EP1379654A2 - Intracellular signaling molecules - Google Patents
Intracellular signaling moleculesInfo
- Publication number
- EP1379654A2 EP1379654A2 EP02713563A EP02713563A EP1379654A2 EP 1379654 A2 EP1379654 A2 EP 1379654A2 EP 02713563 A EP02713563 A EP 02713563A EP 02713563 A EP02713563 A EP 02713563A EP 1379654 A2 EP1379654 A2 EP 1379654A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polynucleotide
- polypeptide
- seq
- amino acid
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Definitions
- This invention relates to nucleic acid and amino acid sequences of intracellular signaling molecules and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, developmental, vesicle trafficking disorders, and viral infections, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of intracellular signaling molecules.
- Cell-cell communication is essential for the growth, development, and survival of multicellular organisms.
- Cells communicate by sending and receiving molecular signals.
- An example of a molecular signal is a growth factor, which binds and activates a specific transmembrane receptor on the surface of a target cell. The activated receptor transduces the signal intracellularly, thus initiating a cascade of biochemical reactions that ultimately affect gene transcription and cell cycle progression in the target cell.
- Intracellular signaling is the process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule.
- Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered.
- the intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules such as cyclic nucleotides, calcium- calmodulin, inositol, and various mitogens that regulate protein phosphorylation.
- a distinctive class of signal transduction molecules are involved in odorant detection.
- the process of odorant detection involves specific recognition by odorant receptors.
- the olfactory mucosa also appears to possess an additional group of odorant-binding proteins which recognize and bind separate classes of odorants.
- cDNA clones from rat have been isolated which correspond to mRNAs highly expressed in olfactory mucosa but not detected in other tissues.
- the proteins encoded by these clones are homologous to proteins that bind lipopolysaccharides or polychlorinated biphenyls, and the different proteins appear to be expressed in specific areas of the mucosal tissue.
- Cells also respond to changing conditions by switching off signals. Many signal transduction proteins are short-lived and rapidly targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Cells also maintain mechanisms to monitor changes in the concentration of denatured or unfolded proteins in membrane-bound extracytoplasmic compartments, including a transmembrane receptor that monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones in the endoplasmic reticulum.
- Certain proteins in intracellular signaling pathways serve to link or cluster other proteins involved in the signaling cascade. These proteins are referred to as scaffold, anchoring, or adaptor proteins.
- scaffold anchoring
- adaptor proteins As many intracellular signaling proteins such as protein kinases and phosphatases have relatively broad substrate specificities, the adaptors help to organize the component signaling proteins into specific biochemical pathways. Many of the above signaling molecules are characterized by the presence of particular domains that promote protein-protein interactions. A sampling of these domains is discussed below, along with other important intracellular messengers.
- Protein kinases and phosphatases play a key role in the intracellular signaling process by controlling the phosphorylation and activation of various signaling proteins.
- the high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule (ATP) to a particular protein by a protein kinase and removed from that protein by a protein phosphatase.
- ATP adenosine triphosphate molecule
- Protein kinases are roughly divided into two groups: those that phosphorylate serine or threonine residues (serine/threonine kinases, STK) and those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK).
- a few protein kinases have dual specificity for serine/threonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Books, Vol 1:7-20, Academic Press, San Diego, CA).
- STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smooth muscle contraction, glycogen breakdown, and neurotransmission; and the mitogen-activated protein kinases (MAP kinases) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades.
- PKA cyclic-AMP dependent protein kinases
- CaM calcium-calmodulin dependent protein kinases
- MAP kinases mitogen-activated protein kinases
- PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.
- Transmembrane PTKs are receptors for most growth factors.
- Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors.
- Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls.
- HPK histidine protein kinase family
- HPKs bear little homology with mammalian STKs or PTKs but have distinctive sequence motifs of their own (Davie, J.R. et al. (1995) J. Biol. Chem. 270: 19861-19867).
- a histidine residue in the N-terminal half of the molecule (region I) is an autophosphorylation site.
- Three additional motifs located in the C-te ⁇ ninal half of the molecule include an invariant asparagine residue in region II and two glycine-rich loops characteristic of nucleotide binding domains in regions III and IN. Recently a branched chain alpha-ketoacid dehydrogenase kinase has been found with characteristics of HPK in rat (Davie et al., supra).
- the two principal categories of protein phosphatases are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine phosphatases (PTPs).
- PPs dephosphorylate phosphoserine/threonine residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508).
- PTPs reverse the effects of protein tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonneau and Tonks, supra).
- PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific inhibition of PTPs can enhance cell transformation (Charbonneau and Tonks, supra). Phospholipid and Inositol-phosphate Signaling
- Inositol phospholipids are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane. This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma membrane to the biphosphate state (PJP 2 ) by inositol kinases. Simultaneously, the G- protein linked receptor binding stimulates a trimeric G-protein which in turn activates a phosphoinositide-specific phospholipase C- ⁇ .
- PI phosphatidylinositol
- Phospholipase C- ⁇ then cleaves PIP 2 into two products, inositol triphosphate (1P 3 ) and diacylglycerol. These two products act as mediators for separate signaling events.
- -P 3 diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), while diacylglycerol remains in the membrane and helps activate protein kinase C, a serine-threonine kinase that phosphorylates selected proteins in the target cell.
- the calcium response initiated by IP 3 is terminated by the dephosphorylation of IP 3 by specific inositol phosphatases. Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation.
- Inositol-phosphate signaling controls tubby, a membrane bound transcriptional regulator that serves as an intracellular messenger of G q -coupled receptors (Santagata et al. (2001) Science 292:2041-2050).
- Members of the tubby family contain a C-terminal tubby domain of about 260 amino acids that binds to double-stranded DNA and an N-terminal transcriptional activation domain.
- Tubby binds to phosphatidylinositol 4,5-bisphosphate, which localizes tubby to the plasma membrane.
- Activation of the G-protein q leads to activation of phospholipase C- ⁇ and hydrolysis of phosphoinositide.
- Loss of phosphatidylinositol 4,5-bisphosphate causes tubby to dissociate from the plasma membrane and to translocate to the nucleus where tubby regulates transcription of its target genes. Defects in the tubby gene are associated with obesity, retinal degeneration, and hearing loss (Boggon, TJ. et al. (1999) Science 286:2119-2125). Cyclic Nucleotide Signaling
- Cyclic nucleotides function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters.
- cyclic-AMP dependent protein kinases PKA
- PKA cyclic-AMP dependent protein kinases
- Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca 2+ -specific channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter.
- adenylyl cyclase which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to ⁇ -adrenergic receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the Ca 2+ -specific channels and recovery of the dark state in the eye.
- transmembrane isoforms of mammalian adenylyl cyclase as well as a soluble form preferentially expressed in testis.
- Soluble adenylyl cyclase contains a P-loop, or nucleotide binding domain, and may be involved in male fertility (Buck, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96:79-84).
- PDEs cyclic nucleotides that hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the opposite of these and other effects mediated by increased cyclic nucleotide levels.
- PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins. At least seven families of mammahan PDEs (PDEl-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, J.A. (1995) Physiol. Rev. 75:725-748). PDE inhibitors have been found to be particularly useful in treating various clinical disorders.
- Rolipram a specific inhibitor of PDE4
- Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000).
- Ca 2+ is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP.
- Ca 2+ can enter the cytosol by two pathways, in response to extracellular signals.
- One pathway acts primarily in nerve signal transduction where Ca 2+ enters a nerve terminal through a voltage-gated Ca + channel.
- the second is a more ubiquitous pathway in which Ca 2+ is released from the ER into the cytosol in response to binding of an extracellular signaling molecule to a receptor.
- Ca 2+ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways.
- Ca 2+ also binds to specific Ca 2+ -binding proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels.
- CBPs Ca 2+ -binding proteins
- CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M.R. et al. (1996) Guidebook to Calcium-binding Proteins, Oxford University Press, Oxford, UK, pp. 15-20).
- Ca 2+ binding proteins are characterized by the presence of one or more EF-hand Ca 2+ binding motifs, which are comprised of 12 amino acids flanked by ⁇ -helices (Celio, supra).
- the regulation of CBPs has implications for the control of a variety of disorders.
- Calcineurin a CaM-regulated protein phosphatase, is a target for inhibition by the immunosuppressive agents cyclosporin and FK506. This indicates the importance of calcineurin and CaM in the immune response and immune disorders (Schwaninger M. et al. (1993) J. Biol Chem. 268:23111-23115).
- the level of CaM is increased several-fold in tumors and tumor- derived cell lines for various types of cancer (Rasmussen, CD. and A.R. Means (1989) Trends Neurosci. 12:433-438).
- the annexins are a family of calcium-binding proteins that associate with the cell membrane
- Annexins reversibly bind to negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium dependent manner.
- Annexins participate in various processes pertaining to signal transduction at the plasma membrane, including membrane-cytoskeleton interactions, phospholipase inhibition, anticoagulation, and membrane fusion.
- Annexins contain four to eight repeated segments of about 60 residues. Each repeat folds into five alpha helices wound into a right-handed superhelix.
- G-proteins are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCRs), and intracellular second messengers such as cAMP and Ca 2+ .
- G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to GTP, inducing an "active" state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca 2+ into the cytosol from the ER, which, in turn, regulate phosphorylation and activation of other intracellular proteins.
- GTPase activity involves hydrolysis of the bound GTP to GDP by a GTPase activity in the G-protein.
- the superfamily of G-proteins consists of several families which may be grouped as translational factors, heterotrimeric G-proteins involved in transmembrane signaling processes, and low molecular weight (LMW) G-proteins including the proto-oncogene Ras proteins and products of rab, rap, rho, rac, smg21, smg25, YPT, SEC4, and ARF genes, and tubulins (Kaziro, Y. et al. (1991) Annu. Rev. Biochem. 60:349-400). In all cases, the GTPase activity is regulated through interactions with other proteins.
- Heterotrimeric G-proteins are composed of 3 subunits, ⁇ , ⁇ , and ⁇ , which in their inactive conformation associate as a trimer at the inner face of the plasma membrane.
- G ⁇ binds GDP or GTP and contains the GTPase activity.
- the ⁇ complex enhances binding of G ⁇ to a receptor.
- G ⁇ is necessary for the folding and activity of G ⁇ (Neer, EJ. et al. (1994) Nature 371:297-300). Multiple homologs of each subunit have been identified in mammalian tissues, and different combinations of subunits have specific functions and tissue specificities (Spiegel, A.M. (1997) J. Inher. Metab. Dis. 20:113-121).
- the alpha subunits of heterotrimeric G-proteins can be divided into four distinct classes.
- the ⁇ -s class is sensitive to ADP-ribosylation by pertussis toxin which uncouples the receptor: G-protein interaction. This uncoupling blocks signal transduction to receptors that decrease cAMP levels which normally regulate ion channels and activate phospholipases.
- the inhibitory ⁇ -I class is also susceptible to modification by pertussis toxin which prevents ⁇ -I from lowering cAMP levels.
- ⁇ -q which activates phospholipase C
- ⁇ -12 which has sequence homology with the Drosophila gene concertina and may contribute to the regulation of embryonic development
- the mammalian G ⁇ and G ⁇ subunits each about 340 amino acids long, share more than 80% homology.
- the G ⁇ subunit also called transducin
- the activity of both subunits may be regulated by other proteins such as calmodulin and phosducin or the neural protein GAP 43 (Clapham, D. and E. Neer (1993) Nature 365:403-406).
- the ⁇ and ⁇ subunits are tightly associated.
- the ⁇ subunit sequences are highly conserved between species, implying that they perform a fundamentally important role in the organization and function of G-protein linked systems (Nan der Noorn, L. (1992) FEBS Lett.
- WD-repeat proteins contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions.
- WD-repeat proteins contain from four to eight copies of a loosely conserved repeat of approximately 40 amino acids which participates in protein-protein interactions. Mutations and variant expression of ⁇ transducin proteins are linked with various disorders. Mutations in LISl, a subunit of the human platelet activating factor acetylhydrolase, cause Miller-Dieker lissencephaly.
- RACKl binds activated protein kinase C
- RbAp48 binds retinoblastoma protein.
- CstF is required for polyadenylation of mammalian pre-mR ⁇ A in vitro and associates with subunits of cleavage-stimulating factor.
- Defects in the regulation of ⁇ -catenin contribute to the neoplastic transformation of human cells.
- the WD40 repeats of the human F-box protein bTrCP mediate binding to ⁇ -catenin, thus regulating the targeted degradation of ⁇ -catenin by ubiquitin ligase ( ⁇ eer, supra; Hart, M. et al. (1999) Curr. Biol. 9:207-210).
- the ⁇ subunit primary structures are more variable than those of the ⁇ subunits.
- the ⁇ subunit has been shown to modulate the activity of isoforms of adenylyl cyclase, phospholipase C, and some ion channels. It is involved in receptor phosphorylation via specific kinases, and has been implicated in the p2 lras-dependent activation of the MAP kinase cascade and the recognition of specific receptors by G-proteins (Clapham and ⁇ eer, supra).
- G-proteins interact with a variety of effectors including adenylyl cyclase (Clapham and ⁇ eer, supra).
- the signaling pathway mediated by cAMP is mitogenic in hormone-dependent endocrine tissues such as adrenal cortex, thyroid, ovary, pituitary, and testes. Cancers in these tissues have been related to a mutationally activated form of a G ⁇ s known as the gsp (Gs protein) oncogene (Dhanasekaran, supra).
- Another effector is phosducin, a retinal phosphoprotein, which forms a specific complex with retinal G ⁇ and G ⁇ (G ⁇ ) and modulates the ability of G ⁇ to interact with retinal G ⁇ (Clapham and Neer, supra).
- Irregularities in the G-protein signaling cascade may result in abnormal activation of leukocytes and lymphocytes, leading to the tissue damage and destruction seen in many inflammatory and autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis.
- Abnormal cell proliferation, including cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins. Mutations in G ⁇ subunits have been found in growth-hormone-secreting pituitary somatotroph tumors, hyperfunctioning thyroid adenomas, and ovarian and adrenal neoplasms (Meij, J.T.A. (1996) Mol.
- LMW G-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the alpha subunit of the heterotrimeric G-proteins, are able to bind to and hydrolyze GTP, thus cycling between an inactive and an active state. LMW G-proteins respond to extracellular signals from receptors and activating proteins by transducing mitogenic signals involved in various cell functions. The binding and hydrolysis of GTP regulates the response of LMW G-proteins and acts as an energy source during this process (Bokoch, G.M. and C.J. Der (1993) FASEB J. 7:750-759).
- At least sixty members of the LMW G-protein superfamily have been identified and are currently grouped into the ras, rho, arf, sari, ran, and rab subfamilies.
- Activated ras genes were initially found in human cancers, and subsequent studies confirmed that ras function is critical in determining whether cells continue to grow or become differentiated.
- Rasl and Ras2 proteins stimulate adenylate cyclase (Kaziro, supra), affecting a broad array of cellular processes. Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplasmic kinases. These kinases translocate to the nucleus and activate key transcription factors that control gene expression and protein synthesis (Barbacid, M.
- Rho G-proteins control signal transduction pathways that link growth factor receptors to actin polymerization, which is necessary for normal cellular growth and division.
- the rab, arf, and sari families of proteins control the translocation of vesicles to and from membranes for protein processing, localization, and secretion.
- v-SNAREs and t-SNAREs bind to each other and dock the vesicle to the acceptor membrane.
- the budding process is regulated by the closely related ADP ribosylation factors (ARFs) and SAR proteins, while rab proteins allow assembly of SNARE complexes and may play a role in removal of defective complexes (Rothman, J. and F. Wieland (1996) Science 272:227-234).
- Ran G-proteins are located in the nucleus of cells and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990) Science 249:635-640; Barbacid, M.
- Rab proteins have a highly variable amino terminus containing membrane-specific signal information and a prenylated carboxy terminus which determines the target membrane to which the Rab proteins anchor. More than 30 Rab proteins have been identified in a variety of species, and each has a characteristic intracellular location and distinct transport function.
- Rabl and Rab2 are important in ER-to-Golgi transport; Rab3 transports secretory vesicles to the extracellular membrane; Rab5 is localized to endosomes and regulates the fusion of early endosomes into late endosomes; Rab6 is specific to the Golgi apparatus and regulates intra-Golgi transport events; Rab7 and Rab9 stimulate the fusion of late endosomes and Golgi vesicles with lysosomes, respectively; and RablO mediates vesicle fusion from the medial Golgi to the trans Golgi. Mutant forms of Rab proteins are able to block protein transport along a given pathway or alter the sizes of entire organelles. Therefore, Rabs play key regulatory roles in membrane trafficking (Schimm ⁇ ller, LS. and S.R. Pfeffer (1998) J. Biol. Chem. 243:22161-22164).
- Rab proteins in vesicular transport requires the cooperation of many other proteins. Specifically, the membrane-targeting process is assisted by a series of escort proteins (Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6264-6268). In the medial Golgi, it has been shown that GTP-bound Rab proteins initiate the binding of VAMP-like proteins of the transport vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently triggers a cascade of protein-binding and membrane-fusion events. After transport, GTPase-activating proteins (GAPs) in the target membrane are responsible for converting the GTP-bound Rab proteins to their GDP-bound state.
- GAPs GTPase-activating proteins
- guanine-nucleotide dissociation inhibitor recruits the GDP-bound proteins to their membrane of origin.
- the cycling of LMW G-proteins between the GTP-bound active form and the GDP-bound inactive form is regulated by a variety of proteins.
- Guanosine nucleotide exchange factors GEFs
- GEFs Guanosine nucleotide exchange factors
- the best characterized is the mammalian homolog of the Drosophila Son-of-Sevenless protein.
- Certain Ras-family proteins are also regulated by guanine nucleotide dissociation inhibitors (GDIs), which inhibit GDP dissociation.
- GTP hydrolysis of the LMW G-proteins is typically very slow, but it can be stimulated by several orders of magnitude by GAPs (Geyer, M. and A. Wittinghofer (1997) Curr. Opin. Struct. Biol. 7:786-792). Both GEF and GAP activity may be controlled in response to extracellular stimuli and modulated by accessory proteins such as RalBPl and POB1. Mutant Ras-family proteins, which bind but cannot hydrolyze GTP, are permanently activated, and cause cell proliferation or cancer, as do GEFs that inappropriately activate LMW G-proteins, such as the human oncogene NETl, a Rho-GEF (Drivas, G.T. et al. (1990) Mol. Cell Biol. 10:1793-1798; Alberts, A.S. and R. Treisman (1998) EMBO J. 14:4075-4085).
- centaurin beta 2 A member of the ARF family of G-proteins is centaurin beta 2, a regulator of membrane traffic and the actin cytoskeleton.
- the centaurin ⁇ family of GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors contain pleckstrin homology (PH) domains, which are activated by phosphoinositides, as well as ankyrin repeats and a conserved zinc-binding motif. These proteins are targets for the receptor-stimulated phospohinositide 3-kinase cascade (Jackson, T.R. et al. (2000) Trends Biochem. Sci. 25:489-495).
- PH domains bind phosphoinositides, implicating PH domains in signaling processes.
- Phosphoinositides have a role in converting Arf-GTP to Arf-GDP via the centaurin ⁇ family and a role in Arf activation (Ka , J.L. et al. (2000) J. Biol. Chem. 275:9653- 9663).
- the rho GAP family is also implicated in the regulation of actin polymerization at the plasma membrane and in several cellular processes.
- the gene ARHGAP6 encodes GTPase-activating protein 6 isoform 4.
- MLS is an X-linked dominant, male- lethal syndrome (Prakash, S.K. et al. (2000) Hum. Mol. Genet. 9:477-488).
- the signal-induced proliferation-associated protein is a mitogen induced GAP.
- Sipal contains a C-terminal leucine zipper and an N-terminal GAP domain homologous to the human RAP1GAP protein.
- the human SIPA1 gene is widely expressed, in fetal as well as adult tissues, but is most highly expressed in lymphoid organs. (OMIM (Online Mendelian Inheritance in Man) Entry 602180; Ebrahimi, S. (1998) Gene 214:215-221.)
- CDC42 A member of the Rho family of G-proteins is CDC42, a regulator of cytoskeletal rearrangements required for cell division.
- CDC42 is inactivated by a specific GAP (CDC42GAP) that strongly stimulates the GTPase activity of CDC42 while having a much lesser effect on other Rho family members.
- CDC42GAP also contains an SH3-binding domain that interacts with the SH3 domains of cell signaling proteins such as p85 alpha and c-Src, suggesting that CDC42GAP may serve as a link between CDC42 and other cell signaling pathways (Barfod, E.T. et al. (1993) J. Biol. Chem. 268:26059-26062).
- GTP-binding proteins are involved in protein biosynthesis and include initiation factor 2 (IF- 2), elongation factor 2 (EF-Tu), and elongation factor G (EF-G), observed in prokaryotes; and initiation factor 2 (eIF-2), elongation factor la (EF-I ⁇ ), elongation factor 2 (EF-2), and release factor 3 (eRF3) observed in eukaryotes (Kaziro, Y. et al. (1991) Ann. Rev. Biochem. 60:349-400).
- JP-2 promotes the GTP-dependent binding of the tRNA to the small subunit of the ribosome, the step that initiates protein translation.
- Elongation factors promote the binding of tRNA and GTP and the displacement of GDP after hydrolysis as protein biosynthesis proceeds.
- eRF3 participates in the recognition of stop codons and the release of nascent proteins from ribosomes.
- the bacterial mutT protein is involved in the GO system (Koonin E.V. (1993) Nucleic Acids Res. 21:4847-4847) responsible for removing an oxidatively damaged form of guanine (8-hydroxyguanine or 7,8-dihydro-8-oxoguanine) from DNA and the nucleotide pool.
- 8-oxo-dGTP is inserted opposite to dA and dC residues of template DNA with almost equal efficiency thus leading to A.T to G.C transversions.
- MutT specifically degrades 8-oxo-dGTP to the monophosphate with the concomitant release of pyrophosphate.
- MutT is a small protein of about 12 to 15 Kd.(PROSITE PDOC00695).
- the Dbl proteins are a family of GEFs for the Rho and Ras G-proteins (Whitehead, I.P. et al. (1997) Biochim. Biophys. Acta 1332:F1-F23). All Dbl family members contain a Dbl homology (DH) domain of approximately 180 amino acids, as well as a pleckstrin homology (PH) domain located immediately C-terminal to the DH domain. Most Dbl proteins have oncogenic activity, as demonstrated by the ability to transform various cell lines, consistent with roles as regulators of Rho- mediated oncogenic signaling pathways.
- the kalirin proteins are neuron-specific members of the Dbl family, which are located to distinct subcellular regions of cultured neurons (Johnson, R.C. (2000) J. Cell Biol. 275:19324-19333).
- RGS G-protein signaling
- Other regulators of G-protein signaling also exist that act primarily by negatively regulating the G-protein pathway by an unknown mechanism (Druey, K.M. et al. (1996) Nature 379:742-746).
- Some 15 members of the RGS family have been identified.
- RGS family members are related structurally through similarities in an approximately 120 amino acid region termed the RGS domain and functionally by their ability to inhibit the interleukin (cy tokine) induction of MAP kinase in cultured mammalian 293T cells (Druey et al., supra).
- the Immuno-associated nucleotide (IAN) family of proteins has GTP-binding activity as indicated by the conserved ATP/GTP-binding site P-loop motif.
- the IAN family includes IAN-1, IAN-4, IAP38, and IAG-1.
- IAN-1 is expressed in the immune system, specifically in T cells and thymocytes. Its expression is induced during thymic events (Poirier, G.M.C. et al. (1999) J. Immunol. 163:4960-4969).
- IAP38 is expressed in B cells and macrophages and its expression is induced in splenocytes by pathogens.
- IAG-1 which is a plant molecule, is induced upon bacterial infection (Krucken, J.
- IAN-4 is a mitochondrial membrane protein which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the bcr/abl oncogene.
- the bcr/abl oncogene is known to be associated with chronic myelogenous leukemia, a clonal myelo-proliferative disorder, which is due to the translocation between the bcr gene on chromosome 22 and the abl gene on chromosome 9.
- Bcr is the breakpoint cluster region gene and abl is the cellular homolog of the transforming gene of the Abelson murine leukemia virus. Therefore, the IAN family of proteins appears to play a role in cell survival in immune responses and cellular transformation (Daheron, L. et al. (2001) Nucleic Acids Res. 29:1308-1316).
- the large GTP-binding proteins having a high-turnover, concentration-dependent GTPase activity and an antiviral effect include the Mx proteins, the dynamin family, and the guanylate- binding proteins (GBPs) such as GBPl and GBP2.
- GBPs guanylate- binding proteins
- the GBPs are characterized by their ability to bind GMP, GDP, and GTP, but not any other nucleotides. Most of these proteins have a relative molecular mass in the range of 50-100kD. GBP expression is induced by interferons, cytokines that have antiviral effects and inhibit tumor cell proliferation. GBPs are the most abundant class of proteins induced by interferon-gamma.
- GBPl and GBP2 are distinguished from the other GTP-binding proteins by the presence of 2 binding motifs rather than 3 (OMJJvl #600411) .
- the enzymatic properties of GTP hydrolysis by these proteins are well documented (Warnock, D.E. et al. (1996) J. Biol. Chem. 271:22310-22314). Although the full range of functions of this group of proteins has yet to be elucidated, the current state of understanding in this area indicates a role in, among other things, vesicle trafficking, cell cycle progression, and antiviral defense (van der Bliek, A.M. supra; Prakash, B. supra).
- Formin-related genes comprise a large family of morphoregulatory genes and have been shown to play important roles in morphogenesis, embryogenesis, cell polarity, cell migration, and cytokinesis through their interaction with Rho family small GTPases.
- Formin was first identified in mouse limb deformity (Id) mutants where the distal bones and digits of all limbs are fused and reduced in size.
- FRL contains formin homology domains FHl, FH2, and FH3. The FHl domain has been shown to bind the Src homology 3 (SH3) domain, WWP/WW domains, and profilin.
- the FH2 domain is conserved and was shown to be essential for formin function as disruption at the FH2 domain results in the characteristic Id phenotype.
- the FH3 domain is located at the N-terminus of FRL, and is required for associating with Rac, a Rho family GTPase (Yayoshi-Yamamoto, S. et al. (2000) Mol. Cell. Biol. 20:6872-6881). Signaling Complex Protein Domains
- PDZ domains were named for three proteins in which this domain was initially discovered. These proteins include PSD-95 (postsynaptic density 95), Dig (Drosophila lethal(l)discs large-1), and ZO-1 (zonula occludens-1). These proteins play important roles in neuronal synaptic transmission, tumor suppression, and cell junction formation, respectively. Since the discovery of these proteins, over sixty additional PDZ-containing proteins have been identified in diverse prokaryotic and eukaryotic organisms. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For a review of PDZ domain-containing proteins, see Ponting, C.P.
- PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels.
- MAGUK membrane-associated guanylate kinase
- PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serine/threonine kinases, G- protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS).
- the glutamate receptor interacting protein contains seven PDZ domains. GRIP is an adaptor that links certain glutamate receptors to other proteins and may be responsible for the clustering of these receptors at excitatory synapses in the brain (Dong, H. et al. (1997) Nature 386:279-284).
- the Drosophila scribble (SCRUB) protein contains both multiple PDZ domains and leucine-rich repeats.
- SCRIB is located at the epithelial septate junction, which is analogous to the vertebrate tight junction, at the boundary of the apical and basolateral cell surface. SCRIB is involved in the distribution of apical proteins and correct placement of adherens junctions to the basolateral cell surface (Bilder, D. and N. Perrimon (2000) Nature 403:676-680).
- the PX domain is an example of a domain specialized for promoting protein-protein interactions. The PX domain is found in sorting nexins and in a variety of other proteins, including the PhoX components of NADPH oxidase and the Cpk class of phosphatidylinositol 3-kinase.
- PX domains contain a polyproline motif which is characteristic of SH3 domain-binding proteins (Ponting, C.P. (1996) Protein Sci. 5:2353-2357).
- SH3 domain-containing cytosolic components of the NADPH oxidase, p47 ⁇ hox and p40phox are shown by analyses of their sequences to contain single copies of the PX (phox) domain.
- Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3-kinase, S. cerevisiae Bemlp, and S. pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues.
- the SH3 domain is defined by homology to a region of the proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase.
- SH3 is a small domain of 50 to 60 amino acids that interacts with proline-rich ligands.
- SH3 domains are found in a variety of eukaryotic proteins involved in signal transduction, cell polarization, and membrane-cytoskeleton interactions. In some cases, SH3 domain-containing proteins interact directly with receptor tyrosine kinases.
- the SLAP- 130 protein is a substrate of the T-cell receptor (TCR) stimulated protein kinase.
- TCR T-cell receptor
- SLAP-130 interacts via its SH3 domain with the protein SLP-76 to affect the TCR-induced expression of interleukin-2 (Musci, M.A. et al. (1997) J. Biol. Chem. 272:11674-11677).
- SH3 domain protein is macrophage actin-associated tyrosine-phosphorylated protein (MAYP) which is phosphorylated during the response of macrophages to colony stimulating factor- 1 (CSF-1) and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton (Yeung, Y.-G. et al. (1998) J. Biol. Chem. 273:30638-30642).
- the structure of the SH3 domain is characterized by two antiparallel beta sheets packed against each other at right angles. This packing forms a hydrophobic pocket lined with residues that are highly conserved between different SH3 domains. This pocket makes critical hydrophobic contacts with proline residues in the ligand (Feng, S. et al. (1994) Science 266:1241-1247).
- a novel domain resembles the SH3 domain in its ability to bind proline-rich ligands.
- This domain was originally discovered in dystrophin, a cytoskeletal protein with direct involvement in Duchenne muscular dystrophy (Bork, P. and M. Sudol (1994) Trends Biochem. Sci. 19:531-533).
- WW domains have since been discovered in a variety of intracellular signaling molecules involved in development, cell differentiation, and cell proliferation.
- the structure of the WW domain is composed of beta strands grouped around four conserved aromatic residues, generally tryptophan.
- SH2 domain is defined by homology to a region of c-Src.
- SH2 domains interact directly with phospho-tyrosine residues, thus providing an immediate mechanism for the regulation and transduction of receptor tyrosine kinase-mediated signaling pathways.
- SH2 domains are capable of binding to phosphorylated tyrosine residues in the activated PDGF receptor, thereby providing a highly coordinated and finely tuned response to ligand- mediated receptor activation.
- the BLNK protein is a linker protein involved in B cell activation, that bridges B cell receptor-associated kinases with SH2 domain effectors that link to various signaling pathways (Fu, C. et al. (1998) Immunity 9:93-103).
- the pleckstrin homology (PH) domain was originally identified in pleckstrin, the predominant substrate for protein kinase C in platelets. Since its discovery, this domain has been identified in over 90 proteins involved in intracellular signaling or cytoskeletal organization. Proteins containing the pleckstrin homology domain include a variety of kinases, phospholipase-C isoforms, guanine nucleotide release factors, and GTPase activating proteins. For example, members of the FGD1 family contain both Rho-guanine nucleotide exchange factor (GEF) and PH domains, as well as a FYVE zinc finger domain.
- GEF Rho-guanine nucleotide exchange factor
- FGD1 is the gene responsible for faciogenital dysplasia, an inherited skeletal dysplasia (Pasteris, N.G. and J.L. Gorski (1999) Genomics 60:57-66). Many PH domain proteins function in association with the plasma membrane, and this association appears to be mediated by the PH domain itself. PH domains share a common structure composed of two antiparallel beta sheets flanked by an amphipathic alpha helix. Variable loops connecting the component beta strands generally occur within a positively charged environment and may function as ligand binding sites (Lemmon, M.A. et al. (1996) Cell 85:621-624).
- Ankyrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular signaling functions.
- ANK repeats are found in proteins involved in cell proliferation such as kinases, kinase inhibitors, tumor suppressors, and cell cycle control proteins.
- kinases proteins involved in cell proliferation
- kinases proteins involved in cell proliferation
- kinases proteins involved in cell proliferation
- kinases proteins involved in cell proliferation
- kinases proteins involved in cell proliferation
- kinases proteins involved in cell proliferation
- kinase inhibitors include tumor suppressors, and cell cycle control proteins.
- cell cycle control proteins See, for example, Kalus, W. et al. (1997) FEBS Lett. 401:127-132; Ferrante, A.W. et al. (1995) Proc. Natl. Acad. Sci. USA 92:1911-1915.
- Myotrophin is an ANK repeat protein that plays a key role in the development of cardiac hypertrophy, a
- TPR tetratricopeptide repeat
- CDC16, CDC23, and CDC27 members of the anaphase promoting complex which targets proteins for degradation at the onset of anaphase.
- Other processes involving TPR proteins include cell cycle control, transcription repression, stress response, and protein kinase inhibition (Lamb, J.R. et al. (1995) Trends Biochem. Sci. 20:257-259).
- the armadillo/beta-catenin repeat is a 42 amino acid motif which forms a superhelix of alpha helices when tandemly repeated.
- the structure of the armadillo repeat region from beta-catenin revealed a shallow groove of positive charge on one face of the superhelix, which is a potential binding surface.
- the armadillo repeats of beta-catenin, plakoglobin, and pl20 cas bind the cytoplasmic domains of cadherins.
- Beta-catenin cadherin complexes are targets of regulatory signals that govern cell adhesion and mobility (Huber, A.H. et al. (1997) Cell 90:871-882).
- G-beta beta-transducin
- alpha, beta, and gamma beta-transducin
- G proteins guanine nucleotide-binding proteins
- betaTRCP is a component of the ubiquitin ligase complex, which recruits specific proteins, including beta-catenin, to the ubiquitin- proteasome degradation pathway.
- BetaTRCP and its isoforms all contain seven WD repeats, as well as a characteristic "F-box" motif.
- the invention features purified polypeptides, intracellular signaling molecules, referred to collectively as “INTSIG” and individually as “INTSIG-1,” 'TNTSIG-2,” “INTSIG-3,” “INTSIG-4,” “INTSIG-5,” “INTSIG-6,” “INTSIG-7,” “INTSIG-8,” “INTSIG-9,” “INTSIG-10,” “INTSIG-11,” “J-NTSIG-12,” “INTSIG-13,” “INTSIG-14,” “J-NTSIG-15,” “INTSIG-16,” “J-NTSIG-17,” and “INTSIG-18.”
- the invention 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence
- the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-18.
- the invention further 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ J-D NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18.
- the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-18.
- the polynucleotide is selected from the group consisting of SEQ ID NO: 19-36.
- the invention 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18
- the invention provides a cell transformed with the recombinant polynucleotide.
- the invention provides a transgenic organism comprising the recombinant polynucleotide.
- the invention also 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18.
- the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell 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.
- the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18.
- the invention further 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: 19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 19-36, 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 comprises at least 60 contiguous nucleotides. Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 19-36, 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 method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
- the probe comprises at least 60 contiguous nucleotides.
- the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO: 19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 19-36, 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 method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
- the invention further provides a composition 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and a pharmaceutically acceptable excipient.
- the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1- 18.
- the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition.
- the invention also 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18.
- the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
- the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
- the invention provides a method of treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition.
- the invention 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: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18.
- the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
- the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
- the invention provides a method of treating a disease or condition associated with overexpression of functional INTSIG, comprising administering to a patient in need of such treatment the composition.
- the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D NO: 1-18.
- 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.
- the invention further 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 ED NO: 1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-18.
- 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.
- the invention further 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: 19-36, the method comprising a) exposing a sample comprising the target polynucleotide to 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.
- the invention further 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 ED NO: 19-36, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 19-36, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
- 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: 19-36, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 19-36, 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 comprises a fragment of a polynucleotide sequence 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 for polypeptides 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 sequences of the invention, 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 sequences of the invention, along with selected fragments of the polynucleotide sequences.
- Table 5 shows the representative cDNA library for polynucleotides of the invention.
- 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 the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
- INTSIG refers to the amino acid sequences of substantially purified INTSIG obtained from any species, particularly a mammalian 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 ENTSIG.
- Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of INTSIG either by directly interacting with ENTSIG or by acting on components of the biological pathway in which INTSIG participates.
- An "allelic variant” is an alternative form of the gene encoding INTSIG. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered rnRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
- altered nucleic acid sequences encoding ENTSIG include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as INTSIG or a polypeptide with at least one functional characteristic of INTSIG. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding INTSIG, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding INTSIG.
- the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent INTSIG.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of INTSIG 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 similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
- Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
- amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
- Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
- PCR polymerase chain reaction
- Antagonist refers to a molecule which inhibits or attenuates the biological activity of INTSIG.
- Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of INTSIG either by directly interacting with ENTSIG or by acting on components of the biological pathway in which INTSIG participates.
- antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
- Antibodies that bind INTSIG polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
- the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
- an animal e.g., a mouse, a rat, or a rabbit
- RNA e.g., a mouse, a rat, or a rabbit
- antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
- 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. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
- Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
- the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
- Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
- Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
- introduction refers to an aptamer which is expressed in vivo.
- a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
- spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
- antisense refers to any composition capable of base-pairing with the "sense"
- Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
- Antisense molecules may be produced by any method including chemical synthesis or transcription.
- the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell 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.
- biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
- immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic INTSIG, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells 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 sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
- the composition may comprise a dry formulation or an aqueous solution.
- Compositions comprising polynucleotide sequences encoding INTSIG or fragments of INTSIG 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, hi hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
- salts e.g., NaCl
- detergents e.g., sodium dodecyl sulfate; SDS
- other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
- Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG, Madison WE) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
- 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 especially 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 generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the 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 chemically 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 allowing acceleration of the evolution of new protein functions.
- a "fragment” is a unique portion of INTSIG or the polynucleotide encoding ENTSIG which is identical in sequence to but shorter in length than the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
- a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be 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 preferentially selected from certain regions of a molecule.
- a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
- these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
- a fragment of SEQ ED NO: 19-36 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ED NO: 19-36, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
- a fragment of SEQ ID NO: 19-36 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ED NO: 19-36 from related polynucleotide sequences.
- the precise length of a fragment of SEQ ID NO: 19-36 and the region of SEQ ID NO: 19-36 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: 1-18 is encoded by a fragment of SEQ ID NO: 19-36.
- a fragment of SEQ ED NO: 1-18 comprises a region of unique amino acid sequence that specifically identifies SEQ ED NO: 1-18.
- a fragment of SEQ ID NO: 1-18 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ED NO: 1-18.
- the precise length of a fragment of SEQ ED NO: 1-18 and the region of SEQ ID NO: 1-18 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 “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
- a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
- “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
- percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
- NCBI National Center for Biotechnology Information
- BLAST Basic Local Alignment Search Tool
- NCBI National Center for Biotechnology Information
- BLAST Basic Local Alignment Search Tool
- the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
- BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences” tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
- Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED 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 similar 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 all encode substantially the same protein.
- percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
- Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and_hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
- NCBI BLAST software suite may be used.
- BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
- Such default parameters may be, for example: Matrix: BLOSUM62
- Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ED 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.
- H-ACs Human artificial chromosomes
- H-ACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for 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 still retains its original binding ability.
- 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 allowing 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 skill 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.
- wash temperatures are typically 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
- RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
- Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
- a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
- a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
- insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
- 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 cellular and systemic defense systems.
- an “immunogenic fragment” is a polypeptide or oligopeptide fragment of INTSIG 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 INTSIG 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, or other chemical compounds on a substrate.
- array element refers to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
- modulate refers to a change in the activity of ENTSIG.
- modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of INTSIG.
- 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.
- PNA peptide nucleic acid
- 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 preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
- Post-translational modification of an INTSIG may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of INTSIG.
- Probe refers to nucleic acid sequences encoding ENTSIG, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
- Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
- Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence.
- 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).
- Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
- the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows 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 oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
- the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
- this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
- the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
- a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
- the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion 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 cell.
- such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
- a “regulatory element” refers to a nucleic acid sequence usually 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 radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
- RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all 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 INTSIG, nucleic acids encoding ENTSIG, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
- binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small 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 will reduce the amount of labeled A that binds to the antibody.
- substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally 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 capillaries.
- the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
- a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
- Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
- a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
- the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, 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 et al. (1989), 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 "allelic” (as defined above), "splice,” “species,” or “polymorphic” variant.
- a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
- the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
- Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally 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 to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
- Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
- the invention is based on the discovery of new human intracellular signaling molecules (INTSIG), the polynucleotides encoding INTSIG, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, developmental, vesicle trafficking disorders, and viral infections.
- Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED).
- Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ED NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ED) as shown.
- Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ED) as shown.
- Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database.
- Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for polypeptides of the invention.
- Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog.
- Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
- Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where applicable, all 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 ED) 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 column 5 shows potential glycosylation sites, as determined by the MOTEFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
- Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
- Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
- SEQ ID NO: 1 is 26% identical, from residue R221 to residue A458, to human F-box and WD-repeats protein beta-TRCP isoform B (GenBank ED g28577) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
- the BLAST probability score is 6.1e-ll, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ID NO: 1 also contains an F-box domain and six WD repeats as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO:l is a WD-repeat protein.
- SEQ ED NO:2 is 91% identical, from residue R146 to residue S613, to rat potential ligand-binding protein from olfactory mucosa (GenBank ID g57732) with a BLAST probability score of 2.5e-222.
- SEQ ED NO:2 also appears to be expressed exclusively in nasal tissues.
- SEQ ED NO:4 is 65% identical, from residue Ml to residue K520, to human centaurin beta2 (GenBank ED g4688902) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.5e-240, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ED NO:4 also contains a GTPase activating protein for Arf domain, as well as a PH domain and ankyrin repeats as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO:4 is a centaurin beta family ArfGAP.
- SEQ ED NO:6 is 70% identical, from residue M22 to residue S638, to mouse purine nucleotide binding protein (GenBank ID gl 174187), 64% identical from residue M22 to K627, to mouse guanylate binding protein (GenBank ED gl93444), and 55% identical from residue S18 to K60I, to human guanylate binding protein 1, interferon-inducible (GenBank ED gl2803663) as determined by the Basic Local Alignment Search Tool (BLAST).
- BLAST Basic Local Alignment Search Tool
- SEQ ED NO:6 also contains a guanylate-binding protein domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ED NOJ is 76% identical, from residue Ml to residue Q386, to Mus musculus HS1 binding protein 3 (GenBank ID g4160304) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.2e-148, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NOJ also contains a PX domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) These data provide corroborative evidence that SEQ ED NOJ is an HS1 binding protein.
- HMM hidden Markov model
- SEQ ED NO:9 is 36% identical, from residue PI 110 to residue L1437, to rat kalirin-9a (GenBank ED g7650388), a neuronal Dbl family member, as determined by the Basic Local
- BLAST Alignment Search Tool
- HMM hidden Markov model
- SEQ ED NO: 11 is 99% identical, from residue M649 to residue S1725, to the human T-cell lymphoma invasion and metastasis 2 polypeptide (GenBank ID g6224676) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ID NO: 11 also contains a PDZ domain, a RhoGEF domain, and two PH domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO: 11 is a member of the Dbl family of guanosine nucleotide exchange factors.
- SEQ ED NO: 14 is 95% identical, from residue Ml to residue L979, to rat PSD-95/SAP90-associated protein-3, a membrane-associated guanylate kinase (GenBank ED gl864091) as determined by the Basic Local Alignment Search Tool (BLAST).
- BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ED NO: 14 is a guanylate kinase.
- SEQ ED NO: 15 is 56% identical, from residue Ml to residue Q162, to rat ras-related protein (GenBank ID g498257) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.8e-46, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ ED NO: 15 also contains a ras family domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- SEQ ID NO: 15 is a ras- related protein.
- SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:8, SEQ ED NO: 10, SEQ ED NO: 12, SEQ ED NO: 13, and SEQ ED NO: 16-18 were analyzed and annotated in a similar manner.
- the algorithms and parameters for the analysis of SEQ ID NO: 1-18 are described in Table 7.
- the full length polynucleotide sequences of the present invention 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 ED) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
- 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 sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO: 19-36 or that distinguish between SEQ ED NO: 19-36 and related polynucleotide sequences.
- the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries 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 polynucleotide sequences.
- the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (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 FL_XXXXX_Nj_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 applied, and YYYYY is the number of the prediction generated by the algorithm, and N 12 ⁇ 3, stamp . if present, represent specific exons that may have been manually edited during analysis (See Example N).
- the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
- a polynucleotide sequence identified as FLXXXXX_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-stietcliing" algorithm was applied, 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).
- NM nucleophilicity parameter sequence
- NP nucleophilicity parameter sequence
- 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 IN and Example V).
- Incyte cD ⁇ A coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cD ⁇ A identification numbers are not shown.
- Table 5 shows the representative cD ⁇ A libraries for those full length polynucleotide sequences which were assembled using Incyte cD ⁇ A sequences.
- the representative cD ⁇ A library is the Incyte cD ⁇ A library which is most frequently represented by the Incyte cD ⁇ A sequences which were used to assemble and confirm the above polynucleotide sequences.
- the tissues and vectors which were used to construct the cD ⁇ A libraries shown in Table 5 are described in Table 6.
- the invention also encompasses ENTSIG variants.
- a preferred INTSIG variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the INTSIG amino acid sequence, and which contains at least one functional or structural characteristic of INTSIG.
- the invention also encompasses polynucleotides which encode INTSIG. hi a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO: 19-36, which encodes INTSIG.
- polynucleotide sequences of SEQ ID NO: 19-36 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
- the invention also encompasses a variant of a polynucleotide sequence encoding ENTSIG.
- a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding INTSIG.
- a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 19-36 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: 19-36.
- Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of INTSIG.
- a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding INTSIG.
- a splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding INTSIG, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
- a splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding INTSIG over its entire length; however, portions of the splice variant will 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 sequence encoding INTSIG. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of ENTSIG.
- nucleotide sequences which encode INTSIG and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring ENTSIG under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding ENTSIG or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
- the invention also encompasses production of DNA sequences which encode ENTSIG and INTSIG derivatives, or fragments thereof, entirely by synthetic chemistry.
- the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
- synthetic chemistry may be used to introduce mutations into a sequence encoding INTSIG or any fragment thereof.
- polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO: 19-36 and fragments thereof under various conditions of stringency.
- Hybridization conditions including annealing and wash conditions, are described in "Definitions.”
- Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
- the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
- sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology. John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
- the nucleic acid sequences encoding INTSIG 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 amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
- Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
- the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
- a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
- capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
- multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
- Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
- primers may be designed using commercially available 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.
- Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
- Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
- capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
- Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
- Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
- polynucleotide sequences or fragments thereof which encode INTSIG may be cloned in recombinant DNA molecules that direct expression of ENTSIG, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express INTSIG.
- nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter ENTSIG-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
- oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
- 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 INTSIG, such as its biological or enzymatic activity or its ability 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 may be 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.
- sequences encoding ENTSIG may be synthesized, in whole or in part, using chemical methods well known in the art.
- chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
- INTSIG itself or a fragment thereof may be synthesized using chemical methods.
- peptide synthesis can be performed using various solution-phase or solid-phase techniques.
- Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of INTSIG, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
- the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
- the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
- the nucleotide sequences encoding INTSIG or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
- these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding ENTSIG. Such elements may vary in their strength and specificity.
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding ENTSIG. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding ENTSIG. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
- yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., baculovirus)
- plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV, or
- Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
- the invention is not limited by the host cell employed.
- cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding ENTSIG.
- routine cloning, subcloning, and propagation of polynucleotide sequences encoding INTSIG can be achieved using a multifunctional E. coli vector such as PBLUESCREPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding INTSIG into the vector's multiple cloning site disrupts the lacL gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
- 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.
- vectors which direct high level expression of INTSIG 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 ENTSIG.
- 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. Iffaddition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
- Plant systems may also be used for expression of INTSIG. Transcription of sequences encoding ENTSIG 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 from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
- viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1311).
- plant promoters such
- constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
- pathogen-mediated transfection See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.
- a number of viral-based expression systems may be utilized.
- sequences encoding ENTSIG may be ligated 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 INTSIG in host cells.
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- SV40 or EBV- based vectors may also be used for high-level protein expression.
- 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 delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
- sequences encoding ENTSIG can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
- the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
- selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ⁇ and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
- dhfr 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.
- Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
- Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase 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. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)
- marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- sequence encoding ENTSIG is inserted within a marker gene sequence
- transformed cells containing - sequences encoding ENTSIG can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding INTSIG under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression, of the tandem gene as well.
- host cells that contain the nucleic acid sequence encoding ENTSIG and that express INTSIG may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of ENTSIG using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on INTSIG is preferred, but a competitive binding assay may be employed.
- assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. EV; Coligan, J.E. et al. (1997) Current Protocols in Immunology. Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) J-mmunochemical Protocols, Humana Press, Totowa NJ.)
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ENTSIG include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- the sequences encoding ENTSIG, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with nucleotide sequences encoding ENTSIG may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode INTSIG may be designed to contain signal sequences which direct secretion of ENTSIG through a prokaryotic or eukaryotic cell membrane.
- a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
- 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 cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
- ATCC American Type Culture Collection
- natural, modified, or recombinant nucleic acid sequences encoding INTSIG may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
- a chimeric INTSIG protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of ENTSIG activity.
- Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
- Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, 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 commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
- a fusion protein may also be engineered to contain a proteolytic cleavage site located between the INTSIG encoding sequence and the heterologous protein sequence, so that ENTSIG may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
- synthesis of radiolabeled INTSIG may be 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.
- INTSIG of the present invention or fragments thereof may be used to screen for compounds that specifically bind to INTSIG.
- At least one and up to a plurality of test compounds may be screened for specific binding to ENTSIG.
- test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
- the compound thus identified is closely related to the natural ligand of INTSIG, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
- the compound can be closely related to the natural receptor to which INTSIG binds, or to at least a fragment of the receptor, e.g., the ligand binding site.
- the compound can be rationally designed using known techniques.
- screening for these compounds involves producing appropriate cells which express ENTSIG, either as a secreted protein or on the cell membrane.
- Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing INTSIG or cell membrane fractions which contain ENTSIG are then contacted with a test compound and binding, stimulation, or inhibition of activity of either ENTSIG 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 INTSIG, either in solution or affixed to a solid support, and detecting the binding of INTSIG 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.
- INTSIG of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of INTSIG.
- Such compounds may include agonists, antagonists, or partial or inverse agonists.
- an assay is performed under conditions permissive for INTSIG activity, wherein INTSIG is combined with at least one test compound, and the activity of INTSIG in the presence of a test compound is compared with the activity of INTSIG in the absence of the test compound. A change in the activity of INTSIG in the presence of the test compound is indicative of a compound that modulates the activity of INTSIG.
- a test compound is combined with an in vitro or cell-free system comprising INTSIG under conditions suitable for INTSIG activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of ENTSIG 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 INTSIG or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
- ES embryonic stem
- Such techniques are well known in the art and are useful for the generation of animal models of human disease.
- mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
- the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
- a marker gene e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
- the vector integrates into the 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 (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
- Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
- the blastocysts are surgically transferred to pseudopregnant da s, 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 INTSIG may also be manipulated in vitro in ES cells derived from human blastocysts.
- Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
- Polynucleotides encoding ENTSIG can also be used to create "knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
- knockin technology a region of a polynucleotide encoding INTSIG is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
- Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
- Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
- a mammal inbred to overexpress INTSIG e.g., by secreting INTSIG in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-
- INTSIG appears to play a role in cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, developmental, vesicle trafficking disorders, and viral infections.
- INTSIG In the treatment of disorders associated with increased INTSIG expression or activity, it is desirable to decrease the expression or activity of INTSIG.
- INTSIG In the treatment of disorders associated with decreased INTSIG expression or activity, it is desirable to increase the expression or activity of INTSIG.
- ENTSIG 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 INTSIG.
- disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,
- compositions comprising a substantially purified INTSIG in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of INTSIG including, but not limited to, those provided above.
- an agonist which modulates the activity of ENTSIG may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ENTSIG including, but not limited to, those listed above.
- an antagonist of INTSIG may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of INTSIG. Examples of such disorders include, but are not limited to, those cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, developmental, vesicle trafficking disorders, and viral infections described above.
- an antibody which specifically binds INTSIG may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express INTSIG.
- a vector expressing the complement of the polynucleotide encoding INTSIG may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ENTSIG including, but not limited to, those described above.
- any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
- the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
- An antagonist of ENTSIG may be produced using methods which are generally known in the art.
- purified INTSIG may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind INTSIG.
- Antibodies to INTSIG may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
- Single chain antibodies may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
- various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with INTSIG or with any fragment or oligopeptide 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, oil emulsions, KLH, and dinitrophenol.
- oligopeptides, peptides, or fragments used to induce antibodies to ENTSIG have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of INTSIG 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 ENTSIG may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
- chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
- techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce ENTSIG-specific single chain antibodies.
- Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
- Antibody fragments which contain specific binding sites for INTSIG may also be generated.
- 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 libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
- immunoassays may be used for screening to identify antibodies having the desired specificity.
- Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
- Such immunoassays typically involve the measurement of complex formation between INTSIG and its specific antibody.
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering INTSIG epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
- K a is defined as the molar concentration of ENTSIG-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 INTSIG epitope, represents a true measure of affinity.
- High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the ENTSIG-antibody complex must withstand rigorous manipulations.
- Low-affinity antibody preparations with K a ranging from about 10 s to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of ENTSIG, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, E L Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & 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 generally employed in procedures requiring precipitation of ENTSIG-antibody complexes.
- Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
- the polynucleotides encoding INTSIG 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 oligonucleotides) to the coding or regulatory regions of the gene encoding INTSIG.
- complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
- antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding ENTSIG. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., 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.
- Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
- polynucleotides encoding INTSIG may be used for somatic or germline gene therapy.
- Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCED)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine-deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
- SCED 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 Plasmodium falciparum and Trvpanosoma cruzi.
- the expression of INTSIG from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
- INTSIG are treated by constructing mammalian expression vectors encoding INTSIG and introducing these vectors by mechanical means into ENTSIG-deficient cells.
- Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-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) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
- Expression vectors that may be effective for the expression of ENTSIG include, but are not limited to, the PCDNA 3.1, EPEEAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMN-SCRIPT, PCMN-TAG, PEGSH/PERN (Stratagene, La Jolla CA), and PTET-OFF, PTET-O ⁇ , PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
- E TSIG may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268: 1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol.
- a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin
- liposome transformation kits e.g., the PERFECT LEPED TRANSFECTION KIT, available from Invitrogen
- PERFECT LEPED TRANSFECTION KIT available from Invitrogen
- transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
- the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
- diseases or disorders caused by genetic defects with respect to INTSIG expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding INTSIG under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus czs-acting RNA sequences and coding sequences required for efficient vector propagation.
- Retrovirus vectors e.g., PFB and PFBNEO
- PFB and PFBNEO are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
- the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J.
- VPCL vector producing cell line
- U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
- an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding INTSIG to cells which have one or more genetic abnormalities with respect to the expression of INTSIG.
- the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art.
- Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268).
- Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
- Adenovirus vectors for gene therapy For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, EM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
- a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding INTSIG to target cells which have one or more genetic abnormalities with respect to the expression of INTSIG.
- the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing INTSIG to cells of the central nervous system, for which HSV has a tropism.
- the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art.
- a replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
- HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
- U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
- HSV vectors see also Goins, W.F. et al. (1999) J. Virol.
- herpesvirus sequences The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
- an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding INTSIG to target cells.
- SFV Semliki Forest Virus
- This subgenomic RNA replicates to higher levels than the full 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 INTSIG into the alphavirus genome in place of the capsid-coding region results in the production of a large number of ENTSIG-coding RNAs and the synthesis of high levels of INTSIG in vector transduced cells.
- alphavirus infection is typically associated with cell lysis within a few days
- the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SEN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
- the wide host range of alphaviruses will allow the introduction of INTSIG into a variety of cell types.
- the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
- the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
- Oligonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Lmmunologic Approaches. Futura Publishing, Mt. Kisco NY, pp.
- a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
- RNA The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
- engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding ENTSIG.
- RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
- the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
- RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
- RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding ENTSIG Such 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 cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half -life.
- flanking sequences at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
- This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
- An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding ENTSIG.
- Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, 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 INTSIG may be therapeutically useful, and in the treatment of disorders associated with decreased INTSIG expression or activity, a compound which specifically promotes expression of the polynucleotide encoding INTSIG may be therapeutically useful.
- At least one, and up to a plurality, of 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 naturally-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 INTSIG 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 INTSIG 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 INTSIG.
- 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. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
- 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 cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13).
- 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 cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Bio
- a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (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). Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
- oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
- vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
- any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
- An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
- Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
- Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
- Such compositions may consist of ENTSIG, antibodies to INTSIG, and mimetics, agonists, antagonists, or inhibitors of ENTSIG.
- compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, 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.
- compositions are generally aerosolized immediately prior to inhalation by the patient, hi the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast- acting formulations is well-known in the art.
- macromolecules e.g. larger peptides and proteins
- Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
- the determination of an effective dose is well within the capability of those skilled in the art.
- compositions may be prepared for direct intracellular delivery of macromolecules comprising INTSIG or fragments thereof.
- liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
- INTSIG or a fragment thereof may be joined to a short cationic N- terminal portion from the HEV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
- the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, 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 therapeutically effective dose refers to that amount of active ingredient, for example ENTSIG or fragments thereof, antibodies of ENTSIG, and agonists, antagonists or inhibitors of INTSIG, which ameliorates the symptoms or condition.
- Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (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 preferred.
- the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
- the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the 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. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
- Normal dosage amounts may vary from about 0.1 ⁇ 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 delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
- antibodies which specifically bind ENTSIG may be used for the diagnosis of disorders characterized by expression of INTSIG, or in assays to monitor patients being treated with INTSIG or agonists, antagonists, or inhibitors of INTSIG.
- Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for INTSIG include methods which utilize the antibody and a label to detect INTSIG in human body fluids or in extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
- a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
- ENTSIG A variety of protocols for measuring ENTSIG, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of ENTSIG expression.
- Normal or standard values for ENTSIG expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to ENTSIG under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of INTSIG expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
- the polynucleotides encoding INTSIG may be used for diagnostic purposes.
- the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
- the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of INTSIG may be correlated with disease.
- the diagnostic assay may be used to determine absence, presence, and excess expression of INTSIG, and to monitor regulation of INTSIG levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding INTSIG or closely related molecules may be used to identify nucleic acid sequences which encode ENTSIG.
- the specificity of the probe whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding ENTSIG, allelic 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 INTSIG encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO: 19-36 or from genomic sequences including promoters, enhancers, and introns of the INTSIG gene.
- Means for producing specific hybridization probes for DNAs encoding ENTSIG include the cloning of polynucleotide sequences encoding INTSIG or ENTSIG derivatives into vectors for the production of mRNA probes.
- Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 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.
- Polynucleotide sequences encoding ENTSIG may be used for the diagnosis of disorders associated with expression of ENTSIG.
- disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pan
- polynucleotide sequences encoding ENTSIG may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered INTSIG expression. Such qualitative or quantitative methods are well known in the art.
- the nucleotide sequences encoding ENTSIG may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
- the nucleotide sequences encoding INTSIG 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 nucleotide sequences encoding ENTSIG 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 established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding ENTSIG, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
- hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
- the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
- the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
- a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
- oligonucleotides designed from the sequences encoding INTSIG may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding ENTSIG, or a fragment of a polynucleotide complementary to the polynucleotide encoding INTSIG, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
- oligonucleotide primers derived from the polynucleotide sequences encoding ENTSIG 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
- oligonucleotide primers derived from the polynucleotide sequences encoding ENTSIG are used to amplify DNA using the polymerase chain reaction (PCR).
- the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
- SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
- the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high- throughput equipment such as DNA sequencing machines.
- sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the 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 may be 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 cell anemia, or chronic granulomatous disease.
- 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 drug, such as life-threatening toxicity.
- a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while 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.
- Methods which may also be used to quantify the expression of INTSIG include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
- radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
- 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 colorimetric response gives rapid quantitation.
- oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
- the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
- the microarray 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.
- therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
- ENTSIG, fragments of INTSIG, or antibodies specific for INTSIG may be used as elements on a microarray.
- the microarray 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 cell type.
- a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, expressly incorporated by reference herein.)
- a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.
- the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
- the resultant transcript image would provide a profile of gene activity.
- Transcript images may be generated using transcripts isolated from tissues, cell 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 cell 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 well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
- the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
- Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding 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.
- Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type.
- proteome refers to the global pattern of protein expression in a particular tissue or cell type.
- proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
- a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
- the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
- the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
- the optical density of each protein spot is generally proportional to the level of the protein in the sample.
- the optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment.
- the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
- the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
- a proteomic profile may also be generated using antibodies specific for INTSIG to quantify the levels of INTSIG expression.
- the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 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 array element.
- Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level.
- There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be 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 corresponding 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.
- methods known in the art See, e.g., 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; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150- 2155; and Heller, M.J. et al.
- nucleic acid sequences encoding INTSIG may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
- sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
- HACs human artificial chromosomes
- YACs yeast artificial chromosomes
- BACs bacterial artificial chromosomes
- PI constructions or single chromosome cDNA libraries.
- nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
- RFLP restriction fragment length polymorphism
- Fluorescent in situ hybridization may be correlated with other physical and genetic map data.
- FISH Fluorescent in situ hybridization
- Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMEM) World Wide Web site. Correlation between the location of the gene encoding INTSIG 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.
- In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to llq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
- ENTSIG its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
- the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between INTSIG 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.
- This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with ENTSIG, or fragments thereof, and washed. Bound INTSIG is then detected by methods well known in the art. Purified INTSIG can also be coated directly onto plates for use in the aforementioned drug screening techniques.
- non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
- antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with INTSIG.
- nucleotide sequences which encode INTSIG may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD database
- Reverse transcription was initiated using oligo d(T) or random primers.
- Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
- the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (A ersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
- cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCREPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
- Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. ⁇ . Isolation of cDNA Clones
- Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
- a Magic or WIZARD Minipreps DNA purification system Promega
- an AGTC Miniprep purification kit Edge Biosystems, Gaithersburg MD
- plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN Et fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
- Incyte cDNA recovered in plasmids as described in Example ⁇ were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
- Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VEH.
- the polynucleotide sequences derived from Incyte cDNAs were 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 were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens.
- Rattus norvegicus Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
- HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R.
- the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
- the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
- GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
- full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences.
- a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide.
- Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, bidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART.
- MACDNASIS PRO Hitachi Software Engineering, South San Francisco CA
- LASERGENE software DNASTAR
- Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
- Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable 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, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
- Putative intracellular signaling molecules were initially identified by running the Genscan gene identification program against public 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 (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. 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.
- the output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb.
- Genscan predicted cDNA sequences encode intracellular signaling molecules.
- Potential intracellular signaling molecules were also identified by homology to Incyte cDNA sequences that had been annotated as intracellular signaling molecules.
- Genscan- predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
- BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
- Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example EH. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
- Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example EV. Partial cDNAs assembled as described in Example EQ were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
- Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis.
- GenBank primate such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases
- the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IN.
- a chimeric protein was generated by using the resultant high-scoring segment pairs (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 pairs
- GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of INTSIG Encoding Polynucleotides
- sequences which were used to assemble SEQ ID NO: 19-36 were compared with sequences from the Incyte LEFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ED NO: 19-36 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped.
- SHGC Stanford Human Genome Center
- WIGR Whitehead Institute for Genome Research
- Map locations are represented by ranges, or intervals, of human chromosomes.
- the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
- centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers.
- cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
- the cM distances are based on genetic markers mapped by Genethon 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 (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above. In this manner, SEQ ED NO:23 was mapped to chromosome 16 within the interval from
- Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
- the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
- the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
- the product score represents a balance between fractional overlap and quality in a BLAST alignment.
- a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared.
- a product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other.
- a product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
- polynucleotide sequences encoding INTSIG are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example EQ). Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
- Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
- the number of libraries in each category is counted and divided by the total number of libraries across all categories.
- each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding INTSIG.
- One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment.
- the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
- Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.).
- the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C
- the parameters for primer pair T7 and SK+ were as follows: Step 1: 94 °C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C
- the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
- the plate was scanned in a Fluoroskan H (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
- a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
- the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
- CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
- sonicated or sheared prior to religation into pUC 18 vector
- the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
- Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
- the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
- a final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
- Certain SNPs were 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 comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
- the African population comprised 194 individuals (97 male, 97 female), all African Americans.
- the Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic.
- the Asian population comprised 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 were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes
- Hybridization probes derived from SEQ ID NO: 19-36 are employed to screen cDNAs, genomic DNAs, or rnRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
- the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot 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 following endonucleases: Ase I, Bgl ⁇ , Eco RI, Pst I, Xba I, or Pvu El (DuPont NEN).
- the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially 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 visualized using autoradiography or an alternative imaging means and compared. XI. Microarrays
- the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
- the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
- a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements.
- Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well 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 array element.
- laser desorbtion and mass spectrometry may be used for detection of hybridization.
- the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
- microarray preparation and usage is described in detail 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 Pharmacia Biotech).
- the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMB RIGHT 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 SPEN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
- Sequences of the present invention are used to generate array elements.
- Each array 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 array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
- Purified array elements are immobilized on polymer-coated glass slides.
- Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
- Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110 °C oven.
- Array elements are applied to the coated glass substrate using a procedure described in U.S.
- Patent No. 5,807,522 incorporated herein by reference.
- 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus.
- the apparatus then deposits about 5 nl of array element sample per slide.
- Microarrays are UV-crosslinked using a STRATALENKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate 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
- 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 array using a 20X microscope objective (Nikon, Inc., Melville NY).
- the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
- the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
- a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array 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 array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
- the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
- a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated 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 corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore' s emission spectrum.
- SEQ ID NO:28 showed differential expression in non-malignant mammary epithelial cells versus various breast carcinoma lines as determined by microarray analysis. The expression of SEQ ID NO:28 was decreased by at least two fold in the breast carcinoma lines relative to non-malignant mammary epithelial cells. Therefore, SEQ ED NO: 28 is useful in diagnostic assays for detection of breast cancer.
- SEQ ED NO:28 showed differential expression in primary prostate epithelial cells versus various prostate carcinoma lines as determined by microarray analysis. The expression of SEQ ID NO:28 was decreased by at least two fold in the prostate carcinoma lines relative to primary prostate epithelial cells. Therefore, SEQ ED NO:28 is useful in diagnostic assays for detection of prostate cancer.
- Sequences complementary to the ENTSIG-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring INTSIG.
- oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
- Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of INTSIG.
- a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
- To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the J-NTSIG-encoding transcript.
- INTSIG 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 limited to, the trp-lac ⁇ tad) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
- Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
- Antibiotic resistant bacteria express INTSIG upon induction with isopropyl beta-D- thiogalactopyranoside ( PTG).
- ENTSIG in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
- AcMNPV Autographica californica nuclear polyhedrosis virus
- the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding INTSIG by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
- Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
- INTSIG is synthesized as a fusion protein with, e.g., glutathione
- GST S-transferase
- FLAG a peptide epitope tag
- GST a 26- kilodalton enzyme from Schistosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
- the GST moiety can be proteolytically cleaved from INTSIG at specifically 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 (1995, supra, ch. 10 and 16). Purified NTSIG obtained by these methods can be used directly in the assays shown in Examples XVII and XVEfl where applicable.
- INTSIG function is assessed by expressing the sequences encoding ENTSIG at physiologically elevated levels in mammalian cell culture systems.
- cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
- Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
- 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 cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
- Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
- FCM Flow cytometry
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
- the influence of INTSIG on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding ENTSIG and either CD64 or CD64-GFP.
- CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
- Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
- mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding ENTSIG and other genes of interest can be analyzed by northern analysis or microarray techniques.
- the INTSIG amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
- oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
- ABI 431 A peptide synthesizer Applied Biosystems
- KLH Sigma- Aldrich, St. Louis MO
- MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
- Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
- Resulting antisera are tested for antipeptide and anti-ENTSIG activity by, for example, binding the peptide or INTSIG to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
- XVI. Purification of Naturally Occurring INTSIG Using Specific Antibodies
- Naturally occurring or recombinant ENTSIG is substantially purified by immunoaffmity chromatography using antibodies specific for INTSIG.
- An immunoaffinity column is constructed by covalently coupling anti-ENTSIG antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
- the resin is blocked and washed according to the manufacturer's instructions.
- Media containing INTSIG are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of INTSIG (e.g., high ionic strength buffers in the presence of detergent).
- the column is eluted under conditions that disrupt antibody/ENTSIG 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 INTSIG is collected.
- a buffer of pH 2 to pH 3 or a high concentration of a chaotrope, such as urea or thiocyanate ion
- ENTSIG or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
- Bolton-Hunter reagent See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
- Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled INTSIG, washed, and any wells with labeled INTSIG complex are assayed. Data obtained using different concentrations of INTSIG are used to calculate values for the number, affinity, and association of ENTSIG with the candidate molecules.
- ENTSIG may also be used in the PATHCALLENG process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101). XVIII.
- INTSIG activity is associated with its ability to form protein-protein complexes and is measured by its ability to regulate growth characteristics of NEH3T3 mouse fibroblast cells.
- a cDNA encoding ENTSIG is subcloned into an appropriate eukaryotic expression vector. This vector is transfected into NEH3T3 cells using methods known in the art. Transfected cells are compared with non-transfected cells for the following quantifiable properties: growth in culture to high density, reduced attachment of cells to the substrate, altered cell morphology, and ability to induce tumors when injected into immunodeficient mice.
- the activity of INTSIG is proportional to the extent of increased growth or frequency of altered cell morphology in NIH3T3 cells transfected with ENTSIG.
- INTSIG activity is measured by binding of ENTSIG to radiolabeled formin polypeptides containing the proline-rich region that specifically binds to SH3 containing proteins (Chan, D.C. et al. (1996) EMBO J. 15: 1045-1054).
- Samples of INTSIG are run on SDS-PAGE gels, and transferred onto nitrocellulose by electroblotting. The blots are blocked for 1 hr at room temperature in TBST (137 mM NaCl, 2.7 mM KC1, 25 mM Tris (pH 8.0) and 0.1% Tween-20) containing non-fat dry milk.
- Blots are then incubated with TBST containing the radioactive formin polypeptide for 4 hrs to overnight. After washing the blots four times with TBST, the blots are exposed to autoradiographic film. Radioactivity is quantitated by cutting out the radioactive spots and counting them in a radioisotope counter. The amount of radioactivity recovered is proportional to the activity of INTSIG in the assay.
- INTSIG protein kinase activity is measured by quantifying the phosphorylation of an appropriate substrate in the presence of gamma-labeled 32 P-ATP.
- INTSIG is incubated with the substrate, 32 P-ATP, and an appropriate kinase buffer.
- the 32 P incorporated into the product is separated from free 32 P-ATP by electrophoresis, and the incorporated 3 P is quantified using a beta radioisotope counter.
- the amount of incorporated 32 P is proportional to the protein kinase activity of INTSIG in the assay.
- a determination of the specific amino acid residue phosphorylated by protein kinase activity is made by phosphoamino acid analysis of the hydrolyzed protein.
- an assay for INTSIG protein phosphatase activity measures the hydrolysis of para-nitrophenyl phosphate (PNPP).
- ENTSIG is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1% ⁇ -mercaptoethanol at 37°C for 60 min.
- the reaction is stopped by the addition of 6 ml of 10 N NaOH, and the increase in light absorbance of the reaction mixture at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer.
- the increase in light absorbance is proportional to the activity of INTSIG in the assay (Diamond, R.H. et al. (1994) Mol. Cell Biol. 14:3752-3762).
- adenylyl cylcase activity of INTSIG is demonstrated by the ability to convert ATP to cAMP (Mittal, C.K. (1986) Meth. Enzymol. 132:422-428).
- INTSIG is incubated with the substrate [ ⁇ - 32 P] ATP, following which the excess substrate is separated from the product cyclic [ 32 P] AMP.
- INTSIG activity is determined in 12 x 75 mm disposable culture tubes containing 5 ⁇ l of 0.6 M Tris-HCl, pH 7.5, 5 ⁇ l of 0.2 M MgCl 2 , 5 ⁇ l of 150 mM creatine phosphate containing 3 units of creatine phosphokinase, 5 ⁇ l of 4.0 mM l-methyl-3-isobutylxanthine, 5 ⁇ l of 20 mM cAMP, 5 ⁇ l 20 mM dithiothreitol, 5 ⁇ l of 10 mM ATP, 10 ⁇ l [ ⁇ - 32 PjATP (2-4 x 10 s cpm), and water in a total volume of 100 ⁇ l.
- the reaction mixture is prewarmed to 30°C
- the reaction is initiated by adding INTSIG to the prewarmed reaction mixture. After 10-15 minutes of incubation at 30°C, the reaction is terminated by adding 25 ⁇ l of 30% ice-cold trichloroacetic acid (TCA). Zero-time incubations and reactions incubated in the absence of INTSIG are used as negative controls. Products are separated by ion exchange chromatography, and cyclic [ 32 P] AMP is quantified using a ⁇ -radioisotope counter. The ENTSIG activity is proportional to the amount of cyclic [ 32 P] AMP formed in the reaction.
- An alternative assay measures ENTSIG-mediated G-protein signaling activity by monitoring the mobilization of Ca 2+ as an indicator of the signal transduction pathway stimulation.
- the assay requires preloading neutrophils or T cells with a fluorescent dye such as FURA-2 or BCECF (Universal Imaging Corp, Westchester PA) whose emission characteristics are altered by Ca 2+ binding.
- Ca 2+ flux takes place. This flux can be observed and quantified by assaying the cells in a fluorometer or fluorescent activated cell sorter. Measurements of Ca 2+ flux are compared between cells in their normal state and those transfected with INTSIG. Increased Ca 2+ mobilization attributable to increased INTSIG concentration is proportional to INTSIG activity.
- activating stimuli e.g., anti-CD3 antibody ligation of the T cell receptor
- physiologically e.g., by allogeneic stimulation
- GTP-binding activity of INTSIG is determined in an assay that measures the binding of ENTSIG to [ ⁇ - 32 P] -labeled GTP.
- Purified INTSIG is first blotted onto filters and rinsed in a suitable buffer. The filters are then incubated in buffer containing radiolabeled [ ⁇ - 32 P]-GTP. The filters are washed in buffer to remove unbound GTP and counted in a radioisotope counter.
- Nonspecific binding is determined in an assay that contains a 100-fold excess of unlabeled GTP. The amount of specific binding is proportional to the activity of INTSIG.
- GTPase activity of ENTSIG is determined in an assay that measures the conversion of [ ⁇ - 32 P]-GTP to [ ⁇ - 32 P]-GDP.
- ENTSIG is incubated with [ ⁇ - 32 P]-GTP in buffer for an appropriate period of time, and the reaction is terminated by heating or acid precipitation followed by centrifugationi
- An aliquot of the supernatant is subjected to polyacrylamide gel electrophoresis (PAGEj to separate GDP and GTP together with unlabeled standards.
- the GDP spot is cut out and counted in a radioisotope counter.
- the amount of radioactivity recovered in GDP is proportional to the GTPase activity of ENTSIG.
- GTP-binding activity is assayed by incubating varying amounts of INTSIG for 10 minutes at
- GTPase activity of INTSIG is determined by incubating INTSIG at 37 °C in 20 mM Pipes, 20 mM Hepes, 2 mM MgCl 2 , 1 mM EGTA, 1 mM dithiothreitol buffer, pH 7.0, fixed in ionic strength at 42 mM, and containing 0.1% BSA and [ ⁇ - 32 P]GTP at a final concentration of 25 mM in a final reaction volume of 20 ⁇ l. The reaction is initiated by the addition of 0.1 ⁇ Ci of [ ⁇ - 32 P]GTP.
- INTSIG activity is measured by quantifying the amount of a non-hydrolyzable GTP analogue, GTP ⁇ S, bound over a 10 minute incubation period. Varying amounts of INTSIG are incubated at 30°C in 50 mM Tris buffer, pH 7.5, containing 1 mM dithiothreitol, 1 mM EDTA and 1 ⁇ M [ 35 S] GTP ⁇ S.
- Samples are passed through nitrocellulose filters and washed twice with a buffer consisting of 50 mM Tris-HCl, pH 7.8, 1 mM NaN 3 , 10 mM MgCl 2 , 1 mM EDTA, 0.5 mM dithiothreitol, 0.01 mM PMSF, and 200 mM NaCl.
- the filter-bound counts are measured by liquid scintillation to quantify the amount of bound [ 35 S]GTP ⁇ S.
- INTSIG activity may also be measured as the amount of GTP hydrolysed over a 10 minute incubation period at 37 °C.
- INTSIG is incubated in 50mM Tris-HCl buffer, pH 7.8, containing ImM dithiothreitol, 2mM EDTA, lO ⁇ M [ ⁇ - 32 P]GTP, and 1 ⁇ M H-rab protein.
- GTPase activity is initiated by adding MgCl 2 to a final concentration of 10 mM. Samples are removed at various time points, mixed with an equal volume of ice-cold 0.5mM EDTA, and frozen. Aliquots are spotted onto polyethyleneimine-cellulose thin layer chromatography plates, which are developed in 1M LiCl, dried, and autoradiographed. The signal detected is proportional to INTSIG activity.
- INTSIG activity may be demonstrated as the ability to interact with its associated LMW GTPase in an in vitro binding assay.
- the candidate LMW GTPases are expressed as fusion proteins with glutathione S-transferase (GST), and purified by affinity chromatography on glutathione-Sepharose.
- GST glutathione S-transferase
- the LMW GTPases are loaded with GDP by incubating 20 mM Tris buffer, pH 8.0, containing 100 mM NaCl, 2 mM EDTA, 5 mM MgCl 2 , 0.2 mM DTT, 100 ⁇ M AMP-PNP and 1Q ⁇ M GDP at 30 °C for 20 minutes.
- INTSIG is expressed as a FLAG fusion protein in a baculovirus system. Extracts of these baculovirus cells containing ENTSIG-FLAG fusion proteins are precleared with GST beads, then incubated with GST-GTPase fusion proteins. The complexes formed are precipitated by glutathione-Sepharose and separated by SDS-polyacrylamide gel electrophoresis. The separated proteins are blotted onto nitrocellulose membranes and probed with commercially available anti-FLAG antibodies. INTSIG activity is proportional to the amount of ENTSIG-FLAG fusion protein detected in the complex.
- yeast two-hybrid system Zalcman, G. et al. (1996) J. Biol. Chem. 271:30366-30374.
- a plasmid such as pGAD1318 which may contain the coding region of INTSIG can be used to transform reporter L40 yeast cells which contain the reporter genes LacZ and HIS3 downstream from the binding sequences for LexA.
- yeast cells have been previously transformed with a pLexA-Rab6-GDP (mouse) plasmid or with a plasmid which contains pLexA-lamin C.
- the pLEXA-lamin C cells serve as a negative control.
- the transformed cells are plated on a histidine-free medium and incubated at 30°C for 3 days. His + colonies are subsequently patched on selective plates and assayed for ⁇ -galactosidase activity by a filter assay.
- INTSIG binding with Rab6-GDP is indicated by positive His + /lacZ + activity for the cells transformed with the plasmid containing the mouse Rab6-GDP and negative His + /lacZ + activity for those transformed with the plasmid containing lamin C.
- ENTSIG activity is measured by binding of INTSIG to a substrate which recognizes WD-40 repeats, such as ElonginB, by coimmunoprecipitation (Kamura, T. et al. (1998) Genes Dev. 12:3872-3881). Briefly, epitope tagged substrate and INTSIG are mixed and immunoprecipitated with commercial antibody against the substrate tag. The reaction solution is run on SDS-PAGE and the presence of ENTSIG visualized using an antibody to the INTSIG tag. Substrate binding is proportional to INTSIG activity. Alternatively, ENTSIG activity is measured by its inclusion in coated vesicles.
- INTSIG can be expressed by transforming a mammalian cell line such as COS7, HeLa, or CHO with a eukaryotic expression vector encoding ENTSIG.
- Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art.
- a small amount of a second plasmid, which expresses any one of a number of marker genes, such as ⁇ -galactosidase, is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA.
- the cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of INTSIG and ⁇ - galactosidase.
- ENTSIG activity is measured by its ability to alter vesicle trafficking pathways.
- Vesicle trafficking in cells transformed with ENTSIG is examined using fluorescence microscopy. Antibodies specific for vesicle coat proteins or typical vesicle trafficking substrates such as transferrin or the mannose-6-phosphate receptor are commercially available. Various cellular components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and the plasmalemma are examined. Alterations in the numbers and locations of vesicles in cells transformed with INTSIG as compared to control cells are characteristic of INTSIG activity. Transformed cells are collected and cell lysates are assayed for vesicle formation.
- a non-hydrolyzable form of GTP, GTP ⁇ S, and an ATP regenerating system are added to the lysate and the mixture is incubated at 37 °C for 10 minutes. Under these conditions, over 90% of the vesicles remain coated (Orci, L. et al. (1989) Cell 56:357- 368).
- Transport vesicles are salt-released from the Golgi membranes, loaded under a sucrose gradient, centrifuged, and fractions are collected and analyzed by SDS-PAGE. Co-localization of INTSIG with clathrin or COP coatamer is indicative of ENTSIG activity in vesicle formation.
- ENTSIG in vesicle formation
- the contribution of ENTSIG in vesicle formation can be confirmed by incubating lysates with antibodies specific for INTSIG prior to GTP ⁇ S addition.
- the antibody will bind to ENTSIG and interfere with its activity, thus preventing vesicle formation.
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Inventor name: CHAWLA, NARINDER, K. Inventor name: EMERLING, BROOKE, M. Inventor name: FORSYTHE, IAN, J. Inventor name: GRIFFIN, JENNIFER, A. Inventor name: SWARNAKAR, ANITA Inventor name: THANGAVELU, KAVITHA Inventor name: YAO, MONIQUE, G. Inventor name: WANG, YUMEI, E. Inventor name: BAUGHN, MARIAH, R. Inventor name: GURURAJAN, RAJAGOPAL Inventor name: ISON, CRAIG, H. Inventor name: HAFALIA, APRIL, J., A. Inventor name: YANG, JUNMING Inventor name: NGUYEN, DANNIEL, B. Inventor name: LAL, PREETI Inventor name: RICHARDSON, THOMAS, W. Inventor name: LEE, SALLY Inventor name: BURFORD, NEIL Inventor name: YUE, HENRY Inventor name: TANG, Y., TOM Inventor name: ELLIOTT, VICKI, S. Inventor name: WARREN, BRIDGET, A. Inventor name: DING, LI |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: CHAWLA, NARINDER, K. Inventor name: EMERLING, BROOKE, M. Inventor name: FORSYTHE, IAN, J. Inventor name: GRIFFIN, JENNIFER, A. Inventor name: SWARNAKAR, ANITA Inventor name: THANGAVELU, KAVITHA Inventor name: YAO, MONIQUE, G. Inventor name: WANG, YUMEI, E. Inventor name: BAUGHN, MARIAH, R. Inventor name: GURURAJAN, RAJAGOPAL Inventor name: ISON, CRAIG, H. Inventor name: HAFALIA, APRIL, J., A. Inventor name: YANG, JUNMING Inventor name: NGUYEN, DANNIEL, B. Inventor name: LAL, PREETI Inventor name: RICHARDSON, THOMAS, W. Inventor name: LEE, SALLY Inventor name: BURFORD, NEIL Inventor name: YUE, HENRY Inventor name: TANG, Y., TOM Inventor name: ELLIOTT, VICKI, S. Inventor name: WARREN, BRIDGET, A. Inventor name: DING, LI |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: CHAWLA, NARINDER, K. Inventor name: EMERLING, BROOKE, M. Inventor name: FORSYTHE, IAN, J. Inventor name: GRIFFIN, JENNIFER, A. Inventor name: SWARNAKAR, ANITA Inventor name: THANGAVELU, KAVITHA Inventor name: YAO, MONIQUE, G. Inventor name: WANG, YUMEI, E. Inventor name: BAUGHN, MARIAH, R. Inventor name: GURURAJAN, RAJAGOPAL Inventor name: ISON, CRAIG, H. Inventor name: HAFALIA, APRIL, J., A. Inventor name: YANG, JUNMING Inventor name: NGUYEN, DANNIEL, B. Inventor name: LAL, PREETI Inventor name: RICHARDSON, THOMAS, W. Inventor name: LEE, SALLY Inventor name: BURFORD, NEIL Inventor name: YUE, HENRY Inventor name: TANG, Y., TOM Inventor name: ELLIOTT, VICKI, S. Inventor name: WARREN, BRIDGET, A. Inventor name: DING, LI |