CA2440618A1 - Immunoglobulin superfamily proteins - Google Patents

Abstract

The invention provides human immunoglobulin superfamily proteins (IGSFP) and polynucleotides which identify and encode IGSFP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of IGSFP.

Description

IMMUNOGLOBULIN SUPERFAMILY PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of immunoglobulin superfamily proteins and to the use of these sequences in the diagnosis, treatment, and prevention of immune system, neurological, developmental, muscle, and cell proliferative disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of immunoglobulin superfamily proteins.
1o BACKGROUND OF THE INVENTION
Most cell surface and soluble molecules that mediate functions such as recognition, adhesion or binding have evolved from a common evolutionary precursor (i.e., these proteins have structural homology). A number of molecules outside the immune system that have similar functions are also derived from this same evolutionary precursor. These molecules are classified as members of the immunoglobulin (Ig) superfamily. The criteria for a protein to be a member of the Ig superfamily is to have one or more Ig domains, which are regions of 70-110 amino acid residues in length homologous to either Ig variable-like (V) or Ig constant-like (C) domains. Members of the Ig superfamily include antibodies (Ab), T cell receptors (TCRs), class I and II major histocompatibility (MHC) proteins, CD2, CD3, CD4, CDB, poly-Ig receptors, Fc receptors, neural cell-adhesion molecule (NCAM) and platelet-derived growth factor receptor (PDGFR).
Ig domains (V and C) are regions of conserved amino acid residues that give a polypeptide a globular tertiary structure called an immunoglobulin (or antibody) fold, which consists of two approximately parallel layers of (3-sheets. Conserved cysteine residues form an intrachain disulfide-bonded loop, SS-75 amino acid residues in length, which connects the two layers of the (3-sheets.
Each (3-sheet has three or four anti-parallel ~i-strands of 5-10 amino acid residues. Hydrophobic and hydrophilic interactions of amino acid residues within the (3-strands stabilize the Ig fold (hydrophobic on inward facing amino acid residues and hydrophilic on the amino acid residues in the outward facing portion of the strands). A V domain consists of a longer polypeptide than a C
domain, with an additional pair of (3-strands in the Ig fold.
3o A consistent feature of Ig superfamily genes is that each sequence of an Ig domain is encoded by a single exon. It is possible that the superfamily evolved from a gene coding for a single Ig domain involved in mediating cell-cell interactions. New members of the superfamily then arose by exon and gene duplications. Modem Ig superfamily proteins contain different numbers of V and/or C

domains. Another evolutionary feature of this superfamily is the ability to undergo DNA
rearrangements, a unique feature retained by the antigen receptor members of the family.
Many members of the Ig superfamily are integral plasma membrane proteins with extracellular Ig domains. The hydrophobic amino acid residues of their transmembrane domains and their cytoplasmic tails are very diverse, with little or no homology among Ig family members or to known signal-transducing structures. There are exceptions to this general superfamily description.
For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. In addition Thy-1 is a glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic tail, but is instead attached to the plasma membrane by a covalent glycophosphatidylinositol linkage.
Another common feature of many Ig superfamily proteins is the interactions between Ig domains which are essential for the function of these molecules. Interactions between Ig domains of a multimeric protein can be either homophilic or heterophilic (i.e., between the same or different Ig domains). Antibodies are multimeric proteins which have both homophilic and heterophilic interactions between Ig domains. Pairing of constant regions of heavy chains forms the Fc region of an antibody and pairing of variable regions of light and heavy chains form the antigen binding site of an antibody.
Heterophilic interactions also occur between Ig domains of different molecules. These interactions provide adhesion between cells for significant cell-cell interactions in the immune system and in the developing and mature nervous system. (Reviewed in Abbas, A.K. et al. (1991) Cellular and Molecular Immunolo~y, W.B. Saunders Company, Philadelphia, PA, pp.142-145.) Antibodies Antibodies are multimeric members of the Ig superfamily which are either expressed on the surface of B-cells or secreted by B-cells into the circulation. Antibodies bind and neutralize foreign antigens in the blood and other extracellular fluids. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the a, 8, s, 'y, and p. H-chain types. There are two types of L-chains, x and ~,, either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure.
H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. The constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains. The amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class. The variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class. Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site.
(Reviewed in Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing, New York, NY, pp. 1206-1213 and 1216-1217.) Both H-chains and L-chains contain the repeated Ig domains of members of the Ig superfamily. For example, a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region.
The immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens. Such antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site-specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population.
Neural Cell Adhesion Proteins Neural cell adhesion proteins (NCAPs) play roles in the establishment of neural networks during development and regeneration of the nervous system (Uyemura et al.
(1996) Essays Biochem.
31:37-48; Brummendorf and Rathjen (1996) C~rr. Opin. Neurobiol. 6:584-593).
NCAP participates in neuronal cell migration, cell adhesion, neurite outgrowth, axonal fasciculation, pathfmding, synaptic target-recognition, synaptic formation, myelination and regeneration. NCAPs are expressed on the surfaces of neurons associated with learning and memory. Mutations in genes encoding NCAPS are linked with neurological diseases, including Charcot-Marie-Tooth disease (a hereditary neuropathy), Dejerine-Sottas disease, X-linked hydrocephalus, MASA syndrome (mental retardation, aphasia, shuffling gait and adducted thumbs), and spastic paraplegia type I. In some cases, expression of NCAP is not restricted to the nervous system. L1, for example, is expressed in melanoma cells and hematopoietic tumor cells where it is implicated in cell spreading and migration, and may play a role in tumor progression (Montgomery et al. (1996) J. Cell Biol. 132:475-485).
NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et al., supra). They are generally linked to the plasma membrane through a transmembrane domain and/or a glycosyl-phosphatidylinositol (GPI) anchor. The GPI linkage can be cleaved by GPI phospholipase C.
Most NCAPs consist of an extracellular region made up of one or more immunoglobulin domains, a membrane spanning domain, and an intracellular region. Many NCAPs contain post-translational modifications including covalently attached oligosaccharide, glucuronic acid, and sulfate. NCAPs fall into three subgroups: simple-type, complex-type, and mixed-type. Simple-type NCAPs contain one or more variable or constant immunoglobulin domains, but lack other types of domains. Members of the simple-type subgroup include Schwann cell myelin protein (SMP), limbic system-associated membrane protein (LAMP) and opiate-binding cell-adhesion molecule (OBCAM). The complex-type NCAPs contain fibronectin type III domains in addition to the immunoglobulin domains. The complex-type subgroup includes neural cell-adhesion molecule (NCAM), axonin-1, F11, Bravo, and Ll. Mixed-type NCAPs contain a combination of immunoglobulin domains and other motifs such as tyrosine kinase, epidermal growth factor-like, sema, and PSI (plexins, semaphorins, and integrins) domains. This subgroup includes Trk receptors of nerve growth factors such as nerve growth factor (NGF) and neurotropin 4 (NT4), Neu differentiation factors such as glial growth factor II (GGFII) and acetylcholine receptor-inducing factor (ARIA), the semaphorin/collapsin family such as semaphorin B
and collapsin, and receptors for members of the semaphorin/collapsin family such as plexin (for plexin, see below).
An NCAP subfamily, the NCAP-LON subgroup, includes cell adhesion proteins expressed on distinct subpopulations of brain neurons. Members of the NCAP-LON subgroup possess three immunoglobulin domains and bind to cell membranes through GPI anchors. Kilon (a kindred of NCAP-LON), for example, is expressed in the brain cerebral cortex and hippocampus (Funatsu et al.
(1999) J. Biol. Chem. 274:8224-8230). Immunostaining localizes Kilon to the dendrites and soma of pyramidal neurons. Kilon has three C2 type immunoglobulin-like domains, six predicted glycosylation sites, and a GPI anchor. Expression of Kilon is developmentally regulated. It is expressed at higher levels in adult brain in comparison to embryonic and early postnatal brains.
Confocal microscopy shows the presence of Kilon in dendrites of hypothalamic magnocellular neurons secreting neuropeptides, oxytocin, or arginine vasopressin (Miyata et al. (2000) J.
Comp. Neurol. 424:74-85).
Arginine vasopressin regulates body fluid homeostasis, extracellular osmolarity and intravascular volume. Oxytocin induces contractions of uterine smooth muscle during child birth and of myoepithelial cells in mammary glands during lactation. In magnocellular neurons, Kilon is proposed to play roles in the reorganization of dendritic connections during neuropeptide secretion.
Sidekick (SDK) is a member of the NCAP family. The extracellular region of SDK
contains six immunoglobulin domains and thirteen fibronectin type ffI domains. SDK is involved in cell-cell interaction during eye development in Drosophila (Nguyen, D. N. T. et al.
(1997) Development 124:
3303).
Synaptic Membrane Glycoproteins Specialized cell junctions can occur at points of cell-cell contact. Among these cell junctions are communicating junctions which mediate the passage of chemical and electrical signals between cells. In the central nervous system, communicating junctions between neurons are known as synaptic junctions. They are composed of the membranes and cytoskeletons of the pre-and post-synaptic neurons. Some glycoproteins, found in biochemically isolated synaptic subfractions such as the synaptic membrane (5M) and postsynaptic density (PSD) fractions, have been identified and their functions established. An example is the SM glycoprotein, gp50, identified as the (32 subunit of the Na+/K*-ATPase.
Two glycoproteins, gp65 and gp55, are major components of synaptic membranes prepared from rat forebrain. They are members of the Ig superfamily containing three and two Ig domains, respectively. As members of the Ig superfamily, it is proposed that a possible function of these proteins is to mediate adhesive interactions at the synaptic junction.
(Langnaese, K. et al. (1997) J.
Biol. Chem.272:821-827.) Lectins Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K. and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9:237-264). This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L. A.
(1991) J. Cell. Biochem.
45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).
Sialic acid binding Ig-like lectins (SIGLECs) are members of the Ig superfamily that bind to sialic acids in glycoproteins and glycolipids. SIGLECs include sialoadhesin, CD22, CD33, myelin-associated glycoprotein (MAG), SIGLEC-5, SIGLEC-6, SIGLEC-7, and SIGLEC-8. The extracellular 3o region of SIGLEC has a membrane distal V-set domain followed by varying numbers of C2-set domains. The sialic acid binding domain is mapped to the V-set domain. Except for MAG which is expressed exclusively in the nervous system, most SIGLECs are expressed on distinct subsets of hemopoietic cells. For example, SIGLEC-8 is expressed exclusively in eosinophils, one form of polymorphonuclear leucocyte (granulocyte) (Floyd, H. et al. (2000) J. Biol.
Chem. 275: 861-866).
Leucine-Rich Repeat Proteins Leucine-rich repeat proteins (LRRPs) are involved in protein-protein interactions. LRRPs such as mammalian neuronal leucine-rich repeat proteins (NLLR-1 and NLLR-2), Drosophila connectin, slit, chaopin, and toll all play roles in neuronal development. The extracellular region of LRRPs contains varying numbers of leucine-rich repeats, immunoglobulin-like domains, and fibronectin type DI domains (Taguchi, A. et al. (1996) Brain Res. Mol. Brain Res. 35:31-40).
In addition to the V and C2 sets of immunoglobulin-like domains, there is a D
set immunoglobulin-like domain, named IPTlTIG (for immunoglobulin-like fold shared by plexins and transcription factors). IPT/TIG containing proteins include plexins, MET/ RON/
SEA (hepatocyte growth factor receptor family), and the transcription factor XCoe2, a transcription factor of the Col/Olf 1/BBF family involved in the specification of primary neurons in Xenopus (Bork, P. et al.
(1999) Trends in Biochem. 24:261-263; Santoro, N. M. et al. (1996) Mol. Cell Biol. 16:7072-7083;
Dubois L. et al. (1998) Curr. Bio1.8:199-209). Plexins such as plexin A and VESPR have been shown to be neuronal semaphorin receptors that control axon guidance (Winberg M. L.
et al. (1998) Cell 95:903-916).
Expression~ro Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
The discovery of new immunoglobulin superfamily proteins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of immune system, neurological, developmental, muscle, and cell proliferative disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of immunoglobulin superfamily proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, immunoglobulin superfamily proteins, referred to collectively as "IGSFP" and individually as "IGSFP-l," "IGSFP-2," "IGSFP-3,"
"IGSFP-4," "IGSFP-5," "IGSFP-6," "IGSFP-7," "IGSFP-8," "IGSFP-9," "IGSFP-10," "IGSFP-11," and "IGSFP-12." In one aspect, 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 117 NO:1-12, 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:l-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ll~ NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ B7 NO:1-12. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID N0:1-12.
to 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 N0:1-12, 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:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-12. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-12. In another alternative, the polynucleotide is selected from the group consisting of SEQ ll~ N0:13-24.
Additionally, 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 N0:1-12, 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 D7 NO:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-12. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, 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:l-12, 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:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >I7 NO:1-12. '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.
Additionally, 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 )D N0:1-12, 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 B7 NO:l-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:l-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
)D N0:1-12.
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
>D N0:13-24, 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 N0:13-24, 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). In one alternative, 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
>D N0:13-24, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:13-24, 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. In one alternative, 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 )T7 N0:13-24, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of SEQ >D N0:13-24, 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:1-12, 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:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ )D NO:1-12. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional IGSFP, 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:1-12, 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:l-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:I-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D NO:1-12. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional IGSFP, comprising administering to a patient in need of such treatment the composition.
Additionally, 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 N0:1-12, 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 N0:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional IGSFP, 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:1-12, 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:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 ll~ N0:1-12, 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:1-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 )D N0:13-24, 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
)D N0:13-24, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )17 N0:13-24, 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)-iv). 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 )D N0:13-24, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )D N0:13-24, 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). Alternatively, 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.

BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME
database homologs, for 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.
2o DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so 3o forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"IGSFP" refers to the amino acid sequences of substantially purified IGSFP
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.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of IGSFP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of IGSFP either by directly interacting with IGSFP or by acting on components of the biological pathway in which IGSFP
participates.
An "allelic variant" is an alternative form of the gene encoding IGSFP.
Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding IGSFP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as IGSFP or a polypeptide with at least one functional characteristic of IGSFP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding IGSFP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding IGSFP. 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 IGSFP. 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 IGSFP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and 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.
The terms "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.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of IGSFP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of IGSFP either by directly interacting with IGSFP or by acting on components of the biological pathway in which IGSFP
participates.
The term "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 IGSFP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used Garners that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLI~. The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "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'-NHZ), 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.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, 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).
The term "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.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. 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. Once introduced into a cell, 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.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic IGSFP, 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'.
A "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 IGSFP or fragments of IGSFP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salinon 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 GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative 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.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions 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.
The term "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 IGSFP or the polynucleotide encoding IGSFP
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 S 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. For example, 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. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:13-24 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:13-24, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:13-24 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ
ID N0:13-24 from related polynucleotide sequences. The precise length of a fragment of SEQ >D
N0:13-24 and the region of SEQ ID N0:13-24 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 >D NO:1-12 is encoded by a fragment of SEQ ID N0:13-24. A
fragment of SEQ ID NO:1-12 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-12. For example, a fragment of SEQ ID NO:1-12 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-12.
The precise length of a fragment of SEQ ID NO:1-12 and the region of SEQ B7 NO:1-12 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.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, 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.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS

8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off.' S0 Expect: 10 Word Size: Il Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode 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.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off. SO
Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire.defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "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.
to Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) 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 p.g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carned out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"'~ ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.
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, SS°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%.
Typically, 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, may also be used under particular circumstances, such as for 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.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" 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.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of IGSFP
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 IGSFP which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of IGSFP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of IGSFP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, 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.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
to "Post-translational modification" of an IGSFP 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 IGSFP.
"Probe" refers to nucleic acid sequences encoding IGSFP, their complements, or fragments 15 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. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target 20 DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 25 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.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold 30 Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Bio- logy, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M.
et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
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 l0 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. Hence, 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, su ra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, 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.
Alternatively, 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 l0 other moieties known in the art.
An "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.
The term "sample" is used in its broadest sense. A sample suspected of containing IGSFP, nucleic acids encoding IGSFP, 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.
The terms "specific 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.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "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.
The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "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. In one alternative, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). 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 transfernng 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 2Ø9 (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. Polyrnorphic 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.
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 2Ø9 (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
The invention is based on the discovery of new human immunoglobulin superfamily proteins (IGSFP), the polynucleotides encoding IGSFP, and the use of these compositions for the diagnosis, treatment, or prevention of immune system, neurological, developmental, muscle, and cell proliferative disorders.
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 ll~). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ 177 NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide 1D) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ D7 NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown. Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns l and 2 show the polypeptide sequence identification number (Polypeptide SEQ D7 NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank 117 NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID
NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, 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 117) 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 MOTIFS
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.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are immunoglobulin superfamily proteins. For example, SEQ ID N0:2 is 50% identical, from residue Q34 to residue P563, to Mus musculus Fca/m receptor (GenBank ID g11071950) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 9.6e-121, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:2 also contains an immunoglobulin domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from additional BLAST analyses provide further corroborative evidence that SEQ D7 N0:2 is an immunoglobulin. In an alternative example, SEQ D7 N0:3 is 40% identical, from residue L30 to residue V176, to surface protein MCA-32 (GenBank LD g1136501) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 6.9e-35, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ
ID N0:3 also contains an immunoglobulin 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.) Data from BLIMPS, MOTIFS, and additional BLAST
analyses provide further corroborative evidence that SEQ ll~ N0:3 is a surface protein.
In an alternative example, SEQ )D N0:8 is 86% identical, from residue Ml to residue 5433, to cell-surface molecule Ly-9 (GenBank ID g10197717) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 7.4e-191, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:8 also contains immunoglobulin domains 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.) Data from additional BLAST analysis provide further corroborative evidence that SEQ )D N0:8 is a cell surface molecule which is a member of the immunoglobulin superfamily. In an alternative example, SEQ ID NO:11 is 52% identical, from residue N43 to residue Q604, to human NEPHl (GenBank ll~ 814572521) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.4e-158, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. As determined by BLAST
analysis using the PROTEOME database, SEQ ID NO:11 is localized to the plasma membrane, is homologous to a human protein which contains an immunoglobulin domain and has a region of low similarity to a region of an opioid-binding cell adhesion molecule, which is a glycosylphosphatidylinositol (GPI)-anchored neural cell adhesion molecule (PROTEOME >D 598720~FLJ10845); SEQ >D N0:11 is also homologous to human Nephrin which is a member of the immunoglobulin superfamily expressed in renal glomeruli which may have a role in the development or function of the kidney filtration barrier.
Mutation of the Nephrin gene causes congenital nephrotic syndrome (PROTEOME ID
340970~NPHS1). SEQ >D N0:11 also contains an immunoglobulin 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.) Data from BLllVIPS, MOTIFS, and additional BLAST analyses provide further corroborative evidence that SEQ ID N0:11 is a member of the immunoglobulin superfamily. SEQ D7 NO:1, SEQ )D N0:4-7, SEQ >D N0:9-10 and SEQ
ID N0:12 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ 117 NO:1-12 are described in Table 7.
As shown in Table 4, 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 117) 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 )D
N0:13-24 or that distinguish between SEQ )D N0:13-24 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. Alternatively, 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. In addition, 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"). Alternatively, 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"). Alternatively, 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. For example, a polynucleotide sequence identified as FL XXXXXX NI NZ_YYYYY N3 NQ represents a "stitched" sequence in which XXxXXX
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 NI,Z,3..., if present, represent specific exons that may have been manually edited during analysis (See Example V).
Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, a polynucleotide sequence identified as ~~1AAAA~BBBBB_1 N is a "stretched" sequence, with ~'~~;~1XXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" 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). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs GNN, GFG,Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

(Computer Genomics Group, The Sanger Centre, Cambridge, UK) GBI Hand-edited analysis of genomic sequences.

FL Stitched or stretched genomic sequences (see Example V).

to INCY Full length transcript and exon prediction from mapping of EST

sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses IGSFP variants. A preferred IGSFP 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 IGSFP amino acid sequence, and which contains at least one functional or structural characteristic of IGSFP.
The invention also encompasses polynucleotides which encode IGSFP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:13-24, which encodes IGSFP. The polynucleotide sequences of SEQ ID N0:13-24, 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 IGSFP. In particular, such 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 IGSFP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:13-24 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 N0:13-24. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of IGSFP.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding IGSFP. A splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding IGSFP, 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 IGSFP 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 IGSFP. For example, a polynucleotide comprising a sequence of SEQ ID N0:14 is a splice variant of a polynucleotide comprising a sequence of SEQ ID N0:24 and a polynucleotide comprising a sequence of SEQ ID N0:16 is a splice variant of a polynucleotide comprising a sequence of SEQ ID N0:17. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of IGSFP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding IGSFP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring IGSFP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode IGSFP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring IGSFP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding IGSFP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding IGSFP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode IGSFP
and IGSFP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the l0 synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding IGSFP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:13-24 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) 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). Preferably, 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 IGSFP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and l0 ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth 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.
When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the S' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate 3o 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.

In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode IGSFP may be cloned in recombinant DNA molecules that direct expression of IGSFP, 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 IGSFP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter IGSFP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
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 IGSFP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. 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. Thus, 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.
In another embodiment, sequences encoding IGSFP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.

7:225-232.) Alternatively, IGSFP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.

55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of IGSFP, 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.) In order to express a biologically active IGSFP, the nucleotide sequences encoding IGSFP 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 IGSFP. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding IGSFP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding IGSFP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. ( 1994) Results Probl.
Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding IGSFP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding IGSFP. 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. (See, e.g., Sambrook, su ra; Ausubel, su ra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and l0 Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) 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. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding IGSFP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding IGSFP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORTl plasmid (Life Technologies). Ligation of sequences encoding IGSFP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of IGSFP are needed, e.g. for the production of antibodies, vectors which direct high level expression of IGSFP may be used.
For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of IGSFP. 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. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, su ra;

Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of IGSFP. Transcription of sequences encoding IGSFP 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.
6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and TechnoloQV
(1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding IGSFP
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 IGSFP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of IGSFP in cell lines is preferred. For example, sequences encoding IGSFP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk 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. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), B glucuronidase and its substrate B-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding IGSFP is inserted within a marker gene sequence, transformed cells containing sequences encoding IGSFP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding IGSFP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding IGSFP
and that express IGSFP 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.
Itnmunological methods for detecting and measuring the expression of IGSFP
using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on IGSFP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in ItnmunoloQV, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Itnmunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding IGSFP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding IGSFP, or any fragments thereof, may be cloned into a vector to for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerise such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding IGSFP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode IGSFP may be designed to contain signal sequences which direct secretion of IGSFP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" 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.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding IGSFP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric IGSFP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of IGSFP
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 IGSFP encoding sequence and the heterologous protein sequence, so that IGSFP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, su ra, ch. 10). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled IGSFP 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, 35S-methionine.
IGSFP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to IGSFP. At least one and up to a plurality of test compounds may be screened for specific binding to IGSFP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of IGSFP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunology 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which IGSFP
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express IGSFP, either as a secreted protein or on the cell-membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coli. Cells expressing IGSFP or cell membrane fractions which contain IGSFP
are then contacted with a test compound and binding, stimulation, or inhibition of activity of either IGSFP 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. For example, the assay may comprise the steps of combining at least one test compound with IGSFP, either in solution or affixed to a solid support, and detecting the binding of IGSFP to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or axed to a solid support.
IGSFP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of IGSFP. Such compounds may inchzde agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for IGSFP
activity, wherein IGSFP is combined with at least one test compound, and the activity of IGSFP in the presence of a test compound is compared with the activity of IGSFP in the absence of the test compound. A change in the activity of IGSFP in the presence of the test compound is indicative of a compound that modulates the activity of IGSFP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising IGSFP under conditions suitable for IGSFP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of IGSFP
may do so indirectly and need not come in direct contact with the test compound. At least one and up 2o to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding IGSFP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) For example, 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). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, 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) Clip. 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 dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding IGSFP 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 IGSFP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region l0 of a polynucleotide encoding IGSFP 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. Alternatively, a mammal inbred to overexpress IGSFP, e.g., by secreting IGSFP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of IGSFP and immunoglobulin superfamily proteins. In addition, the expression of IGSFP is closely associated with brain, colon, diseased skin, diseased lung, hippocampus, spleen, and diseased 2o vermis tissues, as well as, CD4+ T and peripheral blood cells. Therefore, IGSFP appears to play a role in immune system, neurological, developmental, muscle, and cell proliferative disorders. In the treatment of disorders associated with increased IGSFP expression or activity, it is desirable to decrease the expression or activity of IGSFP. In the treatment of disorders associated with decreased IGSFP expression or activity, it is desirable to increase the expression or activity of IGSFP.
Therefore, in one embodiment, IGSFP 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 IGSFP. Examples of such disorders include, but are not limited to, an immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with C~shing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helininthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyefinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a developmental disorder such as renal tubular acidosis, anemia, C~shing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy;
and 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, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing IGSFP 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 IGSFP including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified IGSFP 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 IGSFP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of IGSFP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IGSFP including, but not limited to, those listed above.
In a further embodiment, an antagonist of IGSFP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IGSFP.
Examples of such disorders include, but are not limited to, those immune system, neurological, developmental, muscle, and cell proliferative disorders described above. In one aspect, an antibody which specifically binds IGSFP 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 IGSFP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding IGSFP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IGSFP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of IGSFP may be produced using methods which are generally known in the art. In particular, purified IGSFP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind IGSFP.
Antibodies to IGSFP may also l0 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 (e.g., from camels or llamas) 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).
For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with IGSFP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to IGSFP 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 IGSFP 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 IGSFP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce IGSFP-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 IGSFP may also be generated.
For example, such fragments include, but are not limited to, F(ab')Z fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between IGSFP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering IGSFP epitopes is generally used, but a competitive binding assay may also be employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for IGSFP. Affinity is expressed as an association constant, Ke, which is defined as the molar concentration of IGSFP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple IGSFP epitopes, represents the average affinity, or avidity, of the antibodies for IGSFP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular IGSFP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the IGSFP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ke ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of IGSFP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRh Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of IGSFP-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. su ra.) In another embodiment of the invention, the polynucleotides encoding IGSFP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, 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 IGSFP. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding IGSFP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. 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. (See, e.g., 3o Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding IGSFP 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 (SCTD)-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 l0 (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
(1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) 15 express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides 20 brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in IGSFP expression or regulation causes disease, the expression of IGSFP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in 25 IGSFP are treated by constructing mammalian expression vectors encoding IGSFP and introducing these vectors by mechanical means into IGSFP-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.
30 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) C~rr. Opin.
Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of IGSFP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
IGSFP
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-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. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V.
and H.M. Blau, su ra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding IGSFP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, 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.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to IGSFP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding IGSFP 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 cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. 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. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") 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. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding IGSFP to cells which have one or more genetic abnormalities with respect to the expression of IGSFP. 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. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding IGSFP to target cells which have one or more genetic abnormalities with respect to the expression of IGSFP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing IGSFP to cells of the central nervous system, for which HSV has a 2o 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). The construction of a 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. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. 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.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding IGSFP to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) C~rr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. 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). Similarly, inserting the coding sequence for IGSFP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of IGSFP-coding RNAs and the synthesis of high levels of IGSFP in vector transduced cells. While 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 (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of IGSFP 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, e.g., between about positions -10 and +10 from the start 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 Itnmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule may also be designed to block translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding IGSFP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared l0 by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding IGSFP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding IGSFP. 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 3o compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased IGSFP
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding IGSFP may be therapeutically useful, and in the treatment of disorders associated with decreased IGSFP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding IGSFP 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 IGSFP 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 IGSFP are assayed by any method commonly known in the art. Typically, 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 IGSFP. 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 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. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) 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 Remin on's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of IGSFP, antibodies to IGSFP, and mimetics, agonists, antagonists, or inhibitors of IGSFP.
The compositions utilized in this invention may be administered by any number of routes l0 including, but not limited to, oral, intravenous, intramuscular, infra-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.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In 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. In the case of macromolecules (e.g. larger peptides and ' proteins), recent developments in the field of pulinonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulinonary 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.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising IGSFP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, IGSFP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, 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 IGSFP
or fragments thereof, antibodies of IGSFP, and agonists, antagonists or inhibitors of IGSFP, 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 EDso (the dose therapeutically effective in 50% of the population) or LDSO (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 LDso/EDso 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 EDso 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 ~cg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind IGSFP may be used for the diagnosis of disorders characterized by expression of IGSFP, or in assays to monitor patients being treated with IGSFP or agonists, antagonists, or inhibitors of IGSFP.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for IGSFP include methods which utilize the antibody and a label to detect IGSFP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring IGSFP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of IGSFP expression. Normal or standard values for IGSFP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to IGSFP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of IGSFP
expressed in l0 subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding IGSFP 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 IGSFP
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of IGSFP, and to monitor regulation of IGSFP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding IGSFP or closely related molecules may be used to identify nucleic acid sequences which encode IGSFP. 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 IGSFP, 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 IGSFP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ )D
N0:13-24 or from genomic sequences including promoters, enhancers, and introns of the IGSFP
gene.
Means for producing specific hybridization probes for DNAs encoding IGSFP
include the cloning of polynucleotide sequences encoding IGSFP or IGSFP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding IGSFP may be used for the diagnosis of disorders associated with expression of IGSFP. Examples of such disorders include, but are not limited to, an S immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SC>D), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), . Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with C~shing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, priors diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood; anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a developmental disorder such as renal tubular acidosis, anemia, C~shing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilins' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy;
and 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, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
The polynucleotide sequences encoding IGSFP 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 IGSFP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding IGSFP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding IGSFP 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 IGSFP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of IGSFP, 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 IGSFP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding IGSFP
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 IGSFP, or a fragment of a polynucleotide complementary to the polynucleotide encoding IGSFP, 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.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding IGSFP 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. In SSCP;
oligonucleotide primers derived from the polynucleotide sequences encoding IGSFP 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. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, 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.
These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, 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. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a.patient's response to a drug, such as life-threatening toxicity.
For example, 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 ALOXS gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) C~rr. Opin.
Neurobiol. 11:637-641.) Methods which may also be used to quantify the expression of IGSFP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C.

et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in 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.
In further embodiments, 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 l0 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. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, IGSFP, fragments of IGSFP, or antibodies specific for IGSFP 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.) Thus 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. In one embodiment, 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.
These fingerprints or signatures are most useful and refined when they contain expression information l0 from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, 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. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. 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. In one embodiment, 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, su ra). 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 IGSFP
to quantify the levels of IGSFP expression. In one embodiment, 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. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the 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.
In another embodiment, 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 l0 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. (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; Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach, M. Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding IGSFP
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 mufti-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop 3o 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).
(See, for example, Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, su ra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding IGSFP 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, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is l0 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 l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, IGSFP, 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 IGSFP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysers, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with IGSFP, or fragments thereof, and washed. Bound IGSFP is then detected by methods well known in the art.
Purified IGSFP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
3o In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding IGSFP specifically compete with a test compound for binding IGSFP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with IGSFP.

In additional embodiments, the nucleotide sequences which encode IGSFP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above and below, 1o including U.S. Ser. No.60/275,249, U.S. Ser. No.60/316,810, U.S. Ser.
No.60/323,977, U.S. Ser.
No.60/348,447, and U.S. Ser. No.60/343,880, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries 15 Incyte cDNAs were derived from cDNA libraries described in the L1FESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with 2o chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles 25 (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the 30 UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, su ra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl 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 XL1-Blue, XLl-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. 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. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4 °C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycfing steps were carned 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 II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosysterns) 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 VIII.
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 norveQicus, Mus musculus, Caenorhabditis ele~ans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM
(Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); 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. (1996) C~rr. Opin.
Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BL>IVVIPS, and HIVIMER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) 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. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, 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, hidden Markov model (HIVI1VI)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 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).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ 1D
N0:13-24. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative immunoglobulin superfamily proteins 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. To determine which of these Genscan predicted cDNA sequences encode immunoglobulin superfamily proteins, the encoded polypeptides were analyzed by querying against PFAM models for immunoglobulin superfamily proteins. Potential immunoglobulin superfamily proteins were also identified by homology to Incyte cDNA sequences that had been annotated as immunoglobulin superfamily proteins.
These selected 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 III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example DI 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. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. 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. 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 IGSFP Encoding Polynucleotides The sequences which were used to assemble SEQ >D N0:13-24 were compared with l0 sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:13-24 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. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ 1D NO:, to that map location.
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. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by 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.nlin.nih.gov/genemap~, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the l0 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. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and SO%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding IGSFP 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 III). 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; heroic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or 3o urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, 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 IGSFP. cDNA sequences and cDNA
library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of IGSFP Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer 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, lnc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mgz+, (NH~)ZSO4, 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. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C, 3 min; Step 2: 94 °C, 15 sec; Step 3: 57 °C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p,1 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 II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A S ~cl to 10 ~1 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). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were relegated 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.
Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in IGSFP Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID N0:13-24 using the LIFESEQ database (Incyte Genomics).
Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. 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, lnc.) 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 Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The to 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 N0:13-24 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~cCi of ~,~ 3zp] 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' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham 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, su ra.), 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), su ra).
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. (See, e.g., Schena, M. et al.
(1995) Science l0 270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A.
and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.) 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.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, 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. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation 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/pl oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/p,l RNase inhibitor, 500 ~M dATP, 500 p.M dGTP, 500 p,M dTTP, 40 p.M
dCTP, 40 p,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 GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium hydroxide and incubated for 20 minutes at 85°C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 u1 5X SSC/0.2% SDS.
Microarray Preparation 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 fig.
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 p,1 of the array element DNA, at an average concentration of 100 ng/pl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER 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 Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 p,1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer (1X SSC, 0.1 %
SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. 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.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, 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 CyS. 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. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, 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 1BM-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.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
For example, SEQ ID N0:19 showed differential expression in toxicology studies as determined by microarray analysis. The expression of SEQ ID N0:19 was decreased by at least two fold in a human C3A liver cell line treated with various drugs (e.g., steroids, steroid hormones) relative to untreated C3A cells. The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The C3A cell line is well established as an in vitro model of the mature human l0 liver (Mickelson et al. (1995) Hepatology 22:866-875; Nageridra et al.
(1997) Am J Physiol 272:G408-G416). Effects upon liver metabolism are important to understanding the pharmacodynamics of a drug. Therefore, SEQ 1D N0:19 is useful for understanding the pharmacodynamics of a drug.
XII. Complementary Polynucleotides Sequences complementary to the IGSFP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring IGSFP.
Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of IGSFP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the IGSFP-encoding transcript.
XIII. Expression of IGSFP
Expression and purification of IGSFP is achieved using bacterial or virus-based expression systems. For expression of IGSFP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the TS 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 IGSFP upon induction with isopropyl beta-D-thiogalactopyranoside (1PTG). Expression of IGSFP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding IGSFP 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 fru 'per erda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, IGSFP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton l0 enzyme from Schistosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from IGSFP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel ( 1995, supra, ch. 10 and 16). Purified IGSFP obtained by these methods can be used directly in the assays shown in Examples XVII and XVI)1 where applicable.
XIV. Functional Assays IGSFP function is assessed by expressing the sequences encoding IGSFP 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 /.cg 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. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. 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 brornodeoxyuridine 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 IGSFP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding IGSFP 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 IGSFP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of IGSFP Specific Antibodies IGSFP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
Alternatively, the IGSFP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
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. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-IGSFP activity by, for example, binding the peptide or IGSFP 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 IGSFP Using Specific Antibodies Naturally occurring or recombinant IGSFP is substantially purified by immunoaffinity chromatography using antibodies specific for IGSFP. An immunoaffinity column is constructed by covalently coupling anti-IGSFP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing IGSFP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of IGSFP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/IGSFP 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 IGSFP is collected.
XVII. Identification of Molecules Which Interact with IGSFP
IGSFP, or biologically active fragments thereof, are labeled with l2sI 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 IGSFP, washed, and any wells with labeled IGSFP complex are assayed. Data obtained using different concentrations of IGSFP are used to calculate values for the number, affinity, and association of IGSFP with the candidate molecules.
Alternatively, molecules interacting with IGSFP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
IGSFP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine 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. Demonstration of IGSFP Activity An assay for IGSFP activity measures the ability of IGSFP to recognize and precipitate antigens from serum. This activity can be measured by the quantitative precipitin reaction. (Golub, E.
S. et al. (1987) ImmunoloQV: A Synthesis, Sinauer Associates, Sunderland, MA, pages 113-115.) IGSFP is isotopically labeled using methods known in the art. Various serum concentrations are added to constant amounts of labeled IGSFP. IGSFP-antigen complexes precipitate out of solution and are collected by centrifugation. The amount of precipitable IGSFP-antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitable IGSFP-antigen complex is plotted against the serum concentration. For various serum concentrations, a characteristic precipitin curve is obtained, in which the amount of precipitable IGSFP-antigen complex initially increases proportionately with increasing serum concentration, peaks at the equivalence point, and then decreases proporkionately with further increases in serum concentration.
Thus, the amount of precipitable IGSFP-antigen complex is a measure of IGSFP
activity which is characterized by sensitivity to both limiting and excess quantities of antigen.
Alternatively, an assay for IGSFP activity measures the expression of IGSFP on the cell surface. cDNA encoding IGSFP is transfected into a non-leukocytic cell line.
Cell surface proteins are labeled with biotin (de la Fuente, M.A. et.al. (1997) Blood 90:2398-2405).
Immunoprecipitations are performed using IGSFP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and imununoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of IGSFP expressed on the cell surface.
Alternatively, an assay for IGSFP activity measures the amount of cell aggregation induced by overexpression of IGSFP. In this assay, cultured cells such as NIH3T3 are transfected with cDNA
encoding IGSFP contained within a suitable mammalian expression vector under control of a strong promoter. Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECI~, is useful for identifying stable transfectants. The amount of cell agglutination, or clumping, associated with transfected cells is compared with that associated with untransfected cells. The amount of cell agglutination is a direct measure of IGSFP activity.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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ioo Table 5 PolynucleotideIncyte ProjectRepresentative Library SEQ ID:
ID NO:

13 ~ 3855123CB BRAHNONOS

15 ' 3939883CB SKINBITO1 17 ~ 8518269CBTLYJTXFO1 ' 18 ( 1592646CBEOSIHET02 o '° a >; s s o ~ ~, ! . ~ ~,-s a~
' i 3 ~o .c c ' ~ ; yv ~ ~a ~ ~ , yo ~ °' o y .° " ° I c o .n 3 ~ o ' ~ ~ c ~ 3 U ~ ~ E
a~ c av~°~~~on. ~ Ian !o~cyF"3yUa~~ i.'~ ~oa~
z .fl e0 ~.. V ,, a n1 i C ' Q E w_ ui ~ e'C e'O ~ ' ~ , Ca ~ .' O
N . b I' I._ C N U G
~UC~~:pcn-OLbE ;~ I'=~O~E-~b'X~C 10 Op"O
cCOf~Q' C cC'wC~ UOEO t T
' ~ 00 t-. U O I C ~ of ' 'fl 00 . t-.
o '' 3 i ~s i °c .5 c ~ ~ o ~ ~E ''_- ~ c Ca w ?~ 3 ~ E
w .E c-xa c .o ~ ~ ~ i w U ca ea Q .E X y ~ . y I ~ _a> fi C U c~3 N S1 .~ ,C ~ T : ~ ' C ~ ~ ~ v ~ Q. U ~" yp .C.
O ~n ~, I p .O e0 ~E O ~"~ c3 :a:.. . ~ C : op ' aS s a .D y .~ -C ~ i ~ ~ ~: V ~ ~ tn ~ C ' cC ' p a 7 O
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<110> INCYTE GENOMICS, INC.
YUE, Henry XU, Yuming THANGAVELU, Kavitha WARREN, Bridget A.
TANG, Y. Tom DUGGAN, Brendan M.
TRAN, Uyen K.
BAUGHN, Mariah R.
HONCHELL, Cynthia D.
BURFORD, Neil FORSYTHE, Ian J.
YANG, Junming MASON, Patricia M.
<120> IMMUNOLGLOBULIN SUPERFAMILY PROTEINS
<130> PF-0925 PCT
<140> To Be Assigned <141> Herewith <150> 60/275,249; 60/316,810; 60/323,977; 60/348,447;
60/343,880 <151> 2001-03-12; 2001-08-31; 2001-09-21; 2001-10-26;

<160> 24 <170> PERL Program <210> 1 <211> 442 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 3855123CD1 <400> 1 Met Thr Thr Glu Pro Gln Ser Leu Leu Val Asp Leu Gly Ser Asp Ala Ile Phe Ser Cys Ala Trp Thr Gly Asn Pro Ser Leu Thr Ile Val Trp Met Lys Arg Gly Ser Gly Val Val Leu Ser Asn Glu Lys Thr Leu Thr Leu Lys Ser Val Arg Gln Glu Asp Ala Gly Lys Tyr Val Cys Arg Ala Val Val Pro Arg Val Gly Ala Gly Glu Arg Glu Val Thr Leu Thr Val Asn Gly Pro Pro Ile Ile Ser Ser Thr Gln Thr Gln His Ala Leu His Gly Glu Lys Gly Gln Ile Lys Cys Phe Ile Arg Ser Thr Pro Pro Pro Asp Arg Ile Ala Trp Ser Trp Lys Glu Asn Val Leu Glu Ser Gly Thr Ser Gly Arg Tyr Thr Val Glu Thr Ile Ser Thr Glu Glu Gly Val Ile Ser Thr Leu Thr Ile Ser Asn Ile Val Arg Ala Asp Phe Gln Thr Ile Tyr Asn Cys Thr Ala Trp Asn Ser Phe Gly Ser Asp Thr Glu Ile Ile Arg Leu Lys Glu 170 175 .180 Gln Gly Ser Glu Met Lys Ser Gly Ala Gly Leu Glu Ala Glu Ser Val Pro Met Ala Val Ile Ile Gly Val Ala Val Gly Ala Gly Val Ala Phe Leu Val Leu Met Ala Thr Ile Val Ala Phe Cys Cys Ala Arg Ser Gln Arg Asn Leu Lys Gly Val Val Ser Ala Lys Asn Asp Ile Arg Val Glu Ile Val His Lys Glu Pro Ala Ser Gly Arg Glu Gly Glu Glu His Ser Thr Ile Lys Gln Leu Met Met Asp Arg Gly Glu Phe Gln Gln Asp Ser Val Leu Lys Gln Leu Glu Val Leu Lys Glu Glu Glu Lys Glu Phe Gln Asn Leu Lys Asp Pro Thr Asn Gly Tyr Tyr Ser Val Asn Thr Phe Lys Glu His His Ser Thr Pro Thr Ile Ser Leu Ser Ser Cys Gln Pro Asp Leu Arg Pro Ala Gly Lys Gln Arg Val Pro Thr Gly Met Ser Phe Thr Asn Ile Tyr Ser Thr Leu Ser Gly Gln Gly Arg Leu Tyr Asp Tyr Gly Gln Arg Phe Val Leu Gly Met Gly Ser Ser Ser Ile Glu Leu Cys Glu Arg Glu Phe Gln Arg Gly Ser Leu Ser Asp Ser Ser Ser Phe Leu Asp Thr Gln Cys Asp Ser Ser Val Ser Ser Ser Gly Lys Gln Asp Gly Tyr Val Gln Phe Asp Lys Ala Ser Lys Ala Ser Ala Ser Ser Ser His His Ser Gln Ser Ser Ser Gln Asn Ser Asp Pro Ser Arg Pro Leu Gln Arg Arg Met Gln Thr His Val <210> 2 <211> 577 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4547188CD1 <400> 2 Met Asp Gly Glu Ala Thr Val Lys Pro Gly Glu Gln Lys Glu Val Val Arg Arg Gly Arg Glu Val Asp Tyr Ser Arg Leu Ile Ala Gly Thr Leu Pro Gln Ser His Val Thr Ser Arg Arg Ala Gly Trp Lys Met Pro Leu Phe Leu Ile Leu Cys Leu Leu Gln Gly Ser Ser Phe Ala Leu Pro Gln Lys Arg Pro His Pro Arg Trp Leu Trp Glu Gly Ser Leu Pro Ser Arg Thr His Leu Arg Ala Met Gly Thr Leu Arg Pro Ser Ser Pro Leu Cys Trp Arg Glu Glu Ser Ser Phe Ala Ala Pro Asn Ser Leu Lys Gly Ser Arg Leu Val Ser Gly Glu Pro Gly Gly Ala Val Thr Ile Gln Cys His Tyr Ala Pro Ser Ser Val Asn Arg His Gln Arg Lys Tyr Trp Cys Cys Leu Gly Pro Pro Arg Trp Ile Cys Gln Thr Ile Val Ser Thr Asn Gln Tyr Thr His His Arg Tyr Arg Asp Arg Val Ala Leu Thr Asp Phe Pro Gln Arg Gly Leu Phe Val Val Arg Leu Ser Gln Leu Ser Pro Asp Asp Ile Gly Cys Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn Met Leu Phe Leu Ser Met Asn Leu Thr Ile Ser Ala Gly Pro Ala Ser Thr Leu Pro Thr 215 220 ~ 225 Ala Thr Pro Ala Ala Gly Glu Leu Thr Met Arg Ser Tyr Gly Thr Ala Ser Pro Val Ala Asn Arg Trp Thr Pro Gly Thr Thr Gln Thr Leu Gly Gln Gly Thr Ala Trp Asp Thr Val Ala Ser Thr Pro Gly Thr Ser Lys Thr Thr Ala Ser Ala Glu Gly Arg Arg Thr Pro Gly Ala Thr Arg Pro Ala Ala Pro Gly Thr Gly Ser Trp Ala Glu Gly Ser Val Lys Ala Pro Ala Pro Ile Pro Glu Ser Pro Pro Ser Lys Ser Arg Ser Met Ser Asn Thr Thr Glu Gly Val Trp Glu Gly Thr Arg Ser Ser Val Thr Asn Arg Ala Arg Ala Ser Lys Asp Arg Arg Glu Met Thr Thr Thr Lys Ala Asp Arg Pro Arg Glu Asp Ile Glu Gly Val Arg Ile Ala Leu Asp Ala Ala Lys Lys Val Leu Gly Thr Ile Gly Pro Pro Ala Leu Val Ser Glu Thr Leu Ala Trp Glu Ile Leu Pro Gln Ala Thr Pro Val Ser Lys Gln Gln Ser Gln Gly Ser Ile Gly Glu Thr Thr Pro Ala Ala Gly Met Trp Thr Leu Gly Thr Pro Ala Ala Asp Val Trp Ile Leu Gly Thr Pro Ala Ala Asp Val Trp Thr Ser Met Glu Ala Ala Ser Gly Glu Gly Ser Ala Ala Gly Asp Leu Asp Ala Ala Thr Gly Asp Arg Gly Pro Gln Ala Thr Leu Ser Gln Thr Pro Ala Val Gly Pro Trp Gly Pro Pro Gly Lys Glu Ser Ser Val Lys Arg Thr Phe Pro Glu Asp Glu Ser Ser Ser Arg Thr Leu Ala Pro Val Ser Thr Met Leu Ala Leu Phe Met Leu Met Ala Leu Val Leu Leu Gln Arg Lys Leu Trp Arg Arg Arg Thr Ser Gln Glu Ala Glu Arg Val Thr Leu Ile Gln Met Thr His Phe Leu Glu Val Asn Pro Gln Ala Asp Gln Leu Pro His Val Glu Arg Lys Met Leu Gln Asp Asp Ser Leu Pro Ala Gly Ala Ser Leu Thr Ala Pro Glu Arg Asn Pro Gly Pro <210> 3 <211> 385 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3939883CD1 <400> 3 Met Gln Thr Ser Ser Lys Pro Ser Asp Phe Leu Asn Leu Ala Lys Lys Lys Arg Lys Phe Ser Glu Leu Leu Thr Thr Val Val Leu Leu Cys Leu Leu Thr Pro Ser Trp Thr Ser Thr Gly Arg Met Trp Ser 35 40 '45 His Leu Asn Arg Leu Leu Phe Trp Ser Ile Phe Ser Ser Val Thr Cys Arg Lys Ala Val Leu Asp Cys Glu Ala Met Lys Thr Asn Glu Phe Pro Ser Pro Cys Leu Asp Ser Lys Thr Lys Val Val Met Lys Gly Gln Asn Val Ser Met Phe Cys Ser His Lys Asn Lys Ser Leu Gln Ile Thr Tyr Ser Leu Phe Arg Arg Lys Thr His Leu Gly Thr Gln Asp Gly Lys Gly Glu Pro Ala Ile Phe Asn Leu Ser Ile Thr Glu Ala His Glu Ser Gly Pro Tyr Lys Cys Lys Ala Gln Val Thr Ser Cys Ser Lys Tyr Ser Arg Asp Phe Ser Phe Thr Ile Val Asp Pro Val Thr Ser Pro Val Leu Asn Ile Met Val Ile Gln Thr Glu Thr Asp Arg His Ile Thr Leu His Cys Leu Ser Val Asn Gly Ser Leu Pro Ile Asn Tyr Thr Phe Phe Glu Asn His Val Ala Ile Ser Pro Ala Ile Ser Lys Tyr Asp Arg Glu Pro Ala Glu Phe Asn Leu Thr Lys Lys Asn Pro Gly Glu Glu Glu Glu Tyr Arg Cys Glu Ala Lys Asn Arg Leu Pro Asn Tyr Ala Thr Tyr Ser His Pro Val Thr Met Pro Ser Thr Gly Gly Asp Ser Cys Pro Phe Cys Leu Lys Leu Leu Leu Pro Gly Leu Leu Leu Leu Leu Val Val Ile Ile Leu Ile Leu Ala Phe Trp Val Leu Pro Lys Tyr Lys Thr Arg Lys Ala Met Arg Asn Asn Val Pro Arg Asp Arg Gly Asp Thr Ala Met Glu Val Gly Ile Tyr Ala Asn Ile Leu Glu Lys Gln Ala Lys Glu Glu Ser Val Pro Glu Val Gly Ser Arg Pro Cys Val Ser Thr Ala Gln Asp Glu Ala Lys His Ser Gln Glu Leu Gln Tyr Ala Thr Pro Val Phe Gln Glu Val Ala Pro Arg Glu Gln Glu Ala Cys Asp Ser Tyr Lys Ser Gly Tyr Vah Tyr Ser Glu Leu Asn Phe <210> 4 <211> 221 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3163819CD1 <400> 4 Met Leu Trp Leu Phe Gln Ser Leu Leu Phe Val Phe Cys Phe Gly Pro Gly Gln Leu Arg Asn Ile Gln Val Thr Asn His Ser Gln Leu 20. 25 30 Phe Gln Asn Met Thr Cys Glu Leu His Leu Thr Cys Ser Val Glu Asp Ala Asp Asp Asn Val Ser Phe Arg Trp Glu Ala Leu Gly Asn Thr Leu Ser Ser Gln Pro Asn Leu Thr Val Ser Trp Asp Pro Arg Ile Ser Ser Glu Gln Asp Tyr Thr Cys Ile Ala Glu Asn Ala Val Ser Asn Leu Ser Phe Ser Val Ser Ala Gln Lys Leu Cys Glu Asp Val Lys Ile Gln Tyr Thr Asp Thr Lys Met Ile Leu Phe Met Val Ser Gly Ile Cys Ile Val Phe Gly Phe Ile Ile Leu Leu Leu Leu Val Leu Arg Lys Arg Arg Asp Ser Leu Ser Leu Ser Thr Gln Arg Thr Gln Gly Pro Ala Glu Ser Ala Arg Asn Leu Glu Tyr Val Ser Val Ser Pro Thr Asn Asn Thr Val Tyr Ala Ser Val Thr His Ser Asn Arg Glu Thr Glu Ile Trp Thr Pro Arg Glu Asn Asp Thr Ile Thr Ile Tyr Ser Thr Ile Asn His Ser Lys Glu Ser Lys Pro Thr Phe Ser Arg Ala Thr Ala Leu Asp Asn Val Val <210> 5 <211> 332 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 8518269CD1 <400> 5 Met Leu Trp Leu Phe Gln Ser Leu Leu Phe Val Phe Cys Phe Gly Pro Gly Asn Val Val Ser Gln Ser Ser Leu Thr Pro Leu Met Val Asn Gly Ile Leu Gly Glu Ser Val Thr Leu Pro Leu Glu Phe Pro Ala Gly Glu Lys Val Asn Phe Ile Thr Trp Leu Phe Asn Glu Thr Ser Leu Ala Phe Ile Val Pro His Glu Thr Lys Ser Pro Glu Ile His Val Thr Asn Pro Lys Gln Gly Lys Arg Leu Asn Phe Thr Gln Ser Tyr Ser Leu Gln Leu Ser Asn Leu Lys Met Glu Asp Thr Gly Ser Tyr Arg Ala Gln Ile Ser Thr Lys Thr Ser Ala Lys Leu Ser Ser Tyr Thr Leu Arg Ile Leu Arg Gln Leu Arg Asn Ile Gln Val Thr Asn His Ser Gln Leu Phe Gln Asn Met Thr Cys Glu Leu His Leu Thr Cys Ser Val Glu Asp Ala Asp Asp Asn Val Ser Phe Arg Trp Glu Ala Leu Gly Asn Thr Leu Ser Ser Gln Pro Asn Leu Thr Val Ser Trp Asp Pro Arg Ile Ser Ser Glu Gln Asp Tyr Thr Cys Ile Ala Glu Asn Ala Val Ser Asn Leu Ser Phe Ser Val Ser Ala Gln Lys Leu Cys Glu Asp Val Lys Ile Gln Tyr Thr Asp Thr Lys Met Ile Leu Phe Met Val Ser Gly Ile Cys Ile Val Phe Gly Phe Ile Ile Leu Leu Leu Leu Val Leu Arg Lys Arg Arg Asp Ser Leu Ser Leu Ser Thr Gln Arg Thr Gln Gly Pro Ala Glu Ser Ala Arg Asn Leu Glu Tyr Val Ser Val Ser Pro Thr Asn Asn Thr Val Tyr Ala Ser Val Thr His Ser Asn Arg Glu Thr Glu Ile Trp Thr Pro Arg Glu Asn Asp Thr Ile Thr Ile Tyr Ser Thr Ile Asn His Ser Lys Glu Ser Lys Pro Thr Phe Ser Arg Ala Thr Ala Leu Asp Asn Val Val <210> 6 <211> 288 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1592646CD1 <400> 6 Met Leu Pro His Phe Leu Gly Gly Glu Arg Val Arg Pro Ser Pro Gly Ser Ser Ser Ser Gly Tyr Leu Pro Thr Met Ala Leu Val Leu Ile Leu Gln Leu Leu Thr Leu Trp Pro Leu Cys His Thr Asp Ile Thr Pro Ser Val Pro Pro Ala Ser Tyr His Pro Lys Pro Trp Leu Gly Ala Gln Pro Ala Thr Val Val Thr Pro Gly Val Asn Val Thr Leu Arg Cys Arg Ala Pro Gln Pro Ala Trp Arg Phe Gly Leu Phe Lys Pro Gly Glu Ile Ala Pro Leu Leu Phe Arg Asp Val Ser Ser Glu Leu Ala Glu Phe Phe Leu Glu Glu Val Thr Pro Ala Gln Gly Gly Ser Tyr Arg Cys Cys Tyr Arg Arg Pro Asp Trp Gly Pro Gly Val Trp Ser Gln Pro Ser Asp Val Leu Glu Leu Leu Val Thr Glu Glu Leu Pro Arg Pro Ser Leu Val Ala Leu Pro Gly Pro Val Val Gly Pro Gly Ala Asn Val Ser Leu Arg Cys Ala Gly Arg Leu Arg Asn Met Ser Phe Val Leu Tyr Arg Glu Gly Val Ala Ala Pro Leu Gln Tyr Arg His Ser Ala Gln Pro Trp Ala Asp Phe Thr Leu Leu Gly Ala Arg Ala Pro Gly Thr Tyr Ser Cys Tyr Tyr His Thr Pro Ser Ala Pro Tyr Val Leu Ser Gln Arg Ser Glu Val Leu Val Ile Ser Trp Glu Asp Ser Gly Ser Ser Asp Tyr Thr Arg Gly Asn Leu Val Arg Leu Gly Leu Ala Gly Leu Val Leu Ile Ser Leu Gly Ala Leu Val Thr Phe Asp Trp Arg Ser Gln Asn Arg Ala Pro Ala Gly Ile Arg Pro <210> 7 <211> 1450 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500191CD1 <400> 7 Met Ala Ala Glu Arg Gly Ala Arg Arg Leu Leu Ser Thr Pro Ser Phe Trp Leu Tyr Cys Leu Leu Leu Leu Gly Arg Arg Ala Pro Gly Ala Ala Ala Ala Arg Ser Gly Ser Ala Pro Gln Ser Pro Gly Ala Ser Ile Arg Thr Phe Thr Pro Phe Tyr Phe Leu Val Glu Pro Val Asp Thr Leu Ser Val Arg Gly Ser Ser Val Ile Leu Asn Cys Ser Ala Tyr Ser Glu Pro Ser Pro Lys Ile Glu Trp Lys Lys Asp Gly Thr Phe Leu Asn Leu Val Ser Asp Asp Arg Arg Gln Leu Leu Pro Asp Gly Ser Leu Phe Ile Ser Asn Val Val His Ser Lys His Asn Lys Pro Asp Glu Gly Tyr Tyr Gln Cys Val Ala Thr Val Glu Ser Leu Gly Thr Ile Ile Ser Arg Thr Ala Lys Leu Ile Val Ala Gly Leu Pro Arg Phe Thr Ser Gln Pro Glu Pro Ser Ser Val Tyr Ala Gly Asn Asn Ala Ile Leu Asn Cys Glu Val Asn Ala Asp Leu Val Pro Phe Val Arg Trp Glu Gln Asn Arg Gln Pro Leu Leu Leu Asp Asp Arg Val Ile Lys Leu Pro Ser Gly Met Leu Val Ile Ser Asn Ala Thr Glu Gly Asp Gly Gly Leu Tyr Arg Cys Val Val Glu Ser Gly Gly Pro Pro Lys Tyr Ser Asp Glu Val Glu Leu Lys Val Leu Pro Asp Pro Glu Val Ile Ser Asp Leu Val Phe Leu Lys Gln Pro Ser Pro Leu Val Arg Val Ile Gly Gln Asp Val Val Leu Pro Cys Val Ala Ser Gly Leu Pro Thr Pro Thr Ile Lys Trp Met Lys Asn Glu Glu Ala Leu Asp Thr Glu Ser Ser Glu Arg Leu Val Leu Leu Ala Gly Gly Ser Leu Glu Ile Ser Asp Val Thr Glu Asp Asp Ala Gly Thr Tyr Phe Cys Ile Ala Asp Asn Gly Asn Glu Thr Ile Glu Ala Gln Ala Glu Leu Thr Val Gln Ala Gln Pro Glu Phe Leu Lys Gln Pro Thr Asn Ile Tyr Ala His Glu Ser Met Asp Ile Val Phe Glu Cys Glu Val Thr Gly Lys Pro Thr Pro Thr Val Lys Trp Val Lys Asn Gly Asp Met Val Ile Pro Ser Asp Tyr Phe Lys Ile Val Lys Glu His Asn Leu Gln Val Leu Gly Leu Val Lys Ser Asp Glu Gly Phe Tyr Gln Cys Ile Ala Glu Asn Asp Val Gly Asn Ala Gln Ala Gly Ala Gln Leu Ile Ile Leu Glu His Ala Pro Ala Thr Thr Gly Pro Leu Pro Ser Ala Pro Arg Asp Val Val Ala Ser Leu Val Ser Thr Arg Phe Ile Lys Leu Thr Trp Arg Thr Pro Ala Ser Asp Pro His Gly Asp Asn Leu Thr Tyr Ser Val Phe Tyr Thr Lys Glu Gly Ile Ala Arg Glu Arg Val Glu Asn Thr Ser His Pro Gly Glu Met Gln Val Thr Ile Gln Asn Leu Met Pro Ala Thr Val Tyr Ile Phe Arg Val Met Ala Gln Asn Lys His Gly Ser Gly Glu Ser Ser Ala Pro Leu Arg Val Glu Thr Gln.Pro Glu Val Gln Leu Pro Gly Pro Ala Pro Asn Leu Arg Ala Tyr Ala Ala Ser Pro Thr Ser Ile Thr Val Thr Trp Glu Thr Pro Val Ser Gly Asn Gly Glu Ile Gln Asn Tyr Lys Leu Tyr Tyr Met Glu Lys Gly Thr Asp Lys Glu Gln Asp Val Asp Val Ser Ser His Ser Tyr Thr Ile Asn Gly Leu Lys Lys Tyr Thr Glu Tyr Ser Phe Arg Val Val Ala Tyr Asn Lys His Gly Pro Gly Val Ser Thr Pro Asp Val Ala Val Arg Thr Leu Ser Asp Val Pro Ser Ala Ala Pro Gln Asn Leu Ser Leu Glu Val Arg Asn Ser Lys Ser Ile Met Ile His Trp Gln Pro Pro Ala Pro Ala Thr Gln Asn Gly Gln Ile Thr Gly Tyr Lys Ile Arg Tyr Arg Lys Ala Ser Arg Lys Ser Asp Val Thr Glu Thr Leu Val Ser Gly Thr Gln Leu Ser Gln Leu Ile Glu Gly Leu Asp Arg Gly Thr Glu Tyr Asn Phe Arg Val Ala Ala Leu Thr Ile Asn Gly Thr Gly Pro Ala Thr Asp Trp Leu Ser Ala Glu Thr Phe Glu Ser Asp Leu Asp Glu Thr Arg Val Pro Glu Val Pro Ser Ser Leu His Val Arg Pro Leu Val Thr Ser Ile Val Val Ser Trp Thr Pro Pro Glu Asn Gln Asn Ile Val Val Arg Gly Tyr Ala Ile Gly Tyr Gly Ile Gly Ser Pro His Ala Gln Thr Ile Lys Val Asp Tyr Lys Gln Arg Tyr Tyr Thr Ile Glu Asn Leu Asp Pro Ser Ser His Tyr Val Ile Thr Leu Lys Ala Phe Asn Asn Val Gly Glu Gly Ile Pro Leu Tyr Glu Ser Ala Val Thr Arg Pro His Thr Asp Thr Ser Glu Val Asp Leu Phe Val Ile Asn Ala Pro Tyr Thr Pro Val Pro Asp Pro Thr Pro Met Met Pro Pro Val Gly Val Gln Ala Ser Ile Leu Ser His Asp Thr Ile Arg Ile Thr Trp Ala Asp Asn Ser Leu Pro Lys His Gln Lys Ile Thr Asp Ser Arg Tyr Tyr Thr Val Arg Trp Lys Thr Asn Ile Pro Ala Asn Thr Lys Tyr Lys Asn Ala Asn Ala Thr Thr Leu Ser Tyr Leu Val Thr Gly Leu Lys Pro Asn Thr Leu Tyr Glu Phe Ser Val Met Val Thr Lys Gly Arg Arg Ser Ser Thr Trp Ser Met Thr Ala His Gly Thr Thr Phe Glu Leu Val Pro Thr Ser Pro Pro Lys Asp Val Thr Val Val Ser Lys Glu Gly Lys Pro Lys Thr Ile Ile Val Asn Trp Gln Pro Pro Ser Glu Ala Asn Gly Lys Ile Thr Gly Tyr Ile Ile Tyr Tyr Ser Thr Asp Val Asn Ala Glu Ile His Asp Trp Val Ile Glu Pro Val Val Gly Asn Arg Leu Thr His Gln Ile Gln Glu Leu Thr Leu Asp Thr Pro Tyr Tyr Phe Lys Ile Gln Ala Arg Asn Ser Lys Gly Met Gly Pro Met Ser Glu Ala Val Gln Phe Arg Thr Pro Lys Ala Ser Gly Ser Gly Gly Lys Gly Ser Arg Leu Pro Asp Leu Gly Ser Asp Tyr Lys Pro Pro Met Ser Gly Ser Asn Ser Pro His Gly Ser Pro Thr Ser Pro Leu Asp Ser Asn Met Leu Leu Val Ile Ile Val Ser Val Gly Val Ile Thr Ile Val Val Val Val Ile Ile Ala Val Phe Cys Thr Arg Arg Thr Thr Ser His Gln Lys Lys Lys Arg Ala Ala Cys Lys Ser Val Asn Gly Ser His Lys Tyr Lys Gly Asn Ser Lys Asp Val Lys Pro Pro Asp Leu Trp Ile His His Glu Arg Leu Glu Leu Lys Pro Ile Asp Lys Ser Pro Asp Pro Asn Pro Ile Met Thr Asp Thr Pro Ile Pro Arg Asn Ser Gln Asp Ile Thr Pro Val Asp Asn Ser Met Asp Ser Asn Ile His Gln Arg Arg Asn Ser Tyr Arg Gly His Glu Ser Glu Asp Ser Met Ser Thr Leu Ala Gly Arg Arg Gly Met Arg Pro Lys Met Met Met Pro Phe Asp Ser Gln Pro Pro Gln Pro Val Ile Ser Ala His Pro Ile His Ser Leu Asp Asn Pro His His His Phe His Ser Ser Ser Leu Ala Ser Pro Ala Arg Ser His Leu Tyr His Pro Gly Ser Pro Trp Pro Ile Gly Thr Ser Met Ser Leu Ser Asp Arg Ala Asn Ser Thr Glu Ser Val Arg Asn Thr Pro Ser Thr Asp Thr Met Pro Ala Ser Ser Ser Gln Thr Cys Cys Thr Asp His Gln Asp Pro Glu Gly Ala Thr Ser Ser Ser Tyr Leu Ala Ser Ser Gln Glu Glu Asp Ser Gly Gln Ser Leu Pro Thr Ala His Val Arg Pro Ser His Pro Leu Lys Ser Phe Ala Val Pro Ala Ile Pro Pro Pro Gly Pro Pro Thr Tyr Asp Pro Ala Leu Pro Ser Thr Pro Leu Leu Ser Gln Gln Ala Leu Asn His His Ile His Ser Val Lys Thr Ala Ser Ile Gly Thr Leu Gly Arg Ser Arg Pro Pro Met Pro Val Val Val Pro Ser Ala Pro Glu Val Gln Glu Thr Thr Arg Met Leu Glu Asp Ser Glu Ser Ser Tyr Glu Pro Asp Glu Leu Thr Lys Glu Met Ala His Leu Glu Gly Leu Met Lys Asp Leu Asn Ala Ile Thr Thr Ala <210> 8 <211> 551 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500099CD1 <400> 8 Met Val Ala Pro Lys Ser His Thr Asp Asp Trp Ala Pro Gly Pro 1 5 10 ~ 15 Phe Ser Ser Lys Pro Gln Arg Ser Gln Leu Gln Ile Phe Ser Ser Val Leu Gln Thr Ser Leu Leu Phe Leu Leu Met Gly Leu Arg Ala Ser Gly Lys Asp Ser Ala Pro Thr Val Val Ser Gly Ile Leu Gly Gly Ser Val Thr Leu Pro Leu Asn Ile Ser Val Asp Thr Glu Ile Glu Asn Val Ile Trp Ile Gly Pro Lys Asn Ala Leu Ala Phe Ala Arg Pro Lys Glu Asn Val Thr Ile Met Val Lys Ser Tyr Leu Gly Arg Leu Asp Ile Thr Lys Trp Ser Tyr Ser Leu Cys Ile Ser Asn Leu Thr Leu Asn Asp Ala Gly Ser Tyr Lys Ala Gln Ile Asn Gln Arg Asn Phe Glu Val Thr Thr Glu Glu Glu Phe Thr Leu Phe Val Tyr Glu Gln Leu Gln Glu Pro Gln Val Thr Met Lys Ser Val Lys Val Ser Glu Asn Phe Ser Cys Asn Ile Thr Leu Met Cys Ser Val Lys Gly Ala Glu Lys Ser Val Leu Tyr Ser Trp Thr Pro Arg Glu Pro His Ala Ser Glu Ser Asn Gly Gly Ser Ile Leu Thr Val Ser Arg Thr Pro Cys Asp Pro Asp Leu Pro Tyr Ile Cys Thr Ala Gln Asn Pro Val Ser Gln Arg Ser Ser Leu Pro Val His Val Gly Gln Phe Cys Thr Asp Pro Gly Ala Ser Arg Gly Gly Thr Thr Gly Glu Thr Val Val Gly Val Leu Gly Glu Pro Val Thr Leu Pro Leu Ala Leu Pro Ala Cys Arg Asp Thr Glu Lys Val Val Trp Leu Phe Asn Thr Ser Ile Ile Ser Lys Glu Arg Glu Glu Ala Ala Thr Ala Asp Pro Leu Ile Lys Ser Arg Asp Pro Tyr Lys Asn Arg Val Trp Val Ser Ser Gln Asp Cys Ser Leu Lys Ile Ser Gln Leu Lys Ile Glu Asp Ala Gly Pro Tyr His Ala Tyr Val Cys Ser Glu Ala Ser Ser Val Thr Ser Met Thr His Val Thr Leu Leu Ile Tyr Arg Pro Glu Arg Asn Thr Lys Leu Trp Ile Gly Leu Phe Leu Met Val Cys Leu Leu Cys Val Gly Ile Phe Ser Trp Cys Ile Trp Lys Arg Lys Gly Arg Cys Ser Val Pro Ala Phe Cys Ser Ser Gln Ala Glu Ala Pro Ala Asp Thr Pro Gly Tyr Glu Lys Leu Asp Thr Pro Leu Arg Pro Ala Arg Gln Gln Pro Thr Pro Thr Ser Asp Ser Ser Ser Asp Ser Asn Leu Thr Thr Glu Glu Asp Glu Asp Arg Pro Glu Val His Lys Pro Ile Ser Gly Arg Tyr Glu Val Phe Asp Gln Val Thr Gln Glu Gly Ala Gly His Asp Pro Ala Pro Glu Gly Gln Ala Asp Tyr Asp Pro Val Thr Pro Tyr Val Thr Glu Val Glu Ser Val Val Gly Glu Asn Thr Met Tyr Ala Gln Val Phe Asn Leu Gln Gly Lys Thr Pro Val Ser Gln Lys Glu Glu Ser Ser Ala Thr Ile Tyr Cys Ser Ile Arg Lys Pro Gln Val Val Pro Pro Pro Gln Gln Asn Asp Leu Glu Ile Pro Glu Ser Pro Thr Tyr Glu Asn Phe Thr <210> 9 <211> 336 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7682434CD1 <400> 9 Met Pro Pro Pro Ala Pro Gly Ala Arg Leu Arg Leu Leu Ala Ala Ala Ala Leu Ala Gly Leu Ala Val Ile Ser Arg Gly Leu Leu Ser Gln Ser Leu Glu Phe Asn Ser Pro Ala Asp Asn Tyr Thr Val Cys Glu Gly Asp Asn Ala Thr Leu Ser Cys Phe Ile Asp Glu His Val Thr Arg Val Ala Trp Leu Asn Arg Ser Asn Ile Leu Tyr Ala Gly Asn Asp Arg Trp Thr Ser Asp Pro Arg Val Arg Leu Leu Ile Asn Thr Pro Glu Glu Phe Ser Ile Leu Ile Thr Glu Val Gly Leu Gly Asp Glu Gly Leu Tyr Thr Cys Ser Phe Gln Thr Arg His Gln Pro Tyr Thr Thr Gln Val Tyr Leu Ile Val His Val Pro Ala Arg Ile Val Asn Ile Ser Ser Pro Val Thr Val Asn Glu Gly Gly Asn Val Asn Leu Leu Cys Leu Ala Val Gly Arg Pro Glu Pro Thr Val Thr Trp Arg Gln Leu Arg Asp Gly Phe Thr Ser Glu Gly Glu Ile Leu Glu Ile Ser Asp Ile Gln Arg Gly Gln Ala Gly Glu Tyr Glu Cys Val Thr His Asn Gly Val Asn Ser Ala Pro Asp Ser Arg Arg Val Leu Val Thr Val Asn Tyr Pro Pro Thr Ile Thr Asp Val Thr Ser Ala Arg Thr Ala Leu Gly Arg Ala Ala Leu Leu Arg Cys Glu Ala Met Ala Val Pro Pro Ala Asp Phe Gln Trp Tyr Lys Asp Asp Arg Leu Leu Ser Ser Gly Thr Ala Glu Gly Leu Lys Val Gln Thr Glu Arg Thr Arg Ser Met Leu Leu Phe Ala Asn Val Ser Ala Arg His Tyr Gly Asn Tyr Thr Cys Arg Ala Ala Asn Arg Leu Gly Ala Ser Ser Ala Ser Met Arg Leu Leu Arg Pro Gly Ser Leu Glu Asn Ser Ala Pro Arg Pro Pro Gly Leu Leu Ala Leu Leu Ser Ala Leu Gly Trp Leu Trp Trp Arg Met <210> 10 <211> 241 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 2202389CD1 <400> 10 Met Lys Thr Leu Pro Ala Met Leu Gly Thr Gly Lys Leu Phe Trp Val Phe Phe Leu Ile Pro Tyr Leu Asp Ile Trp Asn Ile His Gly Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr Cys Val Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn Ile Ser Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn Gly Ser Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser His Ser Thr Thr Leu Tyr Val Thr Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val Tyr Ala Ser Leu Asn His Ser Val Ile Gly Leu Asn Ser Arg Leu Ala Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg Ser <210> 11 <211> 766 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503597CD1 <400> 11 Met Lys Pro Phe Gln Leu Asp Leu Leu Phe Val Cys Phe Phe Leu Phe Ser Gln Glu Leu Gly Leu Gln Lys Arg Gly Cys Cys Leu Val Leu Gly Tyr Met Ala Lys Asp Lys Phe Arg Arg Met Asn Glu Gly Gln Val Tyr Ser Phe Ser Gln Gln Pro Gln Asp Gln Val Val Val Ser Gly Gln Pro Val Thr Leu Leu Cys Ala Ile Pro Glu Tyr Asp Gly Phe Val Leu Trp Ile Lys Asp Gly Leu Ala Leu Gly Val Gly Arg Asp Leu Ser Ser Tyr Pro Gln Tyr Leu Val Val Gly Asn His Leu Ser Gly Glu His His Leu Lys Ile Leu Arg Ala Glu Leu Gln Asp Asp Ala Val Tyr Glu Cys Gln Ala Ile Gln Ala Ala Ile Arg Ser Arg Pro Ala Arg Leu Thr Val Leu Val Pro Pro Asp Asp Pro Val Ile Leu Gly Gly Pro Val Ile Ser Leu Arg Ala Gly Asp Pro Leu Asn Leu Thr Cys His Ala Asp Asn Ala Lys Pro Ala Ala Ser Ile Ile Trp Leu Arg Lys Gly Glu Val Ile Asn Gly Ala Thr Tyr Ser Lys Thr Leu Leu Arg Asp Gly Lys Arg Glu Ser Ile Val Ser Thr Leu Phe Ile Ser Pro Gly Asp Val Glu Asn Gly Gln Ser Ile Val Cys Arg Ala Thr Asn Lys Ala Ile Pro Gly Gly Lys Glu Thr Ser Val Thr Ile Asp Ile Gln His Pro Pro Leu Val Asn Leu Ser Val Glu Pro Gln Pro Val Leu Glu Asp Asn Val Val Thr Phe His Cys Ser Ala Lys Ala Asn Pro Ala Val Thr Gln Tyr Arg Trp Ala Lys Arg Gly Gln Ile Ile Lys Glu Ala Ser Gly Glu Val Tyr Arg Thr Thr Val Asp Tyr Thr Tyr Phe Ser Glu Pro Val Ser Cys Glu Val Thr Asn Ala Leu Gly Ser Thr Asn Leu Ser Arg Thr Val Asp Val Tyr Phe Gly Pro Arg Met Thr Thr Glu Pro Gln Ser Leu Leu Val Asp Leu Gly Ser Asp Ala Ile Phe Ser Cys Ala Trp Thr Gly Asn Pro Ser Leu Thr Ile Val Trp Met Lys Arg Gly Ser Gly Val Val Leu Ser Asn Glu Lys Thr Leu Thr Leu Lys Ser Val Arg Gln Glu Asp Ala Gly Lys Tyr Val Cys Arg Ala Val Val Pro Arg Val Gly Ala Gly Glu Arg Glu Val Thr Leu Thr Val Asn Gly Pro Pro Ile Ile Ser Ser Thr Gln Thr Gln His Ala Leu His Gly Glu Lys Gly Gln Ile Lys Cys Phe Ile Arg Ser Thr Pro Pro Pro Asp Arg Ile Ala Trp Ser Trp Lys Glu Asn Val Leu Glu Ser Gly Thr Ser Gly Arg Tyr Thr Val Glu Thr Ile Ser Thr Glu Glu Gly Val Ile Ser Thr Leu Thr Ile Ser Asn Ile Val Arg Ala Asp Phe Gln Thr Ile Tyr Asn Cys Thr Ala Trp Asn Ser Phe Gly Ser Asp Thr Glu Ile Ile Arg Leu Lys Glu Gln Glu Ser Val Pro Met Ala Val Ile Ile Gly Val Ala Val Gly Ala Gly Val Ala Phe Leu Val Leu Met, Ala Thr Ile Val Ala Phe Cys Cys Ala Arg Ser Gln Arg Asn Leu Lys Gly Val Val Ser Ala Lys Asn Asp Ile Arg Val Glu Ile Val His Lys Glu Pro Ala Ser Gly Arg Glu Gly Glu Glu His Ser Thr Ile Lys Gln Leu Met Met Asp Arg Gly Glu Phe Gln Gln Asp Ser Val Leu Lys Gln Leu Glu Val Leu Lys Glu Glu Glu Lys Glu Phe Gln Asn Leu Lys Asp Pro Thr Asn Gly Tyr Tyr Ser Val Asn Thr Phe Lys Glu His His Ser Thr Pro Thr Ile Ser Leu Ser Ser Cys Gln Pro Asp Leu Arg Pro Ala Gly Lys Gln Arg Val Pro Thr Gly Met Ser Phe Thr Asn Ile Tyr Ser Thr Leu Ser Gly Gln Gly Arg Leu Tyr Asp Tyr Gly Gln Arg Phe Val Leu Gly Met Gly Ser Ser Ser Ile Glu Leu Cys Glu Arg Glu Phe Gln Arg Gly Ser Leu Ser Asp Ser Ser Ser Phe Leu Asp Thr Gln Cys Asp Ser Ser Val Ser Ser Ser Gly Lys Gln Asp Gly Tyr Val Gln Phe Asp Lys Ala Ser Lys Ala Ser Ala Ser Ser Ser His His Ser Gln Ser Ser Ser Gln Asn Ser Asp Pro Ser Arg Pro Leu Gln Arg Arg Met Gln Thr His Val <210> 12 <211> 88 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503603CD1 <400> 12 Met Asp Gly Glu Ala Thr Val Lys Pro Gly Glu Gln Lys Glu Val Val Arg Arg Gly Arg Glu Val Asp Tyr Ser Arg Leu Ile Ala Gly Thr Leu Pro Gln Ser His Val Leu Leu Ser Pro Phe His Lys Lys Asp Pro Ile Arg Asp Gly Cys Gly Arg Ala Leu Ser Pro Pro Gly Pro Ile Ser Gly Pro Trp Glu His Ser Gly Leu Pro Arg Pro Ser Ala Gly Gly Arg Arg Ala Pro Leu Gln Leu Gln Ile His <210> 13 <211> 2691 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3855123CB1 <400> 13 ctccactggt caacccttct cggtggagcc acagccaagt gctggaggac atacgtcgtc 60 actttccact gctcttgcaa aggccaaccc agctgtcacc cagtacaggt ggccaatgcg 120 gggccagatc atcaaggagg catctggaga ggtgtacagg accacagtgg actacacgta 180 cttctcagag cccgtctcct gtgaggtgac caacgcctgg gcagcaccaa cctcagccgc 240 acggttgacg tctactttgg gccccggatg accacagaac cccaatcctt gctcgtggat 300 ctgggctctg atgccatctt cagctgcgcc tggaccggca acccatccct gaccatcgtc 360 tggatgaagc ggggctccgg agtggtcctg agcaatgaga agaccctgac cctcaaatcc 420 gtgcgccagg aggacgcggg caagtacgtg tgccgggctg tggtgccccg tgtgggagcc 480 ggggagagag aggtgaccct gaccgtcaat ggacccccca tcatctccag cacccagacc 540 cagcacgccc tccacggcga gaagggccag atcaagtgct tcatccggag cacgccgccg 600 ccggaccgca tcgcctggtc ctggaaggag aacgttctgg agtcgggcac atcggggcgc 660 tatacggtgg agaccatcag caccgaggag ggcgtcatct ccaccctgac catcagcaac 720 atcgtgcggg ccgacttcca gaccatctac aactgcacgg cctggaacag cttcggctcc 780 gacactgaga tcatccggct caaggagcaa ggttcggaaa tgaagtcggg agccgggctg 840 gaagcagagt ctgtgccgat ggccgtcatc attggggtgg ccgtaggagc tggtgtggcc 900 ttcctcgtcc ttatggcaac catcgtggcg ttctgctgtg cccgttccca gagaaatctc 960 aaaggtgttg tgtcagccaa aaatgatatc cgagtggaaa ttgtccacaa ggaaccagcc 1020 tctggtcggg agggtgagga gcactccacc atcaagcagc tgatgatgga ccggggtgaa 1080 ttccagcaag actcagtcct gaaacagctg gaggtcctca aagaagagga gaaagagttt 1140 cagaacctga aggaccccac caatggctac tacagcgtca acaccttcaa agagcaccac 1200 tcaaccccga ccatctccct ctccagctgc cagcccgacc tgcgtcctgc gggcaagcag 1260 cgtgtgccca caggcatgtc cttcaccaac atctacagca ccctgagcgg ccagggccgc 1320 ctctacgact acgggcagcg gtttgtgctg ggcatgggca gctcgtccat cgagctttgt 1380 gagcgggagt tccagagagg ctccctcagc gacagcagct ccttcctgga cacgcagtgt 1440 gacagcagcg tcagcagcag cggcaagcag gatggctatg tgcagttcga caaggccagc 1500 aaggcttctg cttcctcctc ccaccactcc cagtcctcgt cccagaactc tgaccccagt 1560 cgacccctgc agcggcggat gcagactcac gtctaaggat cacacaccgc gggtggggac 1620 gggccaggga agaggtcagg gcacgttctg gttgtccagg gacgaggggt actttgcaga 1680 ggacaccaga attggccact tccaggacag cctcccagcg cctctgccac tgccttcctt 1740 cgaagctctg atcaagcaca aatctgggtc cccaggtgct gtgtgccaga ggtgggcggg 1800 tggggagaca gacagaggct gcggctgagt gcgctgtgct tagtgctgga cacccgtgtc 1860 cccggccctt tcctggaggc ccctctacca cctgctctgc ccacaggcac aagtggcagc 1920 tataactctg ctttcatgaa actgcggtcc actctctggt ctctctgtgg gctctacccc 1980 tcactgacca caagctctac ctacccctgt gcctgtgctc ccatacagcc ctggggagaa 2040 ggggatgacg tcttcccagc actgagctgc cccagaaacc ccggctcccc actgctgctc 2100 atagcccata ccctggaggc tgacaagcca gaaatggcct tggctaaagg agcctctctc 2160 tcaccaggct ggccgggagc ccacccccaa tttgtttggt gttttgtgtc catactcttg 2220 cagttctgtc cttggacttg atgccgctga actctgcggt gggaccggtc ccgtcagagc 2280 ctggtgtact ggggggaggg agggaggagg gagcctgtgc tgacggagca cctcgccggg 2340 tgtgcccctc ctgggctgtg tgaccccagc ctccccaccc acctcctgct ttgtgtactc 2400 ctcccctccc cctcagcaca atcggagttc atataagaag tgcgggagct tctctggtca 2460 gggttctctg aacacttatg gagagagtgc ttcctgggaa gtgtggcgtt tgaaggggct 2520 ggagggcagg tctttaagat ggcgagactg cccttctcag ctgataaaca caagaacggc 2580 gatcctgtct tcagtaaggc tccacgagaa gagaggaagt atatctacac ctcaaccctc 2640 ctagtcacca cctgaaataa atgttaggga cactacaaaa aaaaaaaaaa a 2691 <210> 14 <211> 2518 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4547188CB1 <400> 14 ggaaggatat ggatcaatgt tttctttttt gaagctactg ttaccactcc tggaaaagtt 60 cttcaggaat aagtgacagt aagaatgaca agggattagg actggcttcc tcttataaat 120 aataaaatcc aaagagaagt gacttgagtc tccaggttta aagaagagca actagaagtc 180 gtccaaacac ctgcatctca taaggagaag aaaagtccac ctggatcttg tttctggact 240 gagatggatg gagaggccac agtgaagcct ggagaacaaa aggaagtggt gaggagagga 300 agagaagtgg actactccag gctcattgct ggcactttac cacaatctca cgtcaccagc 360 aggagggcag gatggaaaat gcccctcttc ctcatactgt gcctgctaca aggttcttct 420 ttcgcccttc cacaaaaaag accccatccg agatggctgt gggagggctc tctcccctcc 480 aggacccatc tccgggccat gggaacactc aggccttcct cgcccctctg ctggcgggag 540 gagagctcct ttgcagctcc aaattcattg aagggctcaa ggctggtgtc aggggagcct 600 ggaggagctg tcaccatcca gtgccattat gccccctcat ctgtcaacag gcaccagagg 660 aagtactggt gctgtctggg gcccccaaga tggatctgcc agaccattgt gtccaccaac 720 cagtatactc accatcgcta tcgtgaccgt gtggccctca cagactttcc acagagaggc 780 ttgtttgtgg tgaggctgtc ccaactgtcc ccggatgaca tcggatgcta cctctgcggc 840 attggaagtg aaaacaacat gctgttctta agcatgaatc tgaccatctc tgcaggtccc 900 gccagcaccc tccccacagc cactccagct gctggggagc tcaccatgag atcctatgga 960 acagcgtctc cagtggccaa cagatggacc ccaggaacca cccagacctt aggacagggg 1020 acagcatggg acacagttgc ttccactcca ggaaccagca agactacagc ttcagctgag 1080 ggaagacgaa ccccaggagc aaccaggcca gcagctccag ggacaggcag ctgggcagag 1140 ggttctgtca aagcacctgc tccgattcca gagagtccac cttcaaagag cagaagcatg 1200 tccaatacaa cagaaggtgt ttgggagggc accagaagct cggtgacaaa cagggctaga 1260 gccagcaagg acaggaggga gatgacaact accaaggctg ataggccaag ggaggacata 1320 gagggggtca ggatagctct tgatgcagcc aaaaaggtcc taggaaccat tgggccacca 1380 gctctggtct cagaaacttt ggcctgggaa atcctcccac aagcaacgcc agtttctaag 1440 caacaatctc agggttccat tggagaaaca actccagctg caggcatgtg gaccttggga 1500 actccagctg cagatgtgtg gatcttggga actccagctg cagatgtgtg gaccagcatg 1560 gaggcagcat ctggggaagg aagcgctgca ggggacctag atgctgccac tggagacaga 1620 ggtccccaag caacactgag ccagaccccg gcagtaggac cctggggacc ccctggcaag 1680 gagtcctccg tgaagcgtac ttttccagaa gatgaaagca gctctcggac cctggctcct 1740 gtctctacca tgctggccct gtttatgctt atggctctgg ttctattgca aaggaagctc 1800 tggagaagga ggacctctca ggaggcagaa agggtcacct taattcagat gacacatttt 1860 ctggaagtga acccccaagc agaccagctg ccccatgtgg aaagaaagat gctccaggat 1920 gactctcttc ctgctggggc cagcctgact gccccagaga gaaatccagg accctgaggg 1980 acagagagat gaactgctca gttaccatgg gagaaggacc aagatcaaag gccttcagga 2040 ccccagcctc tttccatcat ccttcctcca cctgtgggaa gagaagctga tgcagccggt 2100 gctccaccca tggaagaaag gctggctgtc cttgggccca agaaagtcaa gcattatcca 2160 cgtccaaagg tgacaagatg actcaaagga gacttcaaga acagtgtatg aaacactgga 2220 agaggtcacc taggaaaagc atgaaatttc cattcctgaa tgtttgcaaa tagaagaggc 2280 ttccaatcag tgtggaaagt gacaaatccc ctatcaacac tcccagccct tgctgggggc 2340 tccttttctg actactgtta gcactcagcc tcccattcac atgtattata tttaagtgta 2400 ccagccttgc cttctcaagt agattctaag ctcctttaag gcagtaattg cattttatct 2460 gtctcatgat gcccccagag aacttccaac tcagtagacc ccaataatac ctgtgtgc 2518 <210> 15 <211> 1522 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3939883CB1 <400> 15 aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg gctaagaaaa 60 agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg accccgtctt 120 ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg agcatatttt 180 cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca aatgaattcc 240 cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat gtatctatgt 300 tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga cgtaagacac 360 acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc atcacagaag 420 cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca aaatacagtc 480 gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac attatggtca 540 ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat ggctcgctgc 600 ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt tccaagtatg 660 acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag gaagagtata 720 ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct gtcaccatgc 780 ttccagcaag actcagtcct gaaacagctg gaggtcctca aagaagagga cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca gggttattac 840 tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa tacaaaacaa 900 gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg gaagttggaa 960 tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa gtgggatcca 1020 ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta cagtatgcca 1080 cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct tataaatctg 1140 gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca tctcaggatg 1200 gagtctcact ctgttgccca ggctggagtt cagtggcgcg atcttggctc acttcaatct 1260 ccatcttccc agttcaagcg attctcatgc ctcgacctcc cgagtagctg ggattgcagg 1320 tgcccgctac cacgcccagc taatttttgt atttttagta gagatggggt ttcactatgg 1380 tggccaggct ggtcttgaac tcctgacctc agatgatctg cctgcctcgg cctcccaaag 1440 tgctggaact acaggcctga gccaccgtgc ccggccctga atcgctttag taagtaaagg 1500 gtctccaaga ataaaaaaaa as 1522 <210> 16 <211> 1084 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3163819CB1 <400> 16 ggaaagcatg ttgtggctgt tccaatcgct cctgtttgtc ttctgctttg gcccaggaca 60 actgaggaac atacaagtta ccaatcacag tcagctattt cagaatatga cctgtgagct 120 ccatctgact tgctctgtgg aggatgcaga tgacaatgtc tcattcagat gggaggcctt 180 gggaaacaca ctttcaagtc agccaaacct cactgtctcc tgggacccca ggatttccag 240 tgaacaggac tacacctgca tagcagagaa tgctgtcagt aatttatcct tctctgtctc 300 tgcccagaag ctttgcgaag atgttaaaat tcaatataca gataccaaaa tgattctgtt 360 tatggtttct gggatatgca tagtcttcgg tttcatcata ctgctgttac ttgttttgag 420 gaaaagaaga gattccctat ctttgtctac tcagcgaaca cagggccccg cagagtccgc 480 aaggaaccta gagtatgttt cagtgtctcc aacgaacaac actgtgtatg cttcagtcac 540 tcattcaaac agggaaacag aaatctggac acctagagaa aatgatacta tcacaattta 600 ctccacaatt aatcattcca aagagagtaa acccactttt tccagggcaa ctgcccttga 660 caatgtcgtg taagttgctg aaaggcctca gaggaattcg ggaatgacac gtcttctgat 720 cccatgagac agaacaaaga acaggaagct tggttcctgt tgttcctggc aacagaattt 780 gaatatctag gataggatga tcacctccag tccttcggac ttaaacctgc ctacctgagt 840 caaacaccta aggataacat catttccagc atgtggttca aataatattt tccaatccac 900 ttcaggccaa aacatgctaa agataacaca ccagcacatt gactctctct ttgataacta 960 agcaaatgga attatggttg acagagagtt tatgatccag aagacaacca cttctctcct 1020 tttagaaagc agcaggattg acttattgag aaataatgca gtgtgttggt tacatgtgta 1080 gtct 1084 <210> 17 <211> 1463 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 8518269CB1 <400> 17 caaaaacatt gactgcctca aggtctcaag caccagtctt caccgcggaa agcatgttgt 60 ggctgttcca atcgctcctg tttgtcttct gctttggccc agggaatgta gtttcacaaa 120 gcagcttaac cccattgatg gtgaacggga ttctggggga gtcagtaact cttcccctgg 180 agtttcctgc aggagagaag gtcaacttca tcacttggct tttcaatgaa acatctcttg 240 ccttcatagt accccatgaa accaaaagtc cagaaatcca cgtgactaat ccgaaacagg 300 gaaagcgact gaacttcacc cagtcctact ccctgcaact cagcaacctg aagatggaag 360 acacaggctc ttacagagcc cagatatcca caaagacctc tgcaaagctg tccagttaca 420 ctctgaggat attaagacaa ctgaggaaca tacaagttac caatcacagt cagctatttc 480 agaatatgac ctgtgagctc catctgactt gctctgtgga ggatgcagat gacaatgtct 540 cattcagatg ggaggccttg ggaaacacac tttcaagtca gccaaacctc actgtctcct 600 gggaccccag gatttccagt gaacaggact acacctgcat agcagagaat gctgtcagta 660 atttatcctt ctctgtctct gcccagaagc tttgcgaaga tgttaaaatt caatatacag 720 ataccaaaat gattctgttt atggtttctg ggatatgcat agtcttcggt ttcatcatac 780 tgctgttact tgttttgagg aaaagaagag attccctatc tttgtctact cagcgaacac 840 agggccccgc agagtccgca aggaacctag agtatgtttc agtgtctcca acgaacaaca 900 ctgtgtatgc ttcagtcact cattcaaaca gggaaacaga aatctggaca cctagagaaa 960 atgatactat cacaatttac tccacaatta atcattccaa agagagtaaa cccacttttt 1020 ccagggcaac tgcccttgac aatgtcgtgt aagttgctga aaggcctcag aggaattcgg 1080 gaatgacacg tcttctgatc ccatgagaca gaacaaagaa caggaagctt ggttcctgtt 1140 gttcctggca acagaatttg aatatctagg ataggatgat cacctccagt ccttcggact 1200 taaacctgcc tacctgagtc aaacacctaa ggataacatc atttccagca tgtggttcaa 1260 ataatatttt ccaatccact tcaggccaaa acatgctaaa gataacacac cagcacattg 1320 actctctctt tgataactaa gcaaatggaa ttatggttga cagagagttt atgatccaga 1380 agacaaccac ttctctcctt ttagaaagca gcaggattga cttattgaga aataatgcag 1440 tgtgttggtt acatgtgtag tct 1463 <210> 18 <211> 1557 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1592646CB1 <400> 18 agcggggcac tcgcgcagaa caaagatgga gccgtggagt gccatagggc tatgacacag 60 tcccccacag gcccccacct cgatactgtc ttccgtaaat gaggatctgg gtctggtttt 120 ctgatgttgc ctcatttcct gggaggggag agggtgcgac caagccctgg ctccagctct 180 agcgggtatc tgcccaccat ggccctggtg ctgatcctcc agctgctgac cctctggcct 240 ctgtgtcaca cagacatcac tccgtctgtc cccccagctt cataccaccc taagccatgg 300 ctgggagctc agccggctac agttgtgacc cctggggtca acgtgacctt gagatgccgg 360 gcaccccaac ccgcttggag atttggactt ttcaagcctg gagagatcgc tccccttctc 420 ttccgggatg tgtcctccga gctggcagaa ttctttctgg aggaggtgac tccagcccaa 480 gggggaagtt accgctgctg ctaccgaagg ccagactggg ggccgggtgt ctggtcccag 540 cccagcgatg tcctggagct gctggtgaca gaggagctgc cgcggccgtc gctggtggcg 600 ctgcccgggc cggtggtggg tcctggcgcc aacgtgagcc tgcgctgcgc gggccgcctg 660 cggaacatga gcttcgtgct gtaccgcgag ggcgtggcgg ccccgctgca gtaccgccac 720 tccgcgcagc cctgggccga cttcacgctg ctgggcgccc gcgcccccgg cacctacagc 780 tgctactatc acacgccctc cgcgccctac gtgctgtcgc agcgcagcga ggtgctggtc 840 atcagctggg aagactctgg ctcctccgac tacacccggg ggaacctagt ccgcctgggg 900 ctggccgggc tggtcctcat ctccctgggc gcgctggtca cttttgactg gcgcagtcag 960 aaccgcgctc ctgctggtat ccgcccctga gccccaggag cactgcagcc cgagacttcc 1020 aacctgagtg gcggagaagc tgggaccctg ggctggactg tcctttcctg cagccccaca 1080 gtcctgctgg ctgagctccg cggaacggtc cttagacccc gctgtgccct gtgctgtagc 1140 ttctttccag gcctttccca aggagtagct gaaaggaaga cgcgattagt ggttaagact 1200 tccaagccag aagacagagg gttcgaatcc cagcactgcc gtctactcac tgtagtagta 1260 gcagctacag aaaggtagta gtgagacgtg aagccagctg gacttcctgg gttgaatggg 1320 gacctggaga acttttctgt cttacaagag gattgtaaaa tggaccaatc agcactctgt 1380 aagatggacc aatcagcgct ctgtaaaatg gaccaatcag caggacatgg gcggggacaa 1440 taagggaata aaagctggcg agcgcggcac cccaccagag tctgcttcca cgctgtggga 1500 gctttgttct cttgctctac acaataaatc ttgctgctgc taaaaaaaaa aaaaagg 1557 <210> 19 <211> 5553 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500191CB1 <400> 19 tgcggccgcg ggagccgagc ttgcagcgag ggaccggctg aggcgcgcgg gagggaagga 60 ggcaagggct ccgcggcgct gtcgccgccg ctgccgctca ctctcgggga agagatggcg 120 gcggagcggg gagcccggcg actcctcagc accccctcct tctggctcta ctgcctgctg 180 ctgctcgggc gccgggcgcc gggcgccgcg gccgccagga gcggctccgc gccgcagtcc 240 ccaggagcca gcattcgaac gttcactcca ttttattttc tggtggagcc ggtggataca 300 ctctcagtta gaggctcttc tgttatatta aactgttcag catattctga gccttctcca 360 aaaattgaat ggaaaaaaga tggaactttt ttaaacttag tatcagatga tcgacgccag 420 cttctcccgg atggatcttt atttatcagc aatgtggtgc attccaaaca caataaacct 480 gatgaaggtt attatcagtg tgtggccact gttgagagtc ttggaactat tatcagtaga 540 acagcgaagc tcatagtagc aggtcttcca agatttacca gccaaccaga accttcctca 600 gtttatgctg ggaacaatgc aattctgaat tgtgaagtta atgcagattt ggtcccattt 660 gtgaggtggg aacagaacag acaacccctt cttctggatg atagagttat caaacttcca 720 agtggaatgc tggttatcag caatgcaact gaaggagatg gcgggcttta tcgctgcgta 780 gtggaaagtg gtgggccacc aaagtatagt gatgaagttg aattgaaggt tcttccagat 840 cctgaggtga tatcagactt ggtatttttg aaacagcctt ctcccttagt cagagtcatt 900 ggtcaggatg tagtgttgcc atgtgttgct tcaggacttc ctactccaac cattaaatgg 960 atgaaaaatg aggaggcact tgacacagaa agctctgaaa gattggtatt gctggcaggt 1020 ggtagcctgg agatcagtga tgttactgag gatgatgctg ggacttattt ttgtatagct 1080 gataatggaa atgagacaat tgaagctcaa gcagagctta cagtgcaagc tcaacctgaa 1140 ttcctgaagc agcctactaa tatatatgct cacgaatcta tggatattgt atttgaatgt 1200 gaagtgactg gaaaaccaac tccaactgtg aagtgggtca aaaatgggga tatggttatc 1260 ccaagtgatt attttaagat tgtaaaggaa cataatcttc aagttttggg tctggtgaaa 1320 tcagatgaag ggttctatca gtgcattgct gaaaatgatg ttggaaatgc acaagctgga 1380 gcccaactga taatccttga acatgcacca gccacaacgg gaccactgcc ttcagctcct 1440 cgggatgtcg tggcctccct ggtctctacc cgcttcatca aattgacgtg gcggacacct 1500 gcatcagatc ctcacggaga caaccttacc tactctgtgt tctacaccaa ggaagggatt 1560 gctagggaac gtgttgagaa taccagtcac ccaggagaga tgcaagtaac cattcaaaac 1620 ctaatgccag cgaccgtgta catctttaga gttatggctc aaaataagca tggctcagga 1680 gagagttcag ctccactgcg agtagaaaca caacctgagg ttcagctccc tggcccagca 1740 cctaaccttc gtgcatatgc agcttcgcct acctccatca ctgttacgtg ggaaacacca 1800 gtgtctggca atggggaaat tcagaattat aaattgtact acatggaaaa ggggactgat 1860 aaagaacagg atgttgatgt ttcaagtcac tcttacacca ttaatgggtt gaaaaaatat 1920 acagagtata gtttccgagt ggtggcctac aataaacatg gtcctggagt ttccacacca 1980 gatgttgctg ttcgaacatt gtcagatgtt cccagtgctg ctcctcagaa tctgtccttg 2040 gaagtgagaa attcaaagag tattatgatt cactggcagc cacctgctcc agccacacaa 2100 aatgggcaga ttactggcta caagattcgc taccgaaagg cctcccgaaa gagtgatgtc 2160 actgagacct tggtaagcgg gacacagctg tctcagctga ttgaaggtct tgatcggggg 2220 actgagtata atttccgagt ggctgctcta acaatcaatg gtacaggccc ggcaactgac 2280 tggctgtctg ctgaaacttt tgaaagtgac ctagatgaaa ctcgtgttcc tgaagtgcct 2340 agctctcttc acgtacgccc gctcgttact agcatcgtag tgagctggac tcctccagag 2400 aatcagaaca ttgtggtcag aggttacgcc attggttatg gcattggcag ccctcatgcc 2460 cagaccatca aagtggacta taaacagcgc tattacacca ttgaaaatct ggatcccagc 2520 tctcactatg tgattaccct gaaagcattt aataacgtgg gtgaaggcat ccccctgtat 2580 gagagtgctg tgaccaggcc tcacacagac acttctgaag ttgatttatt tgttattaat 2640 gctccataca ctccagtgcc agatcccact cccatgatgc caccagtggg agttcaggct 2700 tccattctga gtcatgacac catcaggatt acgtgggcag acaactcgct gcccaagcac 2760 cagaagatta cagactcccg atactacacc gtccgatgga aaaccaacat cccagcaaac 2820 accaagtaca agaatgcaaa tgcaaccact ttgagttatt tggtgactgg tttaaagccg 2880 aatacactct atgaattctc tgtgatggtg accaaaggtc gaagatcaag tacatggagt 2940 atgacagccc atgggaccac ctttgaatta gttccgactt ctccacccaa ggatgtgact 3000 gttgtgagta aagaggggaa acctaagacc ataattgtga attggcagcc tccctctgaa 3060 gccaatggca aaattacagg ttacatcata tattacagta cagatgtgaa tgcagagata 3120 catgactggg ttattgagcc tgttgtggga aacagactga ctcaccagat acaagagtta 3180 actcttgaca caccatacta cttcaaaatc caggcacgga actcaaaggg catgggaccc 3240 atgtctgaag ctgtccaatt cagaacacct aaagcctcag ggtctggagg gaaaggaagc 3300 cggctgccag acctaggatc cgactacaaa cctccaatga gcggcagtaa cagccctcat 3360 gggagcccca cctctcctct ggacagtaat atgctgctgg tcataattgt ttctgttggc 3420 gtcatcacca tcgtggtggt tgtgattatc gctgtctttt gtacccgtcg taccacctct 3480 caccagaaaa agaaacgagc tgcctgcaaa tcagtgaatg gctctcataa gtacaaaggg 3540 aattccaaag atgtgaaacc tccagatctc tggatccatc atgagagact ggagctgaaa 3600 cccattgata agtctccaga cccaaacccc atcatgactg atactccaat tcctcgcaac 3660 tctcaagata tcacaccagt tgacaactcc atggacagca atatccatca aaggcgaaat 3720 tcatacagag ggcatgagtc agaggacagc atgtctacac tggctggaag gcgaggaatg 3780 agaccaaaaa tgatgatgcc ctttgactcc cagccacccc agcctgtgat tagtgcccat 3840 cccatccatt ccctcgataa ccctcaccat catttccact ccagcagcct cgcttctcca 3900 gctcgcagtc atctctacca cccgggcagc ccatggccca ttggcacatc catgtccctt 3960 tcagacaggg ccaattccac agaatccgtt cgaaataccc ccagcactga caccatgcca 4020 gcctcttcgt ctcaaacatg ctgcactgat caccaggacc ctgaaggtgc taccagctcc 4080 tcttacttgg ccagctccca agaggaagat tcaggccaga gtcttcccac tgcccatgtt 4140 cgcccttccc acccattgaa gagcttcgcc gtgccagcaa tcccgcctcc aggacctccc 4200 acctatgatc ctgcattgcc aagcacacca ttactgtccc agcaagctct gaaccatcac 4260 attcactcag tgaagacagc ctccatcggg actctaggaa ggagccggcc tcctatgcca 4320 gtggttgttc ccagtgcccc tgaagtgcag gagaccacaa ggatgttgga agactccgag 4380 agtagctatg aaccagatga gctgaccaaa gagatggccc acctggaagg actaatgaag 4440 gacctaaacg ctatcacaac agcatgacga ccttcaccag gacctgactt caaacctgag 4500 tctggaagtc ttggaactta acccttgaaa acaaggaatt gtacagagta cgagaggaca 4560 gcacttgaga acacagaatg agccagcaga ctggccagcg cctctgtgta gggctggctc 4620 caggcatggc cacctgcctt cccctggtca gcctggaaga agcctgtgtc gaggcagctt 4680 ccctttgcct gctgatattc tgcaggactg ggcaccatgg gccaaaattt tgtgtccagg 4740 gaagaggcga gaagtgcaac ctgcatttca ctttgtggtc aggccgtgtc tttgtgctgt 4800 gactgcatca cctttatgga gtgtagacat tggcatttat gtacaatttt atttgtgtct 4860 tattttattt taccttcaaa aacaaaaacg ccatccaaaa ccaaggaagt ccttggtgtt 4920 ctccacaagt ggttgacatt tgactgcttg ttccaattat gtatggaaag tctttgacag 4980 tgtgggtcgt tcctggggtt ggcttgtttt ttggtttcat ttttattttt taattctgag 5040 tcattgcatc ctctaccagc tgttaatcca tcactctgag ggggaggaaa tgttgcattg 5100 ctgtttgtaa gcttttttta ttattttttt attataatta ttaaaggcct gactctttcc 5160 tctcatcact gtgagattac agatctattt gaattgaatg aaatgtaaca ttgaaaagac 5220 ttgtttgttg ctttctgtgc agtttcagta ttggggcggg tggggggctg ggggttggta 5280 ataggaaatg gaggggctgc tgaggtcctg tgaatgtttc tgtcattgta ctttcttcca 5340 gaagcctgca gagaatggaa gcatcttctt tattgtcctt tcctggcatg tccatcctta 5400 ttgtcactac gttgcaactg gagtttgatt tggatctggt tttaaaattc ttctgtgcaa 5460 tagatgggtt tgaggattta gcggccctga tgtcttggtc atagcctggt aagaatgtcc 5520 atgctgagga gccacatgtt gtatttctaa ctg 5553 <210> 20 <211> 1849 <212> DNA

<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500099CB1 <400> 20 aatagatcat catggtggca ccaaagagtc acacagatga ctgggctcct gggcctttct 60 ccagtaagcc acagaggagt cagctgcaaa tattctcttc tgttctacag acctctctcc 120 tcttcctgct catgggacta agagcctctg gaaaggactc agccccaaca gtggtgtcag 180 ggatcctagg gggttccgtg actctccccc taaacatctc agtagacaca gagattgaga 240 acgtcatctg gattggtccc aaaaatgctc ttgctttcgc acgtcccaaa gaaaatgtaa 300 ccattatggt caaaagctac ctgggccgac tagacatcac caagtggagt tactccctgt 360 gcatcagcaa tctgactctg aatgatgcag gatcctacaa agcccagata aaccaaagga 420 attttgaagt caccactgag gaggaattca ccctgttcgt ctatgagcag ctgcaggagc 480 cccaagtcac catgaagtct gtgaaggtgt ctgagaactt ctcctgtaac atcactctaa 540 tgtgctccgt gaagggggca gagaaaagtg ttctgtacag ctggacccca agggaacccc 600 atgcttctga gtccaatgga ggctccattc ttaccgtctc ccgaacacca tgtgacccag 660 acctgccata catctgcaca gcccagaacc ccgtcagcca gagaagctcc ctccctgtcc 720 atgttgggca gttctgtaca gatccaggag cctccagagg aggaacaacg ggggagactg 780 tggtaggggt cctgggagag ccagtcaccc tgccacttgc actcccagcc tgccgggaca 840 cagagaaggt tgtctggttg tttaacacat ccatcattag caaagagagg gaagaagcag 900 caacggcaga tccactcatt aaatccaggg atccttacaa gaacagggtg tgggtctcca 960 gccaggactg ctccctgaag atcagccagc tgaagataga ggacgccggc ccctaccatg 1020 cctacgtgtg ctcagaggcc tccagcgtca ccagcatgac acatgtcacc ctgctcatct 1080 accgacctga gagaaacaca aagctttgga ttgggttgtt cctgatggtt tgccttctgt 1140 gcgttgggat cttcagctgg tgcatttgga agcgaaaagg acggtgttca gtcccagcct 1200 tctgttccag ccaagctgag gccccagcgg atacaccagg atatgagaag ctggacactc 1260 ccctcaggcc tgccaggcaa cagcctacac ccacctcaga cagcagctct gacagcaacc 1320 tcacaactga ggaggatgag gacaggcctg aggtgcacaa gcccatcagt ggaagatatg 1380 aggtatttga ccaggtcact caggagggcg ctggacatga cccagcccct gagggccaag 1440 cagactatga tcccgtcact ccatatgtca cggaagttga gtctgtggtt ggagagaaca 1500 ccatgtatgc acaagtgttc aacttacagg gaaagacccc agtttctcag aaggaagaga 1560 gctcagccac aatctactgc tccatacgga aacctcaggt ggtgccacca ccacaacaga 1620 atgatcttga gattcctgaa agtcctacct atgaaaattt cacctgaaag gaaaagcagc 1680 tgctgcctct ctcctgggac cgtggggttg gaaagtcagc tggacctcat ggggcctggg 1740 gctcgcagac agaagcacct cagaatttcc ttcagtgcct cagagatgcc tggatgtggc 1800 ccctccccct ccttctcacc cttaaggact cccaaaccca ttaatagtt 1849 <210> 21 <211> 1427 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7682434CB1 <400> 21 cgccgcctct gccgcgatgc ccccccctgc gcccggggcc cggctccggc ttctcgccgc 60 cgccgccctg gccggcttgg ccgtcatcag ccgagggctg ctctcccaga gcctggagtt 120 caactctcct gccgacaact acacagtgtg tgaaggtgac aacgccaccc tcagctgctt 180 catcgacgag cacgtgaccc gcgtggcctg gctgaaccgc tccaacatcc tgtatgccgg 240 caatgaccgc tggaccagcg acccgcgggt gcggctgctc atcaacaccc ccgaggagtt 300 ctccatcctc atcaccgagg tggggctcgg cgacgagggc ctctacacct gctccttcca 360 gacccgccac cagccgtaca ccactcaggt ctacctcatt gtccacgtcc ctgcccgcat 420 tgtgaacatc tcgtcgcctg tgacggtgaa tgaggggggc aatgtgaacc tgctttgcct 480 ggccgtgggg cggccagagc ccacggtcac ctggagacag ctccgagacg gcttcacctc 540 ggagggagag atcctggaga tctctgacat ccagcggggc caggccgggg agtatgagtg 600 cgtgactcac aacggggtta actcggcgcc cgacagccgc cgcgtgctgg tcacagtcaa 660 ctatcctccg accatcacgg acgtgaccag cgcccgcacc gcgctgggcc gggccgccct 720 cctgcgctgc gaagccatgg cggttccccc cgcggatttc cagtggtaca aggatgacag 780 actgctgagc agcggcacgg ccgaaggcct gaaggtgcag acggagcgca cccgctcgat 840 gcttctcttt gccaacgtga gcgcccggca ttacggcaac tatacgtgtc gcgccgccaa 900 ccgactggga gcgtccagcg cctccatgcg gctcctgcgc ccaggatccc tggagaactc 960 agccccgagg cccccagggc tcctggccct cctctccgcc ctgggctggc tgtggtggag 1020 aatgtaggcg caacccagtg gagctcacct ccccctgcag ggggcctcag gccaagagtg 1080 agagaaacgg gggagcaaga gccgtgggtc tcgtgggggc agaagagctc tcggccacca 1140 aggaagaaga gagaggagaa gaggaggagg cagaggaaga aagatcttca gagaacccat 1200 cactgtgagg gataacgcaa aattatgcat ctttctacag ccattctcgc cacccgttca 1260 cgtttccgat tgtgacccac tcccgccacc ccatacccct ctctcttagc tcaggctgtc 1320 aactggcttg tgtgggtgtg ggtgtgtgag tgtgagcctg catgcatgtg taggtgtctg 1380 tgtctctgtt tgtgtgtgtg tgggggggtg ggctggggga agggact 1427 <210> 22 <211> 1014 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2202389CB1 <400> 22 cacagatgcc actggggtag gtaaactgac ccaactctgc agcactcaga agacgaagca 60 aagccttcta cttgagcagt ttttccatca ctgatatgtg caggaaatga agacattgcc 120 tgccatgctt ggaactggga aattattttg ggtcttcttc ttaatcccat atctggacat 180 ctggaacatc catgggaaag aatcatgtga tgtacagctt tatataaaga gacaatctga 240 acactccatc ttagcaggag atccctttga actagaatgc cctgtgaaat actgtgctaa 300 caggcctcat gtgacttggt gcaagctcaa tggaacaaca tgtgtaaaac ttgaagatag 360 acaaacaagt tggaaggaag agaagaacat ttcatttttc attctacatt ttgaaccagt 420 gcttcctaat gacaatgggt cataccgctg ttctgcaaat tttcagtcta atctcattga 480 aagccactca acaactcttt atgtgacagg aaagcaaaat gaactctctg acacagcagg 540 aagggaaatt aacctggttg atgctcacct taagagtgag caaacagaag caagcaccag 600 gcaaaattcc caagtactgc tatcagaaac tggaatttat gataatgacc ctgacctttg 660 tttcaggatg caggaagggt ctgaagttta ttctaatcca tgcctggaag aaaacaaacc 720 aggcattgtt tatgcttccc tgaaccattc tgtcattgga ctgaactcaa gactggcaag 780 aaatgtaaaa gaagcaccaa cagaatatgc atccatatgt gtgaggagtt aagtctgttt 840 ctgactccaa cagggaccac tgaatgatca gcatgttgac atcattgtct gggctcaaca 900 ggatgtcaaa taatatttct caatttgaga atttttactt tagaaatgtt catgttagtg 960 cttgggtctt aagggtccat aggataaatg attaaaattt ctctcagaaa ctta 1014 <210> 23 <211> 3695 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503597CB1 <400> 23 cccgcctgag gaagccgtgt gcctgggatg ccaagagcca gagaatggat cttctccgag 60 tggggacatt gctgacaatc ccggcttccc gaggcggcta agaacaggca gtttgtgtcg 120 gctggctgca gatacccaga ggcacaaaga gaccgaagcc acccggaggg acccacggac 180 ggacagatgg taggcgcgaa cccgagagga ccggcggagg ctgagcaccg agagccgcca 240 aggaagagaa actaaccaca gccaagttac cccgccggct ttccttcgct gcactaagga 300 atgaaaccct tccagctcga tctgctcttc gtctgcttct tcctcttcag tcaagagctg 360 ggcctccaga agagaggatg ctgtctggtg ctgggctaca tggccaagga caagtttcgg 420 agaatgaatg aaggccaagt ctattccttc agccagcagc cccaggacca ggtggtggtg 480 tcgggacagc cagtgacgct actttgcgcc atccccgaat acgatggctt cgttctgtgg 540 atcaaggacg gcttggctct gggtgtgggc agggacctct caagttaccc acagtacctg 600 gtggtaggga accacctgtc aggggagcac cacctgaaga tcctgagggc agagctgcaa 660 gacgatgcgg tgtacgagtg ccaggccatc caggccgcca tccgctcccg ccccgcacgc 720 ctcacagtcc tggtgccgcc tgatgacccc gtcatcctgg ggggccctgt gatcagcctg 780 cgtgcggggg accctctcaa cctcacctgc cacgcagaca atgccaagcc tgcagcctcc 840 atcatctggt tgcgaaaggg agaggtcatc aatggggcca cctactccaa gaccctgctt 900 cgggacggca agcgggagag catcgtcagc accctcttca tctcccctgg tgacgtggag 960 aatggccaga gcatcgtgtg tcgtgccacc aacaaagcca tccccggagg aaaggagacg 1020 tcggtcacca t.tgacatcca gcaccctcca ctggtcaacc tctcggtgga gccacagcca 1080 gtgctggagg acaacgtcgt cactttccac tgctctgcaa aggccaaccc agctgtcacc 1140 cagtacaggt gggccaagcg gggccagatc atcaaggagg catctggaga ggtgtacagg 1200 accacagtgg actacacgta cttctcagag cccgtctcct gtgaggtgac caacgccctg 1260 ggcagcacca acctcagccg cacggttgac gtctactttg ggccccggat gaccacagaa 1320 ccccaatcct tgctcgtgga tctgggctct gatgccatct tcagctgcgc ctggaccggc 1380 aacccatccc tgaccatcgt ctggatgaag cggggctccg gagtggtcct gagcaatgag 1440 aagaccctga ccctcaaatc cgtgcgccag gaggacgcgg gcaagtacgt gtgccgggct 1500 gtggtgcccc gtgtgggagc cggggagaga gaggtgaccc tgaccgtcaa tggacccccc 1560 atcatctcca gcacccagac ccagcacgcc ctccacggcg agaagggcca gatcaagtgc 1620 ttcatccgga gcacgccgcc gccggaccgc atcgcctggt cctggaagga gaacgttctg 1680 gagtcgggca catcggggcg ctatacggtg gagaccatca gcaccgagga gggcgtcatc 1740 tccaccctga ccatcagcaa catcgtgcgg gccgacttcc agaccatcta caactgcacg 1800 gcctggaaca gcttcggctc cgacactgag atcatccggc tcaaggagca agagtctgtg 1860 ccgatggccg tcatcattgg ggtggccgta ggagctggtg tggccttcct cgtccttatg 1920 gcaaccatcg tggcgttctg ctgtgcccgt tcccagagaa atctcaaagg tgttgtgtca 1980 gccaaaaatg atatccgagt ggaaattgtc cacaaggaac cagcctctgg tcgggagggt 2040 gaggagcact ccaccatcaa gcagctgatg atggaccggg gtgaattcca gcaagactca 2100 gtcctgaaac agctggaggt cctcaaagaa gaggagaaag agtttcagaa cctgaaggac 2160 cccaccaatg gctactacag cgtcaacacc ttcaaagagc accactcaac cccgaccatc 2220 tccctctcca gctgccagcc cgacctgcgt cctgcgggca agcagcgtgt gcccacaggc 2280 atgtccttca ccaacatcta cagcaccctg agcggccagg gccgcctcta cgactacggg 2340 cagcggtttg tgctgggcat gggcagctcg tccatcgagc tttgtgagcg ggagttccag 2400 agaggctccc tcagcgacag cagctccttc ctggacacgc agtgtgacag cagcgtcagc 2460 agcagcggca agcaggatgg ctatgtgcag ttcgacaagg ccagcaaggc ttctgcttcc 2520 tcctcccacc actcccagtc ctcgtcccag aactctgacc ccagtcgacc cctgcagcgg 2580 cggatgcaga ctcacgtcta aggatcacac accgcgggtg gggacgggcc agggaagagg 2640 tcagggcacg ttctggttgt ccagggacga ggggtacttt gcagaggaca ccagaattgg 2700 ccacttccag gacagcctcc cagcgcctct gccactgcct tccttcgaag ctctgatcaa 2760 gcacaaatct gggtccccag gtgctgtgtg ccagaggtgg gcgggtgggg agacagacag 2820 aggctgcggc tgagtgcgct gtgcttagtg ctggacaccc gtgtccccgg ccctttcctg 2880 gaggcccctc taccacctgc tctgcccaca ggcacaagtg gcagctataa ctctgctttc 2940 atgaaactgc ggtccactct ctggtctctc tgtgggctct acccctcgct gaccagaagc 3000 tctacctacc cctgtgcctg tgctcccata cagccctggg gagaagggga tgacgtcttc 3060 ccagcactga gctgccccag aaaccccggc tccccactgc tgctcatagc ccataccctg 3120 gaggctgaca agccagaaat ggccttggct aaaggagcct ctctctcacc aggctggccg 3180 ggagcccacc cccaatttgt ttggtgtttt gtgtccatac tcttgcagtt ctgtccttgg 3240 acttgatgcc gctgaactct gcggtgggac cggtccggtc agagcctggt gtactggggg 3300 gagggaggga ggagggagcc tgtgctgacg gagcacctcg ccgggtgtgc ccctcctggg 3360 ctgtgtgacc ccagcctccc cacccacctc ctgctttgtg tactcctccc ctccccctca 3420 gcacaatcgg agttcatata agaagtgcgg gagcttctct ggtcagggtt ctctgaacac 3480 ttatggagag agtgcttcct gggaagtgtg gcgtttgaag gggctggagg gcaggtcttt 3540 aagatggcga gactgccctt ctcagctgat aaacacaaga acggcgatcc tgtcttcagt 3600 aaggctccac gagaagagag gaagtatatc tacacctcaa ccctcctagt caccacctga 3660 aataaatgtt agggacacta ctccaaaaaa aaaaa 3695 <210> 24 <211> 2403 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503603CB1 <400> 24 caggaataag tgacagtaag aatgacaagg gattaggact ggcttcctct tataaataat 60 aaaatccaaa gagaagtgac ttgagtctcc aggtttaaag gagagcaact agaagtcgtc 120 caaacacctg catctcataa ggagaagaaa agtccacctg gatcttgttt ctggactgag 180 atggatggag aggccacagt gaagcctgga gaacaaaagg aagtggtgag gagaggaaga 240 gaagtggact actccaggct cattgctggc actttaccac aatctcacgt tcttctttcg 300 cccttccaca aaaaagaccc catccgagat ggctgtggga gggctctctc ccctccagga 360 cccatctccg ggccatggga acactcaggc cttcctcgcc cctctgctgg cgggaggaga 420 gctcctttgc agctccaaat tcattgaagg gctcaaggct ggtgtcaggg gagcctggag 480 gagctgtcac catccagtgc cattatgccc cctcatctgt caacaggcac cagaggaagt 540 actggtgccg tctggggccc ccaagatgga tctgccagac cattgtgtcc accaaccagt 600 atactcacca tcgctatcgt gaccgtgtgg ccctcacaga ctttccacag agaggcttgt 660 ttgtggtgag gctgtcccaa ctgtccccgg atgacatcgg atgctacctc tgcggcattg 720 gaagtgaaaa caacatgctg ttcttaagca tgaatctgac catctctgca ggtcccgcca 780 gcaccctccc cacagccact ccagctgctg gggagctcac catgagatcc tatggaacag 840 cgtctccagt ggccaacaga tggaccccag gaaccaccca gaccttagga caggggacag 900 catgggacac agttgcttcc actccaggaa ccagcaagac tacagcttca gctgagggaa 960 gacgaacccc aggagcaacc aggccagcag ctccagggac aggcagctgg gcagagggtt 1020 ctgtcaaagc acctgctccg attccagaga gtccaccttc aaagagcaga agcatgtcca 1080 atacaacaga aggtgtttgg gagggcacca gaagctcggt gacaaacagg gctagagcca 1140 gcaaggacag gagggagatg acaactacca aggctgatag gccaagggag gacatagagg 1200 gggtcaggat agctcttgat gcagccaaaa aggtcctagg aaccattggg ccaccagctc 1260 tggtctcaga aactttggcc tgggaaatcc tcccacaagc aacgccagtt tctaagcaac 1320 aatctcaggg ttccattgga gaaacaactc cagctgcagg catgtggacc ttgggaactc 1380 cagctgcaga tgtgtggatc ttgggaactc cagctgcaga tgtgtggacc agcatggagg 1440 cagcatctgg ggaaggaagc gctgcagggg acctagatgc tgccactgga gacagaggtc 1500 cccaagcaac actgagccag accccggcag taggaccctg gggaccccct ggcaaggagt 1560 cctccgtgaa gcgtactttt ccagaagatg aaagcagctc tcggaccctg gctcctgtct 1620 ctaccatgct ggccctgttt atgcttatgg ctctggttct attgcaaagg aagctctgga 1680 gaaggaggac ctctcaggag gcagaaaggg tcaccttaat tcagatgaca cattttctgg 1740 aagtgaaccc ccaagcagac cagctgcccc atgtggaaag aaagatgctc caggatgact 1800 ctcttcctgc tggggccagc ctgactgccc cagagagaga aatccaggac cctgagggac 1860 agagagatga actgctcagt taccatggga gaaggaccaa gatcaaaggc cttcaggacc 1920 ccagcctctt tccatcatcc ttcctccacc tgtgggaaga gaagctgatg cagccggtgc 1980 tccacccatg gaagaaaggc tggctgtcct tgggcccaag aaagtcaagc attatccacg 2040 tccaaaggtg acaagatgac tcaaaggaga cttcaagaac agtgtatgaa acactggaag 2100 aggtcaccta ggaaaagcat gaaatttcca ttcctgaatg tttgaaaata gaagaggctt 2160 ccaatcagtg tggaaagtga caaatcccct atcaacactc ccagcccttg ctgggggctc 2220 cttttctgac tactgttagc actcagcctc ccattcacat gtattatatt taagtgtacc 2280 agccttgcct tctcaagtag attctaagct cctttaaggc agtaattgca ttttatctgt 2340 ctcatgatgc ccccagagaa cttccaactc agtaggaacc catttaatac ctgtgtctga 2400 ttg 2403

Claims (79)

What is claimed is:

1. 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:1-12, 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:1-6 and SEQ ID NO:8-12, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:13-24.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method of producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.

11. An isolated antibody which specifically binds to a polypeptide of claim 1.

12. 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:13-24, 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:13-18 and SEQ ID NO:20-24, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 94% identical to the polynucleotide sequence of SEQ ID NO:19, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and e) an RNA equivalent of a)-f).

13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
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.

15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
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.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.

19. A method for treating a disease or condition associated with decreased expression of functional IGSFP, comprising administering to a patient in need of such treatment the composition of claim 17.

20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.

22. A method for treating a disease or condition associated with decreased expression of functional IGSFP, comprising administering to a patient in need of such treatment a composition of claim 21.

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional IGSFP, comprising administering to a patient in need of such treatment a composition of claim 24.

26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:

a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, 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.

29. A method of assessing toxicity of a test compound, the 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 of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, 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.

30. A diagnostic test for a condition or disease associated with the expression of IGSFP in a biological sample, the method comprising:
a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11, wherein the antibody is:
a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')2 fragment, or e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of IGSFP in a subject, comprising administering to said subject an effective amount of the composition of claim 32.

34. A composition of claim 32, wherein the antibody is labeled.

35. A method of diagnosing a condition or disease associated with the expression of IGSFP in a subject, comprising administering to said subject an effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-12, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-12.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-12 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-12 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-12 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-12.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 13.

47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising:
a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An array of claim 48, which is a microarray.

53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.

56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:1.

57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:2.

58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:3.

59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:4.

60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:5.

61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:8.

64. A polypeptide of claim l, comprising the amino acid sequence of SEQ ID
NO:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:10.

66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:11.

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:12.

68. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:13.

69. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:14.

70. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:15.

71. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:16.

72. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:17.

73. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ 1D NO:18.

74. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:19.

75. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:20.

76. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:21.

77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:24.