WO2004013292A2 - Messagers extracellulaires - Google Patents
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- WO2004013292A2 WO2004013292A2 PCT/US2003/024084 US0324084W WO2004013292A2 WO 2004013292 A2 WO2004013292 A2 WO 2004013292A2 US 0324084 W US0324084 W US 0324084W WO 2004013292 A2 WO2004013292 A2 WO 2004013292A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to novel nucleic acids, extracellular messengers encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of autoiinmune/irjflammatory disorders, cell proliferative disorders, and endocrine disorders.
- the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and extracellular messengers.
- Intercellular communication is essential for the growth and survival of multicellular organisms, and in particular, for the function of the endocrine, nervous, and immune systems.
- intercellular communication is critical for developmental processes such as tissue construction and organogenesis, in which cell proliferation, cell differentiation, and morphogenesis must be spatially and temporally regulated in a precise and coordinated manner.
- Cells communicate with one another through the secretion and uptake of diverse types of signaling molecules such as hormones, growth factors, neuropeptides, and cytokines.
- Hormones include hormones, growth factors, neuropeptides, and cytokines.
- Hormones are signaling molecules that coordmately regulate basic physiological processes from embryogenesis throughout adulthood. These processes include metabolism, respiration, reproduction, excretion, fetal tissue differentiation and organogenesis, growth and development, homeostasis, and the stress response. Hormonal secretions and the nervous system are tightly integrated and interdependent. Hormones are secreted by endocrine glands, primarily the hypothalamus and pituitary, the thyroid and parathyroid, the pancreas, the adrenal glands, and the ovaries and testes.
- Hormones are often secreted in diurnal, pulsatile, and cyclic patterns. Hormone secretion is regulated by perturbations in blood biochemistry, by other upstream-acting hormones, by neural impulses, and by negative feedback loops. Blood hormone concentrations are constantly monitored and adjusted to maintain optimal, steady-state levels. Once secreted, hormones act only on those target cells that express specific receptors.
- hyposecretion often occurs when a hormone's gland of origin is damaged or otherwise impaired. Hypersecretion often results from the proliferation of tumors derived fromhormone- secreting cells. Inappropriate hormone levels may also be caused by defects in regulatory feedback loops or in the processing of hormone precursors. Endocrine malfunction may also occur when the target cell fails to respond to the hormone.
- Hormones can be classified biochemically as polypeptides, steroids, eicosanoids, or amines.
- Polypeptides which include diverse hormones such as insulin and growth hormone, vary in size and function and are often synthesized as inactive precursors that are processed intracellularly into mature, active forms.
- Amines which include epinephrine and dopamine, are amino acid derivatives that function in neuroendocrine signaling.
- Steroids which include the cholesterol-derived hormones estrogen and testosterone, function in sexual development and reproduction.
- Eicosanoids which include prostaglandins and prostacyclins, are fatty acid derivatives that function in a variety of processes.
- polypeptides and some amines are soluble in the circulation where they are highly susceptible to proteolytic degradation within seconds after their secretion. Steroids and lipids are insoluble and must be transported in the circulation by carrier proteins. The following discussion will focus primarily on polypeptide hormones.
- Hypothalamic hormones include thyrotropin-releasing hormone, gonadotropin-releasing hormone, somatostatin, growth-hormone releasing factor, corticotropin-releasing hormone, substance P, dopamine, and prolactin-releasing hormone. These hormones directly regulate the secretion of hormones from the anterior lobe of the pituitary.
- Hormones secreted by the anterior pituitary include adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone, somatotropic hormones such as growth hormone and prolactin, glycoprotein hormones such as thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), ⁇ -lipotropin, and ⁇ endorphins.
- ACTH adrenocorticotropic hormone
- melanocyte-stimulating hormone such as growth hormone and prolactin
- glycoprotein hormones such as thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), ⁇ -lipotropin, and ⁇ endorphins.
- FSH follicle-stimulating hormone
- ⁇ -lipotropin ⁇ -lipotropin
- disorders of the hypothalamus and pituitary often result from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma. Such disorders have profound effects on the function of other endocrine glands.
- disorders associated with hypopituitarism include hypogonadism, Sheehan syndrome, diabetes insipidus,
- Kallman's disease Hand-SchuUer-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism.
- Disorders associated with hyperpituitarism include acromegaly, giantism, and syndrome of inappropriate ADH secretion (SIADH), often caused by benign adenomas.
- SIADH inappropriate ADH secretion
- Thyroid hormones secreted by the thyroid and parathyroid primarily control metabolic rates and the regulation of serum calcium levels, respectively.
- Thyroid hormones include calcitonin, somatostatin, and thyroid hormone.
- the parathyroid secretes parathyroid hormone.
- Disorders associated with hypothyroidism include goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism.
- Disorders associated with hyperthyroidism include thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease.
- Disorders associated with hyperparathyroidism include Conn disease (chronic hypercalemia) leading to bone resorption and parathyroid hyperplasia.
- Pancreatic hormones secreted by the pancreas regulate blood glucose levels by modulating the rates of carbohydrate, fat, and protein metabolism.
- Pancreatic hormones include insulin, glucagon, amylin, ⁇ - aminobutyric acid, gastrin, somatostatin, and pancreatic polypeptide.
- the principal disorder associated with pancreatic dysfunction is diabetes mellitus caused by insufficient insulin activity. Diabetes mellitus is generally classified as either Type I (insulin-dependent, juvenile diabetes) or Type II (non-insulin-dependent, adult diabetes). The treatment of both forms by insulin replacement therapy is well known.
- Diabetes mellitus often leads to acute complications such as hypoglycemia (insulin shock), coma, diabetic ketoacidosis, lactic acidosis, and chronic complications leading to disorders of the eye, kidney, skin, bone, joint, cardiovascular system, nervous system, and to decreased resistance to infection.
- hypoglycemia insulin shock
- coma coma
- diabetic ketoacidosis lactic acidosis
- chronic complications leading to disorders of the eye, kidney, skin, bone, joint, cardiovascular system, nervous system, and to decreased resistance to infection.
- Growth factors are secreted proteins that mediate intercellular communication. Unlike hormones, which travel great distances via the circulatory system, most growth factors are primarily local mediators that act on neighboring cells. Most growth factors contain a hydrophobic N-terminal signal peptide sequence which directs the growth factor into the secretory pathway. Most growth factors also undergo post-translational modifications within the secretory pathway. These modifications can include proteolysis, glycosylation, phosphorylation, and intramolecular disulfide bond formation. Once secreted, growth factors bind to specific receptors on the surfaces of neighboring target cells, and the bound receptors trigger intracellular signal transduction pathways. These signal transduction pathways elicit specific cellular responses in the target cells. These responses can include the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility.
- Growth factors fall into at least two broad and overlapping classes.
- the broadest class includes the large polypeptide growth factors, which are wide-ranging in their effects. These factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor- ⁇ (TGF- ⁇ ), insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF), each defining a family of numerous related factors.
- the large polypeptide growth factors act as mitogens on diverse cell types to stimulate wound healing, bone synthesis and remodeling, extracellular matrix synthesis, and proliferation of epithelial, epidermal, and connective tissues.
- TGF- ⁇ , EGF, and FGF families also function as inductive signals in the differentiation of embryonic tissue.
- NGF functions specifically as a neurotrophic factor, promoting neuronal growth and differentiation.
- Another class of growth factors includes the hematopoietic growth factors, which are narrow in their target specificity. These 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. These factors include the colony-stimulating factors (G-CSF, M-CSF, GM-CSF, and CSF1-3), erythropoietin, and the cytokines. The cytokines are specialized hematopoietic factors secreted by cells of the immune system and are discussed in detail below.
- Growth factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Overexpression of the large polypeptide growth factors promotes the proliferation and transformation of cells in culture. Inappropriate expression of these growth factors by tumor cells in vivo may contribute to tumor vascularization and metastasis. Inappropriate activity of hematopoietic growth factors can result in anemias, leukemias, and lymphomas. Moreover, growth factors are both structurally and functionally related to oncoproteins, the potentially cancer- causing products of proto-oncogenes. Certain FGF and PDGF family members are themselves homologous to oncoproteins, whereas receptors for some members of the EGF, NGF, and FGF families are encoded by proto-oncogenes.
- NP VM Small Peptide Factors - Neuropeptides and Vasomediators
- NP VM Small Peptide Factors - Neuropeptides and Vasomediators
- neuropeptides and neuropeptide hormones such asbombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signahng molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin, gastrin, and many of the peptide hormones discussed above.
- neuropeptide hormones such asbombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation
- NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in signahng 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, New York, NY, pp. 57-62.) Cytokines
- Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system such as B- and T-lymphocytes, monocytes, macrophages, and granulocytes. Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which alter gene expression patterns. There is considerable potential for the use of cytokines in the treatment of inflammation and immune system disorders.
- Cytokine structure and function have been extensively characterized in vitro. Most cytokines are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamilies include the interferons (IFN- , - ⁇ , and - ⁇ ), the interleukins (IL1-IL13), the tumor necrosis factors (TNF- and - ⁇ ), and the chemokines. Many cytokines have been produced using recombinant DNA techniques, and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte proliferation, differentiation, and motility.
- IFN- , - ⁇ , and - ⁇ interferons
- IL1-IL13 interleukins
- TNF- and - ⁇ tumor necrosis factors
- chemokines chemokines.
- Many cytokines have been produced using recombinant DNA techniques, and the activities of individual
- Cytokines interact with a target through receptors expressed on the surface of the responsive cell. Cytokines bind with hemopoietin receptors, receptor kinases, and tumor necrosis factor (TNF)/nerve growth factor (NGF) receptors by bringing together two receptor subunits. This dimerization of receptor subunits transmits a signal through the plasma membrane to the cell cytoplasm. In the case of protein kinase receptors, such as the receptors for epidermal growth factor (EGF) and insulin, the juxtaposition of the two receptor subunit cytoplasmic domains activates their intrinsic tyrosine kinase activity. As a result, the subunits phosphorylate each other.
- EGF epidermal growth factor
- SH2 src homology 2
- SH2 containing proteins that interact with phosphorylated receptor molecules include phosphatidylinositol 3 '-kinase, src kinase family members, GRB2, and she. These SH2 containing proteins are often associated with other cytoplasmic proteins, such as members of the small, monomeric GTP-binding protein families Ras and Rho, and phosphatases, such as the phosphotyrosine phosphatase SHP-2.
- the signaling complexes formed by these interactions can initiate signal cascades, such as the kinase cascade involving raf and mitogen activated protein (MAP) kinase, which result in transcriptional regulation and cytoskeleton reorganization.
- MAP mitogen activated protein
- Hemopoietin and TNF/NGF receptors though they have no intrinsic kinase activity, still activate many of the same signal cascades within responding cells.
- Eps8 is a protein which associates with and is phosphorylated by the EGF receptor.
- Eps8 Human tumor cell lines contain high constitutive levels of tyrosine-phosphorylated Eps8, and overexpression of Eps8 in NIH3T3 cells expressing the EGF receptor (EGFR) leads to an enhanced mitogenic response and cell overgrowth (Provenzano, C. et al. (1998) Exp. Cell Res. 242:186-200).
- a family of molecules which include ABI (Abl interactor protein)-l and ABI-2/e3Bl, interact with tyrosine kinases, such as the src-like kinase Abl, and Eps8.
- ABI-2/e3Bl Overexpression of ABI-2/e3Bl in NIH3T3 cells expressing EGFR inhibits the mitogenic response and cell growth.
- the ABI family of proteins function as negative regulators of cytokine signaling (Ziemnicka-Kotula, D. et al. (1998) J. Biol. Chem. 273:13681-13692).
- SHP-1 and SHP-2 are involved in cytokine signaling.
- SHP-1 the hemopoietic cell phosphatase
- SHP-2 is a positive signal transducer for several cytokines.
- a family of transmembrane glycoproteins, called SIRPs (signal regulatory proteins), are substrates of tyrosine kinases.
- Phosphorylated SIRPs bind to SHP-2 and have a negative effect on cell response induced by cytokines, including an inhibition of growth factor-induced DNA synthesis.
- This inhibition correlates with reduced MAP kinase activation in SIRP-transfected NIH3T3 cells stimulated with insulin or EGF.
- SIRP overexpression also suppressed transformation of NIH3T3 cells by a retrovirus carrying the v-fms oncogene (Kharitonenkov, A. et al. (1997) Nature 386:181-186).
- the activity of an individual cytokine in vitro may not reflect the full scope of that cytokine's activity in vivo.
- Cytokines are not expressed individually in vivo but are instead expressed in combination with a multitude of other cytokines when the organism is challenged with a stimulus. Together, these cytokines collectively modulate the immune response in a manner appropriate for that particular stimulus. Therefore, the physiological activity of a cytokine is determined by the stimulus itself and by complex interactive networks among co-expressed cytokines which may demonstrate both synergistic and antagonistic relationships.
- ANUP anti-neoplastic urinary protein
- ANUP N-terminal 22 amino acids of ANUP comprise a signal peptide which is cleaved from the mature protein.
- the first nine amino acids of the mature protein retain about 10% of the anti-tumor activity.
- ANUP contains a Ly-6/u-PAR sequence motif that is typical of certain cell surface gfycoproteins. This motif is characterized by a distinct pattern of six cysteine residues within a 50-residue consensus sequence.
- the Ly-6/u-PAR motif is found in the Ly-6 T-lymphocyte surface antigen and in the receptor (u-PAR) for urokinase-type plasminogen activator, an extracellular serine protease.
- Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in Wells, T.N.C. and M.C. Peitsch (1997) J. Leukoc. Biol. 61 :545-550.) Chemokines were initially identified as chemotactic proteins that recruit monocytes and macrophages to sites of inflammation. Recent evidence indicates that chemokines may also play key roles in hematopoiesis and HIV-1 infection. Chemokines are small proteins which range from about 6-15 kilodaltons in molecular weight. Chemokines are further classified as C, CC, CXC, or CX 3 C based on the number and position of certain cysteine residues.
- the CC chemokines for example, each contain a conserved motif consisting of two consecutive cysteines followed by two additional cysteines which occur downstream at 24- and 16-residue intervals, respectively (ExPASy PROSITE database, documents PS00472 and PDOC00434).
- the presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly.
- a conserved tyrosine located about 15 residues downstream of the cysteine doublet seems to be important for chemotactic activity.
- Most of the human genes encoding CC chemokines are clustered on chromosome 17, although there are a few examples of CC chemokine genes that map elsewhere.
- chemokines include lymphotactin (C chemokine); macrophage chemotactic and activating factor (MCAF MCP-1; CC chemokine); platelet factor 4 and IL-8 (CXC chemokines); and fractalkine and neurotractin (CX 3 C chemokines).
- C chemokine lymphotactin
- MCAF MCP-1 macrophage chemotactic and activating factor
- CXC chemokines platelet factor 4 and IL-8
- CX 3 C chemokines fractalkine and neurotractin
- Signal transduction is a general process in which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions beginning with the binding of the signal molecule to a cell membrane receptor and ending with an effect on an intracellular target molecule. Intermediate steps in this process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases and the translocation of some of these activated proteins to the cell nucleus, where the transcription of specific genes is affected.
- the signal transduction process regulates all types of cell functions, including cell proliferation, differentiation, and gene transcription. Many of the cytokine receptors, including those for the growth factors EGF, PDGF, and FGF exhibit intrinsic protein kinase activity.
- SH2 domains are found in a variety of signaling molecules and oncogenic proteins, such as phospholipase C-g, Ras GTP-ase activating protein, and GRB2 (Lowenstein, EJ. et al. (1992) Cell 70:431-442).
- SH2-containing proteins are induced in murine lymphoid cells by various cytokines, including IL-2, IL-3, IL-6, Interferon- ⁇ , and EPO (Yoshimura, A. et al. (1995) EMBO Journal 14:2816-2826; Starr, R. et al. (1997) Nature 387:917-921 ; and Naka, T. et al. (1997) Nature 387:924-929).
- a common property of these proteins is the ability to suppress growth and differentiation in murine cells.
- the four proteins share little sequence homology in their N-terminal regions, but all contain a central SH2 domain and a conserved C-terminal region designated the "SOCS box."
- the function of the SOCS box is unknown.
- a conserved core triplet sequence (K/R) (D/E) (Y/F) within the SOCS box is similar to the tyrosine phosphorylation site recognized by the JAK kinase family. This similarity suggests that the SOCS box may provide a site for interaction with, and inhibition of, JAK kinases.
- the finding that SOCS-1 interacts with the catalytic region of JAK kinases supports this hypothesis (Endo, T.A. et al. (1997) Nature 387:921-24).
- CIS binds to tyrosine-phosphorylated residues in the beta-chain of the IL-3 and EPO receptors and provides another possible mechanism for suppressing cell signahng by preventing the binding of other signaling proteins (Yoshimura et al., supra).
- each of the proteins contains a C-terminal SOCS box and a distinctive motif N- terminal of the SOCS box.
- three additional classes of SOCS proteins were found containing WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3), or ankyrin repeats (ASB-1 to -3).
- a class of small GTPases (Rar proteins) that contain the SOCS box were also identified.
- WSB WSB
- SSB SSB
- ASB proteins The function of WSB, SSB, and ASB proteins are as yet unknown. However, like SH2 domains, WD-40 repeats, ankyrin repeats, and SPRY domains have been implicated in protein-protein interactions (Hilton et al., supra). Defects or alterations in the activity of signaling proteins such as CIS may play a role in the development of various proliferative disorders and diseases such as cancer. Loss or rearrangement of the putative human gene encoding CIS is associated with the development of renal cell carcinomas and lung cancer (Yoshimura et al., supra). This association suggests that CIS may function as a tumor suppressor gene. Expression profiling
- Microarrays are analytical tools used in bioanalysis.
- a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support.
- Microarrays of polypeptides, polynucleotides, and or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
- array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
- arrays are employed to detect the expression of a specific gene or its variants.
- arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
- Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologicaUy distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). The fourth group of cancers is referred to as small cell lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K- ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease.
- Familial adenomatous polyposis is caused by mutations in the adenomatous polyposis coli gene (APC), resulting in truncated or inactive forms of the protein.
- APC adenomatous polyposis coli gene
- This tumor suppressor gene has been mapped to chromosome 5q.
- Hereditary nonpolyposis colorectal cancer is caused by mutations in mis-match repair genes.
- somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer.
- erbB receptors such as HER- 2/neu, HER-3, and HER-4
- Hgands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:S13-S24).
- Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, I.I. et al. (1998) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
- adipose tissue is the major energy reserve in periods of excess energy use. Its primary purpose is mobilization during energy deprivation. Understanding how various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity.
- Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked and present interesting relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance. The majority of research in adipocyte biology to date has been done using transformed mouse preadipocyte cell lines.
- the culture condition which stimulates mouse preadipocyte differentiation is different from that for inducing human primary preadipocyte differentiation.
- primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines. Understanding the gene expression profile during adipogenesis in humans will lead to an understanding of the fundamental mechanism of adiposity regulation. Furthermore, through comparing the gene expression profiles of adipogenesis between donor with normal weight and donor with obesity, identification of crucial genes, potential drug targets for obesity and type II diabetes, will be possible.
- Thiazohdinediones act as agonists for the peroxisome-prohferator-activated receptor gamma (PPAR ⁇ ), a member of the nuclear hormone receptor superfamily. TZDs reduce hyperglycemia, hyperinsuhnemia, and hypertension, in part by promoting glucose metabohsm and inhibiting gluconeogenesis. Roles for PPAR ⁇ and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.
- PPAR ⁇ peroxisome-prohferator-activated receptor gamma
- PPAR ⁇ peroxisome-prohferator-activated receptor gamma
- Roles for PPAR ⁇ and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancer
- TZDs and other PPAR ⁇ agonists enhance insulin sensitivity is not fully understood, but may involve the abihty of PPAR ⁇ to promote adipogenesis.
- PPAR ⁇ When ectopically expressed in cultured preadipocytes, PPAR ⁇ is a potent inducer of adipocyte differentiation.
- TZDs, in combination with insulin and other factors can also enhance differentiation of human preadipocytes in culture (Adams et al. (1997) J. Clin. Invest. 100:3149-3153).
- the relative potency of different TZDs in promoting adipogenesis in vitro is proportional to both their insulin sensitizing effects in vivo, and their abihty to bind and activate PPAR ⁇ in vitro.
- adipocytes derived from omental adipose depots are refractory to the effects of TZDs. It has therefore been suggested that the insulin sensitizing effects of TZDs may result from their abihty to promote adipogenesis in subcutaneous adipose depots (Adams et al., supra). Further, dominant negative mutations in the PPAR ⁇ gene have been identified in two non-obese subjects with severe insulin resistance, hypertension, and overt non-insulin dependent diabetes mellitus (NIDDM) (Barroso et al. (1998) Nature 402:880-883).
- NIDDM non-insulin dependent diabetes mellitus
- NIDDM is the most common form of diabetes mellitus, a chronic metabolic disease that affects 143 million people worldwide. NIDDM is characterized by abnormal glucose and lipid metabohsm that results from a combination of peripheral insulin resistance and defective insulin secretion. NIDDM has a complex, progressive etiology and a high degree of heritabihty. Numerous complications of diabetes including heart disease, stroke, renal failure, retinopathy, and peripheral neuropathy contribute to the high rate of morbidity and mortality.
- PPAR ⁇ functions as a ligand activated transcription factor.
- RXR retinoid X receptor
- PPRE ⁇ response element PPRE
- Many genes important in lipid storage and metabohsm contain PPREs and have been identified as PPAR ⁇ targets, including PEPCK, aP2, LPL, ACS, and FAT-P (Auwerx, J. (1999) Diabetologia 42:1033-1049). Multiple hgands for PPAR ⁇ have been identified.
- the prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand for PPAR ⁇ .
- PPAR ⁇ is very high in adipose but barely detectable in skeletal muscle, the primary site for insulin stimulated glucose disposal in the body. PPAR ⁇ is also moderately expressed in large intestine, kidney, hver, vascular smooth muscle, hematopoietic cells, and macrophages. The high expression of PPAR ⁇ in adipose tissue suggests that the insulin sensitizing effects of TZDs may result from alterations in the expression of one or more PPAR ⁇ regulated genes in adipose tissue. Identification of PPAR ⁇ target genes will contribute to better drug design and the development of novel therapeutic strategies for diabetes, obesity, and other conditions.
- array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
- arrays are employed to detect the expression of a specific gene or its variants.
- arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder.
- the potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with diabetes may be compared with the levels and sequences expressed in normal tissue.
- Ovarian cancer Ovarian cancer
- Ovarian cancer is the leading cause of death from a gynecologic cancer.
- the majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rate for this disease is very low. Identification of early-stage markers for ovarian cancer would si nificantly increase the survival rate. Genetic variations involved in ovarian cancer development include mutation of p53 and microsatelhte instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of autoimmune/inflammatory disorders, cell proliferative disorders, and endocrine disorders.
- ⁇ XMES' purified polypeptides, extracellular messengers, referred to collectively as ⁇ XMES' and individuahy as ⁇ XMES-1,' ⁇ XMES-2,' ⁇ XMES-3,' 'EXMES-4,' ⁇ XMES-5,' ⁇ XMES-6,' ⁇ XMES-7,' ⁇ XMES-8,' ⁇ XMES-9,' ⁇ XMES-10,' ⁇ XMES-11,' ⁇ XMES-12,' ⁇ XMES-13,' ⁇ XMES-14,' ⁇ XMES-15,' ⁇ XMES-16,' ⁇ XMES-17,' ⁇ XMES-18,' ⁇ XMES-19,' ⁇ XMES-20,' ⁇ XMES-21,' ⁇ XMES-22,' ⁇ XMES-23,' 'EXMES-24,' and ⁇ XMES-25' and methods for using these proteins and then encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions.
- Embodiments also provide methods for utilizing the purified extracellular messengers and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
- Related embodiments provide methods for utilizing the purified extracellular messengers and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
- An 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:l- 25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-25.
- 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:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-25.
- the polynucleotide is selected from the group consisting of SEQ ID NO:26-50.
- StiU another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a 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:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting
- Another embodiment provides a ceU transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing 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: 1-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
- Yet another embodiment provides an isolated antibody which specificaUy 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:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-25.
- StiU yet another embodiment 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:26-50, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, 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)-d).
- the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
- Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, 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)-d).
- a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleot
- 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 specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
- the method can include detecting the amount of the hybridization complex.
- the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
- StiU yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, 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)-d).
- a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynu
- 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.
- the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
- compositions comprising an effective amount 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: 1-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, and a pharmaceutically acceptable excipient
- the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional EXMES, comprising administering to a patient in need of such treatment the composition.
- Yet 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:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-25.
- the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting agonist activity in the sample.
- Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
- Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional EXMES, comprising administering to a patient in need of such treatment the composition.
- StiU yet 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:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting antagonist activity in the sample.
- Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
- Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional EXMES, comprising administering to a patient in need of such treatment the composition.
- Another embodiment provides a method of screening for a compound that specificaUy 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:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
- Yet 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 acid sequence selected from the group consisting of SEQ ID NO:l-25, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25.
- the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the 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 of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
- StiU 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 sequence selected from the group consisting of SEQ ID NO:26-50, the method comprising a) contacting a sample comprising the target polynucleotide with a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the 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) 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:26-50, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of
- 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)- iv).
- the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
- Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
- Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
- Table 4 lists the cDNA and or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
- Table 5 shows representative cDNA libraries for polynucleotide embodiments.
- Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
- Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
- Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
- EXMES refers to the amino acid sequences of substantiaUy purified EXMES obtained from any species, particularly a mammahan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
- agonist refers to a molecule which intensifies or mimics the biological activity of EXMES.
- Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of EXMES either by directly interacting with EXMES or by acting on components of the biological pathway in which EXMES participates.
- an "aUelic variant” is an alternative form of the gene encoding EXMES. AUelic 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 aUelic variants of its naturaUy occurring form. Common mutational changes which give rise to aUelic variants are generaUy 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 EXMES include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as EXMES or a polypeptide with at least one functional characteristic of EXMES. Included within this definition are polymorphisms which may or may not be readUy detectable using a particular oligonucleotide probe of the polynucleotide encoding EXMES, and improper or unexpected hybridization to aUelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding EXMES.
- the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionaUy equivalent EXMES.
- Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of EXMES is retained.
- negatively charged amino acids may include aspartic acid and glutamic acid
- positively charged amino acids may include lysine and arginine.
- Amino acids with uncharged polar side chains having simUar hydrophihcity values may include: asparagine and glutamine; and serine and threonine.
- Amino acids with uncharged side chains having similar hydrophihcity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
- amino acid and amino acid sequence can refer to an ohgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules.
- amino acid sequence is recited to refer to a sequence of a naturaUy occurring protein molecule
- amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
- “AmpUfication” relates to the production of additional copies of a nucleic acid.
- Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies weU known in the art.
- PCR polymerase chain reaction
- Antagonist refers to a molecule which inhibits or attenuates the biological activity of EXMES.
- Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of EXMES either by directly interacting with EXMES or by acting on components of the biological pathway in which EXMES participates.
- antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
- Antibodies that bind EXMES polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the irnmunizing antigen.
- the polypeptide or oligopepti.de used to immunize an animal e.g., a mouse, a rat, or a rabbit
- chemicaUy Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
- antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
- a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specificaUy to antigenic determinants (particular regions or three-dimensional structures on the protein).
- An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
- aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a 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.
- 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 may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer hfetime in blood.
- Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
- Aptamers may be specificaUy cross-linked to their cognate hgands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
- the term "intramer” 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 naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
- antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
- Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; ohgonucleotides having modified sugar groups such as 2 -methoxyethyl sugars or 2'-methoxyethoxy sugars; or ohgonucleotides having modified bases such as 5-methyl cytosine, 2 -deoxyuracU, or 7-deaza-2 - deoxyguanosine.
- Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
- the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
- biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
- immunologicalaUy active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic EXMES, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
- Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
- composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
- the composition may comprise a dry formulation or an aqueous solution.
- Compositions comprising polynucleotides encoding EXMES or fragments of EXMES may be employed as hybridization probes.
- the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
- the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry ilk, salmon sperm DNA, etc.).
- salts e.g., NaCl
- detergents e.g., sodium dodecyl sulfate; SDS
- other components e.g., Denhardt's solution, dry ilk, salmon sperm DNA, etc.
- Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (Applied
- Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
- the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
- Conservative amino acid substitutions generaUy 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 site of the substitution, and or (c) the bulk of the side chain.
- a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
- derivative refers to a chemicaUy modified polynucleotide or polypeptide.
- Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
- a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
- a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
- a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
- “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 may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
- “Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
- a “fragment” is a unique portion of EXMES or a polynucleotide encoding EXMES which can be 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 about 5 to about 1000 contiguous nucleotides or amino acid residues.
- a fragment used as a probe, primer, 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 nucleotides or amino acid residues in length. Fragments may be preferentiaUy 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, tables, and figures, may be encompassed by the present embodiments.
- a fragment of SEQ ID NO:26-50 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:26-50, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
- a fragment of SEQ ID NO:26-50 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:26-50 from related polynucleotides.
- the precise length of a fragment of SEQ ID NO:26-50 and the region of SEQ ID NO:26-50 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
- a fragment of SEQ ID NO:l-25 is encoded by a fragment of SEQ ID NO:26-50.
- a fragment of SEQ ID NO: 1-25 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO: 1-25.
- a fragment of SEQ ID NO: 1-25 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO:l-25.
- the precise length of a fragment of SEQ ID NO:l-25 and the region of SEQ ID NO:l-25 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
- a “fuU length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
- a “full length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
- Homology refers to sequence simharity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
- percent identity and % identity refer to the percentage of identical nucleotide 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 ahgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
- Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence ahgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5:151- 153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191).
- BLAST 215:403-410 which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/.
- the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also avaUable is a tool caUed "BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nhiLn ⁇ h.gov/gorf/bl2.html.
- BLAST 2 Sequences can be used for both blastn and blastp (discussed below).
- BLAST programs are commonly used with gap and other parameters set to default settings.
- BLAST 2 Sequences Version 2.0.12 (April-21-2000) set at default parameters.
- Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50
- 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 the tables, figures, or Sequence Listing, 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 simUar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
- the phrases "percent identity” and "% identity,” as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence ahgnment are weU-known. Some ahgnment methods take into account conservative amino acid substitutions.
- NCBI BLAST software suite may be used.
- BLAST 2 Sequences Version 2.0.12 (April-21-2000) withblastp set at default parameters.
- Such default parameters maybe, for example:
- 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 the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
- HACs Human artificial chromosomes
- HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome replication, segregation and maintenance.
- humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abihty.
- Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s).
- the washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
- Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
- GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
- wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (T m ) 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 of the 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, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 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, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
- Organic solvent such as formamide at a concentration of about 35-50% v/v
- Organic solvent such as formamide at a concentration of about 35-50% v/v
- Useful variations on these wash conditions wiU be readhy apparent to those of ordinary skiU 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 simUar role for the nucleotides and their encoded polypeptides.
- hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
- a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
- insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
- Immuno 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 ceUular and systemic defense systems.
- an “immunogenic fragment” is a polypeptide or oligopepti.de fragment of EXMES 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 EXMES which is useful in any of the antibody production methods disclosed herein or known in the art.
- microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
- array element refers to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
- modulate refers to a change in the activity of EXMES. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of EXMES.
- nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
- “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
- PNA protein nucleic acid
- PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine.
- the terminal lysine confers solubility to the composition.
- PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their hfespan in the cell.
- "Post-translational modification" of an EXMES may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic milieu of EXMES.
- Probe refers to nucleic acids encoding EXMES, their complements, or fragments thereof, which are used to detect identical, aUelic or related nucleic acids.
- Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, hgands, chemUuminescent agents, and enzymes.
- Primmers are short nucleic acids, usuaUy DNA ohgonucleotides, 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 amphfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous 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, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
- PCR primer pairs can be 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).
- Ohgonucleotides 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 ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 Idlobases. Similar primer selection programs have incorporated additional features for expanded capabihties. For example, the PrirnOU primer selection program (avaUable to the public from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
- OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides
- Primer3 primer selection program (avaUable to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides 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 PrimeGen program (avaUable to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence ahgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments.
- ohgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
- a "recombinant nucleic acid” is a nucleic acid that is not naturaUy 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 and RusseU (supra).
- the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the 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 ceU.
- 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 r mmal.
- a "regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
- Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuchdes; enzymes; fluorescent, chemttuminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
- RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aU occurrences 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.
- a sample suspected of containing EXMES, nucleic acids encoding EXMES, or fragments thereof may comprise a bodUy fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
- binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wUl reduce the amount of labeled A that binds to the antibody.
- substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy associated.
- substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
- Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capiUaries.
- the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
- a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
- Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
- 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 nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C et al. (2002) Science 295:868-872).
- a recombinant viral vector such as a lentiviral vector
- 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, plants and animals.
- the isolated DNA of the 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 of the present invention into such organisms are widely known and provided in references such as Sambrook and RusseU (supra).
- a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% 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 50%, at least 60%, at least 70%, at least 80%, at least 85%, 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.
- a variant may be described as, for example, an "aUehc” (as defined above), “sphce,” “species,” or “polymorphic” variant.
- a sphce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate splicing during mRNA processing.
- the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
- Species variants are polynucleotides that vary from one species to another.
- the resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
- a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
- SNPs single nucleotide polymorphisms
- a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity 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 85%, 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 or sequence similarity over a certain defined length of one of the polypeptides.
- EXMES extraceUular messengers
- polynucleotides encoding EXMES polynucleotides encoding EXMES
- Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID).
- Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
- Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
- Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
- Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
- Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
- Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
- Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, aU of which are expressly incorporated by reference herein.
- Table 3 shows various structural features of the polypeptides of the invention.
- Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
- Column 3 shows the number of amino acid residues in each polypeptide.
- Column 4 shows potential phosphorylation sites and potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accelrys, Burlington MA), and amino acid residues comprising signature sequences, domains, and motifs.
- Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
- SEQ ID NO:l is 100% identical, from residue Ml to residue S 150, to human pre-pro-orphanin FQ (GenBank ID gl 185010) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.)
- the BLAST probability score is l.le-77, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ ID NO:l also has homology to proteins that are extraceUular neuropeptides related to opioid peptide precursors as determined by BLAST analysis using the PROTEOME database.
- SEQ ID NO:l also contains a vertebrate endogenous opioids neuropeptide domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein farnUies/domains.
- HMM hidden Markov model
- SEQ ID NO:l is related to an opioid neuropeptide precursor.
- SEQ ID NO:6 is a sphce variant of human transforming growth factor beta 1 precursor (GenBank ID gl212989) as dete ⁇ nined by the Basic Local Ahgnment Search Tool (BLAST).
- the BLAST probability score is 6.7E-185, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ ID NO:6 also has homology to human transforming growth factor beta 1, as determined by BLAST analysis using the PROTEOME database.
- SEQ ID NO:6 also contains a fransforming growth factor-beta (TGF-beta) domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based SMART database of conserved protein famUies/domains, as weU as a transforming growth factor beta like domain and a TGF-beta propeptide domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein famUies/domains.
- HMM hidden Markov model
- HMM hidden Markov model
- SEQ ID NO:6 is a fransforming growth factor.
- SEQ ID NO: 16 is 74% identical, from residue Ml to residue D128, to human preprovasoactive intestinal polypeptide (GenBank ID g340280) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 2.2e-42, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- BLAST Basic Local Ahgnment Search Tool
- SEQ ID NO: 16 also has homology to vasoactive intestinal peptide precursor which produces a neuropeptide hormone that signals via G protein-coupled receptors and may modulate smooth muscle function and electrolyte balance, ceU proliferation and survival and inflammatory responses, neuronal ceU survival, and oxidative stress response, as determined by BLAST analysis using the PROTEOME database.
- SEQ ID NO: 16 also contains a glucagon-like hormone and peptide hormone domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PF AM/SMART databases of conserved protein famihes/dornains. (See Table 3.) Data from BLIMPS and MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO: 16 is a vasoactive intestinal polypeptide (VIP) family member.
- VIP vasoactive intestinal polypeptide
- SEQ ID NO:22 is 98% identical, from residue Ml to residue S256, to human fransforming growth factor-beta (GenBank ID g339564) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.7e-199, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:22 also has homology to proteins that regulate ceU proliferation, differentiation, and apoptosis, and are bone morphogenetic proteins, as determined by BLAST analysis using the PROTEOME database.
- SEQ ID NO:22 also contains a fransforming growth factor-beta-like domain and a TGF-beta propeptide, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein famihes/dornains.
- HMM hidden Markov model
- SEQ ID NO:22 is a member of the transforming growth factor (TGF)-beta famUy of extraceUular signaling molecules.
- SEQ ID NO:2-5, SEQ ID NO:7-15, SEQ ID NO:17-21 and SEQ ID NO:23-25 were analyzed and annotated in a sirrular manner.
- the algorithms and parameters for the analysis of SEQ ID NO:l-25 are described in Table 7.
- the full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
- Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence inbasepairs.
- Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:26-50 or that distinguish between SEQ ID NO:26-50 and related polynucleotides.
- the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNAlibrari.es or from pooled cDNA libraries.
- the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
- the polynucleotide fragments described in column 2 may identify sequences derived from the ENS EMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
- the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e. , those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e. , those sequences including the designation "NP”).
- the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
- a polynucleotide sequence identified as ⁇ _XXXXX_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was apphed, and YYYYY is the number of the prediction generated by the algorithm, and N ,3 ..., if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
- the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
- a polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was apphed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
- a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e. , gBBBBB).
- GenBank identifier i.e. , gBBBBB.
- a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
- Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
- Table 5 shows the representative cDNA libraries for those fuU length polynucleotides which were assembled using Incyte cDNA sequences.
- the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
- the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
- Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
- Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention.
- Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID).
- Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB 1 SNP).
- Column 7 shows the allele found in the EST sequence.
- Columns 8 and 9 show the two aUeles found at the SNP site.
- Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST.
- Columns 11- 14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n/a (not avaUable) indicates that the allele frequency was not determined for the population.
- EXMES variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the EXMES amino acid sequence, and can contain at least one functional or structural characteristic of EXMES.
- polynucleotides which encode EXMES encompass polynucleotides which encode EXMES.
- the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:26-50, which encodes EXMES.
- the invention also encompasses variants of a polynucleotide encoding EXMES.
- a variant polynucleotide whl have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding EXMES.
- a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:26-50 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:26-50.
- Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of EXMES.
- a polynucleotide variant of the invention is a sphce variant of a polynucleotide encoding EXMES.
- a sphce variant may have portions which have significant sequence identity to a polynucleotide encoding EXMES, but whl generaUy have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate sphcing during mRNA processing.
- a sphce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding EXMES over its entire length; however, portions of the sphce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding EXMES.
- a polynucleotide comprising a sequence of SEQ ID NO:29 and a polynucleotide comprising a sequence of SEQ ID NO:30 are sphce variants of each other;
- a polynucleotide comprising a sequence of SEQ ID NO:34 and a polynucleotide comprising a sequence of SEQ ID NO:43 are sphce variants of each other;
- a polynucleotide comprising a sequence of SEQ ID NO:32, a polynucleotide comprising a sequence of SEQ ID NO:35, and a polynucleotide comprising a sequence of SEQ ID NO:45 are sphce variants of each other.
- Any one of the sphce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of EXMES.
- polynucleotides which encode EXMES and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring EXMES under appropriately selected conditions of stringency, it maybe advantageous to produce polynucleotides encoding EXMES or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturaUy occurring sequence.
- the invention also encompasses production of polynucleotides which encode EXMES and
- EXMES derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic polynucleotide may be inserted into any of the many avaUable expression vectors and ceU systems using reagents weU known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding EXMES or any fragment thereof.
- Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO:26-50 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.”
- Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
- the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Apphed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Invifrogen, Carlsbad CA).
- sequence preparation is automated with machines such as the MICROLAB 2200 hquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Apphed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A.
- the nucleic acids encoding EXMES may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- restriction-site PCR uses universal and nested primers to amphfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Apphc. 2:318-322).
- Another method, inverse PCR uses primers that extend in divergent directions to amphfy unknown sequence from a circularized template.
- the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
- a third method, capture PCR involves PCR amphfication of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Apphc. 1:111-119).
- multiple restriction enzyme digestions and hgations maybe used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
- Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res.
- primers may be designed using commerciaUy avaUable software, such as OLIGO 4.06 primer analysis software (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 template at temperatures of about 68°C to 72°C
- commerciaUy avaUable software such as OLIGO 4.06 primer analysis software (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 template at temperatures of about 68°C to 72°C
- hbraries that have been size-selected to include larger cDNAs.
- random-primed hbraries which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a fuH-length cDNA.
- Genomic hbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
- CapiUary electrophoresis systems which are commerciaUy avaUable may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
- caphlary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
- Output/hght intensity may be converted to electrical signal using appropriate software (e.g. , GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display maybe computer controUed.
- Capillary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
- polynucleotides or fragments thereof which encode EXMES may be cloned in recombinant DNA molecules that direct expression of EXMES , or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides maybe produced and used to express EXMES.
- the polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter EXMES -encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides may be used to engineer the nucleotide sequences.
- oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
- the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of EXMES, such as its biological or enzymatic activity or its abihty to bind to other molecules or compounds.
- MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C
- 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. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
- genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations maybe recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be 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.
- polynucleotides encoding EXMES maybe synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
- EXMES itself or a fragment thereof may be synthesized using chemical methods known in the art.
- peptide synthesis can be performed using various solution-phase or sohd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp.
- the peptide may be substantiaUy purified by preparative high performance hquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
- the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
- the polynucleotides encoding EXMES or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
- these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotides encoding EXMES. Such elements may vary in then strength and specificity.
- Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding EXMES. Such signals include the ATG initiation codon and adjacent sequences, e.g.
- Methods which are weU known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding EXMES and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).
- a variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding EXMES. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU 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 ceU systems (Sambrook and Russell, supra; Ausubel et al., supra; Van Heeke, G.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
- yeast transformed with yeast expression vectors insect ceU systems infected with viral expression vectors (e.
- Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239- 242).
- the invention is not limited by the host ceU employed.
- a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding EXMES.
- routine cloning, subcloning, and propagation of polynucleotides encoding EXMES can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invifrogen).
- PBLUESCRIPT Stratagene, La JoUa CA
- PSPORT1 plasmid Invifrogen
- these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
- vectors which direct high level expression of EXMES may be used.
- vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
- Yeast expression systems may be used for production of EXMES.
- a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
- such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
- Plant systems may also be used for expression of EXMES. Transcription of polynucleotides encoding EXMES may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl.
- CeU Differ. 17:85-105 These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection (The McGraw HUl Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196).
- a number of viral-based expression systems may be utilized.
- polynucleotides encoding EXMES may be hgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses EXMES in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammahan host ceUs.
- SV40 or EBV-based vectors may also be used for high-level protein expression.
- HACs Human artificial chromosomes
- HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
- HACs of about 6 kb to 10 Mb are constructed and dehvered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
- liposomes, polycationic amino polymers, or vesicles for therapeutic purposes.
- polynucleotides encoding EXMES can be transformed into ceU 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.
- ceUs may be aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
- the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfully express the introduced sequences.
- Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type. Any number of selection systems may be used to recover transformed ceU lines.
- herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes for use in tk ⁇ and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823).
- antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
- dhfi' confers resistance to methotrexate
- neo confers resistance to the aminoglycosides neomycin and G-418
- als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
- trpB and hisD which alter ceUular requirements for metabohtes
- Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; BD Clontech), ⁇ -gfucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131). Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- transformed ceUs containing polynucleotides encoding EXMES can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding EXMES under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
- host ceUs that contain the polynucleotide encoding EXMES and that express EXMES may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amphfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of EXMES using either specific polyclonal or monoclonal antibodies are known in the art.
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated ceU sorting
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding EXMES include ohgolabeling, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide.
- polynucleotides encoding EXMES, or any fragments thereof ma be cloned into a vector for the production of an mRNA probe.
- Such vectors are known in the art, are commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commerciaUy avaUable kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuchdes, enzymes, fluorescent, chemhuminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host ceUs transformed with polynucleotides encoding EXMES maybe cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
- the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and or the vector used.
- expression vectors containing polynucleotides which encode EXMES may be designed to contain signal sequences which direct secretion of EXMES through a prokaryotic or eukaryotic ceU membrane.
- a host ceU strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
- Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
- Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are avaUable from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
- ATCC American Type Culture CoUection
- natural, modified, or recombinant polynucleotides encoding EXMES may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
- a chimeric EXMES protein containing a heterologous moiety that can be recognized by a commerciaUy avaUable antibody may facilitate the screening of peptide hbraries for inhibitors of EXMES activity.
- Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commerciaUy avaUable affinity matrices.
- Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
- GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
- FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy avaUable monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
- a fusion protein may also be engineered to contain a proteolytic cleavage site located between the EXMES encoding sequence and the heterologous protein sequence, so that EXMES maybe cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16).
- a variety of commerciaUy avaUable kits may also be used to facilitate expression and purification of fusion proteins.
- synthesis of radiolabeled EXMES maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
- EXMES, fragments of EXMES, or variants of EXMES may be used to screen for compounds that specificaUy bind to EXMES.
- One or more test compounds may be screened for specific binding to EXMES.
- 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to EXMES.
- Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., hgands or receptors), or smaU molecules.
- variants of EXMES can be used to screen for binding of test compounds, such as antibodies, to EXMES, a variant of EXMES, or a combination of EXMES and/or one or more variants EXMES.
- a variant of EXMES can be used to screen for compounds that bind to a variant of EXMES , but not to EXMES having the exact sequence of a sequence of SEQ ID NO:l-25.
- EXMES variants used to perform such screening can have a range of about 50% to about 99% sequence identity to EXMES, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
- a compound identified in a screen for specific binding to EXMES can be closely related to the natural ligand of EXMES, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
- the compound thus identified can be a natural ligand of a receptor EXMES (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
- a compound identified in a screen for specific binding to EXMES can be closely related to the natural receptor to which EXMES binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket.
- the compound may be a receptor for EXMES which is capable of propagating a signal, or a decoy receptor for EXMES which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. Cell Biol. 11 :255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
- the compound can be rationally designed using known techniques.
- Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGi (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611-616).
- TNF tumor necrosis factor
- two or more antibodies having simUar or, alternatively, different specificities can be screened for specific binding to EXMES, fragments of EXMES, or variants of EXMES.
- the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of EXMES.
- an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of EXMES.
- an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of EXMES.
- anticalins can be screened for specific binding to EXMES, fragments of EXMES , or variants of EXMES.
- Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275).
- the protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end.
- loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
- the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
- screening for compounds which specifically bind to, stimulate, or inhibit EXMES involves producing appropriate cells which express EXMES, either as a secreted protein or on the cell membrane.
- Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli.
- Cells expressing EXMES or cell membrane fractions which contain EXMES are then contacted with a test compound and binding, stimulation, or inhibition of activity of either EXMES or the compound is analyzed.
- An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
- the assay may comprise the steps of combining at least one test compound with EXMES, either in solution or affixed to a solid support, and detecting the binding of EXMES to the compound.
- the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
- the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
- An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
- assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No.
- one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural hgands (Matthews, DJ. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
- one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982- 10988).
- EXMES, fragments of EXMES, or variants of EXMES may be used to screen for compounds that modulate the activity of EXMES.
- Such compounds may include agonists, antagonists, or partial or inverse agonists.
- an assay is performed under conditions permissive for EXMES activity, wherein EXMES is combined with at least one test compound, and the activity of EXMES in the presence of a test compound is compared with the activity of EXMES in the absence of the test compound. A change in the activity of EXMES in the presence of the test compound is indicative of a compound that modulates the activity of EXMES.
- test compound is combined with an in vitro or ceU-free system comprising EXMES under conditions suitable for EXMES activity, and the assay is performed.
- a test compound which modulates the activity of EXMES may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
- polynucleotides encoding EXMES or their mammahan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
- ES embryonic stem
- Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
- mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
- the ES ceUs 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).
- the vector integrates into the corresponding region of the 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 (Marfh, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
- Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
- the blastocysts are surgicaUy transfened to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
- Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
- Polynucleotides encoding EXMES may also be manipulated in vitro in ES ceUs derived from human blastocysts. Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types.
- ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
- Polynucleotides encoding EXMES can also be used to create "knockin" humanized animals
- pigs pigs
- transgenic animals e or rats
- knockin technology a region of a polynucleotide encoding EXMES is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
- Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
- Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
- a mammal inbred to overexpress EXMES e.g., by secreting EXMES in its milk, may also serve as a convenient source of that protein (Jaime, J. et al. (1998) Biotechnol.
- EXMES appears to play a role in autoimmune/inflammatory disorders, cell proliferative disorders, and endocrine disorders.
- EXMES or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of EXMES.
- disorders include, but are not limited to, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetahs, erythema nodosum, atrophic
- AIDS acquired immuno
- composition comprising a substantiaUy purified EXMES in conjunction with a suitable pharmaceutical canier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of EXMES including, but not limited to, those provided above.
- an agonist which modulates the activity of EXMES may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of EXMES including, but not limited to, those hsted above.
- an antagonist of EXMES may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of EXMES. Examples of such disorders include, but are not limited to, those autoimmune/inflammatory disorders, ceU prohferative disorders, and endocrine disorders described above.
- an antibody which specificaUy binds EXMES may be used directly as an antagonist or indhectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express EXMES.
- a vector expressing the complement of the polynucleotide encoding EXMES may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of EXMES including, but not limited to, those described above.
- any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
- the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
- An antagonist of EXMES may be produced using methods which are generaUy known in the art. In particular, purified EXMES may be used to produce antibodies or to screen hbraries of pharmaceutical agents to identify those which specificaUy bind EXMES.
- Antibodies to EXMES may also be generated using methods that are weU known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeuticaUy.
- Single chain antibodies e.g., from camels or Uamas
- various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others may be immunized by injection with EXMES or with any fragment or ohgopeptide thereof which has immunogenic properties.
- various adjuvants may be used to increase immunological response.
- adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oU emulsions, KLH, and dinitrophenol.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum are especiaUy preferable.
- the oligopeptides, peptides, or fragments used to induce antibodies to EXMES have an amino acid sequence consisting of at least about 5 amino acids, and generaUy whl consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natural protein. Short stretches of EXMES amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced. Monoclonal antibodies to EXMES may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture.
- chimeric antibodies such as the sphcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
- techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce EXMES-specific single chain antibodies.
- Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin hbraries (Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin hbraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
- Antibody fragments which contain specific binding sites for EXMES may also be generated.
- such fragments include, but are not limited to, F(ab ! ) 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
- Fab expression hbraries ma be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
- Various immunoassays may be used for screening to identify antibodies having the desired specificity.
- K a is defined as the molar concentration of EXMES -antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
- K a association constant
- the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular EXMES epitope represents a true measure of affinity.
- High-affinity antibody preparations with K influence ranging from about 10 9 to 10 12 L/mole are prefened for use in immunoassays in which the EXMES -antibody complex must withstand rigorous manipulations.
- Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are prefened for use in immunopurification and simUar procedures which ultimately require dissociation of EXMES, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Whey & Sons, New York NY).
- polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
- a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of EXMES -antibody complexes.
- Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generaUy avaUable (Catty, supra; Cohgan et al, supra).
- polynucleotides encoding EXMES may be used for therapeutic purposes.
- modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified ohgonucleotides) to the coding or regulatory regions of the gene encoding EXMES.
- complementary sequences or antisense molecules DNA, RNA, PNA, or modified ohgonucleotides
- antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding EXMES (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
- 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 (Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) FASEB J. 9:1288- 1296).
- Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D.
- polynucleotides encoding EXMES may be used for somatic or germline gene therapy.
- Gene therapy may be performed to (i) conect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 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
- hepatitis B or C virus HBV, HCV
- fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
- protozoan parasites such as Plasmodiumfalcipa m and Tr ⁇ panosoma cruzi.
- diseases or disorders caused by deficiencies in EXMES are treated by constructing mammahan expression vectors encoding EXMES and introducing these vectors by mechanical means into EXMES -deficient ceUs.
- Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA micrornjection into individual ceUs, (ii) baUistic gold particle dehvery, (hi) hposome-mediated transfection, (iv) receptor- mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Cun. Opin. Biotechnol. 9:445-450).
- Expression vectors that may be effective for the expression of EXMES include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
- EXMES may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, Ihymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H.
- a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, Ihymidine kinase (TK), or ⁇ -actin genes
- an inducible promoter e.g., the tetracycline-regulated promoter (Gossen, M. and H.
- CommerciaUy avaUable liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, avaUable from Invifrogen
- aUow one with ordinary skUl in the art to dehver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
- transformation is performed using the calcium phosphate method (Graham, F.L. and AJ. Eb (1973) Virology 52:456-467), or by elecfroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
- the introduction of DNA to primary ceUs requires modification of these standardized mammahan transfection protocols.
- diseases or disorders caused by genetic defects with respect to EXMES expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding EXMES under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (hi) a Rev-responsive element (RRE) along with additional retrovirus s-acting RNA sequences and coding sequences required for efficient vector propagation.
- Retrovirus vectors e.g., PFB and PFBNEO
- Retrovirus vectors are commerciaUy avaUable (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl.
- the vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs 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 A.D. MUler (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol.
- VPCL vector producing ceU line
- U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T- ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skhled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J.
- an adenovirus-based gene therapy dehvery system is used to dehver polynucleotides encoding EXMES to ceUs which have one or more genetic abnormahties with respect to the expression of EXMES.
- adenovirus-based vectors The construction and packaging of adenovirus-based vectors are weU known to those with ordinary skUl in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding i munoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).
- a herpes-based, gene therapy dehvery system is used to dehver polynucleotides encoding EXMES to target ceUs which have one or more genetic abnormahties with respect to the expression of EXMES.
- the use of herpes simplex virus (HSV)-based vectors may be especiaUy valuable for introducing EXMES to ceUs of the central nervous system, for which HS V has a tropism.
- the construction and packaging of herpes-based vectors are weU known to those with ordinary skiU in the art.
- HSV herpes simplex virus
- 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transfened to a ceU 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).
- herpesvirus sequences The manipulation of cloned herpesvirus sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of ceUs with herpesvirus are techniques weU known to those of ordinary skill in the art.
- an alphavirus (positive, single-stranded RNA virus) vector is used to dehver polynucleotides encoding EXMES to target ceUs.
- SFV Semhki Forest Virus
- SFV Semhki Forest Virus
- This subgenomic RNA rephcates to higher levels than the fuU length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
- enzymatic activity e.g., protease and polymerase.
- inserting the coding sequence for EXMES into the alphavirus genome in place of the capsid-coding region results in the production of a large number of EXMES-coding RNAs and the synthesis of high levels of EXMES in vector transduced ceUs.
- WhUe alphavirus infection is typicaUy associated with ceU lysis within a few days, the ability to establish a persistent infection in hamster normal kidney ceUs (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 apphcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
- the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
- a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
- RNA The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
- engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding EXMES.
- Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC.
- RNA sequences of between 15 and 20 ribonucleotides may be evaluated for secondary structural features which may render the ohgonucleotide inoperable.
- the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using ribonuclease protection assays.
- RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding EXMES .
- DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
- these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU lines, ceUs, or tissues.
- RNA molecules may be modified to increase intraceUular stability and half-hfe. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
- This concept is inherent in the production of PNAs and can be extended in aU of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easUy recognized by endogenous endonucleases.
- nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easUy recognized by endogenous endonucleases.
- RNAi RNA interference
- PTGS post-franscriptional gene sUencing
- RNAi is a post-franscriptional mode of gene sUencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specificaUy suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene.
- dsRNA double-stranded RNA
- PTGS can also be accomplished by use of DNA or DNA fragments as weU.
- RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T. (2000; Nature 404:804-808).
- PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene dehvery and/or viral vector dehvery methods described herein or known in the art.
- RNAi can be induced in mammahan ceUs by the use of smaU interfering RNA also known as siRNA.
- siRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
- siRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs.
- the use of siRNA for inducing RNAi in mammahan ceUs is described by Elbashir, S.M. et al.
- siRNA can be generated indirectly by introduction of dsRNA into the targeted ceU.
- siRNA can be synthesized dhectly and introduced into a ceU by transfection methods and agents described herein or known in the art (such as hposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods).
- Suitable siRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occunence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being prefened.
- Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
- the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison dgoritirms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.
- the selected siRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy avaUable methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX). In alternative embodiments, long-term gene sUencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
- shRNAs hairpin RNAs
- methods known in the art see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958.
- shRNAs can be dehvered to target ceUs using expression vectors known in the art.
- An example of a suitable expression vector for dehvery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion).
- the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis.
- Expression levels of the mRNA of a targeted gene can be determined, for example, by northern analysis methods using the NORTHERNMAX-GLY kit (Ambion); by microanay methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein.
- Expression levels of the protein encoded by the targeted gene can be determined, for example, by microanay methods; by polyacrylamide gel electrophoresis; and by Western analysis using standard techniques known in the art.
- An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding EXMES.
- Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non- macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
- a compound which specifically inhibits expression of the polynucleotide encoding EXMES may be therapeutically useful, and in the treatment of disorders associated with decreased EXMES expression or activity, a compound which specifically promotes expression of the polynucleotide encoding EXMES may be therapeutically useful.
- test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
- a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturaUy-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
- a sample comprising a polynucleotide encoding EXMES is exposed to at least one test compound thus obtained.
- the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
- Alterations in the expression of a polynucleotide encoding EXMES are assayed by any method commonly known in the art.
- the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding EXMES.
- the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
- a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
- a particular embodiment of the present invention involves screening a combinatorial library of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
- ohgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
- vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466). Any of the therapeutic methods described above may be apphed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
- An additional embodiment of the invention relates to the administration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
- Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
- Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Pubhshing, Easton PA).
- Such compositions may consist of EXMES, antibodies to EXMES, and mimetics, agonists, antagonists, or inhibitors of EXMES.
- compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, infrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, infrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generaUy aerosohzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol dehvery of fast-acting formulations is weU-known in the art.
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
- the determination of an effective dose is weU within the capability of those skilled in the art.
- Specialized forms of compositions may be prepared for direct intraceUular dehvery of macromolecules comprising EXMES or fragments thereof.
- liposome preparations containing a ceU-imperrneable macromolecule may promote ceU fusion and intraceUular dehvery of the macromolecule.
- EXMES or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
- the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
- ceU culture assays e.g., of neoplastic ceUs
- animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
- An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- a therapeuticaUy effective dose refers to that amount of active ingredient, for example EXMES or fragments thereof, antibodies of EXMES, and agonists, antagonists or inhibitors of EXMES, which ameliorates the symptoms or condition.
- Therapeutic efficacy and toxicity may be dete ⁇ r ned by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD S0 (the dose lethal to 50% of the population) statistics.
- the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio. Compositions which exhibit large therapeutic indices are prefened.
- the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
- the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity.
- the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
- Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination ⁇ ), reaction sensitivities, and response to therapy. Long-acting compositions may be a ⁇ jninistered every 3 to 4 days, every week, or biweekly depending on the half-hfe and clearance rate of the particular formulation.
- Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
- Guidance as to particular dosages and methods of dehvery is provided in the hterature and generaUy avaUable to practitioners in the art. Those skilled in the art whl employ different formulations for nucleotides than for proteins or then inhibitors. SimUarly, dehvery of polynucleotides or polypeptides whl be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
- antibodies which specificaUy bind EXMES may be used for the diagnosis of disorders characterized by expression of EXMES , or in assays to monitor patients being treated with EXMES or agonists, antagonists, or inhibitors of EXMES.
- Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for EXMES include methods which utilize the antibody and a label to detect EXMES in human body fluids or in extracts of ceUs or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
- a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
- EXMES EXMES
- ELISAs RIAs
- FACS FACS-activated cytoplasmic plasmic senorization
- normal or standard values for EXMES expression are estabhshed by combining body fluids or ceU extracts taken from normal mammahan subjects, for example, human subjects, with antibodies to EXMES under conditions suitable for complex formation.
- the amount of standard complex formation may be quantitated by various methods, such as photometric means.
- Quantities of EXMES expressed in subject, control, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.
- polynucleotides encoding EXMES may be used for diagnostic purposes.
- the polynucleotides which may be used include ohgonucleotides, complementary RNA and DNA molecules, and PNAs.
- the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of EXMES may be conelated with disease.
- the diagnostic assay may be used to determine absence, presence, and excess expression of EXMES, and to monitor regulation of EXMES levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding EXMES or closely related molecules may be used to identify nucleic acid sequences which encode EXMES.
- the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amphfication wUl determine whether the probe identifies only naturaUy occurring sequences encoding EXMES, aUehc variants, or related sequences.
- Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the EXMES encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:26-50 or from genomic sequences including promoters, enhancers, and introns of the EXMES gene.
- Means for producing specific hybridization probes for polynucleotides encoding EXMES include the cloning of polynucleotides encoding EXMES or EXMES derivatives into vectors for the production of mRNA probes.
- Such vectors are known in the art, are commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuchdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
- Polynucleotides encoding EXMES may be used for the diagnosis of disorders associated with expression of EXMES.
- disorders include, but are not limited to, an autoinirriune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyhtis, a yloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrmopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetahs, erythema nodosum, atrophic gastritis,
- Polynucleotides encoding EXMES maybe used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microanays utilizing fluids or tissues from patients to detect altered EXMES expression. Such quahtative or quantitative methods are weU known in the art.
- polynucleotides encoding EXMES may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
- Polynucleotides complementary to sequences encoding EXMES may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding EXMES in the sample indicates the presence of the associated disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
- a normal or standard profile for expression is estabhshed. This may be accomphshed by confining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding EXMES, under conditions suitable for hybridization or amphfication.
- Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
- hybridization assays may be repeated on a regular basis to dete ⁇ rrine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
- the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
- the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
- a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
- ohgonucleotides designed from the sequences encoding EXMES may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
- Ohgomers wUl preferably contain a fragment of a polynucleotide encoding EXMES, or a fragment of a polynucleotide complementary to the polynucleotide encoding EXMES, and wiU be employed under optimized conditions for identification of a specific gene or condition.
- Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
- ohgonucleotide primers derived from polynucleotides encoding EXMES may be 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 fluorescent SSCP (fSSCP) methods.
- SSCP single-stranded conformation polymorphism
- fSSCP fluorescent SSCP
- ohgonucleotide primers derived from polynucleotides encoding EXMES are used to amphfy DNA using the polymerase chain reaction (PCR).
- the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodUy 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 ohgonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
- AdditionaUy sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
- SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
- SNPs maybe used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drag, such as hfe-threatening toxicity.
- N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, whUe a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway.
- Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and then migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med.
- EXMES EXMES
- Methods which may also be used to quantify the expression of EXMES include radiolabeling or biotinylating nucleotides, coamphfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C et al. (1993) Anal. Bioche 212:229-236).
- the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorirnetric response gives rapid quantitation.
- ohgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microanay.
- the microanay can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
- the microanay may also be used to identify genetic variants, mutations, and polymorphisms.
- This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
- this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
- EXMES EXMES, fragments of EXMES, or antibodies specific for EXMES may be used as elements on a microanay.
- the microanay may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
- a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
- a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and then relative abundance under given conditions and at a given time (SeUhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
- a transcript image may be generated by hybridizing the polynucleotides of the present invention or then complements to the totality of transcripts or reverse transcripts of a particular tissue or ceU type.
- the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurahty of elements on a microarray.
- the resultant transcript image would provide a profile of gene activity.
- Transcript images may be generated using transcripts isolated from tissues, ceU lines, biopsies, or other biological samples.
- the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU line.
- Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring envhonmental compounds.
- AU 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.
- test compound has a signature simUar to that of a compound with known toxicity, it is likely to share those toxic properties.
- These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
- IdeaUy a genome-wide measurement of expression provides the highest quahty signature.
- genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. WhUe the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press
- the toxicity of a test compound can be 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 of the present invention, so that transcript levels conesponding 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 ceU 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 ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU 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, typicaUy by staining the gel with an agent such as Coomassie Blue or sUver or fluorescent stains.
- the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
- the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
- the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
- the identity of the protein in a spot may be dete ⁇ nined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
- a proteomic profile may also be generated using antibodies specific for EXMES to quantify the levels of EXMES expression.
- the antibodies are used as elements on a microanay, and protein expression levels are quantified by contacting the microanay with the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety 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 anay element.
- Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
- There is a poor conelation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. SeUhamer (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.
- 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.
- 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 conesponding 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 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 polypeptides 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 with antibodies specific to the polypeptides 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.
- Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; BaldeschweUer et al. (1995) PCT apphcation W095/25116; Shalon, D. et al. (1995) PCT apphcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662).
- Various types of microanays are weU known and thoroughly described in Schena, M., ed. (1999; DNA Microanays: A Practical Approach, Oxford University Press, London).
- nucleic acid sequences encoding EXMES may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
- Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences.
- conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
- the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA hbraries (Harrington, J.J. et al. (1997) Nat. Genet.
- nucleic acid sequences may be used to develop genetic linkage maps, for example, which conelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357) .
- RFLP restriction fragment length polymorphism
- Fluorescent in situ hybridization may be conelated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendehan Inheritance in Man (OMIM) World Wide Web site. Conelation between the location of the gene encoding EXMES on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
- FISH Fluorescent in situ hybridization
- In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
- physical mapping techniques such as linkage analysis using estabhshed chromosomal markers
- linkage analysis using estabhshed chromosomal markers may be used for extending genetic maps.
- the placement of a gene on the chromosome of another mammahan species, such as mouse may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
- any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580).
- the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, earner, or affected individuals.
- EXMES its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening hbraries of compounds in any of a variety of drug screening techniques.
- the fragment employed in such screening may be free in solution, affixed to a sohd support, borne on a ceU surface, or located intraceUularly.
- the formation of binding complexes between EXMES and the agent being tested may be measured.
- Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT apphcation WO84/03564). In this method, large numbers of different smaU test compounds are synthesized on a sohd substrate.
- test compounds are reacted with EXMES, or fragments thereof, and washed. Bound EXMES is then detected by methods weU known in the art. Purified EXMES can also be coated dhectly onto plates for use in the aforementioned drag screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a sohd support.
- antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with EXMES .
- nucleotide sequences which encode EXMES may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cunently known, including, but not limited to, such properties as the triplet genetic code and specific base pah interactions.
- RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
- RNA is treated with DNase.
- poly(A)+ RNA is isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
- RNA is isolated dhectly from tissue lysates using other RNA isolation kits, e.g. , the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
- Stratagene is provided with RNA and constructs the conesponding cDNA hbraries. Otherwise, cDNA is synthesized and cDNA hbraries are constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invifrogen), using the recommended procedures or simUar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription is initiated using ohgo d(T) or random primers. Synthetic ohgonucleotide adapters are hgated to double stranded cDNA, and the cDNA is digested with the appropriate restriction enzyme or enzymes.
- the cDNAis size-selected 300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
- cDNAs are hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invifrogen, Carlsbad CA), PCDNA2.1 plasmid (Invifrogen), PBK-CMV plasmid (Stratagene), PCR2- TOPOTA plasmid (Invifrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo Alto CA), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof.
- Recombinant plasmids are transformed into competent E. coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invifrogen.
- Plasmids obtained as described in Example I are recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids are purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
- Plasmids are resuspended in 0.1 ml of distiUed water and stored, with or without lyophilization, at 4°C
- plasmid DNA is amplified from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps are carried out in a single reaction mixture.
- Samples are processed and stored in 384- weU plates, and the concentration of amphfied plasmid DNA is quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
- Incyte cDNA recovered in plasmids as described in Example II are sequenced as follows. Sequencing reactions are processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (HamUton) liquid transfer system. cDNA sequencing reactions are prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
- Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides are carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Apphed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences are identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences are selected for extension using the techniques disclosed in Example VIII.
- Polynucleotide sequences derived from Incyte cDNAs are validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
- the Incyte cDNA sequences or translations thereof are then queried against a selection of public databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus nor egicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte, Palo Alto CA); hidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM (Haft, D.H.
- HMM hidden Markov model
- HMM is a probabilistic approach which analyzes consensus primary stractures of gene families; see, for example, Eddy, S.R. (1996) Cun. Opin. Struct. Biol. 6:361-365.
- the queries are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
- the Incyte cDNA sequences are assembled to produce fuU length polynucleotide sequences.
- GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences are used to extend Incyte cDNA assemblages to full length.
- the fuU length polynucleotide sequences are translated to derive the conesponding full length polypeptide sequences.
- a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide.
- FuU length polypeptide sequences are subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. FuU length polynucleotide sequences are also analyzed using GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. FuU length polynucleotide sequences are also analyzed using the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DO
- MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR).
- Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between aligned sequences.
- Table 7 summarizes tools, programs, and algorithms used for the analysis and assembly of
- Incyte cDNA and fuU length sequences and provides apphcable descriptions, references, and threshold parameters.
- the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty value, the greater the identity between two sequences).
- Putative extraceUular messengers are initiaUy identified by ranning the Genscan gene identification program against pubhc genomic sequence databases (e.g., gbpri and gbhtg).
- Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C and S. Karlin (1998) Cun. Opin. Struct. Biol. 8:346-354).
- the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
- Genscan is a FASTA database of polynucleotide and polypeptide sequences.
- the maximum range of sequence for Genscan to analyze at once is set to 30 kb.
- the encoded polypeptides are analyzed by querying against PFAM models for extraceUular messengers. Potential extraceUular messengers are also identified by homology to Incyte cDNA sequences that have been annotated as extraceUular messengers. These selected Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri pubhc databases.
- Genscan-predicted sequences are then edited by comparison to the top BLAST hit from genpept to conect enors in the sequence predicted by Genscan, such as extra or omitted exons.
- BLAST analysis is also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
- Incyte cDNA coverage is avaUable, this information is used to conect or confirm the Genscan predicted sequence.
- FuU length polynucleotide sequences are obtained by assembling Genscan- predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example III.
- fuU length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences.
- Partial cDNA sequences are extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III are mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster is analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible sphce variants that are subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval is present on more than one sequence in the cluster are identified, and intervals thus identified are considered to be equivalent by transitivity.
- Partial DNA sequences are extended to fuU length with an algorithm based on BLAST analysis.
- First, partial cDNAs assembled as described in Example III are queried against pubhc databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases using the BLAST program.
- the nearest GenBank protein homolog is then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
- a chimeric protein is generated by using the resultant high-scoring segment pahs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
- HSPs high-scoring segment pahs
- GenBank protein homolog the chimeric protein, or both are used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences are therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences are examined to determine whether they contain a complete gene. VI. Chromosomal Mapping of EXMES Encoding Polynucleotides
- sequences used to assemble SEQ ID NO:26-50 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 matched SEQ ID NO:26-50 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data avaUable from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genefhon are used to determine if any of the clustered sequences have been previously mapped. Inclusion of a mapped sequence in a cluster results in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
- SHGC Stanford Human Genome Center
- WIGR Whitehead Institute for Genome Research
- Genefhon Genefhon
- Map locations are represented by ranges, or intervals, of human chromosomes.
- the map position of an interval, in centiMorgans, is measured relative to the te ⁇ riinus of the chromosome's p- arm.
- centiMorgan cM
- centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 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.
- the cM distances are based on genetic markers mapped by Genefhon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site
- 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 ceU type or tissue have been bound (Sambrook and RusseU, supra, ch. 7; Ausubel et al., supra, ch. 4).
- Analogous computer techniques applying BLAST are used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte). This analysis is much faster than multiple membrane-based hybridizations.
- the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or sirnUar.
- the basis of the search is the product score, which is defined as:
- the product score takes into account both the degree of simUarity between two sequences and the length of the sequence match.
- the product score is a normalized value between 0 and 100, and is calculated as foUows: the BLAST score is multiphed 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 pah (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 pah with the highest BLAST score is used to calculate the product score.
- the product score represents a balance between fractional overlap and quahty in a BLAST ahgnment. For example, 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.
- polynucleotides encoding EXMES are analyzed with respect to the tissue sources from which they are derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III).
- Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
- Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ ceUs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
- the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
- each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
- the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding EXMES.
- cDNA sequences and cDNA library/tissue information are found in the LIFESEQ database (Incyte, Palo Alto CA). VIII. Extension of EXMES Encoding Polynucleotides
- FuU length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using ohgonucleotide primers designed from this fragment.
- One primer is synthesized to initiate 5' extension of the known fragment, and the other primer is synthesized to initiate 3' extension of the known fragment.
- the initial primers are designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations is avoided.
- Selected human cDNA hbraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed.
- the plate is scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
- a 5 l to 10 ⁇ l ahquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose gel to determine which reactions are successful in extending the sequence.
- the extended nucleotides are desalted and concentrated, transfened to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Biosciences).
- CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
- sonicated or sheared prior to rehgation 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 were religated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fUl-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb hquid media.
- the ceUs are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing 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 dUuted with 20% dirnethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
- Preliminary fUters remove the majority of basecall enors by requiring a minirnu ⁇ i Phred quality score of 15, and remove sequence alignment enors and enors resulting from improper trimming of vector sequences, chimeras, and sphce variants.
- An automated procedure of advanced chromosome analysis is apphed to the original chromatogram files in the vicinity of the putative SNP.
- Clone enor filters use statisticaUy generated algorithms to identify enors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
- Clustering enor fUters use statistically generated algorithms to identify enors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removes duplicates and SNPs found in immunoglobulins or T-cell receptors. Certain SNPs are selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
- the Caucasian population comprises 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
- the African population comprises 194 individuals (97 male, 97 female), all African Americans.
- the Hispanic population comprises 324 individuals (162 male, 162 female), all Mexican Hispanic.
- the Asian population comprises 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies are first analyzed in the Caucasian population; in some cases those SNPs which show no allelic variance in this population are not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes
- Hybridization probes derived from SEQ ID NO:26-50 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
- Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 ⁇ Ci of
- [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
- the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An ahquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
- the DNA from each digest is fractionated on a 0.7% agarose gel and transfened to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuahzed using autoradiography or an alternative imaging means and compared.
- the linkage or synthesis of anay elements upon a microanay can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing; see, e.g., BaldeschweUer et al., supra), mechanical microspotting technologies, and derivatives thereof.
- the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena, M., ed. (1999) DNA Microanays : A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass shdes, glass chips, and silicon wafers.
- a procedure analogous to a dot or slot blot may also be used to anange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
- a typical anay may be produced using avaUable methods and machines weU known to those of ordinary skUl in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
- FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microanay. Fragments or ohgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
- the array elements are hybridized with polynucleotides in a biological sample.
- the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
- a fluorescence scanner is used to detect hybridization at each anay element.
- laser desorbtion and mass spectrometry may be used for detection of hybridization.
- microanay preparation and usage is described in detad below.
- Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
- Each poly(A) + RNA sample 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 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
- the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
- Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, 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.
- Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (BD Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
- SpeedVAC SpeedVAC
- Sequences of the present invention are used to generate array elements.
- Each anay 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 amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified anay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
- Purified anay elements are immobilized on polymer-coated glass slides.
- Glass microscope shdes (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 Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma-Aldrich, St. Louis MO) in 95% ethanol.
- Coated shdes are cured in a 110°C oven. Anay elements are apphed to the coated glass substrate using a procedure described in U.S.
- Patent No. 5, 807,522 incorporated herein by reference.
- 1 ⁇ l of the anay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capUlary printing element by a high-speed robotic apparatus.
- the apparatus then deposits about 5 nl of anay element sample per shde.
- Microanays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distUled water. Non-specific binding sites are blocked by incubation of microanays in 0.2% casein in phosphate 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. Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
- the chamber containing the anays is incubated for about 6.5 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.
- Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an
- 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 anay using a 20X microscope objective (Nikon, Inc., MelvUle NY).
- the slide containing the anay 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 anay used in the present example is scanned with a resolution of 20 micrometers.
- a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) conesponding to the two fluorophores. Appropriate filters positioned between the anay and the photomultiplier tubes are used to filter the signals.
- the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
- Each anay 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 typicaUy cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
- a specific location on the anay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1 : 100,000.
- the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
- the output of the photomultiplier tube is digitized using a 12-bit RTI-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 signal).
- the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first conected 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 integrated to obtain a numerical value conesponding to the average intensity of the signal.
- the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Anay elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed. Expression
- SEQ ID NO:28 showed tissue-specific expression as determined by microarray analysis.
- RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
- the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microanay.
- SEQ ID NO:28 Since these hybridization experiments were conducted using a common reference sample, differential expression values are dhectly comparable from one tissue to another. The expression of SEQ ID NO:28 was increased by at least two-fold in brain tissues as compared to the reference sample. Therefore, SEQ ID NO:28 can be used as a tissue marker for brain.
- SEQ ID NO:29 and SEQ ID NO:30 showed differential expression associated with breast cancer as determined by microarray analysis.
- a tumor from the right breast of a 43-year-old female diagnosed with invasive lobular carcinoma was compared to grossly uninvolved breast tissue from the same donor (Huntsman Cancer Institute, Salt Lake City, UT). The tumor is described as weU differentiated and metastatic to 2 of 13 lymph nodes.
- Expression of SEQ ID NO:29 and SEQ ID NO:30 was decreased at least two-fold in cancerous tissue from the patient with breast cancer compared to matched microscopicaUy normal tissue from the same donor.
- SEQ ID NO:29 and SEQ ID NO:30 can each be used for one or more of the foUowing: i) monitoring treatment of breast cancer, h) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer.
- SEQ ID NO:29 and SEQ ID NO:30 showed differential expression associated with colon cancer as determined by microanay analysis.
- Gene expression profUes were obtained by comparing normal sigmoid colon tissue from a 48-year-old female to a sigmoid colon tumor originating from a metastatic gastric sarcoma (stromal tumor) from the same donor (Huntsman Cancer Institute, Salt Lake City, UT).
- Expression of SEQ ID NO:29 and SEQ ID NO:30 was increased at least two-fold in cancerous tissue from the patient with colon cancer compared to matched microscopicaUy normal tissue from the same donor.
- SEQ ID NO:29 and SEQ ID NO:30 can each be used for one or more of the foUowing: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and hi) developing therapeutics and/or other treatments for colon cancer.
- SEQ ID NO:29 and SEQ ID NO:30 showed differential expression associated with lung cancer as determined by microarray analysis. Moderately differentiated adenocarcinoma of the right lung was compared to grossly uninvolved lung tissue from a 60 year-old donor (Huntsman Cancer Institute). Expression of SEQ ID NO:29 and SEQ ID NO:30 was decreased at least two-fold in cancerous tissue from the patient with lung cancer compared to matched microscopicaUy normal tissue from the same donor.
- SEQ ID NO:29 and SEQ ID NO:30 can each be used for one or more of the foUowing: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and hi) developing therapeutics and or other treatments for lung cancer.
- SEQ ID NO:29 and SEQ ID NO:30 showed differential expression in comparisons of differentiating preadipocytes from adipose tissue of obese and normal donors as determined by microanay analysis.
- Primary subcutaneous preadipocytes were isolated from adipose tissue of a 28-year-old healthy female with body mass index (BMI) of 23.59.
- BMI body mass index
- the preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in differentiation medium containing active components PPAR- ⁇ agonist and human insulin (Zen-Bio).
- Thiazolidinediones or PPAR- ⁇ agonists can bind and activate an orphan nuclear receptor, PPAR- ⁇ , and some of them have been proven to be able to induce human adipocyte differentiation.
- the preadipocytes were treated with human insulin and PPAR- ⁇ agonist for 3 days and subsequently were switched to medium containing insulin for a variety of time periods ranging from one to 20 days before the ceUs were coUected for analysis. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. Between 80% and 90% of the preadipocytes finaUy differentiated to adipocytes as observed under phase contrast microscope.
- primary subcutaneous preadipocytes were isolated from adipose tissue of a 40-year-old healthy female with a body mass index (BMI) of 32.47.
- the preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in differentiation medium containing active components PPAR- ⁇ agonist and human insulin (Zen-Bio).
- Thiazohdinediones or PPAR- ⁇ agonists can bind and activate an orphan nuclear receptor, PPAR- ⁇ , and some of them have been proven to be able to induce human adipocyte differentiation.
- the preadipocytes were treated with human insulin and PPAR- ⁇ agonist for 3 days and subsequently were switched to medium containing insulin alone for a variety of time periods ranging from one to 20 days before the ceUs were coUected for analysis.
- Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. Between 80% and 90% of the preadipocytes finaUy differentiated to adipocytes as observed under phase contrast microscope. Expression of SEQ ID NO:29 and SEQ ID NO:30 was increased at least two-fold in differentiating adipocytes compared to untreated preadipocytes from the obese donor at 24 hours, 48 hours, 4 days, and 1.7 weeks.
- SEQ ID NO:29 and SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring freatment of diabetes mellitus, obesity, hypertension, and atherosclerosis, h) diagnostic assays for diabetes mellitus, obesity, hypertension, and atherosclerosis, and hi) developing therapeutics and/or other treatments for diabetes mellitus, obesity, hypertension, and atherosclerosis.
- SEQ ID NO:47 showed differential expression in breast cancer ceU lines, as determined by microanay analysis.
- the gene expression profile of a nonmalignant mammary epithelial ceU line (HMEC) was compared to the gene expression profUes of breast carcinoma lines at different stages of tumor progression.
- HMEC nonmalignant mammary epithelial ceU line
- expression of SEQ ID NO:47 was increased at least 3.5-fold in MCF7 ceUs grown under optimal media conditions, and was increased at least 4-fold in BT-474 ceUs grown under optimal media conditions. Expression of SEQ ID NO:47 was also increased at least 2.5-fold in starved BT-474 ceUs, when compared to expression levels in starved HMEC ceUs. Expression levels of SEQ ID NO:47 also increased at least 6-fold in untreated Sk-BR-3 ceUs, when compared to the levels of gene expression in untreated HMEC ceUs. In addition, expression of SEQ ID NO:47 was examined in comparison to MCF-10A ceUs.
- SEQ ID NO:47 was increased at least 4.5-fold in Sk-BR-3 ceUs grown in two different types of defined serum-free media, when compared to the expression levels in MCF-10A ceUs grown under the same media conditions. Also, expression of SEQ ID NO:47 was increased at least 7.5-fold in untreated Sk-BR-3 ceUs, when compared to untreated MCF-10A ceUs. Therefore, in various embodiments, SEQ ID NO:47 can be used for one or more of the foUowing: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and hi) developing therapeutics and/or other treatments for breast cancer.
- SEQ ID NO:47 showed differential expression in ovarian tumor tissue, as determined by microanay analysis.
- the expression levels of SEQ ID NO:47 were examined in tissue from a normal ovary from a 79 year-old female donor, and compared to expression levels in tissue from an ovarian tumor from the same donor (Huntsman Cancer Institute, Salt Lake City, UT).
- SEQ ID NO:47 expression was increased at least 3-fold in the tumor tissue, when compared to the expression levels in the normal ovarian tissue.
- SEQ ID NO:47 can be used for one or more of the foUowing: i) monitoring freatment of ovarian cancer, ii) diagnostic assays for ovarian cancer, and hi) developing therapeutics and/or other treatments for ovarian cancer.
- Sequences complementary to the EXMES -encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring EXMES.
- ohgonucleotides comprising from about 15 to 30 base pahs
- essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
- Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of EXMES.
- a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
- a complementary ohgonucleotide is designed to prevent ribosomal binding to the EXMES -encoding transcript.
- EXMES expression and purification of EXMES is achieved 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 hmited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
- Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
- Antibiotic resistant bacteria express EXMES upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
- EXMES Errorless Endometrial bacterium sarcoma
- baculovirus recombinant Autographica califomica nuclear polyhedrosis viras
- the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding EXMES by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Vhal infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
- baculovirus Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (EngeUiard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum Gene Ther. 7:1937- 1945).
- EXMES 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 ceU lysates.
- GST a 26- Irilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobUized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
- the GST moiety can be proteolyticaUy cleaved from EXMES at specificaUy engineered sites.
- 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 et al. (supra, ch. 10 and 16).
- Purified EXMES obtained by these methods can be used dhectly in the assays shown in Examples XVII and XVIII, where apphcable. XIV. Functional Assays
- EXMES function is assessed by expressing the sequences encoding EXMES at physiologicatty elevated levels in mammahan ceU culture systems.
- cDNA is subcloned into a mammahan expression vector containing a strong promoter that drives high levels of cDNA expression.
- Vectors of choice include PCMV SPORT plasmid (Invifrogen, Carlsbad CA) and PCR3.1 plasmid (Invifrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endothelial or hematopoietic ceU line, using either liposome formulations or elecfroporation.
- 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
- Expression of a marker protein provides a means to distinguish transfected ceUs fromnontransfected ceUs and is a reliable predictor of cDNA expression from the recombinant vector.
- Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; BD Clontech), CD64, or a CD64-GFP fusion protein.
- Flow cytometry (FCM) an automated, laser optics-based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward hght scatter and 90 degree side hght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular 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 ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
- EXMES The influence of EXMES on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding EXMES and either CD64 or CD64-GFP.
- CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG).
- Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
- mRNA can be purified from the ceUs using methods weU known by those of skill in the art. Expression of mRNA encoding EXMES and other genes of interest can be analyzed by northern analysis or microanay techniques.
- XV Production of EXMES Specific Antibodies EXMES substantiaUy purified using polyacrylamide gel electrophoresis (PAGE; see, e.g.,
- EXMES amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
- LASERGENE software DNASTAR
- Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are weU described in the art (Ausubel et al., supra, ch. 11).
- oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleinidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the ohgopeptide-KLH complex in complete Freund's adjuvant.
- Resulting antisera are tested for antipeptide and anti- EXMES activity by, for example, binding the peptide or EXMES to a substrate, blocking with 1 % BS A, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
- the column is eluted under conditions that disrupt antibody EXMES 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 EXMES is coUected.
- a buffer of pH 2 to pH 3 or a high concentration of a chaotrope, such as urea or thiocyanate ion
- EXMES or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem J. 133:529-539).
- Candidate molecules previously anayed in the weUs of a multi-weU plate are incubated with the labeled EXMES, washed, and any weUs with labeled EXMES complex are assayed. Data obtained using different concentrations of EXMES are used to calculate values for the number, affinity, and association of EXMES with the candidate molecules.
- EXMES molecules interacting with EXMES are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially avaUable kits based on the two-hybrid system, such as the MATCHMAKER system (BD Clontech).
- EXMES may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
- PATHCALLING process CuraGen Corp., New Haven CT
- EXMES activity is measured by one of several methods. Growth factor activity is measured by the stimulation of DNA synthesis in Swiss mouse 3T3 ceUs. (McKay, I. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York, NY.) Initiation of DNA synthesis indicates the ceUs' entry into the mitotic cycle and their commitment to undergo later division. 3T3 ceUs are competent to respond to most growth factors, not only those that are mitogenic, but also those that are involved in embryonic induction. This competence is possible because the in vivo specificity demonstrated by some growth factors is not necessarily inherent but is determined by the responding tissue.
- EXMES for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is dhectly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold EXMES concentration range is indicative of growth factor activity.
- One unit of activity per mUhhter is defined as the concentration of EXMES producing a 50% response level, where 100% represents nraximal incorporation of [ 3 H]thymidine into acid- precipitable DNA .
- an assay for cytokine activity measures the prohferation of leukocytes.
- the amount of tritiated thymidine incorporated into newly synthesized DNA is used to estimate proliferative activity.
- Varying amounts of EXMES are added to cultured leukocytes, such as granulocytes, monocytes, or lymphocytes, in the presence of [ 3 H]thymidine, a radioactive DNA precursor.
- EXMES for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA.
- a linear dose-response curve over at least a hundred-fold EXMES concentration range is indicative of EXMES activity.
- One unit of activity per mUhhter is conventionaUy defined as the concentration of EXMES producing a 50% response level, where 100% represents maximal incorporation of [ 3 H]thymidine into acid-precipitable DNA.
- EXMES cytokine activity utilizes a Boyden micro chamber (Neuroprobe, Cabin John MD) to measure leukocyte chemotaxis (Vicari, A.P. et al. (1997) Immunity 7:291-301).
- a Boyden micro chamber Neroprobe, Cabin John MD
- leukocyte chemotaxis Vicari, A.P. et al. (1997) Immunity 7:291-301.
- about 10 5 migratory ceUs such as macrophages or monocytes are placed in ceU culture media in the upper compartment of the chamber.
- Varying dUutions of EXMES are placed in the lower cornpartment.
- the two compartments are separated by a 5 or 8 micron pore polycarbonate filter (Nucleopore, Pleasanton CA). After incubation at 37°C for 80 to 120 minutes, the filters are fixed in methanol and stained with appropriate labeling agents.
- CeUs which migrate to the other side of the filter are counted using standard microscopy.
- the chemotactic index is calculated by dividing the number of migratory ceUs counted when EXMES is present in the lower compartment by the number of migratory ceUs counted when only media is present in the lower compartment.
- the chemotactic index is proportional to the activity of EXMES.
- cell lines or tissues transformed with a vector encoding EXMES can be assayed for EXMES activity by irnmunoblotting.
- CeUs are denatured in SDS in the presence of b-mercaptoethanol, nucleic acids removed by ethanol precipitation, and proteins purified by acetone precipitation.
- Pellets are resuspended in 20 mM tris buffer at pH 7.5 and incubated with Protein G-Sepharose pre-coated with an antibody specific for EXMES. After washing, the Sepharose beads are boiled in electrophoresis sample buffer, and the eluted proteins subjected to SDS-PAGE.
- the SDS-PAGE is transferred to a nitrocellulose membrane for irnmunoblotting, and the EXMES activity is assessed by visualizing and quantifying bands on the blot using the antibody specific for EXMES as the primary antibody and 125 I-labeled IgG specific for the primary antibody as the secondary antibody.
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Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU2003257111A AU2003257111A1 (en) | 2002-08-02 | 2003-07-30 | Extracellular messengers |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
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US40081002P | 2002-08-02 | 2002-08-02 | |
US60/400,810 | 2002-08-02 | ||
US41219702P | 2002-09-19 | 2002-09-19 | |
US60/412,197 | 2002-09-19 | ||
US41600402P | 2002-10-04 | 2002-10-04 | |
US60/416,004 | 2002-10-04 | ||
US42486202P | 2002-11-08 | 2002-11-08 | |
US60/424,862 | 2002-11-08 |
Publications (2)
Publication Number | Publication Date |
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WO2004013292A2 true WO2004013292A2 (fr) | 2004-02-12 |
WO2004013292A3 WO2004013292A3 (fr) | 2007-08-02 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US2003/024084 WO2004013292A2 (fr) | 2002-08-02 | 2003-07-30 | Messagers extracellulaires |
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AU (1) | AU2003257111A1 (fr) |
WO (1) | WO2004013292A2 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009091810A1 (fr) * | 2008-01-14 | 2009-07-23 | Genentech, Inc. | Procédés pour inhiber l'angiogenèse au moyen d'antagonistes egfl8 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5821219A (en) * | 1995-08-11 | 1998-10-13 | Oregon Health Sciences University | Opioid antagonists and methods of their use |
-
2003
- 2003-07-30 AU AU2003257111A patent/AU2003257111A1/en not_active Abandoned
- 2003-07-30 WO PCT/US2003/024084 patent/WO2004013292A2/fr not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5821219A (en) * | 1995-08-11 | 1998-10-13 | Oregon Health Sciences University | Opioid antagonists and methods of their use |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009091810A1 (fr) * | 2008-01-14 | 2009-07-23 | Genentech, Inc. | Procédés pour inhiber l'angiogenèse au moyen d'antagonistes egfl8 |
US9549971B2 (en) | 2008-01-14 | 2017-01-24 | Genentech, Inc. | Methods for inhibiting angiogenesis using EGFL8 antagonists |
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Publication number | Publication date |
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WO2004013292A3 (fr) | 2007-08-02 |
AU2003257111A8 (en) | 2004-02-23 |
AU2003257111A1 (en) | 2004-02-23 |
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