Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• the present invention relates to secretory molecules and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases associated with, as well as effects of exogenous compounds on, the expression of secretory molecules.
• Protein transport is mediated by a signal peptide located at the amino terminus of the protein to be transported or secreted.
• the signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the riboso e to a particular membrane bound compartment such as the endoplasmic reticulum (ER).
• ER endoplasmic reticulum
• 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.
• Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues.
• Proteins that are secreted from the cell are generally synthesized as inactive precursors that are activated by post-translational processing events during transit through the secretory pathway. Such events include glycosylation, 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 he nascent protein and interaction of the protein with a receptor or pore complex. Examples of secretory proteins with amino terminal signal peptides are discussed below and include proteins with important roles in cell-to-cell signaling.
• G-protein coupled receptors comprise a superfamily of integral membrane proteins which transduce extracellular signals. Not all GPCRs contain N-terminal signal peptides.
• GPCRs include receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic-type), acetylcholine (muscarinic-type), 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.
• biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic-type), acetylcholine (muscarinic-type), and serotonin
• lipid mediators of inflammation such as prostaglandins, platelet activating factor, and leukotrienes
• 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
• the large third intracellular loop interacts with G proteins to activate second messengers such as cyclic AMP, phospholipase C, inositol triphosphate, or ion channels.
• receptors include cell surface antigens identified on leukocytic cells of he 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 "cluster of differentiation" or "CD” designation.
• CD antigens Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques. CD antigens have been 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. (1995) The Leucocyte Antigen Facts Book, Academic Press, San Diego CA, pp. 17-20.)
• GPI glycosylphosphatidylinositol
• MPs Matrix proteins
• the expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases.
• 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, hi addition, MPs may be heavily glycosylated and may contain an Arginine-Glycine- Aspartate (RGD) tripeptide motif which may play a role in adhesive interactions.
• MPs include extracellular proteins such as fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B; and cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and integrins.
• Cytokines are secreted by hematopoietic cells in response to injury or infection. Interleukins, neurotrophins, growth factors, interferons, and chemokines all define cytokine families that work in conjunction with cellular receptors to regulate cell proliferation and differentiation. In addition, cytokines effect activities such as leukocyte migration and function, hematopoietic cell proliferation, temperature regulation, acute response to infection, tissue remodeling, and apoptosis.
• Chemokines 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. (Ca ⁇ lard, R. and Gearing, A. (1994) The Cytokine Facts Book, Academic Press, New York NY, 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).
• 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).
• CSFs colony stimulating factors
• 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 H, 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 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.
• Proteolytic enzymes or proteases either activate or deactivate proteins byhydrolyzing 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.
• Ion channels, ion pumps, and transport proteins mediate the transport of molecules across cellular membranes.
• Transport can occur by a passive, concentration-dependent mechanism or can be linked to an energy source such as ATP hydrolysis.
• Symporters and antiporters transport ions and small molecules such as amino acids, glucose, and drugs.
• Symporters transport molecules and ions unidirectionally, and antiporters transport molecules and ions 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.
• Ion channels are formed by transmembrane proteins which create a lined passageway across the membrane through which water and ions, such as Na + , K + , Ca 2+ , and CI", enter and exit the cell.
• 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 Ca 2+ ATPase and NaVK + ATPase and function in transporting H + , Na + , K + , and Ca 2+ ions.
• P-class pumps consist of two ⁇ and two ⁇ transmembrane subunits.
• the V- and F-class ion pumps have similar structures 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.
• the proteins in this family contain a highly conserved, large transmembrane domain comprised of 12 ⁇ -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 ofthe 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.)
• 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 5 compositions and mechanisms of action, hormones can be grouped into two categories.
• One category includes 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 includes l o hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane.
• 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.
• catecholamines amino acid derivatives
• peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin.
• Neuropeptides and vasomediators 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
• 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, CR. et al. (1985) Endocrine Physiology, Oxford University Press,
• Embodiments ofthe invention relate to nucleic acid sequences comprising human polynucleotides encoding secretory polypeptides that can contain signal peptides and/or transmembrane domains.
• human polynucleotides as presented in the Sequence Listing uniquely identify partial or full length genes encoding structural, functional, and regulatory polypeptides involved in cell signaling.
• An embodiment of the invention provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• the polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567.
• the polynucleotide comprises at least 30 contiguous nucleotides of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; b) a polynucleotide comprising a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d
• the polynucleotide comprises at least 60 contiguous nucleotides of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; b) a polynucleotide comprising a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• compositions for the detection of expression of secretory polynucleotides comprising at least one isolated polynucleotide comprising a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d); and a detectable label.
• An embodiment of the invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a polynucleotide sequence of a polyneucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence of a polynucleotide selected from the group consisting of SEQ ID NO: 1-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
• Another embodiment also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a polynucleotide sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
• the invention provides a composition comprising a target polynucleotide ofthe method, wherein said probe comprises at least 30 contiguous nucleotides.
• the invention provides a composition comprising a target polynucleotide ofthe method, wherein said probe comprises at least 60 contiguous nucleotides.
• Another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• the invention provides a cell transformed with the recombinant polynucleotide.
• the invention provides a transgenic organism comprising the recombin
• Yet another embodiment provides a method for producing a secretory polypeptide, the method comprising a) culturing a cell under conditions suitable for expression ofthe secretory polypeptide, wherein said cell is transformed with a recombinant polynucleotide, said recombinant polynucleotide comprising an isolated polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; iii) a polynucleotide 5 complementary to the polynucleotide of i); iv) a polynucleotide complementary to the polynucleotide of ii);
• Still another embodiment provides an isolated secretory polypeptide (SPTM) encoded by at 0 least one polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567.
• SPTM secretory polypeptide
• the invention further provides a method of screening for a test compound that specifically binds to the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• the method comprises a) combining the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146 with at least one test compound 5 under suitable conditions, and b) detecting binding of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146 to the test compound, thereby identifying a compound that specifically binds to the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• Still yet another embodiment provides a microarray wherein at least one element of the o microarray is an isolated polynucleotide comprising at least 30 contiguous nucleotides of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; c) a polynucleotide complementary to the 5 polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• the invention also provides a method for generating a transcript image of a sample which contains polynucleotides.
• the method comprises a) labeling the polynucleotides of he sample, b) contacting the elements of the microarray with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the o expression of the polynucleotides in the sample.
• Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-567; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
• a target polynucleotide comprises a polynucleotide selected from the group consisting of
• the method comprises a) exposing a sample comprising the target 5 polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression ofthe target polynucleotide in the presence of varying amounts ofthe compound and in the absence of the compound.
• Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) l o hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567; iii) a polynucleotide
• Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence
• the target polynucleotide comprises a polynucleotide sequence of a
• Another embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-l 146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-l 146, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• the polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• the polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:l-567.
• Another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:568-l 146, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO.568-1146.
• compositions comprising a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-l 146, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ TD NO:568-l 146, and a pharmaceutically acceptable excipient.
• the composition comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146.
• Another embodiment provides a method of treating a disease or condition associated with decreased expression of functional SPTM, comprising administering to a patient in need of such treatment the composition.
• Another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence 5 selected from the group consisting of SEQ ID NO:568-l 146, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568- 1146, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-l 146.
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
• the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
• the invention provides a method of treating a disease or condition associated with decreased expression of functional SPTM, comprising administering to a patient in need of such treatment the composition.
• Another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:568-l 146, c) a biologically active fragment of a o polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:568-
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
• the invention provides a composition comprising an antagonist compound identified by the 5 method and a pharmaceutically acceptable excipient.
• the invention provides a method of treating a disease or condition associated with overexpression of functional SPTM, comprising administering to a patient in need of such treatment the composition.
• Another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino o acid sequence selected from the group consisting of SEQ ID NO.568-1146, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:568-1146, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO.568- 1146, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from 5 the group consisting of SEQ ID NO:568-l 146.
• the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity ofthe polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence ofthe test compound, wherein a change in the activity of he polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
• Table 1 shows the sequence identification numbers (SEQ ID NO:s) and template identification numbers (template IDs) co ⁇ esponding to the polynucleotides of the present invention, along with the sequence identification numbers (SEQ ID NO:s) and open reading frame identification numbers (ORF IDs) co ⁇ esponding to polypeptides encoded by the template ID.
• Table 2 shows the sequence identification numbers (SEQ ID NO:s) and template identification numbers (template IDs) corresponding to the polynucleotides ofthe present invention, along with polynucleotide segments of each template sequence as defined by the indicated “start” and “stop” nucleotide positions.
• the reading frames of the polynucleotide segments are shown, and the polypeptides encoded by the polynucleotide segments constitute either signal peptide (SP) or transmembrane (TM) domains, as indicated.
• SP signal peptide
• TM transmembrane
• Table 3 shows the sequence identification numbers and template identification numbers
• template IDs co ⁇ esponding to the polynucleotides ofthe present invention, along with component sequence identification spans corresponding to each template.
• the component sequences, which were used to assemble the template sequences, are defined by the spans indicating the nucleotide positions along each template.
• Table 4 shows the tissue distribution profiles for the templates of the invention.
• Table 5 shows the sequence identification numbers (SEQ ID NO:s) corresponding to the polypeptides of the present invention, along with the reading frames used to obtain the polypeptide segments, the lengths ofthe polypeptide segments, the "start” and “stop” nucleotide positions ofthe polynucleotide sequences used to define the encoded polypeptide segments, the GenBank hits (GI Numbers), probability scores, and functional annotations co ⁇ esponding to the GenBank hits.
• Table 6 summarizes the bioinformatics tools which are useful for analysis ofthe polynucleotides of the present invention.
• the first column of Table 6 lists analytical tools, programs, and algorithms, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, 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 score, the greater the homology between two sequences).
• sptm refers to a nucleic acid sequence
• SPTM amino acid sequence encoded by sptm
• a “full-length” sptm refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in human tissue.
• adjuvants are materials such as Freund's adjuvant, mineral gels (aluminum hydroxide), and surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol) which maybe administered to increase a host's immunological response.
• Alleles refers to an alternative form of a nucleic acid sequence. Alleles result from a “mutation,” a change or an alternative reading of the genetic code. Any given gene may have none, one, or many allelic forms. Mutations which give rise to alleles include deletions, additions, or substitutions of nucleotides. Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence.
• the present invention encompasses allelic sptm.
• allelic variant is an alternative form of the gene encoding SPTM. 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. A gene may have none, one, or many allelic variants of its naturally occurring form. 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 SPTM include those sequences with deletions, 5 insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SPTM or a polypeptide with at least one functional characteristic of SPTM. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding SPTM, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding SPTM.
• the 0 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 SPTM.
• Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature ofthe residues, as long as the biological or immunological activity of SPTM is retained.
• negatively charged amino acids may 5 include aspartic acid and glutamic acid
• positively charged amino acids may include lysine and arginine.
• Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
• Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
• Amino acid sequence refers to a peptide, a polypeptide, or a protein of either natural or synthetic origin. The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a fragment, epitope, variant, or derivative of a protein expressed by a nucleic acid sequence.
• Amplification refers to the production of additional copies of a sequence and is carried out 5 using polymerase chain reaction (PCR) technologies well known in the art.
• PCR polymerase chain reaction
• 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 SPTM polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or peptide used to immunize an animal e.g., a o mouse, a rat, or a rabbit
• an animal e.g., a o mouse, a rat, or a rabbit
• 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.
• aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a 5 specific molecular target.
• Aptamers are derived from an in vitro evolutionary process (e.g. , SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
• Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
• the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2 -OH group of a ribonucleotide maybe replaced by 2 -F or 2 -NH-,), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
• Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance ofthe aptamer from the circulatory system.
• Aptamers maybe specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
• introduction refers to an aptamer which is expressed in vivo.
• a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
• spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
• Antisense sequence refers to a sequence capable of specifically hybridizing to a target sequence.
• the antisense sequence may include DNA, RNA, or any nucleic acid mimic or analog such as peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified base.
• PNA peptide nucleic acid
• Antisense technology refers to any technology which relies on the specific hybridization of an antisense sequence to a target sequence.
• a 'bin is a portion of computer memory space used by a computer program for storage of data, and bounded in such a manner that data stored in a bin may be retrieved by the program.
• Bioly active refers to an amino acid sequence having a structural, regulatory, or biochemical function of a naturally occu ⁇ ing amino acid sequence.
• “Clone joining” is a process for combining gene bins based upon the bins' containing sequence information from the same clone. The sequences may assemble into a primary gene transcript as well as one or more splice variants.
• Component sequence is a nucleic acid sequence selected by a computer program such as PHRED and used to assemble a consensus or template sequence from one or more component sequences.
• a "consensus sequence” or “template sequence” is a nucleic acid sequence which has been assembled from overlapping sequences, using a computer program for fragment assembly such as the GELVIEW fragment assembly system (Genetics Computer Group (GCG), Madison WI) or using a relational database management system (RDMS).
• GELVIEW fragment assembly system Genetics Computer Group (GCG), Madison WI
• RDMS relational database management system
• Constant amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, i. e. , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
• the table below shows amino acids which maybe substituted for an original amino acid in a protein and which are regarded as conservative substitutions.
• Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• “Deletion” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or amino acid residue, respectively, is absent.
• “Derivative” refers to the chemical modification of a nucleic acid sequence, such as by replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group.
• “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons maybe carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample. 5
• element and “a ⁇ ay element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microa ⁇ ay.
• modulate refers to a change in the activity of SPTM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SPTM.
• E-value refers to the statistical probability that a match between two sequences occurred by chance.
• Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain ofthe encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the 5 evolution of new protein functions.
• a “fragment” is a unique portion of sptm or SPTM which is identical in sequence to but shorter in length than the parent sequence.
• a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
• a fragment may comprise from 10 to 1000 contiguous amino acid residues or nucleotides.
• a fragment used as a probe, primer, 0 antigen, therapeutic molecule, or for other purposes maybe at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues or nucleotides in length. Fragments may be preferentially selected from certain regions of a molecule.
• a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
• these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing and the figures, maybe encompassed by the present embodiments.
• a fragment of sptm comprises a region of unique polynucleotide sequence that specifically identifies sptm, for example, as distinct from any other sequence in the same genome.
• a fragment of sptm is useful, for example, in hybridization and amplification technologies and in analogous methods o that distinguish sptm from related polynucleotide sequences.
• the precise length of a fragment of sptm and the region of sptm to which the fragment co ⁇ esponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
• a fragment of SPTM is encoded by a fragment of sptm.
• a fragment of SPTM comprises a region of unique amino acid sequence that specifically identifies SPTM.
• a fragment of 5 SPTM is useful as an immunogenic peptide for the development of antibodies that specifically recognize SPTM.
• the precise length of a fragment of SPTM and the region of SPTM to which the fragment co ⁇ esponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
• a “full length” nucleotide sequence is one containing at least a start site for translation to a protein sequence, followed by an open reading frame and a stop site, and encoding a "full length” polypeptide.
• “Hit” refers to a sequence whose annotation will be used to describe a given template. Criteria for selecting the top hit are as follows: if the template has one or more exact nucleic acid matches, the top hit is the exact match with highest percent identity. If the template has no exact matches but has significant protein hits, the top hit is the protein hit with the lowest E-value. If the template has no significant protein hits, but does have significant non-exact nucleotide hits, the top hit is the nucleotide hit with the lowest E-value.
• Homology refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of an sptm or between a reference amino acid sequence and a fragment of an SPTM.
• Hybridization refers to the process by which a strand of nucleotides anneals with a complementary strand through base pairing. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under defined annealing conditions, and remain hybridized after the "washing" step.
• the defined hybridization conditions include the annealing conditions and the washing step(s), the latter of which is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid probes that are not perfectly matched.
• Permissive conditions for annealing of nucleic acid sequences are routinely determinable and maybe consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency.
• stringency of hybridization is expressed with reference to the temperature under which the wash step is carried out.
• wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (T, for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, or 55°C may be used. SSC concentration may be varied from about 0.2 to 2 x SSC, with SDS being present at about 0.1%.
• blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200 ⁇ g/ml. Useful variations on these conditions will be readily apparent to those skilled in the art.
• Hybridization, particularly under high stringency conditions may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their resultant proteins.
• RNA:DNA hybridizations may also be used under particular circumstances, such as RNA:DNA hybridizations. Appropriate hybridization conditions are routinely determinable by one of ordinary skill in the art.
• Immunologically active or “immunogenic” describes the potential for a natural, recombinant, or synthetic peptide, epitope, polypeptide, or protein to induce antibody production in appropriate animals, cells, or cell lines.
• Immunogenic 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.
• An "immunogenic fragment” is a polypeptide or oligopeptide fragment of SPTM which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
• the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of SPTM which is useful in any of the antibody production methods disclosed herein or known in the art.
• “Insertion” or “addition” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or residue, respectively, is added to the sequence.
• Labeling refers to the covalent or noncovalent joining of a polynucleotide, polypeptide, or antibody with a reporter molecule capable of producing a detectable or measurable signal.
• “Microarray” is any a ⁇ angement of nucleic acids, amino acids, antibodies, etc., on a substrate.
• the substrate maybe a solid support such as beads, glass, paper, nitrocellulose, nylon, or an appropriate membrane.
• Linkers are short stretches of nucleotide sequence which maybe added to a vector or an sptm to create restriction endonuclease sites to facilitate cloning.
• Polylinkers are engineered to incorporate multiple restriction enzyme sites and to provide for the use of enzymes which leave 5' or 3' overhangs (e.g., BamHI, EcoRI, and Hindlll) and those which provide blunt ends (e.g., EcoRV, SnaBI, and Stul).
• Nucleic acid sequence refers to the specific order of nucleotides joined by phosphodiester bonds in a linear, polymeric arrangement. Depending on the number of nucleotides, the nucleic acid sequence can be considered an oligomer, oligonucleotide, or polynucleotide.
• the nucleic acid can be DNA, RNA, or any nucleic acid analog, such as PNA, may be of genomic or synthetic origin, may be either double-stranded or single-stranded, and can represent either the sense or antisense (complementary) strand.
• Oligomers refers to a nucleic acid sequence of at least about 6 nucleotides and as many as about 60 nucleotides, preferably about 15 to 40 nucleotides, and most preferably between about 20 and 30 nucleotides, that may be used in hybridization or amplification technologies. Oligomers may be used as, e.g., primers for PCR, and are usually chemically synthesized.
• "Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
• PNA protein nucleic acid
• PNA refers to a DNA mimic in which nucleotide bases are attached to a pseudopeptide backbone to increase stability.
• PNAs also designated antigene agents, can prevent gene expression by targeting complementary messenger RNA.
• percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison ofthe two sequences.
• NCBI National Center for Biotechnology Information
• BLAST Basic Local Alignment Search Tool
• NCBI National Center for Biotechnology Information
• BLAST Basic Local Alignment Search Tool
• the BLAST software suite includes various sequence analysis programs including "BLASTN,” that is used to determine alignment between a known polynucleotide sequence and other sequences on a variety of databases.
• BLAST 2 Sequences are used for direct pairwise comparison of two nucleotide sequences.
• "BLAST 2 Sequences” can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2/.
• the "BLAST 2 Sequences” tool can be used for both BLASTN and BLASTP (discussed below).
• BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use BLASTN with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters. Such default parameters maybe, for example:
• Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured.
• Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
• the phrases "percent identity” and "% identity”, as applied to polypeptide sequences refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity of the substituted residue, thus preserving the structure (and therefore function) ofthe folded polypeptide.
• NCBI BLAST software suite may be used.
• BLAST 2 Sequences Version 2.0.9 (May-07-1999) with BLASTP set at default parameters.
• BLASTP default parameters
• Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured.
• Probe refers to sptm or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
• Primer pairs are short nucleic acids, usually DNA 5 oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• Probes and primers as used in the present invention typically comprise at least 15 contiguous 0 nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers maybe considerably longer than these examples, and it is understood that any length supported by the specification, including the figures and Sequence Listing, maybe used. 5 Methods for preparing and using probes and primers are described in the references, for example Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2 nd ed., vol.
• PCR primer pairs can be o derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
• Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 5 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope. The Primer3 primer o selection program (available to the public from the Whitehead Institute/MIT Center for Genome
• Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays.
• the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
• the 5 PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
• this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
• the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microa ⁇ ay elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
• “Purified” refers to molecules, either polynucleotides or polypeptides that are isolated or separated from their natural environment and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other compounds with which they are naturally associated.
• a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
• the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion ofthe nucleic acid.
• a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
• such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
• Regulatory element refers to a nucleic acid sequence from nontranslated regions of a gene, and includes enhancers, promoters, introns, and 3 ' untranslated regions, which interact with host proteins to carry out or regulate transcription or translation.
• Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, an amino acid, or an antibody. They include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
• RNA equivalent in reference to a DNA sequence, is composed ofthe same linear sequence of nucleotides as the reference DNA sequence with the exception that all occu ⁇ ences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose. "Sample” is used in its broadest sense.
• Samples may contain nucleic or amino acids, antibodies, or other materials, and maybe derived from any source (e.g., bodily fluids including, but not limited to, saliva, blood, and urine; chromosome(s), organelles, or membranes isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; and cleared cells or tissues or blots or imprints from such cells or tissues).
• source e.g., bodily fluids including, but not limited to, saliva, blood, and urine; chromosome(s), organelles, or membranes isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; and cleared cells or tissues or blots or imprints from such cells or tissues).
• Specific binding or “specifically binding” refers to the interaction between a protein or peptide and its agonist, antibody, antagonist, or other binding partner. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic dete ⁇ ninant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing 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.
• Substitution refers to the replacement of at least one nucleotide or amino acid by a different nucleotide or amino acid.
• Substrate refers to any suitable rigid or semi-rigid support including, e.g., membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles or 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.
• a “transcript image” refers to the collective pattern of gene expression by a particular tissue or cell type under given conditions at a given time.
• Transformation refers to a process by which exogenous DNA enters a recipient cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed.
• Transformants include 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 cells which transiently express inserted DNA or RNA.
• a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
• the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
• the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
• the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals.
• the isolated DNA ofthe present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA ofthe present invention into such organisms 5 are widely known and provided in references such as Sambrook et al. (1989), supra.
• a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 25% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using BLASTN with the "BLAST 2 Sequences" tool Version 2.0.9 (May- 07-1999) set at default parameters.
• Such a pair of nucleic acids may show, for example, at least 30%, 0 at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
• the variant may result in "conservative" amino acid changes which do not affect structural and/or chemical properties.
• a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymorphic” 5 variant.
• 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 co ⁇ esponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
• Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid o 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. 5
• variants of the polynucleotides of the present invention may be generated through recombinant methods.
• One possible method is a DNA shuffling technique such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S.
• DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties.
• prefe ⁇ ed variants may then be pooled and further subjected to recursive rounds of DNA shuffling and 5 selection/screening.
• genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
• fragments of a given gene maybe recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
• a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using BLASTP with the "BLAST 2 Sequences" tool Version 2.0.9 (May- 07-1999) set at default parameters.
• Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
• cDNA sequences derived from human tissues and cell lines were aligned based on nucleotide sequence identity and assembled into "consensus" or "template” sequences which are designated by the template identification numbers (template IDs) in column 2 of Table 2.
• template IDs template identification numbers
• SEQ ID NO:s sequence identification numbers co ⁇ esponding to the template IDs are shown in column 1.
• Segments ofthe template sequences are defined by the "start” and “stop” nucleotide positions listed in columns 3 and 4. These segments, when translated in the reading frames indicated in column 5, have similarity to signal peptide (SP) or transmembrane (TM) domain consensus sequences, as indicated in column 6.
• the invention incorporates the nucleic acid sequences of these templates as disclosed in the Sequence Listing and the use of these sequences in the diagnosis and treatment of disease states characterized by defects in cell signaling.
• the invention further utilizes these sequences in hybridization and amplification technologies, and in particular, in technologies which assess gene expression patterns correlated with specific cells or tissues and their responses in vivo or in vitro to pharmaceutical agents, toxins, and other treatments. In this manner, the sequences ofthe present invention are used to develop a transcript image for a particular cell or tissue.
• cDNA was isolated from libraries constructed using RNA derived from normal and diseased human tissues and cell lines.
• the human tissues and cell lines used for cDNA library construction were selected from a broad range of sources to provide a diverse population of cDNAs representative of gene transcription throughout the human body. Descriptions of the human tissues and cell lines used for cDNA library construction are provided in the LIEESEQ database (Incyte Genomics, Inc. (Incyte), Palo Alto CA).
• Human tissues were broadly selected from, for example, cardiovascular, dermatologic, endocrine, gastrointestinal, hematopoietic/immune system, musculoskeletal, neural, reproductive, and urologic sources.
• Cell lines used for cDNA library construction were derived from, for example, leukemic cells, teratocarcinomas, neuroepitheliomas, cervical carcinoma, lung fibroblasts, and endothelial cells.
• Such cell lines include, for example, THP-1, Jurkat, HUVEC, hNT2, WI38, HeLa, and other cell lines commonly used and available from public depositories (American Type Culture Collection, Manassas VA).
• cell lines Prior to mRNA isolation, cell lines were untreated, treated with a pharmaceutical agent such as 0 5 -aza-2'-deoxycytidine, treated with an activating agent such as lipopolysaccharide in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress.
• a pharmaceutical agent such as 0 5 -aza-2'-deoxycytidine
• an activating agent such as lipopolysaccharide in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress.
• Chain termination reaction products may be electrophoresed on urea- polyacrylamide gels and detected either by autoradiography (for radioisotope-labeled nucleotides) or by fluorescence (for fluorophore-labeled nucleotides).
• Automated methods for mechanized reaction 5 preparation, sequencing, and analysis using fluorescence detection methods have been developed.
• Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system (Hamilton Company (Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc. (MJ Research), Watertown MA), and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing can be carried out using, for example, the ABI 373 or 377 (Applied o Biosystems) or MEGABACE 1000 (Molecular Dynamics, Inc. (Molecular Dynamics), Sunnyvale
• CA DNA sequencing systems, or other automated and manual sequencing systems well known in the art.
• nucleotide sequences of he Sequence Listing have been prepared by current, state-of- the-art, automated methods and, as such, may contain occasional sequencing errors or unidentified 5 nucleotides. Such unidentified nucleotides are designated by an N. These infrequent unidentified bases do not represent a hindrance to practicing the invention for those skilled in the art.
• Several methods employing standard recombinant techniques maybe used to co ⁇ ect errors and complete the missing sequence information. (See, e.g., those described in Ausubel, F.M. et al. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY; and Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY.)
• Human polynucleotide sequences may be assembled using programs or algorithms well known in the art. Sequences to be assembled are related, wholly or in part, and may be derived from a single or many different transcripts. Assembly of the sequences can be performed using such programs as PHRAP (Phils Revised Assembly Program) and the GELVIEW fragment assembly system (GCG), or other methods known in the art.
• PHRAP Phils Revised Assembly Program
• GCG GELVIEW fragment assembly system
• cDNA sequences are used as "component" sequences that are assembled into “template” or “consensus” sequences as follows. Sequence chromatograms are processed, verified, and quality scores are obtained using PHRED. Raw sequences are edited using an editing pathway known as Block 1 (See, e.g., the LIEESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA). A series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) are replaced by "n's", or masked, to prevent spurious matches. Mitochondrial and ribosomal RNA sequences are also removed.
• Block 1 See, e.g., the LIEESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA).
• a series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide
• the processed sequences are then loaded into a relational database management system (ROMS) which assigns edited sequences to existing templates, if available.
• RDMS relational database management system
• a process is initiated which modifies existing templates or creates new templates from works in progress (i.e., nonfinal assembled sequences) containing queued sequences or the sequences themselves.
• the templates can be merged into bins. If multiple templates exist in one bin, the bin can be split and the templates reannotated.
• bins are "clone joined" based upon clone information. Clone joining occurs when the 5' sequence of one clone is present in one bin and the 3' sequence from the same clone is present in a different bin, indicating that the two bins should be merged into a single bin. Only bins which share at least two different clones are merged.
• a resultant template sequence may contain either a partial or a full length open reading frame, or all or part of a genetic regulatory element. This variation is due in part to the fact that the full length cDNAs of many genes are several hundred, and sometimes several thousand, bases in length. With current technology, cDNAs comprising the coding regions of large genes cannot be cloned because of vector limitations, incomplete reverse transcription of the mRNA, or incomplete "second strand" synthesis. Template sequences maybe extended to include additional contiguous sequences derived from the parent RNA transcript using a variety of methods known to those of skill in the art. Extension may thus be used to achieve the full length coding sequence of a gene.
• the cDNA sequences are analyzed using a variety of programs and algorithms which are well known in the art. (See, e.g., Ausubel, 1997, supra. Chapter 7.7; Meyers, R.A. (Ed.) (1995) Molecular Biology and Biotechnolo y, Wiley VCH, New York NY, pp. 856-853; and Table 6.) These analyses comprise both reading frame determinations, e.g., based on triplet codon periodicity for 0 particular organisms (Fickett, J.W. (1982) Nucleic Acids Res. 10:5303-5318); analyses of potential start and stop codons; and homology searches.
• BLAST Basic Local Alignment Search Tool
• BLAST is especially useful in determining exact matches and comparing two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user (Karlin, S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:841-845).
• GenBank e.g., GenBank
• SwissProt e.g., GenBank
• BLOCKS e.g., GenBank
• PFAM e.g., PFAM
• other databases e.g., GenBank, SwissProt, BLOCKS, PFAM and other databases maybe o searched for sequences containing regions of homology to a query sptm or SPTM of the present invention.
• Protein hierarchies can be assigned to the putative encoded polypeptide based on, e.g., motif, BLAST, or biological analysis. Methods for assigning these hierarchies are described, for example, in o "Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence
• the sptm ofthe present invention maybe used for a variety of diagnostic and therapeutic 5 purposes.
• an sptm maybe used to diagnose a particular condition, disease, or disorder associated with cell signaling.
• Such conditions, diseases, and disorders include, but are not limited to, a cell proliferative disorder 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 5 adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer ofthe adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder
• the sptm can be used to detect the presence of, or to quantify the amount of, an sptm- related polynucleotide in a sample. This information is then compared to information obtained from appropriate reference samples, and a diagnosis is established.
• a polynucleotide complementary to a given sptm can inhibit or inactivate a therapeutically relevant gene related to the sptm.
• the expression of sptm may be routinely assessed by hybridization-based methods to determine, for example, the tissue-specificity, disease-specificity, or developmental stage-specificity of sptm expression.
• the level of expression of sptm may be compared among different cell types or tissues, among diseased and normal cell types or tissues, among cell types or tissues at different developmental stages, or among cell types or tissues undergoing various treatments.
• This type of analysis is useful, for example, to assess the relative levels of sptm expression in fully or partially differentiated cells or tissues, to determine if changes in sptm expression levels are co ⁇ elated with the development or progression of specific disease states, and to assess the response of a cell or tissue to a specific therapy, for example, in pharmacological or toxicological studies.
• Methods for the analysis of sptm expression are based on hybridization and amplification technologies and include membrane-based procedures such as northern blot analysis, high-throughput procedures that utilize, for example, microarrays, and PCR-based procedures.
• the sptm, their fragments, or complementary sequences may be used to identify the presence of and/or to determine the degree of similarity between two (or more) nucleic acid sequences.
• the sptm may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropriately selected temperatures and salt concentrations.
• Hybridization with a probe based on the nucleic acid sequence of at least one of he sptm allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or related to the sptm ofthe Sequence Listing.
• Probes may be selected from non-conserved or unique regions of at least one of the polynucleotides of SEQ ID NO:l-567 and tested for their ability to identify or amplify the target nucleic acid sequence using standard protocols.
• Polynucleotide sequences that are capable of hybridizing, in particular, to those shown in SEQ ID NO: 1 -567 and fragments thereof, can be identified using 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.) Hybridization conditions are discussed in "Definitions.”
• a probe for use in Southern or northern hybridization may be derived from a fragment of an sptm sequence, or its complement, that is up to several hundred nucleotides in length and is either 5 single-stranded or double-stranded. Such probes may be hybridized in solution to biological materials such as plasmids, bacterial, yeast, or human artificial chromosomes, cleared or sectioned tissues, or to artificial substrates containing sptm. Microa ⁇ ays are particularly suitable for identifying the presence of and detecting the level of expression for multiple genes of interest by examining gene expression co ⁇ elated with, e.g., various stages of development, treatment with a drug or compound, or disease 0 progression.
• An array analogous to a dot or slot blot may be used to arrange and link polynucleotides to the surface of a substrate using one or more ofthe following: mechanical (vacuum), chemical, thermal, or UV bonding procedures.
• mechanical (vacuum), chemical, thermal, or UV bonding procedures Such an a ⁇ ay may contain any number of sptm and may be produced by hand or by using available devices, materials, and machines.
• Microa ⁇ ays may be prepared, used, and analyzed using methods known in the art. (See, e.g., 5 Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
• Probes may be labeled by either PCR or enzymatic techniques using a variety of o commercially available reporter molecules.
• commercial kits are available for radioactive and chencdluminescent labeling (Amersham Pharmacia Biotech) and for alkaline phosphatase labeling (Life Technologies).
• sptm may be cloned into commercially available vectors for the production of RNA probes.
• Such probes may be transcribed in the presence of at least one labeled nucleotide (e.g., 32 P-ATP, Amersham Pharmacia Biotech).
• polynucleotides of SEQ ID NO: 1-567 or suitable fragments thereof can be used to isolate full length cDNA sequences utilizing hybridization and/or amplification procedures well known in the art, e.g., cDNA library screening, PCR amplification, etc.
• the molecular cloning of such full length cDNA sequences may employ the method of cDNA library screening with probes using the hybridization, stringency, washing, and probing strategies described above and in Ausubel, supra, o Chapters 3, 5, and 6. These procedures may also be employed with genomic libraries to isolate genomic sequences of sptm in order to analyze, e.g., regulatory elements.
• Gene identification and mapping are important in the investigation and treatment of almost all 5 conditions, diseases, and disorders. Cancer, cardiovascular disease, Alzheimer's disease, arthritis, diabetes, and mental illnesses are of particular interest. Each of these conditions is more complex than the single gene defects of sickle cell anemia or cystic fibrosis, with select groups of genes being predictive of predisposition for a particular condition, disease, or disorder.
• cardiovascular disease may result from malfunctioning receptor molecules that fail to clear cholesterol from the bloodstream, and diabetes may result when a particular individual's immune system is activated by an infection and attacks the insulin-producing cells of the pancreas.
• Alzheimer's disease has been linked to a gene on chromosome 21; other studies predict a different gene and location.
• Mapping of disease genes is a complex and reiterative process and generally proceeds from genetic linkage analysis to physical mapping.
• a genetic linkage map traces parts of chromosomes that are inherited in the same pattern as the condition.
• Statistics link the inheritance of particular conditions to particular regions of chromosomes, as defined by RFLP or other markers.
• RFLP Radio Resource Linearity
• markers and their locations are known from previous studies. More often, however, the markers are simply stretches of DNA that differ among individuals. Examples of genetic linkage maps can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site.
• sptm sequences may be used to generate hybridization probes useful in chromosomal mapping of naturally occurring genomic sequences. Either coding or noncoding sequences of sptm may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of an sptm coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
• 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 PI constructions, or single chromosome cDNA libraries.
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• bacterial PI constructions or single chromosome cDNA libraries.
• Fluorescent in situ hybridization may be co ⁇ elated with other physical chromosome mapping techniques and genetic map data.
• Co ⁇ elation between the location of sptm 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 sptm sequences may also be used to detect polymorphisms that are genetically linked to the inheritance of a particular condition, disease, or disorder.
• In situ hybridization of chromosomal preparations and genetic mapping techniques such as linkage analysis using established chromosomal markers, maybe used for extending existing genetic maps.
• any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• the nucleotide sequences ofthe subject 0 invention may also be used to detect differences in chromosomal architecture due to translocation, inversion, etc., among normal, carrier, or affected individuals.
• a disease-associated gene is mapped to a chromosomal region, the gene is cloned in order to identify mutations or other alterations (e.g., translocations or inversions) that maybe co ⁇ elated with disease.
• This process includes a physical map of the chromosomal region containing 5 the disease-gene of interest along with associated markers. A physical map is necessary for determining the nucleotide sequence of and order of marker genes on a particular chromosomal region. Physical mapping techniques are well known in the art and involve the generation of overlapping sets of cloned DNA fragments from a particular organelle, chromosome, or genome. These clones are analyzed to reconstruct and catalog their order. Once the position of a marker is o determined, the DNA from that region is obtained by consulting the catalog and selecting clones from that region. The gene of interest is located through positional cloning techniques using hybridization or similar methods.
• embodiments of sptm described herein can be configured to be used to design probes useful in diagnostic assays known to those skilled in the art. Such assays can be configured to be used to diagnose, detect, confirm or quantitate conditions, disorders, or diseases associated with abnormal levels of sptm expression. Labeled probes developed from sptm sequences are added to a sample under hybridizing conditions of desired stringency. In some instances, sptm, or o fragments or oligonucleotides derived from sptm, may be used as primers in amplification steps prior to hybridization. The amount of hybridization complex formed is quantified and compared with standards for a selected cell or tissue type.
• Indication of the presence of the condition, disorder, or disease can be determined by variation of the amount of sptm expression from the assay standard.
• the amount of variation indicative of the condition can be above or below the standard, can be selected by the 5 medical practioner, can be determined from known patient populations and/or amounts known in the medical arts and/or associated standards organizations or regulations (e.g. CLIA).
• Examples of qualitative or quantitative diagnostic assays or methods may include but are not limited to northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, enzyme-linked immunosorbent assay (ELISA)-like, pin, chip-based assays and the like.
• the probes described above may also be used to monitor the progress of conditions, disorders, or diseases associated with abnormal levels of sptm expression, or to evaluate the efficacy of a particular therapeutic treatment.
• the candidate probe maybe identified from the sptm that are specific to a given human tissue and have not been observed in GenBank or other genome databases. Such a probe may be used in animal studies, preclinical tests, clinical trials, or in monitoring the 0 treatment of an individual patient.
• standard expression is established by methods well known in the art for use as a basis of comparison, samples from patients affected by the disorder or disease are combined with the probe to evaluate any deviation from the standard profile, and a therapeutic agent is administered and effects are monitored to generate a treatment profile.
• Efficacy is evaluated by determining whether the expression progresses toward or returns to the standard 5 normal pattern. Treatment profiles may be generated over a period of several days or several months. Statistical methods well known to those skilled in the art may be use to determine the significance of such therapeutic agents.
• the polynucleotides are also useful for identifying individuals from minute biological samples, for example, by matching the RFLP pattern of a sample's DNA to that of an individual's DNA.
• the o polynucleotides of the present invention can also be used to detenriine the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, an individual can be identified through a unique set of DNA sequences. Once a unique ID database is established for an individual, positive identification of that individual can be made from 5 extremely small tissue samples.
• oligonucleotide primers derived from the sptm of the invention maybe used to detect single nucleotide polymorphisms (SNPs).
• SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
• Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and o fluorescent SSCP (fSSCP) methods.
• SSCP single-stranded conformation polymorphism
• fSSCP o fluorescent SSCP
• oligonucleotide primers derived from sptm are used to amplify DNA using the polymerase chain reaction (PCR).
• the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
• SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
• the 5 oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high- throughput equipment such as DNA sequencing machines.
• sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequences of individual overlapping DNA fragments which assemble into a common consensus sequence.
• SNPs maybe detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
• DNA-based identification techniques can be used in forensic technology. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be amplified using, e.g., PCR, to identify individuals. (See, e.g., Erlich, H.
• polynucleotides ofthe present invention can be used as polymorphic markers.
• reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
• polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a particular cell type, in the creation of subtracted cDNA libraries which aid in the discovery of novel polynucleotides, in selection and synthesis of oligomers for attachment to an array or other support, and as an antigen to elicit an immune response.
• the polynucleotides encoding SPTM or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
• ES embryonic stem
• Such techniques are well known in the art and are useful for the generation of animal models of human disease.
• mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
• the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
• a marker gene e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
• the vector integrates into the co ⁇ esponding region ofthe host genome by homologous recombination.
• homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
• Transformed ES cells are identified and microinjected into mouse cellblastocysts such as those from the C57BL/6 mouse strain.
• the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
• Transgenic animals thus generated maybe tested with potential therapeutic or toxic agents.
• the polynucleotides encoding SPTM may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types.
• the polynucleotides encoding SPTM of the invention can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of sptm is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
• Transgenic progeny or inbred lines are studied and treated with potential 5 pharmaceutical agents to obtain info ⁇ nation on treatment of a human disease.
• a mammal inbred to overexpress sptm resulting, e.g., in the secretion of SPTM in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
• Screening Assays o SPTM encoded by polynucleotides of the present invention may be used to screen for molecules that bind to or are bound by the encoded polypeptides.
• the binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the bound molecule.
• Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
• the molecule is closely related to the natural ligand of the polypeptide, e.g., a ligand or fragment thereof, a natural substrate, or a structural or functional mimetic.
• the molecule can be closely related to the natural receptor to which the polypeptide binds, or to at least a fragment ofthe receptor, e.g., the active site. In either case, the molecule can be rationally designed using known techniques.
• the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila. or E. coli.
• Cells expressing the polypeptide or cell membrane fractions which contain the expressed polypeptide are then contacted with a test compound and binding, stimulation, or inhibition of activity of either the polypeptide or the molecule is analyzed.
• An assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. Alternatively, the assay may assess binding in the presence of a labeled competitor.
• the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures.
• the assay may also simply 5 comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
• an ELISA assay using, e.g., a monoclonal or polyclonal antibody can measure polypeptide level in a sample.
• the antibody can measure polypeptide level by either binding, directly 0 or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
• All of the above assays can be used in a diagnostic or prognostic context.
• the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule.
• the assays can discover agents which may inhibit or enhance the production of the polypeptide from 5 suitably manipulated cells or tissues.
• a transcript image represents the global pattern of gene expression by a particular tissue or cell o type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al, "Comparative Gene Transcript Analysis," U.S. Patent Number 5,840,484, expressly incorporated by reference herein.)
• a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a 5 particular tissue or cell type.
• the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
• the resultant transcript image would provide a profile of gene activity pertaining to cell signaling.
• Transcript images which profile sptm expression may be generated using transcripts isolated o from tissues, cell lines, biopsies, or other biological samples.
• the transcript image may thus reflect sptm expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
• Transcript images which profile sptm expression may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of 5 industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153- 159; Steiner, S. and Anderson, N. L. (2000) Toxicol. Lett. 112-113:467-71, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
• the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
• Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides ofthe present invention, so that transcript levels co ⁇ esponding to the polynucleotides of the present invention may be quantified.
• the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
• proteome refers to the global pattern of protein expression in a particular tissue or cell type.
• proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
• a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
• the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
• the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
• the optical density of each protein spot is generally proportional to the level of the protein in the sample.
• the optical densities of equivalently positioned protein spots from different samples for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment.
• - 5 spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
• the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
• a proteomic profile may also be generated using antibodies specific for SPTM to quantify the levels of SPTM expression. In one embodiment, the antibodies are used as elements on a microa ⁇ ay, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
• Detection may be performed by a variety 5 of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino- reactive fluorescent compound and detecting the amount of fluorescence bound at each a ⁇ ay element.
• Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor 0 co ⁇ elation between transcript and protein abundances for some proteins in some tissues (Anderson, N. L. and Seilhamer, J. (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases. 5 In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
• Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
• the amount of each protein is compared to the amount of the co ⁇ esponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the o test compound in the treated sample.
• Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the SPTM encoded by polynucleotides of the present invention.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated 5 with antibodies specific to the SPTM encoded by polynucleotides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Transcript images may be used to profile sptm expression in distinct tissue types. This process can be used to determine cell signaling activity in a particular tissue type relative to this activity in a different tissue type.
• Transcript images may be used to generate a profile of sptm expression characteristic of diseased tissue. Transcript images of tissues before and after treatment may be used for diagnostic purposes, to monitor the progression of disease, and to monitor the efficacy of drug treatments for diseases which affect cell signaling activity.
• Transcript images of cell lines can be used to assess cell signaling activity and/or to identify cell lines that lack or misregulate this activity. Such cell lines may then be treated with pharmaceutical agents, and a transcript image following treatment may indicate the efficacy of these agents in restoring desired levels of this activity. A similar approach may be used to assess the toxicity of pharmaceutical agents as reflected by undesirable changes in cell signaling activity. Candidate pharmaceutical agents maybe evaluated by comparing their associated transcript images with those of pharmaceutical agents of known effectiveness.
• Antisense Molecules The polynucleotides of the present invention are useful in antisense technology. Antisense technology or therapy relies on the modulation of expression of a target protein through the specific binding of an antisense sequence to a target sequence encoding the target protein or directing its expression.
• Antisense technology or therapy relies on the modulation of expression of a target protein through the specific binding of an antisense sequence to a target sequence encoding the target protein or directing its expression.
• Agrawal, S., ed. 1996 Antisense Therapeutics, Humana Press Inc., Totawa NJ; Alama, A. et al. (1997) Pharmacol. Res. 36(3):171-178; Crooke, S.T. (1997) Adv. Pharmacol. 40:1-49; Sharma, H.W. and R.
• An antisense sequence is a polynucleotide sequence capable of specifically hybridizing to at least a portion of the target sequence. Antisense sequences bind to cellular mRNA and/or genomic DNA, affecting translation and/or transcription. Antisense sequences can be DNA, RNA, or nucleic acid mimics and analogs. (See, e.g., Rossi, J.J. et al. (1991) Antisense Res. Dev. l(3):285-288; Lee, R. et al.
• the binding which results in modulation of expression occurs through hybridization or binding of complementary base pairs.
• Antisense sequences can also bind to DNA duplexes through specific interactions in the major groove of the double helix.
• the polynucleotides of the present invention and fragments thereof can be used as antisense sequences to modify the expression of the polypeptide encoded by sptm.
• antisense sequences can be produced ex vivo, such as by using any of the ABI nucleic acid synthesizer series (Applied Biosystems) or other automated systems known in the art. Antisense sequences can also be produced biologically, such as by transforming an appropriate host cell with an expression vector containing the 5 sequence of interest. (See, e.g., Agrawal, supra.)
• Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
• Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
• expression 0 In order to express a biologically active SPTM, the nucleotide sequences encoding SPTM or fragments thereof maybe 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.
• 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.
• Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding SPTM and appropriate transcriptional and 5 translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, supra, Chapters 4, 8, 16, and 17; and Ausubel, supra, Chapters 9, 10, 13, and 16.)
• a variety of expression vector/host systems may be utilized to contain and express sequences encoding SPTM. These include, but are not limited to, microorganisms such as bacteria transformed o 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 (mammalian) cell systems.
• microorganisms such as bacteria transformed o 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
• Expression vectors derived from retroviruses, adeno viruses, or herpes or vaccinia viruses, or from various bacterial plasmids maybe used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
• sequences encoding SPTM 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. Any number of selection systems may be used to recover transformed cell lines.
• the polynucleotides encoding SPTM of the invention may be used for somatic or germline gene therapy.
• Gene therapy may be performed to (i) co ⁇ ect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
• SCID severe combined immunodeficiency
• ADA adenosine deaminase
• sptm hepatitis B or C virus
• fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
• protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi
• diseases or disorders caused by deficiencies in sptm are treated by constructing mammalian expression vectors comprising sptm and introducing these vectors by mechanical means into sptm-deficient cells.
• Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct D ⁇ A microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of D ⁇ A transposons (Morgan, R.A. and Anderson, W.F. (1993) Annu. Rev. Biochem. 62:191- 217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and Recipon, H (1998) Curr. Opin. Biotechnol. 9:445-450).
• Expression vectors that may be effective for the expression of sptm include, but are not limited to, the PCD ⁇ A 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-O ⁇ , PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
• the sptm of the invention maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H. (1992) Proc. ⁇ atl. Acad. Sci. U.S.A. 89:5547-5551; Gossen, M. et al., (1995) Science 268:1766-1769; Rossi, F.M.V. and Blau, H.M.
• a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin
• TRANSFECTION KIT available from Invitrogen
• transformation is performed using the calcium phosphate method (Graham, F.L. and Eb, A J. (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
• the introduction of DNA to primary cells involves modification of these standardized mammalian transfection protocols.
• diseases or disorders caused by genetic defects with respect to sptm expression are treated by constructing a retiovirus vector consisting of (i) sptm under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
• Retrovirus vectors e.g., PFB and PFBNEO
• Retrovirus vectors are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. U.S.A.
• the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and Miller, A.D. (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J.
• VPCL vector producing cell line
• U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
• an adenovirus-based gene therapy delivery system is used to deliver sptm to cells which have one or more genetic abnormalities with respect to the expression of sptm.
• the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27 :263-268). Potentially useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy”), hereby incorporated by reference.
• herpes-based, gene therapy delivery system is used to deliver sptm to target cells which have one or more genetic abnormalities with respect to the expression of sptm.
• HSV herpes simplex virus
• herpes-based vectors are well known to those with ordinary skill in the art.
• a replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res.l69:385-395).
• the construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
• Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W. F. et al. 1999 J. Virol. 73:519-532 and Xu, H. et al., (1994) Dev. Biol. 163:152-161, hereby incorporated by reference.
• herpesvirus sequences The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
• an alphavirus (positive, single-stranded RNA virus) vector is used to deliver sptm to target cells.
• SFV Semliki Forest Virus
• SFV Semliki Forest Virus
• alphavirus infection is typically associated with cell lysis within a few days
• BHK-21 hamster normal kidney cells
• SIN Sindbis virus
• the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
• the wide host range of alphaviruses will allow the introduction of sptm into a variety of cell types.
• the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
• the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
• Anti-SPTM antibodies may be used to analyze protein expression levels. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, and Fab fragments. For descriptions of and protocols of antibody technologies, see, e.g., Pound J.D. (1998) Immunochemical Protocols, Humana Press, Totowa, NJ.
• amino acid sequence encoded by the sptm of the Sequence Listing may be analyzed by appropriate software (e.g., LASERGENE NAVIGATOR software, DNASTAR) to determine 5 regions of high immunogenicity.
• appropriate software e.g., LASERGENE NAVIGATOR software, DNASTAR
• the optimal sequences for immunization are selected from the C- terminus, the N-terminus, and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the polypeptide is in its natural conformation. Analysis used to select appropriate epitopes is also described by Ausubel (1997, supra. Chapter 11.7). Peptides used for antibody induction do not need to have biological activity; however, they should be o antigenic.
• Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids, and most preferably at least 15 amino acids.
• a peptide which mimics an antigenic fragment of the natural polypeptide may be fused with another protein such as keyhole limpet hemocyanin (KLH; Sigma, St. Louis MO) for antibody production.
• KLH keyhole limpet hemocyanin
• a peptide encompassing ah antigenic region may be expressed from an sptm, synthesized 5 as described above, or purified from human cells.
• mice, goats, and rabbits may be immunized by injection with a peptide.
• various adjuvants maybe used to increase immunological response.
• peptides about 15 residues in length maybe synthesized using an ABI 431 A 0 ' peptide synthesizer (Applied Biosystems) using frnoc-chemistry and coupled to KLH (Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, 1995, supra).
• Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
• the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin (BSA), reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG.
• BSA bovine serum albumin
• Antisera with antipeptide activity are tested for anti-SPTM activity using protocols well known in the art, including ELISA, radioimmunoassay (RIA), and immunoblotting.
• isolated and purified peptide may be used to immunize mice (about 100 ⁇ g of peptide) or rabbits (about 1 mg of peptide). Subsequently, the peptide is radioiodinated and used to screen the immunized animals' B-lymphocytes for production of antipeptide antibodies. Positive o cells are then used to produce hybridomas using standard techniques. About 20 mg of peptide is sufficient for labeling and screening several thousand clones. Hybridomas of interest are detected by screening with radioiodinated peptide to identify those fusions producing peptide-specific monoclonal antibody.
• wells of a multi-well plate (FAST, Becton-Dickinson, Palo Alto, CA) are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species IgG) antibodies at 5 10 mg/ml.
• the coated wells are blocked with 1% BSA and washed and exposed to supematants from hybridomas. After incubation, the wells are exposed to radiolabeled peptide at 1 mg/ml.
• Clones producing antibodies bind a quantity of labeled peptide that is detectable above background. Such clones are expanded and subjected to 2 cycles of cloning. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A (Amersham Pharmacia Biotech). Several procedures for the production of monoclonal antibodies, including in vitro production, are described in Pound (supra). Monoclonal antibodies with antipeptide activity are tested for anti-SPTM activity using protocols well known in the art, including ELISA, RIA, and immunoblotting.
• Antibody fragments containing specific binding sites for an epitope may also be generated.
• such fragments include, but are not limited to, the Ffab ⁇ fragments produced by pepsin digestion ofthe antibody molecule, and the Fab fragments generated by reducing the disulfide bridges ofthe F(ab')2 fragments.
• construction of Fab expression libraries in filamentous bacteriophage allows rapid and easy identification of monoclonal fragments with desired specificity (Pound, supra, Chaps. 45-47).
• Antibodies generated against polypeptide encoded by spt can be used to purify and characterize full-length SPTM protein and its activity, binding partners, etc.
• Anti-SPTM antibodies may be used in assays to quantify the amount of SPTM found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions.
• the peptides and antibodies of the invention may be used with or without modification or labeled by joining them, either covalently or noncovalently, with a reporter molecule.
• Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS). Such immunoassays typically involve the formation of complexes between the SPTM and its specific antibody and the measurement of such complexes. These and other assays are described in Pound (supra).
• RNA was purchased from CLONTECH Laboratories, Inc. (Palo Alto CA) or isolated from various tissues. 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 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.
• RNA was treated with DNase.
• poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega Corporation (Promega), Madison WI), OLIGOTEX latex particles (QIAGEN, Inc. (QIAGEN), Valencia CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
• RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Inc., Austin TX).
• Stratagene was provided with RNA and constructed the co ⁇ esponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene Cloning Systems, Inc. (Stratagene), La Jolla CA) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, Chapters 5.1 through 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.
• 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., PBLUESCRTPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
• Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies.
• Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system
• Plasmids were purified using at least one of the following: the Magic or WIZARD Minipreps DNA purification system (Promega); the AGTC Miniprep purification kit (Edge BioSystems, Gaithersburg MD); and the QIAWELL 8, QIAWELL 8 Plus, and QIAWELL 8 Ultra plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit (QIAGEN). Following 0 precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
• plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format.
• Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 5 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Inc. (Molecular Probes), Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy . Helsinki, Finland).
• cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 thermal cycler (Applied Biosystems) or the PTC- 200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific Corp., Sunnyvale CA) or the MICROLAB 2200 liquid transfer system (Hamilton).
• cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or 5 supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
• Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence o analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, Chapter 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VHL
• sequences from chromatograms were subject to PHRED analysis and assigned a quality score.
• the sequences having at least a required quality score were subject to various preprocessing editing pathways to eliminate, e.g., low quality 3 ' ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, bacterial contamination sequences, and sequences smaller than 50 base pairs.
• low-information sequences and repetitive 5 elements e.g., dinucleotide repeats, Alu repeats, etc.
• sequences were then subject to assembly procedures in which the sequences were assigned to gene bins (bins). Each sequence could only belong to one bin. Sequences in each gene bin were assembled to produce consensus sequences (templates). Subsequent new sequences were 0 added to existing bins using BLASTN (v.1.4 WashU) and CROSSMATCH. Candidate pairs were identified as all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using a version of PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP.
• each assembled template 5 was determined based on the number and orientation of its component sequences. Template sequences as disclosed in the sequence listing co ⁇ espond to sense strand sequences (the "forward" reading frames), to the best determination. The complementary (antisense) strands are inherently disclosed herein.
• the component sequences which were used to assemble each template consensus sequence are listed in Table 3 by their positions along the template nucleotide sequences. o Bins were compared against each other and those having local similarity of at least 82% were combined and reassembled. Reassembled bins having templates of insufficient overlap (less than 95% local identity) were re-split.
• Assembled templates were also subject to analysis by ST ⁇ TCHER/EXON MAPPER algorithms which analyze the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced 5 genes across tissue types or disease states, etc. These resulting bins were subject to several rounds of the above assembly procedures.
• bins were clone joined based upon clone information. If the 5' sequence of one clone was present in one bin and the 3' sequence from the same clone was present in a different bin, it was likely that the two bins actually o belonged together in a single bin. The resulting combined bins underwent assembly procedures to regenerate the consensus sequences.
• the template sequences were further analyzed by translating each template in all three forward reading frames and searching each translation against the Pfam database of hidden Markov model-based protein families and domains using the HMMER software package (available to the public from Washington University School of Medicine, St. Louis MO). (See also World Wide Web site http://pfam.wustl.edu/for detailed descriptions of Pfam protein domains and families.)
• the template sequences were translated in all three forward reading frames, and each translation was searched against hidden Markov models for signal peptides using the HMMER software package. Construction of hidden Markov models and their usage in sequence analysis has been described. (See, for example, Eddy, S.R. (1996) Curr. Opin. Str. Biol. 6:361-365.) Only those signal peptide hits with a cutoff score of 11 bits or greater are reported. A cutoff score of 11 bits or greater corresponds to at least about 91-94% true-positives in signal peptide prediction.
• Template sequences were also translated in all three forward reading frames, and each translation was searched against TMHMMER, a program that uses a hidden Markov model (HMM) to delineate transmembrane segments on protein sequences and determine orientation (Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. Conf. On Intelligent Systems for Mol. Biol., Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence (AAAI) Press, Menlo Park, CA, and MIT Press, Cambridge, MA, pp. 175-182.) Regions of templates which, when translated, contain similarity to signal peptide or transmembrane consensus sequences are reported in Table 2.
• HMM hidden Markov model
• Template sequences are further analyzed using the bioinformatics tools listed in Table 6, or using sequence analysis software known in the art such as MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases.
• polypeptide sequences were translated to derive the corresponding longest open reading frame as presented by the polypeptide sequences as reported in Table 5.
• a polypeptide of the invention may begin at any of the methionine residues within the full length translated polypeptide.
• Polypeptide sequences were subsequently analyzed by querying against the GenBank protein database (GENPEPT, (GenBank version 128)).
• Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
• Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
• Table 5 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (GENPEPT) database.
• Column 1 shows the polypeptide sequence identification number (SEQ ID NO:) for the polypeptide segments of the invention.
• Column 2 shows the reading frame used in the translation of the polynucleotide sequences encoding the polypeptide segments.
• Column 3 shows the length of the translated polypeptide segments.
• Columns 4 and 5 show the start and stop nucleotide positions of the polynucleotide sequences encoding the polypeptide segments.
• Column 6 shows the GenBank identification number (GI Number) of the nearest GenBank homolog.
• Column 7 shows the probability score for the match between each polypeptide and its GenBank homolog.
• Column 8 shows the annotation ofthe GenBank homolog.
• 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.)
• the product score takes into account both the degree of similarity between two sequences and the length ofthe sequence match.
• the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
• the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
• the product score represents a balance between fractional overlap and quality in a BLAST alignment.
• a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared.
• a product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other.
• a product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
• polynucleotide sequences encoding SPTM are analyzed with respect to the tissue sources from which they were derived.
• Polynucleotide sequences encoding SPTM were assembled, at least in part, with overlapping Incyte cDNA sequences.
• Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
• Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
• the number of libraries in each category for each polynucleotide sequence encoding SPTM is counted and divided by the total number of libraries across all categories for each polynucleotide sequence encoding SPTM.
• each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category for each polynucleotide sequence encoding SPTM is counted and divided by the total number of libraries across all categories for each polynucleotide sequence encoding SPTM. The resulting percentages reflect the tissue-specific and disease-specific expression of cDNA encoding SPTM. Percentage values of tissue-specific expression are reported in . cDNA sequences and cDNA library/tissue information are found in the LEPESEQ GOLD database (Incyte Genomics, Palo Alto CA). VI. Tissue Distribution Profiling
• a tissue distribution profile is determined for each template by compiling the cDNA library tissue classifications of its component cDNA sequences.
• Each component sequence is derived from a cDNA library constructed from a human tissue.
• Each human tissue is classified into one of the 5 following categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
• Template sequences, component sequences, and cDNA library/tissue information are found in the LIFESEQ GOLD 0 database (Incyte Genomics, Palo Alto CA). shows the tissue distribution profile for the templates of the invention. For each template, the three most frequently observed tissue categories are shown in column 2, along with the percentage of component sequences belonging to each category. Only tissue categories with percentage values of ⁇ 10% are shown. A tissue distribution of "widely distributed" in column 2 indicates percentage values 5 of ⁇ 10% in all tissue categories.
• Transcript images are generated as described in Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent Number 5,840,484, incorporated herein by reference. 0
• Oligonucleotide primers designed using an sptm ofthe Sequence Listing are used to extend the nucleic acid sequence.
• One primer is synthesized to initiate 5' extension of the template, and the other primer, to initiate 3' extension ofthe template.
• the initial primers maybe designed using OLIGO 5 4.06 software (National Biosciences, Inc. (National Biosciences), Plymouth MN), 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 are avoided.
• Selected human cDNA libraries are used to extend the sequence. If more than one o extension is necessary or desired, additional or nested sets of primers are designed.
• PCR is performed in 96-well plates using the PTC-200 thermal cycler (MJ Research).
• the reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH ⁇ SO.*., and ⁇ - mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life 5 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
• the parameters for primer pair T7 and SK+ are as follows: Step 1: 94 °C, 3 min; Step 2: 94°C, 15
• the concentration of DNA in each well is determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v); Molecular Probes) dissolved in IX Tris-EDTA (TE) and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Incorporated (Corning), Corning NY), allowing the DNA to bind to the reagent.
• the plate is scanned in a ELUOROSKAN II (Labsystems Oy) to measure the fluorescence ofthe sample and to quantify the concentration of
• a 5 ⁇ l to 10 ⁇ l aliquot ofthe reaction mixture is analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions are successful in extending the sequence.
• the extended nucleotides are desalted and concentrated, transfe ⁇ ed to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
• CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
• sonicated or sheared prior to religation into pUC 18 vector
• the digested nucleotides are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are excised, and agar digested with AGAR ACE (Promega).
• Extended clones are religated using T4 ligase (New England Biolabs, Inc., Beverly MA) into pUC 18 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 are selected on antibiotic-containing media, individual colonies are picked and cultured overnight at 37 °C in 384-well plates in LB/2x carbenicillin liquid media.
• the cells are lysed, and DNA is 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 is quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the same conditions as described above.
• Samples are diluted with 20% dimethysulfoxide (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 (Applied Biosystems).
• the sptm is used to obtain regulatory sequences (promoters, introns, and enhancers) using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
• Hybridization probes derived from the sptm of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA.
• the labeling of probe nucleotides between 100 and 1000 nucleotides in length is specifically described, but essentially the same procedure may be used with larger cDNA fragments.
• Probe sequences are labeled at room temperature for 30 minutes using a T4 polynucleotide kinase, ⁇ 32 P-ATP, and 0.5X One-Phor-All Plus (Amersham Pharmacia Biotech) buffer and purified using a ProbeQuant G-50 Microcolumn (Amersham Pharmacia Biotech).
• the probe mixture is diluted to 10 7 dpm/ ⁇ g/ml hybridization buffer and used in a typical membrane-based hybridization analysis.
• the DNA is digested with a restriction endonuclease such as Eco RV and is electrophoresed through a 0.7% agarose gel.
• the DNA fragments are transferred from the agarose to nylon membrane (NYTRAN Plus, Schleicher & Schuell, Inc., Keene NH) using procedures specified by the manufacturer of the membrane.
• Prehybridization is carried out for three or more hours at 68 °C, and hybridization is carried out overnight at 68 °C
• blots are sequentially washed at room temperature under increasingly stringent conditions, up to 0.
• RNA lx saline sodium citrate (SSC) and 0.5% sodium dodecyl sulfate. After the blots are placed in a PHOSPHORIMAGER cassette (Molecular Dynamics) or are exposed to autoradiography film, hybridization patterns of standard and experimental lanes are compared. Essentially the same procedure is employed when screening RNA.
• the cDNA sequences which were used to assemble SEQ ID NO:l-567 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that match SEQ ID NO: 1-567 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as PHRAP (Table 6). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any ofthe clustered sequences have been previously mapped.
• SHGC Stanford Human Genome Center
• WIGR Whitehead Institute for Genome Research
• Genethon Genethon
• a mapped sequence in a cluster will result in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
• the genetic map locations of SEQ ID NO:l-567 are described as ranges, or intervals, of human chromosomes.
• the map position of an interval, in centiMorgans, is measured relative to the terminus ofthe chromosome's p-arm.
• centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers.
• cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
• Mb megabase
• the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each ofthe clusters.
• Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and polyA + RNA is purified using the oligo (dT) cellulose method.
• Each polyA + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-dT primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 0 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
• the reverse transcription reaction is performed in a 25 ml volume containing 200 ng polyA + RNA with GEMBRIGHT kits (Incyte).
• Specific control polyA + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished).
• the control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 5 1:1000, 1:100 (w/w) to sample mRNA respectively.
• the control mRNAs are diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA differential expression patterns.
• each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
• Probes are purified using two o successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
• reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.
• the probe is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS. 5
• Sequences of the present invention are used to generate a ⁇ ay elements.
• Each a ⁇ ay element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
• PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
• Array elements are o amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
• Amplified a ⁇ ay elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
• Purified a ⁇ ay elements are immobilized on polymer-coated glass slides.
• Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
• Glass slides are etched in 4% hydrofluoric acid (VWR 5 Scientific Products Corporation (VWR), West Chester, PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
• Array elements are applied to the coated glass substrate using a procedure described in US Patent No. 5,807,522, incorporated herein by reference.
• 1 ⁇ l of the a ⁇ ay element DNA, at an average 5 concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus.
• the apparatus then deposits about 5 nl of a ⁇ ay element sample per slide.
• Microarrays are UV-crosslinked using a STRATALINKER UN-crosslihker (Stratagene). Microa ⁇ ays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate o buffered saline (PBS) (Tropix, Inc., Bedford, MA) for 30 minutes at 60° C followed by washes in 0.2% SDS and distilled water as before.
• PBS phosphate o buffered saline
• Hybridization reactions contain 9 ⁇ l of probe mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
• the probe mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
• the arrays are transfe ⁇ ed to a waterproof chamber having a cavity just slightly larger than a microscope slide.
• the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5x SSC in a co er of the chamber.
• the chamber containing the arrays is incubated for about 6.5 0 hours at 60° C.
• the arrays are washed for 10 min at 45°C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
• Reporter-labeled hybridization complexes are detected with a microscope equipped with an 5 Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
• the excitation laser light is focused on the a ⁇ ay using a 20X microscope objective (Nikon, Inc., Melville NY).
• the slide containing the a ⁇ ay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
• the 1.8 cm x 1.8 cm array used in the present example is scanned with a o resolution of 20 micrometers.
• a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) co ⁇ esponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
• the 5 emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each a ⁇ ay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
• the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the probe mix at a known concentration.
• a specific location on the 5 a ⁇ ay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be co ⁇ elated with a weight ratio of hybridizing species of 1:100,000.
• the calibration is done by labeling samples ofthe calibrating cDNA with the two fluorophores and adding i o identical amounts of each to the hybridization mixture.
• the output ofthe photomultiplier tube is digitized using a 12-bit RTT-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood, MA) installed in an IBM-compatible PC computer.
• the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high 15 signal).
• the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
• a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
• the fluorescence signal within each element is then
• the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
• Sequences complementary to the sptm are used to detect, decrease, or inhibit expression of 25 the naturally occurring nucleotide.
• the use of oligonucleotides comprising from about 15 to 30 base pairs is typical in the art. However, smaller or larger sequence fragments can also be used.
• Appropriate oligonucleotides are designed from the sptm using OLIGO 4.06 software (National Biosciences) or other appropriate programs and are synthesized using methods standard in the art or ordered from a commercial supplier. To inhibit transcription, a complementary oligonucleotide is
• a complementary oligonucleotide is designed to prevent ribosomal binding and processing ofthe transcript.
• SPTM expression and purification of SPTM is accomplished using bacterial or virus-based expression systems.
• cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
• promoters include, but are not limited to, the trp-lac (tad) 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 SPTM upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
• SPTM in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
• AcMNPV Autographica californica nuclear polyhedrosis virus
• the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding SPTM 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 e.g., Engelhard, supra; and Sandig, supra.)
• SPTM 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 glutathione S- transferase
• a peptide epitope tag such as FLAG or 6-His
• FLAG an 8-amino acid peptide
• 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. Chapters 10 and 16). Purified SPTM obtained by these methods can be used directly in the following activity assay.
• XIV. Demonstration of SPTM Activity An assay for SPTM activity measures the expression of SPTM on the cell surface.
• cDNA encoding SPTM 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 NTH3T3.
• Cell surface proteins are labeled with biotin using methods known in the art.
• Immunoprecipitations are performed using SPTM-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 SPTM expressed on the cell surface.
• an assay for SPTM activity measures the amount of SPTM 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 SPTM-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques.
• the concentration of SPTM in secretory organelles relative to SPTM in total cell lysate is proportional to the amount of SPTM in transit through the secretory pathway.
• SPTM function is assessed by expressing sptm 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 Corporation, Carlsbad CA), both of which contain the cytomegalovirus promoter.
• 5-10 ⁇ g 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 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
• 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.
• FCM Flow cytometry
• 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 NY.
• the influence of SPTM on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding SPTM 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, Inc., Lake Success NY).
• mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding SPTM and other genes of interest can be analyzed by northern analysis or microarray techniques.
• SPTM substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, can be used to immunize rabbits and to produce antibodies using standard protocols.
• PAGE polyacrylamide gel electrophoresis
• the SPTM amino acid sequence is analyzed using LASERGENE software
• peptides 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using fmoc-chemistry and coupled to KLH (Sigma) by reaction with N-maleiimdobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
• Rabbits are immunized with the peptide-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 radioiodinated goat anti-rabbit IgG. Antisera with antipeptide activity are tested for anti-SPTM activity using protocols well known in the art, including ELISA, RIA, and immunoblotting.
• Naturally occurring or recombinant SPTM is substantially purified by immunoaffinity chromatography using antibodies specific for SPTM.
• An knmunoaffinity column is constructed by covalently coupling anti-SPTM 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 SPTM are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SPTM (e.g., high ionic strength buffers in the presence of detergent).
• the column is eluted under conditions that disrupt antibody/SPTM 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 SPTM is collected.
• disrupt antibody/SPTM binding e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion
• SPTM or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
• Bolton-Hunter reagent See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
• Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled SPTM, washed, and any wells with labeled SPTM complex are assayed. Data obtained using different concentrations of SPTM are used to calculate values for the number, affinity, and association of SPTM with the candidate molecules.
• molecules interacting with SPTM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (CLONTECH).
• SPTM may also be used in the PATHCALLTNG process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
• LG:1138554.16:2001MAR30 1 227 forward 3 TM Non-Cytbsolic 3 LG:1138554.16:2001MAR30 228 250 forward 3 TM Transmembrane 3 LG: 1138554.16.-2001MAR30 251 256 forward 3 TM Cytosolic 3 LG:1138554.16:2001MAR30 257 279 forward 3 TM Transmembrane 3 LG:1138554.16:2001MAR30 280 293 forward 3 TM Non-Cytosolic 3 LG:1138554.16:2001MAR30 294 316 forward 3 TM Transmembrane
• LG:1397614.15:2001MAR30 1 forward 1 TM Cytosolic 5 LG:1397614.15:2001MAR30 2 21 forward 1 TM Transmembrane 5 LG: 1397614.15:2001MAR30 22 35 forward 1 TM Non-Cytosolic 5 LG:1397614.15:2001MAR30 36 58 forward 1 TM Transmembrane 5 LG:1397614.15:2001MAR30 59 193 forward 1 TM Cytosolic 5 LG: 1397614.15:2001MAR30 194 216 forward 1 TM Transmembrane 5 LG:1397614.15:2001MAR30 217 225 forward 1 TM Non-Cytosolic 5 LG:1397614.15:2001MAR30 226 243 forward 1 TM Transmembrane TABLE 2

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