1.ApplicationNumber: US-73990700-A
1.PublishNumber: US-2001012889-A1
2.Date Publish: 20010809
3.Inventor: LAFLEUR DAVID W.
SOPPET DANIEL R.
OLSEN HENRIK
RUBEN STEVEN M.
NI JIAN
ROSEN CRAIG A.
BREWER LAURIE A.
DUAN ROXANNE
EBNER REINHARD

4.Inventor Harmonized: LAFLEUR DAVID W(US)
SOPPET DANIEL R(US)
OLSEN HENRIK(US)
RUBEN STEVEN M(US)
NI JIAN(US)
ROSEN CRAIG A(US)
BREWER LAURIE A(US)
DUAN ROXANNE(US)
EBNER REINHARD(US)

5.Country: US
6.Claims:
(en)The present invention relates to 36 novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

7.Description:
(en)FIELD OF THE INVENTION
[0001] This invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides, and their production.
BACKGROUND OF THE INVENTION
[0002] Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eucaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals,” which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.
[0003] One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.
[0004] Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a “linker” holding the protein to the membrane.
[0005] Despite the great progress made in recent years, only a small number of genes encoding human secreted proteins have been identified. These secreted proteins include the commercially valuable human insulin, interferon, Factor VIII, human growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in light of the pervasive role of secreted proteins in human physiology, a need exists for identifying and characterizing novel human secreted proteins and the genes that encode them. This knowledge will allow one to detect, to treat, and to prevent medical disorders by using secreted proteins or the genes that encode them.
SUMMARY OF THE INVENTION
[0006] The present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting disorders related to the polypeptides, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of the polypeptides.
DETAILED DESCRIPTION
[0007] Definitions
[0008] The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
[0009] In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
[0010] In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
[0011] In specific embodiments, the polynucleotides of the invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length. In a further embodiment, polynucleotides of the invention comprise at least 15 contiguous nucleotides of the coding sequence, but do not comprise all or a portion of any intron. In another embodiment, the nucleic acid comprising the coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene in the genome).
[0012] As used herein , a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
[0013] In the present invention, the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection (“ATCC”). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
[0014] A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.
[0015] Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37° C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH 2 PO 4 ; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).
[0016] Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
[0017] Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
[0018] The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
[0019] The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched , for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
[0020] “SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences identified by an integer specified in Table 1.
[0021] “A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)
[0022] Polynucleotides and Polypeptides of the Invention
[0023] Features of Protein Encoded by Gene No: 1
[0024] This gene was shown to have homology to the human T-cell receptor interacting molecule (TRIM) protein which is thought to be important in modulating the immune response to antigens (See Genbank Accession No. gi|3402216|emb|CAA12178.1; all references available through this accession are hereby incorporated herein by reference; for example, J. Exp. Med. 188 (3), 561-575 (1998)).
[0025] Preferred polypeptides of the invention comprise the following amino acid sequence: IKISLKKRS (SEQ ID NO: 101). Polynucleotides encoding these polypeptides are also provided.
[0026] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: IKISLKKRSMSGISGCPFFLWGLLALLGLA LVISLIFNISHYVEKQRQDKMYSYSSDHTRVDEYYIEDTPIYGNLDDMISEPMD ENCYEQMKARPEKSVN KMQEATPSAQATNETQMCYASLDHSVKGKRRSPGNRELISQTRMEMSNYMQ (SEQ ID NO: 102). Polynucleotides encoding these polypeptides are also provided.
[0027] When tested against U937 cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activating sequence) promoter element. Thus, it is likely that this gene activates promyeloid cells, or more generally, immune or other cells or cell types through the JAK-STAT signal transduction pathway. GAS is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
[0028] The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor-bound protein 2 (Grb2), phospholipase Cgammal, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)-CD3-zeta complex are not very well understood. The protein of the present invention is a novel transmembrane adaptor protein which associates and comodulates with the TCR-CD3-zeta complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19-amino acid transmembrane region, and a 159-amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain-mediated protein-protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR.
[0029] This gene is expressed primarily in normal, apoptotic, and transformed T-cells.
[0030] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune, hematopoietic, inflammatory, and neoplastic diseases and/or conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the lymphatic system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, inflammatory, neoplastic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 56 as residues: Glu-35 to Asp-53, Met-82 to Gln-107, Val-117 to Gly-125. Polynucleotides encoding said polypeptides are also provided.
[0031] The tissue distribution in apoptotic and transformed T-cells, combined with the homology with the TRIM protein and detected GAS biological activity indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of inflammation, blood disorders and neoplasms. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
[0032] Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions, particularly in T-cell directed immune responses. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatou's Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0033] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1699 of SEQ ID NO: 11, b is an integer of 15 to 1713, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 11, and where b is greater than or equal to a +14.
[0034] Features of Protein Encoded by Gene No: 2
[0035] The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 13-29 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type II membrane proteins.
[0036] This gene is expressed primarily in placenta, and to a lesser extent, in ovarian and testis tumor, and in T cells.
[0037] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune, developmental, reproductive, vascular and/or neoplastic conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and reproductive systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, developmental, reproductive, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0038] The tissue distribution in testis and ovarian tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of immune and reproductive disorders and cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene Moreover, since the gene is also expressed in T-cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatou's Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Alternatively, the protein is useful in the detection, treatment, and/or prevention of vascular conditions, which include, but are not limited to, microvascular disease, vascular leak syndrome, aneurysm, stroke, atherosclerosis, arteriosclerosis, or embolism. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0039] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2048 of SEQ ID NO: 12, b is an integer of 15 to 2062, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 12, and where b is greater than or equal to a +14.
[0040] Features of Protein Encoded by Gene No: 3
[0041] This gene was found to have homology to the conserved Homo sapiens mRNA for a human 36 kDa phosphothyrosine protein (See Genbank Accession No.gb|AJ223280|HSAJ3280, and J. Exp Med 1998 Apr 6; 187(7): 1157-61 which is hereby incorporated by reference) which is thought to be involved in modulation of the immune response, potentially in signaling events during T and NK cell activation. Moreover, the protein was also found to have homology to the human LAT protein (See Genbank Accession No. gb|AAC39637.1| (AF036906); all references available through this accession, including the accession referenced above, are hereby incorporated herein by reference; for example, Cell 92 (1), 83-92 (1998)) which is thought to be the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.
[0042] Preferred polypeptides of the invention comprise the following amino acid sequence: GTRGLSTVSWTHTQPSKRGDPSREPRGGHSCLLPGSPATWCLPAPCSLPGPVL TPSSSGLDSALEGPRGA ASLLRAPLQ (SEQ ID NO: 103), HTQPSKRGDPSREPRGGHSCLLP (SEQ ID NO: 104), and/or VLTPSSSGLDSALEGPRGA ASL (SEQ ID NO: 105). Polynucleotides encoding these polypeptides are also provided.
[0043] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: GTRGLSTVSWTHTQPSKRGDPSREPRGGHSCLLPGSPATWCLPAPCSLPGPVL TPSSSGLDSALEGPRGAASLLRAPLQMEEAILVPCVLGLLLLPILAMLMALCV HCHRLPGSYDSTSSDSLYPKGHPVQTASHGCPLATCLPTCHLLPTPEPARPAP HPKIPAAPWGLPPDAIFPAGF (SEQ ID NO: 106). Polynucleotides encoding these polypeptides are also provided. Included in this invention as preferred domains are cytochrome C domains, which were identified using the ProSite analysis tool. In proteins belonging to cytochrome c family [1], the heme group is covalently attached by thioether bonds to two conserved cysteine residues. The consensus sequence for this site is Cys-X-X-Cys-His and the histidine residue is one of the two axial ligands of the heme iron. This arrangement is shared by all proteins known to belong to cytochrome c family, which presently includes cytochromes c, c′, c1 to c6, c550 to c556, cc3/Hmc, cytochrome f and reaction center cytochrome c. The concensus pattern is as follows: C-{CPWHF}-{CPWR}-C-H-{CFYW}.
[0044] Preferred polypeptides of the invention comprise the following amino acid sequence: CVHCHR (SEQ ID NO: 107). Polynucleotides encoding these polypeptides are also provided. Further preferred are polypeptides comprising the cytochrome C domain of the sequence referenced in Table for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence. The additional contiguous amino acid residues is N-terminal or C-terminal to the cytochrome C domain. Alternatively, the additional contiguous amino acid residues is both N-terminal and C-terminal to the cytochrome C domain. wherein the total N- and C-terminal contiguous amino acid residues equal the specified number.
[0045] When tested against Jurket cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activating sequence) promoter element. Thus, it is likely that this gene activates T-cells, or more generally immune cells or other cells and cell types, through the JAK-STAT signal transduction pathway. GAS is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
[0046] This gene is expressed primarily in various T-cell and macrophage lines, and to a lesser extent in endothelial cells.
[0047] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune or hematopoietic disorders, particularly inflammation, infections, or immunodeficiencies. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 58 as residues: Pro-33 to His-49, Glu-74 to Lys-83. Polynucleotides encoding said polypeptides are also provided.
[0048] The tissue distribution in T-cells and macrophages, combined with the homology to the conserved phosphothyrosine protein and detected GAS biological activity indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of inflammatory and general immune disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
[0049] Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatou's Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0050] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1210 of SEQ ID NO: 13, b is an integer of 15 to 1224, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 13, and where b is greater than or equal to a +14.
[0051] Features of Protein Encoded by Gene No: 4
[0052] Preferred polypeptides of the invention comprise the following amino acid sequence: AGSRTNNEQIE (SEQ ID NO: 108). Polynucleotides encoding these polypeptides are also provided.
[0053] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: AGSRTNNEQEEMSCIGRMR LICFULRICGLEHLFGNMGLGXKNGHLPGHYGHSLEFF (SEQ ID NO: 109). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 3-19 of the amino acid sequence referenced in Table 1 for this gene. characteristics, it is believed that the protein product of this gene shares structural features to type II membrane proteins.
[0054] This gene is expressed primarily in aortic endothelial, cardiac, and adipose cells.
[0055] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, metabolic, and vascular diseases and/or conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the cardiovascular system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., metabolic, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0056] The tissue distribution in aortic endothelial and cardiac tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of cardiovascular and metabolic disorders. More generally, the protein is useful in the detection, treatment, and/or prevention of vascular conditions, which include, but are not limited to, microvascular disease, vascular leak syndrome, aneurysm, stroke, atherosclerosis, arteriosclerosis, or embolism. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0057] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1607 of SEQ ID NO: 14, b is an integer of 15 to 1621, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a +14.
[0058] Features of Protein Encoded by Gene No: 5
[0059] The translation product of this gene was found to have homology to the human antigen NY-CO-38 (See Genbank Accession No.gi|3170200 (AF039700)) which is thought to be important in modulation of the immune response. Polynucleotides of the invention do not comprise the polynucleotide sequence shown as Genbank Accession No.gi|3170200, which is hereby incorporated herein by reference.
[0060] Preferred polypeptides of the invention comprise the following amino acid sequence: GTSTSSRGRLHACGHS (SEQ ID NO: 110), PSSEVQKGKPNSPLGNSELRPBLVNTKPRTSLERGHTIPFLWPSEFGLSQLWG TP SLNPNKTPLESLSLFPSPLPSALIAARIVTPNLTLSSLIK (SEQ ID NO: 111), PNSPLGNSELRPHLVNTKPRT (SEQ ID NO: 112), LSLHPSPLPSALIAARIVTPNLT (SEQ ID NO: 113), PGSQGAAAGRELFMTDRERLAEARQRELQRQELLMQKRLAMESNKILQEQQ EMERQRRKEIAQKAAEENERYRKEMEQIVEEEEKFKKQWEEDWGSKEQLLL PKTITAEVHPVPLRKPKYDQGVEPELEPADDLDGGTEEQGEQDFRKYEEGFD PYSMFTPEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVVVSAVYERGAA ERHGGIVKGDEIMAINGKIVTDYTLAEADAALQKAWNQGGDWIDLVVAVCP PKEYDDELTFF (SEQ ID NO: 114), GRELFMTDRERLAEARQRELQRQ (SEQ ID NO: 115), QQEMERQRRKEIAQKAAEENER (SEQ ID NO: 116), KPKYDQGVEPELEPADDLDGGTEEQ (SEQ ID NO: 117), and/or IVTDYTLAEADAALQKAWNQGGDWI (SEQ ID NO: 118). Polynucleotides encoding these polypeptides are also provided.
[0061] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: GTSTSSRGRLHACGHSMILLLSLFQGVRGSLGSPGNRENKEKKVFISLVGSRG LGCSISSGPIQKPGIFI SHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHKELQLAGSCS (SEQ ID NO: 119). Polynucleotides encoding these polypeptides are also provided.
[0062] This gene is expressed primarily in epididiymus, and, to a lesser extent, in monocytes.
[0063] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, reproductive or immune diseases and/or disorders, particularly autoimmune conditions, and/or infertility. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive and immune systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., reproductive, immune, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 60 as residues: Pro-18 to Lys-26. Polynucleotides encoding said polypeptides are also provided.
[0064] The tissue distribution in the epididiymus and monocytes, combined with the homology to the NY-CO-38 antigen indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of abnormalties of the epididiymus, including infertility. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein is used in the development of contraceptives, modulating the immune response (e.g. activating or inhibiting), or inflammatory conditions. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0065] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1406 of SEQ ID NO: 15, b is an integer of 15 to 1420, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 15, and where b is greater than or equal to a +14.
[0066] Features of Protein Encoded by Gene No: 6
[0067] This gene is expressed primarily in neutrophils.
[0068] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune or hematopoietic disorders, particularly acute inflammatory reactions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 61 as residues: Gly-18 to Ser-27, Gly-46 to Asp-51. Polynucleotides encoding said polypeptides are also provided.
[0069] The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of neutrophil mediated disease such as septic shock and acute inflammatory conditions. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
[0070] Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatou's Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0071] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1021 of SEQ ID NO: 16, b is an integer of 15 to 1035, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 16, and where b is greater than or equal to a +14.
[0072] Features of Protein Encoded by Gene No: 7
[0073] Included in this invention as preferred domains are ATP/GTP-binding site motif A (P-loop), which was identified using the ProSite analysis tool. From sequence comparisons and crystallographic data analysis it has been shown that an appreciable proportion of proteins that bind ATP or GTP share a number of more or less conserved sequence motifs. The best conserved of these motifs is a glycine-rich region, which typically forms a flexible loop between a beta-strand and an alpha-helix. This loop interacts with one of the phosphate groups of the nucleotide. This sequence motif is generally referred to as the ‘A’ consensus sequence or the ‘P-loop’. There are numerous ATP- or GTP-binding proteins in which the P-loop is found. Not all ATP- or GTP-binding proteins are picked-up by this motif. A number of proteins escape detection because the structure of their ATP-binding site is completely different from that of the P-loop. Examples of such proteins are the E1-E2 ATPases or the glycolytic kinases. In other ATP- or GTP-binding proteins the flexible loop exists in a slightly different form; this is the case for tubulins or protein kinases. A special mention must be reserved for adenylate kinase, in which there is a single deviation from the P-loop pattern: in the last position Gly is found instead of Ser or Thr. The concensus pattern is as follows: [AG]-x(4)-G-K-[ST].
[0074] Preferred polypeptides of the invention comprise the following amino acid sequence which was determined to represent a consensus a ATP/GTP-binding site motif A (P-loop) domain: GIIAQGKS (SEQ ID NO: 120). Polynucleotides encoding these polypeptides are also provided. Further preferred are polypeptides comprising the ATP/GTP-binding site motif A (P-loop) domain of the sequence referenced in Table for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence. The additional contiguous amino acid residues is N-terminal or C-terminal to the ATP/GTP-binding site motif A (P-loop) domain. Alternatively, the additional contiguous amino acid residues is both N-terminal and C-terminal to the ATP/GTP-binding site motif A (P-loop) domain, wherein the total N- and C-terminal contiguous amino acid residues equal the specified number.
[0075] This gene is expressed primarily in brain frontal cortex.
[0076] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, neural disorders, particularly diseases of the central nervous system (CNS) and brain frontal cortex. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the CNS, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., neural, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 62 as residues: Asn-17 to Arg-29, Gly-37 to Cys-45. Polynucleotides encoding said polypeptides are also provided.
[0077] The tissue distribution in brain frontal cortex indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis and treatment of diseases of the CNS. Moreover, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0078] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0079] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 844 of SEQ ID NO: 17, b is an integer of 15 to 858, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, and where b is greater than or equal to a +14.
[0080] Features of Protein Encoded by Gene No: 8
[0081] A preferred polypeptide fragment of the invention comprises the following amino acid sequence: MFVFLSVLYSLSLEYMFLFVFGKKISFTSLHSDQLGKKKA (SEQ ID NO: 92). Polynucleotides encoding these polypeptides are also provided.
[0082] Preferred polypeptides of the invention comprise the following amino acid sequence: HTML PLKIAAPYLLENCSCPIYISTSPELFLST (SEQ ID NO: 121). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 27-43 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 44 to 71 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.
[0083] The gene encoding the disclosed cDNA is believed to reside on chromosome 20. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 20.
[0084] This gene is expressed primarily in T cells.
[0085] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune or hematopoietic disorders, particularly leukemias, lymphomas, hematopoeitic disorders, auto-immunities and immunodeficiencies. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 63 as residues: His-62 to Ala-71. Polynucleotides encoding said polypeptides are also provided.
[0086] The tissue distribution in T cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic disorders. In addition this gene product is applicable in conditions of general microbial infection, inflammation or cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
[0087] Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatou's Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0088] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 18 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 867 of SEQ ID NO: 18, b is an integer of 15 to 881, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 18, and where b is greater than or equal to a +14.
[0089] Features of Protein Encoded by Gene No: 9
[0090] Preferred polypeptides of the invention comprise the following amino acid sequence: FSILFAFVLFYPGSFFTLP (SEQ ID NO: 122). Polynucleotides encoding these polypeptides are also provided.
[0091] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: FSILFAFVLFYPGSFFTLLPMYMKQVVACRDQLILVL WLIELLCIQGFCKSKSDFSSRIYS (SEQ ID NO: 123). Polynucleotides encoding these polypeptides are also provided.
[0092] This gene is expressed primarily in pineal gland, bone marrow, fetal liver and placenta.
[0093] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, endocrine, immune, hematopoietic, or reproductive disorders, particularly cancers of the blood forming cells. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., endocrine, immune, hematopoietic, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 64 as residues: Cys-29 to Ser-41. Polynucleotides encoding said polypeptides are also provided.
[0094] The tissue distribution in bone marrow and fetal liver indicates that polynucleotides and polypeptides corresponding to this gene are useful for disorders of the blood including lymphomas and leukemias. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0095] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Alternatively, the protein is useful in the detection, treatment, and/or prevention of a variety of endocrine or reproductive disorders, particularly infertility, and biological clock and ion homeostasis disorders. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0096] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 599 of SEQ ID NO: 19, b is an integer of 15 to 613, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 19, and where b is greater than or equal to a +14.
[0097] Features of Protein Encoded by Gene No: 10
[0098] The translation product of this gene shares sequence homology with the kidney injury molecule-1 of Rattus norvegicus which is thought to play an important role in the restoration of the morphological integrity and function to postischemic kidney (See Genbank Accession No. gi|2665892 (AF035963)). Polynucleotides and polypeptides of the present invention are useful to promote growth of new tissue and survival of damaged tissue. Recombinant polypeptides of the present invention can be expressed in prokaryotic and eukaryotic host cells using a claimed process. Soluble variants fused to a toxin, imageable compound or radionuclide, and IgG fusion proteins are also claimed.
[0099] Polynucleotides and polypeptides of the present invention or an agonist, can be used to treat renal disease and to promote the growth of new tissue or the survival of damaged tissue, generally in conditions where the binding of specific ligand to the present invention stimulates cell growth, maintains cellular differentiation or reduces apoptosis, e.g. in cases of renal failure, nephritis, kidney transplants, toxic or hypoxic injury. A monoclonal antibody specific for polynucleotides and polypeptides of the present invention can be used to treat renal disease, e.g. where binding of the invention to a ligand results in neoplasia, loss of cellular function, susceptibility to apoptosis or promotion of inflammation, deliver imaging agents to the cells expressing the present invention in vivo or in vitro and measure the concentration of the present invention by immunoassay. Damage/regeneration of renal cells can be determined by measuring the present invention, particularly to diagnose or monitor the progress of disease or therapy. The tumour cells expressing the present invention can be inhibited by treatment with a fusion protein comprising a ligand of the present invention or MAb with a toxin or radionuclide, and tumour cells that express the present invention ligand can be inhibited with similarly tagged polypeptides of the present invention or anti-present invention ligand antibody.
[0100] Preferred polypeptides of the invention comprise the following amino acid sequence: HESTVK (SEQ ID NO: 124). Polynucleotides encoding these polypeptides are also provided.
[0101] This gene is expressed primarily in infant brain and fetal liver, and to a lesser extent in neoplastic cell lines and endocrine organs.
[0102] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune, neural, endorcine, and growth disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and endocrine systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, neural, endorcine, growth, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, bile, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 65 as residues: Ser-44 to Ser-51, Cys-53 to Cys-64, Val-76 to Lys-83, Pro-102 to Gly-108, Arg-133 to Thr-162, Thr-169 to Lys-183. Polynucleotides encoding said polypeptides are also provided.
[0103] The tissue distribution in fetal liver and infant brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of immune and developmental conditions. Moreover, the expression within infant and fetal tissues and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Alternatively, the protein product of this gene could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0104] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 557 of SEQ ID NO:20, b is an integer of 15 to 571, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a +14.
[0105] Features of Protein Encoded by Gene No: 11
[0106] Preferred polypeptides of the invention comprise the following amino acid sequence: LENLGTHKKKDSFSVKTVGICCCFBLN (SEQ ID NO: 125). Polynucleotides encoding these polypeptides are also provided.
[0107] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: LENLGTHKKKDSFSVKTVGICCCFBLNMLYFCSSIWFGIYFVALITVFLKT LPPLTVGKGPFSGKFVAFFFFLKESCSLLSIVF (SEQ ID NO: 126). Polynucleotides encoding these polypeptides are also provided.
[0108] The gene encoding the disclosed cDNA is believed to reside on chromosome 14. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 14.
[0109] This gene is expressed primarily in infant brain, and to a lesser extent, in monocytes.
[0110] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, neural, immune, or developmental disorders, particularly abnormalities of the infant brain. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the nervous system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., neural, immune, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0111] The tissue distribution in infant brain and monocytes indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of abnormalities of the central nervous system. Moreover, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0112] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. The protein may also be useful in modulating the immune response towards self-antigens. In addition, the expression within embryonic tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0113] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2010 of SEQ ID NO:21, b is an integer of 15 to 2024, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a +14. FEATURES OF PROTEIN ENCODED BY GENE NO: 12
[0114] Preferred polypeptides of the invention comprise the following amino acid sequence: FTKCFH (SEQ ID NO: 127). Polynucleotides encoding these polypeptides are also provided.
[0115] This gene is expressed primarily in activated monocytes.
[0116] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune or hematopoietic disorders, particularly mononucleosis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 67 as residues: Ser-27 to Arg-39, Pro-91 to Arg-100. Polynucleotides encoding said polypeptides are also provided.
[0117] The tissue distribution in monocytes indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of diseases of monocytes such as mononucleosis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0118] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0119] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:22 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 561 of SEQ ID NO:22, b is an integer of 15 to 575, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a +14.
[0120] Features of Protein Encoded by Gene No: 13
[0121] Preferred polypeptides of the invention comprise the following amino acid sequence: QNMNDYNI (SEQ ID NO. 128). Polynucleotides encoding these polypeptides are also provided.
[0122] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: QNMNDYNIMFYLYSIFQVLVWLCQAKHLSQISARSSRRLWRLSLITFPPYLAT SLSHGPHVCLQTLGYE SCEHTDLCFLHD (SEQ ID NO: 129). Polynucleotides encoding these polypeptides are also provided.
[0123] This gene is expressed primarily in a bone marrow cell line.
[0124] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune or hematopoietic disorders, particularly immunodeficiencies. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO 68 as residues: Arg-26 to Trp-32. Polynucleotides encoding said polypeptides are also provided.
[0125] The tissue distribution in bone marrow cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of abnormalities associated with the bone marrow, such as immunodeficiencies. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0126] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0127] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:23 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1167 of SEQ ID NO:23, b is an integer of 15 to 1181, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:23, and where b is greater than or equal to a +14.
[0128] Features of Protein Encoded by Gene No: 14
[0129] Preferred polypeptides of the invention comprise the following amino acid sequence: PARHLWTPSPVCKPSIKPHVSFAGSGSLWRLEPYAFPIEVNRGSAQHWVPG (SEQ ID NO: 130), and/or VCKPSIKPHVSFAGSGSLWRLEPYAFPIE (SEQ ID NO: 131). Polynucleotides encoding these polypeptides are also provided.
[0130] A preferred polypeptide fragment of the invention comprises the following amino acid sequence: MQPCLFMFVLMGIMWATGILPKIMPSRKRCLSIDIPAAPQAGMCLLIL (SEQ ID NO: 132). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 93-109 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 1-14 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.
[0131] This gene is expressed primarily in fetal liver and brain.
[0132] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, neural, metabolic, or developmental disorders, particularly fetal immunodeficiencies. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., neural, metabolic, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 69 as residues: Met-113 to Arg-118. Polynucleotides encoding said polypeptides are also provided.
[0133] The tissue distribution in fetal liver indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of abnormalities of the fetal liver such as fetal immunodeficiencies. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0134] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Alternatively, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions which include, but are not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0135] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0136] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2276 of SEQ ID NO:24, b is an integer of 15 to 2290, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:24, and where b is greater than or equal to a +14.
[0137] Features of Protein Encoded by Gene No: 15
[0138] Preferred polypeptides of the invention comprise the following amino acid sequence: QFSFLSAKGLHWALFVFFYFLSTACQRWAWGL (SEQ ID NO: 133). Polynucleotides encoding these polypeptides are also provided.
[0139] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: QFSFLSAKGLHWALFVFFYFLSTACQRWAWGLMRTLALLVLLFCSCTHSSM GWGRQAWGVALGEVRS PPAQDTVAKT (SEQ ID NO: 134). Polynucleotides encoding these polypeptides are also provided.
[0140] The gene encoding the disclosed cDNA is believed to reside on chromosome 1. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 1.
[0141] This gene is expressed primarily in placenta, breast, testes, fetal liver, and to a lesser extent, in endocrine organs.
[0142] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, reproductive and hormonal conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the gonadal and endocrine systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., reproductive, endocrine, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, seminal fluid, breast milk, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 70 as residues: Thr-15 to Trp-21, Val-33 to Gln-39. Polynucleotides encoding said polypeptides are also provided.
[0143] The tissue distribution placenta, breast, and testes indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of reproductive and hormonal disorders. Representative uses are described here and elsewhere herein. Moreover, the protein is useful in the diagnosis, prevention, and/or treatment of various metabolic disorders such as Tay-Sach's Disease, phenylkenonuria, galactosemia, hyperlipidemias, porphyrias, and Hurler's syndrome. In addition, the expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:25 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 877 of SEQ ID NO:25, b is an integer of 15 to 891, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:25, and where b is greater than or equal to a +14.
[0144] Features of Protein Encoded by Gene No: 16
[0145] The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 6-22 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.
[0146] When tested against Jurket cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activating sequence) promoter element. Thus, it is likely that this gene activates T-cells, or more generally, immune cells or other cells or cell types, through the JAK-STAT signal transduction pathway. GAS is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells. This gene is expressed primarily in CD34-depleted cord blood.
[0147] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune and hemopoietic conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and hemopoietic systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hemopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 71 as residues: Gln-26 to His-34. Polynucleotides encoding said polypeptides are also provided.
[0148] The tissue distribution in CD34-depleted cord blood, combined with the detected GAS biological activity indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of immunological and blood disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0149] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0150] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:26 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 451 of SEQ ID NO:26, b is an integer of 15 to 465, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a +14.
[0151] Features of Protein Encoded by Gene No: 17
[0152] The gene encoding the disclosed cDNA is believed to reside on chromosome 10. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 10.
[0153] This gene is expressed primarily in prostate cancer, and to a lesser extent, in brain.
[0154] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, reproductive or neural disorders, particularly prostate cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., reproductive, neural, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, seminal fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 72 as residues: His-34 to Asn-40. Polynucleotides encoding said polypeptides are also provided.
[0155] The tissue distribution in prostate and brain tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of prostate cancer. Moreover, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0156] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. In addition, the protein is useful in the development of novel contraceptives. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0157] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:27 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 769 of SEQ ID NO:27, b is an integer of 15 to 783, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:27, and where b is greater than or equal to a +14.
[0158] Features of Protein Encoded by Gene No: 18
[0159] Preferred polypeptides of the invention comprise the following amino acid sequence: HEPGRCGPENLALQATQRGTRFSVPMCKSSRQYTYTSVHMCQCACERVEWR GSLTPARALHNHLTEQWFP HGFPFLSRFFTY (SEQ ID NO: 135), ENLALQATQRGTRFSVPMCKSSRQ (SEQ ID NO: 136), and/or MCQCACERVEWRGSLTPARALHNHLT (SEQ ID NO: 137). Polynucleotides encoding these polypeptides are also provided.
[0160] This gene is expressed primarily in fetal brain and CD34 depleted cord blood.
[0161] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, neural, immune, or reproductive conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and nervous systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., neural, immune, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 73 as residues: Ser-35 to Asp-48. Polynucleotides encoding said polypeptides are also provided.
[0162] The tissue distribution in fetal brain and CD34 depleted cord blood indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of nervous system and immune disorders. Moreover, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0163] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. The protein is also useful in the modulation of the immune response, particularly auto-antigens. The expression within embryonic tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0164] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 456 of SEQ ID NO:28, b is an integer of 15 to 470, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a +14.
[0165] Features of Protein Encoded by Gene No: 19
[0166] Preferred polypeptides of the invention comprise the following amino acid sequence: LRRASCPIWSKD (SEQ ID NO: 138). Polynucleotides encoding these polypeptides are also provided.
[0167] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: LRRASCPIWSKDGKTLYLPVCLSFLHSPASTFLPWNQGFLSPFAFSTLGTPGAK QFSI (SEQ ID NO: 139). Polynucleotides encoding these polypeptides are also provided.
[0168] The gene encoding the disclosed cDNA is believed to reside on the X chromosome. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for the X chromosome.
[0169] This gene is expressed primarily in fetal liver spleen and retina, and to a lesser extent in, placenta and fetal lung.
[0170] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, developmental, hemopoietic, retinal, or vascular disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the retinal, hemopoietic and placental tissues and systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., developmental, hemopoietic, retinal, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, bile, pulmonary surfactant and sputum, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0171] The tissue distribution fetal liver spleen and retina indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis and treatment of retinal, hemopoietic, and developmental disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0172] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The protein is also useful in the modulation of the immune response, particularly to auto-antigens in autoimmune disorders. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0173] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:29 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1307 of SEQ ID NO:29, b is an integer of 15 to 1321, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:29, and where b is greater than or equal to a +14.
[0174] Features of Protein Encoded by Gene No: 20
[0175] This gene shares sequence homology with the flatfish growth hormone (see Genbank gb|E02221|E02221) which is thought to be important in growth regulation.
[0176] Preferred polypeptides of the invention comprise the following amino acid sequence: GTSTKLPYCRENVCLAYGSEWSVYAVGSQAHVSFLDPRQPSYNVKSVCSRER GSGIRSVSFYEHIITVGTGQGSLLFYDIRAQRFLEERLSACYGSKPRLAGENLK LTTGKGWLNHDETWRNYFSDIDFFPNAVYTHCYDSSGTKLFVAGGPLPSGLH GNYAGLWS (SEQ ID NO: 140), CRENVCLAYGSEWSVYAVGSQA (SEQ ID NO: 141), PSYNVKSVCSRERGSGIRSVSFYE (SEQ ID NO: 142), DIRAQRFLEERLSACYGSKPRLAGENLKL (SEQ ID NO: 143), KLTTGKGWLNHDETWRNYFSDIDFFP (SEQ ID NO: 144), and/or YDSSGTKLFVAGGPLPSGLHG (SEQ ID NO: 145). Polynucleotides encoding these polypeptides are also provided.
[0177] The gene encoding the disclosed cDNA is believed to reside on chromosome 9. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 9.
[0178] This gene is expressed primarily in infant brain, fetal liver spleen and to a lesser extent, in pancreas tumor and bone marrow.
[0179] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, growth, developmental, immune, neural, or metabolic disorders, particularly cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the endocrine and hemopoietic tissues, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., growth, developmental, neural, immune, metabolic, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 75 as residues: Gly-3 1 to Pro-37, His-46 to Ala-52. Polynucleotides encoding said polypeptides are also provided.
[0180] The tissue distribution in infant brain, combined with the homology to flatfish growth hormone indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, treatment and diagnosis of growth disorders such as cancer particularly pancreas tumor. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the distribution in fetal liver spleen and bone marrow indicates that the protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0181] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. In addition, the expression within fetal and infant tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0182] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:30 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 606 of SEQ ID NO:30, b is an integer of 15 to 620, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:30, and where b is greater than or equal to a +14.
[0183] Features of Protein Encoded by Gene No: 21
[0184] The translation product of this gene shares sequence homology with Dictyostelium discoideum random slug cDNA 25 protein.
[0185] Preferred polypeptides of the invention comprise the following amino acid sequence: KPQRFRRPFFFNHPKPSSBPGLHSRPTLHSHPAFHSBPELQQPTQTSPVPLTPES PLFQNFSGYUIGVGRADCTGQVADINLMGYGKSGQNAQGILTRLYSRAFIMA EPDGSNRTVFVSIDIGMVSQRLRLEVLNRLQSKYGSLYRRDNVILSGTHTHSG PAGYFQYTVFVIASEGFSNQTFQHMVTGILKSIDIAHTNMKPGKIFINKGNVD GVQINRSPYSYLQNPQSERARYSSNTDKEMIVLKMVDLNGDDLGLISFSFSKS ALG TYYEPRNTSLE (SEQ ID NO: 146), KPSSHPGLHSRPTLHSHPAFHSBPELQQPT (SEQ ID NO: 147), RADCTGQVADINLMGYGKSGQNAQGI (SEQ ID NO: 148), RAFIMAEPDGSNRTVFVSIDIGMV (SEQ ID NO: 149), RLQSKYGSLYRRDNVILSGTHTHSGPA (SEQ ID NO: 150), ASEGFSNQTFQHMVTGILKSIDI (SEQ ID NO: 151), IFINKGNVDGVQINRSPYSYLQNP (SEQ ID NO: 152), and/or TDKEMIVLKMVDLNGDDLGLISFSFSKSAL (SEQ ID NO: 153). Polynucleotides encoding these polypeptides are also provided.
[0186] A preferred polypeptide fragment of the invention comprises the following amino acid sequence: MAKRTFSNLETFLIFLLVMMSAITVALLSLLFITSGTIENHKDLGGBFFSTTQSP PATQGSTAAQRSTATQHSTATQSSNSQLKLLQCL (SEQ ID NO: 154). Polynucleotides encoding these polypeptides are also provided.
[0187] A preferred polypeptide varient of the invention comprises the following amino acid sequence: MQPSGLEGPGTFGRWPLLSLLLLLLLLQPVTCAYTTPGPPRALTTLGAPRAHT NMPGTYAPSTTLSSPSTQGLQEQARALMRDFPLVDGHNDLPLVLRQVYQKGL QDVNLRNFSYGQTSLDRLRDGLVGAQFWSAYVPCQTQDRDALRLTLEQEDLI RRMCASYSELELVTSAKALNDTQKLACLIGVEGGHSLDNSLSILRTFYMLGV RYLTLTHTCNTPWAESSAKGVHSFYNNISGLTDFGEKVVAEMNRLGMMVDL SHVSDAVARRALEVSQAPVIFSHSAARGVCNSARNVPDDILQLLKKNGGVV MVSLSMGVIQCNPSANVSTVADHFDFHKAVIGSKFIGIGGDYDGAGKFPQGLE DVSTYPVLIEELLSRGWSEEELQGVLRGNLLRVFRQVEKVQEENKWQSPLED KFPDEQLSSSCHSDLSRLRQRQSLTSGQELTEIPIHWTAKLPAKWSVSESSPHN APVLAVVATFPVLILWL (SEQ ID NO: 155). Polynucleotides encoding these polypeptides are also provided.
[0188] When tested against Jurket cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activating sequence) promoter element. Thus, it is likely that this gene activates T-cells, or more generally, immune cells or other cells or cell types, through the JAK-STAT signal transduction pathway. GAS is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
[0189] This gene is expressed primarily in liver (hepatoma).
[0190] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, hepatic disorders, particularly liver diseases. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the hepatic system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., hepatic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, bile, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 76 as residues: Glu-39 to Gly-45, Thr-51 to Gly-60, Ala-63 to Gln-77, Gly-122 to Asn-129, Leu-175 to Ser-181, Thr-193 to Pro-199, Thr-236 to Gly-241, Asn-256 to Lys-279, Glu-311 to Leu-317. Polynucleotides encoding said polypeptides are also provided.
[0191] The tissue distribution in liver, combined with the homology to the Dictyostelium discoideum random slug cDNA 25 protein and the detected GAS biological activity indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, treatment and diagnoisis of liver diseases and disorders. Representative uses are described in the “Hyperproliferative Disorders”, “Infectious Disease”, and “Binding Activity” sections below, in Example 11, and 27, and elsewhere herein. Briefly, the protein product of this gene is useful for the detection and treatment of liver disorders and cancers (e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells). Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0192] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:31 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1208 of SEQ ID NO:31, b is an integer of 15 to 1222, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:31, and where b is greater than or equal to a +14.
[0193] Features of Protein Encoded by Gene No: 22
[0194] Preferred polypeptides of the invention comprise the following amino acid sequence: BLTQFCVIQLLPTHL (SEQ ID NO: 156), MLASXSVLCDPAPANPSDELLVHSPCFLLSPPVAPVPFFPCAKLIPAPRPVRYF SPPDLRLGNT PAPSEITYTPSVHFCHPIAKLLCLKVRNLCEGVLSAAFPKA (SEQ ID NO: 157), ANPSDELLVHSPCFLLSPPVAPVPFFP (SEQ ID NO: 158), and/or FSPPDLRLGNT PAPSEITYTPSVIFCBPIAKLLC (SEQ ID NO: 159). Polynucleotides encoding these polypeptides are also provided.
[0195] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: HLTQFCVIQLLPTHLMSSWFTLLASCFBLLWPLSRSSHVPSSFQPPDLSATFLL QILG (SEQ ID NO: 160). Polynucleotides encoding these polypeptides are also provided.
[0196] This gene is expressed primarily in liver (hepatoma), fetal dura mater and fetal brain.
[0197] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, liver and central nervous system (CNS) diseases or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the hepatic system and CNS, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., hepatic, neural, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, bile, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 77 as residues: Leu-18 to His-23, Pro-25 to Asp-32. Polynucleotides encoding said polypeptides are also provided.
[0198] The tissue distribution in liver, fetal dura mater, and fetal brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis and treatment of diseases of the liver and CNS. Moreover, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0199] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. The protein is also useful for the detection and treatment of liver disorders and cancers (e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells). The expression within embryonic tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0200] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:32 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 815 of SEQ ID NO:32, b is an integer of 15 to 829, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:32, and where b is greater than or equal to a +14.
[0201] Features of Protein Encoded by Gene No: 23
[0202] Preferred polypeptides of the invention comprise the following amino acid sequence: LIFHFVYL (SEQ ID NO: 161), PTPRVILQVGSRIADRVYDIPRNFPLALDLGCGRGYIAQYLNKLQLFHCRKLL ESFSKLTLQKMLCLHWVNDLPRALEQIHYILKPDGVFIGANTGGDTLYELRCS LQLAETEREGGFSPHISPFTAVNDLGHLLGRAGFNTLTVDTDEIQVNYPGMFE LMEDLQEQKSRMLT (SEQ ID NO: 162), LQVGSRIADRVYDIPRNFPLALDL (SEQ ID NO: 163), GYIAQYLNKLQLFHCRKLLESFSK (SEQ ID NO: 164), VNDLPRALEQIHYILKPDGVFIGAMFG (SEQ ID NO: 165), YELRCSLQLAETEREGGFSPHISPFTAV (SEQ ID NO: 166), and/or NTLTVDTDEIQVNYPGMFELME (SEQ ID NO: 167). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 31-47 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.
[0203] The gene encoding the disclosed cDNA is believed to reside on chromosome 20. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 20.
[0204] This gene is expressed primarily in eosinophils and fetal lung, and to a lesser extent, in bone marrow.
[0205] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune, hemopoietic and respiratory disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune, hemopoietic and respiratory systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, hemopoietic, respiratory, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, pulmonary surfactant and sputum, amniotic fluid, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0206] The tissue distribution in eosinophils and bone marrow indicates that polynucleotides and polypeptides corresponding to this gene are useful for study, diagnosis and treatment of immune, hemopoietic and respiratory diseases. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, protein product of this gene is useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
[0207] The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0208] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:33 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1322 of SEQ ID NO:33, b is an integer of 15 to 1336, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a +14.
[0209] Features of Protein Encoded by Gene No: 24
[0210] The translation product of this gene shares sequence homology with C. elegans transient receptor potential protein and with human O3O gene (PCT patent application, publication no. WO9630389), which is thought to be differentially expressed on tumor cells.
[0211] Preferred polypeptides of the invention comprise the following amino acid sequence: MRQLFYDPDECGLMKKGGLYFSDFWNKLDVGAILLFVAGLTCRLIPATLYPG RVILSLDFILFCLRLMHE FTISKTLGPKIIIVKR (SEQ ID NO: 168), DECGLMKKGGLYFSDFWNKLDVGAILL (SEQ ID NO: 169), TLYPGRVILSLDFELFCLRLMHI FT (SEQ ID NO: 170), VPRERRDAAEPAFPEWLTVLLLCLYLLFTNILLLNLLIAMWNYTFQQVQEHTD QIWKFQRHDLIEEYHGRPAVPPPLILFSBLQLFIKRVVLKTPAKRHKQLKNKL EKNEEAALLSWEIYLKENYLQNRQFQQKQRPEQKIEDISNKVDAMVDLLDLD PLKRSGSMEQRLASLEEQVAQTARALHWIVRTLRASGFSSEADVPTLASQKA AEEPDAEPGGRKKTEEPGDSYHVNARELLYPNCPVTRFPVPNEKVPWETEFLI YDPPFYTAERK (SEQ ID NO: 171), QIWKFQRHDLIEEYHGRPAVPPPLILFS (SEQ ID NO: 172), LQNRQFQQKQRPEQKIEDISNKVDAMVD (SEQ ID NO: 173), VPTLASQKAAEEPDAEPGGRKKTE (SEQ ID NO: 174), and/or PNEKVPW ETEFLIYDPPFYT (SEQ ID NO: 175). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 44-60, 114-130, 138-154, 175-191, and 265-281 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIa membrane proteins.
[0212] Preferred polypeptides of the invention comprise the following amino acid sequence: MMKDVFFFLFLLAVWVVSFGVAKQAILIHNERRVDWLFRGPSTTPTSPSSGR SRATSTV (SEQ ID NO: 176). Polynucleotides encoding these polypeptides are also provided.
[0213] This gene is expressed primarily in dendritic cells and adult brain, and to a lesser extent, in cerebellum.
[0214] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, neural, immune, or hematopoietic disorders, particularly of the central nervous system (CNS) and diseases of the brain, such as tumors. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the CNS, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., neural, immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 79 as residues: Arg-15 to Val-22. Polynucleotides encoding said polypeptides are also provided.
[0215] The tissue distribution in adult brain and cerebellum, combined with the homology to C. elegans transient receptor potential protein indicates that polynucleotides and polypeptides corresponding to this gene are useful for study, diagnosis and treatment of disorders of the CNS such as tumors. Moreover, the protein product of this gene is useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0216] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Alternatively, This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
[0217] Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatou's Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0218] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:34 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1621 of SEQ ID NO:34, b is an integer of 15 to 1635, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:34, and where b is greater than or equal to a +14.
[0219] Features of Protein Encoded by Gene No: 25
[0220] Preferred polypeptides of the invention comprise the following amino acid sequence: PESFNFCFGPGVPMPWCLLPVLSVLHWSTEDTRSCGAQGGGPPLPPRG (SEQ ID NO: 177). Polynucleotides encoding these polypeptides are also provided.
[0221] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: PESFNFCFGPGVPMPWCLLPVLSVLHWSTEDTRSCGAQGGGPPLPPRGMAGT VLGVGAGVFILALLWVAVLLLCVLLSRAS GAARFSVIFYSSVL (SEQ ID NO: 178). Polynucleotides encoding these polypeptides are also provided.
[0222] The gene encoding the disclosed cDNA is thought to reside on chromosome 11. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 11.
[0223] This gene is expressed primarily in infant brain and osteoblastic tissue, and to a lesser extent in leukocytic cell types.
[0224] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, developmental and neurodegenerative diseases of the brain, osteosarcoma, osteoporosis and osteonecrosis, hematopoeitic disorders and other immune deficiencies. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the central nervous and immune systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, neurodegenerative, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0225] The tissue distribution in brain, immune, and osteoblast tissues indicates that the protein product of this gene is useful for the treatment and diagnosis of conditions affecting the central nervous, skeletal and immune systems. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of neurodegenerative diseases of the brain, as well as behavioral or other nervous system disorders, such as depression, schizophrenia, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, mania, dementia, paranoia, addictive behavior and sleep disorders. Expression of this gene product in osteoblastic tissue would suggest a role in the treatment and diagnosis of osteoperosis, fracture, osteosarcoma, ossification, osteonecrosis, arthritis and trauma. Immune and hematopoietic disorders of potential applicability would include leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g. AIDS) and immuno-supressive conditions (transplantation). In addition this gene product is applicable in conditions of general microbial infection, inflammation and cancer. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0226] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:35 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1250 of SEQ ID NO:35, b is an integer of 15 to 1264, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:35, and where b is greater than or equal to a +14.
[0227] Features of Protein Encoded by Gene No: 26
[0228] The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 3-19 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 20 to 47 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.
[0229] This gene is expressed primarily in endometrial tissue and to a lesser extent in breast cancer tissues.
[0230] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, developmental anomalies, fetal deficiencies, female infertility, ovarian, breast and endometrial cancers. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the female reproductive system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., endometrium, breast, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, breast milk, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0231] The tissue distribution in endometrium and breast cancer tissues indicates that the protein product of this gene is useful for the treatment and diagnosis of developmental anomalies or fetal deficiencies, as well as breast, ovarian and endometrial cancers, in addition to cancers of other tissues where expression is observed. Furthermore, the tissue distribution indicates that the protein product of this gene is useful for treating female infertility. The protein product is likely involved in preparation of the endometrium for implantation and could be administered either topically or orally. Alternatively, this gene could be transfected in gene-replacement treatments into the cells of the endometrium and the protein products could be produced. Similarly, these treatments could be performed during artificial insemination for the purpose of increasing the likelihood of implantation and development of a healthy embryo. In both cases this gene or its gene product could be administered at later stages of pregnancy to promote heathy development of the endometrium. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0232] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:36 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 674 of SEQ ID NO:36, b is an integer of 15 to 688, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:36, and where b is greater than or equal to a +14.
[0233] Features of Protein Encoded by Gene No: 27
[0234] The translation product of this gene shares sequence homology with amyloid precursor protein protease which is thought to be important in the processing or clearance of amyloid precursor protein to form beta-amyloid peptide. The translation product of this gene also shares significant homology with Enamel Matrix Serine Proteinase I from Sus scrofa. Based upon the above homologies, in addition to others, this protein is thought to be a novel member of the kallekrein family. Kallikreins are a large family of homologous serine proteases that act in a variety of circulatory and immune system functions. This protein and cDNA are useful in the treatment of conditions such as hypertension, cardiac hypertrophy, arthritis, inflammatory disorders and blood clotting disorders.
[0235] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: SQRHAXQGTGPGIPGSTHASAGEVSVRPAPTSFSVPSLPCLEAARLLSSEFYSA WVSAQCRWWPVLVVPLYLGKSGAAAMATARPPWMWVLCALITALLLGVT EHVLANNDVSCDHPSNTVPSGSNQDLGAGAGEDARSDDSSSRIINGSDCDMH TQPWQAALLLRPNQLYCGAVLVHPQWLLTAAHCRKKVFRVRLGHYSLSPVY ESGQQMFQGVKSIPHPGYSHPGHSNDLMLIKLNRRIRPTKDVRPINVSSHCPS AGTKCLVSGWGTTKSPQVEFPKVLQCLNISVLSQKRCEDAYPRQEDDTMFCA GDKAGRDSCQGDSGGPVVCNGSLQGLVSWGDYPCARPNRPGVYTNLCKFT KWIQETIQANS (SEQ ID NO: 179). Polynucleotides encoding these polypeptides are also provided. The conserved active site domain comprises the following polypeptide sequence: CDMHTQPWQAALLLRPNQLYCGAVLVHPQWLLTAAHCRKKVFRVRLGHYS LSPVYESGQQMFQGVKSIPHPGYSHPGHSNDLMLIKLNRRIRPTKDVRPINVS SHCPSAGTKCLVSGWGTTKSPQVBFPKVLQCLNISVLS QKRCEDAYPRQIDD TMFCAGDKAGRDSCQGDSGGPVVCNGSLQGLVSWGDYPCARPNRPGVYTN LCKFTKWIQETIQAN (SEQ ID NO: 180). Polynucleotides encoding these polypeptides are also provided.
[0236] Contact of cells with supernatant expressing the product of this gene has been shown to increase the permeability of the plasma membrane of THP-1 cells to calcium. Thus it is likely that the product of this gene is involved in a signal transduction pathway that is initiated when the product binds a receptor on the surface of the plasma membrane of monocytes, and to a lesser extent in other cell-lines or tissue cell types. Thus, polynucleotides and polypeptides have uses which include, but are not limited to, activating monocytes. Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium and sodium, as well as alter pH and membrane potential. Alterations in small molecule concentration can be measured to identify supernatants which bind to receptors of a particular cell.
[0237] This gene is expressed primarily in keratinocytes.
[0238] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to; skin cancers (melanomas), eczema, psoriasis or other disorders of the skin, as well as amyloid protein related conditions such as transmissible spongiform encephalopathy (TSE), Creutzfeldt-Jakob disease (CJD) and Alzheimer's Disease. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the skin and brain, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., skin, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 82 as residues: Asn-31 to Thr-41, Pro-43 to Asp-49, Glu-56 to Arg-66, Ser-71 to Trp-80, Asn-160 to Val-169, Thr-192 to Val-198, Lys-215 to Asp-226, Asp-234 to Gly-246, Pro-265 to Gly-273. Polynucleotides encoding said polypeptides are also provided.
[0239] The tissue distribution in skin indicates that the protein product of this gene is useful for the treatment and diagnosis of skin cancers (melanomas), eczema, psoriasis or other disorders of the skin. The homology to amyloid precursor protein-cleaving protease indicates that this gene product cold have a role in the processing or clearance of amyloid precursor protein to form beta-amyloid peptide and therefore useful for treating or preventing conditions associated with beta-amyloid peptide such as Alzheimer's Disease, transmissible spongiform encephalopathy (TSE) and Creutzfeldt-Jakob disease (CJD). Representative uses are described in the “Biological Activity”, “Hyperproliferative Disorders”, “Infectious Disease”, and “Regeneration” sections below, in Example 11, 19, and 20, and elsewhere herein. Briefly, the protein is useful in detecting, treating, and/or preventing congenital disorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen's Disease, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's Disease, mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation of the skin (i.e. wounds, rashes, prickly heat disorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema, photosensitivity, autoimmune disorders (i.e. lupus erythematosus, vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus), keloids, striae, erythema, petechiae, purpura, and xanthelasma. In addition, such disorders may predispose increased susceptibility to viral and bacterial infections of the skin (i.e. cold sores, warts, chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis, erysipelas, impetigo, tinea, althletes foot, and ringworm). Moreover, the protein product of this gene may also be useful for the treatment or diagnosis of various connective tissue disorders (i.e., arthritis, trauma, tendonitis, chrondomalacia and inflammation, etc.), autoimmune disorders (i.e., rheumatoid arthritis, lupus, scleroderma, dermatomyositis, etc.), dwarfism, spinal deformation, joint abnormalities, amd chondrodysplasias (i.e. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0240] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1502 of SEQ ID NO:37, b is an integer of 15 to 1516, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:37, and where b is greater than or equal to a +14.
[0241] Features of Protein Encoded by Gene No: 28
[0242] Preferred polypeptides of the invention comprise the following amino acid sequence: LWTMNPASDGGTSESIFDLDYASWGIRSTL (SEQ ID NO: 181). Polynucleotides encoding these polypeptides are also provided.
[0243] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: LWTMNPASDGGT SESIFDLDYASWGIRSTLMVAGFVFYLGVFVVCHQLSSSLNATYRSLVAREK VFWDLAATRAVFGVQSTA AAVGSAGGPCAACRQGAWPAELVLVSHHDSNGILLL (SEQ ID NO: 182). Polynucleotides encoding these polypeptides are also provided.
[0244] A preferred polypeptide varient of the invention comprises the following amino acid sequence: MPLTVLWWPERRSSGTWRPRVQSLVFRAQPQLWALLGDPVLHADKARGQQ NWCWFHITTATGFFCFENVAVHLSNLIFRTFDLFLVIHHLFAFLGFLGCLVNL QAGHYLAMTTLLLEMSTPFTCVSWMLLKAGWSESLFWKLNQWLMIHMFHC RMVLTYHMWWVCFWHWDGLVSSLYLPBLTLFLVGLALLTLIINPYWTHKK TQQLLNPVDWNFAQPEAKSRPEGNGQLLRKKRP (SEQ ID NO: 183). Polynucleotides encoding these polypeptides are also provided.
[0245] This gene is expressed primarily in pulmonary, endothelial, ubilical cord, and fetal tissues.
[0246] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, asthma, pulmonary edema, atherosclerosis, restenosis, stroke, thrombosis and hypertension. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the pulmonary and cardiovascular systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., vascular tissues, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0247] The tissue distribution in pulmonary and endothelial tissues indicates that the protein product of this gene is useful for the treatment and diagnosis of cardiovasular and respiratory or pulmonary disorders such as asthma, pulmonary edema, pneumonia, atherosclerosis, restenosis, stroke, angina, thrombosis hypertension, inflammation and wound healing. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0248] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1253 of SEQ ID NO:38, b is an integer of 15 to 1267, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:38, and where b is greater than or equal to a +14.
[0249] Features of Protein Encoded by Gene No: 29
[0250] The translation product of this gene shares sequence homology with cathepsin b, a cysteine protease which is thought to be important in demyelination, emphysema, rheumatoid arthritis, and neoplastic infiltration. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with cathepsin and cystein protease proteins. Such activities are known in the art, some of which are described elsewhere herein. Specifically, this sequence is thought to represent a human glucocorticoid inducible suppressor protein. As such, this protein is an intracellular signal transmission inhibitor, and inhibits the activation of interleukin-8 (IL-8) promoter activity in response to a specific extracellular stimulus (especially by interleukin-1 beta). The invention can be used in the treatment and prevention of disorders in which IL-8 expression is involved, such as inflammatory diseases, bronchial asthma, allergy and rheumatism.
[0251] Preferred polypeptides of the invention comprise the following amino acid sequence: GTSGGARASLGPSPKLHQGPGAT (SEQ ID NO: 184). Polynucleotides encoding these polypeptides are also provided.
[0252] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: GTSGGARASLGPSPHLHQGPGATMWRCPLGLLLLLPLAGHLALGAQQGRGR RELAPGLBLRGIRDAGGRYCQEQDLCCRGRADDCALPYLGAICYCDLFCNRT VSDCCPDFWDFCLGVPPPFPPIQGCMHGGRIYPVLGTYWDNCNRCTCQENRQ WQCDQEPCLVDPDMIKAINQGNYGWQAGXHSAFWGMTLDEGIRYRLGTIRP SSSV MNMHEIYTVLNPGEVLPTAFEASEXXPXXFXSLXTKATVQAPGPSPQQLWHP IVSQSILWDT (SEQ ID NO: 185). Polynucleotides encoding these polypeptides are also provided.
[0253] This gene is expressed primarily in endothelial cells.
[0254] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, demyelination, emphysema, rheumatoid arthritis, neoplastic infiltration, atherosclerosis, restenosis, thrombosis and inflammation. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the endothelium, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., endothelial tissues, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 84 as residues: Gln-23 to Glu-30, Ala-43 to Leu-53, Tyr-118 to Gln-137, Gln-152 to Trp-157. Polynucleotides encoding said polypeptides are also provided. The homology to the cysteine protease cathepsin b indicates that the protein product of this gene is useful for the treatment and diagnosis of pathologies such as demyelination, emphysema, rheumatoid arthritis and neoplastic infiltration. In addition, the expression of this gene in endothelial tissues and cells indicates that it is useful in the diagnosis and treatment of cardiovascular and pulmonary conditions such as asthma, pneumonia, atherosclerosis, restenosis, stroke, angina, thrombosis, hypertension, inflammation and wound healing. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0255] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:39 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2189 of SEQ ID NO:39. b is an integer of 15 to 2203, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:39, and where b is greater than or equal to a +14.
[0256] Features of Protein Encoded by Gene No: 30
[0257] When tested against U937 and Jurkat cell lines, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates myeloid and T-cells, or more generally, immune cells, in addition to other cells or cell types, through the Jak-STAT signal transduction pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
[0258] Preferred polypeptides of the invention comprise the following amino acid sequence: YLVIFFLKC (SEQ ID NO: 186). Polynucleotides encoding these polypeptides are also provided.
[0259] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: YLVIFFLKCMYTKLMLNKVLLFWQIVKCKVLVDQYCYNFGAKLLHADWLW DLVJFLRTNVEFEKTP (SEQ ID NO: 187). Polynucleotides encoding these polypeptides are also provided.
[0260] This gene is expressed primarily in placenta, spleen and a variety of blood cell types.
[0261] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune and hematopoietic disorders, as well as those affecting reproduction and fetal development. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and reproductive systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, reproductive, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0262] The tissue distribution in immune tissues indicates that the protein product of this gene is useful for the diagnosis and treatment of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic disorders. In addition, this gene product is applicable in conditions of general microbial infection, inflammation or cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene in placenta would suggest a potential application in the treatment and diagnosis of developmental anomalies or fetal deficiencies. Specific expression within the placenta indicates that this gene product may play a role in the proper establishment and maintenance of placental function. Alternately, this gene product is produced by the placenta and then transported to the embryo, where it may play a crucial role in the development and/or survival of the developing embryo or fetus. Expression of this gene product in a vascular-rich tissue such as the placenta also indicates that this gene product is produced more generally in endothelial cells or within the circulation. In such instances, it may play more generalized roles in vascular function, such as in angiogenesis. It may also be produced in the vasculature and have effects on other cells within the circulation, such as hematopoietic cells. It may serve to promote the proliferation, survival, activation, and/or differentiation of hematopoietic cells, as well as other cells throughout the body. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0263] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:40 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention -are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1712 of SEQ ID NO:40, b is an integer of 15 to 1726, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:40, and where b is greater than or equal to a +14.
[0264] Features of Protein Encoded by Gene No: 31
[0265] Preferred polypeptides of the invention comprise the following amino acid sequence: GSQVNGV (SEQ ID NO: 188). Polynucleotides encoding these polypeptides are also provided.
[0266] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: YLVIFFLKCMYTKLMLNKVLLFWQIVKCKVLVDQYCYNFGAKLLHADWLW DLVFLRTNVEFEKTP (SEQ ID NO: 189). Polynucleotides encoding these polypeptides are also provided.
[0267] This gene is expressed primarily in neutrophils.
[0268] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, auto-immunities, immunodeficiencies, hematopoeitic disorders, general microbial infection, inflammation and cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 86 as residues: Gly-15 to Gly-21, Pro-32 to Trp-38. Polynucleotides encoding said polypeptides are also provided.
[0269] The tissue distribution in neutrophils indicates that the protein product of this gene is useful for the diagnosis and treatment of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g. AIDS), immuno-supressive conditions (e.g. transplantation) and hematopoeitic disorders. In addition this gene product is applicable in conditions of general microbial infection, inflammation and cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product in neutrophils also strongly indicates a role for this protein in immune function and immune surveillance. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0270] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1088 of SEQ ID NO:41, b is an integer of 15 to 1102, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:41, and where b is greater than or equal to a +14.
[0271] Features of Protein Encoded by Gene No: 32
[0272] Preferred polypeptides of the invention comprise the following amino acid sequence: RPTRPLNCGR (SEQ ID NO: 190). Polynucleotides encoding these polypeptides are also provided.
[0273] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: RPTRPLNCGRMRGSVECTWGWGHCAPSPLLLWTLLLFAAPFGLLGEKTRQLL EFDSTNVSDTAAKPLGRPYPPYSLADFSWNNITDSLDPATLSATFQGHPMNDP TRTFANGSLAFRVQAFSRSSRPAQPPRLLHTADTCQLEVALIGASPRGNRSLF GLEVATLGQGPDCPSMQEQHSXER (SEQ ID NO: 191). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 19-35 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 1 to 18 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type II membrane proteins.
[0274] The gene encoding the disclosed cDNA is thought to reside on chromosome 1. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 1.
[0275] This gene is expressed in a wide vareity of tissues suggesting its presence in the vasculature. Those tissues include tumors such as rhabdomyosarcoma, hepatoma, hepatocellular carcinoma uterine cancer, hemangiopericytoma, lymphoma, and testes tumor, but it is also present in T-cell fractions, dendritic cells, endothelial cells, bone marrow stromal cells and melanocytes melanocytes.
[0276] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, highly vascularized tumors, and cardiovascular diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the vascular system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., vascular tissues, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 87 as residues: Pro-56 to Pro-63, Met-92 to Thr-98, Ser-112 to Pro-120, Pro-162 to Ser-169. Polynucleotides encoding said polypeptides are also provided.
[0277] The tissue distribution in vascular tissues indicates that the protein product of this gene is useful for treating disorders of the vasculature and highly vascularized tumors, as well as tumors of other tissues where expression has been observed. Representative uses are described here and elsewhere herein. Furthermore, the tissue distribution in vascular tissues indicates that the protein product of this gene is useful for the diagnosis and treatment of conditions and pathologies of the cardiovascular system, such as heart disease, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0278] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1383 of SEQ ID NO:42, b is an integer of 15 to 1397, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:42, and where b is greater than or equal to a +14.
[0279] Features of Protein Encoded by Gene No: 33
[0280] The gene encoding the disclosed cDNA is thought to reside on chromosome 11. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 11.
[0281] This gene is widely expressed including expression in several libraries from fetal tissues. The gene is also expressed in placenta, lung, synovium, bone marrow, testes and several regions of the brain.
[0282] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, vascular degenerative conditions or tumor associated neo-vascularization. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the vascular system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., vascular, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 88 as residues: Asp-40 to Glu-50, Ser-59 to Gly-69, Ala-98 to His-105, Arg-108 to Glu-114, Pro-124 to Ser-138, Ala-143 to Gly-154. Polynucleotides encoding said polypeptides are also provided.
[0283] The tissue distribution in vascular tissues indicates that the protein product of this gene is useful for treating conditions pertaining to abnormal vascular states such as certain retinopathies, diabetes induced eschemia, or vascularization associated with certain types of tumors. Furthermore, The translation product of this gene is useful in the treatment and diagnosis of conditions and pathologies of the cardiovascular system, such as heart disease, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. Expression of this gene product in a vascular-rich tissue such as the placenta also indicates that this gene product is produced more generally in endothelial cells or within the circulation. In such instances, it may play more generalized roles in vascular function, such as in angiogenesis. It may also be produced in the vasculature and have effects on other cells within the circulation, such as hematopoietic cells. It may serve to promote the proliferation, survival, activation, and/or differentiation of hematopoietic cells, as well as other cells throughout the body. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0284] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:43 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1725 of SEQ ID NO:43, b is an integer of 15 to 1739, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:43, and where b is greater than or equal to a +14.
[0285] Features of Protein Encoded by Gene No: 34
[0286] The translation product of this gene shares homology with a C. elegans protein, in addition to weak similarity to glucarate transporters. (>sp|Q11073|YT45). Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with transporter proteins. Such activities are known in the art, some of which are described elsewhere herein.
[0287] Preferred polypeptides of the invention comprise the following amino acid sequence: VWGPPSVAAALELVDPPGCREFGTSNIEDRDELAYIHSI (SEQ ID NO: 192). Polynucleotides encoding these polypeptides are also provided.
[0288] In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: VWGPPSVAAALELVDPPGCREFGTSNIEDRDELAYHISIMFYIIGGVATLLLIL VIIVFKEKPKYPPSRAQSLSYALTSPDASYLGSIARLFKNLNFVLLVITYGLNA GAFYALSTLLNRMVIWHYPGEEVNAGRIGLTIVIAGMLGAVISGIWLDRSKTY KETTLVVYIMTLVGMVVYTFTLNLGHLWVVFITAGTMGFFMTGYLPLGFEF AVELTYPESEGISSGLLNISAQVFGIIFTISQGQIIDNYGTKPGNIFLCVFLTLGA ALTAFIKADLRR QKANKETLENKLQEEEEESNTSKVPTAVSEDHL (SEQ ID NO: 193). Polynucleotides encoding these polypeptides are also provided. The polypeptide of this gene has been determined to have seven transmembrane domains at about amino acid position 4-20, 59-75, 97-113, 126-142, 149-165, 187-203, and 219-235 of the amino acid sequence referenced in Table 1 for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIb membrane proteins.
[0289] The gene encoding the disclosed cDNA is thought to reside on chromosome 14. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 14.
[0290] When tested against U937 Myeloid cell lines, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates myeloid cells, or more generally, immune cells, or other cells or cell types, through the Jak-STAT signal transduction pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
[0291] This gene is expressed primarily in liver and fetal liver, and to a lesser extent in several other tissue and organs including tissue from Alzheimer's Disease.
[0292] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, metabolic, liver and neural diseases. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the metabolic and neural systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., liver, spleen, neurological, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 89 as residues: Lys-21 to Gln-32, Asp-117 to Glu-124, Tyr-179 to Gly-184, Asn-211 to Gly-217, Leu-239 to Lys-264. Polynucleotides encoding said polypeptides are also provided.
[0293] The tissue distribution in fetal liver/spleen and Alzheimer's tissues indicates that the protein product of this gene is useful for the diagnosis and treatment of metabolic and neural diseases and cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product in fetal liver/spleen indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
[0294] Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0295] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3047 of SEQ ID NO:44, b is an integer of 15 to 3061, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:44, and where b is greater than or equal to a +14.
[0296] Features of Protein Encoded by Gene No: 35
[0297] The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 22-38 of the amino acid sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail encompassing amino acids 39 to 82 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.
[0298] This gene is expressed primarily in brain medulloblastoma, and to a lesser extent in the placenta.
[0299] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, brain medulloblastoma. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the nervous system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., brain, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 90 as residues: Pro-53 to Thr-65. Polynucleotides encoding said polypeptides are also provided.
[0300] The tissue distribution in brain medulloblastoma indicates that the protein product of this gene is useful for the diagnosis and treatment of brain medulloblastoma and other disorders of the CNS, as well as cancers of other tissues where expression has been indicated. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function.
[0301] Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0302] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 960 of SEQ ID NO:45, b is an integer of 15 to 974, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:45, and where b is greater than or equal to a +14.
[0303] Features of Protein Encoded by Gene No: 36
[0304] The translation product of this gene shares sequence homology with mouse oligodendrocyte specific protein and Clostridium endotoxin receptor which is thought to be important in host response to bacterial infections. The translation product of this gene also shares significant homology with the conserved claudin-1 of Mus musculus and the human senescence-associated epithelial membrane protein, which is thought to be located within tight junctions, but the function of which has not yet been deduced (See Genbank Accession Nos gb|AAD16433.1| (AF101051) and gb|AAD22962.1|AF115546 — 1; all references available through these accessions are hereby incorporated herein by reference; for example, Gene 226, 285-295 (1999), and J. Cell Biol. 141 (7), 1539-1550 (1998)).
[0305] Preferred polypeptides of the invention comprise the following amino acid sequence: LPPRGPATFGSPGCPPANSPPSAPATPEPA RAPERV (SEQ ID NO: 194). Polynucleotides encoding these polypeptides are also provided. n another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: LPPRGPATFGSPGCPPANSPPSAPATPEPA RAPERVMANAGLQLLGFELAFLGWIGAIVSTALPQWRIYSYAGDNIVTPRPCT RGCGCPACRRAPGRSSA KSLTPC (SEQ ID NO: 195). Polynucleotides encoding these polypeptides are also provided.
[0306] A preferred polypeptide fragment of the invention comprises the following amino acid sequence: MANAGLQLLGFILAFLGWIGAIVSTALPQWRIYSYAGDNIVTPRPCTRGCGCP ACRRAPGRSSAKSLTPC (SEQ ID NO: 196). Polynucleotides encoding these polypeptides are also provided.
[0307] This gene is expressed primarily in thymic stromal cells, heart, dendritic and colon carcinoma cells.
[0308] Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, Infectious conditions and neoplasms. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and gastrointestinal systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, gastrointestinal, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
[0309] The tissue distribution in immune and gastrointestinal tissues indicates that the protein product of this gene is useful for the study and treatment of immune, Infectious , and cancerous disorders. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses). Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
[0310] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1242 of SEQ ID NO:46, b is an integer of 15 to 1256, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a +14.
5′ NT NT of AA First Last ATCC SEQ 5′ NT 3′ NT 5′ NT First SEQ AA AA First Last Deposit ID Total of of 5 of AA of ID of of AA of AA Gene cDNA Nr and NO: NT Clone Clone Start Signal NO: Sig Sig Secreted of No. Clone ID Date Vector X Seq. Seq. Seq. Codon Pep Y Pep Pep Portion ORF  1 HTGAU75 209563 Uni-ZAP XR 11 1713 1 1713 149 149 56 1 33 34 142 12/18/97  2 HTTDP47 209563 Uni-ZAP XR 12 2062 1 2062 133 133 57 1 21 22 50 12/18/97  3 HTXJQ11 209563 Uni-ZAP XR 13 1224 1 1224 238 238 58 1 29 30 101 12/18/97  4 HADCO45 209563 pSport1 14 1621 1 1621 187 187 59 1 19 20 47 12/18/97  5 HMIAL37 209563 Uni-ZAP XR 15 1420 1 1420 49 49 60 1 13 14 97 12/18/97  6 HNGDU40 209563 Uni-ZAP XR 16 1035 1 1035 333 333 61 1 17 18 51 12/18/97  7 HFXBO84 209563 Lambda ZAP 17 858 1 858 9 9 62 1 18 19 51 12/18/97 II  8 HLLAX19 209563 pCMVSport 1 18 881 112 881 328 328 63 1 53 54 71 12/18/97  8 HLLAX19 209563 pCMVSport 1 47 936 1 936 298 298 92 1 22 23 40 12/18/97  9 HPMAG94 209563 Uni-ZAP XR 19 613 1 613 160 160 64 1 31 32 41 12/18/97 10 HSVAK93 209563 Uni-ZAP XR 20 571 1 571 21 21 65 1 24 25 183 12/18/97 11 HMQBO88 209563 Uni-ZAP XR 21 2024 1 2024 141 141 66 1 31 32 57 12/18/97 12 HMQBU45 209563 Uni-ZAP XR 22 575 1 575 275 275 67 1 24 25 100 12/18/97 13 HMWAJ53 209563 Uni-ZAP XR 23 1181 1 1181 238 238 68 1 17 18 73 12/18/97 14 HNGBQ56 209563 Uni-ZAP XR 24 2290 1618 2290 1519 1519 69 1 30 31 137 12/18/97 14 HNGBQ56 209563 Uni-ZAP XR 48 1765 1 1765 155 155 93 1 21 22 48 12/18/97 15 HAUAU73 209563 Uni-ZAP XR 25 891 1 869 316 316 70 1 25 26 45 12/18/97 16 HCUGO12 209563 ZAP Express 26 465 1 465 133 133 71 1 25 26 63 12/18/97 17 HPFCX44 209563 Uni-ZAP XR 27 783 1 783 85 85 72 1 23 24 47 12/18/97 18 HCUBV79 209563 ZAP Express 28 470 1 470 203 203 73 1 21 22 48 12/18/97 19 HLQBV04 209563 Lambda ZAP 29 1321 1 1321 268 74 1 23 24 46 12/18/97 II 20 HMADW66 209563 Uni-ZAP XR 30 620 1 620 60 60 75 1 19 20 58 12/18/97 21 HLDBE54 209563 pCMVSport 31 1222 1 1222 155 155 76 1 38 39 318 12/18/97 3.0 21 HLDBE54 209563 pCMVSport 49 1194 1 1194 130 130 94 1 26 27 89 12/18/97 3.0 21 HDTAB58 203081 pCMVSport 50 2334 1874 2334 133 133 95 1 33 34 486 07/30/98 2.0 22 HFTAB66 209563 Uni-ZAP XR 32 829 21 762 72 72 77 1 21 22 43 12/18/97 23 HEOMQ63 209563 pSport1 33 1336 1 1336 123 123 78 1 23 24 47 12/18/97 24 HDPJM30 209563 pCMVSport 34 1635 308 1633 59 59 79 1 59 60 525 12/18/97 3.0 24 HDPJM30 209563 pCMVSport 51 1314 1 1313 259 259 96 1 20 21 59 12/18/97 3.0 25 HJABT47 209563 pBluescript 35 1264 231 1264 324 324 80 1 36 37 47 12/18/97 SK- 26 IIJMAG88 209563 pCMVSport 36 688 1 688 20 20 81 1 23 24 47 12/18/97 3.0 27 HKAAH36 209563 pCMVSport 37 1516 1 1516 254 254 82 1 29 30 293 12/18/97 2.0 27 HKAAH36 209563 pCMVSport 52 1381 196 1381 129 129 97 1 29 30 293 12/18/97 2.0 27 HKAAH36 209563 pCMVSport 53 1439 1 1439 189 189 98 1 29 30 61 12/18/97 2.0 28 HMADS41 209563 Uni-ZAP XR 38 1267 1 1267 267 267 83 1 21 22 88 12/18/97 29 HMEFT85 209563 Lambda ZAP 39 2203 1 2203 70 70 84 1 23 24 249 12/18/97 II 30 HMSBX80 209563 Uni-ZAP XR 40 1726 1 1726 169 169 85 1 19 20 57 12/18/97 31 HNGCL23 209563 Uni-ZAP XR 41 1102 1 1102 269 269 86 1 18 19 48 12/18/97 32 HNTBI26 209563 pCMVSport 42 1397 1 1397 32 32 87 1 35 36 172 12/18/97 3.0 32 HNTBI26 209563 pCMVSport 54 1368 1 1368 16 16 99 1 35 36 131 12/18/97 3.0 33 HPIBO15 209563 Uni-ZAP XR 43 1739 1 1739 127 127 88 1 18 19 173 12/18/97 34 HCYBG92 209563 pBluescript 44 3061 1 2661 118 118 89 1 21 22 274 12/18/97 SK- 35 HMDAQ29 209563 Uni-ZAP XR 45 974 1 974 180 180 90 1 48 49 82 12/18/97 36 HCYBI36 209683 pBluescript 46 1256 148 1256 235 235 91 1 23 24 211 03/20/98 SK- 36 HSYBI49 209563 pCMVSport 55 1446 1 1446 230 230 100 1 21 22 70 12/18/97 3.0

[0311] Table 1 summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table 1 and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.
[0312] The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” Some of the deposits contain multiple different clones corresponding to the same gene. “Vector” refers to the type of vector contained in the cDNA Clone ID.
[0313] “Total NT Seq.” refers to the total number of nucleotides in the contig identified by “Gene No.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.”
[0314] The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
[0315] The first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” The predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion.” Finally, the amino acid position of SEQ ID NO:Y of the last amino acid in the open reading frame is identified as “Last AA of ORF.”
[0316] SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to the secreted proteins encoded by the cDNA clones identified in Table 1.
[0317] Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
[0318] Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
[0319] The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
[0320] Also provided in the present invention are species homologs. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
[0321] The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
[0322] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
[0323] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:3140 (1988). Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.
[0324] Signal Sequences
[0325] Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
[0326] In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1.
[0327] As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues (i.e., + or −5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
[0328] Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
[0329] Polynucleotide and Polypeptide Variants
[0330] “Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
[0331] By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown in Table 1, the ORF (open reading frame), or any fragement specified as described herein.
[0332] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght of the subject nucleotide sequence, whichever is shorter.
[0333] If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
[0334] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignement of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
[0335] By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
[0336] As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences shown in Table 1 or to the amino acid sequence encoded by deposited DNA clone can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
[0337] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is becuase the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
[0338] For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
[0339] The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli ).
[0340] Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
[0341] Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
[0342] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual EL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” (See, Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
[0343] Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
[0344] Thus, the invention further includes polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
[0345] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
[0346] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
[0347] As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
[0348] Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
[0349] For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)
[0350] A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
[0351] Polynucleotide and Polypeptide Fragments
[0352] In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in the deposited clone or shown in SEQ ID NO:X. The short nucleotide fragments are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in the deposited clone or the nucleotide sequence shown in SEQ ID NO:X. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.
[0353] Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X or the cDNA contained in the deposited clone. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein.
[0354] In the present invention, a “polypeptide fragment” refers to a short amino acid sequence contained in SEQ ID NO:Y or encoded by the cDNA contained in the deposited clone. Protein fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
[0355] Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.
[0356] Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotide fragments encoding these domains are also contemplated.
[0357] Other preferred fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
[0358] Epitopes & Antibodies
[0359] In the present invention, “epitopes” refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human. A preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment. A region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” In contrast, an “immunogenic epitope” is defined as a part of a protein that elicits an antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).)
[0360] Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)
[0361] In the present invention, antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983).)
[0362] Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope includes the secreted protein. The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)
[0363] As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
[0364] Fusion Proteins
[0365] Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
[0366] Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
[0367] Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
[0368] Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)
[0369] Similarly, EP-A-Q 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
[0370] Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)
[0371] Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
[0372] Vectors, Host Cells, and Protein Production
[0373] The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
[0374] The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
[0375] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[0376] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
[0377] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
[0378] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
[0379] A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“IPLC”) is employed for purification.
[0380] Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
[0381] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[0382] Uses of the Polynucleotides
[0383] Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
[0384] The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
[0385] Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:X will yield an amplified fragment.
[0386] Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
[0387] Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).
[0388] For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.
[0389] Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
[0390] Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
[0391] Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.
[0392] In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.
[0393] Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.
[0394] The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
[0395] The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
[0396] Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
[0397] There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
[0398] In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
[0399] Uses of the Polypeptides
[0400] Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
[0401] A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[0402] In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
[0403] A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)
[0404] Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
[0405] Moreover, polypeptides of the present invention can be used to treat disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B), to inhibit the activity of a polypeptide (e.g., an oncogene), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth).
[0406] Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
[0407] At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.
[0408] Biological Activities
[0409] The polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.
[0410] Immune Activity
[0411] A polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
[0412] A polynucleotide or polypeptide of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells. A polypeptide or polynucleotide of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
[0413] Moreover, a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotide or polypeptide of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotide or polypeptide of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart attacks (infarction), strokes, or scarring.
[0414] A polynucleotide or polypeptide of the present invention may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
[0415] Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
[0416] Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
[0417] A polynucleotide or polypeptide of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
[0418] Similarly, a polypeptide or polynucleotide of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
[0419] Hyperproliferative Disorders
[0420] A polypeptide or polynucleotide can be used to treat or detect hyperproliferative disorders, including neoplasms. A polypeptide or polynucleotide of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polypeptide or polynucleotide of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
[0421] For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
[0422] Examples of hyperproliferative disorders that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
[0423] Similarly, other hyperproliferative disorders can also be treated or detected by a polynucleotide or polypeptide of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
[0424] Infectious Disease
[0425] A polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, the polypeptide or polynucleotide of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
[0426] Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention. Examples of viruses, include, but are not limited to the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox , hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
[0427] Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
[0428] Moreover, parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
[0429] Preferably, treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
[0430] Regeneration
[0431] A polynucleotide or polypeptide of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
[0432] Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
[0433] Moreover, a polynucleotide or polypeptide of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
[0434] Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotide or polypeptide of the present invention.
[0435] Chemotaxis
[0436] A polynucleotide or polypeptide of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
[0437] A polynucleotide or polypeptide of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
[0438] It is also contemplated that a polynucleotide or polypeptide of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis.
[0439] Binding Activity
[0440] A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules.
[0441] Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
[0442] Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
[0443] The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
[0444] Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
[0445] Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
[0446] All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.
[0447] Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity , and (b) determining if a biological activity of the polypeptide has been altered.
[0448] Other Activities
[0449] A polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
[0450] A polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
[0451] A polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
[0452] A polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
[0453] Other Preferred Embodiments
[0454] Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1.
[0455] Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
[0456] Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
[0457] Similarly preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
[0458] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
[0459] Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
[0460] A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
[0461] A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X.
[0462] Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
[0463] Also preferred is a composition of matter comprising a DNA molecule which comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table 1 for said cDNA Clone Identifier.
[0464] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone identified by a cDNA Clone Identifier in Table 1, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table 1.
[0465] Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone.
[0466] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
[0467] A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
[0468] A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said human cDNA clone.
[0469] A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
[0470] Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
[0471] A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0472] The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
[0473] Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0474] The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
[0475] Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
[0476] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1.
[0477] Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:Y in the range of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.
[0478] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.
[0479] Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.
[0480] Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y.
[0481] Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0482] Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a secreted portion of the secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0483] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0484] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0485] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0486] Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0487] Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.
[0488] Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0489] Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
[0490] Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0491] Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
[0492] Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0493] In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.
[0494] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0495] Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
[0496] Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
[0497] Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
[0498] Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1 and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y is defined in Table 1; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The isolated polypeptide produced by this method is also preferred.
[0499] Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.
[0500] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.



EXAMPLES
Example 1
[0501] Isolation of a Selected cDNA Clone From the Deposited Sample
[0502] Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector. Table 1 identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The table immediately below correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 1 as being isolated in the vector “Lambda Zap,” the corresponding deposited clone is in “pBluescript.”
Vector Used to Construct Library Plasmid Corresponding Deposited Lambda Zap pBluescript (PBS) Uni-Zap XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 20 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1 pCR ® 2.1

[0503] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S” is for SacI and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the f1 origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the f1 ori generates sense strand DNA and in the other, antisense.
[0504] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).) Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 1, as well as the corresponding plasmid vector sequences designated above.
[0505] The deposited material in the sample assigned the ATCC Deposit Number cited in Table 1 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table 1. Typically, each ATCC deposit sample cited in Table 1 comprises a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; but such a deposit sample may include plasmids for more or less than 50 cDNA clones, up to about 500 cDNA clones.
[0506] Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNAs cited for that clone in Table 1. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO:X.
[0507] Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32 P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.
[0508] Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5′ NT and the 3′ NT of the clone defined in Table 1) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl 2 , 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
[0509] Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7): 1683-1684 (1993).)
[0510] Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.
[0511] This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
[0512] This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.
Example 2
[0513] Isolation of Genomic Clones Corresponding to a Polynucleotide
[0514] A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO:X., according to the method described in Example 1. (See also, Sambrook.)
Example 3
[0515] Tissue Distribution of Polypeptide
[0516] Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 1 is labeled with P 32 using the rediprime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.
[0517] Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (Clontech) are examined with the labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT1 190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70° C. overnight, and the films developed according to standard procedures.
Example 4
[0518] Chromosomal Mapping of the Polynucleotides
[0519] An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95° C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.
Example 5
[0520] Bacterial Expression of a Polypeptide
[0521] A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
[0522] The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan r ). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
[0523] Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.
[0524] Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4° C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
[0525] Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
[0526] The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4° C. or frozen at −80° C.
[0527] In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operator sequences are made synthetically.
[0528] DNA can be inserted into the pHEa by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
[0529] The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.
Example 6
[0530] Purification of a Polypeptide from an Inclusion Body
[0531] The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10° C.
[0532] Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10° C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
[0533] The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0534] The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4° C. overnight to allow further GuHCl extraction.
[0535] Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4° C. without mixing for 12 hours prior to further purification steps.
[0536] To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 μm membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
[0537] Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 MM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A 280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
[0538] The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7
[0539] Cloning and Expression of a Polypeptide in a Baculovirus Expression System
[0540] In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
[0541] A Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
[0542] Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified in Table 1, is amplified using the PCR protocol described in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
[0543] The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
[0544] The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
[0545] The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
[0546] Five μg of a plasmid containing the polynucleotide is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of the plasmid are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27° C. for four days.
[0547] After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C.
[0548] To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 μCi of 35 S-methionine and 5 μCi 35 S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
[0549] Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.
Example 8
[0550] Expression of a Polypeptide in Mammalian Cells
[0551] The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
[0552] Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
[0553] Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
[0554] The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and a Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
[0555] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DBFR gene under control of the SV40 early promoter.
[0556] Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
[0557] A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
[0558] The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
[0559] The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli BB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
[0560] Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five ,g of the expression plasmid pC6 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9
[0561] Protein Fusions
[0562] The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.
[0563] Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.
[0564] For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
[0565] If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
[0566] Human IgG Fc region:
[0567] GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGT GGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO:1)
Example 10
[0568] Production of an Antibody from a Polypeptide
[0569] The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) For example, cells expressing a polypeptide of the present invention is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the secreted protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
[0570] In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/mil of penicillin, and about 100 μg/ml of streptomycin.
[0571] The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide.
[0572] Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.
[0573] It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
[0574] For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)
Example 11
[0575] Production Of Secreted Protein For High-Throughput Screening Assays
[0576] The following protocol produces a supernatant containing a polypeptide to be tested. This supernatant can then be used in the Screening Assays described in Examples 13-20.
[0577] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.
[0578] Plate 293T cells (do not carry cells past P+20) at 2×10 5 cells/well in 0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/1×Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
[0579] The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8 or 9, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150 ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.
[0580] Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a12-channel pipetter with tips on every other channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37° C. for 6 hours.
[0581] While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1x penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO 4 -5H 2 O; 0.050 mg/L of Fe(NO 3 ) 3 -9H 2 O; 0.417 mg/L of FeSO 4 -7H 2 O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl 2 ; 48.84 mg/L of MgSO 4 ; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO 3 ; 62.50 mg/L of NaH 2 PO 4 —H 2 O; 71.02 mg/L of Na 2 HPO4; 0.4320 mg/L of ZnSO 4 -7H 2 O; 0.002 mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H 2 O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H 2 O; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H 2 O; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalanine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H 2 O; 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin, 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin B1 2 ; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal) with 2 mm glutamine and 1×penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15 ml polystyrene conical.
[0582] The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each well. Incubate at 37° C. for 45 or 72 hours depending on the media used: 1%BSA for 45 hours or CHO-5 for 72 hours.
[0583] On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 13-20.
[0584] It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide directly (e.g., as a secreted protein) or by the polypeptide inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.
Example 12
[0585] Construction of GAS Reporter Construct
[0586] One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.
[0587] GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
[0588] The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.
[0589] The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, EL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID NO:2)).
[0590] Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway.
[0591] Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.
JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISRE IFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotrophic) + + + ? 1, 3 GAS (IRF1 > Lys6 > IFP) Il-11 (Pleiotrophic) ? + ? ? 1, 3 OnM (Pleiotrophic) ? + + ? 1, 3 LIF (Pleiotrophic) ? + + ? 1, 3 CNTF (Pleiotrophic) −/+ + + ? 1, 3 G-CSF (Pleiotrophic) ? + ? ? 1, 3 IL-12 (Pleiotrophic) + − + + 1, 3 g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growth hormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS (B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

[0592] To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 13-14, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is: 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG: 3′ (SEQ ID NO:3)
[0593] The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3′ (SEQ ID NO:4)
[0594] PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence: 5′:+E,uns CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA TGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCG CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT CCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC TCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT AGGCTTTTGCAAA+E,uns AAGCTT: 3′ (SEQ ID NO:5)
[0595] With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
[0596] The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
[0597] Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14.
[0598] Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 15 and 16. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 13
[0599] High-Throughput Screening Assay for T-cell Activity.
[0600] The following protocol is used to assess T-cell activity by identifying factors, such as growth factors and cytokines, that may proliferate or differentiate T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
[0601] Jurkat T-cells are lymphoblastic CD4+Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.
[0602] Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.
[0603] During the incubation period, count cell concentration, spin down the required number of cells (10 7 per transfection), and resuspend in OPTI-MEM to a final concentration of 10 7 cells/ml. Then add 1 ml of 1×10 7 cells in OPTI-MEM to T25 flask and incubate at 37° C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.
[0604] The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing a polypeptide as produced by the protocol described in Example II.
[0605] On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.
[0606] Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100, 000 cells per well).
[0607] After all the plates have been seeded, 50 ul of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.
[0608] The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at −20° C. until SEAP assays are performed according to Example 17. The plates containing the remaining treated cells are placed at 4° C. and serve as a source of material for repeating the assay on a specific well if desired.
[0609] As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.
[0610] The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.
Example 14
[0611] High-Throughput Screening Assay Identifying Myeloid Activity
[0612] The following protocol is used to assess myeloid activity by identifying factors, such as growth factors and cytokines, that may proliferate or differentiate myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
[0613] To transiently transfect U937 cells with the GASISEAP/Neo construct produced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e 7 U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.
[0614] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na 2 HPO 4 .7H 2 O, 1 mM MgCl 2 , and 675 uM CaCl 2 . Incubate at 37° C. for 45 min.
[0615] Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37° C. for 36 hr.
[0616] The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages.
[0617] These cells are tested by harvesting 1×10 8 cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×10 5 cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10 5 cells/well).
[0618] Add 50 ul of the supernatant prepared by the protocol described in Example 11. Incubate at 37° C .for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 17.
Example 15
[0619] High-Throughput Screening Assay Identifying Neuronal Activity.
[0620] When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed.
[0621] Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells can be assessed.
[0622] The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:
[0623] 5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:6)
[0624] 5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′ (SEQ ID NO:7)
[0625] Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.
[0626] To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.
[0627] PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.
[0628] Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamine protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages.
[0629] To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
[0630] The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×10 5 cells/ml.
[0631] Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1×10 5 cells/well). Add 50 ul supernatant produced by Example 11, 37° C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 17.
Example 16
[0632] High-Throughput Screening Assay for T-cell Activity
[0633] NF-κB (Nuclear Factor κB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-κB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-κB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.
[0634] In non-stimulated conditions, NF-κB is retained in the cytoplasm with I-κB (Inhibitor κB). However, upon stimulation, I-κB is phosphorylated and degraded, causing NF-κB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-κB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.
[0635] Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-κB promoter element are used to screen the supernatants produced in Example 11. Activators or inhibitors of NF-κB would be useful in treating diseases. For example, inhibitors of NF-κB could be used to treat those diseases related to the acute or chronic activation of NF-κB, such as rheumatoid arthritis.
[0636] To construct a vector containing the NF-κB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-κB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site: 5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC TTTCCATCCTGCCATCTCAATTAG: 3′ (SEQ ID NO:9)
[0637] The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGClTGCAAAGCCTAGGC: 3′ (SEQ ID NO:4)
[0638] PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence: 5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGA CTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTA TTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT: 3′ (SEQ ID NO: 10)
[0639] Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
[0640] In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-κB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-κB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.
[0641] Once NF-κB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 13. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.
Example 17
[0642] Assay for SEAP Activity
[0643] As a reporter molecule for the assays described in Examples 13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
[0644] Prime a dispenser with the 2.5×Dilution Buffer and dispense 15 μl of 2.5×dilution buffer into Optiplates containing 35 μl of a supernatant. Seal the plates with a plastic sealer and incubate at 65° C. for 30 min. Separate the Optiplates to avoid uneven heating.
[0645] Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 μL Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the table below). Add 50 μl Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on luminometer, one should treat 5 plates at each time and start the second set 10 minutes later.
[0646] Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation # of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 18
[0647] High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability
[0648] Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
[0649] The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo4 (Molecular Probes, Inc.; catalog no. F-14202), used here.
[0650] For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO 2 incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
[0651] A stock solution of 1 mg/ml fluo4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37° C. in a CO 2 incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.
[0652] For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×10 6 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37° C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×10 6 cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final volume.
[0653] For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The supernatant is added to the well, and a change in fluorescence is detected.
[0654] To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event which has resulted in an increase in the intracellular Ca ++ concentration.
Example 19
[0655] High-Throughput Screening Assay Identifying Tyrosine Kinase Activity
[0656] The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
[0657] Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[0658] Because of the wide range of known factors capable of stimulating tyrosine kinase activity, the identification of novel human secreted proteins capable of activating tyrosine kinase signal transduction pathways are of interest. Therefore, the following protocol is designed to identify those novel human secreted proteins capable of activating the tyrosine kinase signal transduction pathways.
[0659] Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4° C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
[0660] To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in Example 11, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4° C. at 16,000×g.
[0661] Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.
[0662] Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
[0663] The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of 5 uM Biotinylated Peptide, then 10 ul ATP/Mg 2+ (5 mM ATP/50 mM MgCl 2 ), then 10 ul of 5×Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl 2 , 5 mM MnCl 2 , 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate (1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30° C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.
[0664] The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.
[0665] Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 370C for 20 min. This allows the streptavadin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD (0.5 u/ml)) to each well and incubate at 37° C. for one hour. Wash the well as above.
[0666] Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 20
[0667] High-Throughput Screening Assay Identifying Phosphorylation Activity
[0668] As a potential alternative and/or compliment to the assay of protein tyrosine kinase activity described in Example 19, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-i or Erk-2 in the following assay.
[0669] Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4° C. until use.
[0670] A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 11 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
[0671] After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation.
Example 21
[0672] Method of Determining Alterations in a Gene Corresponding to a Polynucleotide
[0673] RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X. Suggested PCR conditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffer solutions described in Sidransky, D., et al., Science 252:706 (1991).
[0674] PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.
[0675] PCR products is cloned into T-tailed vectors as described in Holton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
[0676] Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson, Cg. et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-i DNA for specific hybridization to the corresponding genomic locus.
[0677] Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.
Example 22
[0678] Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample
[0679] A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
[0680] For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.
[0681] The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.
[0682] Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.
[0683] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.
Example 23
[0684] Formulating a Polypeptide
[0685] The secreted polypeptide composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the secreted polypeptide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.
[0686] As a general proposition, the total pharmaceutically effective amount of secreted polypeptide administered parenterally per dose will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the secreted polypeptide is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
[0687] Pharmaceutical compositions containing the secreted protein of the invention are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
[0688] The secreted polypeptide is also suitably administered by sustained-release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (198 1), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomally entrapped polypeptides. Liposomes containing the secreted polypeptide are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal secreted polypeptide therapy.
[0689] For parenteral administration, in one embodiment, the secreted polypeptide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
[0690] Generally, the formulations are prepared by contacting the polypeptide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
[0691] The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutaric acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
[0692] The secreted polypeptide is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
[0693] Any polypeptide to be used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[0694] Polypeptides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered I% (w/v) aqueous polypeptide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized polypeptide using bacteriostatic Water-for-Injection.
[0695] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
Example 24
[0696] Method of Treating Decreased Levels of the Polypeptide
[0697] It will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a pharmaceutical composition comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.
[0698] For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 23.
Example 25
[0699] Method of Treating Increased Levels of the Polypeptide
[0700] Antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer.
[0701] For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided in Example 23.
Example 26
[0702] Method of Treatment Using Gene Therapy
[0703] One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37° C. for approximately one week.
[0704] At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
[0705] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
[0706] The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.
[0707] The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
[0708] Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.
[0709] The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
Example 27
[0710] Method of Treatment Using Gene Therapy—In Vivo
[0711] Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al. (1997) Cardiovasc. Res. 35(3): 470-479, Chao J et al. (1997) Pharmacol. Res. 35(6): 517-522, Wolff J.A. (1997) Neuromuscul. Disord. 7(5): 314-318, Schwartz B. et al. (1996) Gene Ther. 3(5): 405-411, Tsurumi Y. et al. (1996) Circulation 94(12): 3281-3290 (incorporated herein by reference).
[0712] The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
[0713] The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(l): 1-7) which can be prepared by methods well known to those skilled in the art.
[0714] The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
[0715] The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated-cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
[0716] For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
[0717] The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
[0718] Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.
[0719] After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 urn cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 28
[0720] Transgenic Animals.
[0721] The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
[0722] Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
[0723] Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
[0724] The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
[0725] Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
[0726] Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
[0727] Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 29
[0728] Knock-Out Animals.
[0729] Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.
[0730] In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
[0731] Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).
[0732] When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
[0733] Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
[0734] It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
[0735] The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.



1


196


1
733
DNA
Homo sapiens

1

gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60

aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120

tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180

tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240

aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300

ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360

agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420

catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480

atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540

ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600

acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660

acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720

gactctagag gat 733



2
5
PRT
Homo sapiens

Site
(3)
Xaa equals any of the twenty naturally ocurring
L-amino acids


2

Trp Ser Xaa Trp Ser
1 5



3
86
DNA
Homo sapiens

3

gcgcctcgag atttccccga aatctagatt tccccgaaat gatttccccg aaatgatttc 60

cccgaaatat ctgccatctc aattag 86



4
27
DNA
Homo sapiens

4

gcggcaagct ttttgcaaag cctaggc 27



5
271
DNA
Homo sapiens

5

ctcgagattt ccccgaaatc tagatttccc cgaaatgatt tccccgaaat gatttccccg 60

aaatatctgc catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 120

gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 180

ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 240

ttttggaggc ctaggctttt gcaaaaagct t 271



6
32
DNA
Homo sapiens

6

gcgctcgagg gatgacagcg atagaacccc gg 32



7
31
DNA
Homo sapiens

7

gcgaagcttc gcgactcccc ggatccgcct c 31



8
12
DNA
Homo sapiens

8

ggggactttc cc 12



9
73
DNA
Homo sapiens

9

gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg 60

ccatctcaat tag 73



10
256
DNA
Homo sapiens

10

ctcgagggga ctttcccggg gactttccgg ggactttccg ggactttcca tctgccatct 60

caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 120

cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 180

ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 240

cttttgcaaa aagctt 256



11
1713
DNA
Homo sapiens

11

ggcacgagca cagataaaga taagttttac tgtcatgctg cttttaacat aacagagcaa 60

catcacctag gaaaaaagtt tgtaggagga tttttaatcc atatatttgt cttatggcta 120

gataaagatt tctctgaaaa aaagaagcat gtcaggaatc tctgggtgcc cctttttcct 180

ctggggactt ctagcattgt tgggcttggc tttggttata tcactgatct tcaatatttc 240

ccactatgtg gaaaagcaac gacaagataa aatgtacagc tactccagtg accacaccag 300

ggttgatgag tattatattg aagacacacc aatttatggt aacttagatg atatgatttc 360

agaaccaatg gatgaaaatt gctatgaaca aatgaaagcc cgaccagaga aatctgtaaa 420

taagatgcag gaagccaccc catctgcaca ggcaaccaat gaaacacaga tgtgctacgc 480

ctcacttgat cacagcgtta aggggaagcg tagaagccca ggaaacagaa tactcatttc 540

tcagacaagg atggagatga gcaactacat gcaatagatg ccagcgtttc taaaccacct 600

tagtagacag tttctccccg aaagccaggc gtagaggaaa acattcatga tgatcccatc 660

gactgtttgg attgatccgt gctaagagaa acctattaac tagctggacc atgatctgtt 720

caatgattgg ctcctattga agatggcttc taagaaaaca agatgcacag aggacacaga 780

aggacttggc agcagggtga tgacctgatc atttgttgat gggatggtgg cttacctctt 840

attcacagct tacacttatg catgccaaat gtaaggccat gaaaatcagt atttcaaata 900

acttaaaaaa tgctttacta ctaaaatgta aaaaattaat gtgctcacct cggcagcaca 960

tatactaaaa attaataaga cccagcttga aaattgagcc tgataacaag attacaaatt 1020

cacaatacct aatacttagg gaaatataaa aatttaagca tgaatgtgtt ctggaacacg 1080

ttagaagaaa aataaaagcc aatgagtttt tttttaattc tcctttctca ccaatgggca 1140

atagcccata attgaaataa atttctgatt gaaaggtata ggaaacatta aaatgcatta 1200

ctaagagaag taatataatt ttcttacaaa gtatttttcc caaagatagc tttactattt 1260

caaaaattgt caaattaatg catgctcctt acaacaaaca aatatcaaaa agagtttagg 1320

aattctacta gccagagata gtcacttgga gaaactttct atatatcctt ctaaatattt 1380

ttctgggcat gcttatgtat gtacatcagt tgtttctttt tattttgaac caaaaatgtg 1440

gtttcttttg tacacattac ttaaactttc tttccagtca acaatatatt gtggatttat 1500

tttcactgtt atatttaact atatataaat acgcatatat tgtaatttta atgtctgctt 1560

agcaccccac tgataaccaa atcacagttt atttaaataa ttttaatgac ttttcaaaaa 1620

caatttattg atgcaaaaag caaggttgag atgacaatgt ttctttcaat aattaaaaaa 1680

tactgcttca ctgtcaaaaa aaaaaaaaaa aaa 1713



12
2062
DNA
Homo sapiens

SITE
(1674)
n equals a,t,g, or c


12

ggcagagcat taattgatat tttaatatgg atagacattg catagattca aataaattaa 60

aatcaatgat aaatgctaaa tattttatct aaatagtttt tcaagaaaca gttatggaaa 120

tgtgtatatt raatggctct aatgtggagc ttgtggtatt tcaactcagt attcattatt 180

agttgtgtgt ctggaaagat tgtacttact tttcctcttt acactacagt ttgctcttat 240

ggggctctaa actgtttaac tgaagaacct tcgtctgtat tttgattgag cataatttag 300

tattttatga tttccaagat gatgttctta tgtctatcaa gtctatgtat caaatttata 360

acatcattta agaaaaagga atttccacag atacttcagt tgcaattttt tgtttcatgc 420

tactgaaaat acatttgttt ctaggggttg gaatattata gaagatgtag gatgaaagaa 480

aacgatagaa caacgaaaga attctgttta tgaaattaca ggaattgtgt ccactatggt 540

aaagcattgt cattttagta cattttctct tagtagtttg gcattttata ctttaaaact 600

tgttttgctt taaaaattgt ttataatgct taccttcttt ctccagtgcc tttagtcttg 660

atttgatatg tttgtaccct cagttaccct ttctattaca tgtttttgat gttttcatag 720

cctaggaaac atcgattcct ttttaataat tgtcaatctg attatttaaa gaggtaacaa 780

ttatctgtta atgctttgga aaaacaagta gggttgcctt tggaggccag gcttcttart 840

tcattcaaaa atattccttg gatttatgcc atgtwttaag catttttagc ccccagtatt 900

acaactgtga accaaacgga taaggcccta accattttca gcattctctt tggatggggt 960

gggattgggg acttaattaa aatagagata tagaaaaata ggcatctaaa taagataata 1020

agtgtggggt tgaaatgaag catctaacaa tagttgaagt tagaagtaat attttacagt 1080

attgtaacct ctatttaagt ttgggtatta gttacagata gcataaaaaa gccttaattt 1140

ttcactttcc ttgctggcaa aggtacattt atttagactg tccatttaaa gtaatgttta 1200

acataaacat tactgtgaaa aacattccat tacatattcc caagcaaatg agctgcatct 1260

tctttactgt attttacaat ttagtacaac agttttaggc ctcaatctta acatcactgg 1320

tattttaaat ttggcaatga atatgaaatt acttttgact tacagattga ttatattatt 1380

actttgaaaa tgcattaatt tcttagaaaa gtttggagcc tctatctttt tttgagttaa 1440

tacttaaatt ctcattactt atattaatag cctgtactaa gtgaaaatat tatttatgca 1500

agtaaacaag tcactatagg cttttaagac ttttctttaa ttttagattt tgtcatcaaa 1560

gtttaaattt tttacctact gtccacttrr atataattta acagtttgta aagtgaaata 1620

gtyttaagta tgatgtatga tgcaccggca tataatgaaa atggcgtgca caangacact 1680

ttactatggg aactgtacng gaagatttat gaaagcatgt gaaattgcac ctaaaattgt 1740

gttattagtg actataagca gcaatgctaa atttattgta cttgatgaat gaatgtattt 1800

agtcacagtt actttggttt aaatgtataa atgtctttag ggtttttttt taaatgtgtt 1860

tgtaatttgt actattgtgg gggtatactt ggactgcagg ggttattgtc aatgtgtgat 1920

ttgtgttttt attttataga atcatctaat gtgatatacc aatttttata agtgatattt 1980

acataattct aataactgta tatttgacaa cctattaaaa tgttttgcat tggaaaaaaa 2040

aaaaaaaaaa aaaaaactcg ta 2062



13
1224
DNA
Homo sapiens

SITE
(1205)
n equals a,t,g, or c


13

ggcacgaggg gactttccac agtcagctgg acgcacactc agcccagtaa aagaggggac 60

ccatcccggg agccccgggg agggcacagc tgcctcctcc cgggctcccc tgccacctgg 120

tgcctacctg ccccctgctc cctgccgggt ccggtcctca ccccatcttc atctggcctt 180

gactctgccc ttgaggggcc taggggtgca gccagcctgc tccgagctcc cctgcagatg 240

gaggaggcca tcctggtccc ctgcgtgctg gggctcctgc tgctgcccat cctggccatg 300

ttgatggcac tgtgtgtgca ctgccacaga ctgccaggct cctacgacag cacatcctca 360

gatagtttgt atccaaaggg gcatccagtt caaacggcct cacacggttg ccccctggcc 420

acctgcctac ccacctgtca cctcctaccc acccctgagc cagccagacc tgctccccat 480

cccaagatcc ccgcagcccc ttgggggctc ccaccggacg ccatcttccc ggcgggattc 540

tgatggtgcc aacagtgtgg cgagctacga aacgaggaac cagcctgtga ggatgcggat 600

gagatgagga cgactatcac aacccaggct acctggtggt gcttcctgac agcaccccgg 660

ccactacact gctgccccat cagctcctgc actcagcacc cctggcatcc gagacagtgc 720

cttctccatg gagtccattg atgattacgt gaacgttccg gagagcgggg agagcgcaga 780

agcgtctctg gatggcagcc gggagtatgt gaatgtgtcc caggaactgc atcctggagc 840

ggctaagact gagcctgccg ccctgagttc ccaggaggca gaggaagtgg aggaagaggg 900

ggctccagat tacgagaatc tgcaggagct gaactgaggg cctgtggagg ccgagtctgt 960

cctggaacca ggcttgcctg ggacggctga gctgggcagc tggaagtggc tctggggtcc 1020

tcacatggcg tcctgccctt gctccagcct gacaacagcc tgagaaatcc ccccgtaact 1080

tattatcact ttggggttcg gcctgtgccc ccgaacgctc tgaccttctg acgcagcctg 1140

agaatgacct gcctggcccc agccctactc tggtaataca ataaaggcct gcgtgtgtct 1200

gtgtnaaaaa aaaaaaaaaa aaaa 1224



14
1621
DNA
Homo sapiens

SITE
(1596)
n equals a,t,g, or c


14

gggcaaccct gccattctcc cctccagtgg aagattctac tttattcagg aaggctggtg 60

gcttgcaggg gctaggctta cacaagccag ccaagattga gaaggtaggg gagtgctttc 120

aatggaggcc atagttgtca tcttttcttc tgagcaggct ctaggacaaa caatgagcaa 180

attgagatgt cctgtattgg gaggatgagg ctgatatgtt tcatcattct cagaatttgt 240

gggctagagc acctttttgg gaatatgggc ctagggraaa agaacggtca cttacctgga 300

cactatggcc atagtcttga gtttttctag tcagcatgtg attggttgta ccttaagttt 360

atgmcaccaa aaatactatt aactctgtta tttttgttct taatcccatc tcaaattagt 420

ttctagagtt agaatcagaa cacaaggatg ctcattttca catggaggga aagttgccaa 480

agcatttaaa aaataacccc aaaagtatat ctagcagtga agcttggtag ataaaggtga 540

actgctaaac agacactggc gtgccacact cctgccaggg cagtctcatt gtgtgtgacg 600

gtgctgcaac actgccagta ttacttgaga tgcagtctct ctcccctgtt ctcagttcct 660

gggctcagcc ctcctccaag gcctgccagt gagtagcwgt ttggaaggca ggccaaggga 720

gatccccaaa gacagttatc agtcttaact tcygctgtct cccgtcaaat acttatacag 780

gcccccatgg gtaataggca agcatgatgg ctgatagaga agtcagtgca tgagttacta 840

tcacatgtcc ccccaacccc tcctgccacc tcccagggct ctggataatt gaatcttcct 900

gagtcccaca gctggagact caaccaggat atagctgtaa atgcccaagt agaatctgac 960

agaataagac agagacactg aaaataaagc cctagaaagg aagaaattgg aagcaaagaa 1020

aaggagaggt gaaaagataa aaagcctcct tcaaggttag gttcaggttc tgttttccat 1080

ttaacctcat gtgccataaa gctgcccagg cacaccagag ccacatcctg aacccgaccc 1140

tccctgacag tgctgctctg ccagtagcaa gccccagatg gaggaagctg ggcccatttc 1200

tggccacttc cacccatttg gagctttgcc agaggagtcg tctatgccaa taatatttct 1260

gcaacagcat attatattat ttgaagatta gtagatcttt ttgggggggk tggggcaggg 1320

gacagtttct atagatgaag aaccagtgtt ggttgtacag ctgttggggg tcatctatcc 1380

catgtgaagc tattcttttt ccaaatcttg ttgtttctgc atttgtgtcc tccaccactc 1440

ccttcttggc tgacatagat atgcctgcca gattktcatc aagggtcata tttcaataaa 1500

aggtgctaag gacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa agggcggccg ttttanagga tccaagntta cgtacncgtg 1620

c 1621



15
1420
DNA
Homo sapiens

15

ggcacgagca caagctcaag aggccgcttg cacgcatgtg gacactccat gattctgctt 60

ctatctctct ttcagggcgt gcgaggcagc ctgggctccc ctggaaatcg ggaaaacaag 120

gagaagaagg tcttcatcag cctggtaggc tcccgaggcc ttggctgcag catttccagc 180

ggccccatcc agaagcctgg catctttatc agccatgtga aacctggctc cctgtctgct 240

gaggtgggat tggagatagg ggaccagatt gtcgaagtca atggcgtcga cttctctaac 300

ctggatcaca aggagctgca gctggccggg agctgttcat gacagaccgg gagcggctgg 360

cagaggcgcg gcagcgtgag ctgcagcggc aggagcttct catgcagaag cggctggcga 420

tggagtccaa caagatcctc caggagcagc aggagatgga gcggcaaagg agaaaagaaa 480

ttgcccagaa ggcagcagag gaaaatgaga gataccggaa ggagatggaa cagattgtag 540

aggaggaaga gaagtttaag aagcaatggg aagaagactg gggctcaaag gaacagctac 600

tcttgcctaa aaccatcact gctgaggtac acccagtacc ccttcgcaag ccaaagtatg 660

atcagggagt ggaacctgag ctcgagcccg cagatgacct ggatggaggc acggaggagc 720

agggagagca ggatttccgg aaatatgagg aaggctttga cccctactct atgttcaccc 780

cagagcagat catggggaag gatgtccggc tcctacgcat caagaaggag ggatccttag 840

acctggccct ggaaggcggt gtggactccc ccattgggaa ggtggttgtt tctgctgtgt 900

atgagcgggg agctgctgag cggcatggtg gcattgtgaa aggggacgag atcatggcaa 960

tcaacggcaa gattgtgaca gactacaccc tggctgaggc tgacgctgcc ctgcagaagg 1020

cctggaatca gggcggggac tggatcgacc ttgtggttgc cgtctgcccc ccaaaggagt 1080

atgacgatga gctgaccttc ttctgaagtc caaaagggga aaccaaattc accgttagga 1140

aacagtgagc tccggcccca cctcgtgaac acaaagcctc ggaccagcct tgagagaggc 1200

cacactattc ctttcctctg gcccagtgaa tttggtctct cccagctctg ggggactcct 1260

tccttgaacc ctaataagac cccactggag tctctctctc tccatccctc tcctctgccc 1320

tctgctctaa ttgctgccag gattgtcact ccaaacctta ctctgagctc attaataaaa 1380

taaacagatt tattttccag cttaaaaaaa aaaaaaaaaa 1420



16
1035
DNA
Homo sapiens

SITE
(55)
n equals a,t,g, or c


16

gagcggataa caaatttcac acaggaaaca gctatgacca tgattacgcc caagntcgaa 60

attaaccttc actaaaggga acaaaagctg gagctccacc gcggtggcgn ccgctctaga 120

actagtggat cccccgggct gcaggaattc ggcacgaggt tacctcctct ctttcagaaa 180

aaagtgttta aatttaataa aaaaatacag acttcctctc tctctgacct gtttctgcac 240

ttctaatttt gtcccattgt tatatctcaa ttctgaaaca agtcccaaac ctttttgtac 300

actcaggctt ttattattta taggtgtctt taatgtggtt tcgctgtttt ttgcttattt 360

ttgtgagcag tgtgactttg acaggtgact ttagaaacat gaagaagcca agcagcctgt 420

gcctctttag acagggcttg atgtctgctt ctgaagttag tggcagcgga agtggagaag 480

gggattgaaa ggtatcttta aattcgraat tatagaaagt aaaaactggt agatgtgagg 540

acagtgggga aactaagatc atagtcgcct aaggttctgt taatacttga gttgaccagg 600

ggggctggtt atgacattga tcatgctaaa ggaaaagatg ccaggaatga gctggggcag 660

agtgaattgg gcagccttcc atcttgacag cacaccaaaa tgtataaatt agcaaaagcc 720

catctttccc taatgccact aagctgtcag tttctggaat tatcatcatt attarattca 780

taatggtttt aatraaggtg tcatccaaac tgacactttg aaaataaagt gagatgatgc 840

ctaaattgga ggcttggaat gaccttagaa aactgctcca ggaaacttga gaatgtccca 900

attacttaaa gaactctgag tcagctacat ggctcattcc attcatttgc tttgcattgg 960

agagatttat ttggattgac acaggttcat gcctcccaga aggctccacc taaaccatca 1020

ctctgctttc tcgag 1035



17
858
DNA
Homo sapiens

17

ggggacatat gtactgtctg tgtggcctat tattacaagc tctcctgaga ctttgtaatg 60

gatacaagac tcaaaagaac catagggagc tgagaatgtg tggtatcatc gcccagggaa 120

aaagccgttg gcagctgcat tgttatccag ggatgaaaag ttagcctgta ttttgcctga 180

gggacgtgat ttgcccttgt tagtacctgt gcctgctttg tcattccagc tgcaggttgg 240

catgctccaa cagcatcatg gacggggcct acacctacgc cgaggtgacc aatgtctcca 300

actgttggat ctgcacaact cttccagcag cagctgggga cagcttgttc tggcacgtgc 360

atccagcttc tatgcagaag tggacatggc tagagacttg gggtcctgca gacaacaggt 420

aggatgcaac acggtgagct ttgaacaggg gttgtcgcag aacccatggc aagcctgccc 480

cctggctgac ttgtagtgtc catgatggat gagcttggct aatgggagaa catgtagtgc 540

ccccattgca ggtaccataa tgtatagagc agcactgtgg taaagtcact gtaagatggt 600

tgcctgctga ggtttgtgca aacaaaacac atgtcaccac aacaagggtg tggtggaaca 660

agcagacttg ccaaggctgg ggccccacgg attttgtgcc ccttgggagt ttatgggtct 720

gtggggacac agaatggcca tctctgccag ccaattggac tggacgttgt acctggaggt 780

ggccctatgt ggctgccact gtgcttccca cctcgtgccg aattcgatat caagcttatc 840

gataccgtcg acctcgag 858



18
881
DNA
Homo sapiens

18

aggaggctga agtgggagga tcacctgggc ccagggaggt caaggatgca gtgagccatg 60

gtctcaccac tgcactctag cctgggtgac agaatgagac cccgtctcaa aaaaaaagaa 120

gaagtagata atctgaatag ccctatatct atagaaactt aatagtgctg ggagatatag 180

gtattattat cctcatttta cagatgtgaa aattgaggct cagagaagta aagtctattg 240

ctcaaggtca tgtggctaga atatggcaga gccatgattc agatccaggt cttctgattc 300

ttattccagt gtcctttcta gcataccatg ttgcctctaa agattgcagc tccttattta 360

ctagaaaatt gttcctgccc aatctacatc tccacctcac cccatctttt cttaagcact 420

atgtttgtgt ttttatcagt attatattca ttgtctttgg aatacatgtt cttgtttgtg 480

tttggaaaaa aaatctcttt taccagcttg cactcggacc aacttggaaa aaaaaaagct 540

taaatgtttt tgctatgtac agtttaaaaa tgtgaagttt gtagctttaa ctttttgtaa 600

gaaaatctaa taacactggc ttaagtgctg acttgaaatg ctattttgta aggtttggat 660

gtaagtaatc aattgaggtc agcagtttgt atgagacata gcttcctcca ttgcccccac 720

tccttttttc ttttttaagt ttgagatgct tcctgtgttt ttatgttaga attgttgttc 780

tccttctttt cttcttccta tacctcatca cgtttgtttt aaataaactg tcctttggac 840

cacaaaccct taaaaaaaaa aaaaaacaaa aaaraaagaa a 881



19
613
DNA
Homo sapiens

SITE
(602)
n equals a,t,g, or c


19

ggcacgaggt catcccttat gtatagtagt taaaggcata aaactgtgac ttttagatat 60

tccacagaac cagacttatt tgatgtggat aataaccaat gatttagcat tttgtttgct 120

tttgttttat tttatccggg ttcatttttt actcttccca tgtacatgaa acaggtggtg 180

gcgtgtagag atcagctgat ccttgtttta tggttaattg aactactttg tatccagggt 240

ttctgcaaat ccaaaagtga tttttcatct aggatctatt cctaacagtc tactccaatc 300

ccactttagt tttccacaat tttaaatctt aatagtgaga attcaaatga aagtcatttc 360

atttgactat tctgatgaca tgattgtggc agaataaatt gggtcttaaa atgccctaga 420

aaatggtaaa tgattaaaaa taatatttta aaattcaacc aaagaaatgg cccattggcc 480

aggtgtggtg gctcacacct gtaatcccag cacttttgga ggctgaggcg ggtggatcac 540

ctgagctcac gagtttgaga ccagcctacc caacatggta aaaccccatc tctacaaaaa 600

annaannnaa aaa 613



20
571
DNA
Homo sapiens

20

ggcacgagtc aaccgtcaaa atgtccaaag aacctctcat tctctggctg atgattgagt 60

tttggtggct ttacctgaca ccagtcactt cagagactgt tgtgacggag gttttgggtc 120

accgggtgac tttgccctgt ctgtactcat cctggtctca caacagcaac agcatgtgct 180

gggggaaaga ccagtgcccc tactccggtt gcaaggaggc gctcatccgc actgatggaa 240

tgagggtgac ctcaagaaag tcagcaaaat atagacttca ggggactatc ccgagaggtg 300

atgtctcctt gaccatctta aaccccagtg aaagtgacag cggtgtgtac tgctgccgca 360

tagaagtgcc tggctggttc aacgatgtaa agataaacgt gcgcctgaat ctacagagag 420

cctcaacaac cacgcacaga acagcaacca ccaccacacg cagaacaaca acaacaagcc 480

ccaccaccac ccgacaaatg acaacaaccc cagctgcact tccaacaacc aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 571



21
2024
DNA
Homo sapiens

SITE
(540)
n equals a,t,g, or c


21

ggcacgaggt taattgcttt caattgagtc agtaaacctg tgatagataa tttatttaac 60

tggaaaacct aggtacccat aagaaaaaag attcattctc tgtgaaaact gtaggaatct 120

gttgttgttt tcatttgaat atgctctact tctgctctag tatttggttt ggaatatatt 180

ttgtggctct aattactgta tttttaaaaa ccctacctcc attaacagtt ggtaaaggcc 240

ccttttcagg aaagtttgtt gctttttttt tttttttaaa ggaaagctgc tctttgctca 300

gtatagtgtt ttgaaagtga acatagtaac aaatacttta aaaataaagg atacacaatt 360

tatatttgaa aataaaaact ttctgctggt gggattattt atagtccttt atttttaaaa 420

aaaatgtttc cctttttata ttgctcttga aagtttaatg agcagaatac aatactggtt 480

ataataaaaa tatggtaacc acacagtact cagcctttca atatgttttg ggtcaaactn 540

catttaggca ctagcattta gaaaaatacc aatcacagtg atgctttggt tatttaatat 600

gaaggaaatg gaactaaaac atttatgtca tcaaatttat ttcactcctt tatatttgac 660

tggctggttg atacataatg gttgatgaac atatatttgc ttaaatcact aatagggatg 720

gttgtaaagt aaatagatca ttggttcaac catttaagtg tttttgccag attgtcaatg 780

aaaccatcat actgaccttt tcctccaaaa ttgccaaatt gactgaactg gttgggtgtt 840

tgttaaaatg aavtcaactg tgtgactaaa aagtcagatg gttgccatat tgtttggaat 900

atgttgaatg tcagtgtatg ccttatgtct ttaattgggt atgcaaaaaa atttttactt 960

aagtagatta aaattttaac ctctagcatg aaaacccagc accaacactg aaagactcca 1020

ttcaggttga agtagcctca aacagtaatt tactttttga taataggctg ttgtttttct 1080

taaataagct taaaacaatt ctatctgaaa ttggtagcat gggttcactt ggctacaact 1140

gagcaaaata gatgcaactt tcttttaatg gggtgctgcc tctttaggac tgactgtact 1200

atccactgac actggtttgg cagttggtac tgctgaacat ttttatacat gctaccatga 1260

agctatatat gttagtattg aagaagctaa cgggtatact atcatttttg atgtgtgggc 1320

tgattataat ttcctgtatt tcctgtacat tgggatgaaa ctactttagc aaagtccaca 1380

gatcagaaac cagacggtag tttttgaagt tgaaaccagc aaaataagaa aaaataaaaa 1440

atcgatttta attttctgcc tctttcccaa attacccttc ccacttgctc gacaaatcta 1500

tgtaaagcag tttgtttttt catgttttaa ctttaccttg ccctgtgtta tggtactggc 1560

tgacatgtct aagactggat gtgtatattt attatggtgt ctaaaaatca tgaagttcat 1620

cacttttcag gagtatagat aaaatcaaat tggtagtaca tcagagttac ttttcagtgc 1680

accatgacat cactaaaatg agtgctgtaa tgttacaggg ctttcaggtt tgtaaaaaca 1740

taaccataaa ttatattgac gtcagatatg agttgagtat ctataaaata tcacgtgtat 1800

ctcaaaatat tggactgctg tttgactgga tattgctgca taattttctt ctattgtccc 1860

atatcctttt ggagagagat ttaatgggat ttgaaatgtg caagctgtct aaataagatg 1920

cagtcaaata aagtatggtt aagttgtgtt tgcatttttc ttttagatac agctgtgtgc 1980

attttatgat tgggttgttt tgcttttacc tactaagtaa aaaa 2024



22
575
DNA
Homo sapiens

22

gaattcggca cgagatttgt gaggcctcag ctcacaaaac ctgaatcagg gtccaacagc 60

atctgcctgt ttttattctg caaagctgag atcaagttaa aaggatgatg ataatcactt 120

gtttatctat tcactcagca aacacttatg ggacatttac tgggtgccag gtcttgagct 180

gggcactgag atgagaagct gcctgaggca tgatggtccc atcccactgc cctccagggc 240

tcaaaatact ttatagttta caaagtgttt tcacatgcaa ttttgtgagc tgtgggtgcc 300

attattatcc acattgctta atacatggca gaacctgacc ttaggttgtc caagtccaga 360

ttctaagtcc aagtccagtc cagatcctag agcctgtcca ctctttcctt ccttcctctc 420

tttctttctt gttagttctt tctttttctt tttctctttt ttctttctct ctctttcttt 480

ctttcttcct ttcttctttc tcttttcttt ctttctctct ctctctcttt ctttttttca 540

agaccctgtc caaaaaaaaa aaaaaaaaac tcgag 575



23
1181
DNA
Homo sapiens

23

ggcacgagtc gccatctacc ctctagaata aagaaatctt atcattcacc gtctaccctc 60

tagagtaaac aaatcttatc attcgccatc taccctctag aatagagaaa tcttatcatt 120

catcatctac cctccagaat aaagaaatgt tatcatttgc catctaccct ctagaataaa 180

gaaatcttat taaggacatt ttcaaagcct taacagaata tgaatgatta caatattatg 240

ttttacctat acagcatctt ccaagttcta gtttggttgt gccaggccaa acatttgagc 300

cagatttcgg caagatcaag caggagactc tggcgtctgt cgctgattac cttcccgcca 360

tacctggcca ccagcctttc ccatggaccc cacgtgtgtc tccagactct tgggtatgaa 420

tcctgtgaac acactgacct ctgctttcta catgactgac aagcactatg aaatttgcat 480

aaatatgaat agaaaatata ctgtccccac atcccctaaa ataaaactga gtcctcaccc 540

atgggctttt gctggattat actaagaaaa aggcaggtct tacaacacac attcccatag 600

actcacatct cagagaagat tccttccaca ggctcagggt cctgaacaca ctgctccact 660

ctcagggctc ggagacactc tgcaggtggg tcttcacccc atccgggagc cctaattcct 720

tcttcctcgg tttttcatac agtgattttt cccagtactg ctgtctactt tcctctgcca 780

gaaattcctt gctggatcca tggcaatctc ctcaggactc ctatttcata aaatggaaat 840

ggaattcttc atatgcctta aaaagtaaaa taattatcca aacaatataa aatacaaaca 900

cccatctagc aaaatatttc ttagtacatc atgaattcaa atatatatcg tacaaaatga 960

ctgaggattt tattaatttt gagcaatatt gaggttttct cataatatct ttttattgtt 1020

aacttcaaac taaataacaa tcatgtctgg gaataaaatg tataagatag tcatttagaa 1080

aaatttggag atttttcctt ccttgtccag taaatactta acattttacg tgcaatgtat 1140

gtcataaata tgggcattcc cccaaaaaaa aaaaaaaaaa a 1181



24
2290
DNA
Homo sapiens

SITE
(146)
n equals a,t,g, or c


24

ggaggaatgg gaagcccgct aagagaacag aagtcaaaaa cagttgggtt ctagatggga 60

ggaggtgtgc gtgcacatgt atgtttgtgt ttcaggtctt ggaatctcag caggtcagtc 120

acattgcagt gtgtcgcttc acctgnctcc ctcttttaaa gattttcctt ccctctttcc 180

aactccctgg gtcctggatc ctccaacagt gtcagggtta gatgcctttt atgggccact 240

tgcattagtg tcctgataga ggcttaatca ctgctcagaa actgccttct gcccactgga 300

aagggaggca ggggaaatac atgattctaa ttaatggtcc aggcagagag gacactcaga 360

atttcaggac tgaagagtat acatgtgtgt gatggtaaat gggcaaaaat catcccttgg 420

cttctcatgc ataatgcatg ggcacacaga ctcaaaccct ctctcacaca catacacata 480

tacattgtta ttccacacac aaggcataat cccagtgtcc agtgncacat gcatacacgc 540

acacattccc ttcctaggcc actgtattgc tttcctaggg catcttctta taagacacca 600

gtcgtataag ggagcccacc ccactcatct gagcttatca accaattaca ttaggaaaga 660

ctgtatttcc tagtaaggtc acattcagta gtactgaggg ttgggacttc aacacagctt 720

tttgggggat cataattcaa cccatgacag ccactgagat tattatatct ccagagaata 780

aatgtgtgga gttaaaagga agatacatgt ggtacaaggg gtggtaaggc aagggtaaaa 840

ggggagggag gggattgaac tagacacaca cagacacatg agcaggactt tggggagtgt 900

gttttatatc tgtcagatgc ctagaacagc acctgaaata tgggactcaa tcattttagt 960

ccccttcttt ctataagtgt gtgtgtgygg atatgtgtgc tagatgttct tgctgtgtta 1020

ggaggtgata aacatttgtc catgttatat aggtggaaag ggtcagacta ctaaattgtg 1080

aagacatcat ctgtctgcat ttattgagaa tgtgaatatg aaacaagctg caagtattct 1140

ataaatgttc actgttatta gatattgtat gtctttgtgt ccttttattc atgaattctt 1200

gcacattatg aagaaagagt ccatgtggtc agtgtcttac ccggtgtagg gtaaatgcac 1260

ctgatagcaa taacttaagc acacctttat aatgacccta tatggcagat gctcctgaat 1320

gtgtgtttcg agctagaaaa tccgggagtg gccaatcgga gattcgtttc ttatctataa 1380

tagacatctg agcccctggc ccatcccatg aaacccaggc tgtagagagg attgaggcct 1440

taagttttgg gttaaatgac agttgccagg tgtcgctcat tagggaaagg ggttaagtga 1500

aaatgctgta taaactgcat gatgtttgca ggcagttgtg gttttcctgc ccagcctgcc 1560

accaccgggc catgcggata tgttgtccag cccaacacca caggaccatt tctgtatgta 1620

agacaattct atccagcccg ccacctctgg actccctccc ctgtatgtaa gccctcaata 1680

aaaccccacg tctcttttgc tggctctggg tctctttggc gtcttgaacc ttatgccttc 1740

cctattgaag ttaatagggg ttcagcacaa cactgggtcc caggcatgca gccttgtctt 1800

tttatgtttg tcctcatggg catcatgtgg gccacaggga tcttacccaa aatcatgcct 1860

agcaggaaga gatgcctctc tattgatatt ccagctgccc cacaagcagg tatgtgccta 1920

ctcattctct gacatgtgtc ctaggtttct tgactgtaam attccgctta ataaataatg 1980

tattgagcac ctgctatatc cagagctctg ctgggcactc tggaagatta gagtttgaag 2040

aacacacaat cgttgtcctc cagaagttca tggtccagta gggagagaag gtcacaaaca 2100

gccakaatct acaagatgag aggggctatw aaagagagct gagaagagtt tcagagccac 2160

tgagaagata ctcattttag tcttggtaac gggaancttt ctggagttgg tggcatttaa 2220

gatgaaccac aaatggagca ggaatttaat gaanangaaa ggaccaggac gtcnaataga 2280

ggacacagca 2290



25
891
DNA
Homo sapiens

25

ggcagagctg gcttgattga aggaaagctt gaaaaggcgc agagccctat acctcatttc 60

ctccatgata aaaggatcca agtgaggccc tgtcacagcc tgtgggtagg ggatgcggcg 120

ggatcctcat tgccatggta ctcaaaggta gaagagcctg gagtttgttg cttctctttg 180

ctattctttc atatcctctt gggcctggtg attaattagc aattctcatt cctctcagcc 240

aaaggcctgc actgggcttt atttgtcttt ttttattttt taagcactgc ctgccagaga 300

tgggcctggg gcctgatgag gaccttagcg ctgctcgttc tccttttctg ttcatgcaca 360

cattcctcca tggggtgggg aaggcaggca tggggtgtgg ccctcggaga agttaggagt 420

cccccagctc aagatacagt ggcaaagacc tagtggtccc ctacccccac ttctctcagt 480

tcctggcatg aggagagaag accctgctct ggtggagctg acaacctttg aggctgggag 540

gagagcagcc tctgggcatc gttcccagtg tccctcacac taaaacggcg tagatggcaa 600

ccccccaccc ccaccccgct gctcaactct tgtgtttgtt gttctgtttg ccccatttat 660

ctgttgctgt ttttgtgttg tcttcccctg tccgcatttt gtaaaatggc ccctggggga 720

gtgtttttgc tggatctgct ccctttcgct ctctcactcc actacttttt ggaacaaagt 780

gatggcagaa tgcggtggtg gtgggggtct tttgtactgt tggattaata aaatgatttt 840

aaaatcccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 891



26
465
DNA
Homo sapiens

26

actagtggat ccaaagaatt cggcacgagc ccagggaagt taagcaactt gcccactcac 60

tcagtctaat tggtgccggg gcagggacag aactcactca attccactgc tttccccagt 120

ttctggtgag tgatgtgtgc ctggcactgt gtgcacctgg cactgtgtgt agtaggaatg 180

ctgtttcttc tgtctgtaac ctcctcccag ttctgtaaac aaagacaaaa ccatgccctg 240

cccttaaagc ctatagggtt caaatgccac ctctttgatg atgctttccc tataacacca 300

tttgatacct ctcatgggac ttagcaagta attgcaacga gaggcatata tgttagttca 360

atatagcaaa cctttgttga gggcccactg tgtgccaggt atgtctcccc tccctgagga 420

ctggaagcaa aaaaagagaa gagaaaaaaa aaaaaaaaaa aaaaa 465



27
783
DNA
Homo sapiens

27

ggcttttttg ggtttatact gggaatttat ttttactaaa ttatttaact tttctaatta 60

tgtaattatg taagctagct tttcatgttt atgtatgtat ggtgtcccct tgtgttattt 120

ttcttcctct tggtttttga attagtgtta aatagaatac tgtctggatt cttaaaatat 180

tttcatttcc atcatggtta taacaaattt gctgcatgcc caaactgaca acagcaatca 240

ctgagggaac aggttttgaa tctttctttt gtgttatgaa gtttatcgtc tctacttgct 300

tgagattttt gttattttgg gggtttgggg gtgctttttg ttttgttttt gccaaatgta 360

acatgaaagc agatgctgca gctttagtct gttatgctga tttagtaaaa aaaaattttt 420

tacatatatt gcttgctttc gatgcttctg tgaaattttt tyctaaagct tttgtgcagc 480

tgtatggtaa aaatatggkg attaattkga agagcttaca ttgaaagaca atgtaatagg 540

aaataaatgt agattgcagt tggtcaagaa ttttgtagag aggataacaa gacttaatta 600

ctgaaaaaca gtaacatagc attttgaaat ataattttta aaatattgat gctttccttt 660

taaatggaaa tttaaawttt atgattaaaa gtttaaacat twatgataat tttcctcatc 720

agttctccca taggaaataa agcatgtgaa agggtaaaaa aaaaaaaaaa aaaaaaactc 780

gag 783



28
470
DNA
Homo sapiens

28

ggcacgagcc agggagatgt ggaccagaaa acctagctct gcaagctacc cagagaggga 60

caagattctc ggtgcctatg tgtaagagtt cacgccagta cacgtataca tcagtgcata 120

tgtgtcagtg tgcgtgtgag cgtgtagagt ggagggggtc actgacacca gccagggcgt 180

tgcataacca cctgacagag caatggtttc cccatggctt ccccttcttg tctcgctttt 240

tcacttactg aattgtctcc gcggtgttgg cacgtctggc cagagcctgg gactgccctc 300

ctcatccttc ccaccaacac cagagcacaa ggccactgcc cgtgactgaa ccacagggat 360

gctgcacacg tccagggatc ctgtcctttc ccttcctttg taagagtgtc ccagcctcct 420

gtcccctaca ctcagaccaa actgttccat ggtggtgact ccagccctga 470



29
1321
DNA
Homo sapiens

29

ggcacgagca ctgagctcta atcactaacc aggcacttgt ccagtcctgg tgggtaggat 60

aagagggtat ggaagtacat ttacctccct gcaggatctt ggaaggaaaa tgattgggac 120

cagagtaatc agtggagaaa aagaactaga caaactgtaa tcagggagtc ttgctgctac 180

aggaggtcat aggaggaaag artcaaagca tgtggtgttg gttggaaata attaaggagg 240

gcttcctgcc caatatggtc taaggatgga aaaactttgt atctcccagt ttgtctttct 300

tttctccata gccctgcctc tactttcctt ccttggaatc aggggttcct tagcccattt 360

gctttctcta ccttggggac cccaggggcc aagcagttct ccatctagtc acaccaaagg 420

caaaaagcct ggctacctcc cccctagcac gtgagtccct actcccctcc cctctgtttc 480

tgcccagctt tgcttatttt ggggatttca aggcagcaga gggtartgak agtaagggga 540

gagcaggmga agsctctgtc ctgtataggc aactgcmtga ctatgcggtg cactagctgt 600

aarccaagat caggtgccca gycctttttg tccattaaca ccccttcttg atctttcaaa 660

ggcagctaat tgctagcaaa tccccccgat tccggtcctt ttccctctat ttctttgtta 720

gaagttttct gtggagctga aaccccagcc tctgtttgac tgggttttca tttaggctta 780

gttgggttct tggagccccc tgtttgttgt tttgtgttgt ttccaatgaa aagcaagttt 840

accctcagag ttatgctttt ccaaagaggc tgatgtcttt gtttttgttt tttttaaatg 900

tttccagggt tctaaagtga aagtgaagtt ggggaagggg gtttgggagt gttaagtaat 960

ccaaggttta gaaacaccca tgagatagtt acccctgatc tccagtccct agctgggggc 1020

tggacagggg gaagggagag aggatttcta ttcaccttta atatattttt acaaaaaaag 1080

caaacaattt aaaaacaagc ccaccgcttc tgtacatgtc taaatatatt tttagaagtg 1140

ggtaggattg tgaatttctg atgcagggcc tttttataaa taggttaggg tagcatcatt 1200

cagacttctc tgttgttttt gtccctgtct ttttcttatg ttgtgttact aatgtaattt 1260

atattttttt tagatcctcc ctttcctata gagataaaag tgatttatct tggcaaatta 1320

t 1321



30
620
DNA
Homo sapiens

30

ggcacgagca ccaaactgcc atattgccgt gagaatgtgt gtctggctta tggtagtgaa 60

tggtcagttt atgcagtggg ctcccaagct catgtctcct tcttggatcc acggcagcca 120

tcatacaacg tcaagtctgt ctgttccagg gagcgaggca gtggaatccg gtcagtgagt 180

ttctacgagc acatcatcac tgtgggaaca gggcagggct ccctgctgtt ctatgacatc 240

cgagctcaga gatttctgga agagaggctc tcagcttgtt atgggtccaa gcccagacta 300

gcaggggaga atctgaaact aaccactggc aaaggctggc tgaatcatga tgaaacctgg 360

aggaattact tttcagacat tgacttcttc cccaatgctg tttacaccca ctgctacgac 420

tcgtctggaa cgaaactctt tgtggcagga ggtcccctcc cttcagggct ccatggaaac 480

tatgctgggc tctggagtta atgacaactc cccaaatgca gagatttcac taacttccaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa 620



31
1222
DNA
Homo sapiens

31

aattcccggg tcgaccacg cgtccgagcc cagcaacgtg caaggggaaa ggggacagga 60

ttctggatgg ccatttgctt cactgggatg caaaacctct tttgagtact agaatcagta 120

tttcttcttc catctctgct gtacctgaga agaaatggcc aaacgcacct tctctaactt 180

ggagacattc ctgattttcc tccttgtaat gatgagtgcc atcacagtgg cccttctcag 240

cctcttgttt atcaccagtg ggaccattga aaaccacaaa gatttaggag gccatttttt 300

ttcaaccacc caaagccctc cagccaccca gggctccaca gccgcccaac gctccacagc 360

cacccagcat tccacagcca cccagagctc cacagccact caaacttctc cagtgccttt 420

aaccccagag tctcctctat ttcagaactt cagtggctac catattggtg ttggacgagc 480

tgactgcaca ggacaagtag cagatatcaa tttgatgggc tatggcaaat ccggccagaa 540

tgcacagggc atcctcacca ggctatacag tcgtgccttc atcatggcag aacctgatgg 600

gtccaatcga acagtgtttg tcagcatcga cataggcatg gtatcccaaa ggctcaggct 660

ggaggtcctg aacagactgc agagtaaata tggctccctg tacagaagag ataatgtcat 720

cctgagtggc actcacactc attcaggtcc tgcaggatat ttccagtata ccgtgtttgt 780

aattgccagt gaaggattta gcaatcaaac ttttcagcac atggtcactg gtatcttgaa 840

gagcattgac ataccacaca caaatatgaa accaggcaaa atcttcatca ataaaggaaa 900

tgtggatggt gtgcagatca acagaagtcc gtattcttac cttcaaaatc cgcagtcaga 960

gagagcaagg tattcttcaa atacagacaa ggaaatgata gttttgaaaa tggtagattt 1020

gaatggagat gacttgggcc ttatcagttt ttcattcagc aagtctgcac tagggaccta 1080

ctatgagcca cgcaatactt ccttggaatg atgtattccc tggccttgaa ataaggaatc 1140

tagtacccat gtttgtgcta ctggaatgaa tccattaaac tctctgagac tcaaaaaaaa 1200

aaaaaaaaaa aaaaaaaagg gc 1222



32
829
DNA
Homo sapiens

SITE
(32)
n equals a,t,g, or c


32

aggtcaaaaa taataataaa tgttagcatc tnactcagtt ctgtgtgatc cagctcctgc 60

caacccatct gatgagctcc tggttcactc tccttgcttc ctgctttcac ctcctgtggc 120

ccctgtcccg ttcttcccat gtgccaagct cattccagcc cccagacctg tccgctactt 180

ttctcctcca gatcttaggt taggtaacac tccagccccc tctgagatca cctacacacc 240

ttctgttcac ttctgtcatc ctattgctaa attactttgt ctcaaggttc gtaatctctg 300

tgagggagtc ctctctgctg cttttcccaa ggcctgacgc acaccgggca cccagttaac 360

atctttaata tattgggctt tcgtgctacc tgagggcttc tagaaagctc tctacatcca 420

agtgagaggc tggctctcca ggaggtacct gtgctctcag cacaaatgga gcccctctgg 480

gaacaggaac ctgcttcagc cctttagcat acacaggcct gccacatgct ggggacaggg 540

ccacctgagt cccttgcctg gtcccatgtg tggtgccagc tccctcccct gatgattgtt 600

ctctctcttt cccaggaccg agatctcact cagcagacag cagcagcccg ggagcctgag 660

ctcaggagga actcttacct ggaaattggg aactgtatgg agactccaaa ctgacttctt 720

tcaaaaaaaa aaaaaaaaaa atttttttag ctttgacaaa cacacaaaag tngtaataaa 780

gagagccctc cttgtcaacc caaaatgtga gccccctgtg gaaaaccan 829



33
1336
DNA
Homo sapiens

33

ccacgcgtcc ggagttccac aaaatttgtt agtcatcaaa taatagagtt tttcttaagt 60

gaccacttac atactcatct acaataaacc ccaactaact aatttttcac tttgtgtacc 120

tcatgctaat atccgtggac agtaatgtgc ctgttgtgtt ccttttgctt ttcatccttg 180

tgatcttatg tcacatggaa tgtaaaggcc acatatatat atgtgtgtgt gtgtgtgtgt 240

atatgtatat ttttaagaat atttagtctg gatttcatga aattgacttc tgaaataatt 300

tgcctcaatt ttgtttcctg gtggtttgag aagaaagttc ctgtggtgaa atgaaaaggg 360

gataaaggga agtacttatt ttaaaacata agtaacttgt ggattgttga atactggaaa 420

aagagtgtta cttccccgtt aacctacgcc tcgtgtaatc cttcaggttg gaagtcggat 480

cgcagaccgt gtatatgaca tacccagaaa tttccccctt gctttggatc ttggttgtgg 540

aagaggttac attgcacaat atttgaataa gcttcagtta ttccattgca ggaaactatt 600

ggaaagtttt tccaagctga cattgcagaa aatgctttgt ttgcattggg tgaatgacct 660

tcctagagca cttgagcaga ttcattatat tttaaaacca gatggagtgt ttatcggtgc 720

aatgtttgga ggcgacacac tctatgaact tcggtgttcc ttacagttag cggaaacgga 780

aagggaagga ggattttctc cacacatttc tcctttcact gctgtcaatg acctgggaca 840

tctgcttggg agagctggct ttaatactct gactgtggac actgatgaaa ttcaagttaa 900

ctatcctgga atgtttgaat tgatggaaga tttacaagaa caaaagtcca gaatgttgac 960

ctaattttac aaaacaagct gcatatcagc tgatgaatgc atgagaaatt ttcaaggctt 1020

tcacagtggt cttaaggtat gggtgagagt aactgtgctt ggaatagaaa agccctgctg 1080

catcgagaca caatgctggc agctgcggca gtgtacagag aaatgtacag aaatgaagat 1140

ggttcagtac ctgctacata ccagatctat tacatgatag gatggaaata tcatgagtca 1200

caggcaagac cagctgaaag aggttccgca actgtgtcat ttggagagct aggaaaaata 1260

aacaacctta tgccaccggg gaaaaaatca caataaatat ttattcagtg ttaaaaaaaa 1320

aaaaaaaaaa aaaaaa 1336



34
1635
DNA
Homo sapiens

SITE
(85)
n equals a,t,g, or c


34

tcgacccacg cgtccgcagc tctccgtgga caatgggctg tggcgtgtga ccctgtgcat 60

gctggccttc ccgctgctcc tcacnggcct catctccttc agggagaaga ggctgcagga 120

tgtgggcacc cccgcggccc gcgcccgtgc cttcttcacc gcacccgtgg tggtcttcca 180

cctgaacatc ctctcctact tcgccttcct ctgcctgttc gcctacgtgc tcatggtgga 240

cttccagcct gtgccctcct ggtgcgagtg tgccatctac ctctggctct tctccttggt 300

gtgcgaggag atgcggcagc tcttctatga ccctgacgag tgcgggctga tgaagaaggc 360

agccttgtac ttcagtgact tctggaataa gctggacgtc ggcgcaatct tgctcttcgt 420

ggcagggctg acctgcaggc tcatcccggc gacgctgtac cccgggcgcg tcatcctctc 480

tctggacttc atcctgttct gcctccggct catgcacatt tttaccatca gtaagacgct 540

ggggcccaag atcatcattg tgaagcggat gatgaaggac gtcttcttct tcctcttcct 600

gctggctgtg tgggtggtgt ccttcggggt ggccaagcag gccatcctca tccacaacga 660

gcgccgggtg gactggctgt tccgargggc cgtctaccac tcctacctca ccatcttcgg 720

gcagatcccg ggctacatcg acggtgtgaa cttcaacccg gagcactgca gccccaatgg 780

caccgacccc tacaagccta agtgccccga gagcgacgcg acgcagcaga ggccggcctt 840

ccctgagtgg ctgacggtcc tcctactctg cctctacctg ctcttcacca acatcctgct 900

gctcaacctc ctcatcgcca tgttcaacta caccttccag caggtgcagg agcacacgga 960

ccagatttgg aagttccagc gccatgacct gatcgaggar taccacggcc gccccgccgc 1020

gccgcccccc ttcatcctcc tcagccacct gcagctcttc atcaagaggg tggtcctgaa 1080

gactccggcc aagaggcaca agcagctcaa gaacaagctg gagaagaacg aggaggcggc 1140

cctgctatcc tgggagatct acctgaagga gaactacctc cagaaccgac agttccagca 1200

aaagcagcgg cccgagcaga agatcgagga catcagcaat aaggttgacg ccatggtgga 1260

cctgctggac ctggacccac tgaagaggtc gggctccatg gagcagaggt tggcctccct 1320

ggaggagcag gtggcccaga cagcccgagc cctgcactgg atcgtgagga cgctgcgggc 1380

cagcggcttc agctcggagg cggacgtccc cactctggcc tcccagaagg ccgcggagga 1440

gccggatgct gagccgggag gcaggaagaa gacggaggag ccgggcgaca gctaccacgt 1500

gaatgcccgg cacctcctct accccaactg ccctgtcacg cgcttccccg tgcccaacga 1560

gaaggtgccc tgggagacgg agttcctgat ctatgaccca cccttttaca cggcagagag 1620

gaaggacgcg gccgc 1635



35
1264
DNA
Homo sapiens

35

tgggctggaa cgcgccggaa tctgaggtgt gagtagagcc tgggggagag tggatccagg 60

tgaagggggc agaggactgg gagttttcrt cctcttgaat aagaactcga caacagagtg 120

ggaactttct gtcttgtgat ccattgcctg gtgagtcaca gctcacacca tggatttaac 180

ctgagagctt caacttctgc tttggccctg gagttcccat gccctggtgt cttctaccag 240

ttcttagtgt gttgcactgg agcacagagg acactcgatc gtgcggcgcg cagggcgggg 300

ggccgccgct gcctccccgc gggatggctg gcactgtgct cggagtcggt gcgggcgtgt 360

tcatcttagc cctgctctgg gtggcagtgc tgctgctgtg tgtgctgctg tccagagcct 420

ccggggcggc gaggttctct gtcatttttt attcttcggt gctgtgatca tcacatcagt 480

tctgttgctt ttcccgcgag ctggtgaatt cccagcccca gaagtggaag ttaagattgt 540

ggatgacttt ttcattggcc gctatgtcct gctggctttc cttagtgcca tcttccttgg 600

aggcctcttc ttggttttaa tccattatgt tctggagccg atctatgcca aaccactgca 660

ctcctactga ccactcttca ggaawacgaa aacctgttct ctccttcatt gtgatgacat 720

tgatgagcag gaaggcacta ttcagagcct tgttttgaca gccctcatgc cttaaggtta 780

gaggagtatc tgtccatcac taagacaaat ctctggagtc ctggcttcca gaaacaggat 840

tgccaaattg tccctgtggg gctagattct taccagctta agaaggatat tgctatcttc 900

ttagtacccg taccttagga tttccaactg ttttgaaagg gaaatagtaa cagtgatctg 960

cttagagtgg attttcactc aagtccttag taagtggatt ttggggaaaa aagcacmtgg 1020

gcttctggtt ctttttgata atatataaaa ttattcatta tgaggttgca gttgtttgca 1080

aaggagaggc actcaaattt gaaaggttat tttaatgtga taatttggaa gacttactca 1140

gatgttggtc attgaccact ctgtgcatat atttctgcag agctctgtga aggcaatgag 1200

tgtcacttcc ctctgctcta ataaagcaat aaataataaa aaaaaaaaaa aaaaaaaact 1260

cgag 1264



36
688
DNA
Homo sapiens

SITE
(607)
n equals a,t,g, or c


36

cccacgcgtc cgggtcccga tgagcctcct gttgcctccg ctggcgctgc tgctgcttct 60

cgcggcgctt gtggccccag ccacagccgc cactgcctac cggccggact ggaaccgtct 120

gagcggccta acccgcgccc gggtagagac ctgcggggga tgacagctga accgcctaaa 180

ggaggtgaag gctttcgtca cgcaggacat tccattctat cacaacctgg tgatgaaaca 240

cctccctggg gccgaccctg agctcgtgct gctgggccgc cgctacgagg aactagagcg 300

catcccactc agtgaaatga cccgcgaaga gatcaatgcg ctagtgcagg agctcggctt 360

taccgcaagg cggcgcccga cgcgcaggtg ccccccgagt acgtgtgggc gcccgcgaag 420

cccccagagg aaacttcgga ccacgctgac ctgtaggtcc gggggcgcgg cggagctggg 480

acctacctgc ctgagtcctg gagacagaat gaagcgctca gcatcccggg aatacttctc 540

ttgctgagag ccgatgcccg tccccgggcc agcagggatg gggttgggga ggttctccca 600

accccanttt cttccttccc cagctccact aaattccctc ctgccttaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 688



37
1516
DNA
Homo sapiens

SITE
(34)
n equals a,t,g, or c


37

ttcacgtgca cactgatcac aacgtcacgc ctgncagggc accggtccgg gaattcccgg 60

gtcgacccac gcgtccgcag gagaggtgtc tgtgcgtcct gcacccacat ctttctctgt 120

cccctccttg ccctgtctgg aggctgctag actcctatct tctgaattct atagtgcctg 180

ggtctcagcg cagtgccgat ggtggcccgt ccttgtggtt cctctctact tggggaaatc 240

aggtgcagcg gccatggcta cagcaagacc cccctggatg tgggtgctct gtgctctgat 300

cacagccttg cttctggggg tcacagagca tgttctcgcc aacaatgatg tttcctgtga 360

ccacccctct aacaccgtgc cctctgggag caaccaggac ctgggagctg gggccgggga 420

agacgcccgg tcggatgaca gcagcagccg catcatcaat ggatccgact gcgatatgca 480

cacccagccg tggcaggccg cgctgttgct aaggcccaac cagctctact gcggggcggt 540

gttggtgcat ccacagtggc tgctcacggc cgcccactgc aggaagaaag ttttcagagt 600

ccgtctcggc cactactccc tgtcaccagt ttatgaatct gggcagcaga tgttccaggg 660

ggtcaaatcc atcccccacc ctggctactc ccaccctggc cactctaacg acctcatgct 720

catcaaactg aacagaagaa ttcgtcccac taaagatgtc agacccatca acgtctcctc 780

tcattgtccc tctgctggga caaagtgctt ggtgtctggc tgggggacaa ccaagagccc 840

ccaagtgcac ttccctaagg tcctccagtg cttgaatatc agcgtgctaa gtcagaaaag 900

gtgcgaggat gcttacccga gacagataga tgacaccatg ttctgcgccg gtgacaaagc 960

aggtagagac tcctgccagg gtgattctgg ggggcctgtg gtctgcaatg gctccctgca 1020

gggactcgtg tcctggggag attacccttg tgcccggccc aacagaccgg gtgtctacac 1080

gaacctctgc aagttcacca agtggatcca ggaaaccatc caggccaact cctgagtcat 1140

cccaggactc agcacaccgg catccccacc tgctgcaggg acagccctga cactcctttc 1200

agaccctcat tccttcccag agatgttgag aatgttcatc tctccagccc ctgaccccat 1260

gtctcctgga ctcagggtct gcttccccca cattgggctg accgtgtctc tctagttgaa 1320

ccctgggaac aatttccaaa actgtccagg gcgggggttg cgtctcaatc tccctggggc 1380

actttcatcc tcaagctcag ggcccatccc ttctctgcag ctctgaccca aatttagtcc 1440

cagaaataaa ctgagaagtg gaatcttaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaagggc ggccgc 1516



38
1267
DNA
Homo sapiens

38

ggcacgagct gcaggggcgg ggcggcgcca agcgcaggga gcccggctga gtggcagccc 60

agattgaaga tggatacgtg acaatcccag ggaccgctgc actgacttca tttccttaga 120

caagacacag tgtagggccc ggcccgtgtt ggccccagga ctcctttgga atatagctgt 180

ggacaatgaa tcctgcgagc gatgggggca catcagagag catttttgac ctggactatg 240

catcctgggg gatccgctcc acgctgatgg tcgctggctt tgtcttctac ttgggcgtct 300

ttgtggtctg ccaccagctg tcctcttccc tgaatgccac ttaccgttct ttggtggcca 360

gagagaaggt cttctgggac ctggcggcca cgcgtgcagt ctttggtgtt cagagcacag 420

ccgcagctgt gggctctgct gggggaccct gtgctgcatg ccgacaaggc gcgtggccag 480

cagaactggt gctggtttca catcacgaca gcaacgggat tcttttgctt tgaaaatgtt 540

gcagtccacc tgtccaactt gatcttccgg acatttgact tgtttctggt tatccaccat 600

ctctttgcct ttcttgggtt tcttggctgc ttggtcaatc tccaagctgg ccactatcta 660

gctatgacca cgttgctcct ggagatgagc acgcccttta cctgcgtttc ctggatgctc 720

ttaaaggcgg gctggtccga gtctctgttt tggaagctca accagtggct gatgattcac 780

atgtttcact gccgcatggt tctaacctac cacatgtggt gggtgtgttt ctggcactgg 840

gacggcctgg tcagcagcct gtatctgcct catttgacac tgttccttgt cggactggct 900

ctgcttacgc taatcattaa tccatattgg acccataaga agactcagca gcttctcaat 960

ccggtggact ggaacttcgc acagccagaa gccaagagca ggccagaagg caacgggcag 1020

ctgctgcgga agaagaggcc atagctgctc cagccggggc tccggggcgg cagcagagct 1080

ggcacaccga ttctgggaag ccccgcgaat gatggctttt gaattaatga ggcagtgaat 1140

gttttgtgtt tacttctaag ggaaatacta actttctttc gcattagtat taattttgaa 1200

gtagctacaa agtattttta agaaattata attttatgac tgtcaaaaaa aaaaaaaaaa 1260

aaaaaaa 1267



39
2203
DNA
Homo sapiens

SITE
(1246)
n equals a,t,g, or c


39

ggcacgagcg gaggcgcgcg ggccagcctg ggccccagcc cacaccttca ccagggccca 60

ggagccacca tgtggcgatg tccactgggg ctactgctgt tgctgccgct ggctggccac 120

ttggctctgg gtgcccagca gggtcgtggg cgccgggagc tagcaccggg tctgcacctg 180

cggggcatcc gggacgcggg aggccggtac tgccaggagc aggacctgtg ctgccgcggc 240

cgtgccgacg actgtgccct gccctacctg ggcgccatct gttactgtga cctcttctgc 300

aaccgcacgg tctccgactg ctgccctgac ttctgggact tctgcctcgg cgtgccaccc 360

ccttttcccc cgatccaagg atgtatgcat ggaggtcgta tctatccagt cttgggaacg 420

tactgggaca actgtaaccg ttgcacctgc caggaraaca ggcagtggca gtgtgaccaa 480

gaaccatgcc tggtggatcc agacatgatc aaagccatca accagggcaa ctatggctgg 540

caggctggga rccacagcgc cttctggggc atgaccctgg atgagggcat tcgstaccgc 600

ctgggcacca tccgcccatc ttcctcggtc atgaacatgc atgaaattta tacagtgctg 660

aacccagggg aggtgcttcc cacagccttc gaggcctctg arartkkycc amcctgwttc 720

awsagcctct wgaccaaggc aactgtgcag gctcctgggc cttctccaca gcagctgtgg 780

catccgatcg tgtctcaatc cattctctgg gacacatgac gcctgtcctg tcgccccaga 840

acctgctgtc ttgtgacacc caccagcagc agggctgccg cggtgggcgt ctcgatggtg 900

cctggtggtt cctgcgtcgc cgaggggtgg tgtctgacca ctgctacccc ttctcgggcc 960

gtgaacgaga cgaggctggc cctgcgcccc cctgtatgat gcacagccga gccatgggtc 1020

ggggcaagcg ccaggccact gcccactgcc ccaacagcta tgttaataac aatgacatct 1080

accaggtcac tcctgtctac cgcctcggct ccaacgacaa ggagatcatg aaggagctga 1140

tggagaatgg ccctgttcca agccctcatg gaggtgcatg aggacttctt cctataacaa 1200

gggargcatc tacagccaca cgccagtgag ccttgggagg ccaganagat accgccggca 1260

tgggacccac tcagtcaaga tcacaggatg gggagaggag acgctgccag atggaatgac 1320

gctcaaatac tggactgcgg ccaactcctg gggcccagcc tggggcgaga ggggccactt 1380

ccgcatcgtg cgcggcgtca atgagtgcga catcgagagc ttcgtgctgg gcgtctgggg 1440

ccgcgtgggc atggaggaca tgggtcatca ctgaggctgc gggcaccacg cggggtccgg 1500

cctgggatcc aggctaaggg ccggcggaag aggccccaat ggggcggtga ccccagcctc 1560

gcccgacaga gcccggggcg caggcgggcg ccagggcgct aatcccggcg cgggttccgc 1620

tgacgcagcg ccccgcctgg gagccgcggg caggcgagac tggcggagcc cccagacctc 1680

ccagtgggga cggggcaggg cctggcctgg gaaaagcaca gctgcagatc ccaggcctct 1740

ggcgccccca ctcaagacta ccaaagccag gacactcaag tctccagccc cactacccca 1800

ccccactcct gtattctttt tttttttttt ttagacaggg tcttgnctcc gttgccccag 1860

gttggagtgc agtggcccat cagggctcac tgtaacctcc gactcctggg ttcaagtgac 1920

cctcccacct cagcctctca agtagctggg actatcaggt gcaccaccac acctggctaa 1980

tttttgtatt ttttgtaaag aggggggtct cactgtgttg cccaggctgg tctcgaactc 2040

ctgggctcaa gcggtccacc tgcctccgcc tcccaaagtg actgggattg ccaggcatga 2100

gccactgcac ccagtccctg tattcttatt cttcagatat ttatttttct tttcactgtt 2160

ttaaaataaa accaaagtat tgataaaaaa aaaaaaaaaa aaa 2203



40
1726
DNA
Homo sapiens

40

cgtctgatta aggtaccttt tgggaaatta aggttctata gaaattactg ggctcaatct 60

agtgatacaa atatgtgttg tttgatttat caacacatta caaaccttaa ctttggagtt 120

ttaatatctg gttatcttta atatctggtt atcttctttc tgaagtgtat gtacacaaaa 180

ttgatgctaa ataaggtctt gttgttttgg caaatagtga aatgcaaggt attggtagat 240

cagtactgtt ataactttgg tgcaaagttg ctgcatgcag attggctgtg ggaccttgtt 300

cattttttga gaactaatgt agagtttgaa aaaacaccgt aagcctgcat tccagaagtt 360

ctggtatgga tagtgtgagc ccagggaatg tgcttagata aaagatcatt taacaaatag 420

gttttgcatt tttttagcaa tcaggctttg tgctgaatat tagagtggtt gtttcagaga 480

gtttgcagca attaggcttt attggtgcac taaggagaag cagagaggag aagcaattct 540

tggtaacttc cttggaagtt gcagctaact ctgaaaagtc tgggttgaac taggtaagta 600

actaattcct agaatcaata aactttgcag gagtccgttt gattgtacat gtagctccct 660

ggaattgcta ttggtcccta aatcatcagt ttgtaatgct ggttttcaaa cttgagtgca 720

catcaagttt tggaggactt gttagaatac agattgctgg gctcaccccc agagtttctg 780

atctggtagg tctggagcgg gacctggtag attgcatttc taaaaagcat ccaggtaata 840

ctgctgctgt ttgggaaagt acctttgaga tcactggctt acagcaatct caaggtgttt 900

ggattttggg caggggtgct gtgcaggcgt tgctgggatc tcttcacagc actccactgc 960

atagaggtga gcctccagat gttttcattc attcaacaaa tatatgtacc tattgtgtgc 1020

tgggcactgc ttaagttgcg aggggatatt gtgaagaaag taagcaaaac ccctttgttt 1080

gtagaatttc agtgagcata gtcctgggtt aacctgacaa cagtcctact gtttattgat 1140

gcttataggt gagcctattt ctctttctag ctttcttcca cttaatttac tttcttttgg 1200

aattcttgaa tttaataata ataatattga tgttattagt catcactata actttttatt 1260

gagtatgtat tttatgtcag acacagtgtg gctaagtgct ttacatacat tatctcatct 1320

aatccttaga aaaaaccctg gtgtattagt cttaatttaa aagatgtact ttggaaaggt 1380

tagtagttta cccaagatta tgcagctagt taaaagtggt gctggggctg ggcttggtgg 1440

ctcacacctg taatcgcagt gctttgggag tctgaggcag gaggatcgct tgacaccagg 1500

agtttgagac taacctggga aacatagcaa gaccccatct ttacaaaaaa taaaaaaatt 1560

agccaggagt gggggtgcac acctgtggtc ccagctactt gggaggctga ggcaggagga 1620

ttgcttgagc cccagaggtt gaggctgcag tgagccatga ttatgccact gcacatctgt 1680

ctgggtgaca gagcaagatc ctgtctccaa aaaaaaaaaa aaaaaa 1726



41
1102
DNA
Homo sapiens

41

ggcacgagtt tttatgctgg ttggtctctt gcagggaggt ggtgctgtcc agagagtatc 60

agctgtggta gtatagagag gaacaggtgg tggatggggc cctagaactc ccaggagtat 120

atgccctttg tcttcagtta ccagggtagg tagggaaggc tgttgggtca gggcagggct 180

aggcatgtct gagctcagac tcttcttggg caggtcttgc tgtggctgct gtgggggatg 240

ggggtgaggt tcccaggtca atggtgttat gttcctagga agtttatgtt tttctctcct 300

gtgtcatgca ggttgtcagg gaagtggggg aaagccagca gtcacaggcc tcacccagct 360

cccacataat ccaaagggct ggtttcactc ccaccatgcc ccccgacccc aatagcaccg 420

agtctgtttt aggcagtgga caagcagggc tgagaacttg ccccaggcta cctgcctccc 480

agctgtgaaa gcaagtttgg ctttccttct tcccctgcct gtagagtctg cacactggat 540

tcaggccctc ccccaagttc tggccaggag acttctcgat tagttcaaat tgttgccaag 600

ttcagctgga gatttccttc tcctgtggcc tttcccagtg cctctggcca ccctaccgaa 660

ggacaatgtg aggccaggca gaaatagttt gctaggggac ccagagagct cacagggctt 720

ttcctctacc cctgtatttt gaggggtaaa ggaaaatctg ctgtttcctc tacccctgta 780

ttttgcttgg ttctctcaat ttactcagct ccaggtaaga tcagcatcat ctcccgtaat 840

ctagaccttc agtttcctta gtggggtgtg tgttcaggag cagacgatct ccctttccca 900

cttccacggt ttgggcactc acagtatttg gatcatctcc tgggtccagc aggagcaatc 960

tgcttccttc agaaggtctg tgggtccccc ccggcttcct gatttattcc tgccgtcatt 1020

gaagacagag gcgacttcct ccttggaaac tttaggcgcc actgctcagt ccgaaaaagc 1080

ccattccatt ttctactaat tg 1102



42
1397
DNA
Homo sapiens

42

tcgacccacg cgtccgctga attgcggccg tatgcgcggc tctgtggagt gcacctgggg 60

ttgggggcac tgtgccccca gccccctgct cctttggact ctacttctgt ttgcagcccc 120

atttggcctg ctgggggaga agacccgcca gctgcttgag tttgacagca ccaacgtgtc 180

cgatacggca gcaaagcctt tgggaagacc atatcctcca tactccttgg ccgatttctc 240

ttggaacaac atcactgatt cattggatcc tgccaccctg agtgccacat ttcaaggcca 300

ccccatgaac gaccctacca ggacttttgc caatggcagc ctggccttca gggtccaggc 360

cttttccagg tccagccgac cagcccaacc ccctcgcctc ctgcacacag cagacacctg 420

tcagctagag gtggccctga ttggagcctc tccccgggga aaccgttccc tgtttgggct 480

ggaggtagcc acattgggcc agggccctga ctgcccctca atgcaggagc agcactccak 540

cgaacgatga atatgcaccg gccgtcttcc agttggacca gctactgtgg ggctccctcc 600

catcaggctt tgcacagtgg cgaccagtgg cttactccca gaagccgggg ggccgagaat 660

cagccctgcc ctgccaagct tcccctcttc atcctgcctt agcatactct cttccccagt 720

cacccattgt ccgagccttc tttgggtccc agaataactt ctgtgccttc aatctgacgt 780

tcggggcttc cacaggccct ggctattggg accaacacta cctcagctgg tcgatgctcc 840

tgggtgtggg cttccctcca gtggacggct tgtccccact agtcctgggc atcatggcag 900

tggccctggg tgccccaggg ctcatgctgc tagggggcgg cttggttctg ctgctgcacc 960

acaagaagta ctcagagtac cagtccataa attaaggccc gctctctgga gggaaggaca 1020

ttactgaacc tgtcttgctg tgcctcgaaa ctctggaggt tggagcatca agttccagcc 1080

ggccccttca ctcccccatc ttgcttttct gtggaacctc agaggccagc ctcgacttcc 1140

tggagacccc caggtggggc ttccttcata ctttgttggg ggactttgga ggcgggcagg 1200

ggacagggct attgataagg tccccttggt gttgccttct tgcatctcca cacatttccc 1260

ttggatggga cttgcaggcc taaatgagag gcattctgac tggttggctg ccctggaagg 1320

caagaaaata gatttatttt ttttcamaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaggg cggccgc 1397



43
1739
DNA
Homo sapiens

43

ggcacgagag atcctcagga tatctttagc caaaggaaaa gctccgcatt cccacctggg 60

gggaaagctg gattgccatg ggcacgaata gtggtgcaga gtccctggcc atcctgaata 120

tccagaatgg tgtttctgaa gttcttctgc atgagtttct tctgccacct gtgtcaaggc 180

tacttcgatg gccccctcta cccagagatg tccaatggga ctctgcacca ctacttcgtg 240

cccgatgggg actatgagga gaacgatgac cccgagaagt gccagctgct cttcagggtg 300

agtgaccaca ggcgctgctc ccagggggag gggagccagg ttggcagcct gctgagcctc 360

accctgcggg aggagttcac cgtgctgggc caccaggtgg aaggatgctg ggcgcgtgct 420

ggagggcatc agcaaaagca tctcctacga cctagacggg gaagagagct atggcaagta 480

cctgcggcgg gagtcccacc agatcggggg atgcctactc caactcggac aaatccctca 540

ctgagctgga gagcaagttc aagcagggcc aggaacagga cagccggcag gagagcaggc 600

tcaacgagga ctttctggga atgctggtcc acaccaggtc cctgctgaag gagacactgg 660

acatctctgt ggggctcagg gacaaatacg agctgctggc cctcaccatt aggaccatgg 720

gacccgacta gtcggctgaa aaatgattat cttaaagtat aggtggaagg atacaaatgc 780

ttagaaagag ggaatcaaat cagccccgtt ttggaaggtg ggggacagaa aatggggcta 840

catttccccc atacctacta tttttttata tcccgatttg cactttgaga atacatctaa 900

ggtcatcttt caaaagagaa aaattggaca cttgagtgac tttgttttta gttttgtttt 960

tgaacattat ttatgtgatt gttatggaat tgtcacctgg aaagaacaat tttaagcaat 1020

gtcatttcta gatgggtttc taattctgca gagacacccg tttcagccac atctaaaaga 1080

gcacagttta tgtggtgcgg aattaaactt ccccatcctg cagattatgt ggaaataccc 1140

aaagataata gtgcatagct cctttcagcc tctagccttc actcctgggc tccaaaagct 1200

atcccagttg cctgtttttc aaatgaggtt caaggtgctg ctttgcatgc ctgccaaccc 1260

atggaagttg tttcttactt cttttctctc ttatttatta accatggtct gagagttgtt 1320

tttgttctat gtaacagtat tgccacaaaa ctataggcaa atcgtgtttg cagggagatt 1380

tctgatgcct ctgtgggtgt gtgtaagtta aagtggccac atttaagaag gccaagcttt 1440

gtagtggttg cacagtcaca ctgatatgct gatttgctct ttctcattgt atgtctatgc 1500

tttgtcatca gtgctatagt aaattacaaa gaaataggta gattgtatga acatacccac 1560

aaatgcctat gatttaggtt accaatgtat tctttctcat ttggggtttt gcttctgtct 1620

gtctgtttat tggaaacttg tacttcaagt agggggaatc ctaattctaa taactcctta 1680

gctaagtttt attattcagg caataaacat gttttcatgt aaaaaaaaaa aaaaaaaaa 1739



44
3061
DNA
Homo sapiens

SITE
(2755)
n equals a,t,g, or c


44

gtgtgggggc caccttcggt ggcggccgct ctagaactag tggatccccc gggctgcagg 60

gaattcggca cgagcaacat tgaagaccgg gacgagcttg cctaccacat cagcatcatg 120

ttctatataa taggaggtgt ggccactctc ctcctcatcc ttgtcatcat tgtgttcaag 180

gagaaaccta aatatccccc cagcagggcc caatccctga gctatgcctt gacctctcct 240

gatgcctcat acttaggttc catcgcccgg ctcttcaaaa atctcaactt tgtgctgctt 300

gtcatcacct atggtctgaa tgctggtgct ttttatgcct tgtccactct tctgaatcgc 360

atggtgatct ggcactaccc gggggaagaa gtgaatgctg gaagaattgg cctgacgatc 420

gtcattgcag gaatgcttgg ggctgtgatc tcaggaatct ggctggatag gtccaaaacc 480

tacaaagaga caaccctggt agtctatatc atgacactgg tgggcatggt ggtgtacacg 540

tttaccttga acctgggaca cctgtgggta gtgttcatca ctgctggcac aatgggcttc 600

tttatgactg gctatctccc actgggattt gagtttgctg tggagctcac gtacccagaa 660

tcagaaggca tctcctccgg cctcctcaac atatctgcac aggtatttgg gatcatcttt 720

accatctccc agggccagat tattgacaac tatggaacca agcctgggaa catcttcctg 780

tgtgtgttcc ttactcttgg agcagccctc actgcattca ttaaggcaga tctccggaga 840

cagaaagcaa acaaagaaac tcttgagaac aaactccaag aggaggagga ggagagcaac 900

accagcaaag tgcccactgc tgtgtcagag gatcatctct gagaggaagg tggtgacaac 960

tcagggaaca cgaacacccc accttttcct tcagcacagc tctcaccgcc agcacaaagg 1020

gcttcgctag agatgttttt ggagggaatc agtgggacta tttgtggcat ggatggccta 1080

ttcctcctag aacccacgta agagcttgga tgatttagtt ggagaaaatt gcacctatca 1140

ccaaatgcaa atttgattcc cacctccacc cccttttagg ttatgggagt tggtgttggg 1200

acagggtggc agagaatatt ggagtcaatc ctagcttggt ctcttgcctt ccctcttttc 1260

ctccatccat cgtggacaat gcctgcaaaa ttttcacagg aagaaagcct attcaggata 1320

ttaacttgaa atttccagtg tcctaagagc ctctcatgaa gcccagttct aataagtggc 1380

aagctgctct gccggggtca tctcctgggt catcggactg attgctcaag ttctgcagga 1440

gaggaagcac cattagaaca actccatcag aacagctcca ccgggacttg tgggcctaaa 1500

ttttcctggc ctaacgggtc tgtctccaaa ccctctttcc taagagctga gcaaaccaac 1560

cataataaac ttgacaaaag actttgttgt ggccatgaca gagataccga ctcaggaggg 1620

ctacctacct aggtgtgatc atgctggggg ctaccttctg agtatatttg tgaaagcaca 1680

tatttgggaa ctctggtagc ttgagttggg aatgggaagg ttctttttta cagaagtact 1740

tccccaggga cttctgtgtg tcacagtcac ctctgatgcc tttatcttga tgttgcattg 1800

ggaatctcag ccatcagccc aagtgcttgt tttattccaa ggcagggtaa tccccgtcaa 1860

cttactctaa cctttgctga aaactaatct tgattcattc tactctgaaa atccaaaggt 1920

gcttctgaga gataagaggg aaggggtaga aggaaaggtg ccccttgaaa tgggaattga 1980

gcctgttaga attaaaagct tatctcacct ctgctgggga cagtatttgc accaccaacc 2040

cctctcctca cctgctttga gcgataatct ttatcagata ttctaaactt aaagggattc 2100

cctttaaacc aactcaagct gatctttcct atctagcctg ctgtttggct gtactcatgg 2160

gctttggtaa tatctcctaa aaatgaggtt ttggtaattt ttcctatgca ttgggcaact 2220

gtgatcgtga ccactgtgct gtcttgctcc agccactgcc ctggcctcag catatcaggg 2280

cagcctgtgc tggctgcaat actgtggtgc ttgggccact gcctgagagg agccaggttt 2340

gtgtgtgtct gcatgtgtgt gtgtgtgtgt ttgtacagat tcaagcaatg gatgcaagga 2400

acatgctgta tgtaatagaa gaaagaagtc cacgttttcg gcagaagtag tgagtcagtg 2460

tggaagagag gtgagggtgt gctttacttt ttgataaaga gaaagatgtt tactcataaa 2520

cccttcaaaa ggtattaaca aaatgtttac caaacctatt gctttatttt aaaaacataa 2580

tttgtgtttt ctatttgtaa gatctgacat ttcgaggcaa taaaaacttc tcagaaaaaa 2640

aaaaaaaaaa aaaaactcga gggggggccc ggtacccaat tcgccctata gtgagtcgta 2700

ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgtnaccca 2760

acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 2820

caccgatcgc ccttcccaac agttgcgcng cctgaatggg cgaatggcaa attgtaagcg 2880

ttaatatttt ggttaaaatt cgcgttaaat ttttgtttna atcagctcat tttttaacca 2940

ataggccgaa atcggcaaaa tcccttatta atcaaaagaa tanaccnaaa tagggttnaa 3000

tgttgttcca tttggaacaa gagtccncta ttaaagacgt ggactccacg tcaaagggcg 3060

a 3061



45
974
DNA
Homo sapiens

45

caggagtaaa gaactttatg agttcatgag aacctaaggc tcagtatttg aaaattactg 60

acttatgaga aagcaggcat gtaaataaaa aataaaaaat gttggcccta gattttgata 120

tgtgtgtggt gtgtggtgta ggagaggccc tgatatttac ctgtaagtgt tagagttgta 180

tgaaaaaggt ggcaagattg agtagcttag ggcatgtggt gtggaggctg tatgctagag 240

ttttggcatt aataacttgt attttctggg ttttggcatt aataatttgt attttcactc 300

cccaaatatt tttcaagcat ctacttcatg ccagaccttg ttctagatac cggagataca 360

acagcaaaaa tacagatctt gcccttatga agcttaaatt gttgaggcag gcagacagtg 420

ataaataaat acatagaatg ttgggaaaga aaataagagg atttgagagg gtggaatggg 480

gaagaaagga ttcactgata agatgccatt cgagctaaga cctgaaaagg tgatctctaa 540

ggtgaggaaa gagctttcta cacagaagga acagctgggg gaagggagca cgcttggaat 600

atttaaggaa tatcaaggag ggaaaagtgg ctagagtaga ggaagagaat ggaagaagtc 660

atgtcaaaca ggtactaatg gaagaagtca tgtcagacag ggtcttgccc attgtaagga 720

ctttggcctt atacctcagc aagctgagca gccgtcggaa tgttttaagc aaaagagtga 780

caccatcttt aaaagggacc ccttgtaagg attcagaaca gacttggagg gaaaacaagt 840

agaagcagca gggggactag ttaggaggct gaggtgggag gattgcttgg gcctgggagg 900

ttgaagctgc agtgagtcat gatcactcca ctgcactcca gcctgggcga cagaaggaga 960

ccctgtctca gaaa 974



46
1256
DNA
Homo sapiens

SITE
(1240)
n equals a,t,g, or c


46

tcgacccacg cgtccgagca accgcagctt ctagtatcca gactccagcg ccgccccggg 60

cgcggacccc aaccccgacc cagagcttct ccagcggcgg cgcacgagca gggctccccg 120

ccttaacttc ctccgcgggg cccagccacc ttcgggagtc cgggttgccc acctgcaaac 180

tctccgcctt ctgcacctgc cacccctgag ccagcgcggg cgcccgagcg agtcatggcc 240

aacgcggggc tgcagctgtt gggcttcatt ctcgccttcc tgggatggat cggcgccatc 300

gtcagcactg ccctgcccca gtggaggatt tactcctatg ccggcgacaa catcgtgacc 360

gcccaggcca tgtacgaggg gctgtggatg tcctgcgtgt cgcagagcac cgggcagatc 420

cagtgcaaag tctttgactc cttgctgaat ctgagcagca cattgcaagc aacccgtgcc 480

ttgatggtgg ttggcatcct cctgggagtg atagcaatct ttgtggccam cgttggcatg 540

aagtgtatga agtgcttgga agacgatgag gtgcagaaga tgaggatggc tgtcattggg 600

ggcgcgatat ttcttcttgc aggtctggct attttagttg ccacagcatg gtatggcaat 660

agaatcgttc aagaattcta tgaccctatg accccagtca atgccaggta cgaatttggt 720

caggctctct tcactggctg ggctgctgct tctctctgcc ttctgggagg tgccctactt 780

tgctgttcct gtccccgaaa aacaacctct tacccaacac caaggcccta tccaaaacct 840

gcaccttcca gcgggaaaga ctacgtgtga cacagaggca aaaggagaaa atcatgttga 900

aacaaaccga aaatggacat tgagatacta tcattaacat taggacctta gaattttggg 960

tattgtaatc tgaagtatgg tattacaaaa caaacaaaca aacaaaaaac ccatgtgtta 1020

aaatactcag tgctaaacat ggcttaatct tattttatct tctttcctca atataggagg 1080

gaagattttt ccatttgtat tactgcttcc cattgagtaa tcatactcaa ctgggggaag 1140

gggtgctcct taaatatata tagatatgta tatatacatg tttttctatt aaaaatagac 1200

agtaaaatwc taaaaaaaaa aaaaaaamcy cggggggggn ccggtaccca ttcgcc 1256



47
936
DNA
Homo sapiens

SITE
(893)
n equals a,t,g, or c


47

gtagataatc tgaatagccc tatatctata gaaacttaat agtgctggga gatataggta 60

ttattatcct cattttacag atgtgaaaat tgaggctcag agaagtaaag tctattgctc 120

aaggtcatgt ggctagaata tggcagagcc atgattcaga tccaggtctt ctgattctta 180

ttccagtgtc ctttctagca taccatgttg cctctaaaga ttgcagctcc ttatttacta 240

gaaaattgtt cctgcccaat ctacatctcc acctcacccc atcttttctt aagcactatg 300

tttgtgtttt tatcagtatt atattcattg tctttggaat acatgttctt gtttgtgttt 360

ggaaaaaaaa tctcttttac cagcttgcac tcggaccaac ttggaaaaaa aaaagcttaa 420

atgtttttgc tatgtacagt ttaaaaatgt gaagtttgta gctttaactt tttgtaagaa 480

aatctaataa cactggctta agtgctgact tgaaatgcta ttttgtaagg tttggatgta 540

agtaatcaat tgaggtcagc agtttgtatg agacatagct tcctccattg cccccactcc 600

ttttttcttt tttaagtttg agatgcttcc tgtgttttta tgttagaatt gttgttctcc 660

ttcttttctt cttcctatac ctcatcacgt ttgttttaaa taaactgtcc tttggaccac 720

aaacccttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aantnaaaaa 900

aaaangaaac naaaaaaaaa aaaaaaaaaa aaaaaa 936



48
1765
DNA
Homo sapiens

48

tccagcccgc cacctctgga ctccctcccc tgtatgtaag ccctcaataa aaccccacgt 60

ctcttttgct ggctctgggt ctctttggcg tcttgaacct tatgccttcc ctattgaagt 120

taataggggt tcagcacaac actgggtccc aggcatgcag ccttgtcttt ttatgtttgt 180

cctcatgggc atcatgtggg ccacagggat cttacccaaa atcatgccta gcaggaagag 240

atgcctctct attgatattc cagctgcccc acaagcaggt atgtgcctac tcattctctg 300

acatgtgtcc taggtttctt gactgtaaaa ttccgcttaa taaataatgt attgagcacc 360

tgctatatcc agagctctgc tgggcactct ggaagattag agtttgaaga acacacaatc 420

gttgtcctcc agaagttcat ggtccagtag ggagagaagg tcacaaacag ccagaatcta 480

caagatgaga ggggctatta aagagagctg agaagagttt cagagccact gagaagatac 540

tcattttagt cttggtaacg ggaagctttc tggagttggt ggcatttaag atgaaccaca 600

aatggagcag gaatttaatg aagaggaaag gaccaggacg ttcaaataga ggacacagca 660

gagttcagac cagatgcagg aaagggtaag tggtttggtt tgctgtgagg tcaggtgcat 720

gatggaaaat ggtgagcagt tagatgacaa agataggtta gggcagttat ggtgggagaa 780

accctgtttt tgaagaaaga tacagtcaga tgggcacttc agcagcatta ttctgacagc 840

aatgtgcaaa taggatggag aggcactgaa gaggcaggat gcaaggagaa caattcagac 900

gcagttgaca tcgcttggga gagaatgagg tactatccag gacagcccca agagagtgga 960

gagccctgtc ctgagattct tgggaaaagg tgccccagta tcaacagcat gtgcccataa 1020

gctccctcag cccacttgtc aggctgggtc agcttgtccc ccaccccacc ccagctctct 1080

cctctcctct cagagcaagc cacctgccct actcactgct ccaccatgat acacctgtcc 1140

tgcctcagcc acgtccccct ctgaccattt cccccttttc aggaaagtgc ttgcagtttg 1200

ctacctccca ttctttgcag cctccttccg caaagctctc accttgtgtc atctttgcct 1260

gtgtgcccta tggagaagga aaacaaagta agcgctgtct tcccagggct gcaggcgtgc 1320

tggcccccta ccttcatgca ctcaaggagc actgactgag catcctgcaa ctctgtcctg 1380

tccccagaag ccatgctttg tgagaagtgg ggtaagcatc acagaagctg gccactggca 1440

ggcgcatgca gggcttcaga cctgctcctg atgtaatctt tgctgcacat tcacctcacc 1500

tgaggacctg tggaaactag cacccaggtc acaggccctg ctaattaaat cagaatgtag 1560

gagccaggca acaataaata gcgctgaaat atccccagct gattccagtg tacagccaag 1620

tttaggaacc gtgtcctggt gtttccctcc tactcttgct aatatgtagc tctacatatc 1680

tgtgtggagt gagactggca gaaaggctgt ctggatgctc cttcctgtat agcttggagg 1740

caaatggact ctatacagct caggg 1765



49
1194
DNA
Homo sapiens

49

gagcccagca acgtgcaagg ggaaagggga caggattctg gatggccatt tgcttcactg 60

ggatgcaaaa cctcttttga gtactagaat cagtatttct tcttccatct ctgctgtacc 120

tgagaagaaa tggccaaacg caccttctct aacttggaga cattcctgat tttcctcctt 180

gtaatgatga gtgccatcac agtggccctt ctcagcctct tgtttatcac cagtgggacc 240

attgaaaacc acaaagattt aggaggccat tttttttcaa ccacccaaag ccctccagcc 300

acccagggct ccacagccgc ccaacgctcc acagccaccc agcattccac agccacccag 360

agctccaaca gccaactcaa acttctccag tgcctttaac cccagagtct cctctatttc 420

agaacttcag tggctaccat attggtgttg gacgagctga ctgcacagga caagtagcag 480

atatcaattt gatgggctat ggcaaatccg gccagaatgc acagggcatc ctcaccaggc 540

tatacagtcg tgccttcatc atggcagaac ctgatgggtc caatcgaaca gtgtttgtca 600

gcatcgacat aggcatggta tcccaaaggc tcaggctgga ggtcctgaac agactgcaga 660

gtaaatatgg ctccctgtac agaagagata atgtcatcct gagtggcact cacactcatt 720

caggtcctgc aggatatttc cagtataccg tgtttgtaat tgccagtgaa ggatttagca 780

atcaaacttt tcagcacatg gtcactggta tcttgaagag cattgacata gcacacacaa 840

atatgaaacc aggcaaaatc ttcatcaata aaggaaatgt ggatggtgtg cagatcaaca 900

gaagtccgta ttcttacctt caaaatccgc agtcagagag agcaaggtat tcttcaaata 960

cagacaagga aatgatagtt ttgaaaatgg tagatttgaa tggagatgac ttgggcctta 1020

tcagtttttc attcagcaag tctgcactag ggacctacta tgagccacgc aatacttcct 1080

tggaatgatg tattccctgg ccttgaaata aggaatctag tacccatgtt tgtgctactg 1140

gaatgaatcc attaaactct ctgagactca aaaaaaaaaa aaaaaaaaaa aaaa 1194



50
2334
DNA
Homo sapiens

SITE
(2278)
n equals a,t,g, or c


50

gggagtgtgg ctgcagaacc caggtggcag ggctttcctc aggcccctta ctcctgacct 60

ggacgaggcc ggggcttcct caaggaggct ctctgactgc cacccctgcc tgcctgcccg 120

gccctgcaca acatgcagcc ctccggcctc gagggtcccg gcacgtttgg tcggtggcct 180

ctgctgagtc tgctgctcct gctgctgctg ctccagcctg taacctgtgc ctacaccacg 240

ccaggccccc ccagagccct caccacgctg ggcgccccca gagcccacac catgccgggc 300

acctacgctc cctcgaccac actcagtagt cccagcaccc agggcctgca agagcaggca 360

cgggccctga tgcgggactt cccgctcgtg gacggccaca acgacctgcc cctggtccta 420

aggcaggttt accagaaagg gctacaggat gttaacctgc gcaatttcag ctacggccag 480

accagcctgg acaggcttag agatggcctc gtgggcgccc agttctggtc agcctatgtg 540

ccatgccaga cccaggaccg ggatgccctg cgcctcaccc tggagcagat tgacctcata 600

cgccgcatgt gtgcctccta ttctgagctg gagcttgtga cctcggctaa agctctgaac 660

gacactcaga aattggcctg cctcatcggt gtagagggtg gccactcgct ggacaatagc 720

ctctccatct tacgtacctt ctacatgctg ggagtgcgct acctgacgct cacccacacc 780

tgcaacacac cctgggcaga gagctccgct aagggcgtcc actccttcta caacaacatc 840

agcgggctga ctgactttgg tgagaaggtg gtggcagaaa tgaaccgcct gggcatgatg 900

gtagacttat cccatgtctc agatgctgtg gcacggcggg ccctggaagt gtcacaggca 960

cctgtgatct tctcccactc ggctgcccgg ggtgtgtgca acagtgctcg gaatgttcct 1020

gatgacatcc tgcagcttct gaagaagaac ggtggcgtcg tgatggtgtc tttgtccatg 1080

ggagtaatac agtgcaaccc atcagccaat gtgtccactg tggcagatca cttcgaccac 1140

atcaaggctg tcattggatc caagttcatc gggattggtg gagattatga tggggccggc 1200

aaattccctc aggggctgga agacgtgtcc acatacccag tcctgataga ggagttgctg 1260

agtcgtggct ggagtgagga agagcttcag ggtgtccttc gtggaaacct gctgcgggtc 1320

ttcagacaag tggaaaaggt acaggaagaa aacaaatggc aaagcccctt ggaggacaag 1380

ttcccggatg agcagctgag cagttcctgc cactccgacc tctcacgtct gcgtcagaga 1440

cagagtctga cttcaggcca ggaactcact gagattccca tacactggac agccaagtta 1500

ccagccaagt ggtcagtctc agagtcctcc ccccacatgg ccccagtcct tgcagttgtg 1560

gccaccttcc cagtccttat tctgtggctc tgatgaccca gttagtcctg ccagatgtca 1620

ctgtagcaag ccacagacac cccacaaagt tcccctgttt gcaggcacaa atatttcctg 1680

aaataaatgt tttggacata gaaaaaaaaa aaaaaaaaag ggcggccgct ctagaggatc 1740

cctcgagggg cccaagctta cgcgtgcatg cgacgtcata gctctctccc tatagtgagt 1800

cgtattataa gctaggcact ggccgtcgtt ttacaacgtc gtgactggga gatctgctag 1860

cttgggatct ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct 1920

acagagattt aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta 1980

gctgcatatg cttgctgctt gagagttttg cttactgagt atgatttatg aaaatattat 2040

acacaggagc tagtgattct aattgtttgt gtattttaga ttcacagtcc caaggctcat 2100

ttcaggcccc tcagtcctca cagtctgttc atgatcataa tcagccatac cacatttgta 2160

gaggttttac ttgctttaaa aaacctycca cacctccccc tgaacctgaa acataaaatg 2220

aatgcaattg gtggtggtaa cttggttaat ggagcttata atggtaccaa taaagcantg 2280

catcacaaan ttcccaaata aagcattttt tcctggaatt taaatggggg ttgg 2334



51
1314
DNA
Homo sapiens

51

atgcggcagc tcttctatga ccctgacgag tgcgggctga tgaagaaggg gggcttgtac 60

ttcagtgact tctggaataa gctggacgtc ggcgcaatct tgctcttcgt ggcagggctg 120

acctgcaggc tcatcccggc gacgctgtac cccgggcgcg tcatcctctc tctggacttc 180

atcctgttct gcctccggct catgcacatt tttaccatca gtaagacgct ggggcccaag 240

atcatcattg tgaagcggat gatgaaggac gtcttcttct tcctcttcct gctggctgtg 300

tgggtggtgt ccttcggggt ggccaagcag gccatcctca tccacaacga gcgccgggtg 360

gactggctgt tccgagggcc gtctaccact cctacctcac catcttcggg cagatcccgg 420

gctacatcga cggtgtgaac ttcaacccgg agcactgcag ccccaatggc accgacccct 480

acaagcctaa gtgccccgag agcgacgcga cgcagcagag ccggccttcc ctgagtggct 540

gacggtcctc ctactctgcc tctacctgct cttcaccaac atcctgctgc tcaacctcct 600

catcgccatg ttcaactaca ccttccagca ggtgcaggag cacacggacc agatttggaa 660

gttccagcgc catgacctga tcgaggagta ccacggccgc cccgccgtgc cgcccccgtt 720

gatcctcttc agccacctgc agctcttcat caagagggtg gtcctgaaga ctccggccaa 780

gaggcacaag cagctcaaga acaagctgga gaagaacgag gaggcggccc tgctatcctg 840

ggagatctac ctgaaggaga actacctcca gaaccgacag ttccagcaaa agcagcggcc 900

cgagcagaag atcgaggaca tcagcaataa ggttgacgcc atggtggacc tgctggacct 960

ggacccactg aagaggtcgg gctccatgga gcagaggttg gcctccctgg aggagcaggt 1020

ggcccagaca gcccgagccc tgcactggat cgtgaggacg ctgcgggcca gcggcttcag 1080

ctcggaggcg gacgtcccca ctctggcctc ccagaaggcc gcggaggagc cggatgctga 1140

gccgggaggc aggaagaaga cggaggagcc gggcgacagc taccacgtga atgcccggca 1200

cctcctctac cccaactgcc ctgtcacgcg cttccccgtg cccaacgaga aggtgccctg 1260

ggagacggag ttcctgatct atgacccacc cttttacacg gcagagagga agga 1314



52
1381
DNA
Homo sapiens

52

gataactcag gcccggtgcc cagagcccag gaggaggcag tggccaggaa ggcacaggcc 60

tgagaagtct gcggctgagc tgggagcaaa tcccccaccc cctacctggg ggacagggtg 120

cagcggccat ggctacagca agacccccct ggatgtgggt gctctgtgct ctgatcacag 180

ccttgcttct gggggtcaca gagcatgttc tcgccaacaa tgatgtttcc tgtgaccacc 240

cctctaacac cgtgccctct gggagcaacc aggacctggg agctggggcc ggggaagacg 300

cccggtcgga tgacagcagc agccgcatca tcaatggatc cgactgcgat atgcacaccc 360

agccgtggca ggccgcgctg ttgctaaggc ccaaccagct ctactgcggg gcggtgttgg 420

tgcatccaca gtggctgctc acggccgccc actgcaggaa gaaagttttc agagtccgtc 480

tcggccacta ctccctgtca ccagtttatg aatctgggca gcagatgttc cagggggtca 540

aatccatccc ccaccctggc tactcccacc ctggccactc taacgacctc atgctcatca 600

aactgaacag aagaattcgt cccactaaag atgtcagacc catcaacgtc tcctctcatt 660

gtccctctgc tgggacaaag tgcttggtgt ctggctgggg gacaaccaag agcccccaag 720

tgcacttccc taaggtcctc cagtgcttga atatcagcgt gctaagtcag aaaaggtgcg 780

aggatgctta cccgagacag atagatgaca ccatgttctg cgccggtgac aaagcaggta 840

gagactcctg ccagggtgat tctggggggc ctgtggtctg caatggctcc ctgcagggac 900

tcgtgtcctg gggagattac ccttgtgccc ggcccaacag accgggtgtc tacacgaacc 960

tctgcaagtt caccaagtgg atccaggaaa ccatccaggc caactcctga gtcatcccag 1020

gactcagcac accggcatcc ccacctgctg cagggacagc cctgacactc ctttcagacc 1080

ctcattcctt cccagagatg ttgagaatgt tcatctctcc agcccctgac cccatgtctc 1140

ctggactcag ggtctgcttc ccccacattg ggctgaccgt gtctctctag ttgaaccctg 1200

ggaacaattt ccaaaactgt ccagggcggg ggttgcgtct caatctccct ggggcacttt 1260

catcctcaag ctcagggccc atcccttctc tgcagctctg acccaaattt agtcccagaa 1320

ataaactgag aagtggaatc ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

a 1381



53
1439
DNA
Homo sapiens

53

cccacgcgtc cgcaggagag gtgtctgtgc gtcctgcacc cacatctttc tctgtcccct 60

ccttgccctg tctggaggct gctagactcc tatcttctga attctatagt gcctgggtct 120

cagcgcagtg ccgatggtgg cccgtccttg tggttcctct ctacttgggg aaatcaggtg 180

cagcggccat ggctacagca agacccccct ggatgtgggt gctctgtgct ctgatcacag 240

ccttgcttct gggggtcaca gagcatgttc tcgccaacaa tgatgtttcc tgtgaccacc 300

cctctaacac cgtgccctct gggagcaacc aggacctggg agctggggcc gggggaagac 360

gcccggtcgg atgacagcag cagccgcatc atcaatggat ccgactgcga tatgcacacc 420

cagccgtggc aggccgcgct gttgctaagg cccaaccagc tctactgcgg ggcggtgttg 480

gtgcatccac agtggctgct cacggccgcc cactgcagga agaaagtttt cagagtccgt 540

ctcggccact actccctgtc accagtttat gaatctgggc agcagatgtt ccagggggtc 600

aaatccatcc cccaccctgg ctactcccac cctggccact ctaacgacct catgctcatc 660

aaactgaaca gaagaattcg tcccactaaa gatgtcagac ccatcaacgt ctcctctcat 720

tgtccctctg ctgggacaaa tgcttggtgt ctggctgggg gacaaccaag acccccaagt 780

gcacttccct aaggtcctcc agtgcttgaa tatcacgtgc taagtcagaa aaggtgcgag 840

gatgcttacc cgagacagat agatgacacc atgttctgcg ccggtgacaa agcaggtaga 900

gactcctgcc agggtgattc tggggggcct gtggtctgca atggctccct gcagggactc 960

gtgtcctggg gagattaccc ttgtgcccgg cccaacagac cgggtgtcta cacgaacctc 1020

tgcaagttca ccaagtggat ccaggaaacc atccaggcca actcctgagt catcccagga 1080

ctcagcacac cggcatcccc acctgctgca gggacagccc tgacactcct ttcagaccct 1140

cattccttcc cagagatgtt gagaatgttc atctctccag cccctgaccc catgtctcct 1200

ggactcaggg tctgcttccc ccacattggg ctgaccgtgt ctctctagtt gaaccctggg 1260

aacaatttcc aaaactgtcc agggcggggg ttgcgtctca atctccctgg ggcactttca 1320

tcctcaagct cagggcccat cccttctctg cagctctgac ccaaatttag tcccagaaat 1380

aaactgagaa gtggaatctt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1439



54
1368
DNA
Homo sapiens

54

ctgaattgcg gccgtatgcg cggctctgtg gagtgcacct ggggttgggg gcactgtgcc 60

cccagccccc tgctcctttg gactctactt ctgtttgcag ccccatttgg cctgctgggg 120

gagaagaccc gccagctgct tgagtttgac agcaccaacg tgtccgatac ggcagcaaag 180

cctttgggaa gaccatatcc tccatactcc ttggccgatt tctcttggaa caacatcact 240

gattcattgg atcctgccac cctgagtgcc acatttcaag gccaccccat gaacgaccct 300

accaggactt ttgccaatgg cagcctggcc ttcaggtcca ggccttttcc aggtccagcc 360

gaccagccca accccctcgc ctcctgcaca cagcagacac ctgtcagcta gaggtggccc 420

tgattggagc ctctccccgg ggaaaccgtt ccctgtttgg gctggaggta gccacattgg 480

gccagggccc tgactgcccc tcaatgcagg agcagcactc catcgacgat gaatatgcac 540

cggccgtctt ccagttggac cagctactgt ggggctccct cccatcaggc tttgcacagt 600

ggcgaccagt ggcttactcc cagaagccgg ggggccgaga atcagccctg ccctgccaag 660

cttcccctct tcatcctgcc ttagcatact ctcttcccca gtcacccatt gtccgagcct 720

tctttgggtc ccagaataac ttctgtgcct tcaatctgac gttcggggct tccacaggcc 780

ctggctattg ggaccaacac tacctcagct ggtcgatgct cctgggtttg ggcttccctc 840

cagtggacgg cttgtcccca ttagtcctgg gcatcatggc agtggcctgg gtgccccagg 900

gctcatgctg ctagggggcg gcttggttct gctgctgcac cacaagaagt actcagagta 960

ccagtccata aattaaggcc cgctctctgg agggaaggac attactgaac ctgtcttgct 1020

gtgcctcgaa actctggagg ttggagcatc aagttccagc cggccccttc actcccccat 1080

cttgcttttc tgtggaacct cagaggccag cctcgacttc ctggagaccc ccaggtgggg 1140

cttccttcat actttgttgg gggactttgg aggcgggcag gggacagggc tattgataag 1200

gtccccttgg tgttgccttc ttgcatctcc acacatttcc cttggatggg acttgcaggc 1260

ctaaatgaga ggcattctga ctggttggct gccctggaag gcaagaaaat agatttattt 1320

tttttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1368



55
1446
DNA
Homo sapiens

55

ccacgcgtcc gagcaaccgc agcttctagt atccagactc cagcgccgcc ccgggcgcgg 60

accccaaccc cgacccagag cttctccagc ggcggcgcac gagcagggct ccccgcctta 120

acttcctccg cggggcccag ccaccttcgg gagtccgggt tgcccacctg caaactctcc 180

gccttctgca cctgccaccc ctgagccagc gcgggcgccc gagcgagtca tggccaacgc 240

ggggctgcag ctgttgggct tcattctcgc cttcctggga tggatcggcg ccatcgtcag 300

cactgccctg ccccagtgga ggatttactc ctatgccggc gacaacatcg tgacgcccag 360

gccatgtacg aggggctgtg gatgtcctgc gtgtcgcaga gcaccgggca gatccagtgc 420

aaagtctttg actccttgct gaatctgagc agcacattgc aagcaacccg tgccttgatg 480

gtggttggca tcctcctggg agtgatagca atctttgtgg ccaccgttgg catgaagtgt 540

atgaagtgct tggaagacga tgaggtgcag aagatgagga tggctgtcat tgggggcgcg 600

atatttcttc ttgcaggtct ggctatttta gttgccacag catggtatgg caatagaatc 660

gttcaagaat tctatgaccc tatgacccca gtcaatgcca ggtacgaatt tggtcaggct 720

ctcttcactg gctgggctgc tgcttctctc tgccttctgg gaggtgccct actttgctgt 780

tcctgtcccc gaaaaacaac ctcttaccca acaccaaggc cctatccaaa acctgcacct 840

tccagcggga aagactacgt gtgacacaga ggcaaaagga gaaaatcatg ttgaaacaaa 900

ccgaaaatgg acattgagat actatcatta acattaggac cttagaattt tgggtattgt 960

aatctgagta tggtatacaa acaacaaaca aacaaaaaac ccatgtgtta aaatactcag 1020

tgctaaacat ggcttaatct tattttatct tctttcctca atataggagg gaagattttt 1080

ccatttgtat tactgcttcc cattgagtaa tcatactcaa atgggggaag gggtgctcct 1140

taaatatata tagatatgta tatatacatg tttttctatt aaaaatagac agtaaaatac 1200

tattctcatt atgttgatac tagcatactt aaaatatctc taaaatagtt aaatgtattt 1260

aattccatat tgatgaagat gtttattggt atattttctt tttcgtcctt atatacatat 1320

gtaacagtca aatatcattt actcttcttc attagctttg ggtgcctttg ccacaagacc 1380

tagcctaatt taccaaggat gaattctttc aattcttcat gcgtgcccag caaaaaaaaa 1440

aaaaaa 1446



56
143
PRT
Homo sapiens

SITE
(143)
Xaa equals stop translation


56

Met Ser Gly Ile Ser Gly Cys Pro Phe Phe Leu Trp Gly Leu Leu Ala
1 5 10 15

Leu Leu Gly Leu Ala Leu Val Ile Ser Leu Ile Phe Asn Ile Ser His
20 25 30

Tyr Val Glu Lys Gln Arg Gln Asp Lys Met Tyr Ser Tyr Ser Ser Asp
35 40 45

His Thr Arg Val Asp Glu Tyr Tyr Ile Glu Asp Thr Pro Ile Tyr Gly
50 55 60

Asn Leu Asp Asp Met Ile Ser Glu Pro Met Asp Glu Asn Cys Tyr Glu
65 70 75 80

Gln Met Lys Ala Arg Pro Glu Lys Ser Val Asn Lys Met Gln Glu Ala
85 90 95

Thr Pro Ser Ala Gln Ala Thr Asn Glu Thr Gln Met Cys Tyr Ala Ser
100 105 110

Leu Asp His Ser Val Lys Gly Lys Arg Arg Ser Pro Gly Asn Arg Ile
115 120 125

Leu Ile Ser Gln Thr Arg Met Glu Met Ser Asn Tyr Met Gln Xaa
130 135 140



57
51
PRT
Homo sapiens

SITE
(51)
Xaa equals stop translation


57

Met Ala Leu Met Trp Ser Leu Trp Tyr Phe Asn Ser Val Phe Ile Ile
1 5 10 15

Ser Cys Val Ser Gly Lys Ile Val Leu Thr Phe Pro Leu Tyr Thr Thr
20 25 30

Val Cys Ser Tyr Gly Ala Leu Asn Cys Leu Thr Glu Glu Pro Ser Ser
35 40 45

Val Phe Xaa
50



58
102
PRT
Homo sapiens

SITE
(102)
Xaa equals stop translation


58

Met Glu Glu Ala Ile Leu Val Pro Cys Val Leu Gly Leu Leu Leu Leu
1 5 10 15

Pro Ile Leu Ala Met Leu Met Ala Leu Cys Val His Cys His Arg Leu
20 25 30

Pro Gly Ser Tyr Asp Ser Thr Ser Ser Asp Ser Leu Tyr Pro Lys Gly
35 40 45

His Pro Val Gln Thr Ala Ser His Gly Cys Pro Leu Ala Thr Cys Leu
50 55 60

Pro Thr Cys His Leu Leu Pro Thr Pro Glu Pro Ala Arg Pro Ala Pro
65 70 75 80

His Pro Lys Ile Pro Ala Ala Pro Trp Gly Leu Pro Pro Asp Ala Ile
85 90 95

Phe Pro Ala Gly Phe Xaa
100



59
48
PRT
Homo sapiens

SITE
(31)
Xaa equals any of the naturally occurring
L-amino acids


59

Met Ser Cys Ile Gly Arg Met Arg Leu Ile Cys Phe Ile Ile Leu Arg
1 5 10 15

Ile Cys Gly Leu Glu His Leu Phe Gly Asn Met Gly Leu Gly Xaa Lys
20 25 30

Asn Gly His Leu Pro Gly His Tyr Gly His Ser Leu Glu Phe Phe Xaa
35 40 45



60
98
PRT
Homo sapiens

SITE
(98)
Xaa equals stop translation


60

Met Ile Leu Leu Leu Ser Leu Phe Gln Gly Val Arg Gly Ser Leu Gly
1 5 10 15

Ser Pro Gly Asn Arg Glu Asn Lys Glu Lys Lys Val Phe Ile Ser Leu
20 25 30

Val Gly Ser Arg Gly Leu Gly Cys Ser Ile Ser Ser Gly Pro Ile Gln
35 40 45

Lys Pro Gly Ile Phe Ile Ser His Val Lys Pro Gly Ser Leu Ser Ala
50 55 60

Glu Val Gly Leu Glu Ile Gly Asp Gln Ile Val Glu Val Asn Gly Val
65 70 75 80

Asp Phe Ser Asn Leu Asp His Lys Glu Leu Gln Leu Ala Gly Ser Cys
85 90 95

Ser Xaa



61
52
PRT
Homo sapiens

SITE
(52)
Xaa equals stop translation


61

Met Trp Phe Arg Cys Phe Leu Leu Ile Phe Val Ser Ser Val Thr Leu
1 5 10 15

Thr Gly Asp Phe Arg Asn Met Lys Lys Pro Ser Ser Leu Cys Leu Phe
20 25 30

Arg Gln Gly Leu Met Ser Ala Ser Glu Val Ser Gly Ser Gly Ser Gly
35 40 45

Glu Gly Asp Xaa
50



62
52
PRT
Homo sapiens

SITE
(52)
Xaa equals stop translation


62

Met Tyr Cys Leu Cys Gly Leu Leu Leu Gln Ala Leu Leu Arg Leu Cys
1 5 10 15

Asn Gly Tyr Lys Thr Gln Lys Asn His Arg Glu Leu Arg Met Cys Gly
20 25 30

Ile Ile Ala Gln Gly Lys Ser Arg Trp Gln Leu His Cys Tyr Pro Gly
35 40 45

Met Lys Ser Xaa
50



63
71
PRT
Homo sapiens

63

Met Leu Pro Leu Lys Ile Ala Ala Pro Tyr Leu Leu Glu Asn Cys Ser
1 5 10 15

Cys Pro Ile Tyr Ile Ser Thr Ser Pro His Leu Phe Leu Ser Thr Met
20 25 30

Phe Val Phe Leu Ser Val Leu Tyr Ser Leu Ser Leu Glu Tyr Met Phe
35 40 45

Leu Phe Val Phe Gly Lys Lys Ile Ser Phe Thr Ser Leu His Ser Asp
50 55 60

Gln Leu Gly Lys Lys Lys Ala
65 70



64
42
PRT
Homo sapiens

SITE
(42)
Xaa equals stop translation


64

Met Tyr Met Lys Gln Val Val Ala Cys Arg Asp Gln Leu Ile Leu Val
1 5 10 15

Leu Trp Leu Ile Glu Leu Leu Cys Ile Gln Gly Phe Cys Lys Ser Lys
20 25 30

Ser Asp Phe Ser Ser Arg Ile Tyr Ser Xaa
35 40



65
183
PRT
Homo sapiens

65

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp
1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu
20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn
35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys
50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys
65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser
85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys
100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg
115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr
130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met
145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Lys Lys Lys Lys Lys Lys
165 170 175

Lys Lys Lys Lys Lys Lys Lys
180



66
58
PRT
Homo sapiens

SITE
(58)
Xaa equals stop translation


66

Met Leu Tyr Phe Cys Ser Ser Ile Trp Phe Gly Ile Tyr Phe Val Ala
1 5 10 15

Leu Ile Thr Val Phe Leu Lys Thr Leu Pro Pro Leu Thr Val Gly Lys
20 25 30

Gly Pro Phe Ser Gly Lys Phe Val Ala Phe Phe Phe Phe Leu Lys Glu
35 40 45

Ser Cys Ser Leu Leu Ser Ile Val Phe Xaa
50 55



67
100
PRT
Homo sapiens

67

Met Gln Phe Cys Glu Leu Trp Val Pro Leu Leu Ser Thr Leu Leu Asn
1 5 10 15

Thr Trp Gln Asn Leu Thr Leu Gly Cys Pro Ser Pro Asp Ser Lys Ser
20 25 30

Lys Ser Ser Pro Asp Pro Arg Ala Cys Pro Leu Phe Pro Ser Phe Leu
35 40 45

Ser Phe Phe Leu Val Ser Ser Phe Phe Phe Phe Phe Ser Phe Phe Phe
50 55 60

Leu Ser Leu Ser Phe Phe Leu Pro Phe Phe Phe Leu Phe Ser Phe Phe
65 70 75 80

Leu Ser Leu Ser Leu Ser Phe Phe Gln Asp Pro Val Gln Lys Lys Lys
85 90 95

Lys Lys Thr Arg
100



68
74
PRT
Homo sapiens

SITE
(74)
Xaa equals stop translation


68

Met Phe Tyr Leu Tyr Ser Ile Phe Gln Val Leu Val Trp Leu Cys Gln
1 5 10 15

Ala Lys His Leu Ser Gln Ile Ser Ala Arg Ser Ser Arg Arg Leu Trp
20 25 30

Arg Leu Ser Leu Ile Thr Phe Pro Pro Tyr Leu Ala Thr Ser Leu Ser
35 40 45

His Gly Pro His Val Cys Leu Gln Thr Leu Gly Tyr Glu Ser Cys Glu
50 55 60

His Thr Asp Leu Cys Phe Leu His Asp Xaa
65 70



69
137
PRT
Homo sapiens

69

Met Met Phe Ala Gly Ser Cys Gly Phe Pro Ala Gln Pro Ala Thr Thr
1 5 10 15

Gly Pro Cys Gly Tyr Val Val Gln Pro Asn Thr Thr Gly Pro Phe Leu
20 25 30

Tyr Val Arg Gln Phe Tyr Pro Ala Arg His Leu Trp Thr Pro Ser Pro
35 40 45

Val Cys Lys Pro Ser Ile Lys Pro His Val Ser Phe Ala Gly Ser Gly
50 55 60

Ser Leu Trp Arg Leu Glu Pro Tyr Ala Phe Pro Ile Glu Val Asn Arg
65 70 75 80

Gly Ser Ala Gln His Trp Val Pro Gly Met Gln Pro Cys Leu Phe Met
85 90 95

Phe Val Leu Met Gly Ile Met Trp Ala Thr Gly Ile Leu Pro Lys Ile
100 105 110

Met Pro Ser Arg Lys Arg Cys Leu Ser Ile Asp Ile Pro Ala Ala Pro
115 120 125

Gln Ala Gly Met Cys Leu Leu Ile Leu
130 135



70
46
PRT
Homo sapiens

SITE
(46)
Xaa equals stop translation


70

Met Arg Thr Leu Ala Leu Leu Val Leu Leu Phe Cys Ser Cys Thr His
1 5 10 15

Ser Ser Met Gly Trp Gly Arg Gln Ala Trp Gly Val Ala Leu Gly Glu
20 25 30

Val Arg Ser Pro Pro Ala Gln Asp Thr Val Ala Lys Thr Xaa
35 40 45



71
64
PRT
Homo sapiens

SITE
(64)
Xaa equals stop translation


71

Met Cys Ala Trp His Cys Val His Leu Ala Leu Cys Val Val Gly Met
1 5 10 15

Leu Phe Leu Leu Ser Val Thr Ser Ser Gln Phe Cys Lys Gln Arg Gln
20 25 30

Asn His Ala Leu Pro Leu Lys Pro Ile Gly Phe Lys Cys His Leu Phe
35 40 45

Asp Asp Ala Phe Pro Ile Thr Pro Phe Asp Thr Ser His Gly Thr Xaa
50 55 60



72
48
PRT
Homo sapiens

SITE
(48)
Xaa equals stop translation


72

Met Phe Met Tyr Val Trp Cys Pro Leu Val Leu Phe Phe Phe Leu Leu
1 5 10 15

Val Phe Glu Leu Val Leu Asn Arg Ile Leu Ser Gly Phe Leu Lys Tyr
20 25 30

Phe His Phe His His Gly Tyr Asn Lys Phe Ala Ala Cys Pro Asn Xaa
35 40 45



73
49
PRT
Homo sapiens

SITE
(49)
Xaa equals stop translation


73

Met Val Ser Pro Trp Leu Pro Leu Leu Val Ser Leu Phe His Leu Leu
1 5 10 15

Asn Cys Leu Arg Gly Val Gly Thr Ser Gly Gln Ser Leu Gly Leu Pro
20 25 30

Ser Ser Ser Phe Pro Pro Thr Pro Glu His Lys Ala Thr Ala Arg Asp
35 40 45

Xaa



74
47
PRT
Homo sapiens

SITE
(47)
Xaa equals stop translation


74

Gly Lys Thr Leu Tyr Leu Pro Val Cys Leu Ser Phe Leu His Ser Pro
1 5 10 15

Ala Ser Thr Phe Leu Pro Trp Asn Gln Gly Phe Leu Ser Pro Phe Ala
20 25 30

Phe Ser Thr Leu Gly Thr Pro Gly Ala Lys Gln Phe Ser Ile Xaa
35 40 45



75
59
PRT
Homo sapiens

SITE
(59)
Xaa equals stop translation


75

Met Val Ser Leu Cys Ser Gly Leu Pro Ser Ser Cys Leu Leu Leu Gly
1 5 10 15

Ser Thr Ala Ala Ile Ile Gln Arg Gln Val Cys Leu Phe Gln Gly Ala
20 25 30

Arg Gln Trp Asn Pro Val Ser Glu Phe Leu Arg Ala His His His Cys
35 40 45

Gly Asn Arg Ala Gly Leu Pro Ala Val Leu Xaa
50 55



76
318
PRT
Homo sapiens

76

Met Ala Lys Arg Thr Phe Ser Asn Leu Glu Thr Phe Leu Ile Phe Leu
1 5 10 15

Leu Val Met Met Ser Ala Ile Thr Val Ala Leu Leu Ser Leu Leu Phe
20 25 30

Ile Thr Ser Gly Thr Ile Glu Asn His Lys Asp Leu Gly Gly His Phe
35 40 45

Phe Ser Thr Thr Gln Ser Pro Pro Ala Thr Gln Gly Ser Thr Ala Ala
50 55 60

Gln Arg Ser Thr Ala Thr Gln His Ser Thr Ala Thr Gln Ser Ser Thr
65 70 75 80

Ala Thr Gln Thr Ser Pro Val Pro Leu Thr Pro Glu Ser Pro Leu Phe
85 90 95

Gln Asn Phe Ser Gly Tyr His Ile Gly Val Gly Arg Ala Asp Cys Thr
100 105 110

Gly Gln Val Ala Asp Ile Asn Leu Met Gly Tyr Gly Lys Ser Gly Gln
115 120 125

Asn Ala Gln Gly Ile Leu Thr Arg Leu Tyr Ser Arg Ala Phe Ile Met
130 135 140

Ala Glu Pro Asp Gly Ser Asn Arg Thr Val Phe Val Ser Ile Asp Ile
145 150 155 160

Gly Met Val Ser Gln Arg Leu Arg Leu Glu Val Leu Asn Arg Leu Gln
165 170 175

Ser Lys Tyr Gly Ser Leu Tyr Arg Arg Asp Asn Val Ile Leu Ser Gly
180 185 190

Thr His Thr His Ser Gly Pro Ala Gly Tyr Phe Gln Tyr Thr Val Phe
195 200 205

Val Ile Ala Ser Glu Gly Phe Ser Asn Gln Thr Phe Gln His Met Val
210 215 220

Thr Gly Ile Leu Lys Ser Ile Asp Ile Pro His Thr Asn Met Lys Pro
225 230 235 240

Gly Lys Ile Phe Ile Asn Lys Gly Asn Val Asp Gly Val Gln Ile Asn
245 250 255

Arg Ser Pro Tyr Ser Tyr Leu Gln Asn Pro Gln Ser Glu Arg Ala Arg
260 265 270

Tyr Ser Ser Asn Thr Asp Lys Glu Met Ile Val Leu Lys Met Val Asp
275 280 285

Leu Asn Gly Asp Asp Leu Gly Leu Ile Ser Phe Ser Phe Ser Lys Ser
290 295 300

Ala Leu Gly Thr Tyr Tyr Glu Pro Arg Asn Thr Ser Leu Glu
305 310 315



77
44
PRT
Homo sapiens

SITE
(44)
Xaa equals stop translation


77

Met Ser Ser Trp Phe Thr Leu Leu Ala Ser Cys Phe His Leu Leu Trp
1 5 10 15

Pro Leu Ser Arg Ser Ser His Val Pro Ser Ser Phe Gln Pro Pro Asp
20 25 30

Leu Ser Ala Thr Phe Leu Leu Gln Ile Leu Gly Xaa
35 40



78
48
PRT
Homo sapiens

SITE
(48)
Xaa equals stop translation


78

Met Leu Ile Ser Val Asp Ser Asn Val Pro Val Val Phe Leu Leu Leu
1 5 10 15

Phe Ile Leu Val Ile Leu Cys His Met Glu Cys Lys Gly His Ile Tyr
20 25 30

Ile Cys Val Cys Val Cys Val Tyr Met Tyr Ile Phe Lys Asn Ile Xaa
35 40 45



79
525
PRT
Homo sapiens

SITE
(210)
Xaa equals any of the naturally occurring
L-amino acids


79

Met Leu Ala Phe Pro Leu Leu Leu Thr Gly Leu Ile Ser Phe Arg Glu
1 5 10 15

Lys Arg Leu Gln Asp Val Gly Thr Pro Ala Ala Arg Ala Arg Ala Phe
20 25 30

Phe Thr Ala Pro Val Val Val Phe His Leu Asn Ile Leu Ser Tyr Phe
35 40 45

Ala Phe Leu Cys Leu Phe Ala Tyr Val Leu Met Val Asp Phe Gln Pro
50 55 60

Val Pro Ser Trp Cys Glu Cys Ala Ile Tyr Leu Trp Leu Phe Ser Leu
65 70 75 80

Val Cys Glu Glu Met Arg Gln Leu Phe Tyr Asp Pro Asp Glu Cys Gly
85 90 95

Leu Met Lys Lys Ala Ala Leu Tyr Phe Ser Asp Phe Trp Asn Lys Leu
100 105 110

Asp Val Gly Ala Ile Leu Leu Phe Val Ala Gly Leu Thr Cys Arg Leu
115 120 125

Ile Pro Ala Thr Leu Tyr Pro Gly Arg Val Ile Leu Ser Leu Asp Phe
130 135 140

Ile Leu Phe Cys Leu Arg Leu Met His Ile Phe Thr Ile Ser Lys Thr
145 150 155 160

Leu Gly Pro Lys Ile Ile Ile Val Lys Arg Met Met Lys Asp Val Phe
165 170 175

Phe Phe Leu Phe Leu Leu Ala Val Trp Val Val Ser Phe Gly Val Ala
180 185 190

Lys Gln Ala Ile Leu Ile His Asn Glu Arg Arg Val Asp Trp Leu Phe
195 200 205

Arg Xaa Ala Val Tyr His Ser Tyr Leu Thr Ile Phe Gly Gln Ile Pro
210 215 220

Gly Tyr Ile Asp Gly Val Asn Phe Asn Pro Glu His Cys Ser Pro Asn
225 230 235 240

Gly Thr Asp Pro Tyr Lys Pro Lys Cys Pro Glu Ser Asp Ala Thr Gln
245 250 255

Gln Arg Pro Ala Phe Pro Glu Trp Leu Thr Val Leu Leu Leu Cys Leu
260 265 270

Tyr Leu Leu Phe Thr Asn Ile Leu Leu Leu Asn Leu Leu Ile Ala Met
275 280 285

Phe Asn Tyr Thr Phe Gln Gln Val Gln Glu His Thr Asp Gln Ile Trp
290 295 300

Lys Phe Gln Arg His Asp Leu Ile Glu Glu Tyr His Gly Arg Pro Ala
305 310 315 320

Ala Pro Pro Pro Phe Ile Leu Leu Ser His Leu Gln Leu Phe Ile Lys
325 330 335

Arg Val Val Leu Lys Thr Pro Ala Lys Arg His Lys Gln Leu Lys Asn
340 345 350

Lys Leu Glu Lys Asn Glu Glu Ala Ala Leu Leu Ser Trp Glu Ile Tyr
355 360 365

Leu Lys Glu Asn Tyr Leu Gln Asn Arg Gln Phe Gln Gln Lys Gln Arg
370 375 380

Pro Glu Gln Lys Ile Glu Asp Ile Ser Asn Lys Val Asp Ala Met Val
385 390 395 400

Asp Leu Leu Asp Leu Asp Pro Leu Lys Arg Ser Gly Ser Met Glu Gln
405 410 415

Arg Leu Ala Ser Leu Glu Glu Gln Val Ala Gln Thr Ala Arg Ala Leu
420 425 430

His Trp Ile Val Arg Thr Leu Arg Ala Ser Gly Phe Ser Ser Glu Ala
435 440 445

Asp Val Pro Thr Leu Ala Ser Gln Lys Ala Ala Glu Glu Pro Asp Ala
450 455 460

Glu Pro Gly Gly Arg Lys Lys Thr Glu Glu Pro Gly Asp Ser Tyr His
465 470 475 480

Val Asn Ala Arg His Leu Leu Tyr Pro Asn Cys Pro Val Thr Arg Phe
485 490 495

Pro Val Pro Asn Glu Lys Val Pro Trp Glu Thr Glu Phe Leu Ile Tyr
500 505 510

Asp Pro Pro Phe Tyr Thr Ala Glu Arg Lys Asp Ala Ala
515 520 525



80
48
PRT
Homo sapiens

SITE
(48)
Xaa equals stop translation


80

Met Ala Gly Thr Val Leu Gly Val Gly Ala Gly Val Phe Ile Leu Ala
1 5 10 15

Leu Leu Trp Val Ala Val Leu Leu Leu Cys Val Leu Leu Ser Arg Ala
20 25 30

Ser Gly Ala Ala Arg Phe Ser Val Ile Phe Tyr Ser Ser Val Leu Xaa
35 40 45



81
48
PRT
Homo sapiens

SITE
(48)
Xaa equals stop translation


81

Met Ser Leu Leu Leu Pro Pro Leu Ala Leu Leu Leu Leu Leu Ala Ala
1 5 10 15

Leu Val Ala Pro Ala Thr Ala Ala Thr Ala Tyr Arg Pro Asp Trp Asn
20 25 30

Arg Leu Ser Gly Leu Thr Arg Ala Arg Val Glu Thr Cys Gly Gly Xaa
35 40 45



82
293
PRT
Homo sapiens

82

Met Ala Thr Ala Arg Pro Pro Trp Met Trp Val Leu Cys Ala Leu Ile
1 5 10 15

Thr Ala Leu Leu Leu Gly Val Thr Glu His Val Leu Ala Asn Asn Asp
20 25 30

Val Ser Cys Asp His Pro Ser Asn Thr Val Pro Ser Gly Ser Asn Gln
35 40 45

Asp Leu Gly Ala Gly Ala Gly Glu Asp Ala Arg Ser Asp Asp Ser Ser
50 55 60

Ser Arg Ile Ile Asn Gly Ser Asp Cys Asp Met His Thr Gln Pro Trp
65 70 75 80

Gln Ala Ala Leu Leu Leu Arg Pro Asn Gln Leu Tyr Cys Gly Ala Val
85 90 95

Leu Val His Pro Gln Trp Leu Leu Thr Ala Ala His Cys Arg Lys Lys
100 105 110

Val Phe Arg Val Arg Leu Gly His Tyr Ser Leu Ser Pro Val Tyr Glu
115 120 125

Ser Gly Gln Gln Met Phe Gln Gly Val Lys Ser Ile Pro His Pro Gly
130 135 140

Tyr Ser His Pro Gly His Ser Asn Asp Leu Met Leu Ile Lys Leu Asn
145 150 155 160

Arg Arg Ile Arg Pro Thr Lys Asp Val Arg Pro Ile Asn Val Ser Ser
165 170 175

His Cys Pro Ser Ala Gly Thr Lys Cys Leu Val Ser Gly Trp Gly Thr
180 185 190

Thr Lys Ser Pro Gln Val His Phe Pro Lys Val Leu Gln Cys Leu Asn
195 200 205

Ile Ser Val Leu Ser Gln Lys Arg Cys Glu Asp Ala Tyr Pro Arg Gln
210 215 220

Ile Asp Asp Thr Met Phe Cys Ala Gly Asp Lys Ala Gly Arg Asp Ser
225 230 235 240

Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn Gly Ser Leu Gln
245 250 255

Gly Leu Val Ser Trp Gly Asp Tyr Pro Cys Ala Arg Pro Asn Arg Pro
260 265 270

Gly Val Tyr Thr Asn Leu Cys Lys Phe Thr Lys Trp Ile Gln Glu Thr
275 280 285

Ile Gln Ala Asn Ser
290



83
89
PRT
Homo sapiens

SITE
(89)
Xaa equals stop translation


83

Met Val Ala Gly Phe Val Phe Tyr Leu Gly Val Phe Val Val Cys His
1 5 10 15

Gln Leu Ser Ser Ser Leu Asn Ala Thr Tyr Arg Ser Leu Val Ala Arg
20 25 30

Glu Lys Val Phe Trp Asp Leu Ala Ala Thr Arg Ala Val Phe Gly Val
35 40 45

Gln Ser Thr Ala Ala Ala Val Gly Ser Ala Gly Gly Pro Cys Ala Ala
50 55 60

Cys Arg Gln Gly Ala Trp Pro Ala Glu Leu Val Leu Val Ser His His
65 70 75 80

Asp Ser Asn Gly Ile Leu Leu Leu Xaa
85



84
250
PRT
Homo sapiens

SITE
(161)
Xaa equals any of the naturally occurring
L-amino acids


84

Met Trp Arg Cys Pro Leu Gly Leu Leu Leu Leu Leu Pro Leu Ala Gly
1 5 10 15

His Leu Ala Leu Gly Ala Gln Gln Gly Arg Gly Arg Arg Glu Leu Ala
20 25 30

Pro Gly Leu His Leu Arg Gly Ile Arg Asp Ala Gly Gly Arg Tyr Cys
35 40 45

Gln Glu Gln Asp Leu Cys Cys Arg Gly Arg Ala Asp Asp Cys Ala Leu
50 55 60

Pro Tyr Leu Gly Ala Ile Cys Tyr Cys Asp Leu Phe Cys Asn Arg Thr
65 70 75 80

Val Ser Asp Cys Cys Pro Asp Phe Trp Asp Phe Cys Leu Gly Val Pro
85 90 95

Pro Pro Phe Pro Pro Ile Gln Gly Cys Met His Gly Gly Arg Ile Tyr
100 105 110

Pro Val Leu Gly Thr Tyr Trp Asp Asn Cys Asn Arg Cys Thr Cys Gln
115 120 125

Glu Asn Arg Gln Trp Gln Cys Asp Gln Glu Pro Cys Leu Val Asp Pro
130 135 140

Asp Met Ile Lys Ala Ile Asn Gln Gly Asn Tyr Gly Trp Gln Ala Gly
145 150 155 160

Xaa His Ser Ala Phe Trp Gly Met Thr Leu Asp Glu Gly Ile Arg Tyr
165 170 175

Arg Leu Gly Thr Ile Arg Pro Ser Ser Ser Val Met Asn Met His Glu
180 185 190

Ile Tyr Thr Val Leu Asn Pro Gly Glu Val Leu Pro Thr Ala Phe Glu
195 200 205

Ala Ser Glu Xaa Xaa Pro Xaa Xaa Phe Xaa Ser Leu Xaa Thr Lys Ala
210 215 220

Thr Val Gln Ala Pro Gly Pro Ser Pro Gln Gln Leu Trp His Pro Ile
225 230 235 240

Val Ser Gln Ser Ile Leu Trp Asp Thr Xaa
245 250



85
58
PRT
Homo sapiens

SITE
(58)
Xaa equals stop translation


85

Met Tyr Thr Lys Leu Met Leu Asn Lys Val Leu Leu Phe Trp Gln Ile
1 5 10 15

Val Lys Cys Lys Val Leu Val Asp Gln Tyr Cys Tyr Asn Phe Gly Ala
20 25 30

Lys Leu Leu His Ala Asp Trp Leu Trp Asp Leu Val His Phe Leu Arg
35 40 45

Thr Asn Val Glu Phe Glu Lys Thr Pro Xaa
50 55



86
49
PRT
Homo sapiens

SITE
(49)
Xaa equals stop translation


86

Met Phe Leu Gly Ser Leu Cys Phe Ser Leu Leu Cys His Ala Gly Cys
1 5 10 15

Gln Gly Ser Gly Gly Lys Pro Ala Val Thr Gly Leu Thr Gln Leu Pro
20 25 30

His Asn Pro Lys Gly Trp Phe His Ser His His Ala Pro Arg Pro Gln
35 40 45

Xaa



87
172
PRT
Homo sapiens

SITE
(170)
Xaa equals any of the naturally occurring
L-amino acids


87

Met Arg Gly Ser Val Glu Cys Thr Trp Gly Trp Gly His Cys Ala Pro
1 5 10 15

Ser Pro Leu Leu Leu Trp Thr Leu Leu Leu Phe Ala Ala Pro Phe Gly
20 25 30

Leu Leu Gly Glu Lys Thr Arg Gln Leu Leu Glu Phe Asp Ser Thr Asn
35 40 45

Val Ser Asp Thr Ala Ala Lys Pro Leu Gly Arg Pro Tyr Pro Pro Tyr
50 55 60

Ser Leu Ala Asp Phe Ser Trp Asn Asn Ile Thr Asp Ser Leu Asp Pro
65 70 75 80

Ala Thr Leu Ser Ala Thr Phe Gln Gly His Pro Met Asn Asp Pro Thr
85 90 95

Arg Thr Phe Ala Asn Gly Ser Leu Ala Phe Arg Val Gln Ala Phe Ser
100 105 110

Arg Ser Ser Arg Pro Ala Gln Pro Pro Arg Leu Leu His Thr Ala Asp
115 120 125

Thr Cys Gln Leu Glu Val Ala Leu Ile Gly Ala Ser Pro Arg Gly Asn
130 135 140

Arg Ser Leu Phe Gly Leu Glu Val Ala Thr Leu Gly Gln Gly Pro Asp
145 150 155 160

Cys Pro Ser Met Gln Glu Gln His Ser Xaa Glu Arg
165 170



88
174
PRT
Homo sapiens

SITE
(174)
Xaa equals stop translation


88

Met Val Phe Leu Lys Phe Phe Cys Met Ser Phe Phe Cys His Leu Cys
1 5 10 15

Gln Gly Tyr Phe Asp Gly Pro Leu Tyr Pro Glu Met Ser Asn Gly Thr
20 25 30

Leu His His Tyr Phe Val Pro Asp Gly Asp Tyr Glu Glu Asn Asp Asp
35 40 45

Pro Glu Lys Cys Gln Leu Leu Phe Arg Val Ser Asp His Arg Arg Cys
50 55 60

Ser Gln Gly Glu Gly Ser Gln Val Gly Ser Leu Leu Ser Leu Thr Leu
65 70 75 80

Arg Glu Glu Phe Thr Val Leu Gly His Gln Val Glu Gly Cys Trp Ala
85 90 95

Arg Ala Gly Gly His Gln Gln Lys His Leu Leu Arg Pro Arg Arg Gly
100 105 110

Arg Glu Leu Trp Gln Val Pro Ala Ala Gly Val Pro Pro Asp Arg Gly
115 120 125

Met Pro Thr Pro Thr Arg Thr Asn Pro Ser Leu Ser Trp Arg Ala Ser
130 135 140

Ser Ser Arg Ala Arg Asn Arg Thr Ala Gly Arg Arg Ala Gly Ser Thr
145 150 155 160

Arg Thr Phe Trp Glu Cys Trp Ser Thr Pro Gly Pro Cys Xaa
165 170



89
275
PRT
Homo sapiens

SITE
(275)
Xaa equals stop translation


89

Met Phe Tyr Ile Ile Gly Gly Val Ala Thr Leu Leu Leu Ile Leu Val
1 5 10 15

Ile Ile Val Phe Lys Glu Lys Pro Lys Tyr Pro Pro Ser Arg Ala Gln
20 25 30

Ser Leu Ser Tyr Ala Leu Thr Ser Pro Asp Ala Ser Tyr Leu Gly Ser
35 40 45

Ile Ala Arg Leu Phe Lys Asn Leu Asn Phe Val Leu Leu Val Ile Thr
50 55 60

Tyr Gly Leu Asn Ala Gly Ala Phe Tyr Ala Leu Ser Thr Leu Leu Asn
65 70 75 80

Arg Met Val Ile Trp His Tyr Pro Gly Glu Glu Val Asn Ala Gly Arg
85 90 95

Ile Gly Leu Thr Ile Val Ile Ala Gly Met Leu Gly Ala Val Ile Ser
100 105 110

Gly Ile Trp Leu Asp Arg Ser Lys Thr Tyr Lys Glu Thr Thr Leu Val
115 120 125

Val Tyr Ile Met Thr Leu Val Gly Met Val Val Tyr Thr Phe Thr Leu
130 135 140

Asn Leu Gly His Leu Trp Val Val Phe Ile Thr Ala Gly Thr Met Gly
145 150 155 160

Phe Phe Met Thr Gly Tyr Leu Pro Leu Gly Phe Glu Phe Ala Val Glu
165 170 175

Leu Thr Tyr Pro Glu Ser Glu Gly Ile Ser Ser Gly Leu Leu Asn Ile
180 185 190

Ser Ala Gln Val Phe Gly Ile Ile Phe Thr Ile Ser Gln Gly Gln Ile
195 200 205

Ile Asp Asn Tyr Gly Thr Lys Pro Gly Asn Ile Phe Leu Cys Val Phe
210 215 220

Leu Thr Leu Gly Ala Ala Leu Thr Ala Phe Ile Lys Ala Asp Leu Arg
225 230 235 240

Arg Gln Lys Ala Asn Lys Glu Thr Leu Glu Asn Lys Leu Gln Glu Glu
245 250 255

Glu Glu Glu Ser Asn Thr Ser Lys Val Pro Thr Ala Val Ser Glu Asp
260 265 270

His Leu Xaa
275



90
83
PRT
Homo sapiens

SITE
(83)
Xaa equals stop translation


90

Met Lys Lys Val Ala Arg Leu Ser Ser Leu Gly His Val Val Trp Arg
1 5 10 15

Leu Tyr Ala Arg Val Leu Ala Leu Ile Thr Cys Ile Phe Trp Val Leu
20 25 30

Ala Leu Ile Ile Cys Ile Phe Thr Pro Gln Ile Phe Phe Lys His Leu
35 40 45

Leu His Ala Arg Pro Cys Ser Arg Tyr Arg Arg Tyr Asn Ser Lys Asn
50 55 60

Thr Asp Leu Ala Leu Met Lys Leu Lys Leu Leu Arg Gln Ala Asp Ser
65 70 75 80

Asp Lys Xaa



91
212
PRT
Homo sapiens

SITE
(99)
Xaa equals any of the naturally occurring
L-amino acids


91

Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu
1 5 10 15

Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile
20 25 30

Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu
35 40 45

Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys
50 55 60

Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr Leu Gln Ala Thr
65 70 75 80

Arg Ala Leu Met Val Val Gly Ile Leu Leu Gly Val Ile Ala Ile Phe
85 90 95

Val Ala Xaa Val Gly Met Lys Cys Met Lys Cys Leu Glu Asp Asp Glu
100 105 110

Val Gln Lys Met Arg Met Ala Val Ile Gly Gly Ala Ile Phe Leu Leu
115 120 125

Ala Gly Leu Ala Ile Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile
130 135 140

Val Gln Glu Phe Tyr Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu
145 150 155 160

Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu
165 170 175

Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser
180 185 190

Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro Ser Ser Gly Lys
195 200 205

Asp Tyr Val Xaa
210



92
41
PRT
Homo sapiens

SITE
(41)
Xaa equals stop translation


92

Met Phe Val Phe Leu Ser Val Leu Tyr Ser Leu Ser Leu Glu Tyr Met
1 5 10 15

Phe Leu Phe Val Phe Gly Lys Lys Ile Ser Phe Thr Ser Leu His Ser
20 25 30

Asp Gln Leu Gly Lys Lys Lys Ala Xaa
35 40



93
49
PRT
Homo sapiens

SITE
(49)
Xaa equals stop translation


93

Met Gln Pro Cys Leu Phe Met Phe Val Leu Met Gly Ile Met Trp Ala
1 5 10 15

Thr Gly Ile Leu Pro Lys Ile Met Pro Ser Arg Lys Arg Cys Leu Ser
20 25 30

Ile Asp Ile Pro Ala Ala Pro Gln Ala Gly Met Cys Leu Leu Ile Leu
35 40 45

Xaa



94
90
PRT
Homo sapiens

SITE
(90)
Xaa equals stop translation


94

Met Ala Lys Arg Thr Phe Ser Asn Leu Glu Thr Phe Leu Ile Phe Leu
1 5 10 15

Leu Val Met Met Ser Ala Ile Thr Val Ala Leu Leu Ser Leu Leu Phe
20 25 30

Ile Thr Ser Gly Thr Ile Glu Asn His Lys Asp Leu Gly Gly His Phe
35 40 45

Phe Ser Thr Thr Gln Ser Pro Pro Ala Thr Gln Gly Ser Thr Ala Ala
50 55 60

Gln Arg Ser Thr Ala Thr Gln His Ser Thr Ala Thr Gln Ser Ser Asn
65 70 75 80

Ser Gln Leu Lys Leu Leu Gln Cys Leu Xaa
85 90



95
486
PRT
Homo sapiens

95

Met Gln Pro Ser Gly Leu Glu Gly Pro Gly Thr Phe Gly Arg Trp Pro
1 5 10 15

Leu Leu Ser Leu Leu Leu Leu Leu Leu Leu Leu Gln Pro Val Thr Cys
20 25 30

Ala Tyr Thr Thr Pro Gly Pro Pro Arg Ala Leu Thr Thr Leu Gly Ala
35 40 45

Pro Arg Ala His Thr Met Pro Gly Thr Tyr Ala Pro Ser Thr Thr Leu
50 55 60

Ser Ser Pro Ser Thr Gln Gly Leu Gln Glu Gln Ala Arg Ala Leu Met
65 70 75 80

Arg Asp Phe Pro Leu Val Asp Gly His Asn Asp Leu Pro Leu Val Leu
85 90 95

Arg Gln Val Tyr Gln Lys Gly Leu Gln Asp Val Asn Leu Arg Asn Phe
100 105 110

Ser Tyr Gly Gln Thr Ser Leu Asp Arg Leu Arg Asp Gly Leu Val Gly
115 120 125

Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gln Thr Gln Asp Arg Asp
130 135 140

Ala Leu Arg Leu Thr Leu Glu Gln Ile Asp Leu Ile Arg Arg Met Cys
145 150 155 160

Ala Ser Tyr Ser Glu Leu Glu Leu Val Thr Ser Ala Lys Ala Leu Asn
165 170 175

Asp Thr Gln Lys Leu Ala Cys Leu Ile Gly Val Glu Gly Gly His Ser
180 185 190

Leu Asp Asn Ser Leu Ser Ile Leu Arg Thr Phe Tyr Met Leu Gly Val
195 200 205

Arg Tyr Leu Thr Leu Thr His Thr Cys Asn Thr Pro Trp Ala Glu Ser
210 215 220

Ser Ala Lys Gly Val His Ser Phe Tyr Asn Asn Ile Ser Gly Leu Thr
225 230 235 240

Asp Phe Gly Glu Lys Val Val Ala Glu Met Asn Arg Leu Gly Met Met
245 250 255

Val Asp Leu Ser His Val Ser Asp Ala Val Ala Arg Arg Ala Leu Glu
260 265 270

Val Ser Gln Ala Pro Val Ile Phe Ser His Ser Ala Ala Arg Gly Val
275 280 285

Cys Asn Ser Ala Arg Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Lys
290 295 300

Lys Asn Gly Gly Val Val Met Val Ser Leu Ser Met Gly Val Ile Gln
305 310 315 320

Cys Asn Pro Ser Ala Asn Val Ser Thr Val Ala Asp His Phe Asp His
325 330 335

Ile Lys Ala Val Ile Gly Ser Lys Phe Ile Gly Ile Gly Gly Asp Tyr
340 345 350

Asp Gly Ala Gly Lys Phe Pro Gln Gly Leu Glu Asp Val Ser Thr Tyr
355 360 365

Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gly Trp Ser Glu Glu Glu
370 375 380

Leu Gln Gly Val Leu Arg Gly Asn Leu Leu Arg Val Phe Arg Gln Val
385 390 395 400

Glu Lys Val Gln Glu Glu Asn Lys Trp Gln Ser Pro Leu Glu Asp Lys
405 410 415

Phe Pro Asp Glu Gln Leu Ser Ser Ser Cys His Ser Asp Leu Ser Arg
420 425 430

Leu Arg Gln Arg Gln Ser Leu Thr Ser Gly Gln Glu Leu Thr Glu Ile
435 440 445

Pro Ile His Trp Thr Ala Lys Leu Pro Ala Lys Trp Ser Val Ser Glu
450 455 460

Ser Ser Pro His Met Ala Pro Val Leu Ala Val Val Ala Thr Phe Pro
465 470 475 480

Val Leu Ile Leu Trp Leu
485



96
60
PRT
Homo sapiens

SITE
(60)
Xaa equals stop translation


96

Met Met Lys Asp Val Phe Phe Phe Leu Phe Leu Leu Ala Val Trp Val
1 5 10 15

Val Ser Phe Gly Val Ala Lys Gln Ala Ile Leu Ile His Asn Glu Arg
20 25 30

Arg Val Asp Trp Leu Phe Arg Gly Pro Ser Thr Thr Pro Thr Ser Pro
35 40 45

Ser Ser Gly Arg Ser Arg Ala Thr Ser Thr Val Xaa
50 55 60



97
293
PRT
Homo sapiens

97

Met Ala Thr Ala Arg Pro Pro Trp Met Trp Val Leu Cys Ala Leu Ile
1 5 10 15

Thr Ala Leu Leu Leu Gly Val Thr Glu His Val Leu Ala Asn Asn Asp
20 25 30

Val Ser Cys Asp His Pro Ser Asn Thr Val Pro Ser Gly Ser Asn Gln
35 40 45

Asp Leu Gly Ala Gly Ala Gly Glu Asp Ala Arg Ser Asp Asp Ser Ser
50 55 60

Ser Arg Ile Ile Asn Gly Ser Asp Cys Asp Met His Thr Gln Pro Trp
65 70 75 80

Gln Ala Ala Leu Leu Leu Arg Pro Asn Gln Leu Tyr Cys Gly Ala Val
85 90 95

Leu Val His Pro Gln Trp Leu Leu Thr Ala Ala His Cys Arg Lys Lys
100 105 110

Val Phe Arg Val Arg Leu Gly His Tyr Ser Leu Ser Pro Val Tyr Glu
115 120 125

Ser Gly Gln Gln Met Phe Gln Gly Val Lys Ser Ile Pro His Pro Gly
130 135 140

Tyr Ser His Pro Gly His Ser Asn Asp Leu Met Leu Ile Lys Leu Asn
145 150 155 160

Arg Arg Ile Arg Pro Thr Lys Asp Val Arg Pro Ile Asn Val Ser Ser
165 170 175

His Cys Pro Ser Ala Gly Thr Lys Cys Leu Val Ser Gly Trp Gly Thr
180 185 190

Thr Lys Ser Pro Gln Val His Phe Pro Lys Val Leu Gln Cys Leu Asn
195 200 205

Ile Ser Val Leu Ser Gln Lys Arg Cys Glu Asp Ala Tyr Pro Arg Gln
210 215 220

Ile Asp Asp Thr Met Phe Cys Ala Gly Asp Lys Ala Gly Arg Asp Ser
225 230 235 240

Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn Gly Ser Leu Gln
245 250 255

Gly Leu Val Ser Trp Gly Asp Tyr Pro Cys Ala Arg Pro Asn Arg Pro
260 265 270

Gly Val Tyr Thr Asn Leu Cys Lys Phe Thr Lys Trp Ile Gln Glu Thr
275 280 285

Ile Gln Ala Asn Ser
290



98
62
PRT
Homo sapiens

SITE
(62)
Xaa equals stop translation


98

Met Ala Thr Ala Arg Pro Pro Trp Met Trp Val Leu Cys Ala Leu Ile
1 5 10 15

Thr Ala Leu Leu Leu Gly Val Thr Glu His Val Leu Ala Asn Asn Asp
20 25 30

Val Ser Cys Asp His Pro Ser Asn Thr Val Pro Ser Gly Ser Asn Gln
35 40 45

Asp Leu Gly Ala Gly Ala Gly Gly Arg Arg Pro Val Gly Xaa
50 55 60



99
132
PRT
Homo sapiens

SITE
(132)
Xaa equals stop translation


99

Met Arg Gly Ser Val Glu Cys Thr Trp Gly Trp Gly His Cys Ala Pro
1 5 10 15

Ser Pro Leu Leu Leu Trp Thr Leu Leu Leu Phe Ala Ala Pro Phe Gly
20 25 30

Leu Leu Gly Glu Lys Thr Arg Gln Leu Leu Glu Phe Asp Ser Thr Asn
35 40 45

Val Ser Asp Thr Ala Ala Lys Pro Leu Gly Arg Pro Tyr Pro Pro Tyr
50 55 60

Ser Leu Ala Asp Phe Ser Trp Asn Asn Ile Thr Asp Ser Leu Asp Pro
65 70 75 80

Ala Thr Leu Ser Ala Thr Phe Gln Gly His Pro Met Asn Asp Pro Thr
85 90 95

Arg Thr Phe Ala Asn Gly Ser Leu Ala Phe Arg Ser Arg Pro Phe Pro
100 105 110

Gly Pro Ala Asp Gln Pro Asn Pro Leu Ala Ser Cys Thr Gln Gln Thr
115 120 125

Pro Val Ser Xaa
130



100
71
PRT
Homo sapiens

SITE
(71)
Xaa equals stop translation


100

Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu
1 5 10 15

Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile
20 25 30

Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Pro Arg Pro Cys Thr Arg
35 40 45

Gly Cys Gly Cys Pro Ala Cys Arg Arg Ala Pro Gly Arg Ser Ser Ala
50 55 60

Lys Ser Leu Thr Pro Cys Xaa
65 70



101
9
PRT
Homo sapiens

101

Ile Lys Ile Ser Leu Lys Lys Arg Ser
1 5



102
151
PRT
Homo sapiens

102

Ile Lys Ile Ser Leu Lys Lys Arg Ser Met Ser Gly Ile Ser Gly Cys
1 5 10 15

Pro Phe Phe Leu Trp Gly Leu Leu Ala Leu Leu Gly Leu Ala Leu Val
20 25 30

Ile Ser Leu Ile Phe Asn Ile Ser His Tyr Val Glu Lys Gln Arg Gln
35 40 45

Asp Lys Met Tyr Ser Tyr Ser Ser Asp His Thr Arg Val Asp Glu Tyr
50 55 60

Tyr Ile Glu Asp Thr Pro Ile Tyr Gly Asn Leu Asp Asp Met Ile Ser
65 70 75 80

Glu Pro Met Asp Glu Asn Cys Tyr Glu Gln Met Lys Ala Arg Pro Glu
85 90 95

Lys Ser Val Asn Lys Met Gln Glu Ala Thr Pro Ser Ala Gln Ala Thr
100 105 110

Asn Glu Thr Gln Met Cys Tyr Ala Ser Leu Asp His Ser Val Lys Gly
115 120 125

Lys Arg Arg Ser Pro Gly Asn Arg Ile Leu Ile Ser Gln Thr Arg Met
130 135 140

Glu Met Ser Asn Tyr Met Gln
145 150



103
79
PRT
Homo sapiens

103

Gly Thr Arg Gly Leu Ser Thr Val Ser Trp Thr His Thr Gln Pro Ser
1 5 10 15

Lys Arg Gly Asp Pro Ser Arg Glu Pro Arg Gly Gly His Ser Cys Leu
20 25 30

Leu Pro Gly Ser Pro Ala Thr Trp Cys Leu Pro Ala Pro Cys Ser Leu
35 40 45

Pro Gly Pro Val Leu Thr Pro Ser Ser Ser Gly Leu Asp Ser Ala Leu
50 55 60

Glu Gly Pro Arg Gly Ala Ala Ser Leu Leu Arg Ala Pro Leu Gln
65 70 75



104
23
PRT
Homo sapiens

104

His Thr Gln Pro Ser Lys Arg Gly Asp Pro Ser Arg Glu Pro Arg Gly
1 5 10 15

Gly His Ser Cys Leu Leu Pro
20



105
22
PRT
Homo sapiens

105

Val Leu Thr Pro Ser Ser Ser Gly Leu Asp Ser Ala Leu Glu Gly Pro
1 5 10 15

Arg Gly Ala Ala Ser Leu
20



106
180
PRT
Homo sapiens

106

Gly Thr Arg Gly Leu Ser Thr Val Ser Trp Thr His Thr Gln Pro Ser
1 5 10 15

Lys Arg Gly Asp Pro Ser Arg Glu Pro Arg Gly Gly His Ser Cys Leu
20 25 30

Leu Pro Gly Ser Pro Ala Thr Trp Cys Leu Pro Ala Pro Cys Ser Leu
35 40 45

Pro Gly Pro Val Leu Thr Pro Ser Ser Ser Gly Leu Asp Ser Ala Leu
50 55 60

Glu Gly Pro Arg Gly Ala Ala Ser Leu Leu Arg Ala Pro Leu Gln Met
65 70 75 80

Glu Glu Ala Ile Leu Val Pro Cys Val Leu Gly Leu Leu Leu Leu Pro
85 90 95

Ile Leu Ala Met Leu Met Ala Leu Cys Val His Cys His Arg Leu Pro
100 105 110

Gly Ser Tyr Asp Ser Thr Ser Ser Asp Ser Leu Tyr Pro Lys Gly His
115 120 125

Pro Val Gln Thr Ala Ser His Gly Cys Pro Leu Ala Thr Cys Leu Pro
130 135 140

Thr Cys His Leu Leu Pro Thr Pro Glu Pro Ala Arg Pro Ala Pro His
145 150 155 160

Pro Lys Ile Pro Ala Ala Pro Trp Gly Leu Pro Pro Asp Ala Ile Phe
165 170 175

Pro Ala Gly Phe
180



107
6
PRT
Homo sapiens

107

Cys Val His Cys His Arg
1 5



108
11
PRT
Homo sapiens

108

Ala Gly Ser Arg Thr Asn Asn Glu Gln Ile Glu
1 5 10



109
58
PRT
Homo sapiens

SITE
(42)
Xaa equals any of the naturally occurring
L-amino acids


109

Ala Gly Ser Arg Thr Asn Asn Glu Gln Ile Glu Met Ser Cys Ile Gly
1 5 10 15

Arg Met Arg Leu Ile Cys Phe Ile Ile Leu Arg Ile Cys Gly Leu Glu
20 25 30

His Leu Phe Gly Asn Met Gly Leu Gly Xaa Lys Asn Gly His Leu Pro
35 40 45

Gly His Tyr Gly His Ser Leu Glu Phe Phe
50 55



110
16
PRT
Homo sapiens

110

Gly Thr Ser Thr Ser Ser Arg Gly Arg Leu His Ala Cys Gly His Ser
1 5 10 15



111
95
PRT
Homo sapiens

111

Pro Ser Ser Glu Val Gln Lys Gly Lys Pro Asn Ser Pro Leu Gly Asn
1 5 10 15

Ser Glu Leu Arg Pro His Leu Val Asn Thr Lys Pro Arg Thr Ser Leu
20 25 30

Glu Arg Gly His Thr Ile Pro Phe Leu Trp Pro Ser Glu Phe Gly Leu
35 40 45

Ser Gln Leu Trp Gly Thr Pro Ser Leu Asn Pro Asn Lys Thr Pro Leu
50 55 60

Glu Ser Leu Ser Leu His Pro Ser Pro Leu Pro Ser Ala Leu Ile Ala
65 70 75 80

Ala Arg Ile Val Thr Pro Asn Leu Thr Leu Ser Ser Leu Ile Lys
85 90 95



112
21
PRT
Homo sapiens

112

Pro Asn Ser Pro Leu Gly Asn Ser Glu Leu Arg Pro His Leu Val Asn
1 5 10 15

Thr Lys Pro Arg Thr
20



113
23
PRT
Homo sapiens

113

Leu Ser Leu His Pro Ser Pro Leu Pro Ser Ala Leu Ile Ala Ala Arg
1 5 10 15

Ile Val Thr Pro Asn Leu Thr
20



114
268
PRT
Homo sapiens

114

Pro Gly Ser Gln Gly Ala Ala Ala Gly Arg Glu Leu Phe Met Thr Asp
1 5 10 15

Arg Glu Arg Leu Ala Glu Ala Arg Gln Arg Glu Leu Gln Arg Gln Glu
20 25 30

Leu Leu Met Gln Lys Arg Leu Ala Met Glu Ser Asn Lys Ile Leu Gln
35 40 45

Glu Gln Gln Glu Met Glu Arg Gln Arg Arg Lys Glu Ile Ala Gln Lys
50 55 60

Ala Ala Glu Glu Asn Glu Arg Tyr Arg Lys Glu Met Glu Gln Ile Val
65 70 75 80

Glu Glu Glu Glu Lys Phe Lys Lys Gln Trp Glu Glu Asp Trp Gly Ser
85 90 95

Lys Glu Gln Leu Leu Leu Pro Lys Thr Ile Thr Ala Glu Val His Pro
100 105 110

Val Pro Leu Arg Lys Pro Lys Tyr Asp Gln Gly Val Glu Pro Glu Leu
115 120 125

Glu Pro Ala Asp Asp Leu Asp Gly Gly Thr Glu Glu Gln Gly Glu Gln
130 135 140

Asp Phe Arg Lys Tyr Glu Glu Gly Phe Asp Pro Tyr Ser Met Phe Thr
145 150 155 160

Pro Glu Gln Ile Met Gly Lys Asp Val Arg Leu Leu Arg Ile Lys Lys
165 170 175

Glu Gly Ser Leu Asp Leu Ala Leu Glu Gly Gly Val Asp Ser Pro Ile
180 185 190

Gly Lys Val Val Val Ser Ala Val Tyr Glu Arg Gly Ala Ala Glu Arg
195 200 205

His Gly Gly Ile Val Lys Gly Asp Glu Ile Met Ala Ile Asn Gly Lys
210 215 220

Ile Val Thr Asp Tyr Thr Leu Ala Glu Ala Asp Ala Ala Leu Gln Lys
225 230 235 240

Ala Trp Asn Gln Gly Gly Asp Trp Ile Asp Leu Val Val Ala Val Cys
245 250 255

Pro Pro Lys Glu Tyr Asp Asp Glu Leu Thr Phe Phe
260 265



115
23
PRT
Homo sapiens

115

Gly Arg Glu Leu Phe Met Thr Asp Arg Glu Arg Leu Ala Glu Ala Arg
1 5 10 15

Gln Arg Glu Leu Gln Arg Gln
20



116
22
PRT
Homo sapiens

116

Gln Gln Glu Met Glu Arg Gln Arg Arg Lys Glu Ile Ala Gln Lys Ala
1 5 10 15

Ala Glu Glu Asn Glu Arg
20



117
25
PRT
Homo sapiens

117

Lys Pro Lys Tyr Asp Gln Gly Val Glu Pro Glu Leu Glu Pro Ala Asp
1 5 10 15

Asp Leu Asp Gly Gly Thr Glu Glu Gln
20 25



118
25
PRT
Homo sapiens

118

Ile Val Thr Asp Tyr Thr Leu Ala Glu Ala Asp Ala Ala Leu Gln Lys
1 5 10 15

Ala Trp Asn Gln Gly Gly Asp Trp Ile
20 25



119
113
PRT
Homo sapiens

119

Gly Thr Ser Thr Ser Ser Arg Gly Arg Leu His Ala Cys Gly His Ser
1 5 10 15

Met Ile Leu Leu Leu Ser Leu Phe Gln Gly Val Arg Gly Ser Leu Gly
20 25 30

Ser Pro Gly Asn Arg Glu Asn Lys Glu Lys Lys Val Phe Ile Ser Leu
35 40 45

Val Gly Ser Arg Gly Leu Gly Cys Ser Ile Ser Ser Gly Pro Ile Gln
50 55 60

Lys Pro Gly Ile Phe Ile Ser His Val Lys Pro Gly Ser Leu Ser Ala
65 70 75 80

Glu Val Gly Leu Glu Ile Gly Asp Gln Ile Val Glu Val Asn Gly Val
85 90 95

Asp Phe Ser Asn Leu Asp His Lys Glu Leu Gln Leu Ala Gly Ser Cys
100 105 110

Ser



120
8
PRT
Homo sapiens

120

Gly Ile Ile Ala Gln Gly Lys Ser
1 5



121
33
PRT
Homo sapiens

121

His Thr Met Leu Pro Leu Lys Ile Ala Ala Pro Tyr Leu Leu Glu Asn
1 5 10 15

Cys Ser Cys Pro Ile Tyr Ile Ser Thr Ser Pro His Leu Phe Leu Ser
20 25 30

Thr



122
19
PRT
Homo sapiens

122

Phe Ser Ile Leu Phe Ala Phe Val Leu Phe Tyr Pro Gly Ser Phe Phe
1 5 10 15

Thr Leu Pro



123
60
PRT
Homo sapiens

123

Phe Ser Ile Leu Phe Ala Phe Val Leu Phe Tyr Pro Gly Ser Phe Phe
1 5 10 15

Thr Leu Pro Met Tyr Met Lys Gln Val Val Ala Cys Arg Asp Gln Leu
20 25 30

Ile Leu Val Leu Trp Leu Ile Glu Leu Leu Cys Ile Gln Gly Phe Cys
35 40 45

Lys Ser Lys Ser Asp Phe Ser Ser Arg Ile Tyr Ser
50 55 60



124
6
PRT
Homo sapiens

124

His Glu Ser Thr Val Lys
1 5



125
27
PRT
Homo sapiens

125

Leu Glu Asn Leu Gly Thr His Lys Lys Lys Asp Ser Phe Ser Val Lys
1 5 10 15

Thr Val Gly Ile Cys Cys Cys Phe His Leu Asn
20 25



126
84
PRT
Homo sapiens

126

Leu Glu Asn Leu Gly Thr His Lys Lys Lys Asp Ser Phe Ser Val Lys
1 5 10 15

Thr Val Gly Ile Cys Cys Cys Phe His Leu Asn Met Leu Tyr Phe Cys
20 25 30

Ser Ser Ile Trp Phe Gly Ile Tyr Phe Val Ala Leu Ile Thr Val Phe
35 40 45

Leu Lys Thr Leu Pro Pro Leu Thr Val Gly Lys Gly Pro Phe Ser Gly
50 55 60

Lys Phe Val Ala Phe Phe Phe Phe Leu Lys Glu Ser Cys Ser Leu Leu
65 70 75 80

Ser Ile Val Phe



127
6
PRT
Homo sapiens

127

Phe Thr Lys Cys Phe His
1 5



128
8
PRT
Homo sapiens

128

Gln Asn Met Asn Asp Tyr Asn Ile
1 5



129
81
PRT
Homo sapiens

129

Gln Asn Met Asn Asp Tyr Asn Ile Met Phe Tyr Leu Tyr Ser Ile Phe
1 5 10 15

Gln Val Leu Val Trp Leu Cys Gln Ala Lys His Leu Ser Gln Ile Ser
20 25 30

Ala Arg Ser Ser Arg Arg Leu Trp Arg Leu Ser Leu Ile Thr Phe Pro
35 40 45

Pro Tyr Leu Ala Thr Ser Leu Ser His Gly Pro His Val Cys Leu Gln
50 55 60

Thr Leu Gly Tyr Glu Ser Cys Glu His Thr Asp Leu Cys Phe Leu His
65 70 75 80

Asp



130
51
PRT
Homo sapiens

130

Pro Ala Arg His Leu Trp Thr Pro Ser Pro Val Cys Lys Pro Ser Ile
1 5 10 15

Lys Pro His Val Ser Phe Ala Gly Ser Gly Ser Leu Trp Arg Leu Glu
20 25 30

Pro Tyr Ala Phe Pro Ile Glu Val Asn Arg Gly Ser Ala Gln His Trp
35 40 45

Val Pro Gly
50



131
29
PRT
Homo sapiens

131

Val Cys Lys Pro Ser Ile Lys Pro His Val Ser Phe Ala Gly Ser Gly
1 5 10 15

Ser Leu Trp Arg Leu Glu Pro Tyr Ala Phe Pro Ile Glu
20 25



132
48
PRT
Homo sapiens

132

Met Gln Pro Cys Leu Phe Met Phe Val Leu Met Gly Ile Met Trp Ala
1 5 10 15

Thr Gly Ile Leu Pro Lys Ile Met Pro Ser Arg Lys Arg Cys Leu Ser
20 25 30

Ile Asp Ile Pro Ala Ala Pro Gln Ala Gly Met Cys Leu Leu Ile Leu
35 40 45



133
32
PRT
Homo sapiens

133

Gln Phe Ser Phe Leu Ser Ala Lys Gly Leu His Trp Ala Leu Phe Val
1 5 10 15

Phe Phe Tyr Phe Leu Ser Thr Ala Cys Gln Arg Trp Ala Trp Gly Leu
20 25 30



134
77
PRT
Homo sapiens

134

Gln Phe Ser Phe Leu Ser Ala Lys Gly Leu His Trp Ala Leu Phe Val
1 5 10 15

Phe Phe Tyr Phe Leu Ser Thr Ala Cys Gln Arg Trp Ala Trp Gly Leu
20 25 30

Met Arg Thr Leu Ala Leu Leu Val Leu Leu Phe Cys Ser Cys Thr His
35 40 45

Ser Ser Met Gly Trp Gly Arg Gln Ala Trp Gly Val Ala Leu Gly Glu
50 55 60

Val Arg Ser Pro Pro Ala Gln Asp Thr Val Ala Lys Thr
65 70 75



135
82
PRT
Homo sapiens

135

His Glu Pro Gly Arg Cys Gly Pro Glu Asn Leu Ala Leu Gln Ala Thr
1 5 10 15

Gln Arg Gly Thr Arg Phe Ser Val Pro Met Cys Lys Ser Ser Arg Gln
20 25 30

Tyr Thr Tyr Thr Ser Val His Met Cys Gln Cys Ala Cys Glu Arg Val
35 40 45

Glu Trp Arg Gly Ser Leu Thr Pro Ala Arg Ala Leu His Asn His Leu
50 55 60

Thr Glu Gln Trp Phe Pro His Gly Phe Pro Phe Leu Ser Arg Phe Phe
65 70 75 80

Thr Tyr



136
24
PRT
Homo sapiens

136

Glu Asn Leu Ala Leu Gln Ala Thr Gln Arg Gly Thr Arg Phe Ser Val
1 5 10 15

Pro Met Cys Lys Ser Ser Arg Gln
20



137
26
PRT
Homo sapiens

137

Met Cys Gln Cys Ala Cys Glu Arg Val Glu Trp Arg Gly Ser Leu Thr
1 5 10 15

Pro Ala Arg Ala Leu His Asn His Leu Thr
20 25



138
12
PRT
Homo sapiens

138

Leu Arg Arg Ala Ser Cys Pro Ile Trp Ser Lys Asp
1 5 10



139
58
PRT
Homo sapiens

139

Leu Arg Arg Ala Ser Cys Pro Ile Trp Ser Lys Asp Gly Lys Thr Leu
1 5 10 15

Tyr Leu Pro Val Cys Leu Ser Phe Leu His Ser Pro Ala Ser Thr Phe
20 25 30

Leu Pro Trp Asn Gln Gly Phe Leu Ser Pro Phe Ala Phe Ser Thr Leu
35 40 45

Gly Thr Pro Gly Ala Lys Gln Phe Ser Ile
50 55



140
166
PRT
Homo sapiens

140

Gly Thr Ser Thr Lys Leu Pro Tyr Cys Arg Glu Asn Val Cys Leu Ala
1 5 10 15

Tyr Gly Ser Glu Trp Ser Val Tyr Ala Val Gly Ser Gln Ala His Val
20 25 30

Ser Phe Leu Asp Pro Arg Gln Pro Ser Tyr Asn Val Lys Ser Val Cys
35 40 45

Ser Arg Glu Arg Gly Ser Gly Ile Arg Ser Val Ser Phe Tyr Glu His
50 55 60

Ile Ile Thr Val Gly Thr Gly Gln Gly Ser Leu Leu Phe Tyr Asp Ile
65 70 75 80

Arg Ala Gln Arg Phe Leu Glu Glu Arg Leu Ser Ala Cys Tyr Gly Ser
85 90 95

Lys Pro Arg Leu Ala Gly Glu Asn Leu Lys Leu Thr Thr Gly Lys Gly
100 105 110

Trp Leu Asn His Asp Glu Thr Trp Arg Asn Tyr Phe Ser Asp Ile Asp
115 120 125

Phe Phe Pro Asn Ala Val Tyr Thr His Cys Tyr Asp Ser Ser Gly Thr
130 135 140

Lys Leu Phe Val Ala Gly Gly Pro Leu Pro Ser Gly Leu His Gly Asn
145 150 155 160

Tyr Ala Gly Leu Trp Ser
165



141
22
PRT
Homo sapiens

141

Cys Arg Glu Asn Val Cys Leu Ala Tyr Gly Ser Glu Trp Ser Val Tyr
1 5 10 15

Ala Val Gly Ser Gln Ala
20



142
24
PRT
Homo sapiens

142

Pro Ser Tyr Asn Val Lys Ser Val Cys Ser Arg Glu Arg Gly Ser Gly
1 5 10 15

Ile Arg Ser Val Ser Phe Tyr Glu
20



143
29
PRT
Homo sapiens

143

Asp Ile Arg Ala Gln Arg Phe Leu Glu Glu Arg Leu Ser Ala Cys Tyr
1 5 10 15

Gly Ser Lys Pro Arg Leu Ala Gly Glu Asn Leu Lys Leu
20 25



144
26
PRT
Homo sapiens

144

Lys Leu Thr Thr Gly Lys Gly Trp Leu Asn His Asp Glu Thr Trp Arg
1 5 10 15

Asn Tyr Phe Ser Asp Ile Asp Phe Phe Pro
20 25



145
21
PRT
Homo sapiens

145

Tyr Asp Ser Ser Gly Thr Lys Leu Phe Val Ala Gly Gly Pro Leu Pro
1 5 10 15

Ser Gly Leu His Gly
20



146
280
PRT
Homo sapiens

146

Lys Pro Gln Arg Phe Arg Arg Pro Phe Phe Phe Asn His Pro Lys Pro
1 5 10 15

Ser Ser His Pro Gly Leu His Ser Arg Pro Thr Leu His Ser His Pro
20 25 30

Ala Phe His Ser His Pro Glu Leu Gln Gln Pro Thr Gln Thr Ser Pro
35 40 45

Val Pro Leu Thr Pro Glu Ser Pro Leu Phe Gln Asn Phe Ser Gly Tyr
50 55 60

His Ile Gly Val Gly Arg Ala Asp Cys Thr Gly Gln Val Ala Asp Ile
65 70 75 80

Asn Leu Met Gly Tyr Gly Lys Ser Gly Gln Asn Ala Gln Gly Ile Leu
85 90 95

Thr Arg Leu Tyr Ser Arg Ala Phe Ile Met Ala Glu Pro Asp Gly Ser
100 105 110

Asn Arg Thr Val Phe Val Ser Ile Asp Ile Gly Met Val Ser Gln Arg
115 120 125

Leu Arg Leu Glu Val Leu Asn Arg Leu Gln Ser Lys Tyr Gly Ser Leu
130 135 140

Tyr Arg Arg Asp Asn Val Ile Leu Ser Gly Thr His Thr His Ser Gly
145 150 155 160

Pro Ala Gly Tyr Phe Gln Tyr Thr Val Phe Val Ile Ala Ser Glu Gly
165 170 175

Phe Ser Asn Gln Thr Phe Gln His Met Val Thr Gly Ile Leu Lys Ser
180 185 190

Ile Asp Ile Ala His Thr Asn Met Lys Pro Gly Lys Ile Phe Ile Asn
195 200 205

Lys Gly Asn Val Asp Gly Val Gln Ile Asn Arg Ser Pro Tyr Ser Tyr
210 215 220

Leu Gln Asn Pro Gln Ser Glu Arg Ala Arg Tyr Ser Ser Asn Thr Asp
225 230 235 240

Lys Glu Met Ile Val Leu Lys Met Val Asp Leu Asn Gly Asp Asp Leu
245 250 255

Gly Leu Ile Ser Phe Ser Phe Ser Lys Ser Ala Leu Gly Thr Tyr Tyr
260 265 270

Glu Pro Arg Asn Thr Ser Leu Glu
275 280



147
30
PRT
Homo sapiens

147

Lys Pro Ser Ser His Pro Gly Leu His Ser Arg Pro Thr Leu His Ser
1 5 10 15

His Pro Ala Phe His Ser His Pro Glu Leu Gln Gln Pro Thr
20 25 30



148
26
PRT
Homo sapiens

148

Arg Ala Asp Cys Thr Gly Gln Val Ala Asp Ile Asn Leu Met Gly Tyr
1 5 10 15

Gly Lys Ser Gly Gln Asn Ala Gln Gly Ile
20 25



149
24
PRT
Homo sapiens

149

Arg Ala Phe Ile Met Ala Glu Pro Asp Gly Ser Asn Arg Thr Val Phe
1 5 10 15

Val Ser Ile Asp Ile Gly Met Val
20



150
27
PRT
Homo sapiens

150

Arg Leu Gln Ser Lys Tyr Gly Ser Leu Tyr Arg Arg Asp Asn Val Ile
1 5 10 15

Leu Ser Gly Thr His Thr His Ser Gly Pro Ala
20 25



151
23
PRT
Homo sapiens

151

Ala Ser Glu Gly Phe Ser Asn Gln Thr Phe Gln His Met Val Thr Gly
1 5 10 15

Ile Leu Lys Ser Ile Asp Ile
20



152
24
PRT
Homo sapiens

152

Ile Phe Ile Asn Lys Gly Asn Val Asp Gly Val Gln Ile Asn Arg Ser
1 5 10 15

Pro Tyr Ser Tyr Leu Gln Asn Pro
20



153
30
PRT
Homo sapiens

153

Thr Asp Lys Glu Met Ile Val Leu Lys Met Val Asp Leu Asn Gly Asp
1 5 10 15

Asp Leu Gly Leu Ile Ser Phe Ser Phe Ser Lys Ser Ala Leu
20 25 30



154
89
PRT
Homo sapiens

154

Met Ala Lys Arg Thr Phe Ser Asn Leu Glu Thr Phe Leu Ile Phe Leu
1 5 10 15

Leu Val Met Met Ser Ala Ile Thr Val Ala Leu Leu Ser Leu Leu Phe
20 25 30

Ile Thr Ser Gly Thr Ile Glu Asn His Lys Asp Leu Gly Gly His Phe
35 40 45

Phe Ser Thr Thr Gln Ser Pro Pro Ala Thr Gln Gly Ser Thr Ala Ala
50 55 60

Gln Arg Ser Thr Ala Thr Gln His Ser Thr Ala Thr Gln Ser Ser Asn
65 70 75 80

Ser Gln Leu Lys Leu Leu Gln Cys Leu
85



155
486
PRT
Homo sapiens

155

Met Gln Pro Ser Gly Leu Glu Gly Pro Gly Thr Phe Gly Arg Trp Pro
1 5 10 15

Leu Leu Ser Leu Leu Leu Leu Leu Leu Leu Leu Gln Pro Val Thr Cys
20 25 30

Ala Tyr Thr Thr Pro Gly Pro Pro Arg Ala Leu Thr Thr Leu Gly Ala
35 40 45

Pro Arg Ala His Thr Met Pro Gly Thr Tyr Ala Pro Ser Thr Thr Leu
50 55 60

Ser Ser Pro Ser Thr Gln Gly Leu Gln Glu Gln Ala Arg Ala Leu Met
65 70 75 80

Arg Asp Phe Pro Leu Val Asp Gly His Asn Asp Leu Pro Leu Val Leu
85 90 95

Arg Gln Val Tyr Gln Lys Gly Leu Gln Asp Val Asn Leu Arg Asn Phe
100 105 110

Ser Tyr Gly Gln Thr Ser Leu Asp Arg Leu Arg Asp Gly Leu Val Gly
115 120 125

Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gln Thr Gln Asp Arg Asp
130 135 140

Ala Leu Arg Leu Thr Leu Glu Gln Ile Asp Leu Ile Arg Arg Met Cys
145 150 155 160

Ala Ser Tyr Ser Glu Leu Glu Leu Val Thr Ser Ala Lys Ala Leu Asn
165 170 175

Asp Thr Gln Lys Leu Ala Cys Leu Ile Gly Val Glu Gly Gly His Ser
180 185 190

Leu Asp Asn Ser Leu Ser Ile Leu Arg Thr Phe Tyr Met Leu Gly Val
195 200 205

Arg Tyr Leu Thr Leu Thr His Thr Cys Asn Thr Pro Trp Ala Glu Ser
210 215 220

Ser Ala Lys Gly Val His Ser Phe Tyr Asn Asn Ile Ser Gly Leu Thr
225 230 235 240

Asp Phe Gly Glu Lys Val Val Ala Glu Met Asn Arg Leu Gly Met Met
245 250 255

Val Asp Leu Ser His Val Ser Asp Ala Val Ala Arg Arg Ala Leu Glu
260 265 270

Val Ser Gln Ala Pro Val Ile Phe Ser His Ser Ala Ala Arg Gly Val
275 280 285

Cys Asn Ser Ala Arg Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Lys
290 295 300

Lys Asn Gly Gly Val Val Met Val Ser Leu Ser Met Gly Val Ile Gln
305 310 315 320

Cys Asn Pro Ser Ala Asn Val Ser Thr Val Ala Asp His Phe Asp His
325 330 335

Ile Lys Ala Val Ile Gly Ser Lys Phe Ile Gly Ile Gly Gly Asp Tyr
340 345 350

Asp Gly Ala Gly Lys Phe Pro Gln Gly Leu Glu Asp Val Ser Thr Tyr
355 360 365

Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gly Trp Ser Glu Glu Glu
370 375 380

Leu Gln Gly Val Leu Arg Gly Asn Leu Leu Arg Val Phe Arg Gln Val
385 390 395 400

Glu Lys Val Gln Glu Glu Asn Lys Trp Gln Ser Pro Leu Glu Asp Lys
405 410 415

Phe Pro Asp Glu Gln Leu Ser Ser Ser Cys His Ser Asp Leu Ser Arg
420 425 430

Leu Arg Gln Arg Gln Ser Leu Thr Ser Gly Gln Glu Leu Thr Glu Ile
435 440 445

Pro Ile His Trp Thr Ala Lys Leu Pro Ala Lys Trp Ser Val Ser Glu
450 455 460

Ser Ser Pro His Met Ala Pro Val Leu Ala Val Val Ala Thr Phe Pro
465 470 475 480

Val Leu Ile Leu Trp Leu
485



156
15
PRT
Homo sapiens

156

His Leu Thr Gln Phe Cys Val Ile Gln Leu Leu Pro Thr His Leu
1 5 10 15



157
105
PRT
Homo sapiens

SITE
(5)
Xaa equals any of the naturally occurring
L-amino acids


157

Met Leu Ala Ser Xaa Ser Val Leu Cys Asp Pro Ala Pro Ala Asn Pro
1 5 10 15

Ser Asp Glu Leu Leu Val His Ser Pro Cys Phe Leu Leu Ser Pro Pro
20 25 30

Val Ala Pro Val Pro Phe Phe Pro Cys Ala Lys Leu Ile Pro Ala Pro
35 40 45

Arg Pro Val Arg Tyr Phe Ser Pro Pro Asp Leu Arg Leu Gly Asn Thr
50 55 60

Pro Ala Pro Ser Glu Ile Thr Tyr Thr Pro Ser Val His Phe Cys His
65 70 75 80

Pro Ile Ala Lys Leu Leu Cys Leu Lys Val Arg Asn Leu Cys Glu Gly
85 90 95

Val Leu Ser Ala Ala Phe Pro Lys Ala
100 105



158
27
PRT
Homo sapiens

158

Ala Asn Pro Ser Asp Glu Leu Leu Val His Ser Pro Cys Phe Leu Leu
1 5 10 15

Ser Pro Pro Val Ala Pro Val Pro Phe Phe Pro
20 25



159
34
PRT
Homo sapiens

159

Phe Ser Pro Pro Asp Leu Arg Leu Gly Asn Thr Pro Ala Pro Ser Glu
1 5 10 15

Ile Thr Tyr Thr Pro Ser Val His Phe Cys His Pro Ile Ala Lys Leu
20 25 30

Leu Cys



160
58
PRT
Homo sapiens

160

His Leu Thr Gln Phe Cys Val Ile Gln Leu Leu Pro Thr His Leu Met
1 5 10 15

Ser Ser Trp Phe Thr Leu Leu Ala Ser Cys Phe His Leu Leu Trp Pro
20 25 30

Leu Ser Arg Ser Ser His Val Pro Ser Ser Phe Gln Pro Pro Asp Leu
35 40 45

Ser Ala Thr Phe Leu Leu Gln Ile Leu Gly
50 55



161
8
PRT
Homo sapiens

161

Leu Ile Phe His Phe Val Tyr Leu
1 5



162
173
PRT
Homo sapiens

162

Pro Thr Pro Arg Val Ile Leu Gln Val Gly Ser Arg Ile Ala Asp Arg
1 5 10 15

Val Tyr Asp Ile Pro Arg Asn Phe Pro Leu Ala Leu Asp Leu Gly Cys
20 25 30

Gly Arg Gly Tyr Ile Ala Gln Tyr Leu Asn Lys Leu Gln Leu Phe His
35 40 45

Cys Arg Lys Leu Leu Glu Ser Phe Ser Lys Leu Thr Leu Gln Lys Met
50 55 60

Leu Cys Leu His Trp Val Asn Asp Leu Pro Arg Ala Leu Glu Gln Ile
65 70 75 80

His Tyr Ile Leu Lys Pro Asp Gly Val Phe Ile Gly Ala Met Phe Gly
85 90 95

Gly Asp Thr Leu Tyr Glu Leu Arg Cys Ser Leu Gln Leu Ala Glu Thr
100 105 110

Glu Arg Glu Gly Gly Phe Ser Pro His Ile Ser Pro Phe Thr Ala Val
115 120 125

Asn Asp Leu Gly His Leu Leu Gly Arg Ala Gly Phe Asn Thr Leu Thr
130 135 140

Val Asp Thr Asp Glu Ile Gln Val Asn Tyr Pro Gly Met Phe Glu Leu
145 150 155 160

Met Glu Asp Leu Gln Glu Gln Lys Ser Arg Met Leu Thr
165 170



163
24
PRT
Homo sapiens

163

Leu Gln Val Gly Ser Arg Ile Ala Asp Arg Val Tyr Asp Ile Pro Arg
1 5 10 15

Asn Phe Pro Leu Ala Leu Asp Leu
20



164
24
PRT
Homo sapiens

164

Gly Tyr Ile Ala Gln Tyr Leu Asn Lys Leu Gln Leu Phe His Cys Arg
1 5 10 15

Lys Leu Leu Glu Ser Phe Ser Lys
20



165
27
PRT
Homo sapiens

165

Val Asn Asp Leu Pro Arg Ala Leu Glu Gln Ile His Tyr Ile Leu Lys
1 5 10 15

Pro Asp Gly Val Phe Ile Gly Ala Met Phe Gly
20 25



166
28
PRT
Homo sapiens

166

Tyr Glu Leu Arg Cys Ser Leu Gln Leu Ala Glu Thr Glu Arg Glu Gly
1 5 10 15

Gly Phe Ser Pro His Ile Ser Pro Phe Thr Ala Val
20 25



167
22
PRT
Homo sapiens

167

Asn Thr Leu Thr Val Asp Thr Asp Glu Ile Gln Val Asn Tyr Pro Gly
1 5 10 15

Met Phe Glu Leu Met Glu
20



168
86
PRT
Homo sapiens

168

Met Arg Gln Leu Phe Tyr Asp Pro Asp Glu Cys Gly Leu Met Lys Lys
1 5 10 15

Gly Gly Leu Tyr Phe Ser Asp Phe Trp Asn Lys Leu Asp Val Gly Ala
20 25 30

Ile Leu Leu Phe Val Ala Gly Leu Thr Cys Arg Leu Ile Pro Ala Thr
35 40 45

Leu Tyr Pro Gly Arg Val Ile Leu Ser Leu Asp Phe Ile Leu Phe Cys
50 55 60

Leu Arg Leu Met His Ile Phe Thr Ile Ser Lys Thr Leu Gly Pro Lys
65 70 75 80

Ile Ile Ile Val Lys Arg
85



169
27
PRT
Homo sapiens

169

Asp Glu Cys Gly Leu Met Lys Lys Gly Gly Leu Tyr Phe Ser Asp Phe
1 5 10 15

Trp Asn Lys Leu Asp Val Gly Ala Ile Leu Leu
20 25



170
25
PRT
Homo sapiens

170

Thr Leu Tyr Pro Gly Arg Val Ile Leu Ser Leu Asp Phe Ile Leu Phe
1 5 10 15

Cys Leu Arg Leu Met His Ile Phe Thr
20 25



171
274
PRT
Homo sapiens

171

Val Pro Arg Glu Arg Arg Asp Ala Ala Glu Pro Ala Phe Pro Glu Trp
1 5 10 15

Leu Thr Val Leu Leu Leu Cys Leu Tyr Leu Leu Phe Thr Asn Ile Leu
20 25 30

Leu Leu Asn Leu Leu Ile Ala Met Phe Asn Tyr Thr Phe Gln Gln Val
35 40 45

Gln Glu His Thr Asp Gln Ile Trp Lys Phe Gln Arg His Asp Leu Ile
50 55 60

Glu Glu Tyr His Gly Arg Pro Ala Val Pro Pro Pro Leu Ile Leu Phe
65 70 75 80

Ser His Leu Gln Leu Phe Ile Lys Arg Val Val Leu Lys Thr Pro Ala
85 90 95

Lys Arg His Lys Gln Leu Lys Asn Lys Leu Glu Lys Asn Glu Glu Ala
100 105 110

Ala Leu Leu Ser Trp Glu Ile Tyr Leu Lys Glu Asn Tyr Leu Gln Asn
115 120 125

Arg Gln Phe Gln Gln Lys Gln Arg Pro Glu Gln Lys Ile Glu Asp Ile
130 135 140

Ser Asn Lys Val Asp Ala Met Val Asp Leu Leu Asp Leu Asp Pro Leu
145 150 155 160

Lys Arg Ser Gly Ser Met Glu Gln Arg Leu Ala Ser Leu Glu Glu Gln
165 170 175

Val Ala Gln Thr Ala Arg Ala Leu His Trp Ile Val Arg Thr Leu Arg
180 185 190

Ala Ser Gly Phe Ser Ser Glu Ala Asp Val Pro Thr Leu Ala Ser Gln
195 200 205

Lys Ala Ala Glu Glu Pro Asp Ala Glu Pro Gly Gly Arg Lys Lys Thr
210 215 220

Glu Glu Pro Gly Asp Ser Tyr His Val Asn Ala Arg His Leu Leu Tyr
225 230 235 240

Pro Asn Cys Pro Val Thr Arg Phe Pro Val Pro Asn Glu Lys Val Pro
245 250 255

Trp Glu Thr Glu Phe Leu Ile Tyr Asp Pro Pro Phe Tyr Thr Ala Glu
260 265 270

Arg Lys



172
28
PRT
Homo sapiens

172

Gln Ile Trp Lys Phe Gln Arg His Asp Leu Ile Glu Glu Tyr His Gly
1 5 10 15

Arg Pro Ala Val Pro Pro Pro Leu Ile Leu Phe Ser
20 25



173
28
PRT
Homo sapiens

173

Leu Gln Asn Arg Gln Phe Gln Gln Lys Gln Arg Pro Glu Gln Lys Ile
1 5 10 15

Glu Asp Ile Ser Asn Lys Val Asp Ala Met Val Asp
20 25



174
24
PRT
Homo sapiens

174

Val Pro Thr Leu Ala Ser Gln Lys Ala Ala Glu Glu Pro Asp Ala Glu
1 5 10 15

Pro Gly Gly Arg Lys Lys Thr Glu
20



175
20
PRT
Homo sapiens

175

Pro Asn Glu Lys Val Pro Trp Glu Thr Glu Phe Leu Ile Tyr Asp Pro
1 5 10 15

Pro Phe Tyr Thr
20



176
59
PRT
Homo sapiens

176

Met Met Lys Asp Val Phe Phe Phe Leu Phe Leu Leu Ala Val Trp Val
1 5 10 15

Val Ser Phe Gly Val Ala Lys Gln Ala Ile Leu Ile His Asn Glu Arg
20 25 30

Arg Val Asp Trp Leu Phe Arg Gly Pro Ser Thr Thr Pro Thr Ser Pro
35 40 45

Ser Ser Gly Arg Ser Arg Ala Thr Ser Thr Val
50 55



177
48
PRT
Homo sapiens

177

Pro Glu Ser Phe Asn Phe Cys Phe Gly Pro Gly Val Pro Met Pro Trp
1 5 10 15

Cys Leu Leu Pro Val Leu Ser Val Leu His Trp Ser Thr Glu Asp Thr
20 25 30

Arg Ser Cys Gly Ala Gln Gly Gly Gly Pro Pro Leu Pro Pro Arg Gly
35 40 45



178
95
PRT
Homo sapiens

178

Pro Glu Ser Phe Asn Phe Cys Phe Gly Pro Gly Val Pro Met Pro Trp
1 5 10 15

Cys Leu Leu Pro Val Leu Ser Val Leu His Trp Ser Thr Glu Asp Thr
20 25 30

Arg Ser Cys Gly Ala Gln Gly Gly Gly Pro Pro Leu Pro Pro Arg Gly
35 40 45

Met Ala Gly Thr Val Leu Gly Val Gly Ala Gly Val Phe Ile Leu Ala
50 55 60

Leu Leu Trp Val Ala Val Leu Leu Leu Cys Val Leu Leu Ser Arg Ala
65 70 75 80

Ser Gly Ala Ala Arg Phe Ser Val Ile Phe Tyr Ser Ser Val Leu
85 90 95



179
372
PRT
Homo sapiens

SITE
(6)
Xaa equals any of the naturally occurring
L-amino acids


179

Ser Gln Arg His Ala Xaa Gln Gly Thr Gly Pro Gly Ile Pro Gly Ser
1 5 10 15

Thr His Ala Ser Ala Gly Glu Val Ser Val Arg Pro Ala Pro Thr Ser
20 25 30

Phe Ser Val Pro Ser Leu Pro Cys Leu Glu Ala Ala Arg Leu Leu Ser
35 40 45

Ser Glu Phe Tyr Ser Ala Trp Val Ser Ala Gln Cys Arg Trp Trp Pro
50 55 60

Val Leu Val Val Pro Leu Tyr Leu Gly Lys Ser Gly Ala Ala Ala Met
65 70 75 80

Ala Thr Ala Arg Pro Pro Trp Met Trp Val Leu Cys Ala Leu Ile Thr
85 90 95

Ala Leu Leu Leu Gly Val Thr Glu His Val Leu Ala Asn Asn Asp Val
100 105 110

Ser Cys Asp His Pro Ser Asn Thr Val Pro Ser Gly Ser Asn Gln Asp
115 120 125

Leu Gly Ala Gly Ala Gly Glu Asp Ala Arg Ser Asp Asp Ser Ser Ser
130 135 140

Arg Ile Ile Asn Gly Ser Asp Cys Asp Met His Thr Gln Pro Trp Gln
145 150 155 160

Ala Ala Leu Leu Leu Arg Pro Asn Gln Leu Tyr Cys Gly Ala Val Leu
165 170 175

Val His Pro Gln Trp Leu Leu Thr Ala Ala His Cys Arg Lys Lys Val
180 185 190

Phe Arg Val Arg Leu Gly His Tyr Ser Leu Ser Pro Val Tyr Glu Ser
195 200 205

Gly Gln Gln Met Phe Gln Gly Val Lys Ser Ile Pro His Pro Gly Tyr
210 215 220

Ser His Pro Gly His Ser Asn Asp Leu Met Leu Ile Lys Leu Asn Arg
225 230 235 240

Arg Ile Arg Pro Thr Lys Asp Val Arg Pro Ile Asn Val Ser Ser His
245 250 255

Cys Pro Ser Ala Gly Thr Lys Cys Leu Val Ser Gly Trp Gly Thr Thr
260 265 270

Lys Ser Pro Gln Val His Phe Pro Lys Val Leu Gln Cys Leu Asn Ile
275 280 285

Ser Val Leu Ser Gln Lys Arg Cys Glu Asp Ala Tyr Pro Arg Gln Ile
290 295 300

Asp Asp Thr Met Phe Cys Ala Gly Asp Lys Ala Gly Arg Asp Ser Cys
305 310 315 320

Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn Gly Ser Leu Gln Gly
325 330 335

Leu Val Ser Trp Gly Asp Tyr Pro Cys Ala Arg Pro Asn Arg Pro Gly
340 345 350

Val Tyr Thr Asn Leu Cys Lys Phe Thr Lys Trp Ile Gln Glu Thr Ile
355 360 365

Gln Ala Asn Ser
370



180
220
PRT
Homo sapiens

180

Cys Asp Met His Thr Gln Pro Trp Gln Ala Ala Leu Leu Leu Arg Pro
1 5 10 15

Asn Gln Leu Tyr Cys Gly Ala Val Leu Val His Pro Gln Trp Leu Leu
20 25 30

Thr Ala Ala His Cys Arg Lys Lys Val Phe Arg Val Arg Leu Gly His
35 40 45

Tyr Ser Leu Ser Pro Val Tyr Glu Ser Gly Gln Gln Met Phe Gln Gly
50 55 60

Val Lys Ser Ile Pro His Pro Gly Tyr Ser His Pro Gly His Ser Asn
65 70 75 80

Asp Leu Met Leu Ile Lys Leu Asn Arg Arg Ile Arg Pro Thr Lys Asp
85 90 95

Val Arg Pro Ile Asn Val Ser Ser His Cys Pro Ser Ala Gly Thr Lys
100 105 110

Cys Leu Val Ser Gly Trp Gly Thr Thr Lys Ser Pro Gln Val His Phe
115 120 125

Pro Lys Val Leu Gln Cys Leu Asn Ile Ser Val Leu Ser Gln Lys Arg
130 135 140

Cys Glu Asp Ala Tyr Pro Arg Gln Ile Asp Asp Thr Met Phe Cys Ala
145 150 155 160

Gly Asp Lys Ala Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro
165 170 175

Val Val Cys Asn Gly Ser Leu Gln Gly Leu Val Ser Trp Gly Asp Tyr
180 185 190

Pro Cys Ala Arg Pro Asn Arg Pro Gly Val Tyr Thr Asn Leu Cys Lys
195 200 205

Phe Thr Lys Trp Ile Gln Glu Thr Ile Gln Ala Asn
210 215 220



181
30
PRT
Homo sapiens

181

Leu Trp Thr Met Asn Pro Ala Ser Asp Gly Gly Thr Ser Glu Ser Ile
1 5 10 15

Phe Asp Leu Asp Tyr Ala Ser Trp Gly Ile Arg Ser Thr Leu
20 25 30



182
118
PRT
Homo sapiens

182

Leu Trp Thr Met Asn Pro Ala Ser Asp Gly Gly Thr Ser Glu Ser Ile
1 5 10 15

Phe Asp Leu Asp Tyr Ala Ser Trp Gly Ile Arg Ser Thr Leu Met Val
20 25 30

Ala Gly Phe Val Phe Tyr Leu Gly Val Phe Val Val Cys His Gln Leu
35 40 45

Ser Ser Ser Leu Asn Ala Thr Tyr Arg Ser Leu Val Ala Arg Glu Lys
50 55 60

Val Phe Trp Asp Leu Ala Ala Thr Arg Ala Val Phe Gly Val Gln Ser
65 70 75 80

Thr Ala Ala Ala Val Gly Ser Ala Gly Gly Pro Cys Ala Ala Cys Arg
85 90 95

Gln Gly Ala Trp Pro Ala Glu Leu Val Leu Val Ser His His Asp Ser
100 105 110

Asn Gly Ile Leu Leu Leu
115



183
236
PRT
Homo sapiens

183

Met Pro Leu Thr Val Leu Trp Trp Pro Glu Arg Arg Ser Ser Gly Thr
1 5 10 15

Trp Arg Pro Arg Val Gln Ser Leu Val Phe Arg Ala Gln Pro Gln Leu
20 25 30

Trp Ala Leu Leu Gly Asp Pro Val Leu His Ala Asp Lys Ala Arg Gly
35 40 45

Gln Gln Asn Trp Cys Trp Phe His Ile Thr Thr Ala Thr Gly Phe Phe
50 55 60

Cys Phe Glu Asn Val Ala Val His Leu Ser Asn Leu Ile Phe Arg Thr
65 70 75 80

Phe Asp Leu Phe Leu Val Ile His His Leu Phe Ala Phe Leu Gly Phe
85 90 95

Leu Gly Cys Leu Val Asn Leu Gln Ala Gly His Tyr Leu Ala Met Thr
100 105 110

Thr Leu Leu Leu Glu Met Ser Thr Pro Phe Thr Cys Val Ser Trp Met
115 120 125

Leu Leu Lys Ala Gly Trp Ser Glu Ser Leu Phe Trp Lys Leu Asn Gln
130 135 140

Trp Leu Met Ile His Met Phe His Cys Arg Met Val Leu Thr Tyr His
145 150 155 160

Met Trp Trp Val Cys Phe Trp His Trp Asp Gly Leu Val Ser Ser Leu
165 170 175

Tyr Leu Pro His Leu Thr Leu Phe Leu Val Gly Leu Ala Leu Leu Thr
180 185 190

Leu Ile Ile Asn Pro Tyr Trp Thr His Lys Lys Thr Gln Gln Leu Leu
195 200 205

Asn Pro Val Asp Trp Asn Phe Ala Gln Pro Glu Ala Lys Ser Arg Pro
210 215 220

Glu Gly Asn Gly Gln Leu Leu Arg Lys Lys Arg Pro
225 230 235



184
23
PRT
Homo sapiens

184

Gly Thr Ser Gly Gly Ala Arg Ala Ser Leu Gly Pro Ser Pro His Leu
1 5 10 15

His Gln Gly Pro Gly Ala Thr
20



185
272
PRT
Homo sapiens

SITE
(184)
Xaa equals any of the naturally occurring
L-amino acids


185

Gly Thr Ser Gly Gly Ala Arg Ala Ser Leu Gly Pro Ser Pro His Leu
1 5 10 15

His Gln Gly Pro Gly Ala Thr Met Trp Arg Cys Pro Leu Gly Leu Leu
20 25 30

Leu Leu Leu Pro Leu Ala Gly His Leu Ala Leu Gly Ala Gln Gln Gly
35 40 45

Arg Gly Arg Arg Glu Leu Ala Pro Gly Leu His Leu Arg Gly Ile Arg
50 55 60

Asp Ala Gly Gly Arg Tyr Cys Gln Glu Gln Asp Leu Cys Cys Arg Gly
65 70 75 80

Arg Ala Asp Asp Cys Ala Leu Pro Tyr Leu Gly Ala Ile Cys Tyr Cys
85 90 95

Asp Leu Phe Cys Asn Arg Thr Val Ser Asp Cys Cys Pro Asp Phe Trp
100 105 110

Asp Phe Cys Leu Gly Val Pro Pro Pro Phe Pro Pro Ile Gln Gly Cys
115 120 125

Met His Gly Gly Arg Ile Tyr Pro Val Leu Gly Thr Tyr Trp Asp Asn
130 135 140

Cys Asn Arg Cys Thr Cys Gln Glu Asn Arg Gln Trp Gln Cys Asp Gln
145 150 155 160

Glu Pro Cys Leu Val Asp Pro Asp Met Ile Lys Ala Ile Asn Gln Gly
165 170 175

Asn Tyr Gly Trp Gln Ala Gly Xaa His Ser Ala Phe Trp Gly Met Thr
180 185 190

Leu Asp Glu Gly Ile Arg Tyr Arg Leu Gly Thr Ile Arg Pro Ser Ser
195 200 205

Ser Val Met Asn Met His Glu Ile Tyr Thr Val Leu Asn Pro Gly Glu
210 215 220

Val Leu Pro Thr Ala Phe Glu Ala Ser Glu Xaa Xaa Pro Xaa Xaa Phe
225 230 235 240

Xaa Ser Leu Xaa Thr Lys Ala Thr Val Gln Ala Pro Gly Pro Ser Pro
245 250 255

Gln Gln Leu Trp His Pro Ile Val Ser Gln Ser Ile Leu Trp Asp Thr
260 265 270



186
9
PRT
Homo sapiens

186

Tyr Leu Val Ile Phe Phe Leu Lys Cys
1 5



187
66
PRT
Homo sapiens

187

Tyr Leu Val Ile Phe Phe Leu Lys Cys Met Tyr Thr Lys Leu Met Leu
1 5 10 15

Asn Lys Val Leu Leu Phe Trp Gln Ile Val Lys Cys Lys Val Leu Val
20 25 30

Asp Gln Tyr Cys Tyr Asn Phe Gly Ala Lys Leu Leu His Ala Asp Trp
35 40 45

Leu Trp Asp Leu Val His Phe Leu Arg Thr Asn Val Glu Phe Glu Lys
50 55 60

Thr Pro
65



188
7
PRT
Homo sapiens

188

Gly Ser Gln Val Asn Gly Val
1 5



189
66
PRT
Homo sapiens

189

Tyr Leu Val Ile Phe Phe Leu Lys Cys Met Tyr Thr Lys Leu Met Leu
1 5 10 15

Asn Lys Val Leu Leu Phe Trp Gln Ile Val Lys Cys Lys Val Leu Val
20 25 30

Asp Gln Tyr Cys Tyr Asn Phe Gly Ala Lys Leu Leu His Ala Asp Trp
35 40 45

Leu Trp Asp Leu Val His Phe Leu Arg Thr Asn Val Glu Phe Glu Lys
50 55 60

Thr Pro
65



190
10
PRT
Homo sapiens

190

Arg Pro Thr Arg Pro Leu Asn Cys Gly Arg
1 5 10



191
182
PRT
Homo sapiens

SITE
(180)
Xaa equals any of the naturally occurring
L-amino acids


191

Arg Pro Thr Arg Pro Leu Asn Cys Gly Arg Met Arg Gly Ser Val Glu
1 5 10 15

Cys Thr Trp Gly Trp Gly His Cys Ala Pro Ser Pro Leu Leu Leu Trp
20 25 30

Thr Leu Leu Leu Phe Ala Ala Pro Phe Gly Leu Leu Gly Glu Lys Thr
35 40 45

Arg Gln Leu Leu Glu Phe Asp Ser Thr Asn Val Ser Asp Thr Ala Ala
50 55 60

Lys Pro Leu Gly Arg Pro Tyr Pro Pro Tyr Ser Leu Ala Asp Phe Ser
65 70 75 80

Trp Asn Asn Ile Thr Asp Ser Leu Asp Pro Ala Thr Leu Ser Ala Thr
85 90 95

Phe Gln Gly His Pro Met Asn Asp Pro Thr Arg Thr Phe Ala Asn Gly
100 105 110

Ser Leu Ala Phe Arg Val Gln Ala Phe Ser Arg Ser Ser Arg Pro Ala
115 120 125

Gln Pro Pro Arg Leu Leu His Thr Ala Asp Thr Cys Gln Leu Glu Val
130 135 140

Ala Leu Ile Gly Ala Ser Pro Arg Gly Asn Arg Ser Leu Phe Gly Leu
145 150 155 160

Glu Val Ala Thr Leu Gly Gln Gly Pro Asp Cys Pro Ser Met Gln Glu
165 170 175

Gln His Ser Xaa Glu Arg
180



192
39
PRT
Homo sapiens

192

Val Trp Gly Pro Pro Ser Val Ala Ala Ala Leu Glu Leu Val Asp Pro
1 5 10 15

Pro Gly Cys Arg Glu Phe Gly Thr Ser Asn Ile Glu Asp Arg Asp Glu
20 25 30

Leu Ala Tyr His Ile Ser Ile
35



193
313
PRT
Homo sapiens

193

Val Trp Gly Pro Pro Ser Val Ala Ala Ala Leu Glu Leu Val Asp Pro
1 5 10 15

Pro Gly Cys Arg Glu Phe Gly Thr Ser Asn Ile Glu Asp Arg Asp Glu
20 25 30

Leu Ala Tyr His Ile Ser Ile Met Phe Tyr Ile Ile Gly Gly Val Ala
35 40 45

Thr Leu Leu Leu Ile Leu Val Ile Ile Val Phe Lys Glu Lys Pro Lys
50 55 60

Tyr Pro Pro Ser Arg Ala Gln Ser Leu Ser Tyr Ala Leu Thr Ser Pro
65 70 75 80

Asp Ala Ser Tyr Leu Gly Ser Ile Ala Arg Leu Phe Lys Asn Leu Asn
85 90 95

Phe Val Leu Leu Val Ile Thr Tyr Gly Leu Asn Ala Gly Ala Phe Tyr
100 105 110

Ala Leu Ser Thr Leu Leu Asn Arg Met Val Ile Trp His Tyr Pro Gly
115 120 125

Glu Glu Val Asn Ala Gly Arg Ile Gly Leu Thr Ile Val Ile Ala Gly
130 135 140

Met Leu Gly Ala Val Ile Ser Gly Ile Trp Leu Asp Arg Ser Lys Thr
145 150 155 160

Tyr Lys Glu Thr Thr Leu Val Val Tyr Ile Met Thr Leu Val Gly Met
165 170 175

Val Val Tyr Thr Phe Thr Leu Asn Leu Gly His Leu Trp Val Val Phe
180 185 190

Ile Thr Ala Gly Thr Met Gly Phe Phe Met Thr Gly Tyr Leu Pro Leu
195 200 205

Gly Phe Glu Phe Ala Val Glu Leu Thr Tyr Pro Glu Ser Glu Gly Ile
210 215 220

Ser Ser Gly Leu Leu Asn Ile Ser Ala Gln Val Phe Gly Ile Ile Phe
225 230 235 240

Thr Ile Ser Gln Gly Gln Ile Ile Asp Asn Tyr Gly Thr Lys Pro Gly
245 250 255

Asn Ile Phe Leu Cys Val Phe Leu Thr Leu Gly Ala Ala Leu Thr Ala
260 265 270

Phe Ile Lys Ala Asp Leu Arg Arg Gln Lys Ala Asn Lys Glu Thr Leu
275 280 285

Glu Asn Lys Leu Gln Glu Glu Glu Glu Glu Ser Asn Thr Ser Lys Val
290 295 300

Pro Thr Ala Val Ser Glu Asp His Leu
305 310



194
36
PRT
Homo sapiens

194

Leu Pro Pro Arg Gly Pro Ala Thr Phe Gly Ser Pro Gly Cys Pro Pro
1 5 10 15

Ala Asn Ser Pro Pro Ser Ala Pro Ala Thr Pro Glu Pro Ala Arg Ala
20 25 30

Pro Glu Arg Val
35



195
106
PRT
Homo sapiens

195

Leu Pro Pro Arg Gly Pro Ala Thr Phe Gly Ser Pro Gly Cys Pro Pro
1 5 10 15

Ala Asn Ser Pro Pro Ser Ala Pro Ala Thr Pro Glu Pro Ala Arg Ala
20 25 30

Pro Glu Arg Val Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile
35 40 45

Leu Ala Phe Leu Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro
50 55 60

Gln Trp Arg Ile Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Pro Arg
65 70 75 80

Pro Cys Thr Arg Gly Cys Gly Cys Pro Ala Cys Arg Arg Ala Pro Gly
85 90 95

Arg Ser Ser Ala Lys Ser Leu Thr Pro Cys
100 105



196
70
PRT
Homo sapiens

196

Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu
1 5 10 15

Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile
20 25 30

Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Pro Arg Pro Cys Thr Arg
35 40 45

Gly Cys Gly Cys Pro Ala Cys Arg Arg Ala Pro Gly Arg Ser Ser Ala
50 55 60

Lys Ser Leu Thr Pro Cys
65 70