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Dosage factors considered include disease severity, general health of the patient, age, weight and sex of the patient, time and frequency of administration, concomitant medications, response susceptibility, and treatment. Contains the response. Long-acting pharmaceutical compositions may be administered every three or four days, once a week, once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0189] The usual dose varies depending on the route of administration, but is about 0.1-100,000 μg.
Up to about 1 gram. Guidance on specific dosages and methods of delivery is provided in the literature and is generally available to practitioners in the art. Those skilled in the art will utilize formulations of nucleotides that are different than the protein or inhibitor. Similarly, delivery of a polynucleotide or polypeptide is
It will be specific to a particular cell, condition, location, etc.

Diagnosis In another example, a patient who is diagnosed with a disease in which an antibody that specifically binds to HTMP is characterized by the expression of HTMP, or is treated with HTMP or an agonist or antagonist of HTMP, or an inhibitor. Used in an assay to monitor Antibodies useful for diagnosis are formulated in the same manner as described in treatment.
HTMP diagnostic assays include methods of detecting HTMP from human body fluids or from cells or tissues using antibodies and labels. These antibodies, used with or without modification, can be labeled by covalent or non-covalent attachment of reporter molecules. Various reporter molecules known in the art are used, some of which have been mentioned above.

Various protocols for measuring HTMP, including ELISA, RIA, and FACS, are well known in the art and provide a basis for diagnosing altered or abnormal levels of HTMP expression. The value of normal or normal HTMP expression is determined by binding a body fluid or cells collected from a subject such as a normal mammal, for example, a human, with an antibody against HTMP under conditions suitable for complex formation. To do. The amount of canonical complex formation can be quantified by various methods such as photometric. The amount of HTMP expression, control and disease in the subject, samples from biopsy tissue are compared to baseline. The deviation between the reference value and the subject is an index for diagnosis.

According to another embodiment, the polynucleotide encoding HTMP can also be used for diagnosis. The polynucleotide used includes an oligonucleotide sequence,
Included are complementary RNA and DNA molecules, and PNAs. This polynucleotide is used to detect and quantify gene expression in biopsied tissues that express HTMP, which can be correlated with disease. Use this diagnostic assay to check for the presence or absence of HTMP and for overexpression,
Monitor the regulation of HTMP levels during treatment.

In certain embodiments, it is possible to identify a nucleic acid sequence encoding HTMP by hybridization with a PCR probe capable of detecting a polynucleotide sequence comprising a gene sequence encoding HTMP or a closely related molecule. is there. The specificity of the probe, whether it is made from a highly specific region, for example the 5'regulatory region, or a region with less specificity, which is a conserved motif, and the stringency of hybridization or amplification, Will identify only the natural sequences that encode HTMP, or will only identify the natural sequences that encode alleles or related sequences.

Probes can also be used to detect related sequences and have at least 50% sequence identity with any sequence encoding HTMP. The desired hybridization probe of the present invention can be DNA or RNA and can be derived from the sequence of SEQ ID NO: 30-58, or the genomic sequence including the promoter, enhancer and intron of the HTMP gene.

As a method for producing a hybridization probe specific to HTMP-encoding DNA, there is a method of cloning a polynucleotide sequence encoding HTMP and an HTMP derivative into a vector for producing an mRNA probe. Such vectors are commercially available, well known to those skilled in the art, and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. Hybridization probes can be labeled with a population of different reporters such as radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase linked to the probe by an avidin / biotin binding system.

Polynucleotide sequences encoding HTMP can be used to diagnose diseases associated with the expression of HTMP. Examples of such disorders include, but are not limited to, cell proliferative disorders, including actinic keratoses and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed Type connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and malformations Cancer, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung,
This includes cancers of muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, uterus, and also immune disorders, among which acquired immune deficiency syndrome ( AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polygland Endocrine candid ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphotoxin-induced temporary lymphopenia, erythroblasts , Erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout,
Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation And viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, and reproductive disorders, including prolactin production abnormalities. , Fallopian tube disease, ovulation abnormality, and endometriosis, estrus, abnormal menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune abnormality, ectopic Pregnancy And infertility, including teratogenesis, breast cancer, fibrocystic mastopathy, and milk leakage, spermatogenesis, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie Diseases, impotence, male breast cancer and gynecomastia, and also smooth muscle disorders, among which stomatitis, anaphylaxis, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, Hypoglycemia, myocardial infarction, migraine, and pheochromocytoma with cardiomyopathy, encephalopathy, epilepsy, Kearns-
Includes muscle disorders such as Seiya syndrome, lactic acidosis, muscular clonic disorders, and ophthalmoplegia, and also includes neurological disorders, among which are epilepsy, ischemic cerebrovascular disorder, stroke, cerebral neoplasm. , Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, hereditary Ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and nerve roots. Inflammation, viral central nervous system diseases and prion diseases including Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia, neurotrophic disease Metabolic disease, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastomatosis, trigeminal vascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomy Nervous system disorders, peripheral nerve diseases, dermatomyositis and polymyositis, hereditary, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic quadriplegia, and mood and anxiety disorders, Psychosis, including schizophrenia, seasonal disorders (SAD)
Amnesia, catatonic disease, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease, progressive supranuclear palsy, corticobasal degeneration And familial frontotemporal amnesia. The HTMP-encoding polynucleotide sequence can be expressed by Southern, Northern, dot-blot, or other membrane-based techniques, PCR, dipstick, pin, ELISA assay, and expression of mutant HTMP. Can be used in a microarray that utilizes body fluids or tissues collected from a patient to detect Such qualitative or quantitative methods are well known in the art.

In certain embodiments, HTMP-encoding nucleotide sequences will be useful in assays to detect related diseases, particularly those mentioned above. The nucleotide sequence encoding HTMP could be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the samples are washed and the signal is quantified and compared to the reference value. If the amount of signal in the patient sample is significantly altered compared to the control sample, the level of mutation of the HTMP-encoding nucleotide sequence in the sample reveals the presence of the associated disease. Such assays can be used to estimate the effectiveness of a particular treatment in animal studies, clinical trials, or monitoring individual patient treatment.

A normal or canonical expression profile is established to provide a basis for diagnosis of diseases associated with expression of HTMP. This can be achieved by combining the HTMP coding sequence or a fragment thereof with a body fluid or cell extracted from a normal subject, either animal or human, under conditions suitable for hybridization or amplification. . Standard hybridizations can be quantitated by comparing the values obtained from normal subjects to those from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared with those obtained from subjects who exhibit symptoms of the disease. The deviation between the reference value and the subject's value is used to determine whether or not the patient is affected.

Once the presence of the disease has been established and the treatment protocol has begun, the hybridization assay is repeated on a regular basis to estimate whether the level of expression in the subject has begun to approach the values shown in normal patients. Is possible. The results of repeated assays can be used to see the effect of treatment over a period of days to months.

In cancer, abnormal amounts of transcripts in living tissue from an individual predispose to the development of the disease and can provide a method of detecting the disease before actual clinical symptoms develop. is there. This type of clearer diagnosis allows medical professionals to use preventative or aggressive therapies early to prevent the development or progression of cancer.

Further diagnostic uses for oligonucleotides designed from sequences encoding HTMP can include the use of PCR. Such oligomers are chemically synthesized,
It can be produced enzymatically or in vitro . The oligomer is preferably
It includes a fragment of a polynucleotide encoding HTMP or a fragment of a polynucleotide complementary to the polynucleotide encoding HTMP, and is used for identifying a specific gene or condition under the optimum conditions. Further, the oligomer can be used for detecting and / or quantifying a closely related DNA or RNA sequence under slightly mild stringent conditions.

Methods that can be used to quantify the expression of HTMP include radiolabelling of nucleotides or biotin labeling, coamplification of regulatory nucleic acids, and standard curves with the results added. (For example, Melby, PC et al. (1993) J. Imm
unol. Methods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The quantitation rate of a large number of samples was accelerated by using a high-throughput assay in which the oligomer of interest was included in various dilutions and the quantitation was rapid by spectrophotometry or non-color response.

In another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein are used as targets in microarrays. Microarrays are used to monitor the expression levels of a large number of genes at the same time and to identify gene mutations, mutations and polymorphisms. This information is useful in determining gene function, interpreting the genetic basis of disease, diagnosing disease, and monitoring and developing the activity of therapeutic agents.

Microarrays are prepared, used, and analyzed by methods well known in the art. (For example, Brennan, TM et al. (1995) US Pat. No. 5,474,796; Schena, M. et al. (1996) Pro.
c. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) PCT application number W
O95 / 251116; Shalon, D. et al. (1995) PCT application number WO95 / 35505; Heller, RA et al. (1
997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Heller, MJ et al. (1997) U.S. Patent No. 5,605,662. Thus, it is possible to generate hybridization probes useful for mapping naturally occurring genomic sequences. This array maps to:
Specific chromosomes, specific regions of chromosomes or artificially generated chromosomes, for example, human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 products or single chromosome cDNA libraries. (For example, Harrington, 1.3. Other (1
997) Nat Genet. 15: 345-355; Price, CM (1993) Blood Rev. 5-87-134, and Trask, BJ (1991) Trends Genet. 7: 149-154) in sit fluorescence hybridization ( FISH) will correlate with other physical chromosome mapping techniques and genetic map data (see, eg, Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.). Examples of genetic map data can be found in various scientific journals or the World Wide Web site of the Online Mendelian Inheritance in Man (OMIM). On a physical chromosome map
The correlation between the location of the gene encoding HTMP and a particular disease, or the predisposition to a particular disease, helps determine the region of DNA associated with such a disease. The nucleotide sequences of the present invention may also be used to detect differences in gene sequences between normal, carrier and infected individuals.

The genetic map can also be extended using physical mapping techniques such as in sit hybridization of chromosomal preparations and binding analysis using established chromosomal markers. By placing the gene on the chromosome of another mammal, such as a mouse,
Relevant markers are often revealed, even if the number or arm of a particular human chromosome is not known. The new array by physical mapping
It can also be assigned to a chromosome arm. This is valuable information for researchers studying genetic disorders using positional cloning or other gene discovery techniques. The location of the disease or syndrome is, for example, 11q22-
Once roughly determined by gene binding to a particular gene region such as 23, any sequence mapping for that region represents a relevant gene or regulatory gene for further investigation (eg, Gatti, RA et al. (1988). Nature 336: 577-580
See). Further, by using the nucleotide sequence of the present invention of interest, a normal person, a holder,
That is, a difference in chromosomal position due to translocation, inversion, etc. between infected persons may be detected.

In another embodiment of the invention, HTMP, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen a library of compounds in any of a variety of drug screening techniques. Fragments used for such screening may be free in solution, immobilized on a solid support, retained on the surface of cells, or present intracellularly. The formation of a complex due to the binding of HTMP with the agent to be tested may be measured.

Another method used for drug screening is used to increase the screening throughput of compounds with suitable binding affinity for the protein of interest (eg, Geysen, et al. (1984) PCT Application No. See WO84 / 03564). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is reacted with HTMP or fragments thereof and then washed. Bound HTMP is then detected by methods well known in the art. Purified HTMP can also be coated directly on the plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another example, competitive drug screening assays can be used in which neutralizing antibodies capable of binding HTMP specifically compete with a test compound for binding HTMP.
In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with HTMP.

In another embodiment, nucleotide sequences encoding HTMP are used in developing molecular biology techniques, including, but not limited to, currently known nucleotides such as the triplet code and specific base pair interactions. New technologies can be provided that depend on the characteristics of the sequence.

Those skilled in the art will be able to make maximum use of the present invention without further explanation given the preceding description. Therefore, the specific preferred embodiments described below are for purposes of illustration and not limitation of the present invention.

All patent applications, patents, publications mentioned above and below, especially US patent application serial numbers 60 / 125,537 and 60 / 139,565, are incorporated herein by reference.

Example 1 Preparation of cDNA Library RNA was purchased from Clontech or isolated from the tissues listed in Table 4. First, while homogenizing a part of this tissue and dissolving it in a guanidinium isothiocyanate solution, homogenizing another part of this tissue and dissolving it in phenol, or TRIZOL (Life Technologies), guanidinium Dissolved in a mixture of suitable modifiers such as a single phase solution of thiocyanate and phenol. The lysate was centrifuged in cesium chloride or extracted with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.

RNA extraction with phenol and precipitation were repeated as many times as necessary to increase the purity of the RNA. In some cases, RNA is treated with a DNA degrading enzyme. Most libraries have oligo d (T) -linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN).
Valencia CA), OLIGOTEX mRNA purification kit (QIAGEN) was used to isolate poly (A +) RNA. Alternatively, another RNA isolation kit such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX) was used to isolate directly from tissue lysates.

[0214] In some cases, RNA was provided to Stratagene and a cDNA library corresponding to Stratagene was created. Otherwise, UNIZAP vector system (Stratagene)
Alternatively, a cDNA library was prepared by using the SUPERSCRIPT plasmid system (Life Technologies) to synthesize cDNA by a recommended method known in the art or a similar method.
(See, eg, Ausubel, 1997, supra, Units 5.1-6.6). Reverse transcription is oligo d (T)
Or started with random primers. A synthetic oligonucleotide adapter was attached to the double-stranded cDNA and the cDNA was digested with a suitable restriction enzyme or restriction enzymes. For most libraries, SEPHACRYL S 1000 or SEPHAROSE
CL2B, SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech
), And the size of the cDNA (300-1000 bp) was selected by agarose gel electrophoresis. PBLUESCRIPT plasmid (Stratagene) or pSPORT1 plasmid (Life Technolo
gies), pcDNA2.1 plasmid (Invitrogen Carlsbad CA), plNCY plasmid (Incyt
The cDNA was attached to the compatible restriction enzyme sites of the polylinker of a suitable plasmid such as ePharmaceuticals, Palo Alto CA). Use this recombinant plasmid with XL1-Blue, XL1-BIueMRF, SOLR from Stratagene, or DH5α or DH from Life Technologies.
10B, ELECTROMAX DH 10B was introduced into competent E. coli cells and incorporated.

2 Isolation of cDNA clones Plasmids were recovered from host cells by in vivo excision utilizing the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Minipreps
DNA purification system (Promega) and AGTC Miniprep purification kit (Edge Biosystems, G
aithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QI
Plasmids were purified using at least one of the AWELL 8 Ultra Plasmid Purification System, REAL Prep 96 Plasmid Kit. After precipitation, it was resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without freeze-drying.

Alternatively, plasmid DNA was amplified from host cell lysates by a high throughput direct ligation PCR method. (Rao, VB (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and transferred to 384-well plates for storage and the concentration of amplified plasmid DNA is quantified using PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). Measured.

3 Sequencing and Analysis cDNA sequencing reactions can be performed using standard methods or ABI CATALYST 800 (
Perkin-Elmer) thermal cycler or PTC-200 thermal cycler (MJ Research)
HYDRA Microdispenser (Robbins Scientific) or MICROLAB 2200 (Ham
ilton) High throughput equipment such as combination with liquid transfer system. For the preparation of the cDNA sequencing reaction, a reagent from Amersham Pharmacia Biotech or a reagent contained in an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer) was used. For electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides, the MEGABACE 1000 DNA Sequencing System (Molecular Dynami
cs), ABI PRISM 37 using standard ABI protocol and base pair calling software
A 3 or 377 sequencing system (Perkin-Elmer), other sequence analysis systems known in the art, was used. The open reading frame of the cDNA sequence is standard (Ausubel, 1997
, Unit 7.7), supra. Some of the cDNA sequences were selected and extended by the method described in 6 of this Example.

The construction and analysis of the polynucleotide sequences obtained from the sequencing of the cDNA was performed in combination with software utilizing algorithms well known to those skilled in the art.
Table 5 outlines the tools, software, algorithms utilized, their description, references, and threshold parameters used. Column 1 of Table 5 is the tools and programs used, algorithms, column 2 is a brief description thereof, column 3 is a reference cited as part of this specification by reference, column 4 is a sequence of two sequences. The score, probability value, and other parameters used in the evaluation of the degree of coincidence are shown (the higher the probability value, the higher the homology between sequences). MACDNASIS PRO software (Hitachi Softwa
re Engineering, S. San Francisco CA) and LASER GENE software (DNASTAR)
Was used.

Confirmation of polynucleotide sequences is accomplished by using BLAST and dynamic programming, programs and algorithms based on dinucleotide nearest neighbor analysis to remove vectors and linkers, poly A sequences and mask ambiguous base pairs. I went there. next,
Using databases based on BLAST, FASTA and BLIMPS, public databases such as GenBank primates and rodents, mammals, vertebrates, eukaryotic databases and BLOCKS, PRINTS, DOMO, PRODOM and PFAM databases. Annotations were obtained by querying these sequences against sequences selected from. These sequences were constructed into full-length polynucleotide sequences using programs based on Phred and Phrap, Consed and screened for open reading frames with programs based on BLAST and FASTA, BLIMPS. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, and the GenBank database (above) and databases such as SwissProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite,
Alternatively, these full-length sequences were analyzed by querying a protein family database based on Hidden Markov Models (HMM) such as PFAM. HMMs use probabilities to analyze consensus primary structure of gene families (eg, Ed
dy, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365).

The programs described above for the construction and analysis of full length polynucleotide and amino acid sequences can also be used to identify fragments of the polynucleotide sequence from SEQ ID NO: 30-58. Fragments of up to about 20-4000 nucleotides are useful for hybridization and amplification and have been described in the invention above.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of transcripts of a gene, labeled nucleotides on the membrane to which RNA from a particular cell type or tissue is bound. With sequence hybridization (see, for example, Sambrook, supra,
Chapter 7; and Ausubel. FM et al., Supra, Chapters 4 and 16).

Using similar computer technology for BLAST, GenBank or LIFESEQ (Inc
Search for identical or related molecules in nucleotide databases such as yte Pharmaceuticals). This assay is much faster than many membrane-based hybridizations. You can also change the sensitivity of the computer search so that any particular match
It can be established whether it is classified as an exact match or a homologous match. The search criterion is the product score defined as (% sequence identity x% maximum BLAST score) / 100. The product score takes into account both the similarity between the two sequences and the length of the sequence match. For example, if the product score is 40, the match is 1 to 2
Exact within% error, at 70 the match would be exact. Similar molecules are usually identified by selecting those molecules that show a product score in the range of 15-40, although lower scores may identify related molecules.

Results of Northern analysis are reported as the percent distribution of the library in which transcripts encoding HTMP occurred. The analysis also includes classification of the cDNA library by organ / tissue and disease. Organ / tissue categories include cardiovascular, skin, development, endocrine,
Includes gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive, urological. Disease categories include cancer, inflammation, trauma, cell proliferation, nerves, pooled. By category, the number of libraries expressing the sequence of interest was counted and divided by the number of libraries in the entire range. Table 3 shows the ratio (percentage) of specific expression in each tissue and the ratio of expression in each disease.

Chromosomal Mapping of a Polynucleotide Encoding 5 HTMP The cDNA sequence used to construct SEQ ID NO: 30-58 was prepared using BLAST and Smith-Water.
An implementation of the man algorithm was used to compare sequences with the Insight LIFESEQ database and the public domain database. Sequences from these databases that match SEQ ID NO: 30-58 were assembled into a cluster of contiguous and overlapping sequences using a construction algorithm such as Phrap (Table 5). Stanford Human
Radiation hybrids available from public sources such as the Genonse Center (SHGC), Whitehead Institute for Genome Research (WIGR) and Genethon.
hybrid) and gene mapping data to see if the clustered sequences have already been mapped. If the cluster contained a sequence that was mapped, all sequences in that cluster (including the particular SEQ ID NO) were assigned to that map position.

SEQ ID NO: 45 and SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53
, SEQ ID NO: 55, SEQ ID NO: 58 gene map positions are described in the (invention) part of this specification as sections or ranges of the human chromosome. The range of map positions shown in centimorgan was measured from the end of the p-arm of the chromosome (centimorgan (c
M) is a unit that represents a distance based on the transfer rate between genes on the same chromosome.
On average, 1 cM is approximately equal to 1 megabase of human chromosomes, but can vary greatly due to the high recombination rate and the low recombination rate.) Distance cM can detect the boundaries of radiation hybrid markers whose sequences are contained in each cluster
Based on genetic markers mapped by Genethon. The diseases related to the sequences of the public and insight companies located in the above-mentioned section are also described in the (invention) section of the present specification.

6 Extension of a Polynucleotide Encoding HTMP The full-length nucleic acid sequence of SEQ ID NO: 30-58 is prepared using oligonucleotide primers designed from suitable fragments of the full-length molecule. The fragment was extended and produced. One primer was synthesized to initiate the 5'extension of the known fragment and the other primer was synthesized to initiate the 3'extension of the known fragment. The starting primer has a GC content of about 50% or more and a length of about 22 to about 30 nucleotides and a pH of about 68 using OLIGO 4.06 software (National Biosciences) or other suitable program. It was designed to anneal to the target sequence at a temperature of ~ 72 ° C. Nucleotides were extended so that hairpin structures and primer-primer dimers did not occur.

This sequence was extended with selected human cDNA libraries. If more than one extension is required or desired, additional or nested primer sets are designed.

Amplification was performed with high fidelity by the PCR method using a method known in the art. PCR is PTC-200
It was performed in a 96-well block plate using a thermal cycler (MJ Research, Inc.). The reaction mixture consisted of template DNA, 200 nmol of each primer, Mg ²⁺ and (NH ₄ ) ₂ SO ₄ and β.
− Buffer containing mercaptoethanol, Taq DNA polymerase (Amersham Pha
rmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (St
ratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 60 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Storage at 4 ° C Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 57 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Store at 4 ° C.

The DNA concentration in each well was dissolved in 1 × TE and 0.5 μl of undiluted PCR product.
100 μl PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugen
e OR) is distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagent and be measured. Fluoroskan this plate
Scan with II (Labsystems Oy, Helsinki, Finland) and measure the fluorescence of the sample to quantify the concentration of DNA. A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reaction succeeded in extending the sequence.

The extended nucleotides are desalted and concentrated before being transferred to a 384 well plate and Cvi
JI Cholera virus endonuclease (Molecular Biology Research, Madison W
Digested with I) and sonicated or sheared before religation to pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel to cut the fragments and the agar was digested with Agar ACE (Promega). T4 Ligase (New England B
The clones extended with iolabs, Beverly MA) were cloned into pUC 18 vector (Amersham Pha
rmacia Biotech) and treated with Pfu DNA polymerase (Stratagene) extension of restriction sites to transfect competent E. coli cells. The transfected cells were selected and transferred to a medium containing antibiotics, each colony was cut off, and cultured in a 384-well plate of LB / 2X carbenicillin culture solution at 37 ° C. overnight.

Cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was PCR amplified using DNA polymerase (Stratagene) according to the following procedure. Step 1 3 minutes at 94 ° C Step 2 15 seconds at 94 ° C Step 3 1 minute at 60 ° C Step 4 2 minutes at 72 ° C Step 5 Repeat steps 2, 3 and 4 29 times Step 6 5 minutes at 72 ° C Step 7 Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with poor DNA recovery were amplified again under the above conditions. Samples were diluted with 20% dimethysulphoxide (1: 2, v / v) and used in the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cycle se.
Sequencing was performed using the quencing ready reaction kit (Perkin-Elmer).

Similarly, following the procedure described above, utilizing the nucleotide sequence of SEQ ID NO: 30-58, using the oligonucleotide designed for this extension and a suitable gene library, 5 '
The regulatory sequence was obtained.

7 Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO: 30-58 are used to screen cDNA, mRNA, or genomic DNA. Special mention is made of labeling of oligonucleotides of about 20 base pairs, but essentially the same procedure is used for larger cDNA fragments. Oligonucleotides were designed using state-of-the-art software, such as OLIGO 4.06 software (National Bioscience), with 50 pmol of each oligomer and 250 μCi of [γ- ³² P] adenosine triphosphate (A
mersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN, Bost)
on MA) and used in combination for labeling. The labeled oligonucleotide was loaded onto a SEPHADEX G-25 ultrafine exclusion dextran bead column (Amersham Phar
Substantially purified using macia Biotech). An aliquot containing the labeled probe at 10 ⁷ counts per minute was added to the following endonucleases: Ase I, Bgl II, Eco R.
Human genome cleaved with one of I, Pst I, Xba1 or Pvu II (DuPont NEN)
Used in typical membrane-based hybridization analysis of DNA.

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are sequentially washed at room temperature, eg, under conditions of max 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. The hybridization patterns are visualized and compared by autoradiography or another imaging means.

It is possible to synthesize array elements on the surface of a substrate using the 8 microarray chemical bonding method and an inkjet device (see eg Baldeschweiler, supra). Using an array similar to dot blotting or slot blotting, heat, UV,
It is placed and bonded to the surface of the substrate using mechanical or chemical bonding methods. A typical array can be made manually or using available methods and machines and can include any suitable number of elements. After hybridization, unhybridized probe is removed and a scanner is used to determine the level and pattern of fluorescence. Scanned images can be analyzed to determine the degree of complementarity and the relative amount / expression level of each probe that hybridizes to the element on the microarray.

Full-length cDNA, expressed gene sequence fragments (ESTs), or fragments thereof can be elements of a microarray. A fragment suitable for hybridization is LASERGEN
Selection can be made using software well known in the art such as E software (DNASTAR). Aligning a full-length cDNA corresponding to one of the nucleic acid sequences of the invention, EST, or fragments thereof, or a cDNA arbitrarily selected from a cDNA library related to the invention, on a suitable substrate such as a glass slide. . The cDNA is fixed to the slide, for example using UV cross-linking, then heat-treated and chemically treated and finally dried (eg Schena, M. et al. (1995) Science 27.
0: 467-470; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used to hybridize to the elements on the substrate. This substrate is analyzed as described above.

9 Complementary Polynucleotides A sequence complementary to the sequence encoding HTMP or any part thereof is used to reduce or inhibit the expression of naturally occurring HTMP. About 15 to about 3
Although the use of oligonucleotides containing 0 base pairs is described, essentially the same method can be used for smaller or larger sequence fragments. Ol
Appropriate oligonucleotides are designed using igo4.06 software (National Biosciences) and the coding sequence of HTMP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5'sequence and used to prevent the promoter from binding to the coding sequence. To prevent translation,
A complementary oligonucleotide is designed to prevent the ribosome from binding to the HTMP-encoding transcript.

10 Expression of HTMP Expression and purification of HTMP can be performed using a bacterial or virus-based expression system. For expression of HTMP in bacteria, the cDNA is subcloned into a suitable vector containing an inducible promoter that enhances antibiotic resistance and the transcription level of the cDNA. Such promoters include T5, which is associated with the lac operator regulatory element.
Alternatively, it includes but is not limited to the T7 bacteriophage promoter and the trp-lac (tac) hybrid promoter. Recombinant vector, BL
Transform into a suitable bacterial host such as 21 (DE3). Bacteria with antibiotic resistance express HTMP when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of HTMP in eukaryotic cells is expressed in insect or mammalian cell lines by the Autographica californica nuclear pleiotropic virus (A), commonly known as baculovirus.
cMNPV). The nonessential polyhedrin gene of baculovirus is replaced with the cDNA encoding HTMP by either homologous recombination or bacterial mediated gene transfer with transfer plasmid mediated. Viral infectivity is maintained and the strong polyhedrin promoter results in high levels of cDNA transcription. Recombinant baculovirus is often used to infect Spodoptera frugiperda (Sf9) insect cells, but can also be used to infect human hepatocytes. In the case of the latter infection, further genetic modification of the baculovirus is required. (For example, Eng
elhard. EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227; Sandig,
V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.).

In most expression systems, HTMP, such as glutathione S transferase (GST)
, Or a fusion protein synthesized with a peptide epitope tag such as FLAG or 6-His, the affinity-based purification of the recombinant fusion protein from crude cell lysate can be performed rapidly in one step. The 26-kilodalton enzyme GST from Schistosoma japonicum allows purification of fusion proteins with immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharma
cia Biotech). After purification, the GST moiety can be proteolytically cleaved from HTMP at specific manipulation sites. FLAG, a peptide of 8 amino acids, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). A stretch of 6 consecutive histidine residues, 6-His, allows purification on a metal chelate resin (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995, supra, ch 10, 16). The following assay can be performed using HTMP directly purified by these methods.

11 Demonstration of HTMP Activity The assay for HTMP activity measures the expression level of HTMP on the cell surface. HT
The cDNA encoding MP is transfected into a suitable mammalian expression vector. Cell surface proteins are labeled with biotin as described above (de la Fuente, MA
(1997) Blood 90: 2398-2405). Immunoprecipitation is performed with an antibody specific for HTMP and the immunoprecipitated sample is analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled to unlabeled immunoprecipitate is proportional to the amount of HTMP expressed on the cell surface.

Another assay for HTMP activity is based on a prototypical assay for ligand / receptor mediated regulation of cell proliferation. This assay measures the amount of newly synthesized DNA in Swiss mouse 3T3 cells expressing HTMP. A suitable mammalian expression vector containing a cDNA encoding HTMP is added to quiescent 3T3 cultured cells using transfection methods well known in the art. Transfected cells are incubated in the presence of [ ³ H] thymidine and varying amounts of HTMP ligand. Acid precipitation possible
[ ³ H] thymidine incorporated into DNA (acid precipitable DNA) is measured at suitable time intervals using a tritium radioisotope counter. The amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose response curve for a concentration range of HTMP ligand of at least 100-fold shows the activity of the receptor. One unit of activity per ml was defined as the concentration of HTMP that resulted in a reaction level of 50%, 100% representing the maximum [ ³ H] thymidine incorporation into acid-precipitable DNA (McKay. I.
and Leigh, I., eds. (1993) Growth Factors: A Practical Aoyroach , Oxford
University Press, New York, NY, p. 73).

Another assay for HTMP activity measures the effect of HTMP expression on the regulation of cell growth. To demonstrate that increased expression levels of HTMP correlate with decreased cell motility and increased cell proliferation, an expression vector encoding HTMP was added to U-937 (A
TCC CRL 1593), HEL 92.1.7 (ATCC TIB 180), MAC 10 and other high motility cell lines are electroporated, and the motility of electroporated cells and control cells is compared. The design and construction of expression vectors capable of expressing HTMP in selected suitable mammalian cell lines is well known in the art. Assays for cell motility in media are well known in the art (Miyake, M. et al. (1991) J. Exp. Med. 174: 1347-1354 and Ikeyama.
, S. et al. (1993) J. Exp. Med. 177: 1231-1237). Transfection with HTMP expression vectors increases the level of HTMP in high motility cell lines and inhibits or reduces the motility of these cell lines. The extent of this inhibition in this assay
It is proportional to the activity of HTMP.

12 Functional Assay HTMP function is at physiologically elevated levels in mammalian cell culture systems.
Evaluation is by expression of the HTMP coding sequence. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels.
Such vectors include pCMV SPORT ^™ (Life Technologies.) And pCR 3.1 (In
Vitrogen, Carlsbad, CA), both containing the cytomegalovirus promoter. For example, 5 to 10 μg of the recombinant vector is transiently transfected into a human cell line derived from endothelium or hematopoiesis by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows to distinguish between transfected and non-transfected cells. In addition, by expressing the labeled protein, the cDNA
The expression from the recombinant vector can be accurately predicted. Such labeled proteins include green fluorescent protein (GFP; Clontech), and CD64 or CD64-GFP fusion protein. Automatic flow cytometry using a technology based on laser optics (F
CM) is used to identify transfected cells expressing GFP or CD64-GFP and to assess their apoptotic status and other cellular characteristics. In addition, FCM detects and weighs uptake of fluorescent molecules that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena include nuclear DNA measured by staining DNA with propidium iodide.
Changes in contents, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, and changes in protein expression on cell surface and intracellular as measured by reactivity with specific antibodies. , Changes in plasma membrane composition measured by binding of fluorescent-complexed annexin V protein to the cell surface. Flow cytometry is by Ormerod, MG (1994) Flow Cytometry Oxford, New York,
It is listed in NY.

The effect of HTMP on gene expression depends on the sequence encoding HTMP and CD64 or CD64-G.
Highly purified cell populations transfected with either FP can be evaluated. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or antibodies against CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods well known in the art. The expression of mRNA encoding HTMP and other genes of interest can be analyzed by Northern analysis or microarray technology.

13 Preparation of Antibodies Specific for HTMP Polyacrylamide gel electrophoresis (PAGE; eg, Harrington, MG (1990)
Methods Enzymol. 1816-3088-495) or other purification techniques and HTMP substantially purified using standard protocols to immunize rabbits to generate antibodies.

Alternatively, the HTMP amino acid sequence may be analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions and the corresponding oligopeptides synthesized and used to determine well known methods to those of skill in the art. To produce antibodies. Selection of suitable epitopes, such as those near the C-terminus or within adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11).

Typically, oligopeptides approximately 15 residues in length were applied to ABI 431A from Applied Biosystems.
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was synthesized by chemistry of fmoc method using a peptide synthesizer (Perkin-Elmer).
) Is used to bind to KLH (Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity and anti-HTMP activity of the obtained antiserum, peptide or HT
MPs are bound to the substrate, blocked with 1% BSA, reacted with rabbit antiserum, washed, and further reacted with radioiodine-labeled goat anti-rabbit IgG.

Purification of Spontaneous HTMP Using 14 Specific Antibodies Spontaneous HTMP or recombinant HTMP is substantially purified by immunoaffinity chromatography using an antibody specific for HTMP. The immunoaffinity column is formed by covalently binding an anti-HTMP antibody with a resin for activation chromatography such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.

A culture solution containing HTMP is passed through an immunoaffinity column, and the column is washed under conditions capable of preferentially adsorbing HTMP (for example, with a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and HTMP (for example, with a buffer of pH 2-3 or with a high concentration of chaotropic ion such as urea or thiocyanate ion), and HTMP is recovered.

Identification of 15 HTMP-Interacting Molecules HTMP or biologically active fragments thereof were labeled with ¹²⁵ I Bolton-Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). Label with. Pre-arranged candidate molecules in multi-well plates are labeled with labeled HTMP
Incubate with, wash and assay all wells with labeled HTMP complex. The data obtained at various HTMP concentrations are used to calculate values for the number, affinity and association of HTMP bound to the candidate molecule.

Alternatively, a molecule that interacts with HTMP is described in Fields, S. and O. Song (1989, Nature
340: 245-246). Yeast two-hybrid syst
em) and MATCHMAKER system (Clontech) for analysis using a commercially available kit based on a 2-hybrid system.

Those skilled in the art will be able to make various modifications of the described methods and systems of the present invention without departing from the scope and spirit of the invention. Although the present invention has been described with reference to particular preferred embodiments, it should be understood that the scope of the invention should not be unduly limited to such particular embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are within the scope of the following claims.

BRIEF DESCRIPTION OF THE TABLES Table 1 lists the polypeptide and nucleotide sequences used to generate the full-length sequences encoding HTMP, SEQ ID NO :, Clone ID, cDNA.
A library and a cDNA fragment are shown.

Table 2 lists the characteristics of each polypeptide sequence, including latent motifs and homologous sequences, and
The method, algorithm, and searchable database used for HTMP analysis are shown.

Table 3 shows selected fragments of each nucleic acid sequence, tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, diseases, disorders and conditions associated with these tissues, and each DNA. Shows the cloned vector.

Table 4 shows the tissues used to create the cDNA library in which the cDNA clone encoding HTMP was isolated.

Table 5 shows the tools, programs, and algorithms used in the analysis of HTMP, as well as their description, references, and threshold parameters.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Sequence list]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61P 1/04 A61P 3/10 4C084 1/16 7/00 4H045 3/10 7/04 7/00 7 / 06 7/04 9/00 7/06 9/10 9/00 9/12 9/10 11/06 9/12 11/16 11/06 13/12 11/16 15/00 13/12 15/06 15 / 00 15/08 15/06 15/10 15/08 17/00 15/10 101 17/00 19/02 101 19/10 19/02 21/04 19/10 25/00 21/04 25/02 25 / 00 103 25/02 25/14 103 25/16 25/14 25/18 25/16 25/28 25/18 29/00 101 25/28 31/04 29/00 101 31/12 31/04 31 / 18 31/12 33/00 31/18 33/02 33/00 35/00 33/02 35/02 35/00 37/02 35/02 37/06 37/02 37/08 37/06 43/00 105 37/08 111 43/00 105 C07K 14/47 111 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1 / 68 A 5/10 01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/53 D G01N 33/15 M 33/50 37/00 102 33/53 C12N 15/00 ZNAA 5/00 A 37/00 102 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, L, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor tongue, Wy Thom USA California 95118 San Jose Runwick Court 4230 (72) Inventor Hillman, Jennifer El United States California 94040 Mountain View # 17 Monrode Live 230 (72) Inventor Lady, Looper United States California 94086 · Sunnyvale West McKinley Dry Eve 1233 (72) Inventor Bandman, Olga California USA 94043 · Mau Tenview Anna Ave 366 (72) Inventor Bogun, Mariah Earl USA California 94577 San Leandro Santiago Road 14244 (72) Inventor Ryu, Dung Aina Em USA California 95123 San Jose Coy Drive 233 (72) ) Inventor Azimzai, Yalda 94552, Castro Valley, Boulder Canyon Drive, California, USA 5518 (72) Inventor Young, Junming 95129, San Jose, Barcrane, 7125 F Term (reference) 2G045 AA25 AA40 BB03 CB01 DA12 DA13 DA14 DA36 DA77 DA80 FB02 FB03 FB07 4B024 AA01 AA11 BA44 BA63 BA80 CA04 CA07 CA09 CA11 DA02 DA05 DA11 EA02 EA03 EA04 FA02 GA11 HA01 HA12 HA15 4B063 QA01 QA08 QA18 CAAQQQQCAQCAQQQQQQQQQQR QRQ QR QR QR QR QR QR QR QR QR QR CA19 CC24 DA01 DA 13 4B065 AA01X AA57X AA90X AA93Y AB01 BA02 CA24 CA25 CA44 CA46 4C084 AA02 AA06 AA07 AA13 AA17 BA01 BA02 BA08 BA16 BA17 BA18 BA23 CA18 CA25 CA53 CA56 ZA ZA 522 ZA 252 ZA ZA ZA ZA ZA ZA ZA752 ZA812 ZA892 ZA902 ZA942 ZA962 ZA972 ZB072 ZB082 ZB112 ZB132 ZB152 ZB212 ZB262 ZB272 ZB332 ZB352 ZB372 ZB382 ZC102 ZC112 ZC352 ZC552 4H045 AA10 BA20 DA50 FA72 FA50 CA41 DA40 CA40 DA41 CA40 DA41 CA40 DA50

Claims (23)

[Claims] 1. An isolated polypeptide comprising: a) SEQ ID NO: 1 to SEQ ID NO: 23 and SEQ ID NO: 25 to SEQ ID NO: 29 (SEQ I
D NO: 1-23, 25-29), an amino acid sequence selected from the group consisting of: b) SEQ ID NO: 1-23, 25-29, and an amino acid sequence selected from the group consisting of at least 90% of the sequence; A naturally occurring amino acid sequence having identity, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23, 25-29, d) SEQ ID NO: 1 An isolated polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -23 and 25-29, and an amino acid sequence selected from the group consisting of. 2. The isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NOs: 1-23, 25-29. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. The isolated polynucleotide of claim 3 selected from the group consisting of SEQ ID NOs: 30-52, 54-58. 5. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 6. A cell transformed with the recombinant polynucleotide of claim 5. 7. A genetically modified organism comprising the recombinant polynucleotide of claim 5. 8. A method of making the polypeptide of claim 1, comprising: a) operably binding to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. The polypeptide of claim 1 comprising the steps of culturing cells transformed with a recombinant polynucleotide containing the expressed promoter sequence, and b) recovering the polypeptide so expressed. Manufacturing method. 9. An isolated antibody that specifically binds to the polypeptide of claim 1. 10. An isolated polynucleotide comprising: a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 30-52, 54-58; and b) SEQ ID NO: 30-52. A naturally occurring polynucleotide sequence having at least 90% sequence identity with a polynucleotide sequence selected from the group consisting of 54-58; c) a polynucleotide sequence complementary to a) above; and d) b) above. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence complementary to e), and e) the RNA equivalent of a) -d) above. 11. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 10. 12. A method of detecting a target polynucleotide in a sample having the polynucleotide sequence of claim 10, comprising: a) at least 16 contiguous sequences comprising a sequence complementary to the target polynucleotide in the sample. In the step of hybridizing a probe containing a nucleotide with the sample, the probe is specific to the target polynucleotide under the condition that a hybridization complex is formed by the probe and the target polynucleotide. Of the target polynucleotide, and b) detecting the presence or absence of the hybridization complex and, if present, optionally measuring the amount thereof. Detection method. 13. The method of claim 12, wherein the probe comprises at least 30 contiguous nucleotides. 14. The method of claim 12, wherein the probe comprises at least 60 contiguous nucleotides. 15. A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 16. A method for treating a disease associated with reduced expression of functional HTMP or a symptom thereof, which comprises administering the pharmaceutical composition according to claim 15 to a patient in need of such treatment. Characterized treatment method. 17. A method of screening a compound effective as an agonist of the polypeptide of claim 1, comprising the steps of: a) exposing a sample containing the polypeptide of claim 1 to the compound; And a step of detecting agonistic activity. 18. A pharmaceutical composition comprising an agonist compound identified by the screening method of claim 17 and a pharmaceutically acceptable excipient. 19. A method for treating a disease associated with decreased expression of functional HTMP or a symptom thereof, which comprises administering the pharmaceutical composition according to claim 18 to a patient in need of such treatment. Characterized treatment method. 20. A method of screening a compound that is effective as an antagonist of the polypeptide of claim 1, comprising the steps of: a) exposing a sample containing the polypeptide of claim 1 to the compound; Detecting the antagonist activity. 21. A pharmaceutical composition comprising an antagonist compound identified by the screening method of claim 20 and a pharmaceutically acceptable excipient. 22. A method for treating a disease associated with overexpression of functional HTMP or a symptom thereof, comprising administering the pharmaceutical composition according to claim 21 to a patient in need of such treatment. Characterized treatment method. 23. A method of screening for a compound that effectively alters the expression of a target polynucleotide comprising the sequence of claim 4, comprising the steps of: a) exposing a sample containing the target polynucleotide to the compound; b) A step of detecting a change in expression of the target polynucleotide.