# Patent

## Title
Antibodies which bind to signal peptide-containing molecules

## Abstract

The invention provides human signal peptide-containing proteins (HSPP) and polynucleotides which identify and encode HSPP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of HSPP.

## Background
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application is a divisional of U.S. patent application Ser. No. 13/397,592, filed Feb. 15, 2012, now U.S. Pat. No. 8,716,445, which is a divisional of U.S. patent application Ser. No. 12/457,389, filed Jun. 9, 2009, now U.S. Pat. No. 8,153,398, which is a continuation of U.S. patent application Ser. No. 11/905,820, filed Oct. 4, 2007, now abandoned, which is a divisional application of U.S. patent application Ser. No. 10/820,474, filed Apr. 7, 2004, now abandoned, which is a divisional of U.S. patent application Ser. No. 09/720,533, filed Mar. 20, 2001, now abandoned, which is the National Phase of International Application No. PCT/US99/14484, filed Jun. 25, 1999, which claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/090,762, filed Jun. 26, 1998, U.S. Provisional Patent Application No. 60/094,983, filed Jul. 31, 1998, U.S. Provisional Patent Application No. 60/102,686, filed Oct. 1, 1998, and U.S. Provisional Patent Application No. 60/112,129, filed Dec. 11, 1998. These applications are all incorporated herein by reference in their entirety.


TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human signal peptide-containing proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative disorders including cancer; inflammation; and cardiovascular, neurological, reproductive, and developmental disorders.
BACKGROUND OF THE INVENTION
Protein transport is essential for cellular function. Transport of a protein may be mediated by a signal peptide located at the amino terminus of the protein itself. The signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to a particular membrane bound compartment such as the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane. Secreted proteins are often synthesized as inactive precursors that are activated by post-translational processing events during transit through the secretory pathway. Such events include glycosylation, phosphorylation, proteolysis, and removal of the signal peptide by a signal peptidase. Other events that may occur during protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex. Examples of secreted proteins with amino terminal signal peptides are discussed below and include receptors, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, neuropeptides, vasomediators, phosphokinases, phosphatases, phospholipases, phosphodiesterases, G and Ras-related proteins, ion channels, transporters/pumps, proteases, and transcription factors. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing, New York, N.Y., pp. 557-560, 582-592.)
G-protein coupled receptors (GPCRs) comprise a superfamily of integral membrane proteins which transduce extracellular signals. GPCRs include receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin; for lipid mediators of inflammation such as prostaglandins, platelet activating factor, and leukotrienes; for peptide hormones such as calcitonin, C5a anaphylatoxin, follicle stimulating hormone, gonadotropin releasing hormone, neurokinin, oxytocin, and thrombin; and for sensory signal mediators such as retinal photopigments and olfactory stimulatory molecules. The structure of these highly conserved receptors consists of seven hydrophobic transmembrane regions, cysteine disulfide bridges between the second and third extracellular loops, an extracellular N-terminus, and a cytoplasmic C-terminus. The N-terminus interacts with ligands, the disulfide bridges interact with agonists and antagonists, and the large third intracellular loop interacts with G proteins to activate second messengers such as cyclic AMP, phospholipase C, inositol triphosphate, or ion channels. (Reviewed in Watson, S, and Arkinstall, S. (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego, Calif., pp. 2-6; and Bolander, F. F. (1994) Molecular Endocrinology, Academic Press, San Diego, Calif., pp. 162-176.)
Other types of receptors include cell surface antigens identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)-based “shot gun” techniques. These techniques have resulted in the production of hundreds of mAbs directed against unknown cell surface leukocytic antigens. These antigens have been grouped into “clusters of differentiation” based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a “CD” number. Some of the genes encoding proteins identified by CD antigens have been isolated and characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A. N. et al. (1993) The Leucocyte Antigen Facts Book, Academic Press, San Diego, Calif., pp. 144-145; Noel, L. S. et al. (1998) J. Biol. Chem. 273:3878-3883.)
Tetraspanins are a superfamily of membrane proteins which facilitate the formation and stability of cell-surface signaling complexes containing lineage-specific proteins, integrins, and other tetraspanins. They are involved in cell activation, proliferation (including cancer), differentiation, adhesion, and motility. These proteins cross the membrane four times, have conserved intracellular—and C-termini and an extracellular, non-conserved hydrophilic domain. Tetraspanins include, e.g., platelet and endothelial cell membrane proteins, leukocyte surface proteins, tissue specific and tumorous antigens, and the retinitis pigmentosa-associated gene peripherin. (Maecker, H. T. et al. (1997) FASEB J. 11:428-442.)
Matrix proteins (MPs) are transmembrane and extracellular proteins which function in formation, growth, remodeling, and maintenance of tissues and as important mediators and regulators of the inflammatory response. The expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases. In addition, MPs affect leukocyte migration, proliferation, differentiation, and activation in the immune response. MPs are frequently characterized by the presence of one or more domains which may include collagen-like domains, EGF-like domains, immunoglobulin-like domains, and fibronectin-like domains. In addition, some MPs are heavily glycosylated. MPs include extracellular proteins such as fibronectin, collagen, and galectin and cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and integrins. (Reviewed in Ayad, S. et al. (1994) The Extracellular Matrix Facts Book, Academic Press, San Diego, Calif., pp. 2-16; Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M. D. and Nelson, W. J. (1997) BioEssays 19:47-55.)
Lectins are proteins characterized by their ability to bind carbohydrates on cell membranes by means of discrete, modular carbohydrate recognition domains, CRDs. (Kishore, U. et al. (1997) Matrix Biol. 15:583-592.) Certain cytokines and membrane-spanning proteins have CRDs which may enhance interactions with extracellular or intracellular ligands, with proteins in secretory pathways, or with molecules in signal transduction pathways. The lipocalin superfamily constitutes a phylogenetically conserved group of more than forty proteins that function by binding to and transporting a variety of physiologically important ligands. (Tanaka, T. et al. (1997) J. Biol. Chem. 272:15789-15795; and van't Hof, W. et al. (1997) J. Biol. Chem. 272:1837-1841.) Selectins are a family of calcium ion-dependent lectins expressed on inflamed vascular endothelium and the surface of some leukocytes. (Rossiter, H. et al. (1997) Mol. Med. Today 3:214-222.)
Protein kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Reversible protein phosphorylation is a key strategy for controlling protein functional activity in eukaryotic cells. The high energy phosphate which drives this activation is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals, cell cycle checkpoints, and environmental or nutritional stresses. Protein kinases may be roughly divided into two groups; protein tyrosine kinases (PTKs) which phosphorylate tyrosine residues, and serine/threonine kinases (STKs) which phosphorylate serine or threonine residues. A few protein kinases have dual specificity. A majority of kinases contain a similar 250-300 amino acid catalytic domain. (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book, Vol I, pp. 7-47, Academic Press, San Diego, Calif.)
Protein phosphatases remove phosphate groups from molecules previously modified by protein kinases thus participating in cell signaling, proliferation, differentiation, contacts, and oncogenesis. Protein phosphorylation is a key strategy used to control protein functional activity in eukaryotic cells. The high energy phosphate is transferred from ATP to a protein by protein kinases and removed by protein phosphatases. There appear to be three, evolutionarily-distinct protein phosphatase gene families: protein phosphatases (PPs); protein tyrosine phosphatases (PTPs); and acid/alkaline phosphatases (APs). PPs dephosphorylate phosphoserine/threonine residues and are an important regulator of many cAMP mediated, hormone responses in cells. PTPs reverse the effects of protein tyrosine kinases and therefore play a significant role in cell cycle and cell signaling processes. Although APs dephosphorylate substrates in vitro, their role in vivo is not well known. (Charbonneau, H. and Tonics, N. K. (1992) Annu. Rev. Cell Biol. 8:463-493.)
Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals, including hormones, light and neurotransmitters. Cyclic nucleotide phosphodiesterases (PDEs) degrade cyclic nucleotides to their corresponding monophosphates, thereby regulating the intracellular concentrations of cyclic nucleotides and their effects on signal transduction. At least seven families of mammalian PDEs have been identified based on substrate specificity and affinity, sensitivity to cofactors and sensitivity to inhibitory drugs. (Beavo, J. A. (1995) Physiological Reviews 75: 725-748.)
Phospholipases (PLs) are enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. PLs play an important role in transmembrane signal transduction and are named according to the specific ester bond in phosphoglycerides that is hydrolyzed, i.e., A1, A2, C or D. PLA2 cleaves the ester bond at position 2 of the glycerol moiety of membrane phospholipids giving rise to arachidonic acid. Arachidonic acid is the common precursor to four major classes of eicosanoids, namely prostaglandins, prostacyclins, thromboxanes and leukotrienes. Eicosanoids are signaling molecules involved in the contraction of smooth muscle, platelet aggregation, and pain and inflammatory responses. (Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing, Inc., New York, N.Y., pp. 85, 211, 239-240, 642-645.)
The nucleotide cyclases, i.e., adenylate and guanylate cyclase, catalyze the synthesis of the cyclic nucleotides, cAMP and cGMP, from ATP and GTP, respectively. They act in concert with phosphodiesterases, which degrade cAMP and cGMP, to regulate the cellular levels of these molecules and their functions. cAMP and cGMP function as intracellular second messengers to transduce a variety of extracellular signals, e.g., hormones, and light and neurotransmitters. (Stryer, L. (1988) Biochemistry W.H. Freeman and Co., New York, pp. 975-980, 1029-1035.)
Cytokines are produced in response to cell perturbation. Some cytokines are produced as precursor forms, and some form multimers in order to become active. They are produced in groups and in patterns characteristic of the particular stimulus or disease, and the members of the group interact with one another and other molecules to produce an overall biological response. Interleukins, neurotrophins, growth factors, interferons, and chemokines are all families of cytokines which work in conjunction with cellular receptors to regulate cell proliferation and differentiation and to affect such activities as leukocyte migration and function, hematopoietic cell proliferation, temperature regulation, acute response to infections, tissue remodeling, apoptosis, and cell survival. Studies using antibodies or other drugs that modify the activity of a particular cytokine are used to elucidate the roles of individual cytokines in pathology and physiology.
Chemokines, in particular, are small chemoattractant cytokines involved in inflammation, leukocyte proliferation and migration, angiogenesis and angiostasis, regulation of hematopoiesis, HIV infectivity, and stimulation of cytokine secretion. Chemokines generally contain 70-100 amino acids and are subdivided into four subfamilies based on the presence of conserved cysteine-based motifs. (Callard, R. and Gearing, A. (1994) The Cytokine Facts Book, Academic Press, New York, N.Y., pp. 181-190, 210-213, 223-227.)
Growth and differentiation factors are secreted proteins which function in intercellular communication. Some factors require oligomerization or association with MPs for activity. Complex interactions among these factors and their receptors trigger intracellular signal transduction pathways that stimulate or inhibit cell division, cell differentiation, cell signaling, and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling). There are three broad classes of growth and differentiation factors. The first class includes the large polypeptide growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and platelet-derived growth factor. The second class includes the hematopoietic growth factors such as the colony stimulating factors (CSFs). Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors. The third class includes small peptide factors such as bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II, vasoactive intestinal peptide, and bradykinin which function as hormones to regulate cellular functions other than proliferation.
Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Inappropriate expression of growth factors by tumor cells may contribute to vascularization and metastasis of melanotic tumors. During hematopoiesis, growth factor misregulation can result in anemias, leukemias, and lymphomas. Certain growth factors such as interferon are cytotoxic to tumor cells both in vivo and in vitro. Moreover, some growth factors and growth factor receptors are related both structurally and functionally to oncoproteins. In addition, growth factors affect transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Reviewed in Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor, Mich., pp. 1-9.)
Proteolytic enzymes or proteases either activate or deactivate proteins by hydrolyzing peptide bonds. Proteases are found in the cytosol, in membrane-bound compartments, and in the extracellular space. The major families are the zinc, serine, cysteine, thiol, and carboxyl proteases.
Zinc proteases, e.g., carboxypeptidase A, have a zinc ion bound to the active site. These proteases recognize C-terminal residues that contain an aromatic or bulky aliphatic side chain, and hydrolyze the peptide bond adjacent to the C-terminal residues. Serine proteases have an active site serine residue and include digestive enzymes, e.g., trypsin and chymotrypsin, components of the complement and blood-clotting cascades, and enzymes that control the degradation and turnover of extracellular matrix (ECM) molecules. Cysteine proteases (e.g. cathepsin) are produced by monocytes, macrophages and other immune cells, and are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Overproduction of these enzymes can cause the tissue destruction associated with rheumatoid arthritis and asthma. Thiol proteases, e.g., papain, contain an active site cysteine and are widely distributed within tissues. Carboxyl proteases, e.g., pepsin, are active only under acidic conditions (pH 2 to 3).
Guanosine triphosphate-binding proteins (G proteins) can be grouped into two major classes: heterotrimeric G proteins and small G proteins. Heterotrimeric G proteins interact with GPCRs that respond to hormones, growth factors, neuromodulators, or other signaling molecules. The interaction between GPCR and G protein allows the G protein to exchange GTP for guanosine diphosphate (GDP). This exchange activates the G protein, allowing it to dissociate from the receptor and interact with the its cognate second messenger-generating protein, e.g., adenylate cyclase, guanylate cyclase, phospholipase C, or ion channels. The hydrolysis of GTP to GDP by the G protein acts as an on-off switch, terminating the action of the G protein and preparing it to interact with another receptor molecule, thus beginning another round of signal transduction.
The small G proteins consist of single 21-30 kDa polypeptides. They can be classified into five subfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor. These proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. In particular, the Ras proteins are essential in transducing signals from receptor tyrosine kinases to serine/threonine kinases which control cell growth and differentiation. Mutant Ras proteins, which bind but can not hydrolyze GTP, are permanently activated and cause continuous cell proliferation or cancer. All five subfamilies share common structural features and four conserved motifs. Most of the membrane-bound G proteins require a carboxy terminal isoprenyl group (CAAX), added posttranslationally, for membrane association and biological activity. The G proteins also have a variable effector region, located between motifs I and II, which is characterized as the interaction site for guanine nucleotide exchange factors or GTPase-activating proteins.
Eukaryotic cells are bound by a membrane and subdivided into membrane-bound compartments. Membranes are impermeable to many ions and polar molecules, therefore transport of these molecules is mediated by ion channels, ion pumps, transport proteins, or pumps. Symporters and antiporters regulate cytosolic pH by transporting ions and small molecules, e.g., amino acids, glucose, and drugs, across membranes; symporters transport small molecules and ions in the same direction, and antiporters, in the opposite direction. Transporter superfamilies include facilitative transporters and active ATP binding cassette transporters involved in multiple-drug resistance and the targeting of antigenic peptides to MHC Class I molecules. These transporters bind to a specific ion or other molecule and undergo conformational changes in order to transfer the ion or molecule across a membrane. Transport can occur by a passive, concentration-dependent mechanism or can be linked to an energy source such as ATP hydrolysis or an ion gradient.
Ion channels, ion pumps, and transport proteins mediate the transport of molecules across cellular membranes. Symporters and antiporters regulate cytosolic pH by transporting ions and small molecules such as amino acids, glucose, and drugs. Symporters transport small molecules and ions unidirectionally, and antiporters, bidirectionally. Transporter superfamilies include facilitative transporters and active ATP-binding cassette transporters which are involved in multiple-drug resistance and the targeting of antigenic peptides to MHC Class I molecules. These transporters bind to a specific ion or other molecule and undergo a conformational change in order to transfer the ion or molecule across the membrane. Transport can occur by a passive, concentration-dependent mechanism or can be linked to an energy source such as ATP hydrolysis. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing, New York, N.Y., pp. 523-546.)
Ion channels are formed by transmembrane proteins which create a lined passageway across the membrane through which water and ions, such as Na+, K+, Ca2+, and Cl—, enter and exit the cell. For example, chloride channels are involved in the regulation of the membrane electric potential as well as absorption and secretion of ions across the membrane. Chloride channels also regulate the internal pH of membrane-bound organelles.
Ion pumps are ATPases which actively maintain membrane gradients. Ion pumps are classified as P, V, or F according to their structure and function. All have one or more binding sites for ATP in their cytosolic domains. The P-class ion pumps include Ca2+ ATPase and Na+/K+ ATPase and function in transporting H+, Na+, K+, and Ca2+ ions. P-class pumps consist of two ÿ and two ÿ transmembrane subunits. The V- and F-class ion pumps have similar structures and but transport only H+. F class H+ pumps mediate transport across the membranes of mitochondria and chloroplasts, while V-class H+ pumps regulate acidity inside lysosomes, endosomes, and plant vacuoles.
A family of structurally related intrinsic membrane proteins known as facilitative glucose transporters catalyze the movement of glucose and other selected sugars across the plasma membrane. The proteins in this family contain a highly conserved, large transmembrane domain comprised of 12 ÿ-helices, and several weakly conserved, cytoplasmic and exoplasmic domains (Pessin, J. E., and Bell, G. I. (1992) Annu. Rev. Physiol. 54:911-930).
Amino acid transport is mediated by Na+ dependent amino acid transporters. These transporters are involved in gastrointestinal and renal uptake of dietary and cellular amino acids and in neuronal reuptake of neurotransmitters. Transport of cationic amino acids is mediated by the system y+ family and the cationic amino acid transporter (CAT) family. Members of the CAT family share a high degree of sequence homology, and each contains 12-14 putative transmembrane domains (Ito, K. and Groudine, M. (1997) J. Biol. Chem. 272:26780-26786).
Proton-coupled, 12 membrane-spanning domain transporters such as PEPT 1 and PEPT 2 are responsible for gastrointestinal absorption and for renal reabsorbtion of peptides using an electrochemical H+ gradient as the driving force. A heterodimeric peptide transporter, consisting of TAP 1 and TAP 2, is associated with antigen processing. Peptide antigens are transported across the membrane of the endoplasmic reticulum so they can be presented to the major histocompatibility complex class I molecules. Each TAP protein consists of multiple hydrophobic membrane spanning segments and a highly conserved ATP-binding cassette. (Boll, M. et al. (1996) Proc. Natl. Acad. Sci. 93:284-289.)
Hormones are secreted molecules that travel through the circulation and bind to specific receptors on the surface of, or within, target cells. Although they have diverse biochemical compositions and mechanisms of action, hormones can be grouped into two categories. One category consists of small lipophilic hormones that diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, and form a complex that alters gene expression. Examples of these molecules include retinoic acid, thyroxine, and the cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone. The second category consists of hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane. Examples of such hormones include amino acid derivatives such as catecholamines and peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin. (See, for example, Lodish et al. (1995) Molecular Cell Biology, Scientific American Books Inc., New York, N.Y., pp. 856-864.)
Neuropeptides and vasomediators (NP/VM) comprise a large family of endogenous signaling molecules. Included in this family are neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin and gastrin. NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in cascades. The effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, C. R. et al. (1985) Endocrine Physiology, Oxford University Press, New York, N.Y., pp. 57-62.)
Regulatory molecules turn individual genes or groups of genes on and off in response to various inductive mechanisms of the cell or organism; act as transcription factors by determining whether or not transcription is initiated, enhanced, or repressed; and splice transcripts as dictated in a particular cell or tissue. Although they interact with short stretches of DNA scattered throughout the entire genome, most gene expression is regulated near the site at which transcription starts or within the open reading frame of the gene being expressed. Many of the transcription factors incorporate one of a set of DNA-binding structural motifs, each of which contains either ÿ helices or β sheets and binds to the major groove of DNA. (Pabo, C. O. and R. T. Sauer (1992) Ann. Rev. Biochem. 61:1053-95.) Other domains of transcription factors may form crucial contacts with the DNA. In addition, accessory proteins provide important interactions which may convert a particular protein complex to an activator or a repressor or may prevent binding. (Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing Co, New York, N.Y. pp. 401-474.)
The discovery of new human signal peptide-containing proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative disorders including cancer; inflammation; and cardiovascular, neurological, reproductive, and developmental disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, proteins with signal peptides, referred to collectively as “HSPP” and individually as “HSPP-1”, “HSPP-2”, “HSPP-3”, “HSPP-4”, “HSPP-5”, “HSPP-6”, “HSPP-7”, “HSPP-8”, “HSPP-9”, “HSPP-10”, “HSPP-11”, “HSPP-12”, “HSPP-13”, “HSPP-14”, “HSPP-15”, “HSPP-16”, “HSPP-17”, “HSPP-18”, “HSPP-19”, “HSPP-20”, “HSPP-21”, “HSPP-22”, “HSPP-23”, “HSPP-24”, “HSPP-25”, “HSPP-26”, “HSPP-27”, “HSPP-28”, “HSPP-29”, “HSPP-30”, “HSPP-31”, “HSPP-32”, “HSPP-33”, “HSPP-34”, “HSPP-35”, “HSPP-36”, “HSPP-37”, “HSPP-38”, “HSPP-39”, “HSPP-40”, “HSPP-41”, “HSPP-42”, “HSPP-43”, “HSPP-44”, “HSPP-45”, “HSPP-46”, “HSPP-47”, “HSPP-48”, “HSPP-49”, “HSPP-50”, “HSPP-51”, “HSPP-52”, “HSPP-53”, “HSPP-54”, “HSPP-55”, “HSPP-56”, “HSPP-57”, “HSPP-58”, “HSPP-59”, “HSPP-60”, “HSPP-61”, “HSPP-62”, “HSPP-63”, “HSPP-64”, “HSPP-65”, “HSPP-66”, “HSPP-67”, “HSPP-68”, “HSPP-69”, “HSPP-70”, “HSPP-71”, “HSPP-72”, “HSPP-73”, “HSPP-74”, “HSPP-75”, HSPP-76”, “HSPP-77”, “HSPP-78”, “HSPP-79”, “HSPP-80”, “HSPP-81”, “HSPP-82”, “HSPP-83”, “HSPP-84”, “HSPP-85”, “HSPP-86”, “HSPP-87”, “HSPP-88”, “HSPP-89”, “HSPP-90”, “HSPP-91”, “HSPP-92”, “HSPP-93”, “HSPP-94”, “HSPP-95”, “HSPP-96”, “HSPP-97”, “HSPP-98”, “HSPP-99”, “HSPP-100”, “HSPP-101”, “HSPP-102”, “HSPP-103”, “HSPP-104”, “HSPP-105”, “HSPP-106”, “HSPP-107”, “HSPP-108”, “HSPP-109”, “HSPP-110”, HSPP-111”, “HSPP-112”, “HSPP-113”, “HSPP-114”, “HSPP-115”, “HSPP-116”, “HSPP-117”, “HSPP-118”, “HSPP-119”, “HSPP-120”, “HSPP-121”, “HSPP-122”, “HSPP-123”, “HSPP-124”, “HSPP-125”, “HSPP-126”, “HSPP-127”, “HSPP-128”, “HSPP-129”, “HSPP-130”, “HSPP-131”, “HSPP-132”, “HSPP-133”, and “HSPP-134”. In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of 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, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, 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, 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:31, SEQ ID NO:32, SEQ ID NO:33, 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, 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, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, 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, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, 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:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134 (SEQ ID NO:1-134), and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO:1-134, and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268 (SEQ ID NO:135-268), and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO:135-268, and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:135-268, and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO:1-134, and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of HSPP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of HSPP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-134, and fragments thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows nucleotide and polypeptide sequence identification numbers (SEQ ID NO), clone identification numbers (clone ID), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding HSPP.
Table 2 shows features of each polypeptide sequence, including predicted signal peptide sequences, and methods and algorithms used for identification of HSPP.
Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis, diseases, disorders, or conditions associated with these tissues, and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which Incyte cDNA clones encoding HSPP were isolated.
Table 5 shows the programs, their descriptions, references, and threshold parameters used to analyze HSPP.
Table 6 shows the regions of the full-length nucleotide sequences of HSPP to which cDNA fragments of Table 1 correspond.


DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
“HSPP” refers to the amino acid sequences of substantially purified HSPP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term “agonist” refers to a molecule which, when bound to HSPP, increases or prolongs the duration of the effect of HSPP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HSPP.
An “allelic variant” is an alternative form of the gene encoding HSPP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
“Altered” nucleic acid sequences encoding HSPP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as HSPP or a polypeptide with at least one functional characteristic of HSPP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HSPP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HSPP. The encoded protein may also be “altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HSPP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of HSPP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
The terms “amino acid” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. In this context, “fragments,” “immunogenic fragments,” or “antigenic fragments” refer to fragments of HSPP which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of HSPP. Where “amino acid sequence” is recited to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
“Amplification” relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term “antagonist” refers to a molecule which, when bound to HSPP, decreases the amount or the duration of the effect of the biological or immunological activity of HSPP. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of HSPP.
The term “antibody” refers to intact molecules as well as to fragments thereof, such as Fab, F(ab′)2, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind HSPP 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 (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term “antigenic determinant” refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term “antisense” refers to any composition containing a nucleic acid sequence which is complementary to the “sense” strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.
The term “biologically active,” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic HSPP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing. For example, the sequence “5′ A-G-T 3′” bonds to the complementary sequence “3′ T-C-A 5′.” Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
A “composition comprising a given polynucleotide sequence” or a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding HSPP or fragments of HSPP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
“Consensus sequence” refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk Conn.) in the 5′ and/or the 3′ direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW Fragment Assembly system (GCG, Madison Wis.). Some sequences have been both extended and assembled to produce the consensus sequence.
The term “correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding HSPP, by northern analysis is indicative of the presence of nucleic acids encoding HSPP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding HSPP.
A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term “derivative” refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
The term “similarity” refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.” A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases “percent identity” or “% identity” refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Madison Wis.) which creates alignments between two or more sequences according to methods selected by the user, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
“Human artificial chromosomes” (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
The term “humanized antibody” refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
“Hybridization” refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words “insertion” or “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
“Immune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
The term “microarray” refers to an arrangement of distinct polynucleotides on a substrate.
The terms “element” or “array element” in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term “modulate” refers to a change in the activity of HSPP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HSPP.
The phrases “nucleic acid” or “nucleic acid sequence,” as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In this context, “fragments” refers to those nucleic acid sequences which, comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:135-268, for example, as distinct from any other sequence in the same genome. For example, a fragment of SEQ ID NO:135-268 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:135-268 from related polynucleotide sequences. A fragment of SEQ ID NO:135-268 is at least about 15-20 nucleotides in length. The precise length of the fragment of SEQ ID NO:135-268 and the region of SEQ ID NO:135-268 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. In some cases, a fragment, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms “operably associated” or “operably linked” refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
The term “oligonucleotide” refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. “Oligonucleotide” is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.
“Peptide nucleic acid” (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
The term “sample” is used in its broadest sense. A sample suspected of containing nucleic acids encoding HSPP, or fragments thereof, or HSPP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope “A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term “stringent conditions” refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
The term “substantially purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
A “substitution” refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
“Substrate” refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
“Transformation” describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed” cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A “variant” of HSPP polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to HSPP. This definition may also include, for example, “allelic” (as defined above), “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
The Invention
The invention is based on the discovery of new human signal peptide-containing proteins (HSPP), the polynucleotides encoding HSPP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders including cancer; inflammation; and cardiovascular, neurological, reproductive, and developmental disorders.
Table 1 lists the Incyte Clones used to derive full length nucleotide sequences encoding HSPP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NO) of the amino acid and nucleic acid sequences, respectively. Column 3 shows the Clone ID of the Incyte Clone in which nucleic acids encoding each HSPP were identified, and column 4, the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones, their corresponding cDNA libraries, and shotgun sequences. The clones and shotgun sequences are part of the consensus nucleotide sequence of each HSPP and are useful as fragments in hybridization technologies.
Table 6 shows the regions of the full-length nucleotide sequences of HSPP to which cDNA fragments of Table 1 correspond. Column 1 lists nucleotide sequence identifiers and column 2 shows the clone ID of the Incyte clone in which nucleic acids encoding each HSPP were identified. Column 3 shows Incyte clones and shotgun sequences which are part of the consensus nucleotide sequence of each HSPP and are useful as fragments in hybridization technologies. Column 4 lists the starting nucleotide position and column 5 the ending nucleotide position of the region of the full-length HSPP to which the cDNA fragment corresponds.
The columns of Table 2 show various properties of the polypeptides of the invention: column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3, potential phosphorylation sites; column 4, potential glycosylation sites; column 5, the amino acid residues comprising signature sequences and motifs; column 6, the identity of each protein; and column 7, analytical methods used to identify each HSPP as a signal peptide-containing protein. Note that in column 5, the first line of each cell lists the amino acid residues comprising predicted signal peptide sequences. Additional identifying motifs or signatures are also listed in column 5. Of particular note is the presence of a glycosyl hydrolase family 9 active site signature in SEQ ID NO:126, a ribosomal protein S18 signature in SEQ ID NO:127, an adrenodoxin family iron-sulfur binding region signature and a cytochrome c family heme-binding site signature in SEQ ID NO:132, and a urotensin II signature sequence in SEQ ID NO:96.
Using BLAST, SEQ ID NO:68 (HSPP-68) has been identified as a TWIK-related acid-sensitive K+ channel, and SEQ ID NO:92 (HSPP-92) has been identified as a tyrosine-specific protein phosphatase. The tyrosine-specific protein phosphatases signature in SEQ ID NO:92 (HSPP-92) from about V328 through about F340 (including the putative active site cysteine residue at C330) was identified using BLOCKS and PRINTS. Also of note is the identification of SEQ ID NO:66 (HSPP-66) as a steroid binding protein using BLAST.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding HSPP. The first column of Table 3 lists the nucleotide sequence identifiers. The second column lists tissue categories which express HSPP as a fraction of total tissue categories expressing HSPP. The third column lists the diseases, disorders, or conditions associated with those tissues expressing HSPP. The fourth column lists the vectors used to subclone the cDNA library. Of particular note is the expression of SEQ ID NO:200, SEQ ID NO:203, and SEQ ID NO:225 in lung tissues; the expression of SEQ ID NO:212, SEQ ID NO:216, and SEQ ID NO:220 in reproductive tissues; the expression of SEQ ID NO:223 in cancerous tissues; the expression of SEQ ID NO:232 in gastrointestinal tissue, specifically the small intestine or colon (fifteen out of sixteen (93.8%) cDNA libraries); and the expression of SEQ ID NO:224 in cancerous and proliferating tissues. Also of particular interest is the tissue-specific expression of SEQ ID NO:252 and SEQ ID NO:257. SEQ ID NO:252 is derived from OVARTUT01, an ovarian tumor cDNA library and is exclusively expressed in reproductive tumor tissue. SEQ ID NO:257 is derived from THP1AZT01, a 5-aza-2′-deoxycytidine treated human promonocyte cDNA library and is exclusively expressed in hematopoietic tissue.
The following fragments of the nucleotide sequences encoding HSPP are useful in hybridization or amplification technologies to identify SEQ ID NO:135-268 and to distinguish between SEQ ID NO:135-268 and related polynucleotide sequences. The useful fragments are the fragment of SEQ ID NO:230 from about nucleotide 75 to about nucleotide 104; the fragment of SEQ ID NO:231 from about nucleotide 210 to about nucleotide 239; the fragment of SEQ ID NO:232 from about nucleotide 157 to about nucleotide 186; the fragment of SEQ ID NO:233 from about nucleotide 268 to about nucleotide 297; the fragment of SEQ ID NO:234 from about nucleotide 160 to about nucleotide 186; the fragment of SEQ ID NO:235 from about nucleotide 201 to about nucleotide 230; the fragment of SEQ ID NO:236 from about nucleotide 165 to about nucleotide 194; the fragment of SEQ ID NO:237 from about nucleotide 366 to about nucleotide 395; the fragment of SEQ ID NO:238 from about nucleotide 714 to about nucleotide 743; the fragment of SEQ ID NO:239 from about nucleotide 1731 to about nucleotide 1760; the fragment of SEQ ID NO:240 from about nucleotide 419 to about nucleotide 448; the fragment of SEQ ID NO:241 from about nucleotide 494 to about nucleotide 523; the fragment of SEQ ID NO:242 from about nucleotide 100 to about nucleotide 129; the fragment of SEQ ID NO:243 from about nucleotide 104 to about nucleotide 133; the fragment of SEQ ID NO:244 from about nucleotide 136 to about nucleotide 165; the fragment of SEQ ID NO:245 from about nucleotide 140 to about nucleotide 169; the fragment of SEQ ID NO:246 from about nucleotide 125 to about nucleotide 154; the fragment of SEQ ID NO:247 from about nucleotide 687 to about nucleotide 758; the fragment of SEQ ID NO:248 from about nucleotide 327 to about nucleotide 398; the fragment of SEQ ID NO:249 from about nucleotide 741 to about nucleotide 785; the fragment of SEQ ID NO:250 from about nucleotide 184 to about nucleotide 255; the fragment of SEQ ID NO:251 from about nucleotide 165 to about nucleotide 242; the fragment of SEQ ID NO:252 from about nucleotide 271 to about nucleotide 342; the fragment of SEQ ID NO:253 from about nucleotide 1081 to about nucleotide 1152; the fragment of SEQ ID NO:254 from about nucleotide 781 to about nucleotide 852; the fragment of SEQ ID NO:255 from about nucleotide 620 to about nucleotide 691; the fragment of SEQ ID NO:256 from about nucleotide 872 to about nucleotide 916; the fragment of SEQ ID NO:257 from about nucleotide 242 to about nucleotide 313; the fragment of SEQ ID NO:258 from about nucleotide 595 to about nucleotide 648; the fragment of SEQ ID NO:259 from about nucleotide 163 to about nucleotide 216; the fragment of SEQ ID NO:260 from about nucleotide 244 to about nucleotide 315; the fragment of SEQ ID NO:261 from about nucleotide 75 to about nucleotide 128; the fragment of SEQ ID NO:262 from about nucleotide 650 to about nucleotide 703; the fragment of SEQ ID NO:263 from about nucleotide 143 to about nucleotide 214; the fragment of SEQ ID NO:264 from about nucleotide 434 to about nucleotide 487; the fragment of SEQ ID NO:265 from about nucleotide 218 to about nucleotide 271; the fragment of SEQ ID NO:266 from about nucleotide 89 to about nucleotide 145; the fragment of SEQ ID NO:267 from about nucleotide 198 to about nucleotide 254; and the fragment of SEQ ID NO:268 from about nucleotide 10 to about nucleotide 54.
The invention also encompasses HSPP variants. A preferred HSPP variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the HSPP amino acid sequence, and which contains at least one functional or structural characteristic of HSPP.
The invention also encompasses polynucleotides which encode HSPP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:135-268, which encodes HSPP.
The invention also encompasses a variant of a polynucleotide sequence encoding HSPP. In particular, such a variant polynucleotide sequence will have at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HSPP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:135-268 which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:135-268. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HSPP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding HSPP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HSPP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode HSPP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HSPP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HSPP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding HSPP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode HSPP and HSPP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HSPP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:135-268 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25° C., more preferably of at least about 42° C., and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase (Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway N.J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg Md.). Preferably, sequence preparation is automated with machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown Mass.) and the ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale Calif.). The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F. M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853.)
The nucleic acid sequences encoding HSPP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68° C. to 72° C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5′ regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode HSPP may be cloned in recombinant DNA molecules that direct expression of HSPP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express HSPP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HSPP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding HSPP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232.) Alternatively, HSPP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of HSPP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York N.Y.)
In order to express a biologically active HSPP, the nucleotide sequences encoding HSPP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ untranslated regions in the vector and in polynucleotide sequences encoding HSPP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HSPP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding HSPP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HSPP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and express sequences encoding HSPP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HSPP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding HSPP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or pSPORT1 plasmid (Life Technologies). Ligation of sequences encoding HSPP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of HSPP are needed, e.g. for the production of antibodies, vectors which direct high level expression of HSPP may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HSPP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra; Grant et al. (1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)
Plant systems may also be used for expression of HSPP. Transcription of sequences encoding HSPP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding HSPP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses HSPP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable expression of HSPP in cell lines is preferred. For example, sequences encoding HSPP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk− or apr− cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides, neomycin and G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding HSPP is inserted within a marker gene sequence, transformed cells containing sequences encoding HSPP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HSPP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding HSPP and that express HSPP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of HSPP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HSPP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HSPP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding HSPP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding HSPP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode HSPP may be designed to contain signal sequences which direct secretion of HSPP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Manassas, Va.) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding HSPP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric HSPP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HSPP activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His (SEQ ID NO: 269), FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His (SEQ ID NO: 269) enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the HSPP encoding sequence and the heterologous protein sequence, so that HSPP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled HSPP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 35S-methionine.
Fragments of HSPP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin-Elmer). Various fragments of HSPP may be synthesized separately and then combined to produce the full length molecule.
Therapeutics
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of HSPP and signal peptide sequences. In addition, chemical and structural similarity, in the context of sequences and motifs, exists between HSPP-66 and prostatic steroid-binding C3 precursor from rat (GI 206453); between HSPP-68 and TWIK-related acid-sensitive K+channel from human (GI 2465542); and between HSPP-92 and tyrosine specific protein phosphatases (PROSITE PDOC00323). In addition, the expression of HSPP is closely associated with proliferative, cancerous, inflamed, cardiovascular, nervous, reproductive, hematopoietic/immune, and developmental tissue. Therefore, HSPP appears to play a role in cell proliferative disorders including cancer; inflammation; and cardiovascular, neurological, reproductive, and developmental disorders. In the treatment of cell proliferative disorders including cancer; inflammation; and cardiovascular, neurological, reproductive, and developmental disorders associated with increased HSPP expression or activity, it is desirable to decrease the expression or activity of HSPP. In the treatment of the above conditions associated with decreased HSPP expression or activity, it is desirable to increase the expression or activity of HSPP.
Therefore, in one embodiment, HSPP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HSPP. Examples of such disorders include, but are not limited to, cell proliferative disorders such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; inflammatory disorders, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; cardiovascular disorders including disorders of the blood vessels such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, and vascular tumors; disorders of the heart such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, and congenital heart disease; and disorders of the lungs such as congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, and pleural tumors; neurological disorders such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, 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 neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis; mental disorders including mood, anxiety, and schizophrenic disorders; akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; reproductive disorders such as disorders of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis; disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea; disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male breast, and gynecomastia; and developmental disorders, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss.
In another embodiment, a vector capable of expressing HSPP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HSPP including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified HSPP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HSPP including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of HSPP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HSPP including, but not limited to, those listed above.
In a further embodiment, an antagonist of HSPP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HSPP. Examples of such disorders include, but are not limited to, those described above. In one aspect, an antibody which specifically binds HSPP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HSPP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HSPP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HSPP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of HSPP may be produced using methods which are generally known in the art. In particular, purified HSPP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HSPP. Antibodies to HSPP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HSPP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to HSPP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HSPP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to HSPP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell. Biol. 62:109-120.)
In addition, techniques developed for the production of “chimeric antibodies,” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HSPP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HSPP may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between HSPP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HSPP epitopes is preferred, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for HSPP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of HSPP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple HSPP epitopes, represents the average affinity, or avidity, of the antibodies for HSPP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular HSPP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the HSPP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 107 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of HSPP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington, D.C.; Liddell, J. E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York N.Y.).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is preferred for use in procedures requiring precipitation of HSPP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding HSPP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding HSPP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HSPP. Thus, complementary molecules or fragments may be used to modulate HSPP activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HSPP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HSPP. (See, e.g., Sambrook, supra; Ausubel, 1995, supra.)
Genes encoding HSPP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding HSPP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the gene encoding HSPP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may 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, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HSPP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HSPP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may 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′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HSPP, antibodies to HSPP, and mimetics, agonists, antagonists, or inhibitors of HSPP. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa.).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of HSPP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An 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.
A therapeutically effective dose refers to that amount of active ingredient, for example HSPP or fragments thereof, antibodies of HSPP, and agonists, antagonists or inhibitors of HSPP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED50 (the dose therapeutically effective in 50% of the population) or LD50 (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the LD50/ED50 ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage varies 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 requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
Diagnostics
In another embodiment, antibodies which specifically bind HSPP may be used for the diagnosis of disorders characterized by expression of HSPP, or in assays to monitor patients being treated with HSPP or agonists, antagonists, or inhibitors of HSPP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HSPP include methods which utilize the antibody and a label to detect HSPP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring HSPP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of HSPP expression. Normal or standard values for HSPP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HSPP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of HSPP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding HSPP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HSPP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of HSPP, and to monitor regulation of HSPP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HSPP or closely related molecules may be used to identify nucleic acid sequences which encode HSPP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding HSPP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the HSPP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:135-268 or from genomic sequences including promoters, enhancers, and introns of the HSPP gene.
Means for producing specific hybridization probes for DNAs encoding HSPP include the cloning of polynucleotide sequences encoding HSPP or HSPP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding HSPP may be used for the diagnosis of disorders associated with expression of HSPP. Examples of such disorders include, but are not limited to, cell proliferative disorders such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; inflammatory disorders, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; cardiovascular disorders including disorders of the blood vessels such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, and vascular tumors; disorders of the heart such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, and congenital heart disease; and disorders of the lungs such as congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, and pleural tumors; neurological disorders such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, 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 neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis; mental disorders including mood, anxiety, and schizophrenic disorders; akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; reproductive disorders such as disorders of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis; disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea; disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male breast, and gynecomastia; and developmental disorders, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss. The polynucleotide sequences encoding HSPP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered HSPP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HSPP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding HSPP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HSPP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of HSPP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HSPP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding HSPP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding HSPP, or a fragment of a polynucleotide complementary to the polynucleotide encoding HSPP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of HSPP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding HSPP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding HSPP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, HSPP, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HSPP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with HSPP, or fragments thereof, and washed. Bound HSPP is then detected by methods well known in the art. Purified HSPP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HSPP specifically compete with a test compound for binding HSPP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HSPP.
In additional embodiments, the nucleotide sequences which encode HSPP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all applications, patents, and publications, mentioned above and below, in particular U.S. Ser. No. 60/090,762, U.S. Ser. No. 60/094,983, U.S. Ser. No. 60/102,686, and U.S. Ser. No. 60/112,129, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Valencia Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), or pINCY (Incyte Corporation, Palo Alto Calif.). Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH55ÿ, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision, using the UNIZAP vector system (Stratagene) or cell lysis. Plasmids were purified using at least one of the following: a MAGIC or WIZARD minipreps DNA purification system (Promega); an AGTC miniprep purification kit (Edge Biosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep 96 plasmid kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V. B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene Oreg.) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols, base calling software, and kits. In one alternative, cDNAs were sequenced using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics). In another alternative, the cDNAs were amplified and sequenced using the ABI PRISM BIGDYE terminator cycle sequencing ready reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were determined using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table 5 summarizes the software programs, descriptions, references, and threshold parameters used. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides a brief description thereof, the third column presents the references which are incorporated by reference herein, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the probability the greater the homology). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco Calif.) and LASERGENE software (DNASTAR).
The polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation, using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM is a probalistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO:135-268. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ database (Incyte Corporation). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:




  
    %
    ⁢
    
      
    
    ⁢
    sequence
    ⁢
    
      
    
    ⁢
    identity
    ×
    %
    ⁢
    
      
    
    ⁢
    maximum
    ⁢
    
      
    
    ⁢
    BLAST
    ⁢
    
      
    
    ⁢
    score
  
  100




The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.

The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding HSPP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation/trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
V. Extension of HSPP Encoding Polynucleotides
Full length nucleic acid sequences of SEQ ID NOs: 135-229 were produced by extension of the component fragments described in Table 1, column 5, using oligonucleotide primers based on these fragments. For each nucleic acid sequence, one primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide. Primers were used to facilitate the extension of the known sequence “outward” generating amplicons containing new unknown nucleotide sequence for the region of interest. The initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences, Plymouth, Minn.), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (GIBCO BRL) were used to extend the sequence. If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region.
High fidelity amplification was obtained by following the instructions for the XL-PCR kit (The Perkin-Elmer Corp., Norwalk, Conn.) and thoroughly mixing the enzyme and reaction mix. PCR was performed using the PTC-200 thermal cycler (MJ Research, Inc., Watertown, Mass.), beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:













Step 1
94° C. for 1 min (initial denaturation)


Step 2
65° C. for 1 min


Step 3
68° C. for 6 min


Step 4
94° C. for 15 sec


Step 5
65° C. for 1 min


Step 6
68° C. for 7 min


Step 7
Repeat steps 4 through 6 for an additional 15 cycles


Step 8
94° C. for 15 sec


Step 9
65° C. for 1 min


Step 10
68° C. for 7:15 min


Step 11
Repeat steps 8 through 10 for an additional 12 cycles


Step 12
72° C. for 8 min


Step 13
 4° C. (and holding)









A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6% to 0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQUICK (QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16° C. Competent E. coli cells (in 40 μl of appropriate media) were transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37° C., the E. coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2× carb). The following day, several colonies were randomly picked from each plate and cultured in 150 μl of liquid LB/2× carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 μl of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 μl from each sample was transferred into a PCR array.
For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
















Step 1
94° C. for 60 sec



Step 2
94° C. for 20 sec



Step 3
55° C. for 30 sec



Step 4
72° C. for 90 sec



Step 5
Repeat steps 2 through 4 for an additional 29 cycles



Step 6
72° C. for 180 sec



Step 7
 4° C. (and holding)










Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and appropriate clones were selected, ligated into plasmid, and sequenced.
The full length nucleic acid sequences of SEQ ID NO:230-268 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5′ extension of the known fragment, and the other primer, to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)2SO4, and ÿ-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C.
The concentration of DNA in each well was determined by dispensing 100 ÿl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1× TE and 0.5 ÿl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly Mass.) into pUC18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37° C. in 384-well plates in LB/2× carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above. Samples were diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID NO:135-268 are used to obtain 5′ regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:135-268 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 μCi of [ÿ-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston Mass.). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba1, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.). Hybridization is carried out for 16 hours at 40° C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1× saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the blots to film for several hours, hybridization patterns are compared visually.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the HSPP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HSPP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of HSPP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HSPP-encoding transcript.
IX. Expression of HSPP
Expression and purification of HSPP is achieved using bacterial or virus-based expression systems. For expression of HSPP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express HSPP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of HSPP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HSPP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E. K. 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, HSPP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from HSPP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch 10 and 16). Purified HSPP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of HSPP Activity
HSPP-68
HSPP-68 activity is measured by determining the potassium current using voltage clamp analysis on single Xenopus laevis oocytes injected with HSPP-68 cRNA. HSPP-68 cRNA is synthesized in vitro from linearized HSPP-68 encoding plasmids using the T7 RNA polymerase and injected into oocytes. Injected oocytes are used two to four days after injection. In a 0.3 ml perfusion chamber, a single oocyte is impaled with two standard microelectrodes (1-2.5 Mÿ) filled with 3 M KCl. The oocyte is maintained under voltage clamp by using a Dagan TEV 200 amplifier, in buffer containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 2 mM MgCl2, 5 mM HEPES, pH 7.4 with NaOH. Stimulation of the preparation, data acquisition, and analysis is performed using a computer. All experiments are performed at room temperature (21-22° C.). Following a depolarizing pulse, the characteristics of the resulting potassium current are measured via the recording electrode. The amount of potassium current that flows in response to a unit depolarization is proportional to the activity of HSPP-68 in the cell. (Duprat, F. et al. (1997) EMBO J. 16:5464-5471.)
HSPP-92
HSPP-92 protein phosphatase activity is measured by the hydrolysis of P-nitrophenyl phosphate (PNPP). HSPP-92 is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1% b-mercaptoethanol at 37° C. for 60 min. The reaction is stopped by the addition of 6 ml of 10 N NaOH and the increase in light absorbance at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in light absorbance is proportional to the activity of PP in the assay. (Diamond R. H. et al (1994) Mol Cell Biol 14:3752-62.)
Alternatively, HSPP, or biologically active fragments thereof, are labeled with 125I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HSPP, washed, and any wells with labeled HSPP complex are assayed. Data obtained using different concentrations of HSPP are used to calculate values for the number, affinity, and association of HSPP with the candidate molecules.
Alternatively, an assay for HSPP activity measures the expression of HSPP on the cell surface. cDNA encoding HSPP is subcloned into an appropriate mammalian expression vector suitable for high levels of cDNA expression. The resulting construct is transfected into a nonhuman cell line such as NIH3T3. Cell surface proteins are labeled with biotin using methods known in the art Immunoprecipitations are performed using HSPP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of HSPP expressed on the cell surface.
Alternatively, an assay for HSPP activity measures the amount of HSPP in secretory, membrane-bound organelles. Transfected cells as described above are harvested and lysed. The lysate is fractionated using methods known to those of skill in the art, for example, sucrose gradient ultracentrifugation. Such methods allow the isolation of subcellular components such as the Golgi apparatus, ER, small membrane-bound vesicles, and other secretory organelles. Immunoprecipitations from fractionated and total cell lysates are performed using HSPP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The concentration of HSPP in secretory organelles relative to HSPP in total cell lysate is proportional to the amount of HSPP in transit through the secretory pathway.
XI. Functional Assays
HSPP function is assessed by expressing the sequences encoding HSPP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad Calif.), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York N.Y.
The influence of HSPP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HSPP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding HSPP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of HSPP Specific Antibodies
HSPP substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HSPP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides 15 residues in length are synthesized using an ABI 431A Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring HSPP Using Specific Antibodies
Naturally occurring or recombinant HSPP is substantially purified by immunoaffinity chromatography using antibodies specific for HSPP. An immunoaffinity column is constructed by covalently coupling anti-HSPP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing HSPP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HSPP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/HSPP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HSPP is collected.
XIV. Identification of Molecules which Interact with HSPP
HSPP, or biologically active fragments thereof, are labeled with 125I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HSPP, washed, and any wells with labeled HSPP complex are assayed. Data obtained using different concentrations of HSPP are used to calculate values for the number, affinity, and association of HSPP with the candidate molecules.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific 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.











TABLE 1





Protein
Nucleotide





SEQ ID NO:
SEQ ID NO:
Clone ID
Library
Fragments



















1
135
443531
MPHGNOT03
443531H1 (MPHGNOT03), 1406807F6 (LATRTUT02), 443531T6






(MPHGNOT03), SBBA00451F1, SBBA00676F1


2
136
632860
NEUTGMT01
632860H1 (NEUTGMT01), 784715R3 (PROSNOT05), 509590H1 (MPHGNOT03)


3
137
670010
CRBLNOT01
670010H1 (CRBLNOT01), 669971R1 (CRBLNOT01), 1553045F1 (BLADTUT04)


4
138
726498
SYNOOAT01
726498H1 (SYNOOAT01), 726498R6 (SYNOOAT01), 866599R3 (BRAITUT03)


5
139
795064
OVARNOT03
795064H1 (OVARNOT03), 4339458H1 (BRAUNOT02), 937605R3






(CERVNOT01), 2381151F6 (ISLTNOT01), 1466346F6 (PANCTUT02)


6
140
924925
BRAINOT04
924925H1 (BRAINOT04), 3268330H1 (BRAINOT20), 759120R3 (BRAITUT02)


7
141
962390
BRSTTUT03
962390H1 (BRSTTUT03), 1907958F6 (CONNTUT01), 023569F1 (ADENINB01),






167282F1 (LIVRNOT01), 1309211F1 (COLNFET02), SAUA00696F1,






SAUA02860F1


8
142
1259405
MENITUT03
1259405H1 (MENITUT03), 2472425H1 (THP1NOT03), 774303R1






(COLNNOT05), 1520779F1 (BLADTUT04), 1693833F6 (COLNNOT23),






1831858T6.comp (THP1AZT01), 1527737T6.comp (UCMCL5T01)


9
143
1297384
BRSTNOT07
1297384H1 (BRSTNOT07), 1269310F6 (BRAINOT09), 1457367F1






(COLNFET02), 415587R1 (BRSTNOT01), SANA02967F1


10
144
1299627
BRSTNOT07
1299627H1 (BRSTNOT07), 1359140F6 (LUNGNOT09), 1349224F1






(LATRTUT02), SBAA01431F1, SBAA02909F1, SBAA01156F1


11
145
1306026
PLACNOT02
1306026H1 (PLACNOT02), 1464088R6 (PANCNOT04), SBAA02496F1,






SBAA04305F1


12
146
1316219
BLADTUT02
1316219H1 (BLADTUT02), 2458603F6 (ENDANOT01), 2504756T6






(CONUTUT01)


13
147
1329031
PANCNOT07
1329031H1 (PANCNOT07), 1329031T6 (PANCNOT07), 1329031F6






(PANCNOT07)


14
148
1483050
CORPNOT02
1483050H1 (CORPNOT02), 855049H1 (NGANNOT01), 077017F1






(SYNORAB01), 1483050F6 (CORPNOT02), 1480024T6 (CORPNOT02),






1483050T6 (CORPNOT02), 759486R1 (BRAITUT02)


15
149
1514160
PANCTUT01
1514160H1 (PANCTUT01), 1866765T7 (SKINBIT01), 782676R1






(MYOMNOT01), 008055X4 (HMC1NOT01), 008055X5 (HMC1NOT01),






1866765F6 (SKINBIT01), SAOA03127F1


16
150
1603403
LUNGNOT15
1603403H1 (LUNGNOT15), 372910F1 (LUNGNOT02), 733299R7 (LUNGNOT03)


17
151
1652303
PROSTUT08
1652303H1 (PROSTUT08), 1671806H1 (BLADNOT05), 1341743T1






(COLNTUT03), 3803812H1 (BLADTUT03), 1878546F6 (LEUKNOT03),






1428640F1 (SINTBST01), 2058609R6 (OVARNOT03), 1331621F1






(PANCNOT07), 1306331T1 (PLACNOT02)


18
152
1693358
COLNNOT23
1693358H1 (COLNNOT23), 2498265H1 (ADRETUT05), 1867125F6






(SKINBIT01), 1693358T6 (COLNNOT23), 2245848R6 (HIPONON02)


19
153
1707711
DUODNOT02
1707711H1 (DUODNOT02), 1484609T1 (CORPNOT02), 1707711F6






(DUODNOT02), 1267959F1 (BRAINOT09), 1484609F1 (CORPNOT02),






SAJA00930F1, SAJA01300R1, SAJA00999R1


20
154
1738735
COLNNOT22
1738735H1 (COLNNOT22), SAJA00944R1, SAJA00137F1, SAJA03629F1


21
155
1749147
STOMTUT02
1749147H1 (STOMTUT02), 1749147F6 (STOMTUT02), 1749147T6






(STOMTUT02)


22
156
1817722
PROSNOT20
1817722H1 (PROSNOT20), 2011085H1 (TESTNOT03)


23
157
1831290
THP1AZT01
1831290H1 (THP1AZT01), 3473958H1 (LUNGNOT27), 1972268F6






(UCMCL5T01), 1301277F1 (BRSTNOT07), 1521574F1 (BLADTUT04),






1561690T6 (SPLNNOT04), 891461R1 (STOMTUT01)


24
158
1831477
THP1AZT01
1831477H1 (THP1AZT01), 1582867H1 (DUODNOT01), 1336769T1






(COLNNOT13), 1933092H1 (COLNNOT16), 1519909F1 (BLADTUT04),






1220946H1 (NEUTGMT01), 809556T1 (LUNGNOT04), 1217559T1






(NEUTGMT01), 1309225F1 (COLNFET02)


25
159
1841607
COLNNOT07
1841607H1 (COLNNOT07), SBHA03588F1


26
160
1852391
LUNGFET03
1852391H1 (LUNGFET03), 734140H1 (TONSNOT01), 1852391F6 (LUNGFET03)


27
161
1854555
HNT3AZT01
1854555H1 (HNT3AZT01), 2511711H1 (CONUTUT01), 782453R1






(MYOMNOT01), 1854555F6 (HNT3AZT01), 1840675T6 (COLNNOT07),






2109736H1 (BRAITUT03)


28
162
1855755
PROSNOT18
1855755H1 (PROSNOT18), 3040236H1 (BRSTNOT16), 1283207F1






(COLNNOT16), 833763T1 (PROSNOT07), 1920926R6 (BRSTTUT01)


29
163
1861434
PROSNOT19
1861434H1 (PROSNOT19), 980291R1 (TONGTUT01), 1861434T6






(PROSNOT19), SARA01525F1, SARA02548F1


30
164
1872334
LEUKNOT02
1872334H1 (LEUKNOT02), 1872334F6 (LEUKNOT02), SBGA03684F1


31
165
1877230
LEUKNOT03
1877230H1 (LEUKNOT03), 2519841H1 (BRAITUT21), 1877230T6






(LEUKNOT03), 1254693F1 (LUNGFET03), 077020R1 (SYNORAB01),






1232336F1 (LUNGFET03), 1004952R6 (BRSTNOT03), SARA01879F1,






SARA02654F1


32
166
1877885
LEUKNOT03
1877885H1 (LEUKNOT03), 508020F1 (TMLR3DT01), 2751126R6 (THP1AZS08),






SARA02571F1


33
167
1889269
BLADTUT07
1889269H1 (BLADTUT07), 1915551H1 (PROSTUT04), 629493X12






(KIDNNOT05), 1441289F1 (THYRNOT03), 1215274X34F1 (BRSTTUT01),






1818447F6 (PROSNOT20), 1208463R1 (BRSTNOT02)


34
168
1890243
BLADTUT07
1890243H1 (BLADTUT07), SARA01884F1, SATA00046F1, SARA03294F1,






SARA02790F1


35
169
1900433
BLADTUT06
1900433H1 (BLADTUT06), SATA00396F1, SATA02742F1


36
170
1909441
CONNTUT01
1909441H1 (CONNTUT01), 1398811F1 (BRAITUT08), 3039939H1






(BRSTNOT16), 3324740H1 (PTHYNOT03), 1442131F6 (THYRNOT03),






2254056H1 (OVARTUT01), 2199453T6 (SPLNFET02), 1692610F6






(COLNNOT23), 1698531H1 (BLADTUT05)


37
171
1932226
COLNNOT16
1932226H1 (COLNNOT16), 2320569H1 (OVARNOT02), 1932226F6






(COLNNOT16), 2469455T6 (THP1NOT03), 2469455F6 (THP1NOT03),






1907140F6 (OVARNOT07), SATA02592F1


38
172
1932647
COLNNOT16
1932647H1 (COLNNOT16), 1492745T1 (PROSNON01), 1492745H1






(PROSNON01), SASA02355F1, SASA00117F1, SASA00192F1


39
173
2124245
BRSTNOT07
2124245H1 (BRSTNOT07), 1235393F1 (LUNGFET03), 1402264F6






(LATRTUT02), 1303990F1 (PLACNOT02), 1402264T6 (LATRTUT02)


40
174
2132626
OVARNOT03
2132626H1 (OVARNOT03), 1723432T6 (BLADNOT06), 2132626R6






(OVARNOT03), 1736723T6 (COLNNOT22), 1504738F1 (BRAITUT07)


41
175
2280639
PROSNON01
2280639H1 (PROSNON01), 1435330H1 (PANCNOT08), 1377560F6






(LUNGNOT10)


42
176
2292356
BRAINON01
2292356H1 (BRAINON01), 4086827H1 (LIVRNOT06), 1754442F6 (LIVRTUT01),






3571126H1 (HEAPNOT01), 1601305F6 (BLADNOT03)


43
177
2349310
COLSUCT01
2349310H1 (COLSUCT01), 2349310T6 (COLSUCT01)


44
178
2373227
ADRENOT07
2373227H1 (ADRENOT07), 3316444H1 (PROSBPT03), 302685R6 (TESTNOT04),






SASA02181F1, SASA01923F1, SASA03516F1


45
179
2457682
ENDANOT01
2457682H1 (ENDANOT01), 2457682F6 (ENDANOT01)


46
180
2480426
SMCANOT01
2480426H1 (SMCANOT01), 2480426F6 (SMCANOT01)


47
181
2503743
CONUTUT01
2503743H1 (CONUTUT01), 1853909H1 (HNT3AZT01), 1517619F1






(PANCTUT01), 1467896F6 (PANCTUT02), 490031F1 (HNT2AGT01), 1208654R1






(BRSTNOT02), 880544R1 (THYRNOT02)


48
182
2537684
BONRTUT01
2537684H1 (BONRTUT01), 2005493H1 (TESTNOT03), 730969H1






(LUNGNOT03), 2537601F6 (BONRTUT01), 916487H1 (BRSTNOT04), 996135R1






(KIDNTUT01), 1920738R6 (BRSTTUT01), 1957710F6 (CONNNOT01)


49
183
2593853
OVARTUT02
2593853H1 (OVARTUT02), 807497H1 (STOMNOT02), 914020R6






(STOMNOT02), 889992R1 (STOMTUT01)


50
184
2622354
KERANOT02
2622354H1 (KERANOT02), 2623992H1 (KERANOT02), 1556510F6






(BLADTUT04)


51
185
2641377
LUNGTUT08
2641377H1 (LUNGTUT08), 4341415H2 (BRAUNOT02), SBCA07049F3


52
186
2674857
KIDNNOT19
2674857H1 (KIDNNOT19), 1872373H1 (LEUKNOT02), 470512R6






(MMLR1DT01), 1728547H1 (PROSNOT14), 3013651F6 (MUSCNOT07),






SBCA01366F1, SBCA00694F1


53
187
2758485
THP1AZS08
2758485H1 (THP1AZS08), 3097533H1 (CERVNOT03), 1578959F6






(DUODNOT01)


54
188
2763296
BRSTNOT12
2763296H1 (BRSTNOT12), 3486025F6 (KIDNNOT31), SBDA07002F3


55
189
2779436
OVARTUT03
2779436H1 (OVARTUT03), 2779436F6 (OVARTUT03), SBDA07009F3


56
190
2808528
BLADTUT08
2808528H1 (BLADTUT08), 2611513F6 (THYMNOT04), SBDA07021T3


57
191
2809230
BLADTUT08
2809230H1 (BLADTUT08), 2213849H1 (SINTFET03), 711706R6 (SYNORAT04),






958323R1 (KIDNNOT05), 030732F1 (THP1NOB01)


58
192
2816821
BRSTNOT14
2816821H1 (BRSTNOT14), 3746964H1 (THYMNOT08), 2816821F6






(BRSTNOT14), 948722T6 (PANCNOT05), 807947R6 (STOMNOT02)


59
193
2817268
BRSTNOT14
2817268H1 (BRSTNOT14), 3591308H1 (293TF5T01), 419522R1 (BRSTNOT01),






2073028F6 (ISLTNOT01), 1308781F6 (COLNFET02)


60
194
2923165
SININOT04
2923165H1 (SININOT04), 2011630H1 (TESTNOT03), 1457250F1 (COLNFET02),






754668R1 (BRAITUT02), 1406510F6 (LATRTUT02)


61
195
2949822
KIDNFET01
2949822H1 (KIDNFET01), SBDA07078F3


62
196
2992192
KIDNFET02
2992192H1 (KIDNFET02), 2534324H2 (BRAINOT18), 2815255T6






(OVARNOT10), 1551107T6 (PROSNOT06), 1551107R6 (PROSNOT06)


63
197
2992458
KIDNFET02
2992458H1 (KIDNFET02), 2618951H1 (GBLANOT01), 1479252F1






(CORPNOT02), 1879054H1 (LEUKNOT03), 1879054F6 (LEUKNOT03),






2215240H1 (SINTFET03), 1535968T1 (SPLNNOT04)


64
198
3044710
HEAANOT01
3044710H1 (HEAANOT01), 3741773H1 (MENTNOT01), 859906X42C1






(BRAITUT03), 1534347F1 (SPLNNOT04), 1421122F1 (KIDNNOT09), 1303865F1






(PLACNOT02), 1704452F6 (DUODNOT02), 1251642F1 (LUNGFET03),






1781694R6 (PGANNON02)


65
199
3120415
LUNGTUT13
3120415H1 (LUNGTUT13), 1360123T1 (LUNGNOT12), 1375015H1






(LUNGNOT10)


66
200
126758
LUNGNOT01
126758H1 (LUNGNOT01), 126758X11 (LUNGNOT01), 811864T1 (LUNGNOT04)


67
201
674760
CRBLNOT01
674760H1 (CRBLNOT01), 3253976H1 (OVARTUN01), SAUA03387F1


68
202
1229438
BRAITUT01
1229438H1 (BRAITUT01), 1230616H1 (BRAITUT01), 1461187R1






(PANCNOT04), 2493039H1 (ADRETUT05), 2891628H1 (LUNGFET04)


69
203
1236935
LUNGFET03
1236935H1 (LUNGFET03), SBAA00983F1, SBAA02057F1, SBAA00170F1


70
204
1359283
LUNGNOT12
1359283H1 (LUNGNOT12), SBAA01213F1, SBAA03934F1


71
205
1450703
PENITUT01
551298F1 (BEPINOT01), 551298R1 (BEPINOT01), 1450703H1 (PENITUT01),






2748715H1 (LUNGTUT11)


72
206
1910668
CONNTUT01
1269346H1 (BRAINOT09), 1380872F1 (BRAITUT08), 1910668F6






(CONNTUT01), 1910668H1 (CONNTUT01), SATA02800F1, SATA03799F1,






SARA02035F1


73
207
1955143
CONNNOT01
1955143F6 (CONNNOT01), 1955143H1 (CONNNOT01)


74
208
1961637
BRSTNOT04
867025H1 (BRAITUT03), 1961637H1 (BRSTNOT04), 2809064T6 (BLADTUT08),






2938714H1 (THYMFET02), 2956402H1 (KIDNFET01), 3808735T6






(CONTTUT01)


75
209
1990762
CORPNOT02
1990762H1 (CORPNOT02), 1990762T3 (CORPNOT02), SBGA04911F1,






SBGA01201F1, SBGA02205F1


76
210
1994131
CORPNOT02
1994131H1 (CORPNOT02), 2645984F6 (OVARTUT04)


77
211
1997745
BRSTTUT03
1752307F6 (LIVRTUT01), 1853730H1 (HNT3AZT01), 1997745H1 (BRSTTUT03),






SAZA00953F1


78
212
2009035
TESTNOT03
2009035H1 (TESTNOT03), 2009035R6 (TESTNOT03)


79
213
2009152
TESTNOT03
2009152H1 (TESTNOT03), 2009152R6 (TESTNOT03), 2783263H1 (BRSTNOT13)


80
214
2061752
OVARNOT03
2061752H1 (OVARNOT03), 2061752T6 (OVARNOT03), 2732805H1






(OVARTUT04), SAZA01310F1, SAZA00830F1


81
215
2061933
OVARNOT03
046580R1 (CORNNOT01), 746061R1 (BRAITUT01), 826996R1 (PROSNOT06),






2061933H1 (OVARNOT03)


82
216
2081422
UTRSNOT08
2081422F6 (UTRSNOT08), 2081422H1 (UTRSNOT08), SBCA04793F1,






SBCA05657F1, SBDA00065F1


83
217
2101278
BRAITUT02
2101278H1 (BRAITUT02), SAXA00399F1, SAXA01284F1, SAXA01227F1


84
218
2121353
BRSTNOT07
341437H1 (NEUTFMT01), 687136H1 (UTRSNOT02), 2121353H1 (BRSTNOT07),






SASA01311F1


85
219
2241736
PANCTUT02
833263H1 (PROSTUT04), 2241736H1 (PANCTUT02), SAZA01148F1,






SASA03299F1, SASA01349F1


86
220
2271935
PROSNON01
2271935H1 (PROSNON01), 2276774H1 (PROSNON01), 2760171T6






(THP1AZS08)


87
221
2295344
BRSTNOT05
2295344H1 (BRSTNOT05), 3288561F6 (BONRFET01), SBGA01801F1


88
222
2303994
BRSTNOT05
905482T1 (COLNNOT08), 1858636F6 (PROSNOT18), 2303994H1 (BRSTNOT05)


89
223
2497805
ADRETUT05
2497805F6 (ADRETUT05), 2497805H1 (ADRETUT05)


90
224
2646362
LUNGTUT11
1754702H1 (LIVRTUT01), 2640776T6 (LUNGTUT08), 2646362H1






(LUNGTUT11), 3356773H1 (PROSTUT16)


91
225
2657146
LUNGTUT09
2657146F6 (LUNGTUT09), 2657146H1 (LUNGTUT09)


92
226
2755786
THP1AZS08
288436R1 (EOSIHET02), 1252824F6 (LUNGFET03), 1305549H1 (PLACNOT02),






1364975R1 (SCORNON02), 2018293H1 (THP1NOT01), 2047320H1






(THP1T7T01), 2184537F6 (SININOT01), 2755786H1 (THP1AZS08), 4111022H1






(PROSBPT07)


93
227
2831245
TLYMNOT03
2831245H1 (TLYMNOT03), SBMA01396F1


94
228
3116250
LUNGTUT13
126263F1 (LUNGNOT01), 2729942H1 (OVARTUT04), 3116250H1






(LUNGTUT13)


95
229
3129630
LUNGTUT12
3129630F6 (LUNGTUT12), 3129630H1 (LUNGTUT12), SBDA06436F1


96
230
007632
HMC1NOT01
007632H1 (HMC1NOT01), 007632R6 (HMC1NOT01), 007632T6 (HMC1NOT01)


97
231
1236968
LUNGFET03
1236968H1 (LUNGFET03), SBAA02713F1, SBAA03203F1, SBAA04196F1


98
232
1334153
COLNNOT13
776410R1 (COLNNOT05), 1334153H1 (COLNNOT13), 1334153T1






(COLNNOT13), 1800085F6 (COLNNOT27), 2701948H1 (OVARTUT10)


99
233
1396975
BRAITUT08
864113H1 (BRAITUT03), 876139R1 (LUNGAST01), 1268313F1 (BRAINOT09),






1351348T1 (LATRTUT02), 1396975H1 (BRAITUT08), 1485768F6






(CORPNOT02), 1815364F6 (PROSNOT20)


100
234
1501749
SINTBST01
079080R1 (SYNORAB01), 1501749H1 (SINTBST01), 1724970H1 (PROSNOT14)


101
235
1575240
LNODNOT03
081858R1 (SYNORAB01), 1575240H1 (LNODNOT03), 3451462R6






(UTRSNON03)


102
236
1647884
PROSTUT09
1647884H1 (PROSTUT09), 1647884T6 (PROSTUT09), 3998922R6 (HNT2AZS07)


103
237
1661144
BRSTNOT09
720941X17 (SYNOOAT01), 1661144H1 (BRSTNOT09), 2181782H1 (SININOT01)


104
238
1685409
PROSNOT15
755203R1 (BRAITUT02), 1226185T1 (COLNNOT01), 1300837F1 (BRSTNOT07),






1685409H1 (PROSNOT15), 1705256H1 (DUODNOT02)


105
239
1731419
BRSTTUT08
1731419H1 (BRSTTUT08), 1731419X319T3 (BRSTTUT08), 1731419X322F1






(BRSTTUT08), 1731419X326F1 (BRSTTUT08), 1731419X329F1 (BRSTTUT08),






1733786F6 (BRSTTUT08), SZAH01494F1


106
240
2650265
BRSTNOT14
1680316T6 (STOMFET01), 2650265H1 (BRSTNOT14), 2650265T6






(BRSTNOT14), 2760588R6 (BRAINOS12)


107
241
2677129
KIDNNOT19
1592129H1 (CARGNOT01), 2645962H1 (OVARTUT04), 2677129F6






(KIDNNOT19), 2677129H1 (KIDNNOT19), 2910973H1 (KIDNTUT15),






4571722H1 (PROSTMT02), 4906791H2 (TLYMNOT08)


108
242
3151073
ADRENON04
3150857T6 (ADRENON04), 3151073H1 (ADRENON04), 3151073R6






(ADRENON04)


109
243
3170095
BRSTNOT18
3170095F6 (BRSTNOT18), 3170095H1 (BRSTNOT18)


110
244
3475168
LUNGNOT27
079680F1 (SYNORAB01), 443811T6 (MPHGNOT03), 1509356T6






(LUNGNOT14), 1873596F6 (LEUKNOT02), 2440867H1 (EOSITXT01),






3475168H1 (LUNGNOT27)


111
245
3836893
DENDTNT01
446637H1 (MPHGNOT03), 1219376R6 (NEUTGMT01), 3735467F6






(SMCCNOS01), 3735467T6 (SMCCNOS01), 3836893H1 (DENDTNT01)


112
246
4072159
KIDNNOT26
2129415T6 (KIDNNOT05), 4072159F6 (KIDNNOT26), 4072159H1






(KIDNNOT26)


113
247
1003916
BRSTNOT03
620937R6 (PGANNOT01), 1003916H1 and 1003916R6 (BRSTNOT03),






1413623H1 (BRAINOT12), 1435945F1 (PANCNOT08), 1479127F1






(CORPNOT02), 1969146R6 (BRSTNOT04), 2517587F6 (BRAITUT21),






2967848H1 (SCORNOT04)


114
248
2093492
PANCNOT04
489651H1 (HNT2AGT01), 1265353T1 (SYNORAT05), 1431505R6 (BEPINON01),






1605237F6 (LUNGNOT15), 2093492H1 and 2093492T6 (PANCNOT04),






4195560H1 (COLITUT02)


115
249
2108789
BRAITUT03
2108789H1 and 2108789R6 (BRAITUT03), 2182008T6 (SININOT01), 3255751R6






and 3255751T6 (OVARTUN01)


116
250
2171401
ENDCNOT03
037241F1 (HUVENOB01), 1821492F6 (GBLATUT01), 2055814T6 (BEPINOT01),






2171401F6 and 2171401H1 (ENDCNOT03), 2668952F6 (ESOGTUT02),






3140313H1 and 3140313T6 (SMCCNOT02), 5031775H1 (EPIBTXT01)


117
251
2212530
SINTFET03
187596R6 and 187596T6 (CARDNOT01), 919634R6 (RATRNOT02), 1992331H1






(CORPNOT02), 2062034H1 (OVARNOT03), 2212530F6 and 2212530H1






(SINTFET03), 2520479H1 (BRAITUT21), 2878284F6 (THYRNOT10), 2992354H1






(KIDNFET02), 4020719F6 (BRAXNOT02)


118
252
2253036
OVARTUT01
2253036H1 and 2253036R6 (OVARTUT01)


119
253
2280161
PROSNON01
482326H1 (HNT2RAT01), 934345H1 (CERVNOT01), 1379358F1 and 1379358T1






(LUNGNOT10), 1438562T1 (PANCNOT08), 1467511F6 (PANCTUT02),






1568138F1 (UTRSNOT05), 1636106T6 (UTRSNOT06), 2134534F6






(ENDCNOT01), 2280161H1 and 2280161X19F1 (PROSNON01), 2789845F6






(COLNTUT16), 3096938H1 (CERVNOT03), 3774621F6 (BRSTNOT25),






4222971H1 (PANCNOT07), 5111983H1 (ENDITXT01), 5324177H1 (FIBPFEN06)


120
254
2287485
BRAINON01
1454588F1 (PENITUT01), 1593332F6 (BRAINOT14), 2287485H1 and 2287485R6






(BRAINON01), 3765992H1 (BRSTNOT24), 4374293H1 (CONFNOT03),






4937931H1 (PROSTUS18), SBCA01722F1


121
255
2380344
ISLTNOT01
2380344F6 and 2380344H1 (ISLTNOT01), 2888536T3 (LUNGFET04),






SASA03644F1, SASA03689F1


122
256
2383171
ISLTNOT01
956296R1 (KIDNNOT05), 1342250F1 (COLNTUT03), 1468046F1 and 1468046T1






(PANCTUT02), 2383171H1 (ISLTNOT01), SBYA05452U1, SBYA01369U1


123
257
2396046
THP1AZT01
2396046F6, 2396046H1 and 2396118T6 (THP1AZT01)


124
258
2456587
ENDANOT01
2456587H1 and 2456587T6 (ENDANOT01), 2872569H1 (THYRNOT10),






SBCA03778F1, SBDA00115F1, SBCA02401F1, SBCA03351F1, SBCA05164F1,






SBCA04783F1, SBCA00155F1, SBCA04141F1


125
259
2484813
BONRTUT01
1234970T1 (LUNGFET03), 1338090F6 (COLNNOT13), 2484813H1






(BONRTUT01), SBCA00053F1, SBCA02064F1, SBCA02151F1, SBCA03770F1,






SBCA04866F1, SBCA03406F1


126
260
2493851
ADRETUT05
2493851H1 (ADRETUT05), 3805916F6 (BLADTUT03), 4500439H1 and






4500748H1 (BRAVTXT02), 5120601H1 (SMCBUNT01)


127
261
2495719
ADRETUT05
603447R1 (BRSTTUT01), 2495719H1 (ADRETUT05), 2917493F6 (THYMFET03),






4647103H1 (PROSTUT20), SBRA04984D1


128
262
2614153
GBLANOT01
1833135R6 (BRAINON01), 1966515R6 (BRSTNOT04), 2331103R6






(COLNNOT11), 2614153H1 (GBLANOT01), 2656691F6 (LUNGTUT09),






3951176H1 (DRGCNOT01)


129
263
2655184
THYMNOT04
2655184H1 (THYMNOT04), SBDA05215F1, SBDA05213F1, SBDA01516F1


130
264
2848362
BRSTTUT13
1297974F1 and 1297974T6 (BRSTNOT07), 2630138F6 (COLNTUT15),






2848362H1 (BRSTTUT13)


131
265
2849906
BRSTTUT13
1541617R1 and 1541617T1 (SINTTUT01), 2684504F6 and 2684504T6






(LUNGNOT23), 2796805H1 (NPOLNOT01), 2849906H1 (BRSTTUT13)


132
266
2899137
DRGCNOT01
2899137H1 (DRGCNOT01), 3026490F6 and 3026490T6 (HEARFET02),






3483359H1 (KIDNNOT31)


133
267
2986229
CARGDIT01
1740227T6 (HIPONON01), 2986229H1 (CARGDIT01)


134
268
3222081
COLNNON03
1754079F6 (LIVRTUT01), 3222081H1 (COLNNON03), 4053813T6






(SPLNNOT13), 4230282H1 (BRAMDIT01), SBDA07029F3






















TABLE 2






Amino
Potential
Potential





Protein
Acid
Phosphorylation
Glycosylation
Signature

Analytical


SEQ ID NO:
Residues
Sites
Sites
Sequences
Identification
Methods





















1
88
T83 S38 T76

M1-A21

Signal Peptide








HMM


2
128
S30 S40 T47 T119

M1-F28

Signal Peptide




W125



HMM


3
111
T70

M1-T18

Signal Peptide








HMM


4
110
S32 T64
N58
M1-A29

Signal Peptide








HMM


5
78
T27 S39 S39 S44

M1-R24

Signal Peptide




S22 T27 S28 S57



HMM


6
88
T55 S30 S40 T55
N34
M1-N21

Signal Peptide








HMM


7
227
S220 S70 S83 T131
N100
M1-Q20

Signal Peptide




S134 S141 T158



HMM




Y123


8
198
S62 T123 S142
N60
M1-A28

Signal Peptide




S189 S62 T100



HMM




Y85


9
65
T48

M1-A29

Signal Peptide








HMM


10
154


M1-A29

Signal Peptide








HMM


11
237
T116 T26 T79 T85
N128
M1-A19

Signal Peptide




T182 T188 T194



HMM




T206 S60 S123




S176 S213


12
225
T158 S128
N166
M1-G27

Signal Peptide








HMM


13
117
S41

M1-A23

Signal Peptide








HMM


14
253
S49 T63 S92 T110
N42 N47 N72
M1-T20

Signal Peptide




S127 T239
N207


HMM


15
171
S43 S94 T114

M88-R112

Signal Peptide








HMM


16
78
S38 S43
N37
M1-G19

Signal Peptide








HMM


17
71
T64 T67

M1-C19

Signal Peptide








HMM


18
188
S36 T58 T133 Y31
N121 N171
M1-A21

Signal Peptide








HMM


19
80
S76

M1-C19

Signal Peptide








HMM


20
80


M1-G25

Signal Peptide








HMM


21
84
S39 S53 S60

M1-G21

Signal Peptide








HMM


22
171
S41 T150

M3-A21

Signal Peptide








HMM


23
243
S3 S44 T75 S86
N97
M1-C25

Signal Peptide




S183 S223 S36 S92



HMM




S205 Y40 Y110


24
311
T5 S76 T82 T93
N49 N91 N108
M1-A32

Signal Peptide




T109 S121 T137
N128 N135


HMM




T170 S184 S11 T53
N190




S75 S84 T132 S223




S274 Y69


25
57


M1-L29

Signal Peptide








HMM


26
82
S46 Y26

M1-S18

Signal Peptide








HMM


27
115


M1-G34

Signal Peptide








HMM


28
327
S93 S50 S167 S233
N138 N206
M1-E25

Signal Peptide




S89 T105 T214



HMM




S302 T318


29
133
S63
N105
M1-E29

Signal Peptide








HMM


30
129
S21 S65 T93

M1-G20

Signal Peptide








HMM


31
472
S164 T32 S42 T141
N61 N179 N353
M1-G20
hematopoietic lineage
Signal Peptide




T154 S155 T235
N356 N396

switch 2 (g3169729)
HMM




T262 T271 T334



BLAST -




T376 S402 S421



GenBank




S435 T441 S19




S29 T327 S378


32
93
T21

M1-A18

Signal Peptide








HMM


33
92
S57 S5

M1-G47

SPScan


34
143
T6 T14 T135

M9-G40

Signal Peptide








HMM


35
89
T15 S58 S66

M1-A19

Signal Peptide








HMM


36
560
T7 T76 S150 T224
N163 N184
M1-E34

SPScan




S228 S257 S358
N379




S474 S529 S539




T186 S219 S368




Y523


37
197
T80 S163

M1-G28

Signal Peptide








HMM


38
437
T47 T146 S233 S391
N46 N189 N382
M1-A21

Signal Peptide




S403 T43 S130 S273



HMM




S339 S364


39
330
S197 T49 T150 S193
N46 N64 N166
M1-G28

Signal Peptide




T214 T215 T49 S111
N191


HMM




S237


40
148
T73 S141
N29 N58 N71
M1-R24
receptor-activity-
Signal Peptide





N103

modifying protein
HMM







(RAMP; g4165368)
BLAST -








GenBank


41
188
S49

M1-V25

Signal Peptide








HMM


42
222
S89 S165 T174 T182

M1-S24

Signal Peptide




T83 S155



HMM


43
111
S54 S29 S98 S50

M1-T23

Signal Peptide




S57 T104



HMM


44
341
T29 S106 T120 S161

M1-G22

Signal Peptide




S195 S37 S47 T51



HMM




S136 S223 S230




S281


45
148
S21 T63 T63 A146
N40
M1-G23

Signal Peptide








HMM


46
87
S65

M1-P18

Signal Peptide








HMM


47
383
T77 S95 S108 S280
N93 N207
M1-P23

Signal Peptide




S351 S121 S124



HMM




S153 T187


48
109
S25 S22

M1-L18

Signal Peptide








HMM


49
185
S62

M1-A20

Signal Peptide








HMM


50
110
T100 T73 S97 Y48
N71
M1-C21

Signal Peptide








HMM


51
126
S17 S110

M1-G18

Signal Peptide








HMM


52
488
S205 T31 S86 T236
N250 N321
M1-L25
putative involvement
Signal Peptide




S7 T447
N463

in cell wall structure
HMM







or biosynthesis (g3738170)
BLAST -








GenBank


53
197
T55 S34 S46 S69

M1-A26

Signal Peptide




T98 S108 T119 T167



HMM




S194 S2 S34 T153


54
84
S65 S36 T41 S51
N39
M1-G25

Signal Peptide




S69 S81



HMM


55
97
S56

M1-A22

Signal Peptide








HMM


56
140
S29

M1-P23

Signal Peptide








HMM


57
285
S53 S108 T216
N153
M1-A25

Signal Peptide




S253 S277



HMM


58
262
S62 T166 S62 S71
N190
M1-G28
3-acylating enzyme
Signal Peptide




Y246


(Q44449)
HMM








BLAST -








GENESEQ


59
189
S120 T154 T34 T37

M1-C22

Signal Peptide




S174



HMM


60
257
S98 T136 T67 S112

M55-E84β

SPScan




S234 S237


61
82
T68
N67
M1-G18

Signal Peptide








HMM


62
202
T21 S117 S120

M1-G27

Signal Peptide








HMM


63
450
S107 S97 S146

M1-G18

Signal Peptide




S339 S440 S245



HMM




T303 S304 S399


64
322
T145 T214 T16 S24
N53 N130 N289
M1-G23

Signal Peptide




S35 S45 T145 T269



HMM




S297 T300 T314




Y87


65
104
S38 S25 S75

M1-A18

Signal Peptide








HMM


66
93


M1 through about S18

SPscan






Transmembrane:

HMM






M1 through about Y17


67
71
S23 S64

M1 through about A24

SPscan








HMM








MOTIFS


68
394
S392 S393 S31
N53
M1 through about S31

SPScan




S127 S179 S334

Transmembrane:

HMM




T338 S358 T383

about M159 through

MOTIFS




Y323

about F178 about F109






through about S127






about F225 through






about V243


69
72
S59
N69
M1 through about S23

SPscan






Transmembrane:

HMM






M1 through about L16

MOTIFS


70
71
S11 T26

M1 through about Q18

SPscan








HMM








MOTIFS


71
247
S41 T79

M1 through about S25

SPscan








HMM








MOTIFS


72
73
S56

M1 through about G27

SPscan








HMM








MOTIFS


73
70


M1 through about G20

SPscan








HMM


74
67


M1 through about G30

SPscan








HMM


75
91


M1 through about G26

SPScan


76
56
T29 S46 T51

M1 through about S19

SPscan








HMM








MOTIFS


77
112
S62 S65

M1 through about G27

SPscan






Transmembrane:

HMM






about W79 through

MOTIFS






about H97


78
54

N48
M1 through about N34

SPscan








HMM








MOTIFS


79
57
T33 R55

M1 through about C18

SPscan








HMM








MOTIFS


80
52
S34

M1 through about S30

SPscan








HMM








MOTIFS


81
64
T43 Y27

M1 through about S41

SPscan








HMM








MOTIFS


82
65
S45

M1 through about A31

SPscan






Transmembrane:

HMM






about L38 through

MOTIFS






about F55


83
56


M1 through about E23

SPscan








HMM


84
120
S69 S109
N89 N95
M1 through about A38

SPscan






Transmembrane: about

HMM






L23 through about T41

MOTIFS


85
67
S28

M1 through about K30

SPscan






Microbodies C-terminal

HMM






targetting signal:

MOTIFS






A65KV


86
62
S29 S42 S46
N40
M1 through about S29

SPscan








HMM








MOTIFS


87
75
S25 S46

M1 through about L19

SPscan






Transmembrane:

HMM






about I3 through

MOTIFS






about G20


88
80
T28

M1 through about A20

SPscan








HMM








MOTIFS


89
50
S11

M1 through about C48

SPscan








HMM








MOTIFS


90
116
S38

M1 through about G22

SPscan








HMM








MOTIFS


91
67
S43

M1 through about P21

SPscan








HMM








MOTIFS


92
538
S415 S52 T77 S97
N226
M1 through about S18

SPScan




T178 T228 S282

Tyrosine specific

BLOCKS




S320 S332 S384

protein phosphatases

PRINTS




T401 T424 S483

signature: about V328

MOTIFS




S207 S230 S357

through about F340




T410 Y263 Y365


93
58


M1 through about S25

SPscan








HMM


94
119
S39

M1 through about S22

SPscan






Transmembrane:

HMM






about V3 through

MOTIFS






about S21


95
128
S91

M1 through about G31

SPscan






Transmembrane:

HMM






about F108 through

MOTIFS






about L126


96
124
T115 T43 S91

M1-S20 P116-V124

SPScan






(urotensin II

HMM






signature)

Motifs








BLOCKS








BLAST


97
182
S28 T70 S172

M1-S23, M1-S25

SPScan




S25 S32 S48



HMM




S108 S131



Motifs


98
237
S55 S88 S121
N45 N73 N107
M1-A16, M1-S21 C40-C198

SPScan




S135
N118 N132
(cysteine spacing

HMM





N172 N175
pattern similar

Motifs





N185
to that of RoBo-1)

BLAST


99
160
S36 S59 T143

M1-A27

SPScan








HMM








Motifs


100
148
T76 S64 Y103

M1-S30, M1-G31

SPScan








HMM








Motifs


101
170
S78 T4 T30 S130

M1-A23, M1-L28

SPScan




S25 S29 T122



HMM








Motifs


102
150
S50 S78 S91

M1-A26, M1-S28

SPScan








HMM








Motifs


103
142
T57 T80

M1-A25, M1-G26

SPScan








HMM








Motifs


104
110
T3

M1-G18, M1-T25

SPScan








HMM








Motifs


105
120
T29 S40 S72

M1-G22, M1-A20

SPScan








HMM








Motifs


106
135
T115 S38 T41
N32 N101
M1-G26, M1-C25

SPScan








HMM








Motifs


107
301
S53 S217 S240

M1-A22

SPScan




S283 T224



HMM








Motifs


108
103
S88 T73 S84

M1-P19, M1-L22

SPScan








HMM








Motifs


109
95
T82 S52 S77
N50
M1-T15, M1-P19

SPScan








HMM








Motifs


110
113
T84 S4

M1-P19, M1-A24

SPScan








HMM








Motifs


111
234
S179 S184 S51
N146 N191
M1-A20
NK cell activating
SPScan




T70 T158 S168
N194

receptor (g4493702)
HMM




T228 Y29



Motifs








BLAST -








GenBank


112
119
S39 T61

M1-G30, M1-G27

SPScan








HMM








Motifs


113
200
S51 T46 S191

M1-G26 Signal
Signal Peptide
SPScan






Peptide
Containing Protein,
Motifs







Homology with KIAA0206
BLAST


114
225


M1-Q29 Signal
Signal Peptide
SPScan






Peptide
Containing Protein


115
155
S29

M1-A20 Signal
Signal Peptide
HMM






Peptide
Containing Protein
Motifs


116
468
S143 T156 T227
N280 N384
M1-G23 Signal
Signal Peptide
SPScan




S235 T271 T293

Peptide
Containing Protein
Motifs




T436 S453 S117




T148 T213 S263




S417 Y73


117
403
S19 S320 S69
N87
M1-A24 Signal
Signal Peptide
HMM




S151 T171 T97 S393

Peptide
Containing Protein
Motifs




Y193 Y378


118
131
T131 S24 T79 T118
N116
M1-G25 Signal
Signal Peptide
SPScan




T123 T127

Peptide
Containing Protein
Motifs


119
556
T176 S192 S196
N62 N79 N127
M1-P21 Signal
Signal Peptide
SPScan




T220 S344 S369
N157 N160
Peptide L226-W244,
Containing Protein,
Motifs




S476 T501 S529

Y402-W422, V375-
Weakly similar to
HMM




S541 T548 T553

L392 and Y355-I376
Putative Transmembrane
BLAST




S48 S115 S121

Transmembrane Domains
Protein (PTM1)




T386 T424 S500


Precursor




Y104


120
514
T457 T80 S86 T141
N100 N168
M1-G24 Signal
Signal Peptide
SPScan




T372 T420 S447
N319
Peptide
Containing Protein,
Motifs




S94 T102 S112




T240 S297 S353




S470


121
109
T46 S78 T12

M1-S15 Signal
Signal Peptide
SPScan






Peptide
Containing Protein
MotifS


122
431
S57 T320 S339

M1-L25 Signal
Signal Peptide
SPScan




S396 S100 S239

Peptide
Containing Protein,
Motifs







Weakly similar to
BLAST







OXA1L


123
142


M1-W16 Signal
Signal Peptide
SPScan






Peptide
Containing Protein


124
643
T8 S28 S77 T169
N251
M1-S28 Signal
Signal Peptide
SPScan




T199 T235 S252

Peptide, D37-C81,
Containing Protein,
Motifs




T320 S402 T413

W380-C437, W440-
Thrombospondin Type
Pfam




S414 S558 S22 T25

C492 and F526-C583
1 Protein
BLAST




S56 S62 S120 T184

Thrombospondin Type




S329 T423 S475

1 Domains




S574 Y226


125
568
S510 T24 T80 S91
N322
M1-T19 Signal
Signal Peptide
SPScan




T153 T165 S232

Peptide
Containing Protein
Motifs




S248 S262 T300




T334 S380 S446 S16




T19 T60 S127 S273




T436 T531 S554




T564 Y135 Y489


126
125
T62 S27 T36

M1-R32 Signal
Signal Peptide
SPScan






Peptide, V4-L53
Containing Protein,
Motifs






Glycosyl Hydrolase
Glycosyl Hydrolase
PROFILE-






Family 9 Active Site
Protein
SCAN






Signature


127
196
T105 T47 T56 S158

M1-S26 Signal
Signal Peptide
SPScan






Peptide, H79-H123
Containing Protein,
Motifs






Ribosomal Protein
Ribosomal Protein
BLAST






S18 Signature
S18
Pfam








PROFILE-








SCAN


128
214
S112 S131
N37 N92
M1-S35 Signal
Signal Peptide
SPScan






Peptide
Containing Protein,
Motifs







Homology with GTP
BLAST







Binding Protein


129
88


M1-S24 Signal
Signal Peptide
HMM






Peptide
Containing Protein


130
260
S146 S179 S192
N50 N109
M1-A48 Signal
Signal Peptide
SPScan




S239 S70 T126 T150

Peptide, G59-S142
Containing Protein,
Motifs






Immunoglobulin
Immunoglobulin
Pfam






Domain
Superfamily Protein


131
295
T176 T56 S72 S179

M1-A30 Signal
Signal Peptide
SPScan




S256 S87

Peptide
Containing Protein
Motifs


132
183
S11 T41 T42 S83

M1-W24 Signal
Signal Peptide
HMM






Peptide, E131-K168
Containing Protein,
Motifs






and C105-H115
Adrenodoxin Family
BLOCKS






Adrenodoxin Iron-
Iron-Sulfur
PRINTS






Sulfur Binding
Binding Protein, and
Pfam






Signature, C111-V116
Cytochrome C Family






Cytochrome C Heme
Heme Binding Protein






Binding Signature,






N69-A162 Iron-






Sulfur Cluster






Binding Domain


133
113
S93 T89 Y9

M1-G30 Signal
Signal Peptide
SPScan






Peptide, V28-L74
Containing Protein,
Motifs






PF00646 F-Box
PF00646 F-Box Protein
Pfam






Domain


134
160
T46 T55 S65 S124

M1-A27 Signal
Signal Peptide
SPScan




T125 T46

Peptide
Containing Protein,
Motifs







F45G2.10 and Yhr122wp
BLAST







Homology



















TABLE 3





Nucleotide

Disease/Condition-Specific Expression



SEQ ID NO:
Tissue Expression (Fraction of Total)
(Total of Fraction)
Vector







135
Hematopoietic/Immune (1.000)
Inflammation (1.000)
pBLUESCRIPT


136
Hematopoietic/Immune (0.750) Cardiovascular (0.250)
Inflammation (0.750) Cancer (0.250)
pSPORT1


137
Nervous (1.000)
Trauma (1.000)
pSPORT1


138
Musculoskeletal (1.000)
Inflammation (1.000)
pSPORT1


139
Gastrointestinal (0.714) Cardiovascular (0.143)
Cancer (0.714) Trauma (0.143)
pSPORT1



Reproductive (0.143)


140
Nervous (1.000)
Neurological (0.500) Trauma (0.500)
pSPORT1


141
Reproductive (0.293) Gastrointestinal (0.146)
Cancer (0.524) Inflammation (0.256)
pSPORT1



Hematopoietic/Immune (0.146)
Fetal (0.146)


142
Reproductive (0.266) Gastrointestinal (0.170)
Cancer (0.479) Inflammation (0.277)
pINCY



Nervous (0.138)
Fetal (0.181)


143
Reproductive (0.417) Nervous (0.292)
Cancer (0.417) Inflammation (0.250)
pINCY



Developmental (0.125)
Fetal (0.167)


144
Reproductive (0.321) Cardiovascular (0.143)
Cancer (0.464) Fetal (0.214)
pINCY



Developmental (0.143)
Inflammation (0.143)


145
Reproductive (0.600) Gastrointestinal (0.400)
Cancer (0.400) Trauma (0.400)
pINCY




Inflammation (0.200)


146
Cardiovascular (0.400) Dermatologic (0.200)
Cancer (0.600) Fetal (0.600)
pINCY



Nervous (0.200)


147
Developmental (0.667) Gastrointestinal (0.333)
Fetal (0.667) Cancer (0.333)
pINCY


148
Reproductive (0.256) Nervous (0.248)
Cancer (0.479) Inflammation (0.214)
pINCY



Cardiovascular (0.137)
Fetal (0.145)


149
Reproductive (0.244) Nervous (0.178)
Cancer (0.433) Inflammation (0.322)
pINCY



Hematopoietic/Immune (0.167)
Fetal (0.156)


150
Cardiovascular (0.923) Developmental (0.077)
Cancer (0.692) Fetal (0.154)
pINCY




Inflammation (0.154)


151
Reproductive (0.215) Nervous (0.190)
Cancer (0.494) Inflammation (0.278)
pINCY



Gastrointestinal (0.177)
Trauma (0.152)


152
Reproductive (0.200) Nervous (0.171)
Inflammation (0.371) Cancer (0.229)
pINCY



Hematopoietic/Immune (0.143)
Fetal (0.200)


153
Reproductive (0.333) Nervous (0.157)
Cancer (0.549) Inflammation (0.176)
pINCY



Hematopoietic/Immune (0.137)
Fetal (0.137)


154
Gastrointestinal (0.500) Urologic (0.167)
Inflammation (0.667) Cancer (0.167)
pINCY




Trauma (0.167)


155
Gastrointestinal (0.429) Reproductive (0.286)
Inflammation (0.429) Cancer (0.286)
pINCY



Nervous (0.143)
Trauma (0.143)


156
Reproductive (1.000)
Cancer (0.500) Inflammation (0.500)
pINCY


157
Hematopoietic/Immune (0.346) Reproductive (0.154)
Cancer (0.404) Inflammation (0.404)
pINCY



Gastrointestinal (0.115)
Fetal (0.212)


158
Reproductive (0.236) Hematopoietic/Immune (0.217)
Cancer (0.415) Inflammation (0.358)
pINCY



Gastrointestinal (0.132)
Fetal (0.142)


159
Gastrointestinal (1.000)
Cancer (1.000)
pSPORT1


160
Developmental (0.500) Hematopoietic/Immune (0.250)
Fetal (0.500) Inflammation (0.250)
pINCY



Nervous (0.250)
Trauma (0.250)


161
Hematopoietic/Immune (0.250) Reproductive (0.250)
Cancer (0.583) Fetal (0.292)
pINCY



Nervous (0.208)
Inflammation (0.250)


162
Gastrointestinal (0.412) Reproductive (0.412)
Cancer (0.735) Inflammation (0.176)
pINCY



Cardiovascular (0.088)
Fetal (0.029)


163
Reproductive (0.298) Cardiovascular (0.170) Nervous
Cancer (0.532) Inflammation (0.213)
pINCY



(0.149)
Fetal (0.191)


164
Gastrointestinal (0.333) Hematopoietic/Immune (0.333)
Cancer (0.667) Inflammation (0.333)
pINCY



Reproductive (0.333)


165
Reproductive (0.295) Gastrointestinal (0.159)
Cancer (0.534) Inflammation (0.284)
pINCY



Nervous (0.148)
Fetal (0.091)


166
Hematopoietic/Immune (0.538) Cardiovascular (0.077)
Inflammation (0.731) Cancer (0.154)
pINCY



Reproductive (0.077)
Fetal (0.154)


167
Reproductive (0.483) Gastrointestinal (0.121)
Cancer (0.672) Inflammation (0.155)
pINCY



Nervous (0.103)


168
Gastrointestinal (0.222) Hematopoietic/Immune (0.222)
Cancer (0.519) Inflammation (0.370)
pINCY



Nervous (0.148)
Fetal (0.259)


169
Urologic (1.000)
Cancer (0.333) Fetal (0.333)
pINCY




Inflammation (0.333)


170
Reproductive (0.214) Gastrointestinal (0.179)
Cancer (0.643) Inflammation (0.143)
pINCY



Nervous (0.143)
Fetal (0.107)


171
Reproductive (0.261) Developmental (0.174)
Cancer (0.391) Fetal (0.304)
pINCY



Nervous (0.174)
Inflammation (0.217)


172
Reproductive (0.357) Gastrointestinal (0.321)
Cancer (0.571) Inflammation (0.286)
pINCY



Cardiovascular (0.071)
Fetal (0.107)


173
Reproductive (0.306) Nervous (0.161)
Cancer (0.387) Inflammation (0.323)
pINCY



Cardiovascular (0.129)
Fetal (0.226)


174
Reproductive (0.229) Nervous (0.188)
Cancer (0.521) Inflammation (0.312)
pSPORT1



Cardiovascular (0.167)
Trauma (0.146)


175
Reproductive (0.444) Developmental (0.167)
Cancer (0.556) Fetal (0.278)
pSPORT1



Cardiovascular (0.111)
Trauma (0.111)


176
Reproductive (0.294) Gastrointestinal (0.176)
Cancer (0.765) Fetal (0.118)
pSPORT1



Cardiovascular (0.118)
Inflammation (0.118)


177
Gastrointestinal (1.000)
Cancer (0.667) Inflammation (0.333)
pINCY


178
Reproductive (0.385) Nervous (0.231)
Cancer (0.385) Inflammation (0.385)
pINCY



Gastrointestinal (0.154)


179
Reproductive (0.500) Cardiovascular (0.167)
Cancer (0.667) Fetal (0.167)
pBLUESCRIPT



Gastrointestinal (0.167)
Inflammation (0.167)


180
Cardiovascular (0.231) Reproductive (0.231)
Cancer (0.615) Inflammation (0.308)
pINCY



Gastrointestinal (0.154)
Fetal (0.154)


181
Reproductive (0.324) Gastrointestinal (0.176)
Cancer (0.519) Inflammation (0.222)
pINCY



Cardiovascular (0.130)
Fetal (0.157)


182
Reproductive (0.320) Nervous (0.180)
Cancer (0.580) Inflammation (0.160)
pINCY



Gastrointestinal (0.120)
Fetal (0.100)


183
Gastrointestinal (0.667) Reproductive (0.333)
Cancer (1.000)
pINCY


184
Urologic (0.667) Dermatologic (0.333)
Cancer (0.667) Fetal (0.333)
pSPORT1


185
Cardiovascular (0.500) Reproductive (0.500)
Cancer (1.000)
pINCY


186
Reproductive (0.393) Developmental (0.107)
Cancer (0.607) Fetal (0.179)
pINCY



Urologic (0.107)
Inflammation (0.107)


187
Cardiovascular (0.400) Reproductive (0.333)
Inflammation (0.467) Cancer (0.267)
pSPORT1



Gastrointestinal (0.133)
Fetal (0.267)


188
Nervous (0.318) Reproductive (0.227) Urologic (0.136)
Cancer (0.636) Inflammation (0.136)
pINCY




Trauma (0.091)


189
Cardiovascular (0.500) Reproductive (0.500)
Cancer (1.000)
pINCY


190
Reproductive (0.318) Nervous (0.227)
Cancer (0.500) Fetal (0.227)
pINCY



Hematopoietic/Immune (0.136)
Inflammation (0.227)


191
Reproductive (0.253) Cardiovascular (0.158)
Cancer (0.463) Inflammation (0.232)
pINCY



Gastrointestinal (0.147)
Fetal (0.200)


192
Reproductive (0.333) Gastrointestinal (0.286)
Cancer (0.571) Inflammation (0.333)
pINCY



Cardiovascular (0.095)
Fetal (0.095)


193
Reproductive (0.304) Cardiovascular (0.217)
Cancer (0.435) Inflammation (0.391)
pINCY



Gastrointestinal (0.130)
Fetal (0.174)


194
Reproductive (0.312) Nervous (0.188)
Cancer (0.438) Inflammation (0.250)
pINCY



Cardiovascular (0.125)
Fetal (0.188)


195
Developmental (1.000)
Fetal (1.000)
pINCY


196
Reproductive (0.233) Cardiovascular (0.209)
Cancer (0.605) Fetal (0.186)
pINCY



Nervous (0.140)
Inflammation (0.116)


197
Reproductive (0.182) Gastrointestinal (0.136)
Cancer (0.477) Inflammation (0.341)
pINCY



Hematopoietic/Immune (0.136)
Fetal (0.182)


198
Gastrointestinal (0.205) Reproductive (0.205)
Inflammation (0.341) Cancer (0.250)
pINCY



Cardiovascular (0.114)
Fetal (0.227)


199
Cardiovascular (0.520) Reproductive (0.280)
Cancer (0.720) Fetal (0.200)
pINCY



Developmental (0.160)
Inflammation (0.080)


200
Lung (0.958) Developmental (0.25)
Cancer (0.583) Fetal or Proliferating
pBLUESCRIPT



Musculoskeletal (0.042)
(0.292) Inflammation (0.167)


201
Reproductive (0.571) Musculoskeletal (0.143)
Cancer (0.429) Inflammation (0.571)
pSPORT1



Nervous (0.143) Urologic (0.143)


202
Endocrine (0.250) Nervous (0.250) Cardiovascular (0.125)
Cancer (0.375) Inflammation (0.625)
pSPORT1



Developmental (0.125) Gastrointestinal (0.125)
Fetal or Proliferating (0.125)



Reproductive (0.125)


203
Lung (1.000)
Fetal or Proliferating (1.000)
pINCY


204
Lung (0.500) Penis (0.500)
Cancer (0.500)
pINCY


205
Cardiovascular (0.231) Dermatologic (0.231)
Fetal or Proliferating (0.385)
pINCY



Reproductive (0.231)
Cancer (0.308)


206
Nervous (0.596) Reproductive (0.154)
Cancer (0.442) Neurological (0.192)
pINCY



Gastrointestinal (0.077)
Inflammation (0.231)


207
Gastrointestinal (1.000)
Inflammation (1.000)
pINCY


208
Reproductive (0.300) Hematopoietic/Immune (0.200)
Cancer (0.450) Inflammation (0.400)
pSPORT1



Nervous (0.150)
Fetal or Proliferating (0.250)


209
Heart (0.500) Brain (0.500)
Neurological (0.500) Inflammation
pINCY




(0.500)


210
Nervous (0.625) Reproductive (0.250)
Cancer (0.750) Fetal or Proliferating
pINCY



Musculoskeletal (0.125)
(0.250) Neurological (0.125)


211
Nervous (0.261) Reproductive (0.304)
Cancer (0.522) Fetal or Proliferating
pSPORT1



Gastrointestinal (0.174)
(0.174) Inflammation (0.130)


212
Testis (1.000)
Inflammation (1.000)
pBLUESCRIPT


213
Nervous (0.400) Reproductive (0.400)
Cancer (0.400) Inflammation (0.400)
pBLUESCRIPT



Gastrointestinal (0.200)
Neurological (0.200)


214
Reproductive (0.476) Gastrointestinal (0.286)
Cancer (0.714) Inflammation (0.286)
pSPORT1



Cardiovascular (0.095)
Neurological (0.048)


215
Reproductive (0.284) Gastrointestinal (0.216)
Cancer (0.486) Inflammation (0.351)
pSPORT1



Nervous (0.176) Hematopoietic/Immune (0.108)
Fetal or Proliferating (0.122)



Cardiovascular (0.108)


216
Uterus (0.500) Prostate (0.500)
Cancer (0.500) Inflammation (0.500)
pINCY


217
Nervous (0.429) Cardiovascular (0.143)
Cancer (0.571) Inflammation (0.429)
pSPORT1



Gastrointestinal (0.143) Hematopoietic/Immune (0.143)
Fetal or Proliferating (0.285)



Reproductive (0.143)


218
Reproductive (0.450) Hematopoietic/Immune (0.200)
Cancer (0.650) Inflammation (0.200)
pINCY



Nervous (0.100) Gastrointestinal (0.100)
Fetal or Proliferating (0.050)


219
Reproductive (0.364) Cardiovascular (0.182)
Cancer (0.636) Fetal or Proliferating
pINCY



Nervous (0.182)
(0.182) Inflammation (0.273)


220
Prostate (1.000)
Inflammation (1.000)
pSPORT1


221
Developmental (0.333) Nervous (0.333)
Cancer (0.667) Fetal or Proliferating
pSPORT1



Reproductive (0.333)
(0.667)


222
Reproductive (0.393) Hematopoietic/Immune (0.180)
Cancer (0.508) Inflammation (0.344)
pSPORT1



Nervous (0.098) Cardiovascular (0.098)
Fetal or Proliferating (0.066)


223
Endocrine (0.333) Gastrointestinal (0.333)
Cancer (1.000)
pINCY



Reproductive (0.333)


224
Cardiovascular (0.200) Developmental (0.200)
Cancer (0.800) Fetal or Proliferating
pINCY



Gastrointestinal (0.200) Reproductive (0.200)
(0.200)



Urologic (0.200)


225
Lung (1.000)
Cancer (1.000)
pINCY


226
Reproductive (0.302) Hematopoietic/Immune (0.254)
Cancer (0.381) Inflammation (0.381)
pSPORT1



Cardiovascular (0.111)
Fetal or Proliferating (0.286)


227
Lymphocytes (1.000)
Inflammation (1.000)
pINCY


228
Cardiovascular (0.531) Reproductive (0.250)
Cancer (0.656) Inflammation (0.250)
pINCY



Urologic (0.094)
Fetal or Proliferating (0.094)


229
Reproductive (0.333) Cardiovascular (0.167)
Cancer (0.500) Fetal or Proliferating
pINCY



Gastrointestinal (0.167) Endocrine (0.167)
(0.167) Inflammation (0.333)



Hematopoietic/Immune (0.167)


230
Hematopoietic/Immune (0.500) Reproductive (0.500)
Cell Proliferation (0.500)
pBLUESCRIPT




Inflammation (0.500)


231
Cardiovascular (0.333) Nervous (0.333)
Cancer (0.500) Cell Proliferation
pINCY



Developmental (0.167)
(0.333) Inflammation (0.167)


232
Gastrointestinal (0.938) Reproductive (0.062)
Cancer (0.500) Inflammation (0.500)
pINCY


233
Nervous (0.324) Reproductive (0.235)
Cancer (0.456) Inflammation (0.235)
pINCY



Hematopoietic/Immune (0.118)
Trauma (0.147)


234
Nervous (0.255) Reproductive (0.255)
Cancer (0.545) Inflammation (0.255)
pINCY



Musculoskeletal (0.182)
Trauma (0.109)


235
Musculoskeletal (0.308) Reproductive (0.231)
Cancer (0.538) Inflammation (0.231)
pINCY



Gastrointestinal (0.154)
Trauma (0.154)


236
Nervous (1.000)
Cancer (1.000)
pINCY


237
Gastrointestinal (0.429)
Cancer (0.571) Cell Proliferation
pINCY



Hematopoietic/Immune (0.143) Nervous (0.143)
(0.143) Trauma (0.143)


238
Reproductive (0.254) Gastrointestinal (0.160)
Cancer (0.453) Inflammation (0.241)
pINCY



Nervous (0.128)
Cell Proliferation (0.175)


239
Nervous (0.333) Dermatologic (0.167)
Trauma (0.333) Cancer (0.167)
pINCY



Endocrine (0.167)
Cell Proliferation (0.167)


240
Nervous (0.273) Reproductive (0.227)
Cancer (0.545) Cell Proliferation
pINCY



Endocrine (0.136)
(0.182) Inflammation (0.182)


241
Reproductive (0.273)
Cancer (0.455) Cell Proliferation
pINCY



Hematopoietic/Immune (0.182) Urologic (0.182)
(0.273) Inflammation (0.273)


242
Endocrine (1.000)
Trauma (1.000)
pSPORT1


243
Reproductive (1.000)
Cancer (1.000)
pINCY


244
Hematopoietic/Immune (0.545)
Inflammation (0.636) Trauma (0.182)
pINCY



Musculoskeletal (0.182) Cardiovascular (0.091)
Cancer (0.091)


245
Hematopoietic/Immune (0.400)
Inflammation (0.650) Cancer (0.300)
pINCY



Musculoskeletal (0.300) Cardiovascular (0.150)


246
Urologic (1.000)
Cancer (0.500) Cell Proliferation
pINCY




(0.500)


247
Nervous (0.292) Reproductive (0.222)
Cell Proliferation (0.625)
pSPORT1



Musculoskeletal (0.125)
Inflammation/Trauma (0.181)


248
Reproductive (0.211) Developmental (0.132)
Cell Proliferation (0.658)
pSPORT1



Nervous (0.132)
Inflammation/Trauma (0.184)


249
Nervous (0.500) Gastrointestinal (0.300)
Cell Proliferation (0.900)
pSPORT1



Hematopoietic/Immune (0.100)
Inflammation/Trauma (0.300)


250
Cardiovascular (0.209) Gastrointestinal (0.140)
Cell Proliferation (0.605)
pINCY



Hematopoietic/Immune (0.140)
Inflammation/Trauma (0.256)


251
Nervous (0.308) Cardiovascular (0.154)
Cell Proliferation (0.616)
pINCY



Gastrointestinal (0.154)
Inflammation/Trauma (0.269)


252
Reproductive (1.000)
Cell Proliferation (1.000)
pSPORT1


253
Reproductive (0.324) Nervous (0.162)
Cell Proliferation (0.641)
pSPORT1



Gastrointestinal (0.113)
Inflammation/Trauma (0.197)


254
Reproductive (0.315) Nervous (0.296)
Cell Proliferation (0.630)
pSPORT1



Developmental (0.093)
Inflammation/Trauma (0.278)


255
Nervous (0.211) Reproductive (0.211)
Cell Proliferation (0.579)
pINCY



Gastrointestinal (0.158)
Inflammation/Trauma (0.298)


256
Reproductive (0.250) Gastrointestinal (0.148)
Cell Proliferation (0.705)
pINCY



Hematopoietic/Immune (0.148)
Inflammation/Trauma (0.193)


257
Hematopoietic/Immune (1.000)
Cell Proliferation (0.400)
pINCY




Inflammation/Trauma (0.600)


258
Cardiovascular (0.333) Reproductive (0.333)
Cell Proliferation (0.833)
pBLUESCRIPT



Developmental (0.167)
Inflammation/Trauma (0.333)


259
Cardiovascular (0.333) Reproductive (0.250)
Cell Proliferation (0.625)
pINCY



Developmental (0.167)
Inflammation/Trauma (0.208)


260
Endocrine (0.500) Cardiovascular (0.250) Nervous (0.250)
Cell Proliferation (0.750)
pINCY




Inflammation/Trauma (0.500)


261
Reproductive (0.252) Cardiovascular (0.155)
Cell Proliferation (0.728)
pINCY



Hematopoietic/Immune (0.136)
Inflammation/Trauma (0.194)


262
Reproductive (0.274) Cardiovascular (0.177)
Cell Proliferation (0.742)
pINCY



Nervous (0.145)
Inflammation/Trauma (0.210)


263
Reproductive (0.267) Cardiovascular (0.160)
Cell Proliferation (0.654)
pINCY



Hematopoietic/Immune (0.127)
Inflammation/Trauma (0.193)


264
Nervous (0.229) Hematopoietic/Immune (0.200)
Cell Proliferation (0.743)
pINCY



Reproductive (0.200)
Inflammation/Trauma (0.286)


265
Hematopoietic/Immune (0.333) Gastrointestinal (0.167)
Cell Proliferation (0.600)
pINCY



Nervous (0.133)
Inflammation/Trauma (0.333)


266
Nervous (0.290) Reproductive (0.258)
Cell Proliferation (0.677)
pINCY



Cardiovascular (0.129)
Inflammation/Trauma (0.194)


267
Reproductive (0.261) Hematopoietic/Immune (0.217)
Cell Proliferation (0.652)
pINCY



Cardiovascular (0.087)
Inflammation/Trauma (0.391)


268
Gastrointestinal (0.227) Reproductive (0.193)
Cell Proliferation (0.731)
pSPORT1



Hematopoietie/Immune (0.168)
Inflammation/Trauma (0.227)



















TABLE 4





Polynucleotide





SEQ ID NO:
Clone ID
Library
Library Description


















135
443531
MPHGNOT03
The library was constructed using RNA isolated





from plastic adherent mononuclear cells isolated





from buffy coat units obtained from unrelated





male and female donors.


136
632860
NEUTGMT01
The library was constructed using RNA isolated





from peripheral blood granulocytes collected by





density gradient centrifugation through Ficoll-





Hypaque. The cells were isolated from buffy coat





units obtained from 20 unrelated male and female





donors. Cells were cultured in 10 nM GM-CSF





for 1 hour before washing and harvesting for RNA





preparation.


137
670010
CRBLNOT01
The library was constructed using RNA isolated





from the cerebellum tissue of a 69-year-old





Caucasian male who died from chronic obstructive





pulmonary disease. Patient history included





myocardial infarction, hypertension, and





osteoarthritis.


138
726498
SYNOOAT01
The library was constructed using RNA isolated





from the knee synovial membrane tissue of an 82-





year-old female with osteoarthritis.


139
795064
OVARNOT03
The library was constructed using RNA isolated





from ovarian tissue removed from a 43-year-old





Caucasian female during removal of the fallopian





tubes and ovaries. Pathology for the associated





tumor tissue indicated grade 2 mucinous





cystadenocarcinoma. Patient history included mitral valve





disorder, pneumonia, and viral hepatitis. Family





history included atherosclerotic coronary artery





disease, pancreatic cancer, cerebrovascular disease,





breast cancer, and uterine cancer.


140
924925
BRAINOT04
The library was constructed using RNA isolated





from the brain tissue of a 44-year-old Caucasian





male with a cerebral hemorrhage. The tissue,





which contained coagulated blood, came from the





choroid plexus of the right anterior temporal





lobe. Family history included coronary artery disease





and myocardial infarction.


141
962390
BRSTTUT03
The library was constructed using RNA isolated





from breast tumor tissue removed from a 58-year-





old Caucasian female during a unilateral extended





simple mastectomy. Pathology indicated





multicentric invasive grade 4 lobular carcinoma.





The mass was identified in the upper outer





quadrant, and three separate nodules were found





in the lower outer quadrant of the left breast.





Patient history included skin cancer, rheumatic





heart disease, osteoarthritis, and tuberculosis.





Family history included cerebrovascular disease,





coronary artery aneurysm, breast cancer, prostate





cancer, atherosclerotic coronary artery disease,





and type I diabetes.


142
1259405
MENITUT03
The library was constructed using RNA isolated





from brain meningioma tissue removed from a 35-





year-old Caucasian female during excision of a





cerebral meningeal lesion. Pathology indicated a





benign neoplasm in the right cerebellopontine





angle of the brain. Patient history included





hypothyroidism. Family history included myocardial





infarction and breast cancer.


143
1297384
BRSTNOT07
The library was constructed using RNA isolated





from diseased breast tissue removed from a 43-





year-old Caucasian female during a unilateral





extended simple mastectomy. Pathology indicated





mildly proliferative fibrocystic changes with





epithelial hyperplasia, papillomatosis, and duct ectasia.





Pathology for the associated tumor tissue indicated





invasive grade 4, nuclear grade 3 mammary





adenocarcinoma with extensive comedo necrosis.





Family history included epilepsy, atherosclerotic





coronary artery disease, and type II diabetes.


144
1299627
BRSTNOT07
The library was constructed using RNA isolated





from diseased breast tissue removed from a 43-





year-old Caucasian female during a unilateral





extended simple mastectomy. Pathology indicated





mildly proliferative fibrocystic changes with





epithelial hyperplasia, papillomatosis, and duct ectasia.





Pathology for the associated tumor tissue indicated





invasive grade 4, nuclear grade 3 mammary





adenocarcinoma with extensive comedo necrosis.





Family history included epilepsy, atherosclerotic





coronary artery disease, and type II diabetes.


145
1306026
PLACNOT02
The library was constructed using RNA isolated





from the placental tissue of a Hispanic female





fetus, who was prematurely delivered at 21 weeks'





gestation. Serologies of the mother's blood were





positive for CMV (cytomegalovirus).


146
1316219
BLADTUT02
The library was constructed using RNA isolated





from bladder tumor tissue removed from an 80-





year-old Caucasian female during a radical





cystectomy and lymph node excision. Pathology





indicated grade 3 invasive transitional cell





carcinoma. Family history included osteoarthritis and





atherosclerosis.


147
1329031
PANCNOT07
The library was constructed using RNA isolated





from the pancreatic tissue of a Caucasian male





fetus, who died at 23 weeks' gestation.


148
1483050
CORPNOT02
The library was constructed using RNA isolated





from diseased corpus callosum tissue removed





from the brain of a 74-year-old Caucasian





male who died from Alzheimer's disease.


149
1514160
PANCTUT01
The library was constructed using RNA isolated





from pancreatic tumor tissue removed from a 65-





year-old Caucasian female during radical subtotal





pancreatectomy. Pathology indicated an invasive





grade 2 adenocarcinoma. Patient history included





type II diabetes, osteoarthritis, cardiovascular





disease, benign neoplasm in the large bowel, and





a cataract. Family history included cardiovascular





disease, type II diabetes, and stomach cancer.


150
1603403
LUNGNOT15
The library was constructed using RNA isolated





from lung tissue removed from a 69-year-old





Caucasian male during a segmental lung resection.





Pathology for the associated tumor tissue





indicated residual grade 3 invasive squamous cell





carcinoma. Patient history included acute





myocardial infarction, prostatic hyperplasia, and





malignant skin neoplasm. Family history included





cerebrovascular disease, type I diabetes, acute





myocardial infarction, and arteriosclerotic coronary





disease.


151
1652303
PROSTUT08
The library was constructed using RNA isolated





from prostate tumor tissue removed from a 60-





year-old Caucasian male during radical prostatectomy





and regional lymph node excision. Pathology





indicated an adenocarcinoma (Gleason grade 3 + 4).





Adenofibromatous hyperplasia was also present.





The patient presented with elevated prostate





specific antigen (PSA). Patient history included a





kidney cyst. Family history included tuberculosis,





cerebrovascular disease, and arteriosclerotic





coronary artery disease.


152
1693358
COLNNOT23
The library was constructed using RNA isolated





from diseased colon tissue removed from a 16-





year-old Caucasian male during a total colectomy





with abdominal/perineal resection. Pathology





indicated gastritis and pancolonitis consistent





with the acute phase of ulcerative colitis. There was





only mild involvement of the ascending and sigmoid





colon, and no significant involvement of the





cecum, rectum, or terminal ileum. Family history





included irritable bowel syndrome.


153
1707711
DUODNOT02
The library was constructed using RNA isolated





from duodenal tissue of a 8-year-old Caucasian





female, who died from head trauma. Serology was





positive for cytomegalovirus (CMV).


154
1738735
COLNNOT22
The library was constructed using RNA isolated





from colon tissue removed from a 56-year-old





Caucasian female with Crohn's disease during a





partial resection of the small intestine. Pathology





indicated Crohn's disease of the ileum and ileal-





colonic anastomosis, causing a fistula at the





anastomotic site that extended into pericolonic





fat. The ileal mucosa showed linear and puncture





ulcers with intervening normal tissue. Previous





surgeries included a partial ileal resection and





permanent ileostomy. Family history included





irritable bowel syndrome.


155
1749147
STOMTUT02
The library was constructed using RNA isolated





from stomach tumor tissue obtained from a 68-





year-old Caucasian female during a partial gastrectomy.





Pathology indicated a malignant lymphoma





of diffuse large-cell type. Patient history





included thalassemia. Family history included acute





leukemia, malignant neoplasm of the esophagus,





malignant stomach neoplasm, and atherosclerotic





coronary artery disease.


156
1817722
PROSNOT20
The library was constructed using RNA isolated





from diseased prostate tissue removed from a 65-





year-old Caucasian male during a radical prostatectomy.





Pathology indicated adenofibromatous





hyperplasia. Pathology for the associated tumor





tissue indicated an adenocarcinoma.


157
1831290
THP1AZT01
The library was constructed using 1 microgram of





polyA RNA isolated from THP-1 promonocyte





cells treated for three days with 0.8 micromolar





5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is





a human promonocyte line derived from peripheral





blood of a 1-year-old Caucasian male with acute





monocytic leukemia.


158
1831477
THP1AZT01
The library was constructed using 1 microgram of





polyA RNA isolated from THP-1 promonocyte





cells treated for three days with 0.8 micromolar





5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is





a human promonocyte line derived from peripheral





blood of a 1-year-old Caucasian male with acute





monocytic leukemia.


159
1841607
COLNNOT07
The library was constructed using RNA isolated





from colon tissue removed from a 60-year-old





Caucasian male during a left hemicolectomy.


160
1852391
LUNGFET03
The library was constructed using RNA isolated





from lung tissue removed from a Caucasian female





fetus, who died at 20 weeks' gestation.


161
1854555
HNT3AZT01
Library was constructed using RNA isolated from





the hNT2 cell line (derived from a human





teratocarcinoma that exhibited properties





characteristic of a committed neuronal precursor). Cells





were treated for three days with 0.35 micromolar





5-aza-2′-deoxycytidine (AZT).


162
1855755
PROSNOT18
The library was constructed using RNA isolated





from diseased prostate tissue removed from a 58-





year-old Caucasian male during a radical cystectomy,





radical prostatectomy, and gastrostomy.





Pathology indicated adenofibromatous hyperplasia.





This tissue was associated with a grade 3





transitional cell carcinoma. Patient history





included angina and emphysema. Family history





included acute myocardial infarction, atherosclerotic





coronary artery disease, and type II diabetes.


163
1861434
PROSNOT19
The library was constructed using RNA isolated





from diseased prostate tissue removed from a 59-





year-old Caucasian male during a radical prostatectomy





with regional lymph node excision.





Pathology indicated adenofibromatous hyperplasia.





Pathology for the associated tumor tissue





indicated an adenocarcinoma (Gleason grade 3 + 3).





The patient presented with elevated prostate-





specific antigen (PSA). Patient history included





colon diverticuli and thrombophlebitis. Family





history included benign hypertension, multiple





myeloma, hyperlipidemia and rheumatoid arthritis.


164
1872334
LEUKNOT02
The library was constructed using RNA isolated





from white blood cells of a 45-year-old female





with blood type O+. The donor tested positive





for cytomegalovirus (CMV).


165
1877230
LEUKNOT03
The library was constructed using RNA isolated





from white blood cells of a 27-year-old female





with blood type A+. The donor tested negative





for cytomegalovirus (CMV).


166
1877885
LEUKNOT03
The library was constructed using RNA isolated





from white blood cells of a 27-year-old female





with blood type A+. The donor tested negative





for cytomegalovirus (CMV).


167
1889269
BLADTUT07
The library was constructed using RNA isolated





from bladder tumor tissue removed from the





anterior bladder wall of a 58-year-old Caucasian





male during a radical cystectomy, radical





prostatectomy, and gastrostomy. Pathology





indicated a grade 3 transitional cell carcinoma





in the left lateral bladder. Patient history





included angina and emphysema. Family history





included acute myocardial infarction,





atherosclerotic coronary artery disease, and





type II diabetes.


168
1890243
BLADTUT07
The library was constructed using RNA isolated





from bladder tumor tissue removed from the





anterior bladder wall of a 58-year-old Caucasian





male during a radical cystectomy, radical





prostatectomy, and gastrostomy. Pathology





indicated a grade 3 transitional cell carcinoma





in the left lateral bladder. Patient history





included angina and emphysema. Family history





included acute myocardial infarction, atherosclerotic





coronary artery disease, and type II diabetes.


169
1900433
BLADTUT06
The library was constructed using RNA isolated





from bladder tumor tissue removed from the





posterior bladder wall of a 58-year-old Caucasian





male during a radical cystectomy, radical





prostatectomy, and gastrostomy. Pathology





indicated grade 3 transitional cell carcinoma in





the left lateral bladder wall. Patient history





included angina and emphysema. Family history





included acute myocardial infarction, atherosclerotic





coronary artery disease, and type II diabetes.


170
1909441
CONNTUT01
The library was constructed using RNA isolated





from a soft tissue tumor removed from the clival





area of the skull of a 30-year-old Caucasian female.





Pathology indicated chondroid chordoma with





neoplastic cells reactive for keratin.


171
1932226
COLNNOT16
The library was constructed using RNA isolated





from sigmoid colon tissue removed from a 62-





year-old Caucasian male during a sigmoidectomy





and permanent colostomy.


172
1932647
COLNNOT16
The library was constructed using RNA isolated





from sigmoid colon tissue removed from a 62-





year-old Caucasian male during a sigmoidectomy





and permanent colostomy.


173
2124245
BRSTNOT07
The library was constructed using RNA isolated





from diseased breast tissue removed from a 43-





year-old Caucasian female during a unilateral





extended simple mastectomy. Pathology indicated





mildly proliferative fibrocystic changes with





epithelial hyperplasia, papillomatosis, and duct ectasia.





Pathology for the associated tumor tissue indicated





invasive grade 4, nuclear grade 3 mammary





adenocarcinoma with extensive comedo necrosis. Family





history included epilepsy, atherosclerotic





coronary artery disease, and type II diabetes.


174
2132626
OVARNOT03
The library was constructed using RNA isolated from





ovarian tissue removed from a 43-year-old





Caucasian female during removal of the fallopian





tubes and ovaries. Pathology for the associated





tumor tissue indicated grade 2 mucinous cystadeno-





carcinoma. Patient history included mitral valve





disorder, pneumonia, and viral hepatitis. Family





history included atherosclerotic coronary artery





disease, pancreatic cancer, cerebrovascular disease,





breast cancer, and uterine cancer.


175
2280639
PROSNON01
The library was constructed and normalized from





4.4 million independent clones from the





PROSNOT11 library. Starting RNA was made from





prostate tissue removed from a 28-year-old





Caucasian male who died from a gunshot wound.





The normalization and hybridization conditions





were adapted from Soares, M. B. et al. (1994)





Proc. Natl. Acad. Sci. USA 91: 9228-9232, using a





longer (19 hour) reannealing hybridization period.


176
2292356
BRAINON01
The library was constructed and normalized from





4.88 million independent clones from the





BRAINOT03 library. Starting RNA was made from brain





tissue removed from a 26-year-old Caucasian male





during cranioplasty and excision of a cerebral





meningeal lesion. Pathology for the associated tumor





tissue indicated a grade 4 oligoastrocytoma in the





right fronto-parietal part of the brain.


177
2349310
COLSUCT01
The library was constructed using RNA isolated





from diseased sigmoid colon tissue obtained from





a 70-year-old Caucasian male during colectomy with





permanent ileostomy. Pathology indicated chronic





ulcerative colitis. Patient history included benign





neoplasm of the colon. Family history included





atherosclerotic coronary artery disease and myocardial





infarctions.


178
2373227
ADRENOT07
The library was constructed using RNA isolated





from adrenal tissue removed from a 61-year-old





female during a bilateral adrenalectomy. Patient





history included an unspecified disorder of the





adrenal glands.


179
2457682
ENDANOT01
The library was constructed using RNA isolated





from aortic endothelial cell tissue from an





explanted heart removed from a male during a





heart transplant.


180
2480426
SMCANOT01
The library was constructed using RNA isolated





from an aortic smooth muscle cell line derived





from the explanted heart of a male during a heart





transplant.


181
2503743
CONUTUT01
The library was constructed using RNA isolated





from sigmoid mesentery tumor tissue obtained





from a 61-year-old female during a total abdominal





hysterectomy and bilateral salpingo-oophorectomy





with regional lymph node excision.





Pathology indicated a metastatic grade 4





malignant mixed mullerian tumor present in the





sigmoid mesentery at two sites.


182
2537684
BONRTUT01
The library was constructed using RNA isolated





from rib tumor tissue removed from a 16-year-old





Caucasian male during a rib osteotomy and a wedge





resection of the lung. Pathology indicated a





metastatic grade 3 (of 4) osteosarcoma, forming





a mass involving the chest wall.


183
2593853
OVARTUT02
The library was constructed using RNA isolated





from ovarian tumor tissue removed from a 51-





year-old Caucasian female during an exploratory





laparotomy, total abdominal hysterectomy,





salpingo-oophorectomy, and an incidental appendectomy.





Pathology indicated mucinous cystadenoma presenting





as a multiloculated neoplasm involving the entire





left ovary. The right ovary contained a follicular





cyst and a hemorrhagic corpus luteum. The uterus





showed proliferative endometrium and a single





intramural leiomyoma. The peritoneal biopsy indicated





benign glandular inclusions consistent with





endosalpingiosis. Family history included atherosclerotic





coronary artery disease, benign hypertension, breast





cancer, and uterine cancer.


184
2622354
KERANOT02
The library was constructed using RNA isolated





from epidermal breast keratinocytes (NHEK).





NHEK (Clontech #CC-2501) is a human breast





keratinocyte cell line derived from a 30-year-old





black female during breast-reduction surgery.


185
2641377
LUNGTUT08
The library was constructed using RNA isolated from





lung tumor tissue removed from a 63-year-





old Caucasian male during a right upper lobectomy





with fiberoptic bronchoscopy. Pathology





indicated a grade 3 adenocarcinoma. Patient history





included atherosclerotic coronary artery





disease, an acute myocardial infarction, rectal





cancer, an asymptomatic abdominal aortic aneurysm,





and cardiac dysrhythmia. Family history included





congestive heart failure, stomach cancer, and lung





cancer, type II diabetes, atherosclerotic coronary





artery disease, and an acute myocardial infarction.


186
2674857
KIDNNOT19
The library was constructed using RNA isolated from





kidney tissue removed a 65-year-old Caucasian male





during an exploratory laparotomy and nephroureterectomy.





Pathology for the associated tumor tissue indicated a





grade 1 renal cell carcinoma within the upper pole of





the left kidney. Patient history included malignant





melanoma of the abdominal skin, benign neoplasm of





colon, cerebrovascular disease, and umbilical hernia.





Family history included myocardial infarction,





atherosclerotic coronary artery disease, cerebrovascular





disease, prostate cancer, myocardial infarction, and





atherosclerotic coronary artery disease.


187
2758485
THP1AZS08
The subtracted THP-1 promonocyte cell line library





was constructed using 5.76 million clones from





a 5-aza-2′-deoxycytidine (AZT) treated THP-1





cell library. Starting RNA was made from THP-1





promonocyte cells treated for three days with 0.8





micromolar AZT. The library was oligo(dT)-





primed, and cDNAs were cloned directionally into the





pSPORT1 vectoring system using Sal1 (5′)





and NotI (3′). The hybridization probe for





subtraction was derived from a similarly constructed





library, made from 1 microgram of polyA RNA isolated





from untreated THP-1 cells. 5.76 million





clones from the AZ-treated THP-1 cell library were





then subjected to two rounds of subtractive





hybridization with 5 million clones from the untreated





THP-1 cell library. Subtractive hybridization





conditions were based on the methodologies of Swaroop





et al. (Nucl. Acids Res. (1991) 19: 1954)





and Bonaldo et al. (Genome Res (1996) 6: 791-806).


188
2763296
BRSTNOT12
The library was constructed using RNA isolated





from diseased breast tissue removed from a 32-





year-old Caucasian female during a bilateral





reduction mammoplasty. Pathology indicated





nonproliferative fibrocystic disease. Family





history included benign hypertension and





atherosclerotic coronary artery disease.


189
2779436
OVARTUT03
The library was constructed using RNA isolated





from ovarian tumor tissue removed from the left





ovary of a 52-year-old mixed ethnicity female





during a total abdominal hysterectomy, bilateral





salpingo-oophorectomy, peritoneal and lymphatic





structure biopsy, regional lymph node excision,





and peritoneal tissue destruction. Pathology





indicated an invasive grade 3 (of 4) seroanaplastic





carcinoma forming a mass in the left ovary.





The endometrium was atrophic. Multiple (2)





leiomyomata were identified, one subserosal and





1 intramural. Pathology also indicated a metastatic





grade 3 seroanaplastic carcinoma involving the





omentum, cul-de-sac peritoneum, left broad





ligament peritoneum, and mesentery colon. Patient





history included breast cancer, chronic peptic





ulcer, and joint pain. Family history included colon





cancer, cerebrovascular disease, breast cancer,





type II diabetes, esophagus cancer, and depressive disorder.


190
2808528
BLADTUT08
The library was constructed using RNA isolated





from bladder tumor tissue removed from a 72-





year-old Caucasian male during a radical cystectomy





and prostatectomy. Pathology indicated an





invasive grade 3 (of 3) transitional cell carcinoma





in the right bladder base. Family history included





myocardial infarction, cerebrovascular disease,





brain cancer, and myocardial infarction.


191
2809230
BLADTUT08
The library was constructed using RNA isolated





from bladder tumor tissue removed from a 72-





year-old Caucasian male during a radical cystectomy





and prostatectomy. Pathology indicated an





invasive grade 3 (of 3) transitional cell carcinoma





in the right bladder base. Patient history included





pure hypercholesterolemia and tobacco abuse. Family





history included myocardial infarction, cerebrovascular





disease, brain cancer, and myocardial infarction.


192
2816821
BRSTNOT14
The library was constructed using RNA isolated from





breast tissue removed from a 62-year-old





Caucasian female during a unilateral extended





simple mastectomy. Pathology for the associated





tumor tissue indicated an invasive grade 3 (of 4),





nuclear grade 3 (of 3) adenocarcinoma, ductal





type. Ductal carcinoma in situ, comedo type,





comprised 60% of the tumor mass. Metastatic





adenocarcinoma was identified in one (of 14)





axillary lymph nodes with no perinodal extension.





The tumor cells were strongly positive for estrogen





receptors and weakly positive for progesterone





receptors. Patient history included a benign colon





neoplasm, hyperlipidemia, and cardiac dysrhythmia.





Family history included atherosclerotic coronary artery





disease, myocardial infarction, colon cancer, ovarian





cancer, lung cancer, and cerebrovascular disease.


193
2817268
BRSTNOT14
The library was constructed using RNA isolated





from breast tissue removed from a 62-year-old





Caucasian female during a unilateral extended





simple mastectomy. Pathology for the associated





tumor tissue indicated an invasive grade 3 (of 4),





nuclear grade 3 (of 3) adenocarcinoma, ductal





type. Ductal carcinoma in situ, comedo type, comprised





60% of the tumor mass. Metastatic adenocarcinoma





was identified in one (of 14) axillary lymph nodes





with no perinodal extension. The tumor cells were





strongly positive for estrogen receptors and weakly





positive for progesterone receptors. Patient history





included a benign colon neoplasm, hyperlipidemia, and





cardiac dysrhythmia. Family history included





atherosclerotic coronary artery disease, myocardial





infarction, colon cancer, ovarian cancer, lung cancer,





and cerebrovascular disease.


194
2923165
SININOT04
The library was constructed using RNA isolated





from diseased ileum tissue obtained from a 26-





year-old Caucasian male during a partial colectomy,





permanent colostomy, and an incidental appendectomy.





Pathology indicated moderately to severely active





Crohn's disease. Family history included enteritis





of the small intestine.


195
2949822
KIDNFET01
The library was constructed using RNA isolated





from kidney tissue removed from a Caucasian





female fetus, who died at 17 weeks' gestation





from anencephalus.


196
2992192
KTDNFET02
The library was constructed using RNA isolated





from kidney tissue removed from a Caucasian male





fetus, who was stillborn with a hypoplastic left





heart and died at 23 weeks' gestation.


197
2992458
KIDNFET02
The library was constructed using RNA isolated





from kidney tissue removed from a Caucasian male





fetus, who was stillborn with a hypoplastic left





heart and died at 23 weeks' gestation.


198
3044710
HEAANOT01
The library was constructed using RNA isolated





from right coronary and right circumflex coronary





artery tissue removed from the explanted heart of





a 46-year-old Caucasian male during a heart





transplantation. Patient history included myocardial





infarction from total occlusion of the left





anterior descending coronary artery, atherosclerotic





coronary artery disease, hyperlipidemia,





myocardial ischemia, dilated cardiomyopathy, and





left ventricular dysfunction. Previous surgeries





included cardiac catheterization. Family history





included atherosclerotic coronary artery disease.


199
3120415
LUNGTUT13
The library was constructed using RNA isolated from





tumorous lung tissue removed from the right





upper lobe of a 47-year-old Caucasian male during





a segmental lung resection. Pathology indicated





invasive grade 3 (of 4) adenocarcinoma. Family





history included atherosclerotic coronary artery





disease, and type II diabetes.


200
126758
LUNGNOT01
The library was constructed at Stratagene using





RNA isolated from the lung tissue of a 72-year-old





male.


201
674760
CRBLNOT01
The library was constructed using RNA isolated





from the cerebellum tissue of a 69-year-old





Caucasian male who died from chronic obstructive





pulmonary disease. Patient history included





myocardial infarction, hypertension, and osteoarthritis.


202
1229438
BRAITUT01
The library was constructed using RNA isolated





from brain tumor tissue removed from a 50-year-





old Caucasian female during a frontal lobectomy.





Pathology indicated recurrent grade 3





oligoastrocytoma with focal necrosis and extensive





calcification. Patient history included a speech





disturbance and epilepsy. The patient's brain had





also been irradiated with a total dose of 5,082 cyg





(Fraction 8). Family history included a brain tumor.


203
1236935
LUNGFET03
The library was constructed using RNA isolated





from lung tissue removed from a Caucasian female





fetus who died at 20 weeks' gestation.


204
1359283
LUNGNOT12
The library was constructed using RNA isolated





from lung tissue removed from a 78-year-old





Caucasian male during a segmental lung resection





and regional lymph node resection. Pathology





indicated fibrosis pleura was puckered, but not





invaded. Pathology for the associated tumor tissue





indicated an invasive pulmonary grade 3 adenocarcinoma.





Patient history included cerebrovascular





disease, arteriosclerotic coronary artery disease,





thrombophlebitis, chronic obstructive pulmonary





disease, and asthma. Family history included





intracranial hematoma, cerebrovascular disease,





arteriosclerotic coronary artery disease, and type





I diabetes.


205
1450703
PENITUT01
The library was constructed using RNA isolated





from tumor tissue removed from the penis of a 64-





year-old Caucasian male during penile amputation.





Pathology indicated a fungating invasive grade





4 squamous cell carcinoma involving the inner wall





of the foreskin and extending onto the glans





penis. Patient history included benign neoplasm of





the large bowel, atherosclerotic coronary artery





disease, angina pectoris, gout, and obesity. Family





history included malignant pharyngeal neoplasm,





chronic lymphocytic leukemia, and chronic liver disease.


206
1910668
CONNTUT01
The library was constructed using RNA isolated from





a soft tissue tumor removed from the clival





area of the skull of a 30-year-old Caucasian female.





Pathology indicated chondroid chordoma with





neoplastic cells reactive for keratin.


207
1955143
CONNNOT01
The library was constructed using RNA isolated from





mesentery fat tissue obtained from a 71-year-





old Caucasian male during a partial colectomy and





permanent colostomy. Family history included





atherosclerotic coronary artery disease, myocardial





infarction, and extrinsic asthma.


208
1961637
BRSTNOT04
The library was constructed using RNA isolated from





breast tissue removed from a 62-year-old





East Indian female during a unilateral extended





simple mastectomy. Pathology for the associated





tumor tissue indicated an invasive grade 3 ductal





carcinoma. Patient history included benign





hypertension, hyperlipidemia, and hematuria. Family





history included cerebrovascular and





cardiovascular disease, hyperlipidemia, and liver





cancer.


209
1990762
CORPNOT02
The library was constructed using RNA isolated





from diseased corpus callosum tissue removed





from the brain of a 74-year-old Caucasian male





who died from Alzheimer's disease.


210
1994131
CORPNOT02
The library was constructed using RNA isolated





from diseased corpus callosum tissue removed





from the brain of a 74-year-old Caucasian male





who died from Alzheimer's disease.


211
1997745
BRSTTUT03
The library was constructed using RNA isolated





from breast tumor tissue removed from a 58-year-





old Caucasian female during a unilateral extended





simple mastectomy. Pathology indicated multicentric





invasive grade 4 lobular carcinoma. The mass was





identified in the upper outer quadrant, and three





separate nodules were found in the lower outer





quadrant of the left breast. Patient history





included skin cancer, rheumatic heart disease,





osteoarthritis, and tuberculosis. Family history





included cerebrovascular disease, coronary artery





aneurysm, breast cancer, prostate cancer,





atherosclerotic coronary artery disease, and type





I diabetes.


212
2009035
TESTNOT03
The library was constructed using polyA RNA





isolated from testicular tissue removed from a 37-





year-old Caucasian male who died from liver disease.





Patient history included cirrhosis, jaundice,





and liver failure.


213
2009152
TESTNOT03
The library was constructed using polyA RNA





isolated from testicular tissue removed from a 37-





year-old Caucasian male who died from liver





disease. Patient history included cirrhosis, jaundice,





and liver failure.


214
2061752
OVARNOT03
The library was constructed using RNA isolated





from ovarian tissue removed from a 43-year-old





Caucasian female during removal of the fallopian





tubes and ovaries. Pathology for the associated





tumor tissue indicated grade 2 mucinous cystadenocarcinoma.





Patient history included mitral valve





disorder, pneumonia, and viral hepatitis. Family





history included atherosclerotic coronary artery





disease, pancreatic cancer, stress reaction,





cerebrovascular disease, breast cancer, and uterine





cancer.


215
2061933
OVARNOT03
The library was constructed using RNA isolated





from ovarian tissue removed from a 43-year-old





Caucasian female during removal of the fallopian





tubes and ovaries. Pathology for the associated





tumor tissue indicated grade 2 mucinous cystadenocarcinoma.





Patient history included mitral valve disorder, pneumonia,





and viral hepatitis. Family history included





atherosclerotic coronary artery disease, pancreatic





cancer, stress reaction, cerebrovascular disease,





breast cancer, and uterine cancer.


216
2081422
UTRSNOT08
The library was constructed using RNA isolated





from uterine tissue removed from a 35-year-old





Caucasian female during a vaginal hysterectomy





with dilation and curettage. Pathology indicated





that the endometrium was secretory phase with a





benign endometrial polyp 1 cm in diameter. The





cervix showed mild chronic cervicitis. Family





history included atherosclerotic coronary artery





disease and type II diabetes.


217
2101278
BRAITUT02
The library was constructed using RNA isolated





from brain tumor tissue removed from the frontal





lobe of a 58-year-old Caucasian male during





excision of a cerebral meningeal lesion. Pathology





indicated a grade 2 metastatic hypernephroma.





Patient history included a grade 2 renal cell





carcinoma, insomnia, and chronic airway obstruction.





Family history included a malignant neoplasm of





the kidney.


218
2121353
BRSTNOT07
The library was constructed using RNA isolated





from diseased breast tissue removed from a 43-





year-old Caucasian female during a unilateral





extended simple mastectomy. Pathology indicated





mildly proliferative fibrocystic changes with





epithelial hyperplasia, papillomatosis, and duct





ectasia. Pathology for the associated tumor





tissue indicated invasive grade 4, nuclear grade





3 mammary adenocarcinoma with extensive comedo





necrosis. Family history included epilepsy, cardiovascular





disease, and type II diabetes.


219
2241736
PANCTUT02
The library was constructed using RNA isolated





from pancreatic tumor tissue removed from a 45-





year-old Caucasian female during radical





pancreaticoduodenectomy. Pathology indicated a





grade 4 anaplastic carcinoma. Family history





included benign hypertension, hyperlipidemia and





atherosclerotic coronary artery disease.


220
2271935
PROSNON01
This normalized prostate library was constructed





from 4.4M independent clones from the PROSNOT11





library. Starting RNA was made from prostate





tissue removed from a 28-year-old Caucasian male





who died from a self-inflicted gunshot wound.





The normalization and hybridization





conditions were adapted from Soares, M. B. et al.





(1994) Proc. Natl. Acad. Sci. USA 91: 9228-9232,





using a longer (19 hour) reannealing hybridization





period.


221
2295344
BRSTNOT05
The library was constructed using RNA isolated





from breast tissue removed from a 58-year-old





Caucasian female during a unilateral extended





simple mastectomy. Pathology for the associated





tumor tissue indicated multicentric invasive grade





4 lobular carcinoma. Patient history included





skin cancer, rheumatic heart disease, osteoarthritis,





and tuberculosis. Family history included





cerebrovascular and cardiovascular disease, breast





and prostate cancer, and type I diabetes.


222
2303994
BRSTNOT05
The library was constructed using RNA isolated





from breast tissue removed from a 58-year-old





Caucasian female during a unilateral extended





simple mastectomy. Pathology for the associated





tumor tissue indicated multicentric invasive





grade 4 lobular carcinoma. Patient history included





skin cancer, rheumatic heart disease, osteoarthritis,





and tuberculosis. Family history included





cerebrovascular and cardiovascular disease, breast





and prostate cancer, and type I diabetes.


223
2497805
ADRETUT05
The library was constructed RNA isolated from





adrenal tumor tissue removed from a 52-year-old





Caucasian female during a unilateral adrenalectomy.





Pathology indicated a pheochromocytoma.


224
2646362
LUNGTUT11
The library was constructed using RNA isolated





from lung tumor tissue removed from the right





lower lobe a 57-year-old Caucasian male during





a segmental lung resection. Pathology indicated an





infiltrating grade 4 squamous cell carcinoma.





Multiple intrapulmonary peribronchial lymph nodes





showed metastatic squamous cell carcinoma.





Patient history included a benign brain neoplasm and





tobacco abuse. Family history included spinal





cord cancer, type II diabetes, cerebrovascular





disease, and malignant prostate neoplasm.


225
2657146
LUNGTUT09
The library was constructed using RNA isolated





from lung tumor tissue removed from a 68-year-





old Caucasian male during segmental lung resection.





Pathology indicated invasive grade 3 squamous





cell carcinoma and a metastatic tumor. Patient





history included type II diabetes, thyroid





disorder, depressive disorder, hyperlipidemia,





esophageal ulcer, and tobacco use.


226
2755786
THP1AZS08
This subtracted THP-1 promonocyte cell line





library was constructed using 5.76 million clones





from a 5-aza-2′-deoxycytidine (AZ) treated





THP-1 cell library. Starting RNA was made from THP-





1 promonocyte cells treated for three days with





0.8 micromolar AZ. The hybridization probe for





subtraction was derived from a similarly constructed





library, made from RNA isolated from





untreated THP-1 cells. 5.76 million clones from





the AZ-treated THP-1 cell library were then





subjected to two rounds of subtractive hybridization





with 5 million clones from the untreated THP-1





cell library. Subtractive hybridization conditions





were based on the methodologies of Swaroop et





al., NAR (1991) 19: 1954, and Bonaldo et al.,





Genome Research (1996) 6: 791. THP-1 (ATCC TIB





202) is a human promonocyte line derived from





peripheral blood of a 1-year-old Caucasian male





with acute monocytic leukemia.


227
2831245
TLYMNOT03
The library was constructed using RNA isolated





from nonactivated Th1 cells. These cells were





differentiated from umbilical cord CD4 T cells





with IL-12 and B7-transfected COS cells.


228
3116250
LUNGTUT13
The library was constructed using RNA isolated





from tumorous lung tissue removed from the right





upper lobe of a 47-year-old Caucasian male during





a segmental lung resection. Pathology indicated





invasive grade 3 (of 4) adenocarcinoma. Family





history included atherosclerotic coronary artery





disease, and type II diabetes.


229
3129630
LUNGTUT12
The library was constructed using RNA isolated





from tumorous lung tissue removed from a 70-





year-old Caucasian female during a lung lobectomy





of the left upper lobe. Pathology indicated





grade 3 (of 4) adenocarcinoma and vascular





invasion. Patient history included tobacco abuse,





depressive disorder, anxiety state, and skin





cancer. Family history included cerebrovascular





disease, congestive heart failure, colon cancer,





depressive disorder, and primary liver.


230
007632
HMC1NOT01
The library was constructed using RNA isolated





from the HMC-1 human mast cell line derived





from a 52-year-old female. Patient history





included mast cell leukemia.


231
1236968
LUNGFET03
The library was constructed using RNA isolated





from lung tissue removed from a Caucasian female





fetus who died at 20 weeks' gestation.


232
1334153
COLNNOT13
The library was constructed using RNA isolated





from ascending colon tissue of a 28-year-old





Caucasian male with moderate chronic ulcerative





colitis.


233
1396975
BRAITUT08
The library was constructed using RNA isolated





from brain tumor tissue removed from the left





frontal lobe of a 47-year-old Caucasian male





during excision of cerebral meningeal tissue.





Pathology indicated grade 4 fibrillary astrocytoma





with focal tumoral radionecrosis. Patient history





included cerebrovascular disease, deficiency anemia,





hyperlipidemia, epilepsy, and tobacco use.





Family history included cerebrovascular disease





and malignant prostate neoplasm.


234
1501749
SINTBST01
The library was constructed using RNA isolated





from ileum tissue removed from an 18-year-old





Caucasian female during bowel anastomosis. Pathology





indicated Crohn's disease of the ileum.





Family history included cerebrovascular disease and





atherosclerotic coronary artery disease.


235
1575240
LNODNOT03
The library was constructed using RNA isolated





from lymph node tissue removed from a 67-year-





old Caucasian male during a segmental lung resection





and bronchoscopy. This tissue was extensively





necrotic with 10% viable tumor. Pathology for the





associated tumor tissue indicated invasive grade





3-4 squamous cell carcinoma. Patient history





included hemangioma. Family history included





atherosclerotic coronary artery disease, benign





hypertension, and congestive heart failure.


236
1647884
PROSTUT09
The library was constructed using RNA isolated





from prostate tumor tissue removed from a 66-





year-old Caucasian male during a radical





prostatectomy, radical cystectomy, and urinary





diversion. Pathology indicated grade 3 transitional





cell carcinoma. Patient history included lung neoplasm,





and benign hypertension. Family history included





malignant breast neoplasm, tuberculosis,





cerebrovascular disease, atherosclerotic coronary





artery disease, and lung cancer.


237
1661144
BRSTNOT09
The library was constructed using RNA isolated





from breast tissue removed from a 45-year-old





Caucasian female during unilateral extended simple





mastectomy. Pathology for the associated





tumor tissue indicated invasive nuclear grade





2-3 adenocarcinoma. Patient history included





valvuloplasty of mitral valve and rheumatic





heart disease. Family history included cardiovascular





disease and type II diabetes.


238
1685409
PROSNOT15
The library was constructed using RNA isolated





from diseased prostate tissue removed from a 66-





year-old Caucasian male during radical prostatectomy





and regional lymph node excision. Pathology indicated





adenofibromatous hyperplasia. Pathology for the





associated tumor tissue indicated adenocarcinoma





(Gleason grade 2 + 3). The patient presented





with elevated prostate specific antigen (PSA).





Family history included prostate cancer, secondary





bone cancer, and benign hypertension.


239
1731419
BRSTTUT08
The library was constructed using RNA isolated





from breast tumor tissue removed from a 45-year-





old Caucasian female during unilateral extended





simple mastectomy. Pathology indicated invasive





nuclear grade 2-3 adenocarcinoma. Patient





history included valvuloplasty of mitral valve and





rheumatic heart disease. Family history included





cardiovascular disease and type II diabetes.


240
2650265
BRSTNOT14
The library was constructed using RNA isolated





from breast tissue removed from a 62-year-old





Caucasian female during a unilateral extended





simple mastectomy. Pathology for the associated





tumor tissue indicated an invasive grade 3 (of 4),





nuclear grade 3 (of 3) adenocarcinoma. Patient





history included a benign colon neoplasm,





hyperlipidemia, cardiac dysrhythmia, and obesity.





Family history included cardiovascular and





cerebrovascular disease and colon, ovary and lung





cancer.


241
2677129
KIDNNOT19
The library was constructed using RNA isolated





from kidney tissue removed a 65-year-old





Caucasian male during an exploratory laparotomy





and nephroureterectomy. Pathology for the





associated tumor tissue indicated grade 1 renal





cell carcinoma within the upper pole of the left





kidney. Patient history included malignant melanoma





of the abdominal skin, benign neoplasm of





colon, cerebrovascular disease, and umbilical





hernia. Family history included myocardial





infarction, atherosclerotic coronary artery





disease, cerebrovascular disease, and prostate





cancer.


242
3151073
ADRENON04
The normalized adrenal gland library was





constructed from 1.36 × 1e6 independent clones





from an adrenal tissue library. Starting RNA was





made from adrenal gland tissue removed from a 20-year-





old Caucasian male who died from head trauma. The





library was normalized in two rounds using





conditions adapted from Soares et al. (PNAS (1994)





91: 9228-9232) and Bonaldo et al. (Genome





Res (1996) 6: 791-806) using a significantly





longer (48-hours/round) reannealing hybridization





period.


243
3170095
BRSTNOT18
The library was constructed using RNA isolated





from diseased breast tissue removed from a 57-





year-old Caucasian female during a unilateral





simple extended mastectomy. Pathology indicated





mildly proliferative breast disease. Patient





history included breast cancer and osteoarthritis.





Family history included type II diabetes,





gallbladder and breast cancer, and chronic





lymphocytic leukemia.


244
3475168
LUNGNOT27
The library was constructed using RNA isolated





from lung tissue removed from a 17-year-old





Hispanic female.


245
3836893
DENDTNT01
The library was constructed using RNA isolated





from treated dendritic cells from peripheral blood.


246
4072159
KIDNNOT26
The library was constructed using RNA isolated





from left kidney medulla and cortex tissue





removed from a 53-year-old Caucasian female





during a nephroureterectomy. Pathology for the





associated tumor tissue indicated grade 2 renal





cell carcinoma involving the lower pole of the





kidney. Patient history included hyperlipidemia,





cardiac dysrhythmia, menorrhagia, cerebrovascular





disease, atherosclerotic coronary artery disease,





and tobacco abuse. Family history included





cerebrovascular disease and atherosclerotic





coronary artery disease.


247
1003916
BRSTNOT03
The library was constructed using RNA isolated





from diseased breast tissue removed from a 54-





year-old Caucasian female during a bilateral





radical mastectomy. Pathology for the associated





tumor tissue indicated residual invasive grade





3 mammary ductal adenocarcinoma. Patient history





included kidney infection and condyloma acuminatum.





Family history included benign hypertension,





hyperlipidemia and a malignant neoplasm of the colon.


248
2093492
PANCNOT04
The library was constructed using RNA isolated





from the pancreatic tissue of a 5-year-old





Caucasian male who died in a motor vehicle accident.


249
2108789
BRAITUT03
The library was constructed using RNA isolated





from brain tumor tissue removed from the left





frontal lobe a 17-year-old Caucasian female during





excision of a cerebral meningeal lesion.





Pathology indicated a grade 4 fibrillary giant





and small-cell astrocytoma. Family history included





benign hypertension and cerebrovascular disease.


250
2171401
ENDCNOT03
The library was constructed using RNA isolated





from dermal microvascular endothelial cells





removed from a neonatal Caucasian male.


251
2212530
SINTFET03
The library was constructed using RNA isolated





from small intestine tissue removed from a





Caucasian female fetus, who died at 20 weeks'





gestation.


252
2253036
OVARTUT01
The library was constructed using RNA isolated





from ovarian tumor tissue removed from a 43-





year-old Caucasian female during removal of the





fallopian tubes and ovaries. Pathology indicated





grade 2 mucinous cystadenocarcinoma involving





the entire left ovary. Patient history included





mitral valve disorder, pneumonia, and viral





hepatitis. Family history included atherosclerotic





coronary artery disease, pancreatic cancer,





stress reaction, cerebrovascular disease, breast cancer,





and uterine cancer.


253
2280161
PROSNON01
The normalized prostate library was constructed





from 4.4M independent clones from the





PROSNOT11 library. Starting RNA was made from





prostate tissue removed from a 28-year-old





Caucasian male who died from a self-inflicted





gunshot wound. The normalization and hybridization





conditions were adapted from Soares, M. B. et al.





(1994) Proc. Natl. Acad. Sci. USA 91: 9228-9232,





using a longer (19 hour) reannealing hybridization





period.


254
2287485
BRAINON01
The library was constructed and normalized from





4.88 million independent clones from the





BRAINOT03 library. RNA was made from brain tissue





removed from a 26-year-old Caucasian male during





cranioplasty and excision of a cerebral meningeal





lesion. Pathology for the associated tumor tissue





indicated a grade 4 oligoastrocytoma in the right





fronto-parietal part of the brain.


255
2380344
ISLTNOT01
The library was constructed using RNA isolated





from a pooled collection of pancreatic islet cells.


256
2383171
ISLTNOT01
The library was constructed using RNA isolated





from a pooled collection of pancreatic islet cells.


257
2396046
THP1AZT01
The library was constructed using RNA isolated





from THP-1 promonocyte cells treated for three





days with 0.8 micromolar 5-aza-2′-deoxycytidine.





THP-1 (ATCC TIB 202)is a human promonocyte line





derived from peripheral blood of a 1-year-old Caucasian





male with acute monocytic leukemia.


258
2456587
ENDANOT01
The library was constructed using RNA isolated





from aortic endothelial cell tissue from an





explanted heart removed from a male during a





heart transplant.


259
2484813
BONRTUT01
The library was constructed using RNA isolated





from rib tumor tissue removed from a 16-year-old





Caucasian male during a rib osteotomy and a wedge





resection of the lung. Pathology indicated a





metastatic grade 3 (of 4) osteosarcoma, forming a





mass involving the chest wall.


260
2493851
ADRETUT05
The library was constructed RNA isolated from





adrenal tumor tissue removed from a 52-year-old





Caucasian female during a unilateral adrenalectomy.





Pathology indicated a pheochromocytoma.


261
2495719
ADRETUT05
The library was constructed RNA isolated from





adrenal tumor tissue removed from a 52-year-old





Caucasian female during a unilateral adrenalectomy.





Pathology indicated a pheochromocytoma.


262
2614153
GBLANOT01
The library was constructed using RNA isolated





from diseased gallbladder tissue removed from a





53-year-old Caucasian female during a cholecystectomy.





Pathology indicated mild chronic cholecystitis and





cholelithiasis with approximately 150 mixed gallstones.





Family history included benign hypertension.


263
2655184
THYMNOT04
The library was constructed using RNA isolated





from thymus tissue removed from a 3-year-old





Caucasian male, who died from anoxia. Serologies





were negative. The patient was not taking any





medications.


264
2848362
BRSTTUT13
The library was constructed using RNA isolated





from breast tumor tissue removed from the right





breast of a 46-year-old Caucasian female during





a unilateral extended simple mastectomy with





breast reconstruction. Pathology indicated an





invasive grade 3 adenocarcinoma, ductal type with





apocrine features and greater than 50% intraductal





component. Patient history included breast





cancer.


265
2849906
BRSTTUT13
The library was constructed using RNA isolated





from breast tumor tissue removed from the right





breast of a 46-year-old Caucasian female during





a unilateral extended simple mastectomy with





breast reconstruction. Pathology indicated an





invasive grade 3 adenocarcinoma, ductal type with





apocrine features and greater than 50% intraductal





component. Patient history included breast





cancer.


266
2899137
DRGCNOT01
The library was constructed using RNA isolated





from dorsal root ganglion tissue removed from the





cervical spine of a 32-year-old Caucasian male





who died from acute pulmonary edema and bronchopneumonia,





bilateral pleural and pericardial effusions, and





malignant lymphoma (natural killer cell type).





Patient history included probable cytomegalovirus,





infection, hepatic congestion and steatosis,





splenomegaly, hemorrhagic cystitis, thyroid hemorrhage,





and Bell's palsy. Surgeries included colonoscopy,





large intestine biopsy, adenotonsillectomy, and





nasopharyngeal endoscopy and biopsy; treatment





included radiation therapy.


267
2986229
CARGDIT01
The library was constructed using RNA isolated





from diseased cartilage tissue. Patient history





included osteoarthritis.


268
3222081
COLNNON03
The normalized colon library was constructed





from 2.84 × 106 independent clones from the





COLNNOT07 library. Starting RNA was made from





colon tissue removed from a 60-year-old





Caucasian male during a left hemicolectomy.





The normalization and hybridization conditions were





adapted from Soares et al. (PNAS (1994) 91: 9228-9232),





Swaroop et al. (Nucl. Acids Res. (1991) 19: 1954)





and Bonaldo et al. (Genome Res (1996) 6: 791-806),





using a significantly longer (48 hour) reannealing





hybridization period.



















TABLE 5





Program
Description
Reference
Parameter Threshold







ABI 
A program that removes vector sequences and 
Perkin-Elmer Applied Biosystems,



FACTURA
masks ambiguous bases in nucleic acid 
Foster City, CA.




sequences.




ABI/PARACEL 
A Fast Data Finder useful in comparing and 
Perkin-Elmer Applied Biosystems,
Mismatch < 50%


FDF
annotating amino acid or nucleic acid sequences.
Foster City, CA; Paracel Inc., Pasadena, CA.



ABI 
A program that assembles nucleic acid 
Perkin-Elmer Applied Biosystems,



AutoAssembler
sequences.
Foster City, CA.



BLAST
A Basic Local Alignment Search Tool useful in 
Altschul, S. F. et al. (1990) J. Mol. Biol.
ESTs: Probability value = 



sequence similarity search for amino acid and 
215: 403-410; Altschul, S. F. et al. (1997)
1.0E−8 or less



nucleic acid sequences. BLAST includes five 
Nucleic Acids Res. 25: 3389-3402.
Full Length sequences:



functions: blastp, blastn, blastx, tblastn, and 

Probability value = 1.0E−10 or less 



tblastx.




FASTA
A Pearson and Lipman algorithm that searches 
Pearson, W. R. and D. J. Lipman (1988) Proc.
ESTs: fasta E value = 1.06E−6



for similarity between a query sequence and a 
Natl. Acad Sci. 85: 2444-2448; Pearson, W. R.
Assembled ESTs: fasta Identity = 



group of sequences of the same type. FASTA 
(1990) Methods Enzymol. 183: 63-98; and
95% or greater and Match



comprises as least five functions: fasta, tfasta, 
Smith, T. F. and M. S. Waterman (1981) Adv.
length = 200 bases or greater; 



fastx, tfastx, and ssearch.
Appl. Math. 2: 482-489.
fastx E value = 1.0E−8 or less





Full Length sequences: fastx





score = 100 or greater


BLIMPS
A BLocks IMProved Searcher that matches a 
Henikoff, S and J. G. Henikoff, Nucl. Acid Res.,
Score = 1000 or greater; Ratio of



sequence against those in BLOCKS, PRINTS, 
19: 6565-72, 1991. J. G. Henikoff and S.
Score/Strength = 0.75 or larger;



DOMO, PRODOM, and PFAM databases to 
Henikoff (1996) Methods Enzymol. 266: 88-105;
and, if applicable, Probability



search for gene families, sequence homology, 
and Attwood, T. K. et al. (1997) J. Chem. Inf.
value = 1.0E−3 or less



and structural fingerprint regions.
Comput. Sci. 37: 417-424.



PFAM
An algorithm for searching a query sequence 
Krogh, A. et al. (1994) J. Mol. Biol., 235: 1501- 
Score = 10-50 bits for PFAM 



against hidden Markov model (HMM)-based 
1531; Sonnhammer, E. L. L. et al. (1988)
hits, depending on individual 



databases of protein family consensus 
Nucleic Acids Res. 26: 320-322.
protein families



sequences, such as PFAM.




ProfileScan
An algorithm that searches for structural and 
Gribskov, M. et al. (1988) CABIOS 4: 61-66;
Score = 4.0 or greater



sequence motifs in protein sequences that 
Gribskov, et al. (1989) Methods Enzymol.




match sequence patterns defined in Prosite.
183: 146-159; Bairoch, A. et al. (1997) Nucleic





Acids Res. 25: 217-221.



Phred
A base-calling algorithm that examines 
Ewing, B. et al. (1998) Genome




automated sequencer traces with high 
Res. 8: 175-185; Ewing, B. and P.




sensitivity and probability.
Green (1998) Genome Res. 8: 186-





194.



Phrap
A Phils Revised Assembly Program including 
Smith, T. F. and M. S. Waterman (1981) Adv.
Score = 120 or greater; Match



SWAT and CrossMatch, programs based on 
Appl. Math. 2: 482-489; Smith, T. F. and M. S.
length = 56 or greater



efficient implementation of the Smith-Waterman 
Waterman (1981) J. Mol. Biol. 147: 195-197;




algorithm, useful in searching sequence 
and Green, P., University of Washington,




homology and assembling DNA sequences.
Seattle, WA.



Consed
A graphical tool for viewing and editing Phrap 
Gordon, D. et al. (1998) Genome




assemblies
Res. 8: 195-202.



SPScan
A weight matrix analysis program that scans 
Nielson, H. et al. (1997) Protein Engineering
Score = 5 or greater



protein sequences for the presence of secretory 
10: 1-6; Claverie, J. M. and S. Audic (1997)




signal peptides.
CABIOS 12: 431-439.



Motifs
A program that searches amino acid sequences 
Bairoch et al. supra; Wisconsin




for patterns that matched those defined in 
Package Program Manual, version




Prosite.
9, page M51-59, Genetics Computer





Group, Madison, WI.




















TABLE 6





Nucleotide

Fragment of
Starting Nucleotide of
Ending Nucleotide of


SEQ ID NO:
Clone ID
SEQ ID NO
Fragment
Fragment



















135
443531
443531H1
1
253




1406807F6
152
336




443531T6
847
355




SBBA00451F1
396
856




SBBA00676F1
546
865


136
632860
632860H1
13
253




784715R3
17
666




509590H1
455
706


137
670010
670010H1
1
263




669971R1
1
633


138
726498
726498H1
13
263




726498R6
13
489




866599R3
7
660


139
795064
795064H1
86
323




4339458H1
4
284




937605R3
86
505




2381151F6
592
1057




1466346F6
857
1241


140
924925
924925H1
111
412




3268330H1
2
239




759120R3
111
629


141
962390
1907958F6
1
478




023569F1
1122
470




167282F1
1216
543




1309211F1
911
1224


142
1259405
1259405H1
46
277




2472425H1
331
354




774303R1
190
743




1520779F1
418
1001




1693833F6
914
1467




1831858T6.comp
1336
1742




1527737T6.comp
1386
1829


143
1297384
1297384H1
402
641




1269310F6
1
492




1457367F1
792
1380




415587R1
1358
1712




SANA02967F1
1143
614


144
1299627
1299627H1
1
250




1359140F6
1004
1573




1349224F1
1330
1731




SBAA01431F1
46
397




SBAA02909F1
868
262




SBAA01156F1
901
1266


145
1306026
1306026H1
1
223




1464088R6
302
829




SBAA02496F1
92
568




SBAA04305F1
366
883


146
1316219
1316219H1
246
491




2458603F6
1
402




2504756T6
980
380


147
1329031
1329031H1
1
264




1329031T6
505
1




1329031F6
1
523


148
1483050
1483050H1
722
931




855049H1
1
267




077017F1
1069
679




1483050F6
722
1215




1480024T6
2063
1315




1483050T6
2068
1535




759486R1
1762
2089


149
1514160
1514160H1
1640
1838




1866765T7
2383
2210




782676R1
1652
1875




008055X4
1090
1804




008055X5
1316
1952




1866765F6
2209
2391




SAOA03127F1
2129
1703


150
1603403
1603403H1
7
224




372910F1
420
44




733299R7
219
420


151
1652303
1652303H1
4
256




1671806H1
1
224




1341743T1
2069
1900




3803812H1
389
697




1878546F6
747
1344




1428640F1
1081
1664




2058609R6
1715
2098




1331621F1
1780
2096




1306331T1
1897
2098


152
1693358
1693358H1
41
125




2498265H1
1
252




1867125F6
205
373




1693358T6
1094
416




2245848R6
737
1103


153
1707711
1707711H1
408
626




1484609T1
2165
1855




1707711F6
408
987




1267959F1
1721
2182




1484609F1
1855
2178




SAJA00930F1
544
1132




SAJA01300R1
1675
1212




SAJA00999R1
1675
1142


154
1738735
1738735H1
7
236




SAJA00944R1
393
5




SAJA00137F1
913
685




SAJA03629F1
435
42


155
1749147
1749147H1
1
276


155

1749147F6
47
457


155

1749147T6
479
1


156
1817722
1817722H1
1
268




2011085H1
344
545


157
1831290
1831290H1
10
257




3473958H1
70
242




1972268F6
163
617




1301277F1
413
852




1521574F1
1024
1602




1561690T6
1729
1058




891461R1
1261
1738


158
1831477
1831477H1
59
337




1582867H1
1
199




1336769T1
1986
1639




1933092H1
525
789




1519909F1
841
1296




1220946H1
1061
1318




809556T1
1983
1687




1217559T1
2002
1445




1309225F1
1747
2001


159
1841607
1841607H1
13
192




SBHA03588F1
13
172


160
1852391
1852391H1
98
367




734140H1
1
225




1852391F6
98
542


161
1854555
1854555H1
1
265




2511711H1
37
58




782453R1
223
712




1854555F6
1
346




1840675T6
1046
860




2109736H1
938
1054


162
1855755
1855755H1
17
224




3040236H1
1
179




1283207F1
306
816




833763T1
1148
835




1920926R6
854
1161


163
1861434
1861434H1
13
253




186143476
872
261




SARA01525F1
426
808




SARA02548F1
587
889


164
1872334
1872334H1
1
229




1872334F6
1
424




SBGA03684F1
358
425


165
1877230
1877230H1
1405
1677




2519841H1
1
251




1877230T6
1903
1405




1254693F1
335
716




077020R1
682
1414




1232336F1
906
1507




1004952R6
1451
1904




SARA01879F1
1545
1921




SARA02654F1
1545
1923


166
1877885
1877885H1
68
323




508020F1
499
51




2751126R6
219
516




SARA02571F1
407
499


167
1889269
1889269H1
757
1020




1915551H1
1
191




629493X12
481
865




1441289F1
693
865




1215274X34F1
1106
1631




1818447F6
1307
1540




1208463R1
1372
1493


168
1890243 
1890243H1
9
268




SARA01884F1
521
168




SATA00046F1
1057
851




SARA03294F1
1329
910




SARA02790F1
1138
1535


169
1900433
1900433H1
1
242




SATA00396F1
409
124




SATA02742F1
1
294


170
1909441
1909441H1
786
1048




1398811F1
1
550




3039939H1
607
876




3324740H1
685
944




1442131F6
787
1232




2254056H1
1423
1522




2199453T6
1955
1351




1698531H1
1968
1796


171
1932226
1932226H1
294
510




2320569H1
1
266




1932226F6
294
685




2469455T6
1475
1071




2469455F6
1034
1492




1907140F6
1158
1482




SATA02592F1
857
518


172
1932647
1932647H1
17
246




1492745T1
1582
1418




1492745H1
1418
1599




SASA02355F1
386
19




SASA00117F1
250
569




SASA00192F1
515
816


173
2124245
2124245H1
45
190




1235393F1
495
895




1402264F6
323
925




1303990F1
682
1240




1402264T6
1613
950


174
2132626
2132626H1
406
651




1723432T6
1299
746




2132626R6
406
904




1736723T6
1292
857




1504738F1
868
1320


175
2280639
2280639H1
28
303




1377560F6
261
777


176
2292356
2292356H1
717
968




4086827H1
1
275




1754442F6
232
577




3571126H1
497
808




1601305F6
808
1464


177
2349310
2349310H1
1
236




2349310T6
682
2


178
2373227
2373227H1
298
524




3316444H1
801
1053




302685R6
1141
1496




SASA02181F1
577
1




SASA01923F1
963
466




SASA03516F1
1102
1249


179
2457682
2457682H1
1
226




2457682F6
1
554


180
2480426
2480426H1
1
213




2480426F6
1
501


181
2503743
2503743H1
6
222




1853909H1
1
272




1517619F1
172
830




1467896F6
540
1112




490031F1
1647
1068




1208654R1
1382
1633




880544R1
1450
1648


182
2537684
2537684H1
434
682




2005493H1
1
194




730969H1
307
547




916487H1
723
989




996135R1
997
1598




1920738R6
1306
1692




1957710F6
1472
1692


183
2593853
2593853H1
1
252




807497H1
2
217




914020R6
284
740




889992R1
416
729


184
2622354
2622354H1
3
266




2623992H1
1
246




1556510F6
81
258


185
2641377
2641377H1
126
369




4341415H2
10
345




SBCA07049F3
126
599


186
2674857
2674857H1
139
393




1872373H1
1
270




470512R6
1486
1502




1728547H1
1285
1508




3013651F6
1423
1987




SBCA01366F1
819
385




SBCA00694F1
973
1198


187
2758485
2758485H1
20
267




3097533H1
1
158




1578959F6
291
771


188
2763296
2763296H1
63
301




3486025F6
1
130




SBDA07002F3
63
687


189
2779436
2779436H1
1
233




2779436F6
1
577




SBDA07009F3
1
608


190
2808528
2808528H1
25
335




2611513F6
2
489




SBDA07021T3
1058
443


191
2809230
2809230H1
409
630




2213849H1
1
133




711706R6
396
691




958323R1
407
800




030732F1
1366
623


192
2816821
2816821H1
210
501




3746964H1
1
307




2816821F6
210
682




948722T6
959
527


193
2817268
2817268H1
42
282




3591308H1
13
264




419522R1
179
808




2073028F6
446
924




1308781F6
869
1112


194
2923165
2923165H1
8
295




2011630H1
18
238




1457250F1
268
856




754668R1
327
878




1406510F6
558
901


195
2949822
2949822H1
1
280




SBDA07078F3
1
606


196
2992192
2992192H1
25
321




2534324H2
1
240




2815255T6
690
219




1551107T6
893
471




1551107R6
471
690


197
2992458
2992458H1
48
362




2618951H1
1
247




1479252F1
163
610




1879054H1
563
840




1879054F6
563
1096




2215240H1
951
1202




1535968T1
1729
1173


198
3044710
3044710H1
652
952




3741773H1
1
283




859906X42C1
94
192




1534347F1
90
268




1421122F1
830
1392




1303865F1
1033
1487




1704452F6
1432
1934




1251642F1
2006
1544




1781694R6
1894
2017


199
3120415
3120415H1
72
363




1360123T1
523
141




1375015H1
380
526

## Claims

1. An isolated antibody or antigen-binding fragment thereof which specifically binds to
a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 43.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the fragment is a Fab, F(ab′)2, or Fv fragment.

3. The isolated antibody or antigen-binding fragment thereof of claim 1, which is humanized.

4. The isolated antibody or antigen-binding fragment thereof of claim 1, which is a monoclonal antibody, polyclonal antibody, chimeric antibody, or single chain antibody.

5. The isolated antibody or antigen-binding fragment thereof of claim 1, which is a neutralizing antibody.

6. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 43.

7. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 43.

8. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, further comprising at least one stabilizing compound.

10. The pharmaceutical composition of claim 8, further comprising at least one active agent, drug, or hormone.

11. The pharmaceutical composition of claim 8, formulated for administration via a method selected from the group consisting of oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, parenteral, intranasal, enteral, topical, sublingual, and rectal means.