JP2003530071A - Intracellular signaling molecule - Google Patents

Abstract

  (57) [Summary] The present invention provides human intracellular signaling molecules (INTRA) and polynucleotides that identify and encode INTRA. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. Furthermore, the present invention also provides a method for diagnosing, treating or preventing a disease associated with the expression of INTRA.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to nucleic acid sequences and amino acid sequences of intracellular signal transduction molecules, and relates to the diagnosis and treatment of cell proliferation disorders, autoimmune / inflammatory diseases, neurological disorders, gastrointestinal disorders, reproductive disorders and developmental disorders. It relates to the use of these sequences in prevention.

BACKGROUND OF THE INVENTION Cell-cell information exchange is essential for the growth, development and survival of multicellular organisms. Cells exchange information by sending and receiving molecular signals. An example of a molecular signal is growth factor, which binds and activates specific transmembrane receptors on the surface of target cells. Activated receptors transduce signals intracellularly, thus initiating a cascade of biochemical reactions that ultimately affect gene transcription and cell cycle progression in target cells.

Intracellular signal transduction begins with the binding of signaling molecules to cell membrane receptors,
It is the process by which cells respond to extracellular signals (hormones, neurotransmitters, growth factors, differentiation factors, etc.) through a cascade of biochemical reactions that result in the activation of intracellular target molecules. Intermediate steps of the process involve activation of various cytoplasmic proteins by phosphorylation at protein kinases and inactivation by protein phosphatases, some of which appear to induce transcription of specific genes of activated proteins. Some are involved in the final translocation to the cell nucleus. Intracellular signaling processes regulate cells of all types, including cell proliferation, cell differentiation and gene transcription, and include various mitogenic factors such as cyclic nucleotides, calcium-calmodulin, inositol and protein phosphorylation. It is involved in the molecular diversity of two messenger molecules, protein kinases and phosphatases.

Intracellular signaling is performed by a variety of molecules that facilitate signal transduction and amplification. For example, binding of a ligand to a transmembrane receptor activates a membrane-associated intracellular protein such as a G protein. G-proteins mediate both the levels of intracellular second messengers such as cyclic AMP and the activity of signaling enzymes such as phospholipase C. Messengers and enzymes then activate the signal transduction pathway. Many of the signal transduction pathways are mediated by the protein kinase cascade. Phosphorylation of proteins in response to extracellular signals, cell cycle checkpoints and environmental or trophic stress is achieved by transferring high energy phosphates from ATP. The second messengers are cyclic AMP, cyclic GMP, inositol triphosphate, cyclic ADP ribose and calcium / calmodulin, the effects of which are mediated by protein kinases. Alternatively, the ligand binds to a transmembrane receptor such as a receptor tyrosine kinase, which induces GTPase activation of a molecular "switch" such as a monomer. In this case, the binding of the ligand to the receptor activates the catalytic domain in the intracellular part of the receptor. The activated domain then turns on the switch of monomeric GTPase activity, such as Ras, usually via an adapter protein.

Cells also respond to changing conditions by switching off the signal. Many signaling proteins are short-lived and are rapidly targeted for degradation by covalent ligation to the highly conserved small protein ubiquitin. Cells also maintain a mechanism to monitor changes in the concentration of denatured or unfolded proteins within the membrane-bound extracytoplasmic compartment, monitoring the effective chaperone molecule concentration in the endoplasmic reticulum and to the cytosol. It contains a transmembrane receptor that activates the transcription of nuclear genes that code for chaperones in the endoplasmic reticulum by transducing the signal of.

Certain proteins within the intracellular signaling pathway serve to bind or cluster other proteins involved in the signaling cascade. Such proteins are referred to as scaffold proteins, fixed proteins or adapter proteins (reviewed by Pawson, T., and Scott, JD (1997) Science 278: 20.
75-2080). Since many intracellular signaling proteins, such as protein kinases and phosphatases, have a relatively broad substrate specificity, adapters help organize component signaling proteins into specific biochemical pathways.

Gangliosides, which are normally associated with the plasma membrane, are also involved in signal transduction. Ectopic ganglioside function has been linked to inflammatory and degenerative diseases in and outside the nervous system, including Tay-Sachs disease, multiple sclerosis, lupus erythematosus and insulin-dependent diabetes mellitus (Misasi. R. et al. (1997) Diabetes. Metab. Rev. 13: 163-179).

Many signaling molecules are characterized by the presence of specific domains that facilitate protein-protein interactions. Sampling of these domains is discussed below, along with other important intracellular messengers.

(Intracellular Signal Transduction Second Messenger Molecule) Phospholipid and Inositol-Phosphate Signal Transduction Inositol phospholipid (phosphoinositide) is a binding of a signaling molecule to a G protein-linked receptor in the plasma membrane. Involved in the intracellular signaling pathway that initiates. It is a phosphatidylinositol (PI) inside the plasma membrane.
) Leads to phosphorylation of residues to the diphosphate state (PIP ₂ ) by inositol kinase. At the same time, G protein-coupled receptor binding stimulates a trimeric G protein that in turn activates the phosphoinositide specific phospholipase C-β. Phospholipase C-β then splits PIP ₂ into two products, inositol triphosphate (IP ₃ ) and diacylglycerol. The two products act as mediators for distinct signaling events. IP ₃ diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), on the other hand, such as diacyl glycerol remains in the film, phosphorylates proteins selected in the target cell STK Assists in the activation of protein kinase C. Calcium response starting with IP ₃ is terminated by dephosphorylation of IP ₃ by specific inositol phosphatases. The cellular response mediated by this pathway is hepatic glycogenolysis in response to vasopressin,
Smooth muscle contraction in response to acetylcholine and thrombin-induced platelet aggregation.

Cyclic Nucleotide Signaling Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers that transmit a variety of extracellular signals including hormones, light and neurotransmitters. In particular, cyclic AMP-dependent protein kinase (PKA) is believed to be responsible for the overall effects of cAMP in most mammalian cells, including various hormone-induced cellular responses. Visible excitation and light transduction of ocular light signals are regulated by cyclic GMP-regulated Ca ²⁺ specific channels. Since cellular levels of cyclic nucleotides are important in mediating these various reactions, controlling the synthesis and degradation of cyclic nucleotides is important. Therefore, adenylyl cyclase, which synthesizes cAMP from AMP, is activated by binding of adrenergic to β-adrenergic receptors to increase muscle cAMP levels, while guanylate at photoreceptors is activated. Cyclase activation and increased cGMP levels lead to reopening of Ca ²⁺ specific channels and restoration of ocular darkness. In contrast, cAM
Hydrolysis of cyclic nucleotides by P and cGMP-specific phosphodiesterases (PDEs) produces the opposite and other effects mediated by increased cyclic nucleotide levels. PDEs appear to be particularly important for the regulation of cyclic nucleotides, given the diversity found in this family of proteins. At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors and sensitivity to inhibitors (Beavo, JA (1995) Physiological Reviews 75: 725- 48).
PDE inhibitors have been found to be particularly useful in the treatment of various clinical disorders. P
Rolipram, a specific inhibitor of DE4, has been used to treat depression. Similar inhibitors have been evaluated as anti-inflammatory agents. Theophylline is a non-specific PDE inhibitor used in the treatment of bronchial asthma and other respiratory disorders (Banner. K
.H. And Page, CP (1995) Eur. Respir. J. 8: 996-1000).

The calcium signaling molecule Ca ²⁺ , another second messenger molecule, is more widely used as an intracellular mediator than cAMP. There are two pathways by which Ca ²⁺ can enter the cytosol in response to extracellular signals. One pathway is primarily, Ca ²⁺ acts in nerve signaling as fall nerve endings through voltage-gated Ca ²⁺ channels. The second pathway is a more ubiquitous pathway in which Ca ²⁺ is released from the ER to the cytosol in response to binding of extracellular signaling molecules to its receptor. Ca ²⁺
Directly activates regulatory enzymes such as protein kinase C that trigger signal transduction pathways. Ca ²⁺ also binds to specific Ca ²⁺ binding proteins (CBPs) such as calmodulin (CaM), which in turn activates multiple target proteins in cells, including enzymes, membrane transport pumps and ion channels. CaM interactions include, but are not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signaling, ion homeostasis, exocytosis and metabolic regulation. Involved in many cellular processes (Celio, MR et al. (1996) Guidebook to Calcium-bindi ng Proteins , Oxford University Press, Oxford, UK, pp. 15-20). There are also Ca ²⁺ binding proteins characterized by the presence of one or several EF-hand Ca ²⁺ binding motifs, which consist of 12 amino acids flanking the α-helix (Celio, supra).
. Control of CBP has implications for controlling various diseases. Calcineurin, a CaM-regulated protein phosphatase, is an immunosuppressant for cyclosporine and FK506.
Is the target of inhibition by. This indicates the importance of calcineurin and CaM in immune reactions and immune disorders (Schwaninger M. et al. (1993) J. Biol Chem. 26.
8: 23111-23115). CaM levels are increased several-fold in tumors and tumor-derived cell lines for various types of cancer (Rasmussen, CD and Means, AR (1989) Tre.
nds in Neuroscience 12: 433-438).

Annexins are a family of calcium-binding proteins associated with cell membranes (Towle, CA and Treadwell, BV (1992) J. Biol. Chem. 267: 5416-23).
. Annexin reversibly binds negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium-dependent manner. Annexin is
It is involved in various processes involved in signal transduction at the plasma membrane, including membrane-cytoskeleton interactions, phospholipase inhibition, anticoagulation and membrane fusion. Annexin contains 4 to 8 repeat segments of approximately 60 residues. Each iteration folds into five α-helices wrapped in a clockwise higher-order helix.

(Signaling Complex Protein Domain) The PDZ domain was named after the three proteins in which it was first discovered. Three proteins are PSD-95 (postsynaptic density 95)
, Dlg (Drosophila lethal (1) discs large-1) and ZO-1 (zonula occludens-1
). Each of these proteins plays an important role in neuronal synaptic transmission, tumor suppression and cell junction formation. As these proteins were discovered, over 60 additional PDZ-containing proteins were identified in a wide variety of prokaryotes and eukaryotes. This domain has been linked to receptor and ion channel clustering and targeting of the multiprotein signaling complex to intrinsically functional regions of the cytosolic surface of the plasma membrane (for a review of PDZ domain-containing proteins, see Ponting, CP et al. (1997) Bioessays 19: 469-479). Most PDZ domains are found in the eukaryotic MAGUK (membrane associated guanylate kinase) protein family, members of which bind to intracellular domains of receptors and channels. However, the PDZ domain is also found in various membrane-localized proteins such as protein tyrosine phosphatases, serine / threonine kinases, G protein cofactors and synapse-related proteins such as syntrophin and neuronal NO synthase (nNOS). Up to 9 PDZ domains have been identified in a single protein, but usually about 1-3 PDZ domains are found in a given protein. The glutamine receptor interacting protein (GRIP) contains several PDZ domains. GRIP is an adapter that links certain glutamine receptors to other proteins and may be responsible for the clustering of such receptors at excitatory synapses in the brain (Dong, H. et al. (1997) Nature. 386: 279-28
Four).

The SH3 domain is defined by homology to a region of the cytoplasmic protein tyrosine kinase, proto-oncogene c-Src. SH3 is a small domain of 50-60 amino acids that interacts with multiple proline ligands. The SH3 domain is found in various eukaryotic proteins involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. In some cases, SH3 domain-containing proteins interact directly with receptor tyrosine kinases. For example, the SLAP-130 protein is a substrate for T cell receptor (TCR) -induced protein kinase. SLAP-130 interacts with the protein SLP-76 through its SH3 domain and influences TCR-induced expression of interleukin 2 (Musci, MA et al. (1997) J. Biol. Chem. 272: 11674-11677). Another recently identified SH3 domain protein is macrophage actin-related tyrosine phosphorylated protein (MAYP), which is a macrophage colony-stimulating factor-1 (CSF-
1) and may play a role in controlling CSF-1-induced rearrangement of the actin cytoskeleton (Yeung, Y.-G. et al. (1998) J. Biol. Chem. 273: 30638- 30
642). The structure of SH3 is characterized by two antiparallel β-sheets placed at right angles to each other. This arrangement forms a hydrophobic pocket lined with highly conserved residues between different SH3 domains. This pocket makes critical hydrophobic contacts with proline residues in the ligand (Feng, S. et al. (1994) Science 266: 1241-47).
. Endophilin is a SH3 domain-containing protein linked to synaptic vesicle endocytosis (Micheva, KD (1997) 272: 27239-
27245).

The novel domain, called the WW domain, is similar to the SH3 domain in its ability to bind multiproline ligands. This domain was originally discovered in dystrophin, a cytoskeletal protein directly involved in Duchenne muscular dystrophy (Bork, P. and Sudol, M. (1994) Trends Biochem. Sci. 19:53.
1-533). The WW domain has since been discovered in a variety of intracellular signaling molecules involved in development, cell differentiation and cell proliferation. The structure of the WW domain is composed of approximately four conserved aromatic residues of the β chain grouping, usually tryptophan.

Like SH3, the SH2 domain is defined by homology to the region of c-Src. The SH2 domain interacts directly with phosphotyrosine residues, thereby providing a direct mechanism for the regulation and transduction of receptor tyrosine kinase-mediated signaling pathways. For example, as many as 10 different SH2 domains can bind to phosphorylated tyrosine residues on activated PDGF receptors, thereby providing a highly coordinated and finely regulated response for ligand-mediated receptor activation ( Review by Schaffhausen, B.
(1995) Biochem. Biophys. Acta. 1242: 61-75).

Homer is an excitatory synapse-enriched neuronal immediate-early gene (Xia
O, B. et al. (1998) Neuron 21: 707-716). Homer proteins form multivalent complexes that bind polyproline motifs at group I metabotropic glutamate receptors and inositol triphosphate receptors, thereby binding these receptors at signaling complexes (Tu , JC (1999) Neuron 23: 583-592).

The pleckstrin homology (PH) domain is originally a protein kinase C in platelets.
Was identified with pleckstrin, which is the predominant substrate for. It was discovered that this domain is involved in intracellular signaling or cytoskeletal organization 90
The above proteins were identified. Proteins with pleckstrin homology domains include various kinases, phospholipase C isoforms, guanine nucleotide terminators and GTPase activating proteins. For example, members of the FGD1 family include not only the FYVE Zn finger domain, but also the Rho-guanine nucleotide exchange factor (GEF) and PH domain. FGD1 is the causative gene of faciogenital dysplasia, a congenital malformation of the skeleton (Pasteris, NG and G
orski, JL (1999) Genomics 60: 57-66). Many PH domain proteins function in association with the plasma membrane, and this association appears to be mediated by the PH domain itself. The PH domain shares a common structure consisting of two antiparallel β sheets flanked by amphipathic α helices. The variable loop that binds the component β chains normally occurs in a positively charged environment and can function as a ligand binding site (Lernmon, MA.
(1996) Cell 85: 621-624). n-Chimaerin is a GAP involved in the formation of lamellipodia and filopodia of neuroblastoma cells (Kozma, R. et al. (1996) Mol. Cell Bid.
. 16: 5069-5080).

Ankyrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular signaling functions. For example, ANK repeat is a kinase,
It is found in proteins involved in cell proliferation such as kinase inhibitors, tumor suppressors and cell cycle regulatory proteins (Kalus, W. et al. (1997) EBBS Lett. 401: 127-132.
, Ferrante, AW et al. (1995) Proc. Natl. Acad. Sci. USA 92: 1911-1915 etc.). These proteins usually contain multiple ANK repeats, each containing approximately 33
Composed of amino acids. Myotrophin is an ANK repeat protein that plays a key role in the development of cardiac hypertrophy, a factor that contributes to many heart diseases. Structural studies show that each myotrophin ANK repeat, like other ANK repeats, forms a helix-turn-heric score preceded by a protruding "tip." These tips are variable sequences and may play a role in protein-protein interactions. The helix-turn-helix regions of ANK repeats stack on top of each other and are stabilized by hydrophobic interactions (Yang. Y. et al. (1998) Structure 6: 619-626).

The tetratricopeptide repeat (TPR) is a 34 amino acid repeat motif found in tissues from bacteria to humans. TPR is expected to form an ampipathic helix and appears to mediate protein-protein interactions. The TPR domain is found in CDC16, CDC23 and CDC27. These are members of the late facilitating complex that target proteins for degradation during late development. Other processes involved in TPR proteins include cell cycle regulation, transcriptional repression, stress response and protein kinase inhibition. (Lamb, JR.
(1995) Trends Biochem. Sci. 20: 257-259).

The armadillo / β-catenin repeat is a 42 amino acid motif that, when repeated in tandem, forms a higher-order helix of the α-helix. The structure of the armadillo repeat region from β-catenin revealed a positively charged shallow groove on one face of a higher-order helix that could serve as a binding surface. β-catenin armadillo repeats, plakoglobin and pl20 ^cas
It binds the cytoplasmic domain of cadherin. The β-catenin / cadherin complex is the target of regulatory signals that govern cell adhesion and mobility (Huber, AH et al. (1997
) Cell 90: 871-882).

The discovery of a novel G protein-coupled receptor and a polynucleotide encoding the same has led to the diagnosis, treatment and prevention of cell proliferation abnormality, autoimmune / inflammatory disease, neuropathy, gastrointestinal disorder, reproductive disorder and developmental disorder, and The need in the art is met by providing new compositions that are useful in calculating the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of G protein coupled receptors.

(Outline of the Invention) The present invention collectively refers to “INTRA”, and individually to “INTRA-1”, “INTRA-2”, and “INTRA-3”.
, "INTRA-4", "INTRA-5", "INTRA-6", "INTRA-7", "INTRA-8", "INTRA-9", "INT
RA-10, INTRA-11, INTRA-12, INTRA-13, INTRA-14, INTRA-15, INT
RA-16, INTRA-17, INTRA-18, INTRA-19, INTRA-20, INTRA-21, INT
RA-22, INTRA-23, INTRA-24, INTRA-25, INTRA-26, INTRA-27, INT
RA-28, INTRA-29, INTRA-30, INTRA-31, INTRA-32, INTRA-33, INT
RA-34, INTRA-35, INTRA-36, INTRA-37, INTRA-38, INTRA-39, INT
RA-40, INTRA-41, INTRA-42, INTRA-43, INTRA-44, INTRA-45, INT
RA-46, INTRA-47, INTRA-48, INTRA-49, INTRA-50, INTRA-51 and I
It is characterized by an intracellular signaling molecule, which is a substantially purified polypeptide, referred to as "NTRA-52". In certain embodiments, the invention provides at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1-52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1-52. Having a naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52, or (d) an immunogenicity of an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 Provided is a substantially isolated polypeptide, including fragments.
In one embodiment, there is provided a substantially isolated polypeptide comprising an amino acid sequence selected from the group having SEQ ID NOs: 1-52.

The present invention also has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
There is provided a substantially isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group comprising In one embodiment, the polynucleotide encodes a polypeptide selected from the group having SEQ ID NOs: 1-52. In another embodiment, the polynucleotide is selected from the group having SEQ ID NOs: 53-104.

The invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
Recombinant polynucleotides having a promoter sequence operably linked to a substantially isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group comprising In one embodiment, the invention provides cells transformed with the recombinant polynucleotide. In another embodiment, the present invention provides a genetic transformant containing the recombinant polynucleotide.

The present invention also has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
There is provided a method for producing a substantially isolated polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group comprising The production method includes (a) a step of culturing cells transformed with a recombinant polynucleotide under conditions suitable for expression of the polypeptide, and (b) a step of receiving the polypeptide so expressed. And the recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.

The present invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
There is provided a substantially isolated antibody which specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group comprising

The present invention further provides at least 90% homology with (a) a polynucleotide sequence selected from the group having SEQ ID NOs: 53 to 104 and (b) a polynucleotide sequence selected from the group having SEQ ID NOS: 53 to 104. A natural polynucleotide sequence having: (c) a polynucleotide sequence complementary to (a), (d) a polynucleotide sequence complementary to (b), or RNA equivalents of (e) (a) to (d). A substantially isolated polynucleotide comprising the same is provided. In one embodiment the polynucleotide has at least 60 contiguous nucleotides.

The invention further provides a method of detecting a target polynucleotide in a sample. Here, the target polynucleotide has at least 90% homology with (a) a polynucleotide sequence selected from the group having SEQ ID NOS: 53 to 104 and (b) a polynucleotide sequence selected from the group having SEQ ID NOS: 53 to 104. A natural polynucleotide sequence having, (c) a polynucleotide sequence complementary to (a), (d) (
Provided is a polynucleotide sequence complementary to b), or a substantially isolated polynucleotide comprising (e) (a)-(d) RNA equivalents. The detection method includes (a) a step of hybridizing the sample with a probe containing at least 20 consecutive nucleotides having a sequence complementary to the target polynucleotide in the sample, and (b) the presence of a hybridization complex. -The probe consists of a process of detecting the absence and optionally detecting the amount of complex, if present, under conditions such that a hybridization complex is formed between the probe and the target polynucleotide. It specifically hybridizes to the target polynucleotide. In one embodiment, the probe comprises at least 60 contiguous nucleotides.

The present invention also provides a method of detecting a target polynucleotide in a sample. Here, the target polynucleotide has at least 90% homology with (a) a polynucleotide sequence selected from the group having SEQ ID NOS: 53 to 104 and (b) a polynucleotide sequence selected from the group having SEQ ID NOS: 53 to 104. A natural polynucleotide sequence having, (c) a polynucleotide sequence complementary to (a), (d) (
Provided is a polynucleotide sequence complementary to b), or a substantially isolated polynucleotide comprising (e) (a)-(d) RNA equivalents. The detection method includes (a) a step of amplifying a target polynucleotide or a fragment thereof using polymerase chain reaction amplification, and (b) detecting the presence / absence of the target polynucleotide or a fragment thereof, and detecting the target polynucleotide or the fragment thereof. If a fragment is present, it optionally includes the step of detecting its amount.

The invention further provides a pharmaceutical ingredient comprising an effective amount of the polypeptide and a pharmaceutically acceptable excipient. The effective amount of the polypeptide is a natural product having at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. Or (c) a biologically active fragment of an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52, or (d) an immunogenic fragment of an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. Including. In one embodiment, the pharmaceutical ingredient comprises an amino acid sequence selected from the group having SEQ ID NOs: 1-52. The present invention further provides a method for treating a disease or medical condition associated with reduced expression of functional INTRA, the method comprising the step of administering a pharmaceutical ingredient to a patient in need of such treatment.

The present invention also has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
A method for screening a compound for confirming the effectiveness as a agonist of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having Nos. The screening method includes (a) exposing a sample having a polypeptide to a compound, and (b) detecting an agonist activity in the sample. In one embodiment, the invention provides a method of treating a disease or condition associated with reduced expression of functional INTRA, the method comprising the step of administering a pharmaceutical ingredient to a patient in need of such treatment. provide.

The present invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
There is provided a method of screening a compound for confirming the effectiveness as a antagonist of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having Nos. The screening method includes (a) exposing a sample containing the polypeptide to a compound, and (b) detecting the agonist activity in the sample. In one embodiment, the invention provides a pharmaceutical ingredient comprising an antagonist compound identified by this method and a pharmaceutically acceptable excipient. In another embodiment, there is provided a method of treating a disease or condition associated with overexpression of functional INTRA, comprising administering a pharmaceutical ingredient to a patient in need of such treatment. .

The invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
There is provided a method for screening a compound which specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having Nos. The screening method comprises: (a) a process of binding the polypeptide to at least one test compound under appropriate conditions; and (b) a compound that detects the binding of the polypeptide to the test compound and thereby specifically binds to the polypeptide. And a process of identifying.

The invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 52. A naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 52, or (d) SEQ ID NO: 1
There is provided a method for screening a compound which regulates the activity of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having 52 to 52. The screening method includes (a) a step of binding the polypeptide to at least one test compound under conditions where the activity of the polypeptide is allowed, and (b) a step of calculating the activity of the polypeptide in the presence of the test compound. And (c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, the change in the activity of the polypeptide in the presence of the test compound. Indicates compounds that modulate the activity of the polypeptide.

The invention further provides a method of screening compounds to confirm the efficacy of mutated expression of a target polynucleotide. The target polynucleotide comprises a sequence selected from the group having SEQ ID NOs: 53-104. The screening method is (
a) exposing a sample containing the target polynucleotide to a compound, and (b) detecting the mutated expression of the target polynucleotide.

(Mode for Carrying Out the Invention) The protein, nucleotide sequence and method of the present invention will be described, but the present invention is not limited to the specific devices, materials and methods described above. It should be understood that modifications can be made. Further, the technical terms used herein are merely used for the purpose of describing specific examples, and are not intended to limit the scope of the present invention which is limited only by the claims. Please understand this as well.

As used in the claims and specification, the singular forms “a” and “the (this)”
It should be noted that the notation of may refer to more than one, unless the context clearly indicates otherwise. Thus, for example, where the phrase "a host cell" is used, there may be more than one such host cell, and "a antibody"
When it is described, it also refers to one or more antibodies, and antibody equivalents known to those skilled in the art.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined. Although any apparatus, material, or method similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable apparatus, materials, or methods are described herein. All publications mentioned in this invention are cited for the purpose of describing and disclosing the cells, protocols, reagents and vectors which were reported in the publications and which may be relevant to the invention. . Nothing in this specification is admitted that the invention is not entitled to antedate such disclosure by virtue of prior art.

The definition "INTRA" is the amino acid sequence of substantially purified INTRA, obtained from any species, especially mammalian species including bovine, ovine, porcine, murine, equine and human. It refers to any natural, synthetic, semi-synthetic or recombinant origin.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of INTRA. Examples of agonists can include proteins, nucleic acids, carbohydrates, small molecules and any other compounds or components, which are either directly interacting with INTRA or the formation of biological pathways involving INTRA. Acts on the element to regulate the activity of INTRA.

“Allelic variants” are another form of the gene encoding INTRA. Allelic variants may be created from at least one mutation in the nucleic acid sequence. It can also be made from mutant RNA or polypeptides. The structure or function of the polypeptide may or may not be mutated. A gene may have no natural allelic variants, or one or several natural allelic variants. Common mutagenic changes that generally give rise to allelic variants are those that result from spontaneous deletions, additions, or substitutions of nucleotides. Each of these changes, alone or in combination with other changes, can occur one or more times within a given sequence.

A “altered” nucleic acid sequence encoding INTRA has a nucleic acid sequence that deletes, inserts or replaces various nucleotides, and is a polypeptide having the same or at least one functional characteristic of INTRA. Produce. This definition includes INTR
A polymorphism that is easily or difficult to detect using a specific oligonucleotide probe of a polynucleotide encoding A and occupies a locus other than the normal chromosomal locus for the polynucleotide sequence encoding INTRA. Inappropriate or unexpected hybridization to allelic variants. The encoded protein may also be "mutated" and may contain deletions, insertions or substitutions of amino acid residues which result in silent changes, resulting in a functionally equivalent INTRA. Planned amino acid substitutions are based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of residues, as long as the biological or immunological activity of INTRA is retained. You can do it. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids having uncharged polar side chains with similar hydrophilicity values include asparagine and glutamine, and serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values include leucine, isoleucine, and valine,
There are glycine and alanine, and phenylalanine and tyrosine.

The term “amino acid” or “amino acid sequence” refers to a sequence of oligopeptides, peptides, polypeptides or proteins or fragments thereof, which refers to natural or synthetic molecules. Here, "amino acid sequence" refers to the amino acid sequence of a naturally occurring protein molecule, and "amino acid sequence" and similar terms limit an amino acid sequence to the complete, native amino acid sequence that associates with the listed protein molecules. Not what you are trying to do.

“Amplification” refers to the additional replication of nucleic acid sequences. Amplification is typically performed using the polymerase chain reaction (PCR) technique well known to those skilled in the art.

The term “antagonist” refers to a molecule that inhibits or attenuates the biological activity of INTRA. Antagonists can include proteins such as antibodies, nucleic acids, carbohydrates, small molecules or any other compound or moiety, which are either directly interacting with INTRA or biologically involved in INTRA. It regulates the activity of INTRA by acting on components of the pathway.

The term “antibody” refers to intact immunoglobulins and fragments thereof, such as Fa, F (ab ′) ₂ and F.
v Fragments, which are capable of binding epitope determinants. Antibodies that bind INTRA polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (mouse, rat, rabbit, etc.) can be derived from translated or chemically synthesized RNA, and can be conjugated to a carrier protein if desired. is there. Commonly used carriers that chemically bond to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The binding peptide is used to immunize the animal.

The term “antigenic determinant” refers to the region of the molecule (ie, the epitope) that is in contact with a particular antibody. When a host animal is immunized with a protein or protein fragment, a large number of regions of the protein are associated with antigenic determinants (specific regions of the protein or 3
Can induce the production of antibodies that specifically bind to the (dimensional structure). An antigenic determinant can compete with an intact antigen for binding to an antibody (ie, an immunogen used to induce an immune response).

The term “antisense” refers to any moiety capable of base pairing with the “sense” strand of a particular nucleic acid sequence. Antisense components include DNA, RNA, peptide nucleic acid (PNA), oligonucleotides having a modified backbone chain such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, 2'-methoxyethyl sugar or 2 '. -Oligonucleotides with modified sugars such as methoxyethoxy sugar, or 5-methylcytosine, 2-deoxyuracil or 7-deaza-2
There are oligonucleotides with modified bases such as'-deoxyguanosine.
Antisense molecules can be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base pairs with the native nucleic acid sequence formed by the cell to form a duplex that interferes with transcription or translation. The term "negative" or "minus (-)" may refer to the antisense strand, and "positive" or "plus (+)" may refer to the sense strand.

The term “biologically active” refers to a protein having the structural, regulatory or biochemical functions of a naturally occurring molecule. Similarly, "immunologically active" is the ability of natural, recombinant or synthetic INTRA, or any oligopeptide thereof, to induce a particular immune response in a suitable animal or cell to induce a specific antibody. Refers to the ability to combine.

The terms “complementary” or “complementarity” refer to the natural association of polynucleotides with each other by base pairing. For example, the sequence "5'AG-T3 '" binds to the complementary sequence "3'TC-A5'". Complementarity between two single-stranded molecules may be "partial," in which only some nucleic acids are attached, or "complete complementarity between single-stranded molecules may exist." It can be "perfect". The degree of complementarity between nucleic acid strands is
It significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is true in amplification reactions that depend on binding between nucleic acid strands and also in peptide nucleic acid (PNA
) Of particular importance in the design and use of the molecule.

“Component consisting of a given polynucleotide sequence” and “component consisting of a given amino acid sequence” generally refer to any component consisting of a given polynucleotide sequence or amino acid sequence. This component may include a dry formulation or an aqueous solution. INTRA
Alternatively, a component consisting of a polynucleotide encoding an INTRA fragment can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be associated with a stabilizer such as sugar. For hybridization, the probe is dispersed in an aqueous solution containing salts (eg NaCl), detergents (eg sodium dodecyl sulfate; SDS) and other constituent elements (eg Denhardt's solution, skim milk powder, salmon sperm DNA etc.). Can be made.

The “consensus sequence” is rearranged in order to separate unnecessary bases, and the XL-PCR kit (PE Biosystems, Foster City CA) is used to select one of the 5 ′ direction and the 3 ′ direction. Refers to nucleic acid sequences that have been extended in both directions and then rearranged. Alternatively, it refers to nucleic acid sequences assembled from overlapping sequences of one or several Incyte clones, optionally one or several public domain ESTs, using a fragment assembly computer program. Examples of computer programs include the GELVIEW fragment assemble system (GCG, Madison WI) and Phrap (Universit
y of Washington, Seattle WA). Some sequences are both extended and assembled to determine the consensus sequence.

A “conservative amino acid substitution” is a substitution that does not impair the properties of the original protein when the substitution is made, that is, the structure and especially the function of the protein are conserved, and there is no major change due to such substitution. Refers to substitution. The table below shows the amino acids that can be substituted for the original amino acids in the protein and the amino acids that are recognized as conservative amino acid substitutions. 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 Conservative amino acid substitutions usually involve (a) a backbone structure of the polypeptide in the substitution region, such as β-sheet or α-helical structure, (b) charge or hydrophobicity of the molecule at the substitution site, and / or (c) side. Holds most of the chains.

“Deletion” refers to a change in an amino acid or nucleotide sequence that results in the loss of one or several amino acids or nucleotides.

The term “derivative” refers to a chemical modification of a polypeptide or polynucleotide sequence. For example, replacement of hydrogen by an alkyl, acyl, hydroxyl or amino group can be included in the chemical modification of the polynucleotide sequence. A polynucleotide derivative encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. Polypeptide derivatives are modified by glycosylation, polyethylene pegylation, or any similar process that retains at least one biological or immunological function from the polypeptide of derived origin. Is a polypeptide that has been deleted.

“Detectable label” refers to a reporter molecule or enzyme that is capable of producing a measurable signal and is covalently or non-covalently attached to a polynucleotide or polypeptide.

“Fragment” refers to a unique portion of INTRA or a polynucleotide encoding INTRA that is identical to the parent sequence but shorter in length than the parent sequence. Fragments
It may have a length that is less than the total length of the defined sequence minus one nucleotide / amino acid residue. For example, a fragment may have 5 to 1000 consecutive nucleotides or amino acid residues. Fragments used as probes, primers, antigens, therapeutic molecules, or for other purposes should be at least 5, 10, 1
It can be 5, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 consecutive nucleotides or amino acid residues in length. Fragments can be preferentially selected from specific regions of the molecule. For example, a polypeptide fragment may have a length of contiguous amino acids selected from the first 250 or 500 amino acids (or the first 25% or 50% of the polypeptide) as shown in the given sequence. These lengths are explicitly given as examples and in the embodiments of the present invention may be of any length supported by the description including the sequence listings, tables and figures.

The fragments of SEQ ID NOs: 53-104 include unique polynucleotide sequence regions. This region uniquely identifies SEQ ID NOS: 53 to 104, and is different from the sequence other than SEQ ID NOS: 53 to 104 in the same genome, for example.
Fragments of SEQ ID NOs: 53-104 are provided, for example, in hybridization and amplification techniques or from related polynucleotide sequences.
Is useful in a similar way to distinguish The exact lengths of the fragments of SEQ ID NOs: 53-104 and the regions of SEQ ID NOs: 53-104 corresponding to the fragments can be routinely determined by one of skill in the art based on the intended purpose for the fragment.

The fragments of SEQ ID NOs: 1 to 52 are encoded by the fragments of SEQ ID NOs: 53 to 104. The fragments of SEQ ID NOS: 1 to 52 include unique amino acid sequence regions that specifically identify SEQ ID NOS: 1 to 52. For example, the fragments of SEQ ID NOS: 1 to 52 are useful as immunogenic peptides for producing antibodies that specifically recognize SEQ ID NOS: 1 to 52. SEQ ID NOS: 1 to 52 and SEQ ID NO: 1 corresponding to the fragments
The exact length of the region 52 to 52 can be routinely determined by one of ordinary skill in the art based on the intended purpose for the fragment.

A “full length” polynucleotide sequence is a sequence that has at least one translation initiation codon (eg, methionine), an open reading frame and a translation stop codon. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence.

The term “homology” refers to sequence similarity, that is, two or more polynucleotide sequences or two.
Compatible sequence identity between sequences of two or more polypeptide sequences.

The term “match rate” or “% match” as applied to a polynucleotide sequence refers to the percentage of residues that match between at least two or more polynucleotide sequences aligned using a standardized algorithm. means. Such algorithms insert gaps in the sequences that are compared to optimize the alignment between the two sequences in a standardized and reproducible manner, allowing the two sequences to be compared significantly more significantly.

The percent match between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm, as incorporated in the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and is a suite of molecular biology analysis programs (DNASTAR, Madison WI).
Is. For CLUSTAL V, Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153 and Higgins, DG et al. (1992) CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as Ktuple = 2, gap penalty = 5, window = 4, "diagonals saved" = 4. Select the "weighted" residue weighting table as the default. CLUS
TAL V reports concordance as the "similarity" between aligned polynucleotide sequence pairs.

Alternatively, the Basic Local Al of the National Center for Biotechnology Information (NCBI)
The ignition search tool (BLAST) is commonly used and provides a set of freely available sequence comparison algorithms (Altschul, SF et al. (1990) J. Mol. B.
iol. 215: 403-410). This algorithm is based on NCBI in Bethesda, MD.
It is available from several sources, including the Internet (http: //www.ncbi.
It is also available on nlm.nih.gov/BLAST/). The BLAST suite of software includes a variety of sequence analysis programs to align a known polynucleotide sequence with another polynucleotide sequence from a variety of databases, "blastn".
Is one of them. In addition, a tool called "BLAST 2 Sequences" used for directly comparing two nucleotide sequences in pairs is also available. "BLAST
2 Sequences "can be used interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The BLAST 2 Sequences tool is bla
It can be used for both stn and blastp (see below). The BLAST program is typically used with gaps and other parameters set to default settings. For example, the "BLAST 2 Sequences" tool Version 2.0.12 (April 21, 2000) set as default parameters for comparing two nucleotide sequences.
May be used to perform blastn. An example of setting default parameters is shown below. 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 Match rate is fully defined It can be measured relative to the sequence length (eg defined by a particular SEQ ID NO). Alternatively, fragments derived from shorter lengths, eg, larger defined sequences (eg, at least 20, 30, 40, 50, 70, 100).
Alternatively, the concordance rate may be determined by comparison with the length of 200 consecutive nucleotide fragments). The lengths listed here are merely exemplary, and using the fragments of any sequence length backed by the sequences described herein, including tables, figures, and sequence listings, the percent match was determined. It should be understood that this may account for the length that can be measured.

Nucleic acid sequences that do not exhibit a high degree of homology may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is to be understood that this degeneracy can be utilized to produce changes in a nucleic acid sequence to produce multiple nucleic acid sequences such that all nucleic acid sequences encode substantially the same protein.

The term “match rate” or “% match” as applied to a polypeptide sequence means the percentage of residues that match between at least two or more polypeptide sequences aligned using a standardized algorithm. To do. Methods for polypeptide sequence alignment are known. There are also alignment methods that consider conservative amino acid substitutions.
Such conservative substitutions, as detailed above, generally preserve the acidity and hydrophobicity of the site of substitution and thus the structure (and thus function) of the polypeptide.

The degree of concordance between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm as incorporated in the MEGALIGN version 3.12e sequence alignment program (see it previously described). CLUSTAL
The default parameters for aligning two pairs of polypeptide sequences using V are set as Ktuple = 1, gap penalty = 3, window = 5, and “diagonals saved” = 5. Select the PAM250 matrix as the default residue weight table. Similar to polynucleotide alignment, CLUSTAL V reports concordance as the "similarity" between aligned polypeptide sequence pairs.

Alternatively, the NCBI BLAST software suite may be used. For example, when comparing polypeptide sequences in pairs, the blastp set as a default parameter
The "BLAST 2 Sequences" tool Version 2.0.12 (April 21, 2000) may be used with. An example of setting default parameters is shown below. Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on It can be measured relative to the length of the polypeptide sequence. The concordance rate is a sequence or shorter length,
For example, a fragment obtained from a larger defined polypeptide sequence (eg, a fragment of at least 15, 20, 30, 40, 50, 70 or 150 contiguous residues).
The match rate may be measured by comparing with the length of the. The lengths listed here are merely exemplary and may be used with any sequence length fragment supported by the sequences described herein, including tables, figures and sequence listings. It should be understood that it can be described as a length, for which a match rate can be measured.

“Human Artificial Chromosome” (HAC) is a linear, small chromosome containing a DNA sequence of 6 kb to 10 Mb in size and containing all elements necessary for stable segregation and maintenance of mitotic chromosomes. Has been.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence in the non-antibody binding region has been mutated so that it is closer to that of a human antibody, and the original binding ability is retained. Point to.

“Hybridization” refers to the process by which a single-stranded polynucleotide anneals to a complementary strand by forming base pairs under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share high homology. The specific hybridization complex is formed under acceptable annealing conditions and remains hybridized after the "wash" step. The wash step is particularly important in determining the stringency of the hybridization process, and more stringent conditions reduce non-specific binding (ie, pair binding between inconsistent nucleic acid strands).
Permissive conditions for the annealing of nucleic acid sequences are routinely determined by those of ordinary skill in the art. Permissive conditions may be constant during the hybridization experiment, but wash conditions can be varied during the experiment to obtain the desired stringency, and thus hybridization specificity. The annealing permissive conditions are satisfied at a temperature of 68 ° C. in the presence of, for example, about 6 × SSC, about 1% (w / v) SDS and about 100 μg / ml denatured salmon sperm DNA.

In general, hybridization stringency can be expressed in part to the temperature at which the wash step is performed. Such washing temperatures are usually selected to be about 5 to 20 ° C. below the melting point (Tm) of the specific 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 hybridization conditions for nucleic acids are well known and are described in Sambrook et al.
989) Molecular Cloning: A Laboratory Manual , 2nd edition, Volume 1-3, Cold Spring H
arbor Press, Plainview NY, see especially Chapter 2 of Volume 2.

High stringency conditions for polynucleotide to polynucleotide hybridization of the invention include wash conditions of about 68 ° C. in the presence of about 0.2 × SSC and about 1% SDS for 1 hour. . Or 65 ℃, 60 ℃, 55
It may be carried out at a temperature of ℃ or 42 ℃. SSC concentration in the presence of about 0.1% SDS,
It can vary in the range of about 0.1 to 2 x SSC. Blockers are usually used to block non-specific hybridization. Such blockers include, for example, about 100-200.
There is μg / ml denatured salmon sperm DNA. Under certain conditions, it is also possible to use organic solvents, for example formamide at a concentration of about 35-50% v / v, for the hybridization of RNA and DNA, for example. Useful variations of wash conditions will be apparent to those of skill in the art. Hybridization can suggest evolutionary similarities between nucleotides, especially under conditions of high stringency. Such similarities strongly suggest a similar role for nucleotides and nucleotide-encoded polypeptides.

The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the ability to form hydrogen bonds between complementary base pairs. A hybridization complex may be formed in solution (C ₀ t or R ₀ t analysis, etc.). Alternatively, one nucleic acid sequence is present in solution and the other nucleic acid sequence is present on a solid support (eg paper, membrane,
It may be formed between two nucleic acid sequences such as filters, chips, pins or glass slides, or other suitable substrate that is immobilized on a substrate on which cells or their nucleic acids are immobilized).

The terms “insertion” and “addition” refer to changes in amino acid or nucleotide sequences that add one or several amino acid residues or nucleotide sequences, respectively.

“Immune response” can refer to a condition associated with inflammation, trauma, immune disorders, contagious diseases or genetic disorders. These conditions can be characterized by the expression of various factors, such as cytokines, chemokines, and other signaling molecules that can act on the cellular and systemic defense system.

“Immune antigenic fragment” is a polypeptide or oligopeptide fragment of INTRA capable of eliciting an immune response when introduced into an organism such as a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of INTRA useful in any method of raising antibodies as disclosed herein or as known in the art. ..

The term “microarray” refers to the composition of multiple polynucleotides, polypeptides or other compounds on a substrate.

The term “element” or “array element” refers to a hybridizable polynucleotide located on the surface of a substrate in the environment of a microarray.

The term “modulate” refers to a change in the activity of INTRA. Modulation can, for example, increase or decrease the protein activity, binding properties or other biological, functional or immunological properties of INTRA.

The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides or fragments thereof. The terms "nucleic acid" and "nucleic acid sequence" refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, or represent the sense or antisense strand, or It may also refer to peptide nucleic acid (PNA) or any DNA-like or RNA-like substance.

“Operably linked” refers to the state in which a first nucleic acid sequence is placed into a functional relationship with a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences may be in close proximity or contiguous if it is necessary to join the two protein coding regions in the same reading frame.

“Peptide nucleic acid” (PNA) is an antisense molecule or anti-gene substance, consisting of an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of lysine-terminated amino acid residues. Refers to something. The terminal lysine confers solubility to the components. PNAs preferentially bind complementary single-stranded DNA or RNA to stop the expansion of transcription, and can be polyethylene glycolated to prolong PNA lifespan in cells.

“Post-translational modifications” of INTRA include lipidation, glycosylation, phosphorylation, acetylation,
Racemization, proteolytic resolution and other modifications known in the art may be included. These processes can occur synthetically or biochemically. Biochemical modification is INTR
It depends on the enzymatic environment of A and can vary by cell type.

“Probe” refers to a nucleic acid sequence that encodes INTRA, the complementary sequence of INTRA, or a fragment thereof, and is used to detect the same nucleic acid sequence, allelic nucleic acid sequence, or related nucleic acid sequences. A probe is an isolated oligonucleotide or polynucleotide attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent reagents and enzymes.

A “primer” is a short nucleic acid, usually a DNA oligonucleotide, that can anneal to a target polynucleotide by forming complementary base pairs. The primer can then extend to the target DNA strand by a DNA polymerase enzyme. Primer pairs are used to amplify (and identify) nucleic acid sequences by, for example, polymerase chain reaction (PCR).
Can be used for.

Probes and primers as used in the present invention usually contain at least 15 contiguous nucleotides of known sequence. Longer probes and primers to increase specificity, eg at least 20, 25, 30, of the disclosed nucleic acid sequences,
Probes and primers that consist of 40, 50, 60, 70, 80, 90, 100 or at least 150 contiguous nucleotides may be used. Some probes and primers are much longer than this. It is understood that any length of nucleotides supported herein, including tables, figures and sequence listings, can be used.

For preparation and use of probes and primers, see Sambrook, J. et al. (1.
989) Molecular Cloning: A Laboratory Manual , 2nd edition, Volume 1-3, Cold Spring H
arbor Press, Plainview NY, Ausubel, FM et al., (1987) Current Protocols in Molecular Biology , Greene Pubi. Assoc. & Wiley-Intersciences, New York.
NY, Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications Ac
See ademic Press, San Diego CA, etc. PCR primer pairs are computer programs for that purpose, such as Primer (Version 0.5, 1991, Whitehe
Ad Institute for Biomedical Research, Cambridge MA).

The selection of oligonucleotides to use as primers is done using software well known in the art for such purposes. For example, OLIGO 4.06 software is useful for selecting PCR primer pairs of up to 100 nucleotides each, and is derived from oligonucleotides and up to 5,000 large polynucleotides from input polynucleotide sequences up to 32 kilobases. Is also useful for analyzing. A similar primer selection program incorporates additional functionality for expanded capacity. For example, the PrimOU primer selection program (available to the public from the Genome Center at the University of Texas Southwestern Medical Center in Dallas, Texas) is capable of selecting a particular primer from a megabase sequence, thus covering the entire genome. Useful for designing primers. Pr
imer3 primer selection program (Whitehead, Cambridge, MA)
(Available to the general public from the Institute / MIT Genome Research Center), the user can input a "mispriming library", and here the user specifies the sequences to be avoided as primer binding sites. Primer3 is particularly useful for the selection of oligonucleotides for microarrays (the source code of the latter two primer selection programs may be modified from individual sources to meet user-specific needs). The PrimerGen Program (UK Human Genome Mapping Project in Cambridge, UK-Publicly available from the Resource Center) designs primers based on multiple sequence alignments, thereby maximally or minimally conserving the aligned nucleic acid sequences. Allows selection of primers to hybridize with any of the regions. Therefore, this program is useful for identifying unique and conserved fragments of oligonucleotides and polynucleotides. Oligonucleotide and polynucleotide fragments identified by any of the above selection methods can be used to identify fully or partially complementary polynucleotides in hybridization techniques, such as PCR or sequencing primers, as microarray elements, or in nucleic acid samples. It is useful as a specific probe. The method for selecting the oligonucleotide is not limited to the above method.

A “recombinant nucleic acid” is a sequence that is either a non-native sequence or a sequence that has been produced by artificially combining two or more segments of a sequence that would be separated unless artificially combined. . This artificial combination is often accomplished by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids, such as by genetic engineering techniques such as those described in Sambrook et al., Supra. To do. The term recombinant nucleic acid also includes mutant nucleic acids in which a portion of the nucleic acid is simply added, substituted or deleted. Frequently, recombinant nucleic acids include a nucleic acid sequence operably linked to a promoter sequence. Such recombinant nucleic acid may be an integral element of the vector, such as that used to transform a cell.

Alternatively, such recombinant nucleic acid may be an integral element of the viral vector, for example based on vaccinia virus. Vaccinia virus is used for vaccination of mammals expressing recombinant nucleic acid and induces a protective immune response in mammals.

A “regulatory element” is a nucleic acid sequence usually derived from the untranslated region of a gene, including enhancers, promoters, introns and 5 ′ and 3 ′ untranslated regions (UTRs).
)including. Regulatory elements interact with host or viral proteins that regulate transcription, translation or RNA stability.

A “reporter molecule” is a chemical or biochemical moiety used to label nucleic acids, amino acids or antibodies. Reporter molecules include radionuclides, enzymes,
Fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles and other ingredients known in the art.

An “RNA equivalent” for a DNA sequence is a reference DNA, except that all of the generated nitrogen base thymine is replaced by uracil and that the sugar backbone is composed of ribose rather than deoxyribose. It is composed of a linear nucleotide sequence identical to the sequence.

The term “sample” is used in its broadest sense. A sample suspected of containing INTRA-encoding nucleic acid or a fragment thereof, or INTRA itself is
Consists of extracts from cells, chromosomes, organelles or membranes isolated from cells, cells, genomic DNA, RNA or cDNA lysed or bound to a substrate, tissues, tissue prints, etc. Can be done.

The term “specific binding” or “specifically binds” refers to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, any natural or synthetic binding moiety. This interaction is meant to depend on the presence or absence of a particular structure of the protein (eg, an antigenic determinant or epitope) that the binding molecule recognizes. For example, if the antibody is specific for epitope "A", the presence of a polynucleotide containing epitope A (ie, free, unlabeled A) in a reaction involving labeled free A and the antibody. Reduces the amount of labeled A bound to the antibody.

The term “substantially purified” is a nucleic acid or amino acid sequence that has been removed from, or isolated or separated from, the natural environment and is at least about 60%, preferably at least 60%, of the other naturally associated components. Indicates at least about 75%, and most preferably at least about 90% free.

“Substitution” means the replacement of one or several amino acids or nucleotides by different amino acids or nucleotides.

“Substrate” refers to any suitable solid or semi-solid support, including membranes, filters, chips, slides, wafers, fibers, magnetic non-magnetic beads, gels, tubes, plates, polymers. , Fine particles, capillaries are included. The substrate can have various surface morphologies such as walls, grooves, pins, channels, pores, etc., and the surface of the substrate is bound by polynucleotides or polypeptides.

“Transcriptional image” refers to the collective pattern of gene expression by a unique cell type or tissue at a given time and condition.

“Transformation” refers to the process by which exogenous DNA enters a host cell and modifies the host cell. Transformation can occur under natural or artificial conditions according to various methods known in the art and is based on any known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. You can The method of transformation is selected according to the type of host cell to be transformed. Transformation methods include, but are not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed" cell includes not only transiently transformed cells that express the inserted DNA or RNA within a limited time, but also stably transformed cells. There is also included one in which the DNA inserted therein is capable of replicating autonomously as a plasmid or as a part of the host chromosome.

As used herein, a “genetic transformant” is any organism, including, but not limited to, animals and plants, in which one or several cells of the organism are associated with human involvement.
For example, having a heterologous nucleic acid introduced by a transformation technique well known in the art. The introduction of the nucleic acid into the cells is carried out directly or indirectly by introducing them into the precursors of the cells, by deliberate genetic manipulation, for example by microinjection or by introduction of recombinant virus. The term genetic engineering does not refer to classical cross breeding or in vitro fertilization, but to the introduction of recombinant DNA molecules.
Genetic transformants contemplated according to the present invention include bacteria, cyanobacteria, fungi and plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, such as infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the invention to such organisms are well known and provided in the references such as Sambrook et al. (1989) supra.

A “variant” of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence homology with the particular nucleic acid sequence over the length of the entire nucleic acid sequence. At that time, "BLAST 2 Sequences" tool Version 2.0 set to default parameters
.9 (May 7, 1999) with blastn. Such nucleic acid pairs may be, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, for a given length.
It may show 95%, 98% or more homology. Certain variants may be described as, for example, "allelic" variants (supra), "splice" variants, "species" variants or "polymorphic" variants. Splice variants may have considerable homology to the reference molecule, but will typically have a large or small number of polynucleotides due to the alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or may lack domains present in the reference molecule. Species variants are polynucleotide sequences that differ from one another. The resulting polypeptides usually have significant amino acid homology to each other. Polymorphic variants differ in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants can also include "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence is altered by one nucleotide base. S
The presence of NPs may indicate, for example, a particular population, medical condition or propensity.

A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence that has at least 40% homology to the particular polypeptide sequence over the length of one of the polypeptide sequences. Here, "BLAST
2 Sequences ”tool Version 2.0.9 (May 7, 1999) to run blastp. Such a polypeptide pair may have, for example, at least 50% for a given length.
, 60%, 70%, 80%, 85%, 90%, 95%, 98% or more homology.

Invention The present invention relates to a novel human intracellular signaling molecule (INTRA), a polynucleotide encoding INTRA, and abnormal cell proliferation, autoimmune / inflammatory disease, neuropathy, gastrointestinal disorder, reproductive disorder and developmental disorder. It is based on the discovery of methods to utilize these sequences for the diagnosis, treatment and prevention of.

Table 1 shows the Incyte used to construct the full-length nucleotide sequence encoding INTRA.
Indicates a clone. Columns 1 and 2 show the SEQ ID NOs for the polypeptides and polynucleotides, respectively (SEQ ID NO). Column 3 shows the clone ID of the Incyte clone and the nucleic acid encoding each INTRA is the one identified here. Column 4 is cDNA
The library is shown and the clone in column 3 was isolated from here. Row 5
Shows an Incyte clone and its corresponding cDNA library. The Incyte clone for which the cDNA library is not shown was obtained from a pooled cDNA library. The Incyte clone in row 5 was used to construct the consensus nucleotide sequence for each INTRA. The Incyte clone in row 5 is useful as a fragment in hybridization techniques.

The columns of Table 2 show 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, column 5 is Sign
ature) amino acid residues having a sequence and a motif, column 6 shows a homologous sequence identified by BLAST analysis, and column 7 shows an analytical method and, in some cases, a searchable database in which the analytical method can be used. The analytical methods in column 7 were used to characterize each polypeptide from sequence homology and protein motifs.

The columns of Table 3 show the tissue specificity and the disease, disorder or condition associated with the nucleotide sequence encoding INTRA. Column 3 of Table 3 shows the nucleotide sequence numbers and column 2 shows the fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification techniques to identify SEQ ID NOS: 53-104 and distinguish the polynucleotide sequences related to SEQ ID NOS: 53-104. Polynucleotides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 shows the tissue categories expressing INTRA as the percentage of INTRA expression in the whole tissue. Column 4 shows diseases, disorders or conditions associated with INTRA expressing tissue as a percentage of total INTRA expressing tissue. Column 5 shows the vector used to subclon each cDNA library. SEQ ID NO: 88 and SEQ ID NO: 9 in reproductive tissue
Of particular interest is the expression of 4, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 103 in hematopoietic / immune tissue, and SEQ ID NO: 96 in cardiovascular tissue.

The columns of Table 4 provide a description of the tissues used to create the cDNA library. INTR
The clone of the cDNA encoding A was isolated from a cDNA library. Row 1
Shows the nucleotide sequence number of the nucleotide, column 2 shows the cDNA library which is the source of clonal isolation, and column 3 shows the collection source of tissue and other bibliographic information related to the cDNA library of column 2.

SEQ ID NO: 58 is located on chromosome 7 within the interval 84.40 to 90.30 centimorgans.
Map to. In this interval, ES highly similar to the thyroid disease virtual self-antigen
Also includes T. SEQ ID NO: 67 maps to chromosome 16 within the 119.20 centimorgan to q-terminal interval. This interval also includes the paraplegin gene whose mutations cause spastic paraplegia and oxidative phosphorylation (OXPHOS) disorders. SEQ ID NO: 70 is located on chromosome 1 within the interval 59.50 to 62.50 centimorgans.
Map to 1. SEQ ID NO: 71 maps to chromosome 7 within the interval 138.0 to 145.8 centimorgans. SEQ ID NO: 73 maps to chromosome 12 within the interval 76.5 to 84.2 centimorgans. SEQ ID NO: 77 is 4.8
To chromosome 7 within the interval from 1 to 10.6 centimorgans and from 56.7 to 6
Maps to chromosome 4 within an interval of 0.5 centimorgan. 4.8 to 10.6
The interval of chromosome 7 of centimorgan also contains genes related to cell proliferation.
The interval between chromosomes 56.7 to 60.5 centimorgans also contains genes involved in cell proliferation. SEQ ID NO: 79 maps to chromosome 15 within the interval of 32.2 to 47.1 centimorgans. This interval also contains genes related to cell proliferation. SEQ ID NO: 80 maps to chromosome 20 within the interval of 50.2 to 53.6 centimorgans. Genes associated with cell differentiation are also included in this interval. SEQ ID NO: 84 is located on chromosome 3 within the interval of 142.2 to 148.7 centimorgans.
Map to. SEQ ID NO: 87 maps to chromosome 5 within the interval of 141.4 to 147.1 centimorgans. SEQ ID NO: 91 maps to chromosome 12 within the interval of 62.7 to 67.3 centimorgans. SEQ ID NO: 95 is 45.5
To chromosome 15 within the interval of 58.8 centimorgans. SEQ ID NO: 97 maps to the X chromosome within the interval 112.8 to 139.4 centimorgans.

The present invention also includes variants of INTRA. A preferred INTRA variant amino acid sequence has at least about 80%, about 90%, or even about 95% concordance with the INTRA amino acid sequence, such that it has at least one functional or structural characteristic of INTRA. It is a mutant.

The invention also includes polynucleotides encoding INTRA. In one example, I
Included in the invention is a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NOs: 53-104 encoding NTRA. The polynucleotide sequences of SEQ ID NOs: 53 to 104 have the same value as the equivalent RNA sequence as shown in the sequence listing, but the appearance of the nitrogen base thymine is replaced by uracil and the sugar backbone is deoxy. It is composed of cilibose rather than ribose.

The present invention also includes variant sequences of the polynucleotide sequence encoding INTRA. Specifically, the variant sequence of such a polynucleotide sequence comprises at least about 80%, or at least about 90% of the polynucleotide sequence encoding INTRA,
Or have a concordance rate of at least about 95%. In some embodiments of the invention, the amino acid sequence selected from the group consisting of SEQ ID NOs: 53-104 and at least about 8
A variant sequence of the polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NOs: 53-104 having 0%, or at least about 90%, or even at least about 95% identity. Variants of any of the above polynucleotides include
It may encode an amino acid sequence having at least one functional or structural characteristic of INTRA.

Those skilled in the art will appreciate that as a result of the degeneracy of the genetic code, a large number of polynucleotide sequences encoding INTRA (polynucleotide sequences having minimal similarity to those of known or naturally occurring genes). Understand that it can produce Thus, the invention may cover any and all possible polynucleotide sequence variants that may be produced by the selection of combinations based on possible codon choices. These combinations are made on the basis of the standard triplet genetic code as applied to the native INTRA polynucleotide sequences, and all such mutations are considered to be explicitly disclosed.

The nucleotide sequence encoding INTRA and variants thereof will generally be hybridizable with the nucleotide sequence of naturally occurring INTRA under suitably selected stringent conditions, although INTRA or a derivative thereof may be substantially It would be beneficial to create nucleotide sequences that code for different codon usages, eg, for inclusion of non-naturally occurring codons. Based on the frequency with which a host utilizes a particular codon, the codon can be selected to increase the expression rate of the peptide occurring in a particular eukaryotic or prokaryotic host. Another reason for substantially altering the nucleotide sequence encoding INTRA and its derivatives without altering the encoded amino acid sequence is that it may be preferable to a transcript made from the native sequence, such as having a longer half-life. There is the production of RNA transcripts that have.

The present invention also includes making DNA sequences encoding INTRA, INTRA derivatives and fragments thereof, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence may be inserted into any of a variety of available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry may be used to induce mutations in the sequence encoding INTRA or any fragment thereof.

Furthermore, the present invention also provides a polynucleotide sequence capable of hybridizing to the polynucleotide sequences according to the claims, particularly the sequences shown in SEQ ID NOs: 53 to 104, and fragments thereof under various stringent conditions. Included (Wahl, GM and SL
. Berger (1987) Methods Enzymol. 152: 399-407, Kimmel. AR (1987) Method
s Enzymol. 152: 507-511.

Methods for DNA sequencing are well known in the art and any of the embodiments of the invention can be performed using DNA sequencing methods. Enzymes can be used in the DNA sequencing method, for example, Klenow fragment of DNA polymerase I, SE
QUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems,
Foster City CA), thermostable T7 polymerase (Amersham, Pharmacia Biotech,
Piscataway NJ) can be used. Alternatively, a polymerase and a proofreading exonuclease can be used in combination, for example as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Suitably, MICROLAB 2200
Liquid transfer system (Hamilton, Reno, NV), PTC200 thermal cycler (MJ Res
earch, Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosyste
automated sequence preparation using a device such as ms). Then ABI 373 or 377 DNA
Sequencing is performed using a Sequencing System (PE Biosystems), MEGABACE 1000 DNA Sequencing System (Molecular Dynamics, Sunnyvale CA) or other methods well known in the art. The resulting sequences are analyzed using various algorithms well known in the art. (Ausubel, FM
. (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New Yo
rk NY, unit 7.7, Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY, pp. 856-853.

The INTRA-encoding nucleic acid sequence is extended using various methods based on the PCR method utilizing partial nucleotide sequences and well known in the art, and upstream sequences such as promoters and regulatory elements are included. Can be detected. For example, the restriction site PCR method, which is one of the methods that can be used, is a method of amplifying an unknown sequence from genomic DNA in a cloning vector using universal primers and nested primers (Sarkar, G. (1993) PCR. See Methods Applic 2: 318-322).
Another method is the inverse PCR method, which is a method of amplifying an unknown sequence from a circularized template using primers extended in a wide range of directions. The template is derived from a restriction fragment consisting of a known genomic locus and its surrounding sequences (Triglia, T. et al. (1988) N
ucleic Acids Res 16: 8186 etc.). The third method is the capture PCR method, which PCRs a DNA fragment adjacent to a known sequence of human and yeast artificial chromosome DNA.
Involved in the method of amplification. (Lagerstrom, M. et al. (1991) PCR Methods Applic
See 1: 111-119 etc. In this method, it is possible to insert a recombinant double-stranded sequence into an unknown sequence region using multiple restriction enzyme digestion and ligation reaction before performing PCR. Also, there are other methods known in the art that can be used to search for unknown sequences. (See Parker, JD et al. (1991) Nucleic Acids Res. 19: 3055-3060 etc.). In addition, PCR, nested primers and PromoterFinder ™ libraries (Clontech, Palo Alto CA) can be used to walk genomic DNA. This procedure is useful for finding intron / exon junctions without the need to screen the library. For all PCR-based methods, use commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another suitable program, approximately 22-30 nucleotides in length, containing a GC. Rate is about 50% or more, temperature is about 68 ° C
Primers can be designed to anneal to the template at 72 ° C.

When screening for full-length cDNAs, it is preferable to use libraries that are size-selected to contain larger cDNAs. In addition, randomly primed libraries often contain sequences with the 5'regions of the genes and contain oligo d (T)
Suitable for situations where the library is unable to produce full-length cDNA. Genomic libraries will be useful for extension of sequences into the 5'non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size of, or confirm the nucleotide sequence of, sequencing or PCR products. Specifically, capillary sequencing consists of a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye, such as is specific for four different nucleotides, and detection of emitted wavelengths. And a CCD camera used for the. Output / light intensity is measured by appropriate software (PE Biosystems GENOT
YPER, SEQUENCE NAVIGATOR, etc.) can be used to convert the electric signal. The entire process from sample loading to computer analysis and electronic data display is computer controllable. Capillary electrophoresis is particularly suitable for sequencing small DNA fragments that may be present in low amounts in a particular sample.

In another embodiment of the invention, a polynucleotide sequence encoding INTRA or a fragment thereof is used to express INTRA, a fragment of INTRA or a functional equivalent thereof in a suitable host cell. Can be cloned in. Due to the unique duplication of the genetic code, other sequences that encode substantially identical or functionally equivalent amino acid sequences
A DNA sequence can be generated and used to express INTRA.

The nucleotide sequences of the invention can be recombined using methods commonly known in the art to alter the sequence encoded by INTRA for a variety of purposes. The purposes herein include, but are not limited to, cloning, processing, and regulation of gene product expression. Nucleotide sequences can be recombined using random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variants, and the like.

Nucleotides of the present invention are described in Molecular Breeding ^™ (Maxygen Inc., Santa Clara).
CA, U.S. Pat.No. 5,837,458, Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:79.
3-797, Christians, FC et al. (1999) Nat. Biotechnol. 17: 259-264, Crameri, A.
(1996) Nat. Biotechnol. 14: 315-319) and other DNA shuffling techniques, and the biological properties of INTRA, such as biological activity, enzymatic activity, or binding to other molecules or compounds. Etc. can be changed or improved. DNA shuffling
The process of generating a library of gene variants using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening to identify the gene variant with the desired property. These suitable variants may then be pooled and further repeated for DNA shuffling and selection / screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene with random point mutations can be recombined, screened, and then shuffled until the desired property is optimized. Alternatively, a given gene can be recombined with a homologous gene from the same gene family, either homologous or heterologous, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner. Can be transformed.

According to another embodiment, INTRA is prepared using chemical methods well known in the art.
Can be synthesized in whole or in part (Caruthers. MH et al. (1980) N
Nucleic. Acids Symp. Ser 7: 215-223, Horn, T. et al. (1980) Nucleic. Acids Symp.
. Ser. 7: 225-232). Alternatively, chemical methods may be used to synthesize INTRA itself or fragments thereof. For example, peptide synthesis can be performed using various liquid or solid phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties , WH Freeman, New York NY, pp. 55-60, Roberge. , JY et al.
(1995) Science 269: 202-204). Automated synthesis can be accomplished using the ABI 431A Peptide Synthesizer (Perkin Elmer). Furthermore, the amino acid sequence of INTRA, or any part thereof, may be altered or mutated during direct synthesis, or while binding to sequences obtained from other proteins or any part thereof, or both. It is possible to produce a type polypeptide.

Peptides can be substantially purified using preparative high performance liquid chromatography (see Chiez, RM and FZ Regnier (1990) Methods Enzymol. 182: 392-421, etc.). The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (see Creighton, pp. 28-53, etc., supra).

In order to express biologically active INTRA, the nucleotide sequence encoding INTRA or a derivative thereof can be inserted into a suitable expression vector. A preferred expression vector is a vector containing the elements necessary for the regulation of transcription and translation of the inserted coding sequence in a suitable host. Required elements are Vector and INTR
There are regulatory sequences such as enhancers, constitutive and expression-inducible promoters in the polynucleotide sequence encoding A, 5'and 3'untranslated regions. Such elements vary in length and specificity. It is also possible to use the unique initiation signal to more efficiently translate sequences encoding INTRA. Such signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. If the INTRA-encoding sequence and its initiation codon, upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be required. However, if only the coding sequence or a fragment thereof is inserted,
Exogenous translational control signals, including the in-frame ATG start codon, should be included in the expression vector. Exogenous translational elements and initiation codons can originate from a variety of natural and synthetic sources. The efficiency of expression can be enhanced by the inclusion of enhancers suitable for the particular host cell line used (Scharf, D.
(1994) Results Probl. Cell Differ. 20: 125-162.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding INTRA and suitable transcriptional and translational regulatory elements. This method includes in vitro recombinant DNA technology, synthetic technology and in vivo gene recombination technology (Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual , Cold.
Spring Harbor Press, Plainview NY, chapters 4, 8, 16-17, Ausubel, FM et al. (199
5) Current Protocols in Molecular Biology , John Wiley & Sons, New York N
(See Chapters 9, 13, 16 of Y).

A variety of expression vector / host systems may be utilized to carry and express sequences encoding INTRA. Such expression vector / host systems include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmids or cosmid DNA expression vectors, yeasts transformed with yeast expression vectors, and viruses. Insect cell lines infected with expression vectors (eg baculovirus), plant cell lines transformed with viral expression vectors (eg cauliflower mosaic virus CaMV or tobacco mosaic virus TMV) or bacterial expression vectors (eg Ti or pBR322 plasmids), There are microorganisms such as animal cell lines (Sambrook, supra, Ausubel, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 550.
3-5509, Bitter, GA et al. (1987) Methods Enzymol. 153: 516-544; Scorer, CA.
(1994) Bio / Technology 12:18 1-184; Engelhard, EK et al. (1994) Proc. N.
atl. Acad. Sci. USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7
: 1937-1945, Takamatsu, N. (1987) EMBOJ. 6: 307-311, Coruzzi, G. et al. (1984)
EMBOJ. 3: 1671-1680, Broglie, R. et al. (1984) Science 224: 838-843, Winter, J.
(1991) Results Probl. Cell Differ. 17: 85-105, The McGraw Hill Yearbook of Science and Technology (1992) McGr
aw Hill New York NY, pp.191-196, Logan, J. and T. Shenk (1984) Proc. Nat
l. Acad. Sci. USA 81: 3655-3659, Harrington, JJ et al. (1997) Nat. Genet. 1
5: 345-355 etc.). Expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses or expression vectors derived from various bacterial plasmids can be used to deliver polynucleotide sequences to target organs, tissues or cell populations (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5 (6)
: 350-356, Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-6344,
Buller, RM et al. (1985) Nature 317 (6040): 813-815; McGregor, DP et al. (1994
) Mol. Immunol. 31 (3): 219-226, Verma, IM and N. Somia (1997) Nature 38.
9: 239-242 etc.). 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 INTRA. For example, multifunctional E. coli vectors such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies) can be used for routine cloning, subcloning and propagation of polynucleotide sequences encoding INTRA. INT
Ligation of the RA coding sequence to multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method to identify transformed bacteria containing recombinant molecules. In addition, these vectors may also be useful for in vitro transcription of cloned sequences, dideoxy sequencing, single-stranded rescue by helper phage, and generation of nested deletions (Va
n Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509. When a large amount of INTRA is required, for example, when producing an antibody, a vector which induces the expression of INTRA at a high level can be used. For example, vectors containing the strong inducible T5 bacteriophage promoter or the inducible T7 bacteriophage promoter can be used.

Yeast expression systems can be used to generate INTRA. A large number of 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 . In addition, such vectors direct the expressed protein either to be secreted or to be retained intracellularly, allowing integration of foreign sequences into the host genome for stable growth (Ausubel (supra. 1995), Bitter, Scorer, etc., supra).

It is also possible to express INTRA using plant systems. Transcription of the INTRA-encoding sequence may be carried by viral promoters, such as alone or TMV (Takamatsu, N. (1987).
EMBO J 6: 307-311) driven by CaMV derived 35S and 19S promoters as used in combination with omega leader sequences. Alternatively, a plant promoter such as the small subunit of RUBISCO or a heat shock promoter may be used (see Coruzzi, supra, Broglie, supra, Winter, supra). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection ( The McGraw Hill Yearbook of Science and Technology (1992) McGraw). Hill New York NY, pp.191-196
Etc.).

In mammalian cells, a number of viral-based expression systems are available. When using adenovirus as an expression vector, the sequence encoding INTRA can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a triple leader sequence. It is possible to insert a viral genome into the essential E1 or E3 region and obtain an infectious virus expressing INTRA in the host cell (Logan, J. and T. She.
nk (1984) Proc. Natl. Acad. Sci. 81: 3655-3659 etc.). In addition, transcription enhancers such as the Rous Sarcoma Virus (RSV) enhancer can be used to increase expression in mammalian host cells. Proteins can also be expressed at high levels using SV40 or EBV based vectors.

Human artificial chromosomes (HACs) can also be used to transport fragments of DNA larger than those contained in and expressed from the plasmid. HACs of about 6 kb to 10 Mb were prepared for therapeutic purposes and supplied by conventional delivery methods (liposomes, polycationic amino polymers or vesicles) (Harrington. JJ et al. (1997) Nat Gen
et.15: 345-355.).

For long-term production of recombinant proteins in mammalian systems, stable expression of INTRA in cell lines is desirable. For example, an expression vector containing a viral origin of a replication expression factor, an endogenous expression factor, or both, and a selectable marker gene on the same or another vector are used to transform the sequence encoding INTRA into cells. It is possible to transform into a strain. Following the introduction of the vector, cells can be allowed to grow for about 1-2 days in enriched medium before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selective medium, and the presence of the selectable marker enables growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Transformed cell lines can be recovered using any number of selection systems. Such selection systems include, but are not limited to, the herpesvirus thymidine kinase gene used for tk ^- simple cells and the adenine phosphoribosyl transferase gene used for apr ^- cells (Wigler, M. Et al (1977) Cell 11: 223-2
32, Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. Eg dh
fr confers resistance to methotrexate, neo confers resistance to aminoglycosid neomycin and G-418, als chlorsulfuron and pa
t confers resistance to phosphinothricin acetyltransferase (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77: 3567-3570, Colbere-
Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). Other selectable genes, such as trpB and hisD that alter cellular requirements for metabolism, have been described in the literature (Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci.
USA 85: 8047-8051 etc.). Visible markers such as anitocyanin, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used to not only identify transformants but also quantify transient or stable protein expression due to a particular vector system (Rhodes, CA (1995) Methods Mol. Biol. . 55: 121-131 etc.).

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

In general, a host cell containing a nucleic acid sequence encoding INTRA and expressing INTRA is
It can be identified using various methods well known to those skilled in the art. Methods well known to those of skill in the art include, but are not limited to, DNA-DNA or DNA-RNA.
There are protein bioassays or immunoassay techniques including hybridization, PCR, membrane-based, solution-based or chip-based techniques for performing nucleic acid or protein detection, quantification, or both.

Immunological methods for detecting and measuring the expression of INTRA using specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA),
Examples include radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS). Using a monoclonal antibody reactive with two non-interfering epitopes on INTRA,
Two-site, monoclonal-based immunoassay
mmunoassay) is preferred, but competitive binding assays can also be used. These and other assays are known in the art (Hampton. R. et al. (19
90) Serological Methods, a Laboratory Manual. APS Press. St Paul. MN, Se
ct. IV, Coligan, JE et al. (1997) Current Protocols in Immunology , Greene
Pub. Associates and Wiley-Interscience, New York NY, Pound, JD (1998)
Immunochemical Protocols , Humans Press, Totowa NJ, etc.).

A wide variety of labeling and conjugation methods are known to those of skill in the art and can be used for various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to a polynucleotide encoding INTRA include oligo-labeling, nick-translation, end-labeling, or labeled nucleotides. PC to use
There is R method. Alternatively, the INTRA coding sequence or any fragment thereof can be cloned into a vector for producing an mRNA probe. Such vectors are known in the art and are commercially available, including T7, T3 or
A suitable RNA polymerase such as SP6 and labeled nucleotides can be added and used to synthesize RNA probes in vitro . Such a method is, for example, Amersha
m Pharmacia Biotech, Promega (Madison WI), US Biochemical, etc. can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, as well as radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents and the like.

Host cells transformed with a nucleotide sequence encoding INTRA can be cultured under conditions suitable for the recovery and expression of the protein from cell culture. Whether a protein produced by a transformed cell is secreted or remains intracellular depends on the sequence used, the vector, or both. As one of ordinary skill in the art would understand,
Expression vectors containing a polynucleotide encoding INTRA can be designed to contain a signal sequence that directs the direct secretion of INTRA across prokaryotic or eukaryotic cell membranes.

In addition, the choice of host cell line may be made by the ability to regulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Modifications of such polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing such as cleaving the "prepro" or "pro" form of the protein can also be utilized to identify protein targeting, folding and / or activity. A variety of host cells (eg, CH 2) with unique cellular machinery and characteristic mechanisms for post-translational activity.
O, HeLa, MDCK, MEK293, WI38, etc.) are American Type Culture Collection (ATC
C, Bethesda, VA) and can be selected to ensure correct modification and processing of foreign proteins.

In another embodiment of the invention, the natural, modified or recombinant nucleic acid sequence encoding INTRA is ligated to a heterologous sequence which results in the translation of the fusion protein in any of the above host systems. You can For example, a chimeric INTRA protein containing a heterologous moiety that can be recognized using commercially available antibodies can facilitate the screening of peptide libraries for inhibitors of INTRA activity. Heterologous protein moieties and heterologous peptide moieties may also facilitate the purification of fusion proteins using commercially available affinity substrates. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (Trx), calmodulin-binding peptide (CBP), 6-His, FLAG, cm.
There are yc and hemagglutinin (HA). GST on immobilized glutathione, MBP on maltose, Trx on phenylarsine oxide, CBP on calmodulin, and 6-His on metal chelate resins allow purification of cognate fusion proteins. F
LAG, c-myc and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. The fusion protein can also be genetically engineered to include a proteolytic cleavage site between the INTRA coding sequence and the heterologous protein sequence so that INTRA can be cleaved from the heterologous moiety after purification. Methods for expressing and purifying fusion proteins are described in Ausubel (1995), supra, Chapter 10.
A variety of commercially available kits can also be used to facilitate expression and purification of the fusion protein.

In yet another embodiment of the invention, TNT rabbit reticulocyte lysate or wheat germ extraction system (Promega) can be used to synthesize radiolabeled INTRA in vitro . These systems couple transcription and translation of protein coding sequences operably linked to the T7, T3 or SP6 promoters. Translation occurs in the presence of radiolabeled amino acid precursors such as ³⁵ S methionine.

INTRA of the present invention or fragments thereof can be used to screen for compounds that specifically bind to INTRA. At least one and more than one test compound can be used to screen for specific binding to INTRA. Examples of test compounds include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

In one example, a compound thus identified is closely related to a natural ligand of INTRA, such as a ligand or fragment thereof, a natural substrate, a structural or functional mimetic or natural binding partner. (See Chapter 5 of Coligan, JE et al. (1991) Current Protocols in Immunology 1 (2)). Similarly, the compound is INT
The RA may be associated with the natural receptor to which it binds, or it may be closely associated with at least a fragment of the receptor, such as the ligand binding site. In any case, the compound may be rationally designed using known techniques. In one example, screening for such compounds is performed by INTRA either as a secreted protein or on the cell membrane.
Is involved in the production of suitable cells that express Suitable cells include cells from mammals, yeast, Drosophila or E. coli. Cells expressing INTRA or cell membrane fragments containing INTRA are contacted with a test compound and binding, stimulation or inhibition of either INTRA or the compound is analyzed.

The assay may simply test bind a test compound to the polypeptide. Here, binding is detected by fluorophores, radioisotopes, enzyme conjugates or other detectable labels. For example, the assay can include the steps of either binding at least one test compound to INTRA or immobilizing it on a solid support in solution and detecting the binding of INTRA to the compound. Alternatively, the assay may detect or measure binding of the test compound in the presence of labeled competitor. Furthermore, the assay can be carried out with cell free formulations, chemical libraries or natural product mixtures, test compounds can be released in solution or immobilized on a solid support.

INTRA of the present invention or fragments thereof can be used to screen for compounds that modulate the activity of INTRA. Such compounds include agonists, antagonists, partial or inverse agonists and the like. In one example, the assay is performed under conditions that permit the activity of INTRA, such that INTRA is bound to at least one test compound, and the activity of INTRA in the presence of the test compound is in the absence of the test compound. Compared to the activity of INTRA in. The change in the activity of INTRA in the presence of the test compound is
The compound which adjusts the activity of TRA is shown. Alternatively, the test compound is bound under conditions suitable for activity of INTRA to an in vitro or cell free system containing FLFXHT and the assay is performed. In any of these assays, the test compound that modulates the activity of INTRA can do so indirectly, eliminating the need for direct contact with the test compound. At least one to multiple test compounds can be screened.

In another example, a polynucleotide encoding INTRA or a mammalian homolog thereof is “knocked out” in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are known in the art and are useful in generating animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). For example, mouse ES cells such as the 129 / SvJ cell line are derived from early mouse embryos and grow in culture. ES cells are transformed with a vector containing a gene of interest divided by a marker gene such as neomycin phosphotransferase gene (neo: Capecchi, MR (1989) Science 244).
: 1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination occurs using the Cre-loxP system, which knocks out the gene of interest in a tissue-specific or developmental stage-specific manner (Marth, JD (1996)
Clin. Invest. 97: 1999-2002; Wagner, KU et al. (1997) Nucleic Acids Res. 2
5: 4323-43 30). Transformed ES cells are identified and microinjected into, for example, mouse cell blastocysts taken from the C57BL / 6 mouse strain. Blastocysts are surgically introduced into pseudopregnant females and the resulting chimeric progeny are determined and propagated to produce heterozygous or homozygous lines. The transgenic animals thus produced may be tested with potential therapeutic or toxic agents.

Polynucleotides encoding INTRA can also be engineered in vitro in human blastocyst-derived ES cells. Human ES cells have the potential to differentiate into at least eight separate cell lineages, including endoderm, mesoderm and ectoderm cell types. This cell lineage differentiates into, for example, neuronal cells, hematopoietic lineages, and cardiomyocytes (Thomson, JA et al.
1998) Science 282: 1145-1147).

Polynucleotides encoding INTRA are "knocked in" to model human diseases.
Humanized animals (pigs) or transgenic animals (mouse or rat) may also be generated. Using knock-in technology, a region of the polynucleotide encoding INTRA is injected into animal ES cells and the injected sequences integrate into the animal cell genome. The transformed cells are injected into the blastula and the blastula is transplanted as described above. Study transgenic offspring or inbreds and treat with powerful pharmaceuticals to obtain information on treatment of human disease. Alternatively, mammals that have been inbred to overexpress INTRA, for example by secreting INTRA into milk, can also serve as a convenient source of protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. . 4: 55-74).

There are chemical and structural similarities between regions of therapeutic INTRA and intracellular signaling molecules, such as in the context of sequences and motifs. Furthermore, INTRA expression is closely associated with hematopoietic / inflammatory, nervous, gastrointestinal and reproductive cancers. Therefore
INTRA is believed to play a role in cell proliferative disorders, autoimmune / inflammatory disorders, neuropathy, gastrointestinal disorders, reproductive and developmental disorders. In treating diseases associated with increased expression or activity of INTRA, it is desirable to reduce expression or activity of INTRA. In addition, in the treatment of diseases associated with decreased expression or activity of INTRA, it is desirable to increase expression or activity of INTRA.

Accordingly, in certain embodiments, INTRA or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of diseases associated with decreased expression or activity of INTRA. Non-limiting examples of such disorders include abnormal cell proliferation, including actinic keratosis, arteriosclerosis, atherosclerosis,
Bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, lymphoma, leukemia and myeloma. Other cancers including hematopoietic cancer, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, adenoma, carcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, Gallium, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterine cancer, etc. Also included are autoimmune / inflammatory diseases, including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, Autoimmune hemolytic anemia, autoimmune thyroiditis, self Epidemic polyendocrine disorder (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphotoxin-induced temporary lymphopenia, erythroblasts Disease, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardium Or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, Thrombocytopenia, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infections, bacterial infections, fungal infections, parasitic infections, protozoa Staining, helminthic infections and trauma, and also gastrointestinal disorders, including dysphagia, peptic esophagitis, esophageal spasm,
Esophageal stricture, esophageal cancer, dyspepsia, dyspepsia, gastritis, gastric cancer, loss of appetite, nausea, vomiting,
Gastroparesis, sinus or pyloric edema, abdominal angina, heartburn, gastroenteritis, ileus,
Intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, liver passive congestion, hepatoma, infectious colitis, Ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colon cancer, colon obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,
Constipation, gastrointestinal bleeding, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hemochromatosis, Wilson disease, α ₁ antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, Hepatic infarction, portal circulatory obstruction and thrombosis, centrilobular necrosis, hepatic purpura, hepatic vein thrombosis, hepatic vein occlusion, preeclampsia, eclampsia, gestational acute liver fat, gestational intrahepatic cholestasis, and nodularity It includes liver cancer including regeneration, and also neuropathy, including epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal diseases. Tract disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, inherited ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meninges Inflammation, brain abscess, subdural Includes abscess, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease and kuru, Creutzfeldt-Jakob disease and Gastmann-Straussler-Scheinker syndrome Prion disease and lethal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastomatosis, brain 3
Cross nerve vascular syndrome, mental retardation and other central nervous system development disorders, cerebral palsy, neuroskeletal abnormalities, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular diseases, peripheral nerve diseases, dermatomyositis and polymyositis And hereditary, metabolic, endocrine and toxic myopathy, myasthenia gravis, periodic quadriplegia, and psychiatric disorders including mood disorders, anxiety disorders and schizophrenia, akathisia, amnesia, catatonia , Diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia, postherpetic neuralgia and Tourette's disease, and also gastrointestinal disorders, including esophagitis, esophageal cancer, Includes gastritis, gastric cancer, inflammatory bowel disease, cholecystitis, intestinal infections, pancreatitis, pancreatic cancer, cirrhosis, hepatitis, hepatoma, colitis, colon cancer and Crohn's disease.

In another example, a vector capable of expressing INTRA or a fragment or derivative thereof is administered to a patient to treat diseases associated with decreased expression or activity of INTRA, including but not limited to those mentioned above. Treatment or prevention is also possible.

In yet another embodiment, a pharmaceutical composition comprising substantially purified INTRA is administered to a patient with a suitable pharmaceutical carrier, including but not limited to the diseases described above.
It is also possible to treat or prevent diseases associated with reduced expression or activity of TRA.

In yet another example, an agonist that modulates the activity of INTRA is administered to a patient,
It is also possible to treat or prevent diseases associated with reduced expression or activity of INTRA, including, but not limited to, those mentioned above.

In yet another embodiment, patients can be administered an antagonist of INTRA to treat or prevent disorders associated with increased expression or activity of INTRA. Examples of such disorders include, but are not limited to, the cell proliferation disorders, autoimmune / inflammatory disorders, neurological disorders, gastrointestinal disorders, reproductive disorders and developmental disorders mentioned above. In one embodiment, an antibody that specifically binds to INTRA can be used directly as an antagonist or indirectly as a targeting or transport mechanism that transports the drug to cells or tissues that express INTRA.

In another example, a vector expressing a complement of a polynucleotide encoding INTRA was administered to a patient and was associated with increased expression or activity of INTRA, including, but not limited to, the disorders described above. It is also possible to treat or prevent the disease.

In another example, any protein, antagonist, antibody, agonist, complementary sequence, or vector of the invention can be administered in combination with another suitable therapeutic agent. Suitable therapeutic agents for use in combination therapy may be selected by those of ordinary skill in the art according to conventional pharmaceutical principles. When combined with a therapeutic agent, a synergistic effect can be brought about in the treatment or prevention of various diseases mentioned above. By using this method, it is possible to obtain a medicinal effect with a small amount of each drug, thereby reducing the possibility of side effects.

INTRA antagonists may be prepared using methods generally known in the art. Specifically, purified INTRA can be used to make antibodies or to screen libraries of therapeutic agents to identify those that specifically bind to INTRA. INTRA antibodies can also be produced using methods generally known in the art. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by Fab expression libraries. Neutralizing antibodies (ie, antibodies that inhibit dimer formation) are usually suitable for treatment.

To produce antibodies, goat, rabbit, rat, mouse, human, etc. can be injected by injecting INTRA, or any fragment or oligopeptide of INTRA having immunogenic properties. A variety of hosts can be immunized, including. Depending on the host species, various adjuvants may be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin,
There are surfactants such as pluronic polyol, polyanion, peptide, oily emulsion, keyhole limpet hemocyanin, and dinitrophenol. Among the adjuvants used in humans, BCG (bacillus Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

Oligopeptides, peptides or fragments used to induce antibodies to INTRA are preferably those having an amino acid sequence of at least about 5 amino acids, generally at least about 10 amino acids. Desirably, these oligopeptides, peptides or fragments are identical to a portion of the amino acid sequence of a naturally occurring protein and include the entire amino acid sequence of a small naturally occurring molecule. Short stretches of INTRA amino acids can be fused to sequences of another protein, such as keyhole limpet hemocyanin, to produce antibodies to the chimeric molecule.

Monoclonal antibodies against INTRA can be produced by continuous cell lines in culture, using any technique that produces antibody molecules. Such techniques include, but are not limited to, hybridoma techniques, human B cell hybridoma techniques and EBV-
Hybridoma technology is available (Kohler, G. et al. (1975) Nature 256: 495-497, Kozb
or, D. et al. (1985) .J. Immunol. Methods 81: 31-42, Cote, RJ et al. (1983) Pro.
c. Natl. Acad. Sci. USA 80: 2026-2030, Cole, SP et al. (1984) Mol. Cell Bio
62.109-120, etc.).

In addition, it is possible to use techniques developed to generate “chimeric antibodies”, such as splicing of a mouse antibody gene into a human antibody gene to obtain a molecule with suitable antigen specificity and biological activity. Possible (Morrison, SL et al. (1984) P
Roc. Natl. Acad. Sci. USA 81: 6851-6855, Neuberger, MS et al. (1984) Nature.
312: 604-608, Takeda, S. et al. (1985) Nature 314: 452-454 etc.). Alternatively,
The techniques described for producing single chain antibodies can be applied to produce INTRA-specific single chain antibodies using methods known in the art. Antibodies with related specificities but differing idiotypic composition can also be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton DR
(1991) Proc. Natl. Acad. Sci. USA 88: 10134-10137).

The production of antibodies may also be carried out by inducing in vivo production in lymphocyte populations or by screening immunoglobulin libraries or panels of high specific binding reagents as disclosed in the literature (Orlandi, R . Et (
1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837, Winter, G. et al. (1991) Nat.
ure 349: 293-299 etc.).

Antibody fragments with specific binding sites for INTRA can also be produced. For example, without limitation, such fragments are made by reducing the disulfide bridges of the F (ab ') ₂ fragment and the F (ab') ₂ fragment produced by pepsin digestion of the antibody molecule. There are Fab fragments. Alternatively, it is possible to rapidly and easily identify a monoclonal Fab fragment with a desired specificity by preparing a Fab expression library (see Huse, WD et al. (1989) Science 256: 1275-1281 etc.).

Various immunoassays can be used to screen to identify antibodies with the desired specificity. Numerous protocols for immunoradiometric or competitive binding assays using either polyclonal or monoclonal antibodies with established specificity are known in the art. Such immunoassays typically involve measuring complex formation between INTRA and its specific antibody. Two-site monoclonal-based immunoassays, such as those using monoclonal antibodies reactive to two non-interfering INTRA epitopes, are commonly used, although competitive binding assays may be used (Pound, supra).

A variety of methods are used in conjunction with radioimmunoassay techniques, such as Scatchard analysis, to assess the affinity of antibodies for INTRA. Affinity is represented by the binding constant Ka. K
a is defined as the molar concentration of INTRA antibody complex divided by the molar concentration of free antibody and free antigen at equilibrium. Polyclonal antibodies have heterogeneous affinities for various INTRA epitopes, and Ka determined for polyclonal antibody reagents.
Represents the average affinity or avidity of the INTRA antibody. Monoclonal antibodies are monospecific for a particular INTRA epitope, and the Ka determined for a monoclonal antibody reagent represents a true measure of affinity. High affinity antibody reagents with Ka values in the range of about 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays where the INTRA antibody complex must withstand vigorous manipulation. Ka value is about 10 ⁶ to 10 ⁷ L
Low affinity antibody reagents, such as those in the range of / mol, are preferably used for immunopurification and similar procedures where INTRA must ultimately dissociate from the antibody in its activated state (Catt.
y, D. (1988) Antibodies, Volume I: A Practical Approach , IRL Press, Wash
Washington, DC, Liddell, JEand Cryer, A. (1991) A Practical Guide to Mono clonal Antibodies , John Wiley & Sons, New York NY).

The titer and avidity of polyclonal antibody reagents can be further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example,
Polyclonal antibody reagents containing at least 1-2 mg / ml, preferably 5-10 mg / ml, of specific antibody are commonly used in processes requiring precipitation of the INTRA antibody complex. Specificity of antibodies, antibody titer, binding activity, and guidelines for antibody quality and use in various applications are publicly available. (See Catty's reference, Coligan's reference, etc.).

In another embodiment of the invention, a polynucleotide encoding INTRA, any fragment of INTRA or a complementary sequence can be used for therapeutic purposes. In one embodiment,
It may be used if the complementary sequence of the polynucleotide encoding INTRA is suitable for blocking the transcription of mRNA. Specifically, cells can be transformed with sequences complementary to the polynucleotide encoding INTRA. Thus, complementary molecules or fragments can be used to regulate INTRA activity or to regulate gene function. Such techniques are already well known in the art and allow sense or antisense oligonucleotides or large fragments to be designed from various positions extending into the coding or control region of the INTRA coding sequence. Is (Agrawa
l, S., ed. (1996) Antisense Therapeutics , Humana Press Inc., Totawa NJ, etc.).

When used in therapy, any gene delivery system suitable for introducing antisense sequences into suitable target cells can be used. Antisense sequences can be transported intracellularly in the form of expression plasmids that express sequences complementary to at least part of the cellular sequence encoding the target protein during transcription (Slater, JE et al. (199
8) J. Allergy Clin. Immunol. 102 (3): 469-475 and Scanlon, KJ et al. (1995).
9 (13): 1288-1296.). Antisense sequences can also be introduced into cells using viral vectors such as retroviruses and adeno-associated viral vectors (Miller, AD (1990) Blood 76: 271, Ausubel, Uckert, W, supra).
and W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347 etc.). Other gene transfer mechanisms include liposomal systems, artificial viral envelopes and other systems known in the art (Rossi, JJ (1995) Br. Med. Bull. 51 (1): 21.
7-225; Boado, RJ et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315, Morris, MC.
Et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736.

In another embodiment of the invention, the polynucleotides encoding INTRA can be used for somatic or germ cell gene therapy. By carrying out gene therapy, (i) gene deficiency (for example, X chromosome chain inheritance (Cavazzana-Calvo, M. et al. (2
000) Science 288: 669-672). Severe combined immunodeficiency (SCID)
-X1), severe combined immunodeficiency associated with congenital adenosine deaminase (ADA) deficiency (Blaese, RM et al. (1995) Science 270: 475-480, Bordignon, C.
(1995) Science 270: 470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell.
75: 207-216: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666, Cryst
al, RG et al. (1995) Hum. Gene Therapy 6: 667-703), thalassemia (thalassam
ia), familial hypercholesterolemia, hemophilia caused by factor VIII or factor IX deficiency (Crystal, 35 RG (1995) Science 270: 404-410, Verma, IM and IM
Somia. N. (1997) Nature 389: 239-242), (ii) expressing a conditionally lethal gene product (eg in the case of cancer due to uncontrolled cell growth), (iii) intracellular Parasites such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Natu
re 335: 395-396, Poescbla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11.
395-11399), B-type or C hepatitis virus (HBV, HCV), fungal parasites, such as Candida albicans及<br/> beauty Paracoccidioides brasiliensis, and the protection against protozoan parasites such as Plasmodium falcipar um and Trypanosoma cruzi The protein possessed can be expressed. When a gene deficiency required for expression or regulation of INTRA causes a disease, expressing INTRA from a suitable population of transduced cells may alleviate the onset of symptoms due to the gene deficiency.

In a further example of the present invention, mammalian expression vectors encoding INTRA are prepared and introduced into INTRA-deficient cells by mechanical means, whereby diseases or abnormalities due to INTRA deficiency are treated. treat. Mechanical transfer techniques used for cells in vivo or ex vitro include (i) direct microinjection of DNA into individual cells, (ii) ballistic gold particle delivery, ( ii
i) Liposome-mediated transfection, (iv) Receptor-mediated gene transfer, and (v) Use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Bioch.
em. 62: 191-217, Ivics, Z. (1997) Cell 91: 501-510; Boulay, JL. and H. Re
cipon (1998) Curr. Opin. Biotechnol. 9: 445-450).

Expression vectors that can influence expression of INTRA include, but are not limited to, P
CDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vector (Invitrogen, Carlsbad CA)
, PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA) and PTET-O
There are FF, PTET-ON, PTRE2, PTRE2-LUC and PTK-HYG (Clontech, Palo Alto CA).
INTRA is (i) a constitutively active promoter (eg cytomegalovirus (CM
V), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK) or β-actin gene), (ii) an inducible promoter (for example, tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. U
.SA 89: 5547-5551, Gossen, M. et al. (1995) Science 268: 1766-1769, Rossi, F.
.MV and HM Blau (1998) Curr. Opin. Biotechnol. 9: 451-456), commercially available In
Vitrogen's T-REX plasmid), ecdysone inducible promoter (I
nVitrogen plasmids PVGRXR and PIND), FK506 / rapamycin inducible promoter or RU486 / mifepristone inducible promoter (Rossi, FMV and HM Blau, supra), or (iii) INTRA from normal individuals. It can be expressed using the natural promoter or the tissue-specific promoter of the endogenous gene encoding the gene.

Commercially available liposome transformation kits (eg PerFec available from Invitrogen)
t Lipid Transfection Kit) allows one of skill in the art to introduce polynucleotides into target cells in culture without resorting to much experience. Alternatively,
Calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52: 456-467
) Or electroporation (Neumann, B. et al. (1982) EMBO J. 1: 841-845). Introducing DNA into primary cells requires modification of standardized mammalian transfection protocols.

In another embodiment of the invention, the disease or disorder caused by a gene deficiency associated with the expression of INTRA encodes INTRA under the control of (i) a retroviral terminal repeat (LTR) promoter or an independent promoter. Polynucleotide, (ii) a suitable RNA packaging signal, and (iii) an additional retroviral cis-acting RN
Retroviral vectors consisting of an A sequence and a Rev responsive element (RRE) with the coding sequences necessary for efficient vector propagation can be made and treated. Retroviral vectors (eg PFB and PFB NEO) are commercially available from Stratagene and published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. US
A. 92: 6733-6737). Reference to the above data is made a part of this specification. The vector is propagated in a suitable vector-producing cell line (VPCL), which expresses an envelope gene with tropism for the receptor on the target cell or a promiscuous envelope protein such as VSVg (Armentano, D. et al. 19
87) J. Virol. 61: 1647-1650, Bender, MA et al. (1987) J. Virol. 61: 1639-164
6, Adam, MA and AD Miller (1988) J. Virol. 62: 3802-3806, Dull, T. et al.
(1998) J. Virol. 72: 8463-8471, Zufferey, R. et al. (1998) J. Virol. 72: 9873.
-9880). U.S. Pat. No. 5,910,434 ("Method for obtaining regi
trovirus packaging cell lines producing high transducing efficiency retr
oviral cells ") disclose methods for obtaining retroviral packaging cell lines, which are incorporated herein by reference. Propagation of retroviral vectors, cell populations (eg CD4 ⁺ Transduction of (T cells) and returning the transduced cells to the patient is a method known to those skilled in the art of gene therapy and has been described in numerous references (Ranga, U. et al. (1997). ) J. Virol.
71: 7020-7029, Bauer, G. et al. (1997) Blood 89: 2259-2267, Bonyhadi, ML (1
997) J. Virol. 71: 4707-4716, Ranga, U. et al. (1998) Proc. Natl. Acad. Sci.
USA 95: 1201-1206, Su, L. (1997) Blood 89: 2283-2290).

Alternatively, an adenovirus-based gene therapy delivery system is used to deliver the polynucleotide encoding INTRA to cells that have one or more genetic abnormalities associated with the expression of INTRA. The construction and packaging of adenovirus-based vectors is known to those of skill in the art. Replication-defective adenovirus vectors have been shown to be variable for introducing genes encoding immunomodulatory proteins into intact pancreatic islets (Csete, ME et al. (1995) Transplantation 27:
263-268). Adenovirus vectors that may be used are described in US Pat. No. 5,707,618 ("Adenovirus vectors for gene therapy") to Armentano, which is incorporated herein by reference. For adenovirus vectors, see Antinozzi, PA et al. (1999) Annu. Rev. Nutr. 19: 511-54.
See also 4 and Verma, IM and N. Somia (1997) Nature 18: 389: 239-242. Both documents are incorporated herein by reference.

Alternatively, the herpes gene therapy delivery system is used to deliver a polynucleotide encoding INTRA to a target cell that has one or more genetic abnormalities associated with the expression of INTRA. The use of herpes simplex virus (HSV) based vectors is particularly useful in introducing INTRA into cells of the central nervous system where HSV is tropic. Construction and packaging of herpes-based vectors is known to those of skill in the art. Vectors of the replication competent herpes simplex virus (HSV) type I system have been used to deliver reporter genes to the primate eye (Liu, X. et al. (1999) Exp. Eye Res. 169: 3.
85-395). Construction of the HSV-1 viral vector is also disclosed in US Pat. No. 5,804,413 (“Herpes simplex virus swains for gene transfer”) assigned to DeLuca, and the citation of the patent is incorporated herein by reference. . U.S. Pat.No. 5,804,
No. 413 describes recombinant HSV d92 containing a genome having at least one endogenous gene introduced into cells under the control of a promoter suitable for purposes including human gene therapy. The above patent also discloses the generation and use of recombinant HSV strains eliminated for ICP4, ICP27 and ICP22. For HSV vectors, Goins, WF et al. (1999) J. Virol. 73: 519-532 and Xu, H.
See also (1994) Dev. Biol. 163: 152-161. Both documents are incorporated herein by reference. Manipulation of cloned herpesvirus sequences, passage of recombinant virus after transfection of multiple plasmids containing different parts of the giant herpesvirus genome, growth and multiplication of herpesvirus, and infection of cells with herpesvirus. Is a technique known to those skilled in the art.

Alternatively, an alphavirus (positive single stranded RNA virus) vector is used to deliver a polynucleotide encoding INTRA to target cells. Extensive biological studies have been conducted on the prototype α virus, Semliki Forest Virus (SFV), and it was found that the gene transfer vector is based on the SFV genome (Garoff, H. and K.-J. Li (1998) Cun. Opin. Biotech. 9: 464-469).
During replication of alphavirus RNA, subgenomic RNA that normally encodes the viral capsid protein is produced. This subgenomic RNA replicates to higher levels than the full-length genomic RNA, and thus has enzymatic activity (eg, protease and polymerase).
The capsid protein is overproduced as compared to the viral protein having Similarly, by introducing a coding sequence for INTRA into the α-virus genome of the capsid coding region, a large number of INTRA-encoding RNAs are produced in vector-transfected cells, and a high level of INTRA is synthesized. The ability to establish a persistent infection of hamster normal kidney cells (BHK-21) with mutants of Sindbis virus (SIN), while alphavirus infection is usually associated with cell lysis within days, is not Have suggested that the lytic replication of α virus can be suitably modified so that it can be applied to gene therapy (Dryga, SA et al. (1997) Virology 228: 74-83). The wide range of alphavirus hosts allows the introduction of INTRA into various cell types. Specific transduction of a subset of cells in a population may require selection of cells prior to transduction. Method for treating α virus infectious cDNA clone, α virus cDNA and RNA
The transfection method and the infection method of α virus are known to those skilled in the art.

It is also possible to inhibit gene expression using, for example, an oligonucleotide derived from a transcription start site that is between about −10 and about +10 counted from the start site. Similarly, inhibition is possible using the triple helix base pair formation method. Triple helix base pairing is useful because it inhibits the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent advances in treatment with triple helix DNA have been described in the literature (Gee, JE
(1994) in: Huber, BEand BI Carr, Molecular and Immunologic Approaches , Futura Publishing Co., Mt. Kisco, NY, pp. 163-177 etc.). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by blocking the binding of transcripts to ribosomes.

Ribozymes are enzymatic RNA molecules and ribozymes can also 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 prior to nucleotide strand scission. For example, a recombinant hammerhead ribozyme molecule
It specifically and effectively catalyzes nucleotide scission of sequences encoding INTRA.

Specific ribozyme cleavage sites within any potential RNA target are first identified by scanning the target molecule for ribozyme cleavage sites, including GUA, GUU, GUC sequences. Once identified, short RNA sequences of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features that render the oligonucleotide dysfunctional. become. The suitability of candidate targets can also be assessed by testing the operability of hybridization with complementary oligonucleotides using ribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the invention can be made using any method well known in the art for nucleic acid molecule synthesis. An optional method is to chemically synthesize an oligonucleotide such as a solid phase phosphoramidite compound. Alternatively, RNA molecules may be produced by in vitro and in vivo transcription of the DNA sequence encoding HRIP. Such DNA sequences can be incorporated into a wide variety of vectors using suitable RNA polymerase promoters such as T7 and SP6. Or complementary
These cDNA products, which synthesize RNA either constitutively or inducibly, can be introduced into cell lines, cells or tissues.

RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5'-end, 3'-end, or both of the molecule, or phosphorothionate or 2 rather than phosphodiesterase linkages within the backbone of the molecule. 'Includes the use of O-methyl. This concept is unique to the production of PNA and can be extended to all these molecules. It includes acetyl-, methyl-, thio-, and similar modifications of adenine, cytidine, guanine, thymine, and uridine, which are not readily recognized by endogenous endonucleases, as well as non-conventional bases such as inosine. , Queosine, wybutosine
) Etc.

Additional examples of the invention include methods of screening for compounds effective for mutated expression of a polynucleotide encoding INTRA. Compounds effective for, but not limited to, variant expression of specific polynucleotides include oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcription control factors, and specific polynucleotide sequences that interact with them. There are non-polymeric chemical entities that can act. Effective compounds may mutate polynucleotide expression by acting as either inhibitors or enhancers of polynucleotide expression. Therefore, in the treatment of diseases associated with increased expression or activity of INTRA, compounds that specifically inhibit the expression of polynucleotides encoding INTRA are of therapeutic benefit and are associated with decreased expression or activity of INTRA. In the treatment of disease, compounds that specifically enhance the expression of polynucleotides encoding INTRA may be of therapeutic benefit.

At least one to a plurality of test compounds may be screened for effectiveness in mutating a specific polynucleotide. The test compound is obtained by any method commonly known in the art. Such methods include mutating the expression of the polynucleotide, selecting from existing, commercially available or proprietary, natural or unnatural compound libraries, and chemical and / or structural analysis of the target polynucleotide. There are chemical modifications of compounds that are known to be effective in the rational design of compounds based on properties and in the selection of combinatorially or randomly generated libraries of compounds. A sample containing a polynucleotide encoding INTRA is exposed to and is thus exposed to at least one test compound. The sample can have, for example, intact cells, permeabilized cells, in vitro cell release or reconstituted biochemical systems. Changes in expression of a polynucleotide encoding INTRA are assayed by any method commonly known in the art. Usually, the expression of a specific nucleotide is detected by hybridization using a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding INTRA. The amount of hybridization can be quantified and thereby form the basis for comparison of the expression of polynucleotides exposed and unexposed to one or more test compounds. Detection of changes in expression of the polynucleotide exposed to the test compound indicates that the test compound is effective in mutating expression of the polynucleotide. For compounds effective for the mutated expression of specific polynucleotides, for example, Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res. 28: E15). Or human cell lines such as HeLa cells (Clarke, ML et al. (2000) Biochem. Biophys.
. Commun. 268: 8-13). Certain embodiments of the invention involve screening combinatorial libraries of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for antisense activity against specific polynucleotide sequences ( Bruice, TW et al. (1997) U.S. Pat.No. 5,686,242, Bruice, TW et al.
(2000) U.S. Pat. No. 6,022,691).

A number of methods are available for introducing vectors into cells or tissues, in vivo
Equally suitable for in vitro and ex vivo use. For ex vivo therapy, the vector can be introduced into hepatocytes taken from a patient, cloned and propagated and autologously transplanted back into the same patient. Transfection, ribosome injection or transport by polycationic aminopolymers can be performed using methods well known in the art (Goldman, CK et al. (1997) Nat. Biotechnol. 15:
462-466. Etc.).

Any of the above treatment methods can be applied to all subjects in need of treatment including mammals such as humans, dogs, cats, cows, horses, rabbits, monkeys and the like.

An additional embodiment of the present invention relates to the administration of pharmaceutical ingredients having the active ingredient usually formulated with pharmaceutically acceptable excipients. Excipients include, for example, cellulose, gums and proteins. Various formulations are commonly known and details are given in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such pharmaceutical ingredients may consist of INTRA, antibodies to INTRA, mimetics, agonists, antagonists, or INTRA inhibitors.

The pharmaceutical ingredients used in the present invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, arachnoid. Intracavitary, intraventricular, pulmonary, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal.

Pulmonary administration pharmaceutical ingredients may be prepared in liquid or dry powder form. Such pharmaceutical ingredients are typically aerosolized shortly before inhalation by the patient. In the case of small molecules (eg traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is known in the art. In the case of macromolecules (eg larger peptides and proteins)
Recent improvements in pulmonary transport through the alveolar region of the lung in the art have enabled drugs such as insulin to be substantially delivered to the blood circulation (Patton, J.
See S. et al., US Pat. No. 5,997,848). Pulmonary transport is superior in that it is administered without needle injection, obviating the need for potentially toxic penetration enhancers.

Pharmaceutical ingredients suitable for use in the present invention include those ingredients that contain the active ingredient in an amount sufficient to achieve the intended purpose. Determination of the effective dose is within the ability of those skilled in the art.

A special form of pharmaceutical ingredient is prepared for direct intracellular delivery of macromolecules, including INTRA or fragments thereof. For example, liposomal formulations containing cell-impermeable macromolecules can facilitate cell fusion and intracellular transport of macromolecules. Alternatively, INTRA or a fragment thereof can be linked from the HIV Tat-1 protein to the cationic N-terminus. The fusion protein thus produced is known to transduce cells of all tissues including the mouse model system brain (Schwarze, SR et al. (1999) Science 2).
85: 1569-1572).

For any compound, in a cell culture assay, eg, a neoplastic cell cell culture assay, or in an animal model, eg, mouse, rabbit, dog or pig, etc., an effective therapeutic dose is first estimated. be able to. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The therapeutically effective dose refers to the amount of active ingredient that ameliorates symptoms and conditions. Examples of such active ingredients are INTRA or fragments thereof, INTRA antibodies, IN
There are agonists, antagonists or inhibitors of TRA. Medicinal efficacy and toxicity are measured by standard drug procedures in cell culture or animal studies, eg by measuring ED ₅₀ (50% pharmaceutically effective dose of the population) or LD ₅₀ (50% lethal dose of the population). Can be decided. The dose ratio relative to the therapeutic effect of toxic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED _50. Pharmaceutical ingredients that exhibit a high therapeutic index are desirable. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in humans. 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 sensitivity of the patient and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Factors relating to the subject include the severity of the disease, the normal health condition of the patient, the age, weight and sex of the patient, time and frequency of administration, drug combination, reaction sensitivity, response to treatment and the like. Long-acting pharmaceutical ingredients may be administered at intervals of once every 3 to 4 days, once every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

A usual dose is about 0.1 to 100,000 μg, depending on the route of administration.
The total is up to about 1g. Guidance regarding specific dosages and delivery methods is available in the literature and is usually available to the onsite physician. Those skilled in the art
A recipe for nucleotides that is different than the recipe for proteins or inhibitors will be utilized. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Diagnosis In another embodiment, for the diagnosis of diseases characterized by the expression of INTRA,
Alternatively, an antibody that specifically binds INTRA may be used in an assay to monitor a patient being treated with INTRA or an INTRA agonist, antagonist or inhibitor. Antibodies useful for diagnostic purposes are formulated in the same manner as described in the treatment section above. INTRA diagnostic assays include methods that utilize antibodies and labels to detect INTRA in human body fluids or in cell or tissue extracts. The antibody may be used with or without modification and may be labeled by covalent or non-covalent attachment of the reporter molecule. A wide variety of reporter molecules are known in the art and can be used. Some reporter molecules have been described above.

Various protocols for measuring INTRA are known in the art, such as ELISA, RIA, FACS, etc., and provide a basis for diagnosing corrected or abnormal levels of INTRA expression. By binding a body fluid or cells collected from a normal mammalian subject such as a human subject with an antibody against INTRA under conditions suitable for complex formation, a normal or standard value for INTRA expression is determined. The amount of standard complex formed can be quantified by various methods such as photometry. The amount of INTRA expressed in the subject, control, and lesion samples from the specimen are compared to standard values. The deviation between the standard value and the subject is the parameter for diagnosing the disease.

According to another embodiment, polynucleotides encoding INTRA can also be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. The polynucleotides can be used to detect and quantify gene expression in a sample such that expression of INTRA in the sample can correlate with disease. Diagnostic assays can be used to measure the absence, presence and overexpression of INTRA, and to monitor the preparation of INTRA levels during therapeutic intervention.

In one embodiment, the use of hybridization with a PCR probe capable of detecting a polynucleotide sequence encoding INTRA or a closely related molecule, including a genomic sequence, to identify a nucleic acid sequence encoding INTRA. You can Whether the probe has a highly specific region, such as the 5'regulatory region, or a low specific region, such as a conserved motif, identifies only the native sequence encoding the INTRA, mutant or related sequences It depends on the specificity of the probe and the stringency of hybridization or amplification.

The probe can also be used for the detection of related sequences, which are INTR
It may have at least 50% homology with any sequence encoding A. The hybridization probe of the present invention can be DNA or RNA and is SEQ ID NO: 53.
To 104 or a genomic sequence containing the INTRA gene promoter, enhancer and intron.

A method of making a specific hybridization probe for DNA encoding INTRA includes the steps of adding a polynucleotide sequence encoding INTRA or an INTRA derivative,
Included is a method of cloning into a vector to make an mRNA probe. mR
Vectors for making NA probes are known to those of skill in the art and are commercially available,
It can be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. Hybridization probes can be labeled with different populations of reporters. Examples of reporter populations include radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase attached to the probe via an avidin / biotin binding system.

The polynucleotide sequence encoding INTRA can be used for the diagnosis of diseases associated with the expression of INTRA. Non-limiting examples of such diseases include abnormal cell proliferation, including actinic keratoses, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, Mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia and hematopoietic cancer, including lymphoma, leukemia and myeloma, adenocarcinoma, leukemia, Other cancers including lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, adenoma, carcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, digestive tract, heart, Kidney, liver, lung, muscle, ovary, pancreas,
Parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterine cancer, etc. are also included, and also autoimmune / inflammatory diseases are included, among them, acquired immune deficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multiple endocrine disorders ( APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema,
Lymphocytic transient lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, hypersensitivity Colorectal syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
Rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis and , Extracorporeal circulation, viral infections, bacterial infections, fungal infections, parasitic infections, protozoal infections, helminthic infections and trauma, and also gastrointestinal disorders, including swallowing Disorders, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal cancer, dyspepsia, dyspepsia, gastritis, gastric cancer, loss of appetite, nausea, vomiting, gastroparesis, sinus or pyloric edema, abdominal angina, heartburn, gastroenteritis , Ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious Colitis, ulcerative Enteritis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colon cancer, colon obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal bleeding, acquired immunodeficiency syndrome (AIDS) enteropathy , Jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hemochromatosis, Wilson's disease, α ₁ antitrypsin deficiency, Rhe syndrome, primary sclerosing cholangitis, hepatic infarction, portal venous obstruction and thrombosis, centrilobular necrosis, liver Includes purpura, hepatic vein thrombosis, hepatic vein occlusion, preeclampsia, eclampsia, gestational acute liver fat, gestational intrahepatic cholestasis, and liver cancer including nodular regeneration, and also neuropathy, Among them are epilepsy, ischemic cerebrovascular disease, 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 radiculitis, viral central nervous system diseases, and kuru, Creutzfeldt-
Prion diseases including Jacob's disease and Gastmann-Straussler-Shineker syndrome, and fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastomatosis ( cerebelloretinal hemangioblastomatosis), cerebral trigeminal vascular syndrome, mental retardation and other central nervous system development disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nerve diseases, Dermatomyositis and polymyositis, hereditary, metabolic, endocrine and toxic myopathy, myasthenia gravis, periodic quadriplegia and psychiatric disorders including mood disorders, anxiety disorders and schizophrenia , Amnesia, catatonic disease, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia, postherpetic neuralgia and Tourette's disease It also includes gastrointestinal disorders, including esophagitis, esophageal cancer, gastritis, gastric cancer, inflammatory bowel disease, cholecystitis, intestinal infections, pancreatitis, pancreatic cancer, cirrhosis, hepatitis, hepatoma, colitis. , Colon cancer and Crohn's disease.
The polynucleotide sequence encoding INTRA can be cloned using Southern, Northern, dot blot and other membrane-based techniques, PCR and dipstick.
k) method, pin and multi-format ELISA-like assays and microarrays that utilize fluid or tissue collected from a patient to detect expression of mutant INTRA. Such qualitative or quantitative methods are known in the art.

In some forms, nucleotide sequences encoding INTRA may be useful in assays that detect related disorders, particularly the disorders described above. The nucleotide sequence encoding INTRA is labeled by standard methods and can be added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. Wash the sample after a suitable incubation period,
The signal is quantified and compared to a standard value. If the amount of signal in the patient sample is significantly altered compared to the control sample, the level of mutation of the nucleotide sequence encoding INTRA in the sample indicates the presence of the associated disease. Such assays can also be used to predict the effects of particular treatments in animal studies, clinical trials, or to monitor the treatment of individual patients.

In order to provide diagnostic criteria for diseases associated with INTRA expression, a normal or standard profile for expression is established. This is a method in which body fluids or cells extracted from a normal animal or human subject are treated under conditions suitable for hybridization or amplification.
It can be achieved by ligating with a sequence encoding TRA or a fragment thereof. Standard hybridizations can be quantitated by comparing the values obtained from experiments performed with known amounts of substantially purified polynucleotide to those obtained from normal subjects. The standard value thus obtained can be compared with the value obtained from a sample obtained from a patient who shows signs of disease. Deviation from standard values is used to establish the presence of disease.

Once the presence of the disease has been established and the treatment protocol has begun, hybridization assays are routinely used to determine whether patient expression levels have begun to approach those observed in normal subjects. Get repeat. The results obtained from the serial assays can be used to show the effect of treatment over a period of days to months.

For cancer, the presence of abnormal amounts of transcripts (under- or over-expression) in living tissue from an individual indicates the predisposition to the disease or detects the disease before actual clinical symptoms appear. May provide a method of doing so. With a clearer diagnosis of this kind,
It allows medical professionals to use preventative or aggressive treatment early to prevent the development or further progression of cancer.

The additional diagnostic utility of oligonucleotides designed from sequences encoding INTRA may involve the use of PCR. These oligomers can be chemically synthesized, enzymatically produced, or produced in vitro . The oligomer preferably contains a fragment of a polynucleotide encoding INTRA or a fragment of a polynucleotide complementary to a polynucleotide encoding INTRA, and is used to identify 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.

In certain embodiments, oligonucleotide primers derived from a polynucleotide sequence encoding INTRA can be used to detect single nucleotide polymorphisms (SNPs). S
NP is a substitution that often causes congenital or acquired genetic disease in humans,
Insertions and deletions. Without limitation, SNP detection methods include restriction enzyme digestion (SSCP) and fluorescent SSCP (fSSCP). In SSCP, an oligonucleotide primer derived from a polynucleotide sequence encoding INTRA is used to amplify DNA using the polymerase chain reaction (PCR). DNA can be derived, for example, from diseased or normal tissue, biopsy samples, body fluids and the like. SNPs in DNA are single-stranded
Differences are made in the secondary and tertiary structure of the PCR product. Differences can be detected using gel electrophoresis in non-denaturing gels. In fSCCP, oligonucleotide primers are fluorescently labeled. This enables the detection of amplimers with high throughput equipment such as DNA sequencing machines. In addition, a sequence database analysis method called in silico SNP, isSNP, identifies polymorphisms by comparing the sequences of individual overlapping DNA fragments that are arranged in a common consensus sequence. obtain. These computer-based methods are DN
Filter out sequence variations due to sequencing errors using A's laboratory adjustments and statistical models and automated analysis of DNA sequence chromatograms. In another aspect, for example, a high throughput MASSARRAY system (Sequenom, Inc
, San Diego CA) to detect and characterize SNPs by mass spectrometry.

Methods that can be used to quantify the expression of INTRA also include radiolabelling or biotin labeling of nucleotides, coamplification of regulatory nucleic acids and interpolation of results obtained from standard curves (Melby, PC et al. 1993) J. Immunol. Methods, 159: 235-
244, Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236). High-throughput format assays in which the oligomer of interest is present in various dilutions and quantified rapidly by spectrophotometric or non-color response can accelerate the quantitation rate of multiple samples .

In yet another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein can be used as targets in microarrays. Microarrays can be used in transcription imaging techniques that simultaneously monitor the associated expression levels of multiple genes. In this regard, Seilhamer, JJ et al., "Comparative Gene Transcrip," of US Pat. No. 5,840,484.
T Analysis "and incorporated herein by reference. Microarrays can also be used to identify genetic variants, mutations and polymorphisms. Use this information To determine gene function, understand genetic basis of disease, diagnose disease, monitor disease progression / regression as a function of gene expression, develop and monitor drug activity in disease treatment In particular, this information can be used to develop a pharmacogenomic profile of a patient to select the most appropriate and effective treatment for the patient, eg, based on the patient's pharmacogenomic profile. On the other hand, it is possible to select a therapeutic agent that is highly effective and shows almost no side effects.

In another example, antibodies specific to INTRA, INTRA or fragments thereof can be used as elements on the microarray. Microarrays can be used to monitor or measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.

Microarrays were prepared and used using methods well known in the art,
And analyze (Brennan, TM et al. (1995) U.S. Pat. No. 5,474,796, Schena,
M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619, Baldeschweiler.
(1995) PCT application No. WO95 / 251116, Shalon, D. et al. (1995) PCT application No. WO95 /
35505, Heller, RA et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155.
, Heller, MJ et al. (1997) US Pat. No. 5,605,662). Various types of microarrays are known and are described in detail in DNA Microarrays: A Practical Approach , M. Schena, ed. (1999) Oxford University Press, London. The document is incorporated herein by reference in its entirety.

In another embodiment of the invention, a nucleic acid sequence encoding INTRA can be used to produce a hybridization probe that is effective in mapping the native genomic sequence. Either coding or non-coding sequences can be used, and in some instances non-coding sequences are preferred over coding sequences. For example, conservation of coding sequences within members of a multigene family can result in unwanted cross-hybridization during chromosome mapping. The nucleic acid sequence may be a specific chromosome, a specific region of the chromosome or an artificially formed chromosome, for example, human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), bacterial P1.
Product, or mapped to a single-chromosomal cDNA library (Harrington
, JJ et al. (1997) Nat Genet. 15: 345-355, Price, CM (1993) Blood Rev. 7:
127-134, Trask, BJ (1991) Trends Genet. 7: 149-154 etc.). Once mapped, the nucleic acid sequences of the invention can be used, for example, to generate genetic linkage maps that correlate the inheritance of a disease condition with a particular chromosomal region of the inheritance or restriction fragment length polymorphism (RFLP).

Fluorescence in situ hybridization (FISH) can be correlated with other physical and genetic map data (see Heinz-Ulrich, et al. (1995) in Meyers, pp. 965-968., Supra). ). Examples of genetic map data are available in various scientific journals or the Online Mendelian Inher
It can be found on the itance in Man (OMIM) website. The correlation between the position of the gene encoding INTRA on the physical chromosomal map and the predisposition to a particular disease may serve to define the region of DNA associated with that disease, and thus It can be an attempt to position clone.

The genetic map can be extended using physical mapping techniques such as binding analysis using established chromosomal markers and chromosomal in situ hybridization methods.
Placing a gene on the chromosome of another mammal, such as a mouse, may reveal related markers even when the number or arm of a particular human chromosome is unknown. This information is valuable to researchers investigating genetic disorders using positional cloning and other gene discovery techniques. Once the disease or syndrome is roughly located by genetic linkage to a particular genetic region, such as the 11q22-23 region of ataxia telangiectasia, any mapping to that region will result in the relevant gene for further investigation. Alternatively, it can represent a regulatory gene (Gatti, RA et al. (1988)
Nature 336: 577-580 etc.). Healthy people, carriers, due to translocation, inversion, etc.
The nucleotide sequences of the present invention may be used to detect differences in chromosomal location among three infected individuals.

In another embodiment of the invention, it is possible to use INTRA, INTRA catalytic fragments, immunogenic fragments, or oligopeptides thereof to screen libraries of compounds using various drug screening techniques. it can. Fragments used for drug screening may be free in solution, immobilized on a solid support, retained on the cell surface, or located intracellularly. The formation of a binding complex between INTRA and the drug under test is measurable.

Another drug screening method is used for high throughput screening of compounds with suitable binding affinity for the protein of interest (Geysen
, Et al. (1984) PCT application number WO84 / 03564, etc.). In this method, a large number of different small test compounds are synthesized on a solid substrate. The test compound is INTRA
Alternatively, it is reacted with the fragment and washed. The bound INTRA is then detected by methods well known in the art. Purified INTRA 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, a competitive drug screening assay can be used. In this assay, neutralizing antibodies capable of binding INTRA specifically compete with the test compound for binding INTRA. In this method, the antibody detects the presence of peptides that share one or several antigenic determinants with INTRA.

In another embodiment, if the new technology relies on currently known characteristics of nucleotide sequences, including but not limited to triplet genetic code and specific base pair interactions, The nucleotide sequence encoding INTRA can be used in any molecular biology technique that is still to be developed.

Without further elaboration, one of ordinary skill in the art will be able to utilize the invention to the full extent from the foregoing description. Therefore, the examples described below are for illustrative purposes only and are not intended to limit the invention in any way.

All patents, patent applications and publications disclosed herein, especially US Pat.
No. 60 / 139,566 (filed on June 16, 1999), No. 60 / 149,640 (filed on August 17, 1999)
And No. 60 / 164,417 (filed on November 9, 1999) 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. Some tissues were homogenized and dissolved in guanidinium isothiocyanate solution,
There are also tissues that have been homogenized and dissolved in a mixture of phenol or a suitable denaturant. The mixture of modifiers is, for example, TRIZOL (Life Technologies), which is a single-phase solution of phenol and guanidinium isothiocyanate. The resulting lysate was either centrifuged in cesium chloride or extracted with chloroform.
RNA was precipitated from the lysates using either isopropanol, sodium acetate and ethanol, or another method.

To increase the RNA purity, RNA extraction with phenol and precipitation was repeated as many times as necessary. In some cases RNA was treated with DNase. Most libraries have oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, V
alencia CA) or OLIGOTEX mRNA purification kit (QIAGEN).
A was isolated. Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

[0227] In some cases, RNA was provided to Stratagene and the corresponding cDNA library was produced by the same. If not, use UNIZAP vector system (Stratagene) or SUPERSCRIPT using recommended methods or similar methods known in the art.
CDNA was synthesized using a plasmid system (Life Technologies) to prepare a cDNA library. (See Ausubel, 1997, unit 5.1-6.6, etc., supra.) Reverse transcription was initiated with oligo d (T) or random primers. The synthetic oligonucleotide adapter was ligated to the double-stranded cDNA, and the cDNA was digested with a suitable restriction enzyme. For most libraries, the cDNA size (300-1000 bp) selection is SE
PHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis was used. The cDNA was ligated to the compatible restriction enzyme sites of the polylinker of the appropriate plasmid. Suitable plasmids include, for example, the PBLUESCRIPT plasmid (Strat
agene), pSPORT1 plasmid (Life Technologies) or plNCY (Incyte Pharm)
aceuticals, Palo Alto CA) etc. The recombinant plasmid is XL from Stratagene.
The cells were transformed into competent E. coli cells containing 1-Blue, XL1-BIueMRF or SOLR, or Life Technologies DH5α, DH10B or ELECTROMAX DH10B.

2 Isolation of cDNA clones Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Magic or WIZARD
Minipreps DNA Purification System (Promega), AGTC Miniprep Purification Kit (Edge Bios
ystems, Gaithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus P
Plasmids were purified using at least one of the lasmid and QIAWELL 8 Ultra Plasmid purification systems, the REAL Prep 96 plasmid kit. After precipitation, resuspend in 0.1 ml distilled water, lyophilized or without lyophilization at 4 ° C.
Stored in.

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

3 Sequencing and Analysis cDNA sequencing reactions may be performed using standard methods or high throughput equipment such as
ABI CATALYST 800 Thermal Cycler (PE Biosystems) or PTC-200 Thermal Cycler (MJ Research) with HYDRA Microdispenser (Robbins Scient)
ific) or MICROLAB 2200 (Hamilton) liquid transfer system. The cDNA sequencing reaction is carried out by using a reagent provided by Amersham Pharmacia Biotech or an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle s.
Preparation was performed using the reagents provided in the equencing ready reaction kit (PE Biosystems). For electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides, use the MEGABACE 1000 DNA Sequencing System (Molecular Dynam
ics) or ABI PRIS using standard ABI protocol and base pair calling software
The M373 or 377 Sequencing Systems (PE Biosystems) or other sequence analysis systems well known in the art were used. Reading frames within the cDNA sequences were determined using standard methods (outlined in Ausubel, 1997, unit 7.7, supra). Some of the cDNA sequences were selected and extended by the method described in Example 6.

Polynucleotide sequences derived from the cDNA sequences were constructed and analyzed using a combination of software utilizing algorithms well known to those skilled in the art. Table 5 shows the outline of tools, programs and algorithms used, applicable explanations, references, and threshold parameters. The tools, programs and algorithms used are listed in column 1 of Table 5.
A brief description of them is given in column 2. Column 3 is the preferred citation, and all references are incorporated by reference in their entirety. If applicable, column 4 shows the score, probability value and other parameters used to assess the strength of the match between the two sequences (the higher the score, the higher the homology between the two sequences). ). Sequence analysis was performed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).

The polynucleotide sequences were validated by removing the vector, linker and polyA sequences and by masking ambiguous base pairs. In doing so, algorithms and programs based on BLAST, dynamic programming, and flanking dinucleotide frequency analysis were used. Then, using BLAST, FASTA and BLIMPS based programs, to obtain annotations in the programs, public databases such as GenBank primates and rodents, mammals, vertebrates, eukaryotic databases, and The array for the choice of BLOCKS, PRINTS, DOMO, PRODOM and PFAM was queried. The sequences were assembled into full-length polynucleotide sequences using programs based on Phred, Phrap and Consed and screened against open reading frames using programs based on GenMark, BLAST and FASTA. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, and then the GenBank database (above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite databases, PFAM and other Hidden Markov Model (HMM ) Full-length sequences were analyzed by querying the base protein family database. HMM is a probabilistic approach for analyzing the consensus primary structure of gene families (see Eddy, 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 NOs: 53-104. About 20 to about 4 useful for hybridization and amplification
Fragments of 000 nucleotides were described in the "Invention" section above.

4 Analysis of Polynucleotide Expression Northern analysis is an experimental technique used to detect the presence of transcribed genetic information, which involves the labeling of labeled nucleotide sequences from RNs from a particular cell type or tissue.
It is involved in hybridization to the membrane to which A is bound. (Sambroo mentioned above
k, Chapter 7, and Ausubel. FM et al., Chapters 4 and 16, etc. ) Using similar computer technology applied to BLAST, GenBank and LifeSeq (Incyt
searching for identical or related molecules in nucleotide databases such as e Pharmaceuticals). Northern analysis is by far faster than many membrane-based hybridizations. Further, the sensitivity of computer searches can be varied to determine whether to classify a particular identity as exact or homologous. The search criterion is the product score, which is defined by the following formula.

[0235]

[Equation 1]

The product score considers both the similarity between the two sequences and the length of sequence matching. The product score is a normalized value of 0 to 100 and is obtained as follows. Multiply the BLAST score by the sequence identity of the nucleotides and multiply the product by 2
Divide by 5 times the length of the shorter of the two sequences. High Scoring Segment Pair (HSP)
The BLAST score is calculated by assigning a score of +5 to each base that matches and a −4 to each mismatch. The two sequences may share more than one HSP (which may be separated by a gap). If there is more than one HSP, calculate the product score using the base pair with the highest BLAST score. The product score represents the balance between fragmentary convolution and quality of BLAST alignment. For example, a product score of 100 is only obtained if there is 100% match over the shorter length of the two sequences compared. The product score 70 has a 100% match at one end and 70%
It is obtained in either case of overlapping, or the other ends are 88% coincident and 100% overlapped. The product score 50 is obtained when either one end is 100% coincident and has 50% overlap, or the other end is 79% coincident and has 100% overlap.

The results of the Northern analysis are reported as the distribution percentage of the library in which transcripts encoding INTRA were produced. Analysis of cDNA by organ / tissue and disease
Involved in the categorization of libraries. Organ / tissue categories include cardiovascular, skin, development, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive and urological. Disease / condition categories include cancer, inflammation, trauma, cell proliferation, nerves, pooled. The number of libraries expressing the target sequence was counted for each category and divided by the number of libraries in all categories. Percentages of tissue-specific and disease / condition-specific expression are shown in Table 3.

5 The cDNA sequence used to sequence the chromosome mapping SEQ ID NOS: 8-14 of ABBR encoding the polynucleotide was prepared using the BLAST and other Smith-Waterman algorithm implementations to
The sequences were compared to sequences obtained from the te LIFE SEQ database and public domain databases. The sequences obtained from the databases corresponding to SEQ ID NOs: 8 to 14 are Phra
Arrays of adjacent and overlapping sequences were clustered using an assembly algorithm such as p (Table 5). Stanford Human Genome Center (SHGC
), Whitehead Genome Institute (WIGR), radiation hybrids and genetic map data available from public sources such as Genethon to determine if the clustered sequences were pre-mapped. As a result of including the mapped sequence in the cluster, the entire sequence of the cluster including the individual sequence number was assigned to the map position.

The positions on the genetic map of SEQ ID NOs: 8 to 14 (fill in the specific SEQ ID NO if all sequences are not mapped) are listed in the section of the invention as a range or spacing of human chromosomes. (If there are two or more positions on the map in any array,
Including the following sentences). Two or more map positions are reported for SEQ ID NOs: 8 to 14 (fill in a specific SEQ ID NO), and are similar to SEQ ID NOS: 8 to 14 (fill in a specific SEQ ID NO) but completely identical Indicates that a pre-mapped sequence that is not assembled into each cluster. The map position of the centimorgan interval is measured relative to the end of the p-arm of the chromosome. (Centimorgan (cM) is a unit of measurement based on the recombination frequency between chromosomal markers. On average, 1 cM is
It is approximately equal to 1 megabase (Mb) of DNA in humans. However, this value varies widely due to recombination hotspots and coldspots. ) CM distance is
Based on genetic markers mapped by Genethon to provide boundaries for radiation hybrid markers such that sequences are contained within each cluster. Human genome map and other publicly available resources such as NCBI's "GeneMap '99"
The website ( http://www.ncbi.n1m.nih.gov/genemap/ ) can be used to determine if the previously identified lesion genetic map is within or near the above intervals.

6 INTRA Extensions Encoding Polynucleotides The full-length nucleic acid sequences of SEQ ID NOs: 53-104 were generated by extending the fragments using oligonucleotide primers designed from the appropriate fragment of the full-length molecule.
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 is designed to have a length of about 22 to 30 nucleotides, a GC content of 50% or more, and a length of about 68 to 7
The OLIGO 4.06 software (Na
designed from cDNA using National Biosciences) or another suitable program. Hairpin structures and primer extension of nucleotides that result in primer-primer dimers were all avoided.

A selected human cDNA library was used to extend the sequence. Additional primers or nested sets of primers were designed when more than one extension was required or desired.

High fidelity amplification was obtained by the PCR method utilizing methods well known to those skilled in the art. PCR was performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture contains DNA template and 200 nm of each primer.
l, a reaction buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, and Taq
DNA polymerase (Amersham Pharmacia Biotech) and ELONGASE enzyme (Life Tech
nologies) and Pfu DNA polymerase (Stratagene). The parameters used for the primer pair PCI A and PCI B are as follows. 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: Steps 2, 3, and 4 are repeated 20 times Step 6: 68 ° C. 5 minutes Step 7: Stored at 4 ° C. For the primer pair T7, SK +, the following parameters were used instead of the above 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: Steps 2, 3, 4 repeated 20 times Step 6: 68 ° C. For 5 minutes Step 7: Store at 4 ° C PICOGREEN quantitative reagent dissolved in 1X TE (0.25% (v / v) PICOGREEN, Molecular
100 μl of Probes, Eugene OR) and 0.5 μl of undiluted PCR product are distributed into each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagent. The DNA concentration in each well was measured by. Fluoroskan II (Lab
systems Oy, Helsinki, Finland). Aliquot 5 of reaction mixture
-10 μl were analyzed by electrophoresis on a 1% agarose minigel to determine which reaction was successful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to a 384-well plate and subjected to CviJ
Cholera virus endonuclease (Molecular Biology Research, Madison W
I) was used to digest and sonicate or shear prior to religation reaction into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments excised and the agar digested with Agar ACE (Promega). The extended clones were religated into pUC 18 vector (Amersham Pharmacia Biotech) using T4 ligase (New England Biolabs, Beverly MA) and Pfu DNA polymerase (Stratag).
ene) to fill the restriction site overhang and transfect competent E. coli cells. Transfected cells were selected on antibiotic containing medium and individual colonies were selected and grown overnight in 384 well plates of LB / 2x carb liquid medium at 37 ° C.

Cells are lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using DNA polymerase (Stratagene). The parameters used at that time are as follows. Step 1: 94 ° C. for 3 minutes Step 2: 94 ° C. for 15 seconds Step 3: 60 ° C. for 1 minute Step 4: 72 ° C. for 2 minutes Step 5: Steps 2, 3 and 4 are repeated 29 times Step 6: 72 ° C. 5 minutes at 7: DNA stored at 4 ° C was quantified by the PICOGREEN reagent (Molecular Probes) described above. Samples with low DNA recovery were reamplified using the same conditions as above. 20 samples
Dilute with% dimethylsulfoxide (1: 2, v / v) and use DYENAMIC energy transfer
sequencing primer and DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE
Biosystems) was used for sequencing.

Similarly, using the above procedure and oligonucleotides designed for extension and an appropriate genomic library, SEQ ID NOs: 53-104 were used to obtain 5'regulatory sequences.
The nucleotide sequence of is used.

7 Labeling and use of individual hybridization probes The hybridization probes obtained from SEQ ID NOs: 53 to 104 are used to screen for cDNA, genomic DNA or mRNA. Although the labeling of oligonucleotides consisting of about 20 base pairs is specifically described, virtually the same procedure is used for larger nucleotide fragments. Oligonucleotides are OLIGO 4.06
Design using the latest software such as software (National Biosciences),
50 pmol of each oligomer and [γ- ³² P] adenosine triphosphate (Amersham Pharmacia
Biotech) 250 μCi and T4 polynucleotide kinase (DuPont NEN, Boston MA
). The labeled oligonucleotide is SEPHADEX G
-25 Ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biot
Substantially purified using ech). 10 ⁷ min -1 in a typical membrane-based hybridization analysis of human genomic DNA digested with any one of Ase I, Bgl II, Eco RI, Pst I, Xba I or Pvu II (DuPont NEN) endonuclease. An aliquot containing a count of labeled probes is used.

The DNA obtained from each digest was fractionated on a 0.7% agarose gel and a nylon membrane (Ny
tran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, eg 0.1
Blots are washed sequentially at room temperature under conditions consistent with sodium citrate saline and 0.5% sodium dodecyl sulfate. Visualize the hybridization pattern using autoradiography or alternative imaging means,
Compare.

8 Microarrays Chaining or synthesis of array elements on the surface of microarrays can be performed by photolithography, piezoelectric printing (inkjet printing, see Baldeschweiler et al., Supra),
It can be achieved using mechanical microspotting techniques and their derivatives. In each of the above techniques, the substrate should be a solid, non-porous solid (Schena (1999). Supra). Recommended substrates include silicon, silica,
There are slide glasses, glass chips, and silicon wafers. Alternatively, an array similar to dot blotting or slot blotting can be used to
Elements may be placed and attached to the surface of the substrate using chemical or mechanical attachment procedures. Regular arrays can be produced manually or using available methods and machines and can have any suitable number of elements (Schena, M. et al. (1995) Scien.
ce 270: 467-470, Shalon. D. et al. (1996) Genome Res. 6: 639-645, Marshall, A.
and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31.).

The full-length cDNA, expressed gene sequence fragment (EST), or fragments or oligomers thereof, may constitute an element of a microarray. Fragments or oligomers suitable for hybridization can be selected using software known in the art such as LASERGENE software (DNASTAR). Array elements are hybridized with a polynucleotide in a biological sample. The polynucleotide in the biological sample is conjugated to a fluorescent label or other molecular tag to facilitate detection. After hybridization, unhybridized nucleotides are removed from the biological sample and hybridization is detected at each array element using a fluorescence scanner. Alternatively, laser desorption and mass spectrometry can also be used to detect hybridization.
The degree of complementarity and relative abundance of each polynucleotide that hybridizes to the elements on the microarray can be calculated. The preparation and use of the microarray in one embodiment is detailed below.

Preparation of Tissue or Cell Samples Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly (A) ⁺ RNA is purified using the oligo (dT) cellulose method. Each poly (A) ⁺ RNA sample contained MMLV reverse transcriptase, 0.05 pg / μl oligo (dT) primer (21mer), 1x
First strand buffer, 0.03 unit / μl RNase inhibitor, 500 μM dATP, 500
Reverse-transcribe using μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is carried out in 25 volume ml containing poly (A) ⁺ RNA using the GEMBRIGHT kit (Incyte). Specific control poly (A) ⁺ RNA is synthesized from non-coding yeast genomic DNA by in vitro transcription after incubation at 370 ° C. for 2 hours. Each reaction sample (one labeled with Cy3 and the other labeled with Cy5) was treated with 2.5 ml of 0.5 M sodium hydroxide and incubated at 850 ° C for 2 hours.
Incubate for 0 minutes to stop the reaction and degrade RNA. Samples were prepared from two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
(CLONTECH), Palo Alto CA). After conjugation, the two reaction samples are ethanol precipitated with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate and 300 ml 100% ethanol. The sample is Sp
Dry and finish with eedVAC (Savant Instruments Inc., Holbrook NY), 1
Resuspend in 4 μl of 5 × SSC / 0.2% SDS.

Microarray Preparation The arrays of the invention are used to generate array elements. Each array element is amplified from vector-containing bacterial cells with a cloned cDNA insert. PCR
Amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in 30 cycles of PCR from an initial amount of 1-2 ng to a final amount greater than 5 μg. The amplified array element is SEPHACRYL-400.
(Amersham Pharmacia Biotech).

The purified array elements are immobilized on polymer-coated glass slides. Microscope glass slides (Corning) are ultrasonically cleaned in and after treatment with a large amount of distilled water wash in 0.1% SDS and acetone. 4% Hydrofluoric acid (VWR Scientific Products Corporation (VWR), Wes)
Chester PA), washed extensively in distilled water, 95%
Coat with 0.05% aminopropylsilane (Sigma) in ethanol. The coated glass slide is cured by heating at 110 ° C.

Array elements are added to a coated glass substrate using the method described in US Pat. No. 5,807,522. The patent is incorporated herein by reference. 1 μl of the array element DNA having an average concentration of 100 ng / μl is filled in the open capillary print element by a high speed robot device. The instrument now deposits approximately 5 nl of array element sample per slide.

For the microarray, use STRATALINKER UV crosslinking agent (Stratagene) for UV.
Crosslink. The microarray is washed once with 0.2% SDS at room temperature and three times with distilled water. After incubating the microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 ° C., 0.2% SDS and Nonspecific binding sites are blocked by washing with distilled water.

Hybridization The hybridization reaction has 9 μl of sample mixture containing 0.2 μg each of the Cy3 and Cy5 labeled cDNA synthesis products in 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65 ° C. for 5 minutes, aliquoted on the microarray surface and covered with a 1.8 cm ² coverslip. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. By adding 140 μl of 5 × SSC to the corner of the chamber, the humidity inside the chamber is reduced.
Hold at 100%. The chamber containing the array is incubated at 60 ° C. for approximately 6.5 hours. Arrays are at 45 ° C. in the first wash buffer (1 × SSC, 0.1% SDS)
Wash for 10 minutes at 50 ° C., then in the second wash buffer (0.1 × SSC) at 45 ° C. for 10 minutes 3 times each and dry.

The detection reporter-labeled hybridization complex is 48 for excitation of Cy3.
Detection with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of producing spectral lines at 8 nm and 632 nm for excitation of Cy3. A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the pump laser light on the array. Computer-controlled microscope with slide containing array
Place it on the XY stage and pass the objective lens for raster scanning. The 1.8 cm × 1.8 cm array used in this example was scanned at a resolution of 20 μm.

Of the two different scans, the mixed gas multi-line laser continuously excites the two phosphors. The emitted light is based on the wavelength depending on the two fluorophores and two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater
NJ). The signal is filtered using a suitable filter placed between the array and the photomultiplier tube. The maximum emission of the phosphor used is 56 for Cy3.
It is 5 nm and 650 nm for Cy5. Although the instrument can record spectra from both phosphors simultaneously, each array is typically scanned twice with a suitable filter in the laser source, once for each phosphor.

The sensitivity of the scan is usually calibrated using the signal intensity produced by the cDNA control species added to a known concentration of sample mixture. A particular location on the array contains a complementary DNA sequence, which correlates the intensity of the signal at that location to a weight ratio of hybridizing species of 1: 100,000. Different sources (eg representing test and control cells)
Labeled samples of calibrated cDNA with two fluorophores when labeled with two fluorophores, each labeled with a different fluorophore, and hybridized to a single array to identify differentially expressed genes Calibrate and add equal amounts of each to the hybridization mixture.

The output of the photomultiplier tube is a 12-bit RTI-835H analog-digital (A / D) conversion board (Analog Device) installed in an IBM compatible PC computer.
s, Inc., Norwood MA). The digitized data is displayed as an image where the signal intensities are mapped using a linear 20 color conversion from the blue (low signal) to the red (high signal) pseudo-color range. The data are also analyzed quantitatively. When exciting and measuring two different fluorophores simultaneously, the emission spectra of each fluorophore are used and the data is first collected by optical magnetic printing between the fluorophores (due to the superposition of the emission spectra). To be

The grid is overlaid on the fluorescent signal image, whereby the signal from each spot is collected at each element of the grid. The fluorescent signals within each element are integrated and a number is obtained depending on the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).

9 A sequence complementary to the sequence encoding the complementary polynucleotide INTRA, or any portion thereof, may be any of the native I
Used to detect, reduce, or inhibit the expression of NTRA. About 15 to 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. Oligo4
Appropriate oligonucleotides are designed using .06 software (National Biosciences) and sequences encoding INTRA. 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 block translation, INTRA
Complementary oligonucleotides are designed so that the ribosome does not bind to the transcript that encodes.

10 Expression of INTRA Expression and purification of INTRA can be carried out using a bacterial or virus based expression system. For expression of INTRA in bacteria, the cDNA is subcloned into a suitable vector containing an antibiotic promoter and an inducible promoter that enhances the transcription level of the cDNA. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host such as BL21 (DE3). INTR when antibiotic-resistant bacteria are induced with isopropyl β-D thiogalactopyranoside (IPTG)
Express A. Expression of INTRA in eukaryotic cells is performed by infecting insect cell lines or mammalian cell lines with Autographica californica nuclear polyhedrosis virus (AcMNPV), which is generally known as baculovirus. The baculovirus indispensable polyhedrin gene is replaced with the cDNA encoding INTRA by either homologous recombination or by bacterial-mediated gene transfer involving the transfer plasmid transfer. Viral infectivity is maintained and high levels of cDNA transcription are driven by the strong polyhedrin promoter. Recombinant baculovirus is often Spodoptera frugiperda
(Sf9) Used to infect insect cells, but also used to infect human hepatocytes. In the case of the latter infection, further genetic modification of the baculovirus is required. (Engelhard.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, for example, glutathione S transferase (GST) or peptide epitope tags such as FLAG can be used to synthesize INTRA as a fusion protein.
Or 6-His is used. By using these, affinity-based purification of recombinant fusion proteins from crude cell lysates can be performed rapidly in one step. GS
T is a 26kDa enzyme from Schistosoma japonicum that allows purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amer
sham Pharmacia Biotech). After purification, the GST portion was
Can be proteolytically cleaved from. FLAG is an 8-amino acid peptide that is commercially available monoclonal and polyclonal anti-FLAG antibody (Eastma
n Kodak) to allow immunoaffinity purification. 6-His, which is a continuous extension of 6 histidine residues, allows purification on a metal chelate resin (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995), chapters 10 and 16 above. Using INTRA purified by these methods directly in Examples 11, 12 and 15
Can be performed.

11 Demonstration of INTRA Activity INTRA activity is related to the ability of INTRA to form protein-protein complexes and is measured by its ability to control the growth characteristics of NIH3T3 mouse fibroblasts. The cDNA encoding INTRA is subcloned into a suitable eukaryotic expression vector. NIH3T3 cells are transfected with this vector using methods known in the art. Transfected cells are compared to non-transfected cells for the following quantifiable properties. Quantifiable properties are growth to high concentrations in culture, reduced attachment of cells to the substrate, morphology of mutant cells and the ability to induce tumors upon injection into immunodeficient mice. The activity of INTRA is proportional to the range of increased growth or frequency of mutant cell morphology in INTRA-transfected NIH3T3 cells.

Alternatively, INTRA activity is measured by binding INTRA to a radiolabeled formin polypeptide containing a multiproline region that specifically binds to 5H3-containing proteins (Ch
an. DC et al. (1996) EMBO J. 15: 1045-54). The INTRA sample is run on an SDS-PAGE gel and transferred to nitrocellulose by electroblotting. Blots were taken for 1 hour at room temperature in TBST (137 mM NaCI, containing skim milk powder,
Inhibits in 2.7 mM Kcl, 25 mM Tris (pH 8.0) and 0.1% Tween 20). Blots are incubated with TBST containing radioactive formin polypeptide for 4 hours to overnight. After washing the blot 4 times with TBST, the blot is exposed to autoradiographic film. Radioactivity is quantified by detaching the radioactive spots and counted with a radioisotope counter. The amount of radioactivity recovered is proportional to the activity of INTRA in the assay.

Alternatively, INT binding by binding INTRA to Ca ²⁺ using the Ca ²⁺ overlay system
Demonstrate RA activity (Weis, K. et al. (1994) J. Biol. Chem. 269: 19142-19150).
The purified INTRA is transferred and immobilized on a nitrocellulose membrane. The membrane is buffered (
Washed 3 times with 60 mM KCl, 5 mM MgCl ₂ , 10 mM imidazole-HCl, pH 6.8) and 1 μCi [ ⁴⁵ Ca ²⁺ ] (NIEN-DuPont, Boston, MA) in this buffer.
Incubate for 0 minutes. Unbound [ ⁴⁵ Ca ²⁺ ] is removed from the membrane by washing with water and the membrane is dried. Membrane-bound [ ⁴⁵ Ca ²⁺ ] is detected by autoradiography and quantified using an image analysis system and software. INTRA activity is proportional to the amount of [ ⁴⁵ Ca ²⁺ ] detected on the membrane.

Alternatively, INTRA activity is assayed by measuring the conversion of ³ H-cAMP to ³ H-adenosine in the presence of INTRA and 5 ′ nucleotidase. 50 mM Tris-HCl pH
Intra was added to a solution containing 7.5, 10 mM MgCl ₂ , 0.1 units of 5 ′ nucleotidase (from Crotalus atrox venom) and 0.0064-2.0 uM ³ H-cAMP, and related to product inhibition. Incubate the reaction at 37 ° C. for a time to cause less than 15% cAMP hydrolysis to avoid non-linearity. Soluble radioactivity associated with ³ H-adenosine is quantified using a β-scintillation counter. The amount of radioactivity recovered is proportional to the activity of INTRA in the reaction.

12 Functional Assays INTRA function is a function of I at physiologically elevated levels in mammalian cell culture systems.
Evaluation is by expression of the NTRA coding sequence. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels.
Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR
3.1 plasmid (Invitrogen) is included, both having the cytomegalovirus promoter. Human cell lines, eg, endothelial-derived or hematopoietic-derived cell lines, are transiently transfected with 5-10 μg of the recombinant vector using liposome formulations or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein provides a means of distinguishing between transfected and untransfected cells. Moreover, the expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. The labeled protein is
For example, it can be selected from green fluorescent protein (GFP; Clontech), CD64 or CD64-GFP fusion protein. Use automated, laser-optics-based technology, flow cytometry (FCM) to identify transfected cells expressing GFP or CD64-GFP and assess their apoptotic status and other cellular characteristics To do. FCM detects and measures the uptake of fluorescent molecules that diagnose phenomena that precede or coincide with cell death. Such phenomena include changes in nuclear DNA content measured by DNA staining with propidium iodide, down-regulation of DNA synthesis measured by reduction of bromodeoxyuridine uptake, and specific antibody There are changes in protein expression on the cell surface and intracellularly measured by reactivity, and changes in plasma membrane composition measured by binding of fluorescent complex annexin V protein to the cell surface. For flow cytometry methods, see Ormerod, MG (1994
) Flow Cytometry Oxford, New York, NY.

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

13 Production of INTRA Specific Antibody Polyacrylamide gel electrophoresis (PAGE; Harrington, MG (1990) Method
S. Enzymol. 182: 488-495 et al.) or INTRA substantially purified using other purification techniques, and rabbits are immunized to produce antibodies using standard protocols.

Alternatively, the INTRA amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions. Then, a corresponding oligopeptide is synthesized, and this oligopeptide is used to generate an antibody by a method well known to those skilled in the art. The selection of appropriate epitopes, for example those near the C-terminus or in adjacent hydrophilic regions, is known in the art (see Ausubel, 1995, supra, chapter 11, etc.).

Normally, oligopeptides of about 15 residues in length are labeled with ABI 43 using Fmoc chemistry.
Synthesized using a 1A Peptide Synthesizer (PE Biosystems) and reacted with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to produce KLH
(Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see A above).
See usubel, 1995, etc.). Rabbits are immunized with the oligopeptide-KLM complex in complete Freund's adjuvant. To test the anti-peptide activity and the anti-INTRA activity of the obtained antiserum, the peptide or INTRA was bound to a substrate and 1% BSA was added.
Blocked with, reacted with rabbit antiserum, washed, and further reacted with goat anti-rabbit IgG labeled with radioactive iodine.

Purification of Natural INTRA Using 14 Specific Antibodies Native or recombinant INTRA is substantially purified by immunoaffinity chromatography using INTRA specific antibodies. Immunoaffinity columns are anti-INTR
The antibody A is activated by a chromatographic resin such as CNBr-activated Sepharose (Ame
rsham Pharmacia Biotech). After combining,
Block and wash the resin according to the manufacturer's instructions.

The culture solution containing INTRA is passed through an immunoaffinity column, and the column is washed under conditions that preferentially adsorb INTRA (eg, high ionic strength buffer in the presence of detergent). The column is eluted under conditions that destroy the binding between the antibody and INTRA (for example, a buffer solution of pH 2-3 or a high-concentration chaotropic agent such as urea or thiocyanate ion), and INTRA is recovered.

Identification of 15 INTRA-Interacting Molecules INTRA or biologically active INTRA fragments are labeled with ¹²⁵ I Bolton-Hunter reagent (Bolton AE and WM Hunter (1973) Biochem. J. 133: 529-539). Etc.). Candidate molecules pre-arranged in the wells of the multiwell plate are incubated with labeled INTRA, washed, and all wells with labeled INTRA complex are assayed. Using the data obtained by changing the INTRA concentration, INT with the candidate molecule
Calculate the number of RA, affinity and association values.

Alternatively, the molecule that interacts with INTRA is described in Fields, S. and O. Song (1989, Natu
re 340: 245-246) or using a commercially available kit based on a two-hybrid system such as the MATCHMAKER system (Clontech).

INTRA has also been used in the PATHCALLING process (CuraGen Corp., New Haven CT) using the yeast two-hybrid system in a high-throughput manner to generate 2
All interactions between the genes encoded in the large library can be determined (Nandabalan, K. et al. (2000) US Pat. No. 6,057,101).

Those skilled in the art can 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 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 intended to be within the scope of the following claims.

BRIEF DESCRIPTION OF THE TABLES Table 1 lists the polypeptide and nucleotide sequences used to assemble the full-length sequence encoding INTRA (SEQ ID NO), clone identification number (clone identification number). ID), cDNA library and cDNA fragment.

Table 2 shows the characteristics of each polypeptide sequence, including latent motifs, homologous sequences, methods used for INTRA analysis, algorithms and searchable databases.

Table 3 lists selected fragments of each nucleic acid sequence, tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, diseases, disorders or conditions associated with these tissues, and each DNA. And the vector to which it is cloned.

Table 4 shows the tissues used for the construction of the cDNA library. CDNA encoding INTRA
The clone was isolated from here.

Table 5 shows the tools, programs and algorithms used in the INTRA analysis, along with applicable explanations, 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]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

[Table 25]

[Table 26]

[Table 27]

[Table 28]

[Table 29]

[Table 30]

[Table 31]

[Table 32]

[Sequence list]

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] October 5, 2001 (2001.10.5)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 25/00 A61P 35/00 4C084 29/00 37/02 4H045 35/00 43/00 105 37/02 111 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 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/53 M G01N 33/15 33/566 33/50 C12N 15/00 ZNAA 33/53 5/00 A 33/566 A61K 37 / 02 (31) Priority claim number 60 / 164,417 (32) Priority date November 9, 1999 (November 1, 1999) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, G B, 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, K E, LS, MW, MZ, 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, IL, 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, L, T J, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Hillman, Jennifer El, California 94040, Mountain View # 12, Monrode Live. 230 (72) Inventor Ral, Pretty United States California 95054 Santa Clara Russ Drive 2382 (72) Inventor Bandman, Olga United States California 94043 Mountain View Anna Avenue 366 (72) Inventor Bogun, Mariah Earl United States California 94577 San Leandro Santiago Road 14244 (72) Inventor Azimzai, Yarda United States California 94545 Hayward Rock Springs Drive 2045 (72) Inventor Young, Junming United States California 9512 9 San Jose Barcrane 7125 (72) Inventor Lady, Looper 94086, California, USA 94086 Sunnyvale # 3 West McKinley Avenue 1233 (72) Inventor Ryu, Dung Aina Em USA 95136 San Jose Park Belmont Place 55F Term (Reference) 2G045 AA34 AA35 BB01 BB07 BB14 BB20 BB46 BB48 BB51 CB01 DA13 DA14 DA36 FB02 FB03 FB05 4B024 AA01 AA11 BA80 CA04 DA02 EA04 GA60AQ4QAQ4QAQ4QAQBQ4QAQBQAQAQQQAQQQAQQQAQQQRQQQQQRQQQQQRQQQRQQQRQQQRQR QRQR QR56 QR66 QR66 QR66 QR66 QR66 QR66 QR66 QR66 QR66 AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA02 AA07 AA17 BA01 BA08 BA20 BA21 BA22 BA23 CA62 DC50 NA14 ZA012 ZA662 ZB072 ZB112 ZB262 ZC022 4H045 AA10 AA11 CA40 DA75 EA20 EA50 FA72 FA74

Claims (27)

[Claims] 1. A substantially isolated polypeptide comprising an amino acid sequence selected from the group having the following (a) to (d): (A) Sequence number 1, sequence number 2, sequence number 3, sequence number 4, sequence number 5, sequence number 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, Sequence number 3
9, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44
, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,
Amino acid sequence selected from the group having SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 (b) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, Sequence number 3
9, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44
, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,
A natural amino acid sequence having at least 90% homology with an amino acid sequence selected from the group having SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52. (c) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, Sequence number 3
9, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44
, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,
A biologically active fragment of an amino acid sequence selected from the group having SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52. (d) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, Sequence number 3
9, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44
, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,
Immunogenic fragments of amino acid sequences selected from the group having SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 2. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence No. 12, SEQ ID No. 13, Sequence No. 14, Sequence No. 15, Sequence No. 16, Sequence No. 17, Sequence No. 18, Sequence No. 19, Sequence No. 20, Sequence No. 21, Sequence No. 22, Sequence No. 23, Sequence No. 24 , SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 3
1, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38
, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
The isolated according to claim 1 selected from the group having SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52. Polypeptides. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. An isolated polynucleotide encoding the polypeptide of claim 2. 5. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 5
6, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61
, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66,
Sequence number 67, sequence number 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72, sequence number 73, sequence number 74, sequence number 75, sequence number 76, sequence number 77, sequence number 78, sequence number 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 9
4, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99
The isolated polynucleotide of claim 4, selected from the group having: SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 7. A cell transformed with the recombinant polynucleotide according to claim 6. 8. A genetic transformant containing the recombinant polynucleotide according to claim 6. 9. A method for producing the polypeptide according to claim 1, comprising the step of: (a) culturing cells transformed with the recombinant polynucleotide under conditions suitable for expression of the polypeptide. And (b) a step of receiving the polypeptide so expressed, wherein the recombinant polynucleotide is a promoter sequence operably linked to the polynucleotide encoding the polypeptide of claim 1. A method comprising: 10. An isolated antibody which specifically binds to the polypeptide of claim 1. 11. A substantially isolated polynucleotide comprising a polynucleotide sequence selected from the group comprising (a) to (d) below: (A) a polynucleotide sequence selected from the group having SEQ ID NOs: 53 to 104. (b) a natural polynucleotide sequence having at least 90% homology with the polynucleotide sequence selected from the group having SEQ ID NOS: 53 to 104. (c) ) Polynucleotide sequence complementary to (a) (d) Polynucleotide sequence complementary to (b) (e) RNA equivalent of (a) to (d) 12. At least 60 of the polynucleotide of claim 11.
An isolated polynucleotide comprising a contiguous nucleotide of. 13. A method for detecting a target polynucleotide having the sequence of the polynucleotide according to claim 11 in a sample, comprising: (a) at least having a sequence complementary to the target polynucleotide in the sample. A step of hybridizing the sample using a probe containing 20 consecutive nucleotides, and (b) detecting the presence or absence of the hybridization complex, and optionally the amount of the complex, if any. And the step of detecting, wherein the probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide. how to. 14. The method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides. 15. A method for detecting a target polynucleotide having the sequence of the polynucleotide according to claim 11 in a sample, comprising: (a) amplifying the target polynucleotide or a fragment thereof using polymerase chain reaction amplification. And (b) detecting the presence / absence of the target polynucleotide or its fragment, and optionally detecting the amount of the target polynucleotide or its fragment, if any. And how to. 16. A pharmaceutical ingredient comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 17. The polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, Sequence number 11, sequence number 12, sequence number 13, sequence number 1
4, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19
, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
Sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, sequence number 43, sequence number 44, sequence number 45, sequence number 46, sequence number 47, sequence number 48, sequence number 49, sequence number 50, sequence number 51 and The pharmaceutical ingredient according to claim 16, characterized in that it comprises an amino acid sequence selected from the group having SEQ ID NO: 52. 18. A method for treating a disease or medical condition associated with reduced expression of functional INTRA, comprising the step of administering the pharmaceutical ingredient according to claim 16 to a patient in need of such treatment. A method comprising. 19. A method of screening a compound for confirming its effectiveness as an agonist of the polypeptide according to claim 1, comprising: (a) exposing a sample containing the polypeptide according to claim 1 to the compound. A method comprising the steps of: (b) detecting an agonist activity in the sample. 20. A pharmaceutical ingredient comprising an agonist compound identified by the method of claim 19 and a pharmaceutically acceptable excipient. 21. A method for treating a disease or medical condition associated with reduced expression of functional INTRA, which comprises the step of administering the pharmaceutical ingredient according to claim 20 to a patient in need of such treatment. A method comprising. 22. A method of screening a compound for confirming its efficacy as an antagonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound. A method comprising the steps of: (b) detecting an antagonist activity in the sample. 23. A pharmaceutical ingredient comprising an antagonist compound identified by the method of claim 22 and a pharmaceutically acceptable excipient. 24. A method for treating a disease or medical condition associated with overexpression of functional INTRA, comprising the step of administering the pharmaceutical ingredient according to claim 23 to a patient in need of such treatment. A method characterized by the following. 25. A method for screening a compound that specifically binds to the polypeptide of claim 1, comprising: (a) binding the polypeptide of claim 1 to at least one test compound under suitable conditions. And a step of (b) detecting the binding of the polypeptide of claim 1 to a test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. And how to. 26. A method for screening a compound that regulates the activity of the polypeptide according to claim 1, which comprises (a) a condition under which the activity of the polypeptide according to claim 1 is allowed. 1
Binding the polypeptide of claim 1 to at least one test compound, (b) calculating the activity of the polypeptide of claim 1 in the presence of the test compound, and (c) in the presence of the test compound. Comparing the activity of the polypeptide of claim 1 with the activity of the polypeptide of claim 1 in the absence of the test compound in the presence of the test compound. 9. A method wherein the altered activity of the polypeptide of claim 1 is indicative of a compound that modulates the activity of the polypeptide of claim 1. 27. A method of screening a compound for confirming the effectiveness of mutant expression of a target polynucleotide having the sequence according to claim 5, comprising: (a) using a sample containing the target polynucleotide as a compound. A method comprising: exposing, and (b) detecting a mutant expression of the target polynucleotide.