METHOD OF MODULATING NEOVASCULARIZATION

Abstract

The invention provides a method of inhibiting neovascularization in a subject. The method comprises administering to the subject an agent that interferes with fibronectin (Fn) matrix assembly in an amount effective to inhibit neovascularization. The invention also provides a method of identifying an agent that inhibits neovascularization. The method comprises detecting fibronectin (Fn) matrix assembly by stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A decrease in Fn matrix assembly in the presence of the agent compared to Fn matrix assembly in the absence of the agent is indicative of an agent that inhibits neovascularization. Alternatively, the method of identifying an agent that inhibits neovascularization comprises detecting changes in nuclear architecture in stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A reduction in nuclear architecture organization identifies an agent that inhibits neovascularization.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/043,610, filed Apr. 9, 2008.

FIELD OF THE INVENTION

[0003] The invention relates to materials and methods of modulating neovascularization.

BACKGROUND OF THE INVENTION

[0004] Neovascularization, or the formation of new blood vessels, is a highly complex and tightly regulated biological process. Neovascularization begins with the enzymatic breakdown of the basement membrane of a blood vessel. Endothelial cells migrate through the area of degradation, invade the surrounding extracellular matrix, and proliferate to form an elongated column of cells. A lumen forms within the solid cell column upon differentiation of endothelial cells and the basement membrane is subsequently regenerated. Eventually, the newly formed vessel structure connects with existing blood vessels (see, for example, Fotsis et al., 1995. J. Nutr., 125: 790S-797S). The newly formed vessel, as well as existing vessels, can further divide to form branches and capillary networks. The division of existing vessels to form capillary networks is called non-sprouting angiogenesis or intussusception.

[0005] Neovascularization is not continuously required on a large scale in adult animals. Indeed, the process for forming blood vessels is often quiescent except in instances of injury and wound repair. Neovascularization is controlled, at least in part, by the body's requirement for a precise combination of signaling molecules, chemical messengers, and mechanical signals to coordinate the biological events necessary for functional blood vessel formation. When vascularization is not stringently controlled, serious pathologies can result. Uncontrolled vascularization is associated with, for instance, tumor growth, edema, diseases of eye (e.g., diabetic retinopathy and the exudative form of age-related macular degeneration), rheumatoid arthritis, psoriasis, and atherosclerosis.

[0006] Several strategies for controlling vascularization have been proposed, and many angiogenesis inhibitors have been identified including angiostatin, endostatin, pigment epithelium-derived factor (PEDF), and protamine. However, a major hurdle in treating or preventing angiogenesis is targeting processes uniquely associated with unwanted neovascularization to avoid side effects. For example, U.S. Pat. No. 6,833,373 proposes administering an "integrin antagonist" to, e.g., impair endothelial cell adhesion via integrins, thereby prompting cell death of proliferating endothelial cells. Bouroulous et al. (J. Cell Biol., 143(1): 267-276 (1998)) reported that a 76 amino acid III₁-C fibronectin fragment, which forms one of fibronectin's self-assembly sites, causes disassembly of fibronectin matrix and inhibited cell migration and proliferation. However, subsequent studies established that the III₁-C fibronectin fragment (also known as "anastellin") did not act by reducing the level of fibronectin present in the extracellular matrix (see, e.g., Ambesi et al. 2005. Cancer Res., 65(1): 148-156). Instead, it has been proposed that anastellin works through a different mechanism, which may include integrin binding (Ambesi, supra). However, integrins are found on many cell types other than endothelial cells, and play a role in other vital physiological processes that would be disrupted by inhibiting integrin function.

[0007] Thus, there exists a need for a means of specifically inhibiting vascularization in an animal.

SUMMARY OF THE INVENTION

[0008] The invention provides a method of inhibiting neovascularization in a subject. The method comprises administering to the subject an agent that interferes with fibronectin (Fn) matrix assembly in an amount effective to inhibit neovascularization. In some embodiments, the agent does not promote apoptosis and/or does not interfere with binding between integrins and soluble Fn. Examples of suitable agents include, but are not limited to, an antibody or fragment thereof that binds Fn, an Fn fragment, and a functional upstream domain (FUD) of Streptococcus pyogenes adhesion F1 protein.

[0009] The invention further provides a method of identifying an agent that inhibits neovascularization. The method comprises detecting fibronectin (Fn) matrix assembly by stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A decrease in Fn matrix assembly in the presence of the agent compared to Fn matrix assembly in the absence of the agent is indicative of an agent that inhibits neovascularization. The invention also provides a method of identifying an agent that inhibits neovascularization, the method comprising detecting changes in nuclear architecture in stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A reduction in nuclear architecture organization identifies an agent that inhibits neovascularization.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The invention provides a method of inhibiting neovascularization in a subject. The method comprises administering to the subject an agent that interferes with fibronectin (Fn) matrix assembly in an amount effective to inhibit neovascularization. The invention is predicated, at least in part, on the surprising discovery that the endothelial cell-dependent unfolding and pericellular polymerization of the soluble glycoprotein, Fn, plays a required--and 3-dimensional (3-D)-specific--role in triggering neovascularization. While not being limited by any particular theory, it is believed that, during neovascularization, endothelial cells embed themselves within a 3-D extracellular matrix (ECM) consisting of crosslinked networks of the clotting protein, fibrin(ogen). Within this extrinsic matrix, endothelial cells are exposed to angiogenic growth factors that initiate neovascularization. Endothelial cells also establish integrin-mediated adhesive interactions with matrix-bound ligands, and undergo shape changes critical to the activation of actomyosin-dependent contractile responses that serve to trigger the motogenic, proliferative, and morphogenic programs underlying neovascularization. In addition, endothelial cell-dependent remodeling of the pericellular ECM controls nuclear compartment organization and architecture, as well as chromatin structure and function, in a 3-D-specific fashion. Growth factor-triggered changes in endothelial cell shape are transmitted to the nuclear envelope via a pathway dependent on F-actin, intermediate filaments, microtubules, actomyosin-generated force and the linker of nucleus and cytoskeleton (LINC) complex embedded within the nuclear membrane. Unexpectedly, the polymerized Fn matrix is necessary for endothelial cells to proliferate, migrate, assemble a functional cytoskeletal-actomyosin complex, and engage the mechanotransduction-sensitive programs that drive 3-D neovessel formation.

[0011] Fn matrix assembly involves converting soluble Fn into insoluble fibrillar matrix (Chemousov et al. 1991. J. Biol. Chem., 266(17): 10851-10858). Fn is a ˜450 kDa glycoprotein composed of two monomers having three types of "modules" (i.e., type I, II, and III repeats) (Tomasini-Johansson et al. 2006. Matrix Biol., 25(5): 282-293). Soluble Fn binds to the endothelial cell surface by displaying a dominant cell-adhesive domain (module III_9,10), a carboxy-terminal heparin-binding domain (module III_12-14), and a 70 kDa amino-terminal domain. The soluble Fn binding sites are recognized by integrins, syndecans, and Fn matrix assembly sites located on the cell surface (Mao and Schwarzbauer. 2005. Matrix Biol, 24(6): 389-399; Tomasini-Johansson 2006. supra). Engagement of cell surface adhesion molecules by soluble Fn dimers triggers endothelial cell signaling cascades. The triggered signaling cascades, in turn, initiate globular Fn glycoprotein unfolding, and the consequent exposure of cryptic domains that serve to support Fn polymerization and matrix assembly (Geiger et al. 2001. Nat Rev Mol Cell Biol, 2(11): 793-805; Mao and Schwarzbauer, supra; Tomasini-Johansson 2006. supra). Fn molecules multimerize and, over time, organize into fibrils which accumulate in the pericellular space to form an insoluble Fn matrix (Chemousov, supra).

Agents

[0012] The inventive method comprises administering an agent that interferes with Fn matrix assembly to a subject. Any agent that inhibits Fn matrix formation is suitable for the invention; the agent is not limited by the particular means by which it impedes Fn polymerization. The agent may interfere with Fn matrix assembly at any point in the polymerization process, such as any fibrillogenesis-related processes described herein. For example, in certain embodiments the agent interrupts unfolding of Fn bound to cell surface molecules. Alternatively or in addition, the agent interferes with polymerization by binding Fn in such a way that blocks association with other Fn molecules. The agent also (or alternatively) hides cryptic binding sites exposed upon Fn unfolding. In this regard, the five N-terminal type 1 modules of Fn, i.e., the estimated 27 kDa N-terminal fragment or Fn modules (see, e.g., McKeown-Longo and Mosher. 1985. J. Cell Biol., 100: 364), are required for polymerization, i.e., assembly of Fn to form an insoluble matrix. The agent may bind this region of Fn and block matrix assembly. Alternatively, the agent comprises the 27 kDa N-terminal Fn region and competes with native, soluble Fn to block polymerization. In various embodiments, the agent binds another region of Fn, e.g., type II or type III modules, to sterically block polymerization or hide cryptic binding sites to prevent further association with Fn fibrils. The agent may selectively inhibit Fn matrix formation, i.e., the agent inhibits Fn matrix formation with minimal disruption of other Fn functions. For example, in one aspect, the agent impedes Fn matrix assembly, but does not interfere with binding between integrins and soluble Fn. Alternatively, an agent may be selected which does not promote apoptosis of, e.g., endothelial cells.

[0013] Examples of agents for use in the invention include, but are not limited to, chemical moieties (e.g., small molecules), proteins, and nucleic acids. In some embodiments, the agent comprises a protein, such as an intact or full-length protein that interferes with Fn matrix formation. Alternatively, the agent is a protein fragment that inhibits Fn matrix assembly. In certain embodiments, the agent is derived from Fn or procured from another source, e.g., an animal protein, a plant protein, a bacterial protein, a viral protein, or a non-native, genetically-engineered protein or fragment thereof. In one aspect, the agent comprises an Fn fragment that blocks Fn matrix assembly. The nucleic acid sequence of human Fn is publicly available as Entrez Gene ID: 2335 (SEQ ID NO: 1). The fibronectin gene is alternatively spliced, and the amino acid sequences of several splice variants are known: Fn1 isoform 3 preproprotein is designated as Entrez Protein ID: NP_--002017.1 (GI: 16933542) (SEQ ID NO: 2), the mature protein spanning residues 32-2355; Fn1 isoform 7 preproprotein is designated as Entrez Protein ID: NP_--473375.2 (GI: 47132547) (SEQ ID NO: 3), the mature protein spanning residues 32-657; Fn1 isoform 6 preproprotein is designated as Entrez Protein ID: NP_--997639.1 (GI: 47132549) (SEQ ID NO: 4), the mature protein spanning residues 32-2176; Fn1 isoform 2 preproprotein is designated as Entrez Protein ID: NP_--997640.1 (GI: 47132551) (SEQ ID NO: 5), the mature protein spanning residues 32-2421; Fn1 isoform 5 preproprotein is designated as Entrez Protein ID: NP_--997641.1 (GI: 47132553) (SEQ ID NO: 6), the mature protein spanning residues 32-2296; Fn1 isoform 4 preproprotein is designated as Entrez Protein ID: NP_--997643.1 (GI: 47132555) (SEQ ID NO: 7), the mature protein spanning residues 32-2330; and Fn1 isoform 1 preproprotein is designated as Entrez Protein ID: NP_--997647.1 (GI: 47132557) (SEQ ID NO: 8), the mature protein spanning residues 32-2477. The amino acid sequence of the precursor of the largest Fn splice variant is designated as Entrez Protein ID: P02751.3 (GI: 2506872) (SEQ ID NO: 9).

[0014] In some embodiments, the agent comprises (or consists of) an Fn fragment comprising a portion of the N-terminal region of Fn that interferes with assembly of Fn matrices, such as the Fn 70 kDa catheptic fragment of the mature Fn polypeptide (i.e., the N-terminal fragment produced by catheptic digestion of Fn). The 70 kDa N-terminal fragment comprises the N-terminal type I modules critical for fibrillogenesis. The binding activity of the Fn 70 kDa fragment is localized to the N-terminal 27 kDa region of Fn, and evidence also suggests that the 70 kDa catheptic Fn fragment binds cryptic assembly sites along the Fn molecule (Tomasini-Johansson et al. 2001. J. Biol. Chem., 276(26): 23430-23439). While the 70 kDa fragment binds to cell monolayers, it lacks domain interactions that allow Fn polymerization to proceed and, therefore, is not incorporated into the insoluble Fn matrix (Tomasini-Johansson 2006, supra; Tomasini-Johansson 2001, supra). The invention expressly excludes the 76 amino acid III₁-C fibronectin fragment (anastellin) as an agent contemplated. Other Fn-derived fragments that inhibit Fn matrix assembly in vivo by, for example, competing for matrix assembly sites while lacking the capacity to mediate Fn unfolding or matrix formation, also may be used. Methods for identifying Fn fragments that inhibit Fn matrix assembly and neovascularization are described below.

[0015] As noted above, the agent need not be derived from Fn; any protein is useful so long as Fn matrix assembly is impeded, resulting in an inhibition of neovascularization. For example, in one aspect, the agent is a bacterial protein that binds Fn and interferes with fibrillogenesis. A number of bacterial proteins bind Fn for adhesion to, and invasion of, host cells. Fn is a ligand for bacterial "microbial surface components recognizing adhesive matrix molecules" (MSCRAMMs) (see, e.g., Jon et al. 1999. Matrix Biol, 18: 211-23). The proteins are generally found on the bacterial surface and have a molecular mass of approximately 100 kDa. Typically, the Fn binding region comprises three to five repeated regions of 40-50 residues each, and is located N-terminal of the cell-wall spanning region of the molecule (Jon, supra). Several MSCRAMMs have been identified including, but not limited to, Sfb and protein F of Streptococcus pyogenes (Talay et al. 1994. Mol. Microbiol., 13: 531-539; Sela et al. 1993. Mol. Microbiol., 10: 1049-1055); FnbpA and FnbpB of Staphylococcus aureas (Signas et al. 1989. PNAS USA, 86: 699-703; Jonsson et al. 1991. Eur. J. Biochem., 2002: 1041-1048); and FnbA and FnbB of Streptococcus dysgalactiae (Lindgren et al. 1993. Eur. J. Biochem., 214: 819-827). MSCRAMMs that bind Fn, but which do not inhibit Fn matrix assembly to the desired degree, can be modified to enhance their inhibitory activity. For instance, the Fn binding region of an MSCRAMM is, in one aspect, conjugated, connected, or fused to, e.g., PEG or an Fc region of an antibody to sterically hinder matrix assembly.

[0016] In some embodiments, the agent is all or part of a Streptococcus pyogenes Fn binding protein, such as an F1 adhesion protein (see, e.g., Tomasini-Johansson 2001, supra). F1 adhesion protein comprises an N-terminal 43-residue upstream non-repetitive domain (UD), followed by five, 37-amino acid tandem repeats (RD5), and a bacterial cell wall attachment region at the C-terminus. In one aspect, the agent comprises the 49-residue functional upstream domain (FUD) of an F1 adhesion protein. FUD comprises the 43-amino acid UD and the first six amino acids of the N-terminal 37-residue repeat. Accordingly, in some embodiments, the FUD comprises the amino acid sequence KDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDT (SEQ ID NO: 10). FUD recognizes Fn's N-terminal ˜27 kDa fragment and adjacent gelatin binding domain (Tomasini-Johansson 2001, supra). Upon binding, FUD interferes with assembly of insoluble Fn matrix and inhibits neovascularization in a subject.

[0017] Other bacterial proteins also are suitable for the invention. The functional repeat domain (FRD) of S. pyogenes binds the most N-terminal ˜27 kDa fragment of Fn. FRD is a 44-residue fragment encompassing the C-terminus of one RD5 repeat through the N-terminus of the next RD5 repeat, flanked at both ends by the sequence MGGQSES (SEQ ID NO: 11) present in each RD repeat. For example, the FRD protein comprises the amino acid sequence MGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPGVLMGGQSES (SEQ ID NO: 12). Although the FRD peptide inhibits fibrillogenesis to a lesser degree than FUD, the FRD peptide may have properties particularly suitable for some embodiments. S. pyogenes F1 adhesion protein and fragments thereof are further described in Tomasini-Johansson 2001, supra.

[0018] In other embodiments, the agent is an antibody or fragment thereof that binds Fn. Any type of antibody is suitable in the context of the invention, including polyclonal, monoclonal, chimeric, humanized, or human versions having full length heavy and/or light chains. Antibodies according to the invention are obtained by immunization and cell fusion procedures as described herein and known in the art. Monoclonal antibodies of the invention are generated using a variety of known techniques (see, for example, Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).

[0019] Likewise, human antibodies are generated by any of a number of techniques including, but not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein. Methods for obtaining human antibodies from transgenic animals are further described, for example, in Green et al. 1994. Nature Genet. 7: 13-21; Lonberg et al. 1994. Nature, 368: 856-859; Taylor et al. 1994. Int. Immun. 6: 579-591; U.S. Pat. No. 5,877,397; Bruggemann et al., 1997. Curr. Opin. Biotechnol. 8: 455-58; and Jakobovits et al. 1995. Ann. N.Y. Acad. Sci., 764: 525-35.

[0020] Antibody fragments include antigen-binding regions and/or effector regions of the antibody, e.g., F(ab')₂, Fab, Fab', Fv (fragments consisting of the variable regions of the heavy and light chains), Fc, and Fd fragments. Antibody fragments are, in various aspects, incorporated into single domain antibodies, single-chain antibodies (immunological molecules wherein light and heavy variable regions are connected by a peptide linker), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis-single chain Fv regions (see, e.g., Hollinger and Hudson. 2005. Nature Biotechnology, 23(9): 1126-1136) to inhibit Fn matrix formation.

[0021] An antibody or antibody fragment is isolated from nature, synthetic, or genetically-engineered. Antibody fragments derived from an antibody are obtained, e.g., by proteolytic hydrolysis of the antibody. For example, papain or pepsin digestion of whole antibodies yields a 5S fragment termed F(ab')₂ or two monovalent Fab fragments and an Fc fragment, respectively. F(ab')₂ can be further cleaved using a thiol reducing agent to produce 3.5S Fab monovalent fragments. Methods of generating antibody fragments are further described in, for example, U.S. Pat. No. 4,331,647; Nisonoff et al. 1960. Arch. Biochem. Biophys., 89: 230-244; Porter. 1959. Biochem. J., 73: 119-127; Edelman et al., in Methods in Enzymology, 1: 422 Academic Press (1967); and by Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5.

[0022] An antibody or fragment thereof also can be genetically engineered such that the antibody or fragment thereof comprises, e.g., a variable region domain generated by recombinant DNA engineering techniques. For example, in one aspect, a specific antibody variable region is modified by insertions, deletions, or changes in or to the amino acid sequences of the antibody to produce an antibody of interest. In this regard, polynucleotides encoding complementarity determining regions (CDRs) of interest are prepared, for example, by using polymerase chain reaction to synthesize variable regions using mRNA of antibody-producing cells as a template (see, for example, Larrick et al. 1991. Methods: A Companion to Methods in Enzymology, 2: 106-110; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)). Current antibody manipulation techniques allow construction of engineered variable region domains containing at least one CDR and, optionally, one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0023] The antibody or fragment thereof binds any region of Fn so long as matrix assembly, and neovascularization, is inhibited. In some embodiments, the method comprises administering Ab 9D2 or Ab L8 (Chemousov et al. 1987. FEBS Lett., 217(1): 124-8; Chemousov et al. 1991. J. Biol. Chem., 266(17): 10851-10858), or antigen-binding fragments thereof, to inhibit neovascularization. Antibody 9D2 binding activity is localized to the first type III module of Fn (Chemousov, supra). The epitope for antibody L8 is found in a region spanning the type I₉ module and type III₁ module, at or near residues 526-675 (Chemousov, supra). Other antibodies which bind Fn and inhibit neovascularization also are suitable in the context of the invention. For example, in various embodiments, the method comprises administering an antibody or fragment thereof that (i) competes for binding with Ab 9D2 or Ab L8, (ii) binds the region of Fn recognized by Ab 9D2 or L8 (i.e., a region spanning the type I₉ module and type III₁ module, or a region comprising the first type III module of Fn), or (iii) binds at or near Fn type I₉ module and type II_1,2 modules, while inhibiting neovascularization. If desired, the agent comprises an Fn-binding peptide comprising all or part of the antigen-binding elements of an antibody, such as Ab L8 or Ab 9D2, but lacking all or part of the framework regions of an antibody. In this regard, the agent comprises an Fn-binding peptide comprising one, two, three, four, five, or six complementary determining regions (CDRs) of an Fn-binding antibody that inhibits neovascularization, e.g., Ab L8 or Ab 9D2. Methods of identifying complementary determining regions and specificity determining regions are known in the art and further described in, for example, Tamura et al. 2000. J. Immunol., 164: 1432-1441.

[0024] The antibody or fragment thereof preferably specifically binds to Fn, meaning that the antibody or fragment thereof binds Fn with greater affinity than it binds to an unrelated control protein. In other words, the antibody or fragment thereof recognizes and bind Fn preferentially and substantially exclusively (i.e., is able to distinguish Fn from other known polypeptides by virtue of measurable differences in binding affinity) in various aspects of the invention. Depending on the embodiment, the antibody or fragment thereof binds to Fn with an affinity that is at least 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein. Screening assays to determine binding specificity/affinity of an antibody, as well as identify antibodies that compete for binding sites, are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. For example, affinity may be determined using a variety of techniques, such as affinity ELISA assay, BIAcore assay, equilibrium/solution assay, and the like. In one aspect, an antibody or fragment thereof has a binding affinity for Fn of less than or equal to 1×10⁷M, less than or equal to 1×10⁸ M, less than or equal to 1×10⁹ M, less than or equal to 1×10 M, less than or equal to 1×10¹¹ M, or less than or equal to 1×10¹² M.

[0025] The agent alternatively comprises a variant or derivative of any of the exemplary agents described herein. By "variant" is meant a peptide or polypeptide wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring (or at least known) amino acid sequence for the agent. In this regard, the agent is a variant of any of the above-described inhibitors of Fn matrix assembly having, e.g., 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the inhibitor. The variant also must retain the ability to interfere with Fn matrix assembly and inhibit neovascularization. For example, in one aspect, the agent is a variant of Fn or a fragment thereof, such as a variant of Fn's 70 kDa catheptic fragment, having 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the 70 kDa catheptic fragment, while retaining inhibitory activity. The terms "identical" or percent "identity," in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence including necessary gaps, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0026] Identity can exist over a region that is at least about 20 residues in length, such as over a region of at least about 50-100 residues or over at least about 150 residues. Regions of identity can span the active domain of the peptide. The active domains and target binding regions of many agents are known in the art and/or provided herein; a practitioner can modify an agent of interest to create a functional variant falling within the scope of the invention. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman. 1981. Adv. Appl. Math., 2: 482; by the homology alignment algorithm of Needleman & Wunsch. 1970. J. Mol. Biol., 48: 443; by the search for similarity method of Pearson & Lipman. 1988. Proc. Natl. Acad. Sci. USA, 85: 2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. One example of a useful algorithm is PILEUP, which uses a simplification of the progressive alignment method of Feng & Doolittle. 1987. J. Mol. Evol., 35: 351-360, and is similar to the method described by Higgins & Sharp. 1989. CABIOS, 5: 151-153. Another algorithm useful for generating multiple alignments of sequences is Clustal W (Thompson et al. 1994. Nucleic Acids Research, 22: 4673-4680). An example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm (Altschul et al. 1990. J. Mol. Biol., 215: 403-410; Henikoff & Henikoff. 1989. Proc. Natl. Acad. Sci. USA, 89: 10915; Karlin & Altschul. 1993. Proc. Natl. Acad. Sci. USA, 90: 5873-5787). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0027] To generate functional variants, one skilled in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a peptide that correspond to amino acid residues that are important for activity or structure in similar peptides. One skilled in the art also can analyze three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. A number of scientific publications have been devoted to the prediction of secondary structure (Moult. 1996. Curr. Op. in Biotech., 7(4): 422-427; Chou et al. 1974. Biochemistry, 13(2): 222-245; Chou et al. 1974. Biochemistry, 113(2): 211-222; Chou et al. 1978. Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148; Chou et al. 1979. Ann. Rev. Biochem., 47: 251-276; Chou et al. 1979. Biophys. J., 26: 367-384; and Holm et al. 1999. Nucl. Acid. Res., 27(1): 244-247). In view of structure information, one skilled in the art predicts the alignment of amino acid residues of a peptide with respect to its three-dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays, such as those known in the art and/or described herein. Such data could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0028] Variants also include fusion proteins wherein a portion of one peptide is fused to another polypeptide, a polypeptide fragment, or amino acids not generally recognized to be part of a protein sequence. In various aspects, a fusion or chimeric peptide comprises the entire amino acid sequences of two or more peptides or, alternatively, can be constructed to comprise portions (fragments) of two or more peptides (e.g., 10, 20, 50, 75, 100, 400, 500, or more amino acid residues). In some instances, it may be desirable to fuse the active domains of two or more factors to generate a fusion peptide having a desired biological activity. In addition to all or part of the Fn matrix inhibitors described herein, a fusion protein, in one aspect, includes all or part of any suitable peptide comprising a desired biological activity/function, such as a therapeutic peptide. Indeed, in some aspects, the fusion protein comprises, for instance, one or more of the following: an immunogenic peptide; a peptide with long circulating half life, such as an immunoglobulin constant region; a marker protein; a peptide that facilitates purification of the agent; a peptide sequence that promotes formation of multimeric proteins (such as leucine zipper motifs that are useful in dimer formation/stability); and fragments of any of the foregoing.

[0029] "Derivatives" include agents that have been chemically modified in some manner distinct from insertion, deletion, or substitution variants. In this regard, the agent is chemically bonded with polymers, lipids, other organic moieties, and/or inorganic moieties. Derivatives are prepared in some embodiments to increase solubility, absorption, circulating half-life, or targeting to particular cells, tissues, or organs. Chemical modification also may eliminate or attenuate any undesirable side effect of the agent, such as immunogenicity. In this regard, agents covalently modified to include one or more water soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol are contemplated herein (U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337). Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of any of the foregoing.

[0030] In addition, in one aspect, the agent competes with, or cross-blocks, one of the exemplary agents described herein to impede Fn matrix assembly. "Cross-block" is meant to refer to the ability of an agent to interfere with the binding of other fibrillogenesis inhibitors, such as those described herein, to Fn and impede (i.e., reduce or prevent) Fn matrix assembly, thereby inhibiting neovascularization. Agents that compete with or cross-block Fn matrix inhibitors and inhibit neovascularization can be determined using any suitable method, such as the binding assays, Fn matrix assembly models, and angiogenesis models described herein.

[0031] In some embodiments, a nucleic acid comprising a coding sequence for an agent of the method is administered. For example, in one aspect, a nucleic acid encoding FUD is incorporated into an expression vector and administered to a subject to inhibit neovascularization. One of ordinary skill in the art will appreciate that any of a number of expression vectors known in the art are suitable for use in the present method, such as, but not limited to, plasmids, plasmid-liposome complexes, and viral vectors. Any of these expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994). Expression vectors, nucleic acid regulatory sequences, and administration methods, are further discussed in U.S. Patent Publication No. 20030045498.

Methods of Identifying/Characterizing an Agent

[0032] The efficacy of the agent to inhibit neovascularization is determined using any of a number of methods, such as those methods known in the art. Screening assays to determine binding specificity/affinity of an agent are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Binding affinity may be determined using a variety of techniques, such as, but not limited to, affinity ELISA assay, surface plasmon resonance (BIAcore®) assay, equilibrium/solution assay, and the like. In addition, several methods of identifying agents that interfere with Fn polymerization are described in the Examples provided herein. In this regard, the invention also provides a method for identifying an agent that interferes with Fn matrix assembly and inhibits neovascularization in vivo. The method comprises detecting fibronectin (Fn) matrix assembly by stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A decrease in Fn matrix assembly in the presence of the agent compared to Fn matrix assembly in the absence of the agent is indicative of an agent that inhibits neovascularization.

[0033] In one aspect, exposure of an endothelial cell to the agent inhibits changes in the nuclear architecture organization required to support angiogenesis. Accordingly, the invention provides a method for identifying an agent that inhibits neovascularization. The method comprises detecting changes in nuclear architecture in stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent, wherein a reduction in nuclear architecture reorganization identifies an agent that inhibits neovascularization. Nuclear envelope morphology is examined in several ways using any of a number of imaging techniques, such as electron microscopy. For example, in one aspect, the nuclear envelope is viewed to detect infoldings and surface irregularities (i.e., exposure to the agent impedes nuclear restructuring that generates a uniform laminar structure in stimulated cells). Alternatively or in addition, the organization of nuclear pore distribution is examined (i.e., exposure to the agent reduces the redistribution of nuclear pores observed in stimulated endothelial cells in 3-D culture). Exemplary imaging techniques are described in, for example, Aebi et al. 1986. Nature, 323:560-564; Gerace et al. 1984. J. Cell Sci. Suppl., 1:137-60, and the Examples.

[0034] Additionally, the ability of an agent to inhibit neovascularization in vivo is determined using any suitable animal angiogenesis model, such as a mouse or rabbit ear model of neovascularization (Frank et al. 1994. Microsurgery, 15(6): 399-404), an animal model of rheumatoid arthritis (Haas et al. 2007. Arthritis Rheum., 56(8): 2535-48), or an in vivo cancer model, such as a mouse melanoma metastasis model (Lee et al. 2006. Cancer Chemother. Pharmacol., 57(6): 761-71) or a canine model of human invasive urinary bladder cancer (Mohammed et al. 2003. Mol. Cancer. Ther., 2(2): 183-188). Methods of monitoring neovascularization in a human patient are well known. Doppler imaging and magnetic resonance imaging detect blood flow or vascularization changes in tissue (see, e.g., Taylor. 2002. Arthritis Res., 4(suppl. 3): S99-S107), and microscopic examination of tissue biopsies detects changes in vessel number or quality. Perfusion computed tomography ("perfusion CT") (Miles et al. 1998. Brit. J. Radiol., 71: 276-281) and dynamic contrast enhanced magnetic resonance imaging (MRI) (Hathout et al. 2007. Transpl. Int., 20(12): 1059-1065) also are effective in evaluating neovascularization. Ocular neovascularization can be detected using fluorecein angiography, color Doppler imaging, and by clinical examination.

[0035] "Inhibiting" neovascularization does not require a 100% abolition of blood vessel formation. Any decrease in unwanted neovascularization constitutes a beneficial biological effect in a subject. In this regard, the invention reduces neovascularization by, e.g., at least about 5%, at least about 10% or at least about 20% compared to levels of neovascularization observed in the absence of the inventive method (e.g., in a biologically-matched control subject or specimen that is not exposed to the agent of the inventive method). In some embodiments, neovascularization is reduced by at least about 30%, at least about 40%, at least about 50%, or at least about 60%. In some embodiments, the inventive method inhibits neovascularization by at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared new blood vessel formation in the absence of the agent of the inventive method.

Administration Considerations

[0036] The inventive method is, in one aspect, performed after it has been determined that a subject is at risk for unwanted neovascularization (e.g., cancer markers are detected) or after neovascularization is detected (e.g., following tumor resection). To this end, the agent is administered before vessel formation is detected to protect, in whole or in part, against unwanted neovascularization. In other aspects, the agent is administered after angiogenesis has begun to prevent, in whole or in part, further unwanted blood vessel formation.

[0037] A particular administration regimen for a particular subject will depend, in part, upon the agent used, the amount of agent administered, the route of administration, and the cause and extent of any side effects. The amount of agent administered to a subject (e.g., a mammal, such as a human) in accordance with the invention should be sufficient to effect the desired response over a reasonable time frame. Dosage typically depends upon a variety of factors, including the particular agent employed, the age and body weight of the subject, as well as the existence or extent of any disease or disorder in the subject. The size of the dose also will be determined by the route, timing, and frequency of administration. Accordingly, the clinician titers the dosage and modifies the route of administration to obtain the optimal therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art. Purely by way of illustration, the inventive method comprises administering, e.g., from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage ranges from 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg; or 10 μg/kg up to about 100 mg/kg. Some conditions or disease states require prolonged treatment, which may or may not entail administering lower doses of agent over multiple administrations.

[0038] Suitable methods of administering a physiologically-acceptable composition, such as a pharmaceutical composition comprising the agent of the invention, are well known in the art. Although more than one route can be used to administer an agent, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition comprising the agent is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation. For example, in certain circumstances, it will be desirable to deliver a pharmaceutical composition comprising the agent orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems, or by implantation devices. If desired, the agent is administered regionally via intraarterial or intravenous administration feeding the region of interest, e.g., via the hepatic artery for delivery to the liver. Alternatively, the composition is administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device is, one aspect, implanted into any suitable tissue or organ, and delivery of the desired molecule is, for example, via diffusion, timed-release bolus, or continuous administration. In other aspects, the agent is administered directly to exposed tissue during tumor resection or other surgical procedures. Therapeutic delivery approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,399,363.

[0039] To facilitate administration, the agent is, in various aspects, formulated into a physiologically-acceptable composition comprising a carrier (i.e., vehicle, adjuvant, or diluent). The particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. Physiologically-acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). Injectable formulations are further described in, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers. eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). A pharmaceutical composition comprising an agent of the invention is, in one aspect, placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.

Combination Therapy

[0040] When appropriate, the agent is administered in combination with other substances (e.g., therapeutics) and/or other therapeutic modalities to achieve an additional (or augmented) biological effect. These other therapeutics/co-treatments include, for example, radiation treatment, hyperthermia, surgical resection, chemotherapy, additional agents that inhibit fibrillogenesis, other anti-angiogenic factors (for instance, soluble growth factor receptors (e.g., sflt), growth factor antagonists (e.g., angiotensin), etc.), antibiotics, hormone therapy, anti-inflammatory agents (e.g., Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or steroidal anti-inflammatory substances), pain relievers, and the like.

[0041] The invention thus includes administering to a subject an agent (or multiple agents) that interferes with Fn matrix assembly, in combination with one or more additionally suitable substances(s), each being administered according to a regimen suitable for that medicament. This aspect includes concurrent administration (i.e., substantially simultaneous administration) and non-concurrent administration (i.e., administration at different times, in any order, whether overlapping or not) of the agent and one or more additionally suitable agents(s). It will be appreciated that different components are, in certain aspects, administered in the same or in separate compositions, and by the same or different routes of administration.

[0042] Chemotherapy treatment for use in conjunction with the invention employ anti-neoplastic agents including, but not limited to, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), and hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; pipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinium coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

[0043] Exemplary cytokines or hematopoietic factors for use in conjunction with the invention include, but are not limited to, Interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), angiopoietins, for example Ang-1, Ang-2, Ang-4, Ang-Y, the human angiopoietin-like polypeptides ANGPTL1 through 7, vitronectin, vascular endothelial growth factor (VEGF), angiogenin, activin A, activin B, activin C, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, bone morphogenic protein receptor II, brain derived neurotrophic factor, cardiotrophin-1, ciliary neutrophic factor, ciliary neutrophic factor receptor, cripto, cryptic, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, β endothelial cell growth factor, endothelin 1, epidermal growth factor, epigen, epiregulin, epithelial-derived neutrophil attractant, fibroblast growth factor (FGF) 4, FGF 5, FGF 6, FGF 7, FGF 8, FGF 8b, FGF 8c, FGF 9, FGF 10, FGF 11, FGF 12, FGF 13, FGF 16, FGF 17, FGF 19, FGF 20, FGF 21, FGF acidic, FGF basic, glial cell line-derived neutrophic factor receptor α1, glial cell line-derived neutrophic factor receptor α2, growth related protein, growth related protein α, growth related protein β, growth related protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, hepatoma-derived growth factor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor a, nerve growth factor, nerve growth factor receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin M (OSM), placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor α, platelet derived growth factor receptor β, pre-B cell growth stimulating factor, stem cell factor (SCF), stem cell factor receptor, tumor necrosis factor (TNF), including TNF0, TNF1, TNF2, transforming growth factor (TGF) α, TGF β, TGF β1, TGF β1.2, TGF β2, TGF β3, TGF β5, latent transforming growth factor β1, TGF β binding protein 1, TGF β binding protein II, TGF β binding protein III, thymic stromal lymphopoietin (TSLP), TNF receptor type I, TNF type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof.

[0044] Additional combination therapies not specifically listed herein are also within the scope of the present invention.

Other Considerations

[0045] It will be appreciated that the materials and methods of the invention are used to treat a number of diseases associated with deregulated or undesired angiogenesis. Such diseases include, but are not limited to, ocular neovascularization, such as retinopathies (e.g., diabetic retinopathy, age-related macular degeneration, choroidal neovascularization, and the like); psoriasis; hemangioblastoma; hemangioma; arteriosclerosis; inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, e.g., arthritis (including rheumatoid arthritis); arterial or post-transplantational atherosclerosis; endometriosis; and neoplastic diseases, e.g., solid tumors and liquid tumors (such as leukemias).

[0046] Many neovascularization-related disease states are monitored using the methods described herein to determine the degree of neovascularization in a subject. When the invention is used to inhibit neovascularization associated with tumor growth, several additional parameters are measured to determine the efficacy of the method to, e.g., alleviate tumor progression in a subject. The proper combination of parameters for a particular situation is established by the clinician. Tumor size is figured, for instance, by measuring tumor dimensions or estimating tumor volume using available computer software, such as FreeFlight software developed at Wake Forest University that enables accurate estimation of tumor volume. Tumor size also can be determined by tumor visualization using, for example, CT, ultrasound, SPECT, spiral CT, MRI, photographs, and the like. Measurement of tumor size, detection of new tumors, tumor antigens, or markers (e.g., CEA, PSA, or CA-125), biopsy, surgical downstaging, PET scans, and the like, can point to the overall progression (or regression) of cancer in a human. Biopsy is particularly useful in detecting a reduction of neovascularization within a tissue.

EXAMPLES

[0047] The invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention.

Example 1

[0048] This Example illustrates the ability of Fn polymerization inhibitors to interfere with Fn matrix formation, cell proliferation, cell migration, and tubulogenesis in three-dimensional cell culture, which simulates in vivo conditions of neovascularization.

[0049] Endothelial cells were isolated from human umbilical cord veins by collagenase digestion and cultured in Medium 199 (Gibco) containing 20% human serum, 50 μg/ml endothelial cell growth supplement (BD Biosciences), 100 U/ml penicillin, and 100 μg/ml streptomycin (Hiraoka et al. 1998. Cell, 95(3): 365-377). For 2-D/3-D culture, endothelial cell monolayers (no later than third passage) were suspended by mild trypsinization and dispersed within or plated atop fibrin (3 mg/ml) or collagen (2.2 mg/ml) gels (prepared as described in Hiraoka, supra, and Hotary et al. 2003. Cell, 114(1): 33-45), and stimulated with a cocktail of growth factors including 100 ng/ml human vascular endothelial growth factor (Genentech), 50 ng/ml human hepatocyte growth factor (Genentech), 10 ng/ml human TGFα (Biosource), 0.5 ng/ml TGFβ1 (R and D Systems), and 100 μg/ml heparin (Sigma). In selected experiments, endothelial cell spheroids were prepared and suspended in 3-D fibrin gels.

[0050] When embedded in a 3-D gel of cross-linked fibrin and stimulated with a cocktail of pro-angiogenic factors in serum-containing media, human endothelial cells assumed a spherical configuration during the first 8-12 hours of culture. No increase in cell number was detected until after 48 hours in culture, whereafter the embedded cells displayed a stretched phenotype. Endothelial cell number subsequently increased after the 2 day lag period, and a tubulogenic program was engaged which led to the formation of an anastomosing network of patent neovessels by day 6. As observed in vivo (Clark et al. 1982. J. Exp. Med., 156(2): 646-651; Risau and Lemmon. 1988. Dev. Biol., 125(2): 441-450; and Neri and Bicknell. 2005. Nat. Rev. Cancer, 5(6): 436-446), endothelial cell morphogenesis occurred in tandem with the assembly of a network of Fn fibrils that not only enmesh the stretched endothelial cells observed at 48 hours, but also ensheath the tubules formed at the end of the 6 day culture period. Thus, three-dimensional cell culture simulates in vivo conditions that allow new blood vessel formation.

[0051] The effect of Fn matrix assembly, or lack thereof, on endothelial cell function during neovascularization was assessed. Human serum was depleted of Fn by gelatin-sepharose affinity chromatography (Amersham) and supplemented with either 20 μg/ml of human plasma Fn (Sigma) or FITC-labeled Fn. Endothelial cells were incubated in 3-D cell culture with:

[0052] i) monoclonal antibodies L8 or 9D2 that are directed against Fn domains embedded within, or near, the Fn III_1,2 modules that are critical for regulating Fn-Fn interactions (final concentration of 100 μg/ml);

[0053] ii) a 70 kDa amino-terminal Fn fragment that interferes with the polymerization of intact Fn dimers by competing for matrix assembly sites on the endothelial cell surface (Sigma; 75 μg/ml);

[0054] iii) FUD, which binds directly to the N-terminal matrix assembly domain of Fn, or Del29, wherein FUD residue 29 is deleted to abrogate Fn binding (250 nM);

[0055] iv) mouse IgG (control; Pierce); Blebbistatin (Calbiochem; 50 μM); or cytochalasin D (Sigma; 10 μM)

(Chemousov et al. 1991. J. Biol. Chem., 266(17): 10851-10858; Tomasini-Johansson et al. 2001. J. Biol. Chem., 276(26): 23430-23439; Mao and Schwarzbauer. 2005. Matrix Biol., 24(6): 389-399; and Tomasini-Johansson et al. 2006. Matrix Biol., 25(5): 282-293). The inhibitors block Fn matrix assembly without affecting the initial binding of soluble Fn binding to α5β1. Cell number in 3-D cultures was determined by hemacytometry after dissolving gels with 2 mg/ml bacterial collagenase (Worthington) while the number of patent tubules was determined in randomly selected cross-sections. Fn matrix assembly and cell morphology were monitored by confocal laser microscopy. To examine cell migration, endothelial cells were embedded in a 100 μl fibrin gel for 2 hours, then placed within a 500 μl gel for 8 days in the presence of Del29, FUD, control IgG, or L8, after which migration from the inner gel was assessed.

[0056] Fn fibrillogenesis inhibitors completely blocked the ability of fibrin-embedded endothelial cells to assemble a Fn matrix. For example, addition of FUD to incubating cells attenuated Fn polymerization, while incubation with Del29 did not negatively impact Fn matrix formation. In the absence of Fn fibrillogenesis--and despite the presence of a surrounding 3-D fibronectin matrix, serum, and exogenously provided pro-angiogenic growth factors--the endothelial cells were unable to undergo the expected shape change and retain a spherical morphology. Coincident with the block in Fn matrix deposition, the 3-D migratory and proliferative responses of embedded endothelial cells were blunted significantly, and tubulogenesis was effectively terminated. Because Fn/α5β1 interactions were left intact, no increase in apoptosis (as assessed by TUNEL staining) was detected in the absence of Fn fibrillogenesis.

[0057] The ability of Fn matrix inhibitors to block neovessel formation is not restricted to the specific use of fibrin gel suspension system. Endothelial cells spheroids were embedded in gels of 3-D fibrin or type 1 collagen matrix and cultured for 6 days in the absence or presence of mAb L8 (100 μg/ul). Tubulogenesis was assessed by phase contrast microscopy, and the assembly of a FITC-labeled Fn matrix was monitored by confocal laser microscopy. Similar, if not identical, results were obtained when neovessel formation was initiated with spheroids of endothelial cells embedded in 3-D fibrin gels (Korff and Augustin. 1998. J. Cell Biol., 143(5): 1341-1352) or alternatively, when type I collagen was used as the supporting matrix.

[0058] This example demonstrates methods to inhibit neovascularization. Endothelial cell responses required for neovascularization were blunted, if not completely prevented, by inhibition of Fn matrix formation.

Example 2

[0059] This Example further demonstrates the effect of inhibiting Fn polymerization on cellular processes associated with angiogenesis.

Blocking Fn Polymerization Interrupts Endothelial Cell Cytoskeletal Organization

[0060] Changes in cell geometry impact the signaling cascades that control cell migration, proliferation, and morphogenesis (Chen et al. 1997. Science, 276(5317): 1425-1428; Tan et al. 2003. Proc. Natl. Acad. Sci. USA, 100(4): 1484-1489; McBeath et al. 2004. Dev. Cell, 6(4): 483-495; and Ingber. 2006. FASEB J., 20(7): 811-827). In vivo, integrins and growth factors collaborate in the activation of MAPK pathways which regulate the angiogenic response (Eliceiri et al. 1998. J. Cell Biol., 140(5): 1255-1263; Geiger et al. 2001. Nat. Rev. Mol. Cell. Biol., 2(11): 793-805; Huang et al. 2004. Proc. Natl. Acad. Sci. USA, 101(7): 1874-1879; and Ingber, supra). To determine the degree to which endothelial cell responses to growth factor and integrin-ligand signals are linked to Fn matrix assembly, the phosphorylation of the MAP kinases, ERK1 and 2, JNK, and p38 were monitored in the absence or presence of fibrillogenesis inhibitors during the 48 hour period that precedes proliferative responses.

[0061] Levels of phosphorylated ERK1/2, JNK, and p38 were determined by immunoblot analysis in lysates of endothelial cells cultured in fibrin gels in the presence of either control IgG or mAb L8 for 0 hours, 2 hours, 1 day, or 2 days. Total ERK1/2 served as the loading control. In control cultures, sustained MAP kinase activation is maintained throughout the 48 hours incubation period in a fashion that recapitulates the in vivo setting (Eliceiri, supra; and Corson et al. 2003. Development, 130(19): 4527-4527). However, independent of the marked changes in endothelial cell morphology and cytoskeletal organization associated with the inhibition of Fn fibrillogenesis, phosphorylation patterns of ERK1/2, JNK, and p38 were largely unaffected.

[0062] Despite the comparable initiation of signal transduction cascades in endothelial cells competent or incompetent for Fn matrix assembly, cell responses to integrin and growth factor-mediated signals also are dictated by the organization of actin cytoskeletal architecture (Chen, supra; Huang, supra; Ingber, supra; and Bershadsky et al. 2006. Curr. Opin. Cell Biol., 18(5): 472-481). To further study the effects of Fn matrix inhibition, additional cytological processes associated with angiogenesis were examined. Endothelial cells were cultured within 3-D or 2-D fibrin gels in the presence of the FUD or Del 29 peptides for 2 days. F-actin cytoskeletal organization was monitored following staining with Alexa 488-conjugated phalloidin. Cells also were stained with an antibody against activated β1 integrin or transfected with a GFP-tagged vinculin expression vector (pRK-vinculin-EGFP) to study distribution. Following counterstaining with Alexa 594-labeled phalloidin, fluorescence was monitored by confocal laser microscopy.

[0063] In tandem with the ability of growth factor-stimulated endothelial cells to adopt an elongated phenotype in 3-D culture, a reticulated pattern of well-organized stress fibers was resolved by F-actin phalloidin staining when cells were cultured in the presence of the Del29 control peptide. In 3-D culture, stress fibers terminate at specialized β1 integrin- and vinculin-rich sites of cell-matrix interactions termed 3-D adhesions (Geiger, supra; Larsen et al. 2006. Curr. Opin. Cell Biol., 18(5): 463-471). As such, endothelial cells transduced with a GFP-tagged vinculin expression vector or alternatively immunostained with an activated β1 integrin-specific monoclonal antibody, established both vinculin and activated β1 integrins into stitch-like structures at the endothelial cell periphery. In the absence of Fn fibrillogenesis, however, stress fiber formation was suppressed completely, and actin staining was confined to the cortical envelope in a punctate network. Further, specific interactions between either activated β1 integrins or vinculin and F-actin networks could no longer be discerned. Endothelial cells alternatively cultured atop fibrin matrices in a 2-D configuration assembled a well-organized stress fiber-focal adhesion network whose organization was unaffected by inhibitors of Fn fibrillogenesis.

Matrix Inhibition Reduces Cellular Tractional Forces and Gene Expression Dependent Thereon

[0064] Adhesive interactions between cells and their surrounding matrix allow for the generation of tractional forces that regulate cell fate and function (McBeath, supra; Discher et al. 2005. Science, 310(5751): 1139-1143; Engler et al. 2006. Cell, 126(4): 677-689; Larsen, supra; Vogel and Sheetz. 2006. Nat. Rev. Mol. Cell. Biol., 7(4): 265-275; and Yamada and Cukierman. 2007. Cell, 130(4): 601-610). The ability of embedded endothelial cells to generate tractional forces on the fibrin matrix was determined in the presence or absence of Fn fibrillogenesis inhibitors. In stressed ECM gels wherein cells are permitted to exert isometric tension, the degree of force exerted by cells on the surrounding fibrillar network can be assessed by monitoring gel contraction after the matrix is released from the surrounding culture dish (Corbett and Schwarzbauer. 1999. J. Biol. Chem., 274 (30): 20943-20948; Even-Ram et al. 2007. Nat. Cell Bio., 9 (3): 299-309). As such, 3-D fibrin gels were cast in individual wells of 24-well plates and cultured alone or in the presence of embedded endothelial cells for 2 days in the presence of control IgG, mAb L8, mAb 9D2, the 70 kDa Fn fragment, or the FUD peptide. Gels were detached from the edges of the culture wells and contraction was monitored after an additional incubation period of 10 hours at 37° C.

[0065] Growth factor-stimulated endothelial cells cultured in control gels for 48 hours were able to actively contract the released fibrin gel. By contrast, each of the Fn fibrillogenesis inhibitors markedly attenuated the ability of the embedded endothelial cells to generate tractional forces under 3-D, but not 2-D, culture conditions.

[0066] Tractional forces exerted at cell-matrix adhesion sites require the activation of an actinomyosin motor complex whose assembly is tightly linked to actin cytoskeleton organization, non-muscle myosin II isoform expression, and the rigidity of the surrounding substratum (Meshel et al. 2005. Nat. Cell Biol., 7(2): 157-164; Engler, supra; and Even-Ram, supra; Yoneda et al. 2007. Mol. Cell. Biol., 19(1): 66-75). β-actin, α-actinin, myosin light chain-2 (MLC2), and non-muscle myosin IIA and IIB isoform (NMMIIA and NMMIIB, respectively) protein levels were monitored in 3-D embedded endothelial cells to determine the effect of Fn matrix inhibition on the expression of gene products critical to the generation of tractional forces. Endothelial cells were cultured in 3-D fibrin gels for 2 days with either the FUD peptide or the control Del29 peptide. Levels of β-actin, α-actinin, NMMIIA, NMMIIB, and MLC2 were measured by Western blot, with ERK1/2 serving as the loading control. As assessed by semi-quantitative densitometry, the levels of β-actin, actinin and MLC2 were 58±7% (n=5; mean±1 SD), 62% (n=2), and 60±12% (n=3; mean±1 SD) of control, respectively. Significantly, whereas each cytoskeletal component is expressed in growth factor-stimulated endothelial cells actively assembling a Fn matrix, endothelial cells cultured in the presence of FUD or Ab L8 express markedly reduced levels of β-actin, α-actinin, and MLC2.

Intracellular Stiffness Decreases Upon Inhibition of Fn Fibrillogenesis

[0067] Endothelial cells cultured atop highly malleable surfaces retain a spherical configuration, fail to organize stress fibers, and are unable to exert tractional forces--a phenotype identical to that observed in 3-D-embedded, Fn matrix assembly-incompetent endothelial cells. A cell's internal stiffness is a viscoelastic property governed by cytoskeletal assembly, actin crosslinking, and the production of actomyosin-dependent stress. Internal stiffness changes as a function of the perceived stiffness of the surrounding substratum (Solon et al. 2007. Biophys. J., 93(12): 4453-4461). Therefore, the micromechanical properties of 3-D-embedded endothelial cells were monitored via intracellular nanorheology.

[0068] Prior to embedding in the 3-D fibrin matrix, endothelial cells were ballistically microinjected with 100 nm polystyrene beads to circumvent the endocytic pathway and subsequent directed motion of the beads. After 3 days of incubation, the beads dispersed in the cytoplasm and their centroids were tracked with high spatial and temporal resolution using fluorescence microscopy. Relative to control endothelial cells, the mean square displacement (MSD) of the beads was significantly increased in cells treated with the FUD peptide, indicating a significant relative cytoplasmic softening compared to that of cells where Fn matrix assembly is intact. Elastic moduli, which quantify the local resistance of the cytoplasm against small random forces acting on the surface of the beads, were derived from MSD curves to quantify cellular mechanical properties. The elastic modulus of the cytoplasm of FUD-treated cells is significantly lower than that of control cells (P<0.001), indicating a pronounced defect in internal stiffness and the cell's ability to sense a sufficiently rigid substratum.

[0069] In the absence of Fn fibrillogenesis, an impaired ability of embedded endothelial cells to generate myosin-dependent forces and increase cytoplasmic stiffness was predicted to affect both Fn unfolding, as well as the ability of the cells to properly register the mechanical properties of the surrounding substratum (Wu et al. 1995. Cell, 83(5): 715-724; Zhong et al. 1998. J. Cell Biol., 141(2): 539-551; Baneyx et al. 2002. Proc. Natl. Acad. Sci. USA, 99(8): 5139-5143; Discher et al. 2005. Science, 310(5751): 1139-1143; Engler, supra; and Yoneda, supra). As such, the rheologic and functional characteristics of endothelial cells were assessed in the presence of the specific myosin ATPase inhibitor, blebbistatin. Endothelial cells were cultured in 3-D fibrin gels for 2 days in the presence or absence of 50 μM±blebbistatin. F-actin organization and Fn matrix assembly was monitored by confocal laser microscopy and staining with Alexa 488-conjugated phalloidin.

[0070] Blebbistatin-treated endothelial cells phenocopied Fn matrix assembly-incompetent cells. In particular, endothelial cells treated with blebbistatin failed to increase cytoplasmic stiffness, failed to undergo cell shape change, failed to assemble a pericellular Fn matrix, and did not reorganize cytoskeletal architecture. Endothelial cell tubulogenesis was blocked completely. Hence, myosin ATPase activity and Fn matrix assembly each play required roles in regulating the endothelial cell's ability to match internal stiffness with that of the surrounding substratum so as to propagate the mechanotransduction-initiated signals critical to neovessel formation.

[0071] This Example illustrates the ability of agents of the inventive method to inhibit cell functions required for neovascularization.

Example 3

[0072] This Example illustrates the ability of the inventive method to inhibit neovascularization in vivo.

[0073] Inhibition of Fn matrix formation is a targeted approach to inhibiting unwanted neovascularization as Fn polymerization is relatively unique to neovessel formation. In this regard, Fn matrix assembly in the context of human tumor angiogenesis was assessed. Renal cell carcinoma (stages GI-III), breast carcinoma, and normal kidney cells were stained for UEA-1 or with mAb L8, which only recognizes unfolded Fn epitopes that are exposed during Fn fibrillogenesis (Chemousov et al. 1991. J. Biol. Chem., 266(17): 10851-10858; Zhong et al. 1998. J. Cell. Biol., 141(2): 539-551). Normal breast cells were stained for FVIIIRAg or with mAb L8. Immunostaining of a series of renal cell carcinomas and invasive ductal breast carcinomas demonstrated that vascular wall L8 immunoreactivity is dramatically increased in tissues undergoing active vascularization/angiogenesis. In both renal cell carcinoma and invasive ductal breast carcinoma specimens, all blood vessels and vascular channels were strongly L8-reactive with additional stromal staining seen in some cases of breast cancer. In normal tissues, immunoreactivity for the L8 Fn epitope was observed infrequently as small streaks in fewer than 10% of the vessels.

[0074] The functional role for fibrillogenesis in tissue sites undergoing active angiogenesis in vivo was assessed. To this end, 3-D composite gels of fibrin and type I collagen were placed atop the chorioallantoic membrane (CAM) of live chicks, and angiogenesis was initiated in the presence of FUD or the Del29 peptide control. In particular, 3-D matrices of type I collagen or a type I collagen/fibrin composite matrix were cast in transwell tissue culture inserts (24 well size) perforated with a 25 gauge needle. A 30 μl Matrigel (BD Biosciences) reservoir was placed atop the matrix containing 200 ng VEGF, 100 ng HGF, and either Del29 or FUD. The entire apparatus was placed atop the dropped CAM of 10-11 day old fertile chicken eggs. Following incubation in a humidified incubator at 37° for 3 days, the matrices were harvested. Vascular ingrowth was monitored by light microscopy following H&E staining. In some experiments, FITC-Fn (McKeown-Longo and Mosher. 1985. J. Cell. Biol., 100(2): 364-74) was supplemented in the matrices during the culture period, and Fn fibrillogenesis within the gels was monitored by confocal laser microscopy.

[0075] Under control conditions, angiogenic vessels infiltrated the extracellular matrix (ECM) construct in tandem with the deposition of a dense network of Fn fibrils. In the presence of FUD, however, Fn matrix assembly was almost completely inhibited and neovessel formation was ablated.

[0076] This Example, as well as the preceding and foregoing Examples, demonstrates that agents that impede Fn matrix formation inhibit neovascularization.

Example 4

[0077] This Example demonstrates that modulating cell shape by, e.g., inhibiting Fn polymerization, also modulates nuclear architecture and function.

Regulation of Nuclear Morphology by 3-D Extracellular Matrix (ECM) Remodeling

[0078] At sites of tissue damage, inflammation or neoplasia, fibrinogen is converted into a 3-D meshwork of fibrin fibrils that serve as a structural support for endothelial cells undergoing neovascularization (Chun et al. 2006. Cell, 125: 577-591; Hiraoka et al. 1998. Cell, 95: 365-377; Schafer and Werner. 2008. Nat. Rev. Mol. Cell. Biol., 9: 628-638; Zhou et al. 2008. Genes Dev., 22: 1231-1243). This process can be recapitulated in vitro by embedding serum-supplemented human endothelial cells within a 3-D fibrin gel in the presence of the pro-angiogenic factors, VEGF and HGF. Human umbilical vein endothelial cells were isolated from umbilical cords by perfusion of the umbilical vein with type 3 collagenase (Worthington, Lakewood, N.J.) and cultured in Medium-199 (Invitrogen, Carlsbad, Calif.) with 20% human serum and 50 μg/ml endothelial cell growth supplement (ECGS; BD Biosciences, Franklin Lakes, N.J.). For 3-D culture, endothelial cells were suspended in a solution of thrombin and aprotinin (Sigma, St. Louis, Mo.) and mixed 1:1 with a solution of 6 mg/ml fibrinogen (Calbiochem, Gibbstown, N.J.). Vasculogenesis was triggered by treatment with 100 ng/ml VEGF, 50 ng/ml HGF (Genentech, San Francisco, Calif.). Fibronectin fibrillogenesis was tracked in 3-D by co-culture with human fibronectin (Sigma) labeled with Alexa-594 (Invitrogen).

[0079] In the presence of VEGF and HGF, endothelial cells i) underwent marked changes in cell morphology from spherical to elongated, ii) mobilized proteinases which degrade the surrounding fibrin, iii) assembled a pericellular meshwork of fibronectin fibrils, and iv) initiated proliferative and tubulogenic responses that result in the formation of an anastomosing network of neovessels over a 6 day culture period (Hiraoka, supra; Saunders et al., 2006. J. Cell Biol. 175: 179-191; Zhou et al., 2008. Proc. Natl. Acad. Sci. USA, 97: 4052-4057). Growth factors, fibrinolytic membrane-type matrix metalloproteinases (MT-MMPs), and fibronectin fibril assembly are each required for the tubulogenic program; endothelial cells suspended in 3-D fibrin gels with serum-supplemented media failed to elongate, proliferate or form neovessels when i) the VEGF/HGF cocktail was omitted (hereafter termed as the baseline or unstimulated condition) or ii) VEGF/HGF-stimulated cells were cultured in the presence of GM6001 (Calbiochem), a synthetic MMP inhibitor, or FUD (Zhou, et al. supra), a fibronectin fibril assembly inhibitor (Chun et al., 2004. Cell, 125: 757-767; Saunders et al., supra; Tomasini-Johansson et al., 2001. J. Biol. Chem., 276: 23430-23439; Zhou et al, supra). Endothelial cells cultured atop--rather than embedded within--fibrin gels (herein referred to as 2-D culture) display distinct growth requirements from those observed in 3-D culture. In 2-D culture, neither GM6001 nor FUD affect the growth of VEGF/HGF-stimulated endothelial cells.

[0080] As the 3-D-specific requirements for MT-MMPs and fibronectin matrix assembly correlate with changes in endothelial cell morphology, efforts were initiated to identify mechanistic routes whereby cell shape changes impact cell function. In particular, the impact of ECM remodeling on nuclear architecture was investigated. Nuclear architecture was tracked by either transfecting cells with a GFP-tagged form of the nuclear matrix filament, lamin A, or by immunostaining for the integral nuclear membrane protein, emerin (Dahl et al., 2008. Circ. Res., 102: 1307-1318; Glynn and Glover, 2005. Hum. Mol. Genet., 14: 2959-2969; Starr, 2009. J. Cell Sci., 122: 577-586; Stewart et al., 2007. Science, 318: 1408-1412). Under 2-D conditions, endothelial cell nuclei assumed an ellipsoid shape when cultured atop fibrin gels in the absence or presence of VEGF/HGF. In marked contrast, under 3-D conditions, endothelial cells cultured within fibrin gels in the absence of VEGF/HGF unexpectedly display a distorted morphology with multiple lamin A matrix and emerin infoldings and surface irregularities. Upon addition of VEGF/HGF, however, the nuclei of fibrin-embedded endothelial cells undergo a dramatic, and 3-D-specific, remodeling to assume a more classic, ovoid morphology. Three-dimensional reconstructions of DAPI-stained nuclei confirm that nuclear architecture transitions between multi-lobed and smooth elliptical shapes in the absence or presence, respectively, of VEGF/HGF.

[0081] Nuclear matrix architecture controls the distribution of the nuclear pore complexes that regulate the trafficking of protein and RNA across the nuclear envelope (Goldman et al., 2004. Proc. Natl. Acad. Sci. USA, 101: 8963-8968; Hawryluk-Gara et al., 2005. Mol. Biol. Cell, 16: 2382-2394; Liu et al., 2007. J. Cell. Biol., 178: 785-798). As such, the localization of nuclear pores was assessed in intact cells by monitoring the localization of the component protein, NUP153. In 3-D culture, unstimulated endothelial cells accumulate NUP153-containing pore complexes at nuclear membrane invaginations. In contrast, the nuclei of VEGF/HGF-stimulated endothelial cells display a uniform distribution of pore complexes in nuclear membranes. Hence, nuclear shape and nuclear pore complex distribution are regulated in tandem by VEGF/HGF signaling in 3-D culture.

[0082] To determine the roles of MMP-dependent proteolysis and fibronectin matrix assembly in mediating nuclear shape and nuclear pore complex changes, fibrin-embedded endothelial cells were stimulated with VEGF/HGF in the presence of FUD or GM6001. Blocking fibronectin fibrillogenesis or MMP activity locked endothelial cell nuclei into the multi-lobular conformation characteristic of unstimulated 3-D-embedded endothelial cells. Further, fibrin-embedded endothelial cells stimulated with VEGF/HGF and treated with either GM6001 or FUD completely failed to redistribute nuclear pore complexes. These findings are not confined to fibrin matrices; similar results are obtained when endothelial cells are embedded within a 3-D ECM comprised of type I collagen fibrils, the major component of interstitial tissues. While abnormalities in nuclear envelope architecture can occur in cells undergoing apoptosis, the formation of multi-lobed nuclei does not correlate with increased TUNEL staining. The observed changes in nuclear shape occur only under 3-D culture conditions; the nuclear architecture of endothelial cells cultured under 2-D conditions is unaffected by inhibiting either MMP activity or fibronectin fibrillogenesis.

3-D ECM Remodeling Regulates Chromatin Structure

[0083] Perturbations in nuclear shape and nuclear pore complexes have not previously been demonstrated in endothelial cells or any other normal cell population. Yet, multi-lobed nuclei and anomalous nuclear pore complex distributions can be observed in laminopathies, a pleiotropic series of genetic disorders wherein mutations in lamin or emerin impact chromatin organization, histone modifications, and transcriptional activity (Csoka et al., 2004. Aging Cell, 3: 235-243; Dechat et al., 2008. Genes Dev, 22: 832-853; Dillon, 2008. Dev Cell, 15: 182-186; Malhas et al., 2007. J. Cell Biol., 176: 593-603; Mendjan et al., 2006. Mol. Cell, 21: 811-823; Shumaker et al., 2006. Proc. Natl. Acad. Sci. USA, 103: 8703-8708; Tang et al., 2008. J. Cell Sci., 121: 1014-1024). To determine whether VEGF/HGF-dependent changes in nuclear shape similarly affect chromatin structure/function, chromatin packaging was tracked at the single-endothelial cell level by monitoring GFP-tagged histone H2B or pericentromeric constitutive heterochromatin localization. H2B and heterochromatin localization was visualized by immunostaining for trimethylated lysine 9 of histone H3 (H3K9me3) (Shumaker, supra; Tang, supra).

[0084] In unstimulated endothelial cells embedded within 3-D fibrin gels, GFP-H2B and H3K9me3 localization revealed chromatin condensation at discrete peripheral locations in the nucleus. By contrast, following exposure to VEGF/HGF for 48 hours, chromatin was dramatically reorganized in elongated endothelial cells, assuming a diffuse distribution with a relative collapse of the interchromatin space. VEGF/HGF-stimulated global chromatin redistribution was completely inhibited by blocking MMP activity or pericellular fibronectin matrix assembly.

[0085] Histone acetylation can be regulated by chromatin positioning within the nucleus (Finian et al., 2008. PLoS Genet, 4: e1000039; Somech et al., 2005. J. Cell Sci., 118: 4017-4025). Thus, the acetylation status of histones H3 and H4 was assessed as functional markers of changes in chromatin organization. Stimulation of endothelial cells in 3-D with VEGF/HGF triggered a significant increase in acetylated histone H3 and H4, changes which are attenuated significantly by blocking MMP activity or fibronectin fibrillogenesis. Under 2-D culture conditions, however, ECM remodeling played no significant role in regulating endothelial cell chromatin organization. Taken together, these data support a model wherein ECM remodeling regulates chromatin organization and structure in a 3-D-specific manner.

[0086] The morphology of nucleoli and nuclear speckles--which serve, respectively, as ribosomal RNA and pre-mRNA processing sites--was assessed by immunostaining for fibrillarin (a pre-rRNA processing protein) or SC-35 (a pre-mRNA splicing protein) (Tang et al., supra). Activating endothelial cells with VEGF/HGF in 3-D resulted in the reorganization of fibrillarin from a single, centrally-located cluster of nucleoli within the interchromatin space, to multiple foci dispersed throughout the nucleus. Likewise, nuclear speckle morphology changed from a small number of large foci to more numerous, smaller foci. In both cases, the patterns of fibrillarin and SC-35 staining observed in VEGF/HGF-stimulated endothelial cells in 3-D culture could be reversed to that resembling unstimulated endothelial cells by blocking MMP activity or fibronectin matrix assembly. By contrast, VEGF/HGF, FUD, and GM6001 did not affect fibrillarin or SC-35 distribution under 2-D culture conditions.

[0087] VEGF/HGF-induced chromatin dispersion, histone acetylation, and nuclear speckle decompaction observed in 3-D culture are consistent with global activation of transcription (Lamond and Spector, 2003. Nat. Rev. Mil. Cell Biol., 4: 605-612; Tang et al., supra). As such, RNA synthesis was quantified in cultured endothelial cells. VEGF/HGF treatment resulted in a marked induction of transcriptional activity, a process attenuated significantly when ECM remodeling events are blocked by either FUD or GM6001. Using low dose actinomysin D to inhibit selectively rRNA synthesis (Ben-Ze'ev et al., 1980. Cell, 21: 365-372), mRNA and rRNA synthesis were observed to be inhibited by ˜50% (data not shown). Under 2-D culture conditions, transcriptional activity proceeded independently of pericellular ECM remodeling and was unaffected by FUD or GM6001. Hence, 3-D ECM remodeling is a regulator of nuclear organization as well as chromatin structure and function.

[0088] This Example demonstrates that ECM-regulated cell shape changes control neovessel morphogenesis by directly impacting nuclear architecture and function. The results show that 3-D-embedded, unstimulated endothelial cells display convoluted nuclei with multiple lobulations and surface invaginations containing nuclear pore complex aggregates. It does not appear that these unusual shapes have previously been described in any normal cell population. Nevertheless, following stimulation with VEGF/HGF, MMP-dependent proteolysis and fibronectin fibrillogenesis allowed endothelial cells to re-sculpt nuclear architecture to generate elliptical nuclei marked by a uniformly distributed array of nuclear pore complexes. By contrast, under standard, 2-D culture conditions atop mechanically rigid, adhesive substrata, cell shape changes--and consequent nuclear shape changes--are not constrained by an encasing 3-D ECM.

[0089] In addition, the observed multi-lobed nuclei and distorted nuclear pore distribution bore striking resemblance to the nuclear shapes observed in cells recovered from human patients bearing mutations in the lamin A/C, emerin or nesprin genes; a family of degenerative diseases termed the laminopathies (Crisp and Burke. 2008. FEBS Lett., 582: 2023-2032; Dechat et al. 2008. Genes Dev., 22: 832-853; Goldman et al. 2004. Proc. Natl. Acad. Sci. USA, 101: 8963-8968; Holaska. 2008. Circ. Res., 103: 16-23). Studies of cells isolated from laminopathy patients or mouse models of human laminopathies, as well as cells engineered to silence lamin expression have demonstrated that disrupted nuclear matrix architecture induces spatial and functional reorganization of the genome, resulting in global alterations in transcription and consequent effects on cell function (Columbaro et al. 2005. Cell Mol. Life. Sci., 62: 2669-2678; Csoka et al. 2004. Aging Cell, 3: 235-243; Dechat, supra; Malhas et al. 2007. J. Cell Biol., 176: 593-603; Meaburn et al. 2007. Aging Cell, 6: 139-153; Shimi et al. 2008. Genes Dev., 22: 3409-3421; Shumaker et al. 2006. Proc. Natl. Acad. Sci. USA, 103: 8703-8708; Tang et al. 2008. J. Cell Sci., 121: 1014-1024). While the specific functional impact of mutations in nuclear envelope proteins on cell function have not been considered to be of necessary relevance to normal cell behavior, the structural changes we observed in endothelial cell nuclei in 3-D culture led us to hypothesize that chromatin reorganization might purposefully accompany the nuclear envelope restructuring that occurs during capillary morphogenesis. Consistent with the proposition that chromatin organization is a critical regulator of endothelial cell function (Haberland et al. 2009. Nat. Rev. Genet., 10: 32-42), VEGF/HGF stimulation induces the translocation of endothelial cell chromatin from a condensed peripheral location to a dispersed, uniform distribution with coincident induction of histone acetylation. Remarkably, these changes in chromatin organization were inhibited almost completely by blocking fibronectin matrix- or MMP-dependent cell shape changes. The observed correlation of reduced histone acetylation with peripheral chromatin distributions is in accord with the observation that peripheral locations in the nuclear envelope contain histone deacetylase activity. Furthermore, changes in nuclear architecture coupled with morphological alterations in nucleoli and nuclear speckles were indicative of significant reductions in ribosomal RNA synthesis and pre-mRNA metabolism, thus connecting ECM-regulated changes in nuclear structure to genome function.

Example 5

[0090] This Example demonstrates that signals generated by cell shape changes mediated by ECM remodeling are transduced to the nucleus via intracellular filaments that tether the cell-ECM interface and the nuclear interior.

Cytoskeleton as a Transducer of ECM Structural Dynamics to the Nuclear Envelope

[0091] Cytoskeletal architecture and tension are closely coupled to cell geometry (Huang et al., 1998. Mol. Biol. Cell, 9: 3179-3193; Nelson et al., 2004. Mol. Biol. Cell, 15: 2943-2953; Tan et al., 2003. Proc. Natl. Acad. Sci. USA, 100: 1484-1489; Wozniak and Chen, 2009. Nat. Rev. Mol. Cell. Biol., 10: 34-43), and can influence nuclear envelope shape (Munter et al., 2006. BMC Cell. Biol., 7: 23; Roca-Cusachs et al., 2008. Biophys. J., 94: 4984-4995; Sarria et al., 1994. J. Cell Sci., 107 (Pt 6): 1593-1607). Hence, cytoskeletal organization in 3-D culture was assessed by examining F-actin, β-tubulin, and vimentin distribution. Treatment of 3-D-embedded endothelial cells with VEGF/HGF induced marked cytoskeletal reorganization, with F-actin, β-tubulin and vimentin redistributing from a diffuse, cortical pattern to a longitudinal fibrous network in tandem with an increase in isometric tension. In contrast, VEGF/HGF-triggered cytoskeletal remodeling and force induction in 3-D culture are inhibited completely by abrogating MMP activity or fibronectin fibrillogenesis, effects that were not observed under 2-D culture conditions.

[0092] Since ECM rigidity regulates cytoskeletal remodeling and force generation, and consequently cell function (Engler et al., 2006. Cell, 126: 677-689; Wozniak and Chen, supra; Zajac and Discher, 2008. Curr. Opin. Cell Biol., 20: 609-615), atomic force microscopy micro-indentation (AFM) was employed to quantify endothelial cell-dependent changes in ECM remodeling. AFM was performed by first washing fibrin gels with phosphate buffered saline (PBS) after removing culture medium. Samples were mechanically characterized using an Asylum MFP-3D atomic force microscope (Asylum Research, Santa Barbara, Calif.). Micro-indentation was performed using a sphere-tipped probe (Novascan, Ames, Iowa) with a sphere diameter of 5 μm and a nominal spring constant of ˜60 pN/nm. The cantilever spring constant was confirmed by thermal fluctuation method (Thundat et al., 1994. Applied Physics Letters, 64: 2894-2896). The AFM system was calibrated by following the manufacturer's recommended procedure before each indentation measurement. AFM micro-indentation was performed in PBS solution at room temperature. Individual force-indentation profile was acquired at an indentation rate of 2 μm/s using deflection trigger mode with a trigger value of 200 nm. The AFM tip was positioned either adjacent to or away from a cell. Shear modulus at each position was calculated from fitting force-indentation data using a Hertz sphere model (Richert et al., 2004, Biomacromolecules, 5. 1908-1916). In the presence of FUD or GM6001, VEGF/HGF-stimulated endothelial cells exhibit significantly lower 3-D pericellular rigidity, consistent with a required role for ECM remodeling events in the regulation of intracellular tension.

[0093] Taken together, cytoskeletal reorganization and contractile tension may serve as the biomechanical effectors that transmit structural and mechanical cues from the pericellular ECM to the nuclear envelope. VEGF/HGF-stimulated endothelial cells were treated with either (a) blebbistatin to inhibit myosin ATPase function, or (b) nocodazole to prevent microtubule assembly under 3-D culture conditions (Salpingidou et al., 2007. J. Cell Biol., 178: 897-904; Zhou et al., supra). Both agents completely inhibit endothelial cell 3-D tubulogenesis while impairing the contractile force exerted on the 3-D ECM. The data indicate that endothelial cell force generation is inhibited to a degree similar to that observed with GM6001 or FUD treatment. Further, compared with the ellipsoid nuclear shapes observed in VEGF/HGF-stimulated endothelial cells in 3-D culture, endothelial cells cultured in the presence of blebbistatin or nocodazole displayed i) multi-lobed nuclei with perturbations in nuclear pore distribution, ii) chromatin condensations at the nuclear periphery with increased interchromatin space and iii) decreased levels of acetylated histones H3 and H4. In toto, these results demonstrate that the ECM-dependent regulation of cytoskeletal organization is a critical determinant of nuclear as well as chromatin architecture.

Nesprins Regulate 3-D Organization of the Nuclear Compartment

[0094] Physical interactions between cytoskeletal networks and the nucleus are mediated by a family of Klarsicht, ANC-1, Syne homology (KASH) domain-containing proteins. The C-terminal domains of the proteins are embedded within the outer nuclear membrane, where they interact with the inner nuclear membrane via members of the SUN protein family (Crisp et al., 2006. J. Cell Biol., 172: 41-53; Dechat et al., supra; Starr, supra; Stewart et al., supra; Wilhelmsen et al., 2006. J. Cell Biol., 171: 799-810). In turn, SUN proteins span the inner nuclear membrane to establish binding interactions with a scaffold of lamin family members and nuclear pore complexes (Dechat et al., supra; Starr, supra; Stewart et al., supra). To determine whether this interaction plays a role in the nuclear morphology regulation by ECM remodeling, expression of nesprins-1 and 2 (also termed Syne-1 and 2) nesprin-3 was examined in 3-D-embedded endothelial cells. Nesprins-1 and 2 bind F-actin, while nesprin-3 indirectly interacts with intermediate filaments via binding of plectin (Crisp and Burke, 2008. FEBS Lett., 582: 2023-2032; Starr, supra; Stewart et al., supra).

[0095] Endothelial cells were observed to express nesprins 1-3 at both the mRNA and protein levels. The nuclear envelope of VEGF/HGF-treated endothelial cells embedded in 3-D fibrin gels displayed uniform nesprin distribution. In contrast, unstimulated endothelial cells, as well as VEGF/HGF-stimulated endothelial cells treated with GM6001 or FUD, displayed irregular nesprin distributions, with nesprin aggregates accumulating at nuclear membrane invaginations.

[0096] Physical interactions between the cytoskeleton, nesprins, SUN proteins and lamins likely dictate nuclear structure because endothelial cells remodel their surrounding ECM as a means to organize cytoskeletal structure (Zhou et al., supra). Hence, the cytoskeleton/nesprin continuum was perturbed by expressing the dominant negative GFP-KASH. GFP-KASH acts as a truncated nesprin protein that binds to SUN proteins without interacting directly with cytoskeletal elements (Crisp and Burke, supra; Crisp et al., supra; Stewart-Hutchinson et al., 2008. Exp. Cell Res., 314: 1892-1905). Endothelial cells were transduced with amphotropic retroviruses encoding GFP-KASH (provided by B. Burke, University of Florida) in the presence of 50 ng/ml VEGF and 6 μg/ml polybrene (Sigma, St. Louis, Mo.). Compared to VEGF/HGF-stimulated endothelial cells cultured in 3-D, endothelial cells expressing GFP-KASH failed to trigger nuclear remodeling and adopt the multi-lobed nuclear shape characteristic of unstimulated endothelial cells. Further, GFP-KASH expression induced peripheral chromatin condensation and perturbed the distribution of nucleoli and nuclear speckles, while inducing marked reductions in acetylated histone H3 or H4 levels. Consistent with nuclear organization regulating endothelial cell function, KASH-expressing cells were unable to participate in a normal tubulogenic response.

Coupling of Cell Geometry with Nuclear Structure/Function

[0097] If ECM-dependent changes in 3-D cell geometry regulate nuclear architecture, then direct modulation of endothelial cell shape would be predicted to impact nuclear organization and chromatin structure. To test this hypothesis, endothelial cells were cultured within 3-D, biomimetic poly(ethylene glycol) (PEG) hydrogels containing RGD peptides incorporated pendantly within a transglutaminase-crosslinked structure engineered to be either susceptible, or resistant, to MMP-mediated degradation (Ehrbar et al., 2007. Biomacromolecules, 8: 3000-3007; Raeber et al., 2007. Acta Biomater., 3: 615-629). In this manner, endothelial cell spreading is controlled as a function of the susceptibility of the 3-D hydrogel to proteolytic remodeling.

[0098] MMP-resistant or MMP-degradable PEG hydrogels were formed by FXIIIa catalyzed reaction as described in Ehrbar et al., supra. Briefly, 8-arm PEG-Gln and PEG-Lys were blended to generate stoichiometrically balanced ([Gln]/[Lys]) PEG precursor solutions. The PEG-Lys component was either chosen to contain a linker peptide that is susceptible (GPQG/IWGQ, with/indicating the cleavage site (SEQ ID NO: 13)) or resistant (GDQGIAGF (SEQ ID NO: 14)) to MMP-mediated degradation. The PEG precursor solutions (1.5, 2.0 and 2% w/v) were cross-linked upon addition of 10 U/mL FXIIIa in presence of 5 0 mM TrisHCl, pH 7.6, 50 mM CaCl₂, 50 μM Lys-RGD (Ac-FKGGRGDSPG-NH2 (SEQ ID NO: 15), NeoMPS Strasbourg, France) and cells suspended in medium 12% (v/v) of the total volume. To form hydrogel discs, 20 μL drops of the still liquid reaction mixture were sandwiched between sterile, hydrophobic glass microscopy slides that were separated by 1 mm spacers and clamped with binder clips. Polymerization was then allowed to take place for 30 minutes at 37° C. in a humidified incubator. To visualize the matrix, 25 μM Lys-FITC (Ac-FKGGGK-FITC-NH2 (SEQ ID NO: 16), NeoMPS Strasbourg, France) was added prior to reaction, leading to a homogenous covalent tethering of FITC to the matrix.

[0099] Within MMP-sensitive gels, endothelial cells were capable of spreading in response to VEGF/HGF. In MMP-resistant gels, endothelial cells remained locked in a spherical shape. Elongated endothelial cells embedded within MMP-sensitive scaffolds displayed oval nuclei observed by GFP-lamin tracking, while spherical cells embedded in MMP-resistant scaffolds exhibit multi-lobed nuclei. Furthermore, genome packaging was regulated as a function of cell shape in 3-D culture with chromatin condensations directed to the nuclear periphery in MMP-resistant hydrogels.

[0100] Alternatively, endothelial shape was modulated independently of proteolysis by culturing cells atop micropatterned fibronectin islands printed onto polymethylsiloxane substrates to generate ECM-adhesive patches surrounded by regions blocked with non-adhesive, Pluronic F108 (Chen et al., 1997. Science, 276: 1425-1428; McBeath et al., 2004. Dev. Cell, 6: 483-495). Microcontact printing techniques were used to fabricate substrates patterned with regions that were coated with fibronectin and regions that resisted such adsorption (Singhvi et al., 1994. Science, 264: 696-698). 1225 μm² islands (35 μm×35 μm) were used to constrain cell spreading, while continuous surfaces of fibronectin allowed for full spreading. Briefly, PDMS stamps bearing the relevant pattern of islands were washed with ethanol, and dried. The stamps were then immersed for 1 hour in an aqueous solution of 25 mg/ml fibronectin, rinsed thoroughly in water, dried, and placed in conformal contact against the culture substrate, blocked with 0.2% Pluronic F127 (BASF), and used under standard culture conditions.

[0101] When endothelial cells are cultured atop microprinted surfaces homogeneously coated with monomeric fibronectin that fully support adhesion and spreading, actin stress fibers were formed and the nucleus assumed a spheroid shape with an ordered distribution of nuclear pore complexes. In contrast, when endothelial cells were plated atop 35×35 μm fibronectin islands permissive for cell adhesion, but not spreading, a cell shape-dependent perturbed regulation of nuclear and chromatin structure was observed (Chen et al., supra; McBeath et al., supra). Hence, ECM-regulated changes in cell geometry directly determine nuclear shape and architecture as well as chromatin structure.

[0102] This Example confirms that the cytoskeletal apparatus acts as a component of the continuous network of protein:protein interactions that link the ECM to the nucleus. The cytoskeletal apparatus tethers cell:ECM adhesion complexes at the cell surface with transmembrane receptors at the nuclear envelope (Dahl et al. 2008. Circ. Res., 102: 1307-1318; Nelson and Bissell. 2006. Annu. Rev. Cell Dev. Biol., 22: 287-309; Starr. 2009. J. Cell Sci., 122: 577-586). Indeed, consistent with the ability of cytoskeletal filament structure to regulate nuclear envelope architecture, VEGF/HGF-induced changes in cytoskeletal organization and pericellular ECM rigidity were lost in the absence of fibronectin fibril deposition or MMP activity. Apparently, ECM remodeling, actin and microtubule assembly as well as myosin-II activity each play a required role adjusting the stiffness of the 3-D microenvironment in a fashion that supports the changes in cell shape and nuclear architecture required for neovessel formation.

[0103] When examining the status of the LINC complex in the 3-D system, nesprin-nuclear envelope distribution was observed to be regulated by the endothelial cell's ability to remodel the 3-D ECM. Furthermore, by uncoupling cytoskeleton-nuclear interactions with the dominant-negative LINC complex protein, GFP-KASH, multi-lobed nuclear shapes appeared in tandem with peripheral chromatin condensation and a reduction in histone acetylation.

[0104] The data of Examples 4 and 5 describe a novel functional mechanism wherein cell shape--modulated as a function of ECM remodeling--controls nuclear envelope organization via the regulation of cytoskeletal architecture and tension. Indeed, by controlling cell metamorphosis in 3-D PEG hydrogels or atop 2-D fibronectin islands that artificially restrain the cell shape changes critical to the generation of tractional forces (Tan et al., 2003. Proc. Natl. Acad. Sci. USA, 100: 1484-1489; Wozniak and Chen. 2009. Nat. Rev. Mol. Cell. Biol., 10: 34-43), nuclear and chromatin organization can be shown to be directly coupled to cell conformation. In vivo, 3-D cell shape is likely regulated not only by ECM remodeling, but by the porosity, mechanical rigidity and adhesive ligand density of the surrounding matrix. Similarly, even under the 2-D-like conditions that exist in the blood vessel lumen, changes in the applied forces exerted by shear flow on the endothelium likely impact nuclear architecture and function (Dahl, supra).

[0105] In addition, no major changes in protein synthesis were detected when endothelial cell shape change in 3-D is prevented by either omitting VEGF/HGF or when growth factor-stimulated cells are cultured with GM6001 or FUD. The results suggest that the coupling of ECM remodeling and shape-induced cytoskeletal tension to the LINC complex and the associated lamin-rich inner nuclear envelope plays a role in translating changes in cell shape into signals for macromolecular metabolism. The laminar network of type A and type B lamins directly or indirectly binds chromatin and DNA, as well as a variety of inner nuclear membrane proteins functionally linked to nuclear architecture and mechanical integrity, chromatin organization, gene regulation, and DNA replication (Crisp and Burke, supra; Dechat et al., supra; Starr, supra).

[0106] The findings described above suggest a new model wherein pericellular remodeling of the ECM represents a required step in transcriptional machinery activation, which is responsible for controlling growth and differentiation. While the results are focused on endothelial cell behavior, it will be appreciated that the findings have broader applications, e.g., cell populations that reside within a 3-D ECM. By linking ECM remodeling to the ordered transmission of mechanical signals to the nuclear envelope, subtle changes in pericellular proteolytic activity would be predicted to profoundly impact phenotype. Indeed, the phenotype of MT1-MMP-null mice--characterized by a markedly shortened lifespan with a profound reduction in growth associated with the onset of severe bone, muscle, vascular and adipose tissue-related defects--bear considerable similarity to mouse models of laminopathy. The overlapping phenotypes raise the possibility that modulating nuclear shape by interfering with ECM remodeling may impact cell function to a degree similar to that observed by directly targeting the nuclear envelope. In summary, the complex changes in gene expression and cell function known to accompany ECM remodeling are interconnected with matrix-derived cues transmitted to the nuclear envelope, chromatin, and transcriptional machinery by a continuum of protein:protein interactions that span from the cell-ECM interface to the nuclear interior.

[0107] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Sequence CWU 1

1

16175615DNAHomo sapiens 1gcccgcgccg gctgtgctgc acagggggag gagagggaac cccaggcgcg agcgggaaga 60ggggacctgc agccacaact tctctggtcc tctgcatccc ttctgtccct ccacccgtcc 120ccttccccac cctctggccc ccaccttctt ggaggcgaca acccccggga ggcattagaa 180gggatttttc ccgcaggttg cgaagggaag caaacttggt ggcaacttgc ctcccggtgc 240gggcgtctct cccccaccgt ctcaacatgc ttaggggtcc ggggcccggg ctgctgctgc 300tggccgtcca gtgcctgggg acagcggtgc cctccacggg agcctcgaag agcaagaggc 360aggctcagca aatggttcag ccccagtccc cggtggctgt cagtcaaagc aagcgtgagt 420actgaccgcg ggctgaaaca ggctgcctca gggatgggac cctaaagccg accaaagttg 480gggctgaagt tttgtgcgcg cgcgtgtgtg cgagtgtgtg cgcgctttac tgagagaaac 540cagctgtgca cacaaaagga ccgagttttg agcacgctgg ttctgagggc ctgggatgat 600aagaccgtgc attggaggac gaggactctg cgactttccc gtgttctaat aaattctgca 660cgttcagatt gtccttctag gaattaacca aaacttgcct ttaaagagaa aaatgatgca 720tgtctataaa ttttccgtct gggattagtg tggtccttac tgctacttat ttccttctgt 780taaataattg gtcaaatatt ttcaacatgg gggtggaaag ggggtattga aatagctgtc 840ttgtttctaa ctaacttgga agagatgtaa ttggttcaga cctctttagg gccgctcagg 900atacttcacc aagaacagag gttggaattc tttccgtttt tcaaagacac accctccttt 960tgctttgaga aagctgctta aagttgtcct ttttgactat tactccaaaa gaatatttaa 1020gttccttgca tgttttaaaa atgtgacttc aattgtctgc cttccaaaat gtttccaact 1080tttttatgta gacccctggc cagatggaaa tgacatcatt gtatataact tttagcaaag 1140ttaaaaggaa aaaaatatgt acgtcaatat tcacatgaag aaaattccat aattttggga 1200aaaggagaaa tgcaaatgta acgttttcct tcaattattt gcagccggtt gttatgacaa 1260tggaaaacac tatcagataa atcaacagtg ggagcggacc tacctaggca atgcgttggt 1320ttgtacttgt tatggaggaa gccgaggttt taactgcgag agtaaacctg aaggtaagtg 1380acaacaagcc ccatagttag tatcttttaa tacatgaagt ggtaattgtt aaactttgca 1440ttagtaagta aaaatacata caccattttt ctaatagaat tacctgtcat ttcctcttaa 1500gtttaaaaac tgcttatatt tgcttttcac atgcttttac ctttaaaaca aagaaacgaa 1560tctttcccaa attagttcct agagtcttct ttttgcttta ctctcccaaa gtttttgatg 1620agaaaaatga aagattttgt gtgtcctccg acaaaaaaaa ttgcttataa aattttaatt 1680tattagaaag cagtctcaaa tcttaaactg ttagtttatg agccagaaaa cactttgggg 1740acttacatcg taaaatgatt tgtcagcggc agttaacaca aaaccattag ccacttcaaa 1800gttctcattc ctttaggacc aatgatattt ttctcataaa ttatagcaac tctgtcagag 1860aagcactgat caggggaaaa tggaaatcat aggattaaca actgtcaagg ccttgtggga 1920ggtggggatc ttcgaattgt ttgtttgttt ttgttttgtt ttgtttttga gacagagtct 1980tgctctgtca ccaggctgga gtgcagtagc actatctcag ctcactgcaa cctctgcctc 2040cagggttcaa gcaattctcc tgcctcagcc tcctgagtag ctgggactac aggcacatgc 2100caccacaccc agctaatttt catattttta gtagagacgg ggtttcaccg tattggtcag 2160ggtggtctcg aattcctgac ctcaggtgat ccacccgcct cagcctccca aagtgctggg 2220attacaggcg tgagccactg tgcccggcca atcgtttgct ttttatgtga accttgcttt 2280gactttctga gtcagagatt ggaatgtgaa acccttcaca aatctagctc tgtcataagt 2340tagtacttta tatggccttt tcctaagagc ctgagatttt tctacaatat gaataattta 2400cagaaaattt gacaatatgt caaggtcaaa aaccatggcc ttattagagc ttaggataaa 2460aatctgtatc tctcacttca ttttattctt tgaggtgtac catgttactt gtggaataga 2520gaagtgggtt ttcctttaga ggggattagt gaactagaaa agcttgtacc taagtgaggc 2580tcacatggac tttccttttc ccctcagctg aagagacttg ctttgacaag tacactggga 2640acacttaccg agtgggtgac acttatgagc gtcctaaaga ctccatgatc tgggactgta 2700cctgcatcgg ggctgggcga gggagaataa gctgtaccat cgcaagtaag gaagagattg 2760tgtaaaatga tgccaaaata tcaaatatga atttctctgt taccatcact gtcattttct 2820gttatccatg actggatatt ccgaactttg aggtttgccc ctggtgacca ggtactctta 2880agtggtcacc caactggttc ctgtgtttct taaagacggg tatgagcaca gatggaatca 2940gtgtttgatg tgtgtgcgtt tatgagtgtg tgtgcattta tgagtgtgtg tgtttctgta 3000catagtagaa ccaagagacc tcttgggttc catttcagta agacatgctt aggggagttg 3060cccattttaa atcacctgga tatcttcaag ctagacaaat catgagactt ttctgcagtg 3120actgggaagg tgttcatgaa gagtgaacca gccatgtgtt gtctggtctt catgtttgca 3180atgcagagac ctcttgcacc tcacagaaac agtctggttt cttggtgacc agtaggttat 3240acccaggaag cagatgtcac tattcctagg gataatacaa aattattaac ccaatagagt 3300ttgctaagga actttgggaa ccgggctgat tctcaactct agtttagcta aggcactctt 3360tccagtatga ttcactgggt taccaataga ttctattaag atagtattta agttttttaa 3420tccattcttt aaatataagt cgtcttaaag acttctattc aaaagaacaa gtcccgtgtg 3480aataggccca atcaactttc cccatatttc atgttagggt ttatccaagt tcacaggcaa 3540atcgcaagag gcaagggtcc atagtgttta caatctagtt cagcatttga atgtgccatt 3600gggcttaaca acttagaaaa ctaccaggat ttccacactt tatatgcata tgtctgtttg 3660ctttccacca aaatgacatt tctatcctag ggtaaaatac aggctctcca tgctcccaaa 3720agctggagtg ctgtgcctga tgtggccttt tcactgaatt agtctcagtc ttagcctgct 3780gtgtatgagt gaagaccaag cctcccagcc tttcttttct gcttaggacc caatttcctg 3840tgatctctct gggaaagcag gattcatgac ctcttccttg ccatccagat ttctctgtgg 3900ttttccattg tgttctaagc aagacactta actgaattga ctccaagtga ccagacctgt 3960taacgtttcc cctgtctctg atgggaaagc tgttgtctgt gtctctactt tagccaacct 4020aagtacctac catgggtgga atatgagacc aaaaaaaaaa atctgttctg ccctctccta 4080acattttcgt tgtatcttca acagaccgct gccatgaagg gggtcagtcc tacaagattg 4140gtgacacctg gaggagacca catgagactg gtggttacat gttagagtgt gtgtgtcttg 4200gtaatggaaa aggagaatgg acctgcaagc ccataggtgt gtgagtctta gggctgagca 4260agagctggga tgcttagttc taatgtgggg ttggaccaga atcacatcta cataggtcat 4320agacctgaat tccagtgaaa accaataaag aaatgggaat tttgtttgaa ataatgaatt 4380attatataat ccatagtctt cttacaggag ttagatcaaa aagtactgac tacacataga 4440agtcttaact ttgcttcaaa agcataaggt agaattgaaa gatttagaat ggagtcattt 4500cttttaccta atagctgatc tcagattcct ccttcgtcaa gatataattt atttaaaaga 4560aaaaaaaatg acactttgga cacatttcta tatggaatgt cctggaccga aacatgaaat 4620agtgtgtgct tgtcacactc tgctcatttc ttttcaaatt aaaagttgtt gagcttcttt 4680ggatctcaat cctcagttga attttgtaag tacaagcctg aaagtttctg gctataaatt 4740ttactctgtt tacttgtctt ctaatttaga ggtttttgtc ttgttttgta ttgttttgct 4800ttccaatatt taaaaatagc tttctttgtc attgtattta ggccactcaa aattcataat 4860tggtcattta taattaagat tggaattttg catatgtagt ctcccacaga ctagatacat 4920acatagatcc ttgctactgg aaatgctgct gggaagtttg gggctcgctg aaaatatgta 4980gtccatgtac ttattaggag aatggaattt ctgcctgcca actcagcttg agctttcttt 5040tgccttggct acttactgtg tgcttagatg ctgggtgtgt cattctttct gaacagagtg 5100ccacttaaaa aaaatgtggc tgaatttttg cttacacact acactttaaa ttacagggag 5160cttgcacaat tcaaaataac ctttttttcc tgtttttctt ccaaatttcc ctacagctga 5220gaagtgtttt gatcatgctg ctgggacttc ctatgtggtc ggagaaacgt gggagaagcc 5280ctaccaaggc tggatgatgg tagattgtac ttgcctggga gaaggcagcg gacgcatcac 5340ttgcacttct agaagtatgt tttacatctt tatgttaaag attaagccag gtattgtttt 5400ctggattcct agagagaagg gtaatactat gttactcaga acacatccag tatatcagca 5460tgctttggta acttctggaa gtcaagaaaa ctttcataac caacttattc cgcatcttca 5520gagaagacta cataaataga aaaacatatc actttgataa ggttcaatct cagctcactg 5580ccactgacat agagttgaac aaaaggttta ggtttccttc tatgtttgaa atttaaatag 5640ggcacattca caggctaaat tgataaaatt aaaaagaatt tatcccataa attaaaatga 5700tttatctact ctggagttag ggatagtgtc tctgacctaa cgcatttgat tagtgctgta 5760aagaagctgg cctctggtgt ctttactgct ccttctaaga ttgtcttggg gtcttaattg 5820ttgcctttgg gtttgaaggc tccttttttg atattgtaaa ctaataacag ctagagagtt 5880tgttgaagta aaacagccat taactactgg tgttgtaaat aagtttaaaa tcaaatccaa 5940ataatttgaa cctgttttat ttatctagct gaacccattt aactaccttt aacatagcca 6000tcatccaaat tcaaattctt tgctaacaaa aataggtctc tcatgaaaag tggtaaccat 6060tttgaccaaa gctttcccag aaacttgctg gtttattaga tattttgcat ttaaaatgtt 6120actgtgatca tcagacttcc aagatctttg tggcaatatt ttagcttaag acaaattaga 6180tgtctgattc aaaccttatc tgttatttag aactctttaa atagcaagtt gggaaaagtt 6240tctcaaagag aagtcattta ttccagaaaa ttttataagg acttactttg ttcaaggtat 6300tataggggtg cagatatgaa atgaacatta gcccagcctt caaagagtac ttagggggtc 6360agggagatga gaaagtctta tacatattta tcatctgcaa aacacagtat taaagatttc 6420aacagaaata ctgaaagtag tgctatggag gttcagggga tgataatact tttcgctggg 6480gatttgggaa aaagctctaa tcagtaatta taccttcatg caaacttcta ttcttgtggt 6540agatggatgt gggtgtgtat ttgtttgaac ctacatcaac tattaatttt ttttctctaa 6600cccaggagtt gcaacaatat ccaattcaca aagacatcag atcctctata ctcacatcgt 6660ggcacagagc aaatttggat tataatttaa ataatctatt taccagataa atgcacgcat 6720agactaatgg tcatttagtt acaaattatc attttatgtt gatcccactc ttccagtgga 6780gggctaacac tgaataattt ggggctattt tgctagtgat ttttaaatac tgtagatgtt 6840tgggtatagg ggaagggaaa ataatatttt agtcaaagaa attgtgcatc ctctacattt 6900tttacataac aaatgaagaa agagatacta ccaccttctt atagcttctt tgtagccatt 6960ggtgaagacc ctttgatacc tgcttgcctc cccattgtta taagcttttt tttgtttgct 7020tgtttttttt gttttgtttt gttttgtttt gtttttgaga cagtctcacc ctgtcgccca 7080ggctggagtg caatggtgtg atctcagctc attgcaacct ccacctcccg ggttcaagcg 7140attctcttgc ctcagcttcc cgagtagctt ggattacagg cgcccgccac cacagccggc 7200taattttttt tggtattttt agtagagacg ggggtttcac catgttgccc aggctggtct 7260tgaactcctg actgcaggtg atccaccctc ctaaagtact aggattacag gcgtgagcca 7320ccgcgcttag cctgttttta gttttctaaa gcaaggtccc tattgaaagg caggccataa 7380acagtgatga ctaagaaaaa tcctggaaga gcctgagaag gaaaaagatg aaatataatg 7440ccagagaatg aagttagtca aaggaacagt gtgaaaacaa taaataaata gataaatgaa 7500aatgttattt gacagagaga tgaaactaga ctaaaccatt cagctgcctt tccactgtaa 7560caaatgtaat ttcatctttc agaagtgtaa taccttgcag caccagagct gaatatgaac 7620atattaccaa aaatagatta ccaggcatag atagcattcc ttttttaagt ttgaattgac 7680cacttgcgac tctcgacctg atgtatgtat gtgcttcctt tgtgacacag atagatgcaa 7740cgatcaggac acaaggacat cctatagaat tggagacacc tggagcaaga aggataatcg 7800aggaaacctg ctccagtgca tctgcacagg caacggccga ggagagtgga agtgtgagag 7860gcacacctct gtgcagacca catcgagcgg tgaggcacag gacgagcagg ggcgggaaat 7920ggggaagcag gtcaagaaat atttccgcaa atccatcttt cctttgacat gccatttgag 7980gataatttgc agtgtttcag ctaataacct aagataattt acacattatt ggttgttaaa 8040acttttttta atgtcaagtt ttaaattttt cagaaaaaaa gaaaaatgac atacaaataa 8100accttagggg gaaaaaagcc agatttatct ccaaaagata aaactgagtt ttaaagaatg 8160ctagcatcat aaaacttacc atggatagat cacgcacaca cgcacacaca cacgtatttt 8220gaatatccaa agttcatttg aaaggaaatg agagttataa ttaattatat gactacctgg 8280ttcttctgct aggaaaggac aaaaaaagtg catttggatt ttttgtttgt ttgtttttga 8340atgaaatata cttccctgtc ccgacattga actctttttg tagtggaaac catcctttat 8400atgtggtttc tatgctctgg caaactttgt tacattctat aaagtaacac acaattattt 8460ccttcatgta ttggcattcg aaattttaga aattcagaga ggacttagag atggccatga 8520aagacatgat atctaagcat tctttttaaa aaacaagttt taatcatttt tggcatgaga 8580aaaagatttt tacgtcataa atgtttcata aaaatctgaa gagagaaata tggccaacaa 8640ggacgtgcac tcctctcatt atttttaata tgttttgatt aactttttac tatatgatgt 8700gccaacatca ttacgtagtg tctcagccat ccttcaatta aaaatattaa ttgttctaat 8760ttttcttctt ttgatgagtt tttgtcttgc tttgagcact tatgaaggtg aacaagatta 8820gatttgataa tatctttgag ttattttatt atcattaata aaattgctac tggccaaaaa 8880aaattataaa catcggccac gcgcggtggc tcacgcctgt aatcccagca ctttgggagg 8940ccgaggcagg cggatcacga ggtcaggaga tcaagaccat cctggctaac acggtgaaac 9000cccatctcta ctaaaaatac aaaaaattaa ccaggcgttg tggcgggcgc ctgtagtccc 9060agctactcgg gaggctgagg caggagaatg gcatgaaccc gggaggtgga gtttgcagtg 9120acccgagatc gcaccactgc actccagcct gggtgataca gcgagacccc atctcaaaaa 9180aataaaataa aataaaaaat aaaaaaaatt ataaatgtca gtctaccaaa atagattaaa 9240agtgtaggtg ggaattaaat ggggataaac actcaataaa tgttagctat atatgaatat 9300tgccaatact gaaaagattc cattgttcaa aaaagtttga gaagcaatgg gttaaacaaa 9360atggaacctg ctctgcagaa tctgtgtgtt cctttacatc atactctcca tggtagagtg 9420taggggatgg gcgccatgtc tccctagtac gtttgacctt gggattcttt gtctgtgaac 9480atctttgggt tctagtgttc agcagcactt agggaggcac tgaattcagt gtacctttgg 9540tctagcctca gccctgattc tgttctgcgg tgggccctgg ccttcaagag aacagatatc 9600taaaagttga aagaaaagat cggccgggcg cggtggctca cgcctgtaat cccagcactt 9660tgggaggcca aggcgggtgg atcacaaggt caggagatcg agaccatcct ggctaacacg 9720gtgaaacccc gtctctacta aaaatacaaa aaattagccg ggcgtggtgg cgggtgcctg 9780tagtcccagc tactcgggag gctgaggcag gagaatggcg tgaacccggg aggcagagct 9840tgcagtgagc cgagattgcg ccactgcact ccagcctggg tgacagagtg agactccgtc 9900tcaaaaaaaa aaaaaaaaaa aaaaagaaaa aagaaaagat caacacatcc tgttgtgtta 9960ttctgaaagg aaagctgtct taagaggatc aattggtttt agaaaaaaca caatagaatc 10020acaaataatc cagaggagaa ataaaatgtg gaaggtggag gtgacctcca gaaaatccag 10080gacagctgct gaaggcaccc tctgatgagc tcggttactc agaagagtga ggatgtgttg 10140aaggtatctg ctgtatggag tggcaggatg atgtctgtga ttgagaaata taatcccggc 10200caggcgaggt ggctcatgcc tgtaatccca gcactttggg aggccgaagc gggtggatcc 10260cctgaggtca ggagtttgag acaggagttt gaggtcagga gtttgccaac atggcaaaac 10320cccgtctcta ctaaaaaata caaaaaaaat cagctgggca tggtggtgcg tgcctgtaat 10380tgcagctact tgggaggttg aggcaggaga atagcttgaa cccaggaggc agaggttgca 10440gtgagccgag accgcgccac tgcactccag cctgggcaac agagtgagac cccatctcaa 10500aaacaaccca aaaaaccaaa aaacaaacaa acaaaaagaa atataatccc agtagcccca 10560gctgagctgg aggatggaga ccacttggta gacacttgtg gattatttcc taggctaaat 10620gcaaaagcta ctgctgaata agggacattt ttttccagtc ccaggccagt agcgacatag 10680atttcagagt gatctctgtg agatcctgaa gatcctgact gcagaaagta gtgaattgtc 10740ttctctcacc cagttttgtg acattccctt ttcatgccat taggatctgg ccccttcacc 10800gatgttcgtg cagctgttta ccaaccgcag cctcaccccc agcctcctcc ctatggccac 10860tgtgtcacag acagtggtgt ggtctactct gtggggatgc agtggctgaa gacacaagga 10920aataagcaaa tgctttgcac gtgcctgggc aacggagtca gctgccaaga gacaggtatg 10980cattatcttt ttgaagaata ggactgatga ctttattatt tagtttttga aggacaatac 11040attttcaatg tgaaacaata aaacaaacaa gaagcctgta atcttaccac cctgtgataa 11100caattagggt tggcatttga aatagtttct tccaatcttt ttaatttatg tattttcttt 11160ctggtcatgg atatcatggg taaaaatttt attgtattta tctgtctaaa gtgttgttac 11220aagagagcta ctttctgaat aatcatcaat gttttatatt ctaaatctca aatttcagca 11280gctttgtgat gtaaacatct tccaataacc taatatatgt attctgcact acaaacatgg 11340tagtcactat ggcaataaca attgctacac aattctcccc cagaatagtc tcatatatta 11400attttatggc atagatatag tcataaatat tatcccaaca tccttaagca gcatccttaa 11460ttgacctgta taaatatagc tttacaaata gagaaactga ggcatggcag cagaagtggt 11520catgaaggac atcagcagaa gaactcaggt gtcgttctat ccacagtaga catggattcc 11580tgagtaatgc attttgactg aaattaacga gatgatcatc tatactcata gcttcttcct 11640ttgagggcac aagctcagta tctcattgaa gccataaata agcagctgct ggtgggagat 11700aaagcatctc tgtttactga cactcttttg attatgattg tagctgtaac ccagacttac 11760ggtggcaact caaatggaga gccatgtgtc ttaccattca cctacaatgg caggacgttc 11820tactcctgca ccacagaagg gcgacaggac ggacatcttt ggtgcagcac aacttcgaat 11880tatgagcagg accagaaata ctctttctgc acagaccaca ctggtgagtg tcccaagggg 11940gagccacaga agtgagaaaa actcactttc atgccctagt tttatttgcc agcattctag 12000ccatttattt tgaacccgcc caagaagcat cgcttttgtt cagtttggac tcaagagatc 12060gcagcgctca cgtaacagct gaggattctt ccatcttccc cagtactgtt gggaaatgac 12120accaagggag tagccttcca gttcatttga tttaacacat tgggattatg atgtgattaa 12180agatacttgt attttggaat cagtagatga tcccacaggg ctgaggaata caaaggatga 12240atgttttagt gccttagctt attttccagt taaaacaatg ttttattcaa agctatcatt 12300taatcttttg tggggggggt gctggggaaa tgacagtgaa agtgggattt aaacctgttt 12360tgaaggtgtg aaggtaaata tgctaagaag cttagaacta tattatcaga cattttttat 12420tctgagatag actgtctgtg aatgagctgc agaaacctgg ctctctcaga ccagtaattc 12480tgtgtacatt ggaaagctca gcggtaatct tttccttctt tgttgtgtat tgttcctggc 12540agttttggtt cagactcgag gaggaaattc caatggtgcc ttgtgccact tccccttcct 12600atacaacaac cacaattaca ctgattgcac ttctgagggc agaagagaca acatgaagtg 12660gtgtgggacc acacagaact atgatgccga ccagaagttt gggttctgcc ccatggctgg 12720taagatgaag cccttgtggg ttgtcttgtt tgacaacaat ttagggagta gagactaaag 12780actagtgtcc agtttactcc catttcattc attaacacaa ttttgagaca acagaaaact 12840tcatgtgaag tgtgtttgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgat gttacatcat 12900atacataagg attgggaaga ataattagat aattatttat ataattttta aacctcattg 12960acatgattta atgtcaaaaa tataattact tatttgtaag tctggaaata tgaatttgca 13020caggtttgtc tttgtaaaga gcacacaact gagtagctta caacatttaa tatatgtatg 13080acggctttag tcacagagct acaatattga cacatggttg tggtttgatg ggcataagct 13140ctatcactta ttaataagtg ccaaagtgac taaaactcaa tgttttctaa caggtaggga 13200atctcactct ttttttaaag gtccccagtt tgtatagatg gcgaacaaat ggaaacgaat 13260accttttact tgttttcaga tttcaagaac cccatagatt ccctttaatt ttccagttgt 13320agaaacaaga gcctgggcgg taggcactgt caagtgtgac tatgagacaa agaaattgct 13380tatactttta tttctttcaa caaaagaaga tgctgagttt agaagaaaaa acccactttt 13440gcttgtaatt ctatatccaa acccatagtt tttatttgat ccagaataaa ctggaactgg 13500gaaaagttat gaagctgtag ttaaatccag gcttctagaa cagcaagaac cctttgtgtg 13560gatgtgtaga tattatctta gtttaacatc ccctaaccct tcctgtaact attttctatg 13620acacgtttgg actacgtttt ctgcctccag ggctcaaaaa ttctacccct tcacctgaca 13680gcacttagat gtctttgatg cacacaaagc ttcttcccaa gtgagaattc ttaggatgac 13740caaactgaac tgatcctttt gcacacatac atgtttagac ctggtgatca tttatcaagt 13800gcatttctta tccatttcca aacagcccac gaggaaatct gcacaaccaa tgaaggggtc 13860atgtaccgca ttggagatca gtgggataag cagcatgaca tgggtcacat gatgaggtgc 13920acgtgtgttg ggaatggtcg tggggaatgg acatgcattg cctactcgca gcttcgaggt 13980atgctggctg attaacaaaa atatttgaga tggcaaaagg tacagaaagg gacacttttt 14040tttatgaaaa cttgcactat gccaaaagca ggggaagaaa tatggaatgc cacgtcattc 14100attagtctac tgtgcatggt aagataagcc tgaaaggctt agcaggcagc ctgctaagac 14160aagcggcata gcaatgctaa tgttctgaaa cactcctagc atgtaagtac ttaggctgag 14220ccaaaaagat ggcttcaaaa gtaagaatga aacatttgat ccattcagct ttaggctatg 14280ccactggatt catgtctaga aaagatagga taatttctgt aaagaaatga agaccttgct 14340attctaaaat cagatcctta cagatccaga tttcaggaaa caaatacata ggggactaac 14400tttccttgtt cagattagtt tttctccttt gcacccagct atataatatg aggaagtatt 14460gactttttaa aagtgtttta gttttccatt tctttgatat gaaaagtaat atttcgggag 14520aaccctgagc tattaataat ctatgtggct agtgcgtaga tattggtctg aatttgttct 14580ccttttgtgg tgtccagtgg gtaacaccat ccgggagtaa taattacatg tggtgttgca 14640gaactgaaag agaccttaat aacacataga gacctcactc tatatagatc aaggagctga 14700gacccaaaaa ggaaaaagta attttctcag gatctctcaa agagtgagca acagagttgg 14760cctaatttat tttagcgttg tgaatactgt tgacatttta tttcccaaat ctaagtatct 14820cctccccttc cccctattcc agagaccaga ccaccacatc atgctgggtg ttagataaat 14880atgtttaatc ttcttcttat ttatcctaac aagcagatat ttaaaggaaa ttatcaacta 14940agcaagaaat tttcagaaag taagacatgt atttgttcaa atactggctt ctcacaggaa 15000agtgtatttt accacattct ttacttgagc atactgtaac

ctctgcaaaa gttacacatt 15060ttgggaagaa aaaaattttt ttggcaaaaa ttgtattact gaccaaactt tgaaaaaaat 15120gttattctat gcttgtagaa aagttatttt agtggaaggt gttgataatt aagtggaagt 15180agttgtatgc tttgagaagc ataccttttt tctttcatca atggaacttt aaaaagtttc 15240tcactcaccc acctgtttcc taaacagatc agtgcattgt tgatgacatc acttacaatg 15300tgaacgacac attccacaag cgtcatgaag aggggcacat gctgaactgt acatgcttcg 15360gtcagggtcg gggcaggtgg aagtgtgatc ccgtcggtga gtagccctat ttccctagat 15420gagtttgcac agggggaatg gttagcaagt ttcagataag aaaagctatg tgaaatcaca 15480tgactgaagt tggctccaga ctttgatcag ttgcttgcaa agaactttgc aaagtcttct 15540ctctaatact ggaccaaaat atctcgatat tggtagtcgt ctggtttttg ctgaatttgg 15600tgacaaattt aggcttattt taattgaatg gaatttattc ttgggtttag aatcataaag 15660ataatccatg ctattaaaag tattctttcc tttttttttt gtttgttttt gtttttgttt 15720ttgttttttt gagagagagt ttcgctcttg ttgcccaggc tggagtgtat ggcacaatct 15780cggctcactg caacctctgc ttcctgggtt caagcaattc tgctgcctca gcctcctgag 15840tagctgggat tacaggcatg cgccaccagg cccagctaat tttgtatctt tagtagagat 15900ggggtttctc catgtgggtc aggctggtct caaactcact tccttaccag ctgtgtaaca 15960gcatgagcaa agggtgtaaa tatcacccac caaaacactc taggtttttt tttggccgcc 16020cttcaaaata gaactaagca aatagtgaag gctgagcctt aaaagagctg tgttaccagc 16080actacaaagt ttaaggtgat ccattactat ttctttacca aaagagacag gttgctcact 16140gagaaaacaa actgataaca tccgtttgtt tgacgtgaga ttaccagaac tgagagagaa 16200gcctgagagg ttttcttaga agctgctcag caggtatact cgtaaagtct agttcattca 16260tttaaatgtc aaacagtttc tttaaatttt gaagaagtaa ggaaaatgaa attattgcag 16320atttttttct tgctatttaa atgttaagcc agttatatta atatgggtaa aaataataac 16380taatatttaa aattaatgtg tagattatca atatacactg aaatctaaat ctttacattt 16440ttatttagaa atattacctt ttagaaaact aaatattcct cctaataggt actttggttt 16500tttttttact acaaactgtc ctgtaaggta aagaatgtga acaaaatatt tttttaactg 16560catatatttg taagaacaat tgcaaatttc tatttaagct aaatgtatgc tctagcaccc 16620tgaaattaaa ttcgtagtta taagtcttca aggctgttta tcttttcctt ccatgtattt 16680tagaccaatg ccaggattca gagactggga cgttttatca aattggagat tcatgggaga 16740agtatgtgca tggtgtcaga taccagtgct actgctatgg ccgtggcatt ggggagtggc 16800attgccaacc tttacagacc tatccaagta agtagctcta ttactgcaag ttgagaactg 16860ccaattgggt tataacaaca gggcagtgat tattaatgct ctcatgccta agttgggggt 16920ctcccctctt tcccaccctt ttctcttgtt attatctaat aatcaattga atttttgatt 16980aaaataattt ttctctcttc ctctatcaag taaaaggtag agaaggctat gaaaatgtgc 17040ctgtttataa ttttacttct taactctgta aaatattctg ttaggttaag acactctggc 17100taatttcatc ttatatccat acatggaaat aaaaaccacc aagtgagtta tgctgggagt 17160aaaggtttgg ggctttatat tatgattctt aacagagaag ctgcatagag agatggcatg 17220aaatgcagca taaggtacgt gttcattcaa catgtcatct aagctccctt tgcatcaaac 17280ttttcatttg tttgatcagt tgccaccagg aacacagtat gttgggccaa gggttgagta 17340acttggtcaa ctctctgccc acacagttca aacactctca aatgtttatt gctgggtttt 17400tccaggtcac aaagacatca tgcctagctg taggtgtaat tagttcattt ggggggaaaa 17460ttgcatttaa atattcactg agtgattata ataaaacatg ttaataaaac atgaaaggct 17520aattaaaagg catcagttat ttgagcaact gctgaggtgc aaagtctcca aagtcttcac 17580taacgtttga ctgaaaatat ggcctacatt cagaaacaaa agagtttcag ggtgtcagaa 17640tctgcatgcg acagaaatta agattaactc tgtgataaaa gattcactgt gacagagaac 17700aagctatggg aacaacttgg ccaaaggtag tgttagccaa gcctcattcc tccatttcct 17760catctgtaat atgggaagat tgtagctgat tacctttaat gttccatcct aaatactaaa 17820cacccagatg caccttttct aggaacttgg aagattctgc ttttcccaac cctcagacac 17880tgtcagtgct ggggaaggtg acttcacttt tgaaggctta tagcacagat tgaccaacct 17940cctaaattgt atttcttgga ggatttaggt gtaggaatca cttaattttt tgtaacttaa 18000tatatattta aatctgattg tggaagtact ataagtatat gaatggtttg tttgtttata 18060tgatgcaaat gatacttaaa tggtagaaac ttctaaaaaa atgctctgtg gtttctatat 18120ttatgattgt tattggtgtt gcaatttgct gaaaatgatt tccttctgaa tgattgagag 18180agatcttcgt ctctctcaac ataaggctgt ccacatagct gcttgctgga agcatttagg 18240tggacatgtt ggagataaaa tctggaaagg aaggaatcct tgagtattgg agtattacat 18300gttgacctta ctcctactct ttaaaaagga gaacagcaag atcccactga gcatagaggt 18360gatttcggag ggaagagatt ggaatttgac ctcagatatg ctctttggtg cttatctgta 18420tgtctggttt gctccgtggc ctagcacgta gggctttaag agtgtggtga gaataaggga 18480acagcagatt accaacagat tgtctctgag tcctgccttg tttgttcctc ctacagagaa 18540cttggtagag ttgttcaaac tagcaaaaga taagaggcat ttggtttgtc aataagcaac 18600tagaaaagca cagatctcag caaaataaat agagaaaaaa aggactgagt caaaaaatca 18660taaatgttta acttctccaa ggacactact attggaaatt attcatttag actttatttg 18720aaactaattt taaaagtgta gacattgtac tatctccttt ttttggtatc atctcaacta 18780ttttattgtt agtttattca tattgaataa gagagggagt aaagatttca caatggcgat 18840agctagtata tgccaattaa gttaatttaa aaagttatac caactaccag tagcgaaaaa 18900ggactgtcaa aagtttaaat ctaaataatg taaaagatgt cataattttt aacttttctg 18960tctttaaagg atacatagta taagctagag taattatacg tagtataagc tagaatatag 19020gaatttaatt gatctaagaa ataactgaca caaagtctct ttacttcctg aacaaaaaca 19080tgctaaattc catgctgttc agtccatttc ctttaaaggt gggctatgcc acagggctag 19140attttaaaac gtggaatttc acaccagtgc tcgaaatctt atgaaagcaa aagggacctc 19200tgtagttgta ctccactttg gcttgagtaa aaaccactgc tgtacccttt ttctttcctt 19260tccctgccca tttttatcct cctccttgtg tccttgtgga catagaaata tgattaggct 19320tagaggtgaa cagtaaaggt catttatgtt atcttttcaa aacttaatag acatttatcc 19380atcataagct tgtcccccct caaaatcatg attgacaaga ctaaataaag tgtatatcag 19440gtgtctcttt atggaggaaa ttgtagtagg atttatttaa aggatcaata tttaaatagc 19500cctatgccaa ttatcataaa taattaagga catgatattc ctagctttcc tgatttacat 19560ggaagtacgt taaatagtca catctccaaa atttttcttg aatagttgtc tttaaaaatg 19620tgtttacatt tgtaaggatc ttcataaaca gaaggggtaa ttcaaagata ggcatggaat 19680tgacttatgc caattgatta aaacaaacat cctgtgtgtt ctgtgaaata catacaactt 19740taaaatgaaa aactcataat tttatgcatg aattttggtg ttcatgtggc ttggaaatat 19800gtgcattaaa tggaattaag attcaaagta tttgctaatc ttcaaccaac tttgaattgt 19860tactggtgtg gaagtgagca tattgtttta gaatttctga atctacatgg atatccaagt 19920tatatatttt ttctgctaca gaaagctttg ttttccaaga gaatttaatg gcttagataa 19980taaagtttga aaatcaatgt attttttttc ctagaagctt tcagaaaact taaatctgtt 20040aataatctgg tgaagtgctt tattacacat acaaaatttt gctctgtttg acagagtgtc 20100agttagaaat tcttgaaaag gttactataa gacacaattt ttatttctag taatttaaac 20160attgactgac atcataaaga tagtgtttta agaaaagata gttttctgtt ctgcaagcat 20220aaattttcta gctatttcat tattatctta aatggagtta acactactta gaaattgatg 20280ctacttccct tattttcctt ttattttaaa caaaagaacc aaaaccatac tttaaatttt 20340gttgataaca gtgatataca tcaaggtgta gaaatactga atttagtata cacttcataa 20400agtcatttct gttgacaact gattttggaa aaaaaataaa tttaatcact taataatttg 20460atggatcaat ggtgtgattt gggagtaaac ttcttgaata aaaataaaac ttgatgtttt 20520ttccaaactg atagactgct tagactgatg agaaataaaa ctaggtcttt aattattacc 20580tttgctattt gtctaagatt ctacccccat ttagaaatgt gtttgtttta catcatctca 20640tatggccttt ggaatgttgt ttccttctta ggtagtagat tcattcttga tgaaaacctt 20700ccaaaattag aatttgcttt aaaagaggct caatacaata agtaaatgaa gtctgtgttc 20760tgtattacat tatttttgta ggatcgtttc acaattttct cacaattctt actttgatag 20820tagagaaaat aaaagcaagc caagagactt ttttttaaaa atatatttta tttgctataa 20880acaactatga tttgcatttc tcatgtgaag aataataatt atcggctaaa aatggatttg 20940ctgcattgat tttcaacgta aatatttaaa agattaatgc cagataattt tattatactc 21000acatttaacg acaagcaaag ctgtttataa tgatgactgt ccatgtacac agttttaagt 21060tgaagtgagt gaatattcaa gataattaaa tgctacattt tcattttcag gctcaagtgg 21120tcctgtcgaa gtatttatca ctgagactcc gagtcagccc aactcccacc ccatccagtg 21180gaatgcacca cagccatctc acatttccaa gtacattctc aggtggagac ctgtgagtat 21240cccacccaga aaccttggat actgagtctc ctaatcttat caattctgat ggtttctttt 21300tttcccagct tttgagccaa caactctgat taactattcc tatagcattt actatatttg 21360tttagtgaac aaacaatatg tggtcaatta aattgacttg tagactgagg ggattttggt 21420tttggttttg ggttttgttt ttttgcggtg ggggggctgg tatttggaag aatttagctc 21480tttatgttac agaaatcttt tttgcaagga cttagaaatg ataatgctta agattgttct 21540tgcccaatgt gggaagagaa tctaaggttt ttatatgtct tgcaacctca tcaaaggaaa 21600attactggca tcattttcat aatttgaaaa aaaaagccaa attaatatat ttcttttttg 21660attcactttt taagtgatca tttttaaaac tttacttttg acccactgaa tttatttaga 21720tagaaggaaa agagatgatg ggagggaagt ttagataaag gatggaagtt ggttttattt 21780aaacaatagc cctgtgattt cctaatgaga agtgactaga aattgaagaa accaaataag 21840gaggatattg gtcaatttag ctttagtttc tcttactctc tcaagcctgc cctgtttaac 21900tccaaagttc atggctcata atttgagaaa cactgtttta aacacaggag aaaaaaatgt 21960ccattttaaa tcatagctat tgaattctac aattacaaag aaacaaacaa acaaaatttg 22020accaacccag gcggttaaat ttaaactctt caggaaaaat ttaagctgtt aaaattattc 22080tttttctaaa tttctaaagt ggagggacag aatttttcag atttaaaagg gcctcctagg 22140tgcccagaaa attagtggaa agaaccacgt ctagacgcat ctttgatgtg tcagagttcc 22200aaggataaaa agaaactttt aaagtcttct atactcagcc aggttatcaa tcaaatatga 22260gggcaaaata atattttcag acagatttta ggcagtttat cttccatata tccttttctt 22320taagggtatt tgtagataca ctccagaaaa acaagagtga aatatgaagg aagttgtggg 22380gtccagcaaa cagtgcttcc aaatcagacc cctgatagag gtggaaaact ttgcaatgca 22440acaactgcgt agctggctta gaggacagcc aatacagatg gaacagaaag atgaggatgg 22500gattgaggga tcagggattg aggtctccaa gaataaaaag ggacttcatg gaaaaagtag 22560gcttgtggat aattaatcac aggggcaaat aatgcagtta aaataacaac atgacaatca 22620ggtggaggaa tgtataataa acccaaatgt ggctgggtag agtggctcac acctgtaatc 22680ccagcacttt gggaggccaa gccgggcaga ttacctgagg tcaggagttc gagaccagct 22740tggccaacat ggcgaaaccc cgtctctact aaaaatacaa aaattagcca ggcttggggg 22800cgcacgcctg tagtcccagc tcctcaggag ctgaggtagg agaatcactt gaacccagga 22860ggcaaaggtt gcagggagtt gagccaagat cgcgccattg caccctagcc tgggcaacag 22920agcgagattc tgtttcaaaa aacccccaag tgtattataa ggcaataatt cctatacgaa 22980gcaaactaaa atgcagcaat attaaggtat aaaaacaaag aggaataatt ccattgaacc 23040ttgattctgg aaactttgat ccacccagca gtcatgatgt tagactcatt gaaaagaatg 23100tatttctaat gcatgatgcg atcggtctat agatgtgtca tggaaacttg gttgcaactt 23160caagacaaaa taaaaagtaa acatttacat gaaaaatggt ggatatggaa ggtggagaag 23220agaggagata acagctttat ctttcaaaat agagaattga gagatggtac caaaagctga 23280tgaagtaaaa aaaaaaaaaa aaaaaaaaga tacttaatat aatactttaa attacaaata 23340taaacacaag aagaacaaat ataatgatac aaatgtcaga cactgggaat gtccaagatt 23400ctggaaggaa agggtggtat tattgagcta aatcctcaac tttgtctggg cacagtggct 23460aaaaattagc cgggcatggt agcatgcacc tgtagtccca gctacttggg aggctgaggc 23520ggaaggatcg cttgagcttg agaggcggaa gttgcagtga gccaagatgg cactactgca 23580ctccagcctg ggagacagag aaagaccctg tgtcaacata aataaatata taaataaatc 23640atcaagtctc atattaaaga ctctgtaaat atgacttatt gttgacaaat gaaacaaata 23700gaggtgtaag catgttgtct acatggaggc aagaccagaa taatagaaaa tggaaacaga 23760ttcccttaaa gaggggaatc gtgtctttct cattggctca atgtagtctc cgtagagtct 23820agaatgcttc agcacctggc acactgctta acaaatggtg aatgaaaaaa aaaaaaagaa 23880aagtcattct ttttcttctt tcaccctatg tccataatct ggccatttgc agaacttgat 23940gtccagtgat cgaaatcaac agcatcagtg catccaatat cttctagtct ctcatcttct 24000tattacatca ttaattttat ttactttaaa attaaggata tccaaagtat tatgtgagac 24060cattgcaatg ggagacttaa aagtggtata aaatgtactt tgggccaggc gcagtggctc 24120acgcctgtaa tcccagcact ttgggaggcc aaggtgggcg gatcacgagg tcaagagatc 24180gagagcatcc tggccaacat ggtgaaaccc cgtctctatt aaaaatacaa aaaaattagc 24240tgggcatggt ggcgcatacc tgtaatccca gctactcggg aagctgaggc aggagaatcg 24300cttgaaacca gaaggcggag gttgcagtga gccaggatca cgccactgca ctccagcctg 24360ggcaacaaga gcgaaactcc atctcaaaaa aaaaaaaaaa aaagtacatt gaattggaaa 24420gtcttcaaaa agcagcagtg atgaattttt tgagattttt aacaattaca aaaattcagg 24480gttttttcta atggatgcca cctgagactt tattttctgt tattttcttg taataactaa 24540ccaaacaagc tcatgttgaa aaatgattac taaatttgag ctaattgcaa tgactggttt 24600caaaattttc cacagtgtat ttgagttaaa atttcactgt gaagagtact acgatcactc 24660tcgcttattc caaaaatata aatggacact tgagtatttg aattattgag gaaatggttg 24720actgggtaat ttttaaaaat cactgggcac aaaaaatata ttttgactta tattagttta 24780gagtatttac acttgaaaga gtctcatctt ttctgaaggg tgtttctttc atacacattt 24840tattgcactg agttttgtga cccatggcat attaatgaag ctgaacagga tgtgaaatat 24900aaactggaag caaaagatta aataaaccaa aattgcattt tttctgtagt cttgtccaaa 24960attgggtaac cacttctgat ggggtagctc atatccaaga atgagtcaca aaaccagact 25020cgttgaacct ggtatatgat gagtcacaaa gcaacattct gcctttgttt tttcaggaca 25080agaaacttga attgtatccc actgagttaa aagataaaat atatggcatt ggcatttctg 25140tacttcagag aggaatatat ctgtttgtgg taggaataaa aaataagtga gagaggcaaa 25200gcttaggttc atcatattat gttactgata taacacaatt aacttggtaa aagtgaaggt 25260gtgggtgggc gtggtggctc acgcttgtaa tcccagcact ttgggagtcc gaggcgggtg 25320gatcacctga ggtcgggagt tcgagaccag cctgaccaac atggagaaac cccatctcta 25380ctaaaaatat aaaattagcc aggcatggtg gcacacgcct ctaatcccag ctactcggga 25440ggctgaggca ggagaagcgc ttaaacctgg gaggtggagt ttgcagtgag ctgagattga 25500gccactgcac tctagcctgg gcaacaagag caaaactctg tctcaaaaaa aaaaaaaaaa 25560agtgaaggtg cccagtgtct gcaactatgt caccccgggc atatcacata tcactctgtt 25620tttccgtctg taaaacggga gcaacaatgc cattgcctta tcatcagaag gatatggaga 25680ctaaatggga gaatgtaggt aaacagcaca gagtatgggt atcagtaagt aaactgcagc 25740agtttgttga tgttaacaat agttagcatt atatctaact atatctaacg atataaccat 25800tgggaatcca gttttccatg attttcctct agaatggagc tgcctaagtc ctgcttaagt 25860catttttctt tgaagattac tgaacatcat cttcaaatgt tcatccttgt aaacacgtgt 25920gtgtgtgtgt gtgtgtgtta atttaaattt tcagaaaaat tctgtaggcc gttggaagga 25980agctaccata ccaggccact taaactccta caccatcaaa ggcctgaagc ctggtgtggt 26040atacgagggc cagctcatca gcatccagca gtacggccac caagaagtga ctcgctttga 26100cttcaccacc accagcacca gcacacctgt gaccagtatg tacacaacca ccctcatgcc 26160tcctaccccc gaggttccta gagctaggct ctcctgaggc aatgctttcc ttctcaattc 26220atattcttcc aggaggggca ccaacgtttt ttaaaatgat gttggcgacg aggacggtaa 26280attttctaga tgactgaagg ctgactttcc cctttctgtg actctctagg caacaccgtg 26340acaggagaga cgactccctt ttctcctctt gtggccactt ctgaatctgt gaccgaaatc 26400acagccagta gctttgtggt ctcctgggtc tcagcttccg acaccgtgtc gggattccgg 26460gtggaatatg agctgagtga ggagggagat gagccacagt acctgggtaa gctcaatatg 26520tcgctcaaga caggttcagg gcagctgctg gaaaactctc cttgtggggg tgggtggcct 26580ctaggcaggt ggtatctgtg gtttggaact ggttgacagc tcagactgaa caaaccaccc 26640tctggcatga ggaagggaag gactgactct ttctaagaag tggccgggtt tttcccaagc 26700cactgtcaca tgttcctggt ccctgatgcc agctgcatca tgcgcctacc tgtgcacaag 26760ttcctacagc aaaagctgtg ttcttggtgg aagtaattac caggactgca gctgacaatg 26820tgagcacagt acggtcactc atacttttca aattgttatg gtgaggggcc tttaaaaaac 26880ttcattggcg cactgaagtg tgtgccatcg taagcactga gttcagtgaa tttgaattct 26940tataaagtga acacaccaca agaccagcac ccagatcaag gaaaagaata tctctctacc 27000ctcaccccat catcttccgc caataatcac taccctgact cttactgcag agagctattt 27060ttaaaaattt ggcattcgat tacaaaaatt atacgtcttt agaaaaaaag ttggaaaaat 27120gaaaggaaga aggaggagga ggagaaagga gaaagacaag aagtggtgtt ttcccatagt 27180acattaataa tcacagatta ggtgggtgcg gtggctaact cctataatct cagcacttcg 27240ggaagctgag gctggtggat cacttgaggc caggagttcg agatcagcct ggccaacatg 27300gtgaacctcc atctctaata aaaatgcaaa aaaaatagcc gggtgtggtg gtgtgcacct 27360gtaatcccag ctactcggga ggccgaggca gaagaattgc ttgaaccgga aaggtagagg 27420atgcagtgag ctgaaattgt gccactgcac tccagcctgg gcaacacagc aagactctgt 27480gtaaaaataa taataataat cactgattag ctattagcac attaccttct agtcgctttt 27540ccctatgaat atataattct taaaatatcc ttcttataag ctgtagagtc atttgaggga 27600ctagtttgct ctgattagtt accttttctt ttcatttcaa agatcttcca agcacagcca 27660cttctgtgaa catccctgac ctgcttcctg gccgaaaata cattgtaaat gtctatcaga 27720tatctgagga tggggagcag agtttgatcc tgtctacttc acaaacaaca ggtacatgtg 27780tgctacatag tgttaaaaga atctttttct gtaaaacaca ggcctgtagt agcacttcct 27840gactgtttgc cccactttct ttctttctta gcgcctgatg cccctcctga cacgactgtg 27900gaccaagttg atgacacctc aattgttgtt cgctggagca gaccccaggc tcccatcaca 27960ggtcagctaa gcgtccccct ctttggctgc tatgttaatc ttaatgacat cagcagggag 28020ggcgcagatt ctgactgcgg acctgcatat cactttaaat ctccaatata atttatggga 28080gaggggtttg tgtgtgtgtg tgtgtgtgtg tgtggcgggg gtgggggagt tattttctat 28140ggcacatttc cccttgaaac catttcacca actcccttat acacacacac cacaacatac 28200acacaacctg taaagccagc tcattggctt attaaagcaa gtgttcccag ggttgaagag 28260gtgtaatttc ctgaaaacgt tgctctaaga tttatcctta aggagaaagc tgagctgtcg 28320tcttagctca ttaggtgatt caactgcctc atcactgaag ttccaaaaag acacacacag 28380tgctagacaa ctctgcttag gctggttcat taattgcttc cctcgtctgg agctcaaaga 28440ggaaaaatca gcttaacatg aatattttca cctaatggca tctctaattg acatttatta 28500aggatgtcag gtcttcaagg atgacattta ttattaaaaa ggttcgcatg actgttctta 28560ttttatcttc gtgctgaata gtcattatta gaagaagtgg caatattcaa agagtcaaaa 28620agtatcactg gctcttcact aatcaagcaa gatgctaagg gatattagaa aagggaggat 28680ttatggtgtt tctaagcctc tgtctcaaag aaaacagtgc atcttacttt tgctcatgaa 28740tctgcagggt acagaatagt ctattcgcca tcagtagaag gtagcagcac agaactcaac 28800cttcctgaaa ctgcaaactc cgtcaccctc agtgacttgc aacctggtgt tcagtataac 28860atcactatct atgctgtgga agaaaatcaa gaaagtacac ctgttgtcat tcaacaagaa 28920accactggca ccccacgctc aggtaacttt tttaagaaga cacttcctat gttatcttat 28980caggattgtt cctgaaggag ggttgttttg tctctgtcaa cagtcctctc attcaagaaa 29040tcttatatat tagtttttcc ctaaacttct gatatttagc tgaaatgtca taagtaactt 29100atcaaagctg gctactggcc tttctgatta aaaactgaca ccataacgtc catctacaaa 29160ttttccccta gaagcttaag ggtcatttca tttttgattc ttaagtatta taaaatgatt 29220cagtaaaaca aaaactgtta cattattttc tgcagttatt tcaaaggctt tttctaaaaa 29280attttttagt atcttttctt ataaccctct ccccccacca ccatatgaca cttcatatgc 29340tggtaactca cttattacct attttagata aaaggttcaa atgtcataat ttaagcacta 29400tgactggacc atgaaaatgt gatctgatta agagacaaaa cttagcaaaa ctctcgaatg 29460agaggctcaa tgaactgcct aacatatcca agaaactggc ttcttaaaag taatcttcag 29520cagagaatgt gaatgaccag ttgactcttg tctgtcagat acagtgccct ctcccaggga 29580cctgcagttt gtggaagtga cagacgtgaa ggtcaccatc atgtggacac cgcctgagag 29640tgcagtgacc ggctaccgtg tggatgtgat ccccgtcaac ctgcctggcg agcacgggca 29700gaggctgccc atcagcagga acacctttgc agaagtcacc gggctgtccc ctggggtcac 29760ctattacttc aaagtctttg cagtgagcca tgggagggag agcaagcctc tgactgctca 29820acagacaacc agtatgtctt ctcctatctc tatctcccct ccaaattctc caccctcact 29880tgcagcctgt gagaaagtgc agtaaaccat tcactcagag gtgtatggct tagagagagg 29940gaaataccca gccggcaagg gaatgcatag tgaacacaaa gcacattaaa cttgaaaaca 30000aaactcagac aagctccatg gatgctaagt ggtaacccat ttctaaaata catgtaccag 30060ctgaagggta ctaagagggg agaactgaag agaatctaat

ttgagtgcat ttttcgtgta 30120actaaatata tctagatcaa agttaaaatg caggatcata acacttagag tagaattcat 30180ttaacaatag caattgtcaa gtgtctagta ttactagcca ccagcttatc tgctcagttt 30240ttacaagcat tattctcata tttactcttt gttttgacct taggaaggaa ggtcttatta 30300ttattatttt atttatttat ttttttgaga tggagtctcg ctctgtcgcc agggctggag 30360tacagtggca ccatctcagc tcactggaac ctctaccacc tgggttcaag caattctcct 30420gcctcagcct cccgagtagc tgggactaca ggcgtgtgcc accatgccct gctaattttt 30480gtgtttttag tagaaatggg tttcgctgtg ttggccaagc tggtctgaaa ctcctgacct 30540caagtgatcc acccactttg gcctcccaaa gtgctgggat tacaggcgtg agccatcgtg 30600cccagccgga aggtcttact agtatccgta ttgaatactt aaagaaactg aggctttaaa 30660aaagttctgc aacttgtagg gtcacagaga taggaaggga tagagctggc tctataacct 30720atgtccgaag cccatgctct caattattat actcgactgc ctcttaaaga tttcctctat 30780ttgaaaggta atttaaattt cggtgggaaa actgctggtt attattctca agaataaact 30840ccacaactta tgtgattctg atagtgcaaa ctcaccagta tcctaccatg aatctgagga 30900tacgttatca ttactgtaat tactgtctaa tctgaaccat gtgaaaataa cttttatttc 30960tctagcaaag ggctattcac agaatattgc ttttgaccca tagagagctt cttcttgctg 31020tcattttagg aggcatatcc ctttttcctt aatctgtttg gctcagagct aactgtgaac 31080ttcagaagtg tttgttttgc ctttttaaaa ataccattgc tttaatgtaa ctataatttc 31140tgagactgat gcgaaagtct tgctggaaaa ttagacttcc caaaggatca cagtcaagca 31200aaatggttcc acaatttctc atgactggca gagttttggc aaagttttgt gtagcactca 31260atctcttact ggctcagttt ttccagggtt ttgactttca catagttaca accttgagga 31320gagaaaactt agacattcaa tcaagtttca ggacttgagt tatgatcatt actgatctaa 31380atatttcttg gcatgtttca tcttttttcc tagaactgga tgctcccact aacctccagt 31440ttgtcaatga aactgattct actgtcctgg tgagatggac tccacctcgg gcccagataa 31500caggataccg actgaccgtg ggccttaccc gaagaggaca gcccaggcag tacaatgtgg 31560gtccctctgt ctccaagtac ccactgagga atctgcagcc tgcatctgag tacaccgtat 31620ccctcgtggc cataaagggc aaccaagaga gccccaaagc cactggagtc tttaccacac 31680gtaagctgaa aattaagtgc cttttcttaa ctatatttac attctctatt cttcatgctt 31740taaaacaaaa caaaacaaaa caaaaaaaac attaaaaaat tagtacataa tttaaatcag 31800tgatactaaa aatgtgctcc atagactggc tgctggcccg ttaattgttt gctgctagtc 31860tgcaacaaga aaaacggttg tgccagaacg taaatcacaa agcacactgc ttagtacagc 31920tgagaatttt tctgaagcca gattttcttg atgaaggaag cagtgtgtta atttgcacac 31980attgccaagc tcgctctttc ctctagggcc agcactttga gtaacatggg tttaaagcag 32040tctgttatta gaaaaattaa attcgattac attaaatgaa tttaccaaac actagttaac 32100gcaagaaaaa attagcacct atgtctacat tctattactt tgggcattga atagtaacta 32160taaatgcaga ataaaaatat ctatggattg aatgggaacc aactaattga acatgaagcc 32220aaggaaatga tttctttatg agtgttggct gcagaagatt aaagtacttt tgcagacgga 32280atcgctcttt tcttaaatta ctcttgaaat tcctcagagg agaaaaatac taacaataat 32340ttttggtcat gtctatcctt ttgctcaaca ttttaaagga agtggtctta aatctcccac 32400atatctacat cacaataaca acctctattc acaaaccgat tcctattaaa tacatttcca 32460tttacattac agagaattat gagactcctt atttctagct gaacatcatt tgttattttc 32520aactcgacat tttgaattat agaagcacct aacataagta ctttttcagc atatattcta 32580accatggact agtttgcaat tttctaagag ctttcaacaa atgttactct tcgactaatt 32640taaaagtatg gatgttaaaa agcattcaaa aagtccatac aagcctagtt tgtaaataac 32700tatggaattg atttcccaaa gaaaatacaa acttttcccc ataagaattc atactttaag 32760aaaaacttac ttccatttaa atttactgta tgaagtttgg ctcatgaagg ctttttccta 32820aataatagtt aatcgtaagc aagtaaaatt cacttttaat ttgcaaataa gcttacttga 32880aaatttggct aaaattttac acggttctaa gatagtctaa gatctactct catgaaatta 32940atgtctttat atttcttgta aatattcatt tcttataaat gtccttcagt gaattagaat 33000ggagatttca gtgaatgcgc cctttcagta gatgtcgtct tttactaaaa tgtagaattc 33060tatagttgtc ttgttcattc cttaacatga gacatatttt atgtagtttc ttttgttgaa 33120cacagtgctt ataaaagaaa aagcattttt aatgatgcta acaataatta agggaaggtg 33180gtgggccaag atatttcaag tacttctgaa gactgatata ttggatatat tattttatgc 33240tttgcataat acattcatat aaaatataat gattttaact gaagtactga tagccaaaac 33300taattttatt agataaaggt taacatagtg tctggaacat tgtatgcttt caaaaactat 33360ttggtgaata gataattgag aaaggaataa taataaaaac agctaaatga agcttatatt 33420taaataaact aatgtaagca gggtattcta caactccatt tgaactttaa gcactcccta 33480aggctgtaaa catccacaag gcttgcatgt ttttgaaatt actaaatttc tgtagttttt 33540tactatctta ctaagctgaa ttctgggagt aacttttctg agttttataa cttgtgctaa 33600attcttaaga gcaaatgtga gaaaagttag gggaaaaagc tgtttctggg gaaaattcag 33660cttagtctta tattgatagg gcaaatttta tttctttaac agctgggctg ttcttcccta 33720acaagacctg ccagaaccca tagctcacac ttagaatcac atcctttgtt tgacatcgtt 33780tgtgggtttg tggtttggtg attttcccat aatggccttc ccaggcagag agcatcatct 33840taaacttggg aagactctag gtggctggct caagcaatag aaaataccta gtcttaaagc 33900ccaggacagt tgaggcgaat ataatttgta aaaaaagttg tggttttcac agatgttcag 33960tgaaagaaac tgactgttct ctgaattgtt ttttgtgggc cattaaaaat ggtcacacag 34020ggctggccat ggcattttgg cacagtcacc agcagtcaag tggtgtataa tttcagaggt 34080actaaaaggc atcgggtcac ccatcccagt catgtccccc caccccccac caaacaccat 34140caaaataata acatacatag atgaaacagc ccactaaacc agtagtacct caattcagcc 34200attagagcta tcattatgaa gtggcccaca catatatttg gctttttctc acacaatatt 34260tttaaaataa aaataaaaaa tggctcctta gaatccagac tgaaaaaaaa atgcgagaat 34320tgcgatgttg attctaaatt cccacatggc aaagaaaaaa aaattgactg gagttgagtt 34380gagactggag tctgaacact ttttaatccc tgtttgtata actctggaga ctaattcttt 34440gtcttgccag ccattcataa tttagtataa atgcattcag aggttttttc ccaatgggaa 34500caaaatttga ttgagatgta aagagaggaa gaattgtgga gatgtcaaac atgtgaccag 34560agctatgaac acacttgata cccttgaact taaacttacc caactcaaaa tatctggcct 34620tctgcgatct tacctttcta catttataat aagatttgca aggttgttgg aaatctctat 34680cttaacttta tatatacatg cctctttctt tctttttctt tcctttcctt tccctttctt 34740tctttctctt tccttccttc cttcctttct ctctctctct tttcttcctt tcctttcctt 34800tccttccttt ctttcttcct ttctttcttt ctttctttct ttctttcttt ctttctttct 34860ttctttcttt tctttctttc tctctcactc tctctctctt tctttctttc ttatttttgg 34920tgctaaaacc caaaacaaat cttttattta aaaataagat tttttttttt tttggccagg 34980tgtgatggct catgcctgta attccagcac tttgggaggc cgaggtgggt agatcaccta 35040aggtcaggag ttcgagactg gcctggccaa catagtgaaa ccccatcttt actaaaaata 35100caaaaattag ctggatgtgg tggtgggcaa ctgtagtccc agctacttgg gagtctgagg 35160caggagaatc acttgaaccc aggaggctga ggttacagtg agatgagatt gcgccactgc 35220actccagcct gggtgacaga gcaagagtcc atctcaaaaa aaaaattgtt taagtaaaat 35280tttattttct ttcttttttt tttttttttt tttttttgag atggagcctt gctctctcac 35340cctggctgga gtgcagtggt gtgatcttgg ctcactgaaa cctccatctc ccaggttcag 35400gtgattctcc tgcctcagct tcccaagcag ctaggattac aggcatccac catcacacct 35460ggctaatttt tatattttta gcagagacaa ggtttcacca tgttggccag gttggtcttg 35520aactcctggc ctcaagtgat tcacctacat cagcctccca aagtgctggg attacaggca 35580tgagccactg cgcctggcca aagtaatatt ttcaataaga aaaataacag tgatgtcagg 35640atgctagaaa atgcaaaata aatttgttat aattcctgat tctggtgata aaatactaaa 35700tttttgcagt attattctga aagaataatc acgtatttaa agtacaaatc tttagacttc 35760aaagtgcatc catggtggca gattttgttt aacttttata tagcatcttt tattgcaacc 35820aaaaatagct gaccattatt gtggaataat tcagcgtaaa gctttttttt tttctttttt 35880tgagacggag tctcattctg tcacccaggc tggagtgcaa tggcatgatc tcggctcact 35940gcaacttcta tctccagggt tcaagcgatt cttgtgtctc agcctcccaa gaagctggga 36000ctacaggcat gagccaccat gacagttaat ttttcatatt tttaatagag acagggtttc 36060accatgtttg ccagcctggt ctcgaactcc tgacctcaag tgatccaccc acctcggctt 36120cccaaagtgc tgggattaca gtcatgagcc accgatcccc gcccagcata aagctgtttt 36180tagatcacct tctataattt accattgttc ttaaattaat ggttaagaaa caatatgata 36240atcagtttgt ggtggccagt tttacatttt ataagggatt ttacactgac gatataggga 36300aatttattgt catcaaagtc aaatcccaac aatcataaaa agacatgaat tagattatat 36360aatttcaatg aaaggaccac cttagcaaac atctaacccc ttgttactca ggtatcattc 36420acgggccagc agaattggta ttatctgtgt ttgtcagaaa tgcaaactcc ctagtttttt 36480gttggacctc cagaaacaga atctgcattt tagccagacc ccagatgatg tgtgtggcac 36540attaaagatt gagaagcctg atcctctccc aattctcacc caatgaaaaa atatgttaca 36600tcctctatcc actgcttggt taaactgagg ttctccataa aaatacttgt tatctatatg 36660ctatgcaatc atctgtgagt ttgagttttg aatatgtgca ttgattctct ctcacagtgc 36720agcctgggag ctctattcca ccttacaaca ccgaggtgac tgagaccacc attgtgatca 36780catggacgcc tgctccaaga attggtttta aggtaaactg cagatgttcc taatctctgt 36840gatacagccc tgagctgtcc ttgtggttcc catgtagtgg aaacagggtg ctcaggagtc 36900aggagacctg ggttttgtca cctgcttctg tccatacatc tttgactaca ttgtcagggc 36960ctaacagtcc ttccctgcct acctcactga attgttggaa gggtagatgg aggctgcgaa 37020agtgttttgc aaaggataaa acattagcac gaagctgctg cttattgtta tcttattttc 37080tctatccttt cctgcaggga attacatttc aaaaaaacat gggaaaactt tatttgatgt 37140gttgttctaa atgagtgtga acaagttcac aaaagccagt ttagggagac cagttaaact 37200cagagtcact taaaaatcgc attttcatcc aatcagtttc atctccaact gttcaaagca 37260ctgagggtga atctcttaat agaagttaag attaaggttt ccctgtggat atctggattc 37320atcttcttta aagtaatgat attagggaag cggtgaatac aaatgaatat gtttaaaaga 37380attccattct ttggcattta gtgtgaagag agaaatattt gttatcgctg gaaatcatga 37440ctcaatcccc ttgatcgttt aaaaaaatac accaaagata aagtttgtaa atggccatat 37500ttatgattat gctactcaaa tatagaagaa ctttctgaag agtgccagta taccttttaa 37560ttcccttaat aatgtcatgc tgactttcag aagccttatg atgtgtgaag gatctctcta 37620gagttgaaca ctattggata acagtgttac ctaagttttt gaaatagaat cttaaaagga 37680ttttaaatta tgggcatagt tattctaatt cttctcttgt aatgtatcat cctgcagttg 37740aagctatgta catatctctt caaaaggtgt gtttttgcaa tacagttgct acaggggctg 37800gtgcctttaa atggcaacta aaaggttaat tgaatgtgaa taactcgtta aagggagagc 37860tcagacattc cttctagcac acacacagaa aaatagaaat gaactaccat gtgacccaca 37920gtccctgtat acgtctggtt tgtaacaaga gattctttat caagcaaaac agtatgtaat 37980gacatttctc tggaaccttc cattacaagc caccttaatg cagtttggaa gatacctccc 38040ccacctgggg gaatttccag ctaaagtata taaaagagtc cccaaatcat ttcccaataa 38100aagtacactg tgcagtttct gaagagttta cactatttaa agcataatca tagcctcaca 38160gcagtaacag tcctctggaa atatttgtcc ttggtgttta ctttgcattc cttcctctag 38220ctgggtgtac gaccaagcca gggaggagag gcaccacgag aagtgacttc agactcagga 38280agcatcgttg tgtccggctt gactccagga gtagaatacg tctacaccat ccaagtcctg 38340agagatggac aggaaagaga tgcgccaatt gtaaacaaag tggtgacacg taagaagaat 38400tttttccctt ttctattagt ttttaaaact gttctacttt tcgaaaaaag ctagtgtcaa 38460tatcactttt tacttatgag aatggcacag gggagatatc ttatccttac ttatattatt 38520aatgctattc ctgaatttgg agtagcaggt tcaattcatg ctttatattt tatggcatca 38580gaaatgttct ttccatgtca agaatattta tgaactggca agatgaaaat aatttcaata 38640gtattgcaaa atcattaatc ataaagaagt ctttgtcaga gaattactgc tgtccatcat 38700attttcataa tgtaacctat atttttatgg gagggaggga gggagggaag aagcatggaa 38760gagagggagg tagggaagga ggaaagatta cacaggtcaa gagctttgtg tcagctttga 38820cttttaaaat gttctttctg catagcattg tctccaccaa caaacttgca tctggaggca 38880aaccctgaca ctggagtgct cacagtctcc tgggagagga gcaccacccc aggtaagttt 38940gggatggatc agagggcaag tatacaccat accttcccaa gacaaagatt ttagaaactg 39000tgtttctttc agagaaagaa gggattcaaa ttacaaatgc ttagctctcc ataaaaacta 39060tagcagtaca tgatgtacat catggagcag cctgcaggat gctttaatgc acgttgactt 39120caatcacagg aagcagaaca accttacact agtctagggg gacaagacag atcctcacac 39180agctgtaggg ctgaagaaaa gcactgtgga aggtggcttt tgctgagtgc attaaaaatt 39240gccaaacaaa tggttgaagt catattacgt aatttccctt ttgtgagttg ttacagagcc 39300taagtttatt attccctgaa gttttatgaa tgttttgtca tgggttgcac cacaaatatt 39360taagggtgat gaaaggcaga aataccttca ttttacagaa taagaaaact gagacttaga 39420aaaaattggc ctactcatag tcacacattt taaatgttac aaaactggga ttgtaactta 39480agtttgtaga ctccttctca cacccaatgt cacatatttg gaatgtaatt tttttttaat 39540tatattctat gcaaactgaa aattctgatt aaggggtttc ctgaccattt ttagagcttt 39600aaatgaagca tttgtctaaa ttccttgttc acatatattt gaaaattatt tataaaatgc 39660taaaatgtat aaagatagtt gttaacaata ttcaaacagc atgacgtact atagcaataa 39720caggaaattt tagataccca ttacttttgc caaaaccaca tggaagtctc aacaaaccta 39780tggagaaaaa tcttaaacaa aaataaaagt tccaattaat gttgattgca ttcttacctc 39840atatacttgt taatttaagg gatatgttta ggttattatt tagctatttc taattttact 39900gtaaaattct tgtgaaattt ttgtttaaaa aaaagtatta tatagttctt atctttgccg 39960gggcacagag ctaaggctat catctctaaa tctgattaat gtatgcaaac acacagaatg 40020aaactagctc agaatatctc ttttaatctc cctctgaagt agagtgattt tggtaaagtt 40080ttcattatct gcggaaacat tgtttaagcc aaagctatac aatttccagc tgagttgctc 40140tgaatttgaa actttaagtt gacaatcttc gtgcttgtta gcagcaggat cattaatatc 40200tcgtctcaat ggcccagccc acacatatgg atgaccacta gcaagtgtaa tgatctcaat 40260atttatttct cattcagttg ggtttccttg tatttgccac attagtgttt accctgttcc 40320taatggcaaa atattctgtc atctccttgc cttttataaa gtttaatata ctttctcatt 40380ttaatctgtc cccacagatc tctagtcatc actgttttta tttgaatgtc tctcatccct 40440ctcaactctt ttactgccca atttctgtga ttcctgaaga cttcaacaat caatactctc 40500tttttttgtt tgttttgttt tttttttttg agacagagtc tcactctgtc acccaggctt 40560ggagtgcagt ggcgccatct caactcactg caacctccgt ctcctgggct caagcgattc 40620tcgtgcctca gcctccccaa gtagctggga ctacagacat gcgccaccaa gcccagctat 40680tttttagatt tttagtagag acagggtttt accgtgttgg ccaggctggt ctcgaactcc 40740caacctcagg tgatctgcct gcctcaacct cccaatcaat actctttcta gaataagtat 40800cagcactttt gtttctcacc ttttctcctt tcttggttct cttcctataa atcccatagt 40860ttcagacctt ttaaattagg agagctctct ggggaatgtg cttaaggtgg agagcgattc 40920tatactaggc aggtagaaag gaatattcct cagctgtctt caaatgattc attaaggaaa 40980agcagggtac agtgatagga ccatgagatt tggaaacaaa gaaagctttg gggaatcact 41040cccctggttc aagatttcct ttaaagtgag gatcttggcg gaggttgaag tgagccaaga 41100tcacaccgct gcactccagc ctgggtgata gagggagact gtctcaaaaa ataaaaataa 41160aaaaataaag tgaggatctt agtactgcct gaaaggattg ttgcaagcat tgaataacag 41220tgacagtgga gtcctcagta aatgccaagt cctgcattcc gccctgtgaa tccatcattg 41280gagtctagtt aaatatgctc tggctcacag atcctctgtg caataacttc ccttttcttt 41340tttctccaga cattactggt tatagaatta ccacaacccc tacaaacggc cagcagggaa 41400attctttgga agaagtggtc catgctgatc agagctcctg cacttttgat aacctgagtc 41460ccggcctgga gtacaatgtc agtgtttaca ctgtcaagga tgacaaggaa agtgtcccta 41520tctctgatac catcatccca ggtaatagaa aaataagctg ctatcctgag agtgacactt 41580ccaataagag tggggattag catcttaatc cccagatgct taagggtgtc aactatattt 41640gggatttaat tccgatctcc cagctgcact ttccaaaacc aagaagtcaa agcagcgatt 41700tggacaaatg cttgctgtta acactgcttt actgtctgtg cttcactggg atgctgtgtg 41760ttgcagcgag tatgtaatgg agtggcagcc atggctttaa ctctgtattg tctgctcaca 41820tggaagtatg actaaaacac tgtcacgtgt ctgtactcag tactgatagg ctcaaagtaa 41880tatggtaaat gcatcccatc agtacatttc tgcccgattt tacaatccat atcaatttcc 41940aacagctgcc tataaaatag ttttgtccct gtatgtgagc actgaaacag catttggttg 42000acacatctag tttttcatct tgcagtttca aatccttctt tttgaaaatt ggattttaaa 42060aaaaagaagt aaaagtcaca ccttcagggt gttctttctt gtggcttgaa agacaacatt 42120gcaaaggcct gtctaaggat aggcttgttt gtccattggg ttataacata atgaaagcat 42180tggacagatc gtgtccccct ttggactctt cagtagaatg cttttactaa cgctaattac 42240atgttttgat tatgaatgaa ctaaaatagt ggcaatggcc ttaaccttag gcctgtcttt 42300cctcagcctg aatgtgcttt tgaatggcac atttcacacc atacattcat aatgcattag 42360cgttatggcc atgatgttgt catgagtttt gtatgggaga aaaaaaatca atttatcacc 42420catttattat tttttaacct tcttcatgca agcttatttt ctactaaaac agttttggaa 42480ttattaaaag cattgctgat acttacttca gatattatgt ctaggctcta agaatggttt 42540tgacatccta aacagccata tgatttttag gaatctgaac agttcaaatt gtacccttta 42600aggatgtttt caaaatgtaa aaaatatata tatatatata ttccctaaaa gaatattcct 42660gtttattctt ctagggaagc aaactgttca tgatgcttag gaagtctttt cagagaattt 42720aaaacagatt gcatattacc atcattgctt taacattcca ccaattttac tactagtaac 42780ctgatataca ctgctttatt ttttcctctt tttttccctc tattttcctt ttgcctcccc 42840ctccctttgc tttgtaactc aatagaggtg ccccaactca ctgacctaag ctttgttgat 42900ataaccgatt caagcatcgg cctgaggtgg accccgctaa actcttccac cattattggg 42960taccgcatca cagtagttgc ggcaggagaa ggtatcccta tttttgaaga ttttgtggac 43020tcctcagtag gatactacac agtcacaggg ctggagccgg gcattgacta tgatatcagc 43080gttatcactc tcattaatgg cggcgagagt gcccctacta cactgacaca acaaacgggt 43140gaattttgaa aacttctgcg tttgagacat agatggtgtt gcatgctgcc accagttact 43200ccggttaaat atggatgttt catgggggaa gtcagcaatt ggccaaagat tcagataggg 43260tggattgggg ggataaggaa tcaaatgcat ctgctaaact gattggagaa aaacacatgc 43320aagtattctt cagtacactc tcatttaaac cacaagtaga tataaagcta gagaaataca 43380gatgtctgct ctgttaaata taaaatagca aatgttcatt caatttgaag acctagaatt 43440tttcgtctta aataccaaac acgaatacca aattgcgtaa gtaccaatta attataagaa 43500atatatcacc aaaatgtacc atcatgatct tccttctacc ctttgataaa ctctaccatg 43560ctccttcttt gtagctaaaa acccatcaaa atttagggta gagtggatgg gcattgtttt 43620gaggtaggag aaaagtaaac ttgggagcat tctaggtttt gttgctgtca ctaggtaaag 43680aaacacctct ttaaccacag tctggggaca agcatgcaac attttaaagg ttctctgctg 43740tgcatgggaa aagaaacatg ctgagaacca atttgcatga acatgttcac ttgtaagtag 43800aattcactga atggaactgt agctctagat atctcacatg gggggaagtt taggaccctc 43860ttgtcttttt gtctgtgtgc atgtatttct ttgtaaagta ctgctatgtt tctctttgct 43920gtgtggcaac ttaagcctct tcggcctggg ataaaataat ctgcagtggt attaataatg 43980tacataaagt caacatattt gaaagtagat taaaattttt tttaaatata tcaatgatgg 44040caaaaaggtt aaagggggcc taacagtact gtgtgtagtg ttttattttt aacagtagta 44100cactataact taaaatagac ttagattaga ctgtttgcat gattatgatt ctgtttcctt 44160tatgcatgaa atattgattt tacctttcca gctacttcgt tagctttaat tttaaaatta 44220cattaactga gtcttccttc ttgttcgaaa ccagctgttc ctcctcccac tgacctgcga 44280ttcaccaaca ttggtccaga caccatgcgt gtcacctggg ctccaccccc atccattgat 44340ttaaccaact tcctggtgcg ttactcacct gtgaaaaatg aggaagatgt tgcagagttg 44400tcaatttctc cttcagacaa tgcagtggtc ttaacaagta agcagttgaa tgtatctgtt 44460ccataaatat taacctagag catagcaaat gaattctaaa ttctcaagta ggaggagcta 44520agagcaagag agctgcaacc aagctacaaa ctaaactctg aattcaatgc acagctccat 44580taattttgaa agatgtaatg tttgttgcta tcttaatata cttttgatat ctacagcttt 44640aaaaaaatca tagtggaaaa acacctgcag gaaagttcca tgacttcaaa caaattctgc 44700ttctaaataa gcacgtaaaa ataagtgaat atcaagagaa attatatgac taaatctaaa 44760tctttagaga aaaaaatgag aactgaaaat agtgtcacca tatgtgcttt attctcattt 44820ttataaaaaa agtgtcagca gttgattgat ttaggatttg aatacttaga aaagtgactg 44880attgtttggt ctagattaga atgttgttgt gaagagagtc agaagtttaa tttgtacttc 44940aaaaagaatc tgttagaagg atttctcaga agactgagag cttagaaaaa aaactgacat 45000taaataaata acaacaattt atggaaattg tctctttcta gtcccaacca ttatagaata 45060gacatctttt gttaaagaat aaaacagtag gctgcaagat ggtgctgtgt ttcacataaa 45120cagtgctttt tattattttc actgtaatag tcaaatatat

aacaacagca aaagattcta 45180cataagggaa aaatagctta catttaggta cattaccaag tattagtctg aaaacatcta 45240cctttcaaac ataatttaga taatgaaaca caaagaagag cagctcagcg tgaccataat 45300cttggtttct tactttgtgg ctgagggcaa gaatatcttt atattggcat atccaccacc 45360ccagggctgt tgcttctgtt ctagagcacc ctggaatcac taattacagc atcacccagt 45420atacaagccc ctgcatcaca atgtctgtcc cttagccgta gacctgtcac atgctaatca 45480tgtgttctaa gaccttatta taatcctaat gctacagatg acctcagggt agcccctctc 45540cctcctagca aagtcattat tatcctcttt taaagatgaa gcaaaccagt gcagtggctc 45600tcgcctgtat aatcccagca ctttgggagg ctgaggtggg ccaattgctt gagcccagga 45660gttcgagacc agcctgggca acacagtgag accaagtctc tacaaaaaat acaaaaatta 45720gccgggcatg gtggtgcaca cctgtggtct cagctatgta ggaagttgag gtaggaggat 45780cacctgagcc tggggaggtt gaggctgcag caagccatga tcgtgccact gcactccagc 45840ctgggtgaca gagtgagacc ctgtctcaaa aaaataaaca aacaaataaa catgaagccg 45900tcgaggtccc cagcagttaa gtaaattgcc actggccagc tagtatggtg gaaatggaat 45960ccaggcatct ggtcctccat tctggcactt ttccaaactt ttgtaggggg ctttatagaa 46020acgctgaagg gcaaatggtg tgcaggggaa tgagggttta gtgagtagga gttccaaatt 46080tagaaacctc actctctgta tcttttagat atacaaatat ttaccatata ttacagttgc 46140ctctagtgtt cagtacagta acatgctgca caggctcata gcctaggagc aataggctag 46200accacatagc ttatagctta ggtgtgtcaa aggctctacc atctaggttt tttgttttgt 46260tttgttttgt tttgtttttg agacggagtc tccctctgtt gcccaggatg gagtgcagtg 46320gcacgatctg ggctcactgc aacctccacc accaaggctc aagtgattct cctgcctcag 46380cctcccgagt agctgggatt acaggcgtga gccaccacgc ccagctaatt tttttgtatt 46440tttagtagac gaggggtttc accatgttgg ccaggctggt ttcaaactcc tgacctcaag 46500tgatccaccc tcctcggcct cccaaagtgc tgggattaca agcccatcta ggtttgtgta 46560ggtacactct atgatgctta cacaataaaa tcaccaaatc acacacttat caaaatgtat 46620ctccaccatt aagctatgcc tgactgtgta tcaaaatgga agaagaagct gggcacagtg 46680gctcacgcct gtaatcccag cactttgaga ggccaaggcg ggtggatcac aaggtcagga 46740tttccagtcc aagcctggtt aacacggtga agccccgtct ctattaaaaa tacaaaaatt 46800agccaggcat ggtggcaggc acctgtaatc ccagctactc aggagtctga ggcaggagaa 46860tcacttgaac ccaggaggca gaggtttcag tgagccaaga tcacaccact gcactccggc 46920ctgggcaaca gagtaaaacc tcgtcaaaaa gaaaaaataa taaaaataaa aaaaaatgga 46980acaagatact cagggatgta tatttaattt tttaaaaaat attctgctct cattttaata 47040tggcagaacc gattgctttc taagtgtggc ttttttccag taaaggttaa ttattaagac 47100cactagtcct ggcctgggtc aatcccagta tgatcctggg caagtaaatt aaagaagata 47160acttctctgt gcctcagttt ttttttgttt tgttttttgt tttttcattt acaaaatgga 47220gataattgta gtaaatcaaa tttttagagg tgataggttt gttcatttct tgaatgcggt 47280gatggtctcc caagtcacac atatgtaaaa acccatcact ttaaagatat gcagtacgtt 47340gtatgacaag aaattgcttt taaaaggagc aaactacctt ccagggttgt tgtgaggcat 47400aaatggcaat ccacagcacc acagcaagga ttatcatgtg ccctccagag acatactctc 47460aggtggatgc gagaaatatc cagctgttgc agcaacttca tcccactcga aatccagctg 47520agtcacactc acaggtggaa tggagggctt cgagaggcca tggggcaagg tgacccttcc 47580ttatcatcta attacagacc ttctcaaggt ctgttcactg aacacttcgc tgtagtgttc 47640acttaggtgt aagtaggcta taggactgga catttggata tttcatcagt tcaaatagtc 47700ctgggcgtgc tttagtttct catgcttttg agcagagttt taaaataagc cccatttgcc 47760cctacagatc tcctgcctgg tacagaatat gtagtgagtg tctccagtgt ctacgaacaa 47820catgagagca cacctcttag aggaagacag aaaacaggtg agtggtgttg gcagtatgac 47880tatccagtag cttttgccta tcaattctgt ataacaaatg aaatgctact tctaaaaata 47940catctccatt ttttgttgtc atggtgtgtg tacctttgtc atcacagtat gattttatcg 48000ctggtctcaa aaactaaaag ataccttact caacaatcac ctagactttc agtcactaac 48060aaattaagaa atttgttgtc tgtcctttta aaaaacattt tctaagaaga tctttgttat 48120ttagatttag cagacattcc ttttcattag gcagctctgt ctaatggctg acccaacact 48180cattgtcatc tatttgtctt cctttactaa gccagcaagt ttacattttc tttttactta 48240ataaaatatg catttactag aaggaagttg aattgaatct cataaatatt acatacttaa 48300atatgaatgc ttttaatttt ttctttcaaa aggtacactt tagtgtattc attaatttat 48360ttatagtcca cttgcttcca aaaaggactt atgatatctt agtttggttt cttattgaaa 48420agaactagta aatgctgtaa ctgaaacaga aatttgctgg aagtcccaga gactaagtga 48480tttgaatttg caacaaactc tgaatttttg tgcatttttg aaaaatgcat ttttcaaaac 48540tgtcaattca cgaggaatta tcagcattgt aatttgtctg ggataatgtc tttagtttca 48600gaaagttttg tgtttggcat cattaccact ctgttgacat ataaatttcc tcttgagctt 48660aggaggcttc tctgagagtc aaacatttac tttgagagtg ggcagatctt gctttacttg 48720gaaggataca cttacaggat agaaacacag aatacttgaa cactgaagaa tttgaaaatg 48780tcaattctca gaagatcttg aacacttatc tccaaatgtg acacagaaac ttactgtaat 48840aacccctaaa atctgcttga attacttagc acaagaaaaa aatgaatgct tgagctggct 48900attttgaatt gagtcaattt aagattttaa aattcatatg tagcttagaa tcagtacatc 48960ttactctttg gtttatggca aatcatggta ttgatgagac aggaacgaaa tgttggatgt 49020acgttaattt cccctacacc ttcctcactt cctaaactgg tggtgtcttt tctttttttt 49080ttctcttcct cccccgggtg ggaaaaacag gtcttgattc cccaactggc attgactttt 49140ctgatattac tgccaactct tttactgtgc actggattgc tcctcgagcc accatcactg 49200gctacaggat ccgccatcat cccgagcact tcagtgggag acctcgagaa gatcgggtgc 49260cccactctcg gaattccatc accctcacca acctcactcc aggcacagag tatgtggtca 49320gcatcgttgc tcttaatggc agagaggaaa gtcccttatt gattggccaa caatcaacag 49380gtaacttttc ttgtctgcaa agaaactcag aagactttcc tacccagttg gtagattctg 49440taaagtagct tgctgttgtc tgtcatcagc tctcaaaaaa aaaaaaaaaa aaaaaaaaaa 49500tagatcattg tcatggtaca tggagaggga agtgagaaaa tgtggagaaa catcttcctt 49560agaatatggt aaagaagccc gggcgtggtg gcagtaaaga agataatttt tttcctctca 49620agaaatttct cacctgattt gggtatttat gcatttctaa taacacaagt tttgttgaaa 49680atgtagaaaa ttggccggac ggggtggctc acgtcagtaa tctcagcact ttgggaggcc 49740gaaatgggca gatcacttga ggtcagaagt tcaagaccag cctggccaac atagtgaaac 49800cccatctcta ctaaatatac aaaaattagc aaggtgtggt ggcatgcacc tgtaatccca 49860gctactgggg aggctgaggc aggagaatct ttgaacctgg gaggcgaagg ttgcagtgag 49920ctgagatcag gccattgcac tccaacctgg gtgacagagc aagaccctgt ctcaaaaaaa 49980aaaaaaaaaa aagggccagg cgcaatggct cacgcttgta atcccagcac tttgggaggc 50040caaggcgggt ggatcacgag gtcaagagat cgagaccatc ctggccaaca tgatgaaacc 50100tcgtctctac taaaaataca aaaattagct gggcgtggtg gcatgcacct gtagtcccag 50160ctactcagga ggctgaggca ggagaattgc ttgaacccag gaggcggagg ttgcagtaag 50220ccaagattgt gtcactgcac tccagcctgg tgacagaggg agactctgtc tcaaaaaaaa 50280aaaaaaaaaa ggtggaaaac tgaacactgt ttcaaagtac ctttaaaaat ataattttag 50340ggtaatagtg tcattgttct tagcagatag aggctgaagt acttacggga acagtagcat 50400catgttatct gtattttagt ctcaagtcgt caagccagag acaaatacct aagggaaggg 50460tatataggtg ttcattgtat tacttttttt tttttttttt tcttcctgaa atggagtctt 50520gctctgtcgc ccaggctgga gtgcaatggt gggatcttgg ctcactgcaa cctctgcctc 50580ccaggctcga gcaattctct tgcctcagcc tcccaagtag ctgggactac aggtgcccgc 50640caccacgccc ggctaatttt tgtattttta gtagagatgg gattttacca tgttggccag 50700gctggttttg aactcctgac ctcaaatgat ccacccgcct cggcctccca aagtgctggg 50760attacaggcg tgagccacca cgcccggcct tgtatcactt tttttttttt tttttttttt 50820aacttttctg tagttttgaa atttttccaa ataaaatgtt gagggaaaaa aactcttccc 50880caaatttgaa ataatcattt tatcacaatt tgaatgggct ctgtaacccc ttatcttgaa 50940ttcgtcataa tataaaattc tgctaattac acgtagtatt tacatgattg tatggaagaa 51000tcattaagac aattatctgg aaaatgaaca aacagtaaat ctgaatattg tttgaaaatt 51060acggatgtga aaagtttccc ttttttttct agtttctgat gttccgaggg acctggaagt 51120tgttgctgcg acccccacca gcctactgat cagctgggat gctcctgctg tcacagtgag 51180atattacagg atcacttacg gagagacagg tacagcagta aaatgctatt ttacactctg 51240attaaatcag attctgttgt ggataacctg aaagcccaac agtgaacaaa gaattaaaga 51300aactttggca agtccattca acggagccct tgttttttcc aagaaaatac gtaagatata 51360gatgatataa tttgttctaa aacccaaata aaaagttgtt tatatactac aactagaggg 51420ggaacggcag agctgaggaa ataaaaggat tgtaaattca caaacatatt atcagtggtg 51480gaaataagtg atttttattt tttcttctct ttacttttct gtattttcca aattttattt 51540aaaaggaatg tattctgtta aaagttttaa aaaggacaca atgcatgcaa tcctgggttg 51600agggcttacc ttctcccact tctaatgcta ctctactact cagtgacatt ttaaagctga 51660aatgttaaaa cagcgctaac tgtaattttc tctcaatgtt tatacactta ccaaggtttg 51720ctacatgcat aaatacccct ttctgttcaa gatagcgctc tttaaaaggg aataagcaag 51780aagatgtgat ttacatgctg ctataaatgt ggtaattcaa ttaatcagta atacccaagt 51840agctctaaac ccctcacact ctgaactaac cctttttcat acaggaggaa atagccctgt 51900ccaggagttc actgtgcctg ggagcaagtc tacagctacc atcagcggcc ttaaacctgg 51960agttgattat accatcactg tgtatgctgt cactggccgt ggagacagcc ccgcaagcag 52020caagccaatt tccattaatt accgaacagg tacaaacttc tactctgggg tgacaccagc 52080ttttacttat tcagatactg ttttgcaatg ttctcccaag gtatttttct aattgtagaa 52140tagattttcc tttttaatga gcaacaacct gcagctagca cctgcagcga acagagtttt 52200gagccagata aagaaggaag caccccaagg gcaggaagtt cagtcagttt tgtcgatata 52260ttccgcatgt ctgcaatacg acaggcatag agagtgttca gtaagtattt gtgggaaaag 52320aatggatgag ttgataaagt aggaagagac acctgcttgt ggaatgtagc ttctttgtga 52380atgaagcaac catctcaaaa ataggaaatg gtattgagat gcctgcccca tccctctaaa 52440agctctctct gtattctttc gagaagaaat acctttctca tgtaagcgat cattcgaata 52500tgtaccagac ctagagagga ggacttgtcc aatcttgtct ccaaggactg gggcttcact 52560ggtttctccc tgcttttatt tgtagaaatt gacaaaccat cccagatgca agtgaccgat 52620gttcaggaca acagcattag tgtcaagtgg ctgccttcaa gttcccctgt tactggttac 52680agagtaacca ccactcccaa aaatggacca ggaccaacaa aaactaaaac tgcaggtcca 52740ggtaagaatc atctgcatct cggccaggtg cggtggctca ctcctataat cccagaactt 52800tgggaggctg atgcgggcag atcacttgag gttaggagtt cgagaccagc ctggccaata 52860tggcgaaacc ccgtctgtac taaaaaatac aaaaaaatta gctgggcatg gtggcttgtg 52920cctgtaatcc cagctactca ggaggctgag gcaggagaat ggcttgaagt ctggaggcag 52980aggttgcagt gagccaagat agccccactg cactccagcc tgggtgacag agtgagagac 53040tccatctcag ggaaaaaaaa aaaaaaagag taatctgcat ctcatataca acaggataga 53100tggggtagga ccacctaata ttctttttta tataaatggc taccttgttg tgagtactat 53160gtattttttt gtcctatgtc atcattgtcc ccattcatga gttcagggct caagatcatt 53220atcaaccctt ttcacagtag aagtcttaag tgcatttctg tttttacatg gatagttcta 53280tttagtgata tggacatctt aaattactag attcaccctt ctggttttgt ttatcattca 53340cactaagaag agataaatgg cctaactgac tttttcagct ctttttagct atgttgtctt 53400tgtttttaaa tagaatactt gtgaaattag gatcttaagg caatttatta gagtcaagtt 53460aattttcatt ttttctgaga gcagtatcac taattgttgg gggcatcata ttaagtttta 53520gatcttatcc ttgagtgtga cttcactccc atatggtaat ttgtattagc aatgaacagg 53580tttgtccaag aggaaatcaa agtctgactc tccatatttt tgttacaatt ctgcaaataa 53640aaattctagg ccaccatatg tttactacca aactctagac gccacttgag gactttatag 53700tggatgacgt ggatgttgca tttgcttttc actccctttg cagatcaaac agaaatgact 53760attgaaggct tgcagcccac agtggagtat gtggttagtg tctatgctca gaatccaagc 53820ggagagagtc agcctctggt tcagactgca gtaaccagta cgtaaccact gcttggtttc 53880cattttcaaa gtcaaatttt gttcttgggt gtctgaatgc ccacgacatg tcttttgcaa 53940ttacacatag ggaaagtgaa cttgttggtt agtttatgtc ttgagctgag ccctttacga 54000acatcttttt tccttctcag tgccaagcga ggaatttaca gagaaagaag ttgtgaaacc 54060accatagtta gttgctgtgc tttgaatttc ttttgctcaa atggcctcag cgaaatctta 54120tttgcctata gcaaatctac aaaaaatttt cctagaccgt cttttctaca actggatggt 54180aaagttgatt gaagtgtgcc tcatgtagct ttatgtttgg ggcatttgaa gggctatggc 54240tggaccagag tgtaatataa atgcttaata gagaggggaa aagaagagtg taagaaccat 54300tatagggctg ggctcacgcc tgtaatccca gcattttggg aggctgaggc aggcggatca 54360cgaggtcagg agttcgagac cagcctgacc aacatggtga aaccccatct ctactaaaaa 54420tacaaaaatt agccagtcgc ggtggcacgt gcctgtaatc ccagctactc aggaggctga 54480ggcagaagaa tcacttggac ccaggaggca gaagttgcag tgagccaaga tcatgcctct 54540gcaccccagc ctaggtgata gagtgagact ccatctcaaa aaaaaacaaa acaaaacaat 54600tataacaatt tgaatctgac attgcaaatc agctttacca cttccaaggt atagaaaatc 54660caggtctatg agactaacat cacattgtaa aaatcaaatc gtggtagaat atctttaaat 54720taatataaat acatccccat tgtggggaca ttttgcaggg tatctgctta tctcacatac 54780acctatgttt taataagtga tgcaacattg catattttct aaaccaagaa aaattaagca 54840agtgtttaag tgatttttcc ttttgatagt gggttaattg gacttcatca aagaaaatgg 54900tatctgcaaa actgctttgc atgttataaa aatgcttatt tcacaacttg cttttcacat 54960aacctcttac cattaatttg cctaacagac attgatcgcc ctaaaggact ggcattcact 55020gatgtggatg tcgattccat caaaattgct tgggaaagcc cacaggggca agtttccagg 55080tacagggtga cctactcgag ccctgaggat ggaatccatg agctattccc tgcacctgat 55140ggtgaagaag acactgcaga gctgcaaggc ctcagaccgg gttctgagta cacagtcagt 55200gtggttgcct tgcacgatga tatggagagc cagcccctga ttggaaccca gtccacaggt 55260atatggttaa ttgcaccacc aggtgcccat gggagcagcg gctttatgcc ctactgaatg 55320aattatgctt cactgggcta ttgattcccg tgtaagggtg aaaaagaatt attaggaaag 55380atcctcttta aagaggaatg gtaagaaaca ataaaactta ggtgatattt aaggaaacaa 55440gtctgattaa aagaaatttt ggagtatcct ggcttataca caagaccata aagcaagaca 55500tttgaagagg atactaaagt tgtggattat ttcctaagct ctgactccct gtgattaccc 55560tcactatgta taaagaaaag aagtttggca ttacagagct tacttataaa aaggaaccca 55620aactcgggca tttcatagca gcatgattct gagcacacgt gggtaagacc tttcttctct 55680ggttagatat catatgctgg tgtataatta gcttaaatga ttgtgattta gacacctagg 55740aaataatcaa tagggcaatt gctttccata atactttatc ttcttgtgct ttatttctga 55800agcagagtag aatgctaaag atgtatccta gtgacagcat aaaccctaga ggtgacagtc 55860tgtattattg cttttcgctt ctcttttctg cttctgttgg gagccagttt tcttcttacg 55920ccgcattaca gagagaacgt caaatttagc agccatatct gccatagggt ccaaataaag 55980agacaataaa aacattattc tctctttttt ggatggaata ctgcgtgaaa tggttatcca 56040tacaaagata ctttatgtag aatagaaaaa ggaggccggg tgcagtggct cacacatgta 56100atcctagtgc tttgggaggc taagccggga gcactgattg aggccaggag ttcatgatca 56160gcctgggcaa tgaagtgaga ccccgtctct acaaaaaaat atgaaaaaat tagcgaggtg 56220tggtgacaca tgcctgtagt cccagctact caagaggctg aggtagagga tcacttgagc 56280ctacgagttc aaggctgcag tgagctatga taactccact gcactgccgc ctggatgaca 56340cagagagacc gtttctaaat taattaatta acaattttaa gaaagaaaaa gggccattgc 56400ttatttttcc atacaaaagt aaaataaatc ataatggcca ataagccaat gtaacttttt 56460tttttaaggg aaagcaaaac ttgtaaaacc taaaatctct tagagttttg gcatttaccc 56520aaatgttttc agtgattctg agaattggtg gatataaaac acatttctca gcaaacactt 56580tcttcatttt gcatccctta ctgtacgtac tttcttgtac tgaatctttg cttgaccagg 56640gaacccacct agcccaacaa gaacaatcca ttctacttct tggaactcac tttattttcc 56700ttttccccca tttcctataa gataacctct aaccaatgac aatctcgaca gctattcctg 56760caccaactga cctgaagttc actcaggtca cacccacaag cctgagcgcc cagtggacac 56820cacccaatgt tcagctcact ggatatcgag tgcgggtgac ccccaaggag aagaccggac 56880caatgaaaga aatcaacctt gctcctgaca gctcatccgt ggttgtatca ggacttatgg 56940taagacatga ccgttgttca ttggaataaa gatggagatc atctctaaca cagtttctaa 57000ggtggtgaaa atataatatc ataataaatc taactgttct tttcctctgc atcaaataat 57060cttattgtaa ttttatatca acggaattcc tttatgttga cctaagtttt ccagatgact 57120attgggacag aattttataa atagctttgg attttgtgca gctcttttag atgtattgtg 57180cttattttaa aaggttgtgg ggggcaattt acatatccat tggttgaatg cataaatcga 57240cttagttatg cattttctga gctctgttac cttggtaaag aatattttac agtttgtacc 57300agtctacctt gagcctaccc tcattaaaac attttaaaat ccttccagac atacatgcag 57360aaaactgcta ggaacctagg ggactgatgt acctcttaac ataaggccaa tttcagggga 57420aactacagaa agagggttca gagacaaaat ggaacattct ctttgcctct ctatgataag 57480gaaaaaatta tgatttacac ctgtcagatc ataaaaaaga aaaatacgct aatacccact 57540tttctcattt tttttaccag cttagtttaa gtatataatc tatggcttac ttaagcttaa 57600ccgctaagag cattttaaaa ttgataaata catttatcac ctgcactgta ggaatgaaat 57660taatctagga attttcaagg ttgtgggttt tgctggtttg tttatttttt attttctaac 57720cattgcattt acctaatgct gtagtgaaac tccttgggtt tcagttgagg acgttgctaa 57780agctcaccat gcccttattt ctctaggtgg ccaccaaata tgaagtgagt gtctatgctc 57840ttaaggacac tttgacaagc agaccagctc agggagttgt caccactctg gagagtaagt 57900aacaaaatgt cttcatatgg acaaaccttc tgtatagaca aaaattaaag aatggtaaat 57960cagtggggtt cagtggctca tgtctaaaat ccaagcactt tgggaagctg aggcgggagc 58020gtcacttgag gccaggagtt tgagacctac ccgggcaaat agcaaggccc tgtctcttaa 58080aaaaaataaa ataaataaaa taaataattt tttagattta tatgttaaca gtggaatgag 58140tcctaatttg aaaatcaatt tgattgcctt tttgacgcat gactgtcatc ttttatactc 58200cttcagaaag gggtctactg acccataaaa tggaatcact tcataagctt ataatgttga 58260tattatggac tatgactgac atctagttta tgctctactt gttagaattt gttttcatag 58320agctaagctt ggggagaccc cactggcttc tgctatatct taacaatgca tattaggcca 58380ttcttgcatt actataaaga aataccggag actgggtaat ttctaaagaa aagaggctta 58440attggcccac aggccagcag gctttacagg aagcatggtg ctggtatctt cttggcttct 58500agggaggcct tgggaagctt actcatggtg gaaggccaag ggggagcagg cacatcacat 58560ggctgtggca aaagcaagac cgagagagag agagagttgg ggggggagga ccttatacat 58620ttaaatgacc cagtctcttg agaactcact gtcataaaga gggcaccaag ccacaaggga 58680tctgccccca tgatccaaac acctctcacc aggccccacc tccagcattg gagattacaa 58740ctcaacagag atttggacag ggacaaatat ccaaattata tcacagcaca gtaaccattg 58800gaccaaatca ggcttagatt ctagtcttct gttatatcaa taccttgatg tatgcctttt 58860caaaagtcag gtaaagtgtc aaagttttat catttataaa agagggatgg cattgtacct 58920gttgagagaa aatacaaaat acttgccgta atattagaca cacacacaca cacacacaca 58980cacacactct ctctctctct cacacacaca tacacacaca cacacacaaa attgttagct 59040ggccatgtta ttgtaactcc taccacacat atttttacat tataatacat taataatttt 59100aatatttatt gaagtatttg tagatactat aaagccagcc ctgggaacca ctggtagtat 59160ctataaagct tttcagctct tcaaaataaa atgtctgaga ggtagatatt ttcctatttt 59220ctaattacag ttgacctttc tctctgaatg ccaaaggaga taatctacac attactagtt 59280atatatttct tgaaatggat gaatttgata tataccaagg aaacgtttta aaataccaaa 59340actttacatg gatgagccaa gcaggcacta atctctagct atgctcctgt gcagatgtca 59400gcccaccaag aagggctcgt gtgacagatg ctactgagac caccatcacc attagctgga 59460gaaccaagac tgagacgatc actggcttcc aagttgatgc cgttccagcc aatggccaga 59520ctccaatcca gagaaccatc aagccagatg tcagaagcta caccatcaca ggtcagggaa 59580ctcattgcac taaccacatt tgttaacaaa tacccacaat gtaaacgggc ttattaactg 59640ttctacgact gacactgata aaatttattt tcagtgttat catcataacc cagttttaga 59700acgttatttt catgctatga tcagaaatag ttttgtcctt tgaatgcctg attttgtgta 59760atatttgtca tggaaattgc gtaagtgtca atcaacaagt ttgatcttcc atcattgtgc 59820cctttcttat ttaaaaaatt gtaacataag gtttaaaact aaaagaaata aaaaacagtg 59880atgtatagat cttagcatta aaaagcatag ttaatataaa agtaaacaat accacttaat 59940aaaggccaaa attgtaaccg aagaatattc aatatctgag gtctttttta gctttttaaa 60000attgtgattc caaggctcaa ctattgacca tctgattacg gtaaagagaa aacctcaata 60060agtggctgac ccccattctg caagagggcc tcttccaaca tagcattttt gcattccaga 60120atttacttta ccagtgtcct tgtctgtatc agtgattcac tttcgagata tgtttcttgt 60180taacagttaa catccatagc atgctctact ttactgttca

aatgtggacc actttggtag 60240tctatataaa tatgggatga tagaagaacc cagaaaaatt gcaggctagc ttgagaattc 60300tcctagtaaa aagcaagaac tgttaaaaat catctcttct caaatcccag gtttacaacc 60360aggcactgac tacaagatct acctgtacac cttgaatgac aatgctcgga gctcccctgt 60420ggtcatcgac gcctccactg gtaactatac cttctactga ggaaatgcca ttgacttgta 60480tgcaatcagt ttcatgaact caaaaaacaa atgtgaggcg tatatttttg tattatagat 60540tccagagaat cttgtttccg gtttacagta ttctcagatt cttttaagtg tgtttagaac 60600ggctcgggag aaaagtgtgg gagtaatttt cttggttatt tgccttctta gagacttaat 60660tttgttttct ttcagccatt gatgcaccat ccaacctgcg tttcctggcc accacaccca 60720attccttgct ggtatcatgg cagccgccac gtgccaggat taccggctac atcatcaagt 60780atgagaagcc tgggtctcct cccagagaag tggtccctcg gccccgccct ggtgtcacag 60840aggctactat tactggtatt gctgcttcca tgctgtcatt ttccttctta ctacctagga 60900cacatgaagt ccttagcaaa ctcccacagc gtctttgata ctgtgtcatg agaatgcgaa 60960actctgttcc tgataacctc aaaaagcatt ctctgtgtag gagtggtaga gcctaataca 61020tcccaaaagg catgagtgaa ggaaaatgca atttcaagac tgtactaatg gcatgactag 61080actcatgttt tcctttcgct gcaagtttgc cagatacctg tcaattcagt cctggagaaa 61140gatatttttc aaagcatact agctgattgt gattctgtca ttacactcag ctctctatag 61200atatggcaat cttgcaggac ttgccagtgc accacctgcc attgaccttg ttgaccacta 61260tcacaggata ggtcttgagg cagagcagtc ccaaccaccc acattggaag aatgcctgga 61320atggggaata agagttgtct accttggtgg gaaagactat aagcctctag tattattttt 61380gcccaagaga tgaaatattt aaactatctg tattagtctg ttttcatact gctataaaga 61440ggtttaattg actcacagtt ccgcatggct ggggaggcct cagaaaatgt acaatcatgg 61500tggaaggaaa agcaggcatg tcttacatgg cagcaggaga gagaagcaca caggaggaac 61560ttccagatac ttacaaaact atcagatctt gtgagaactc actatcatga gaacagcctg 61620ggggaaccac cccatgatcc aatcacctcc tctcctcaat acatggggat tacaattcca 61680gatgagattt ggatgcagac acagagccaa accatatcag tctgtataga gtatcacctg 61740gactttaaaa ttcccacaga acatacagac attagaagga gacactggct ttttagaatt 61800ggggggaaca ggaaaataga agcagacatg agaggaattg aactagacac ttcccacaga 61860ggctgcacaa acactggcca atctctccta cccttcactt gcctttagtt tcacttttca 61920ttgatctgcc actgaggact gcttggttat taggcctaag tagattcatg tatataatct 61980gcaggactct ctttcaaatt tatattccag tggtggtata tgatgctgat agattttctt 62040aaattcaaaa aggcaaataa gaccacgtta aaagaatacc ctggaaaggc caggcgcggt 62100ggctcacgcc tgtaatccca gcactttggg aggccgaggc aggcagatca caaggtcagg 62160agatcgagac catcctggct aacacggtga aaccccatct ctactaaaaa tacaaaaaca 62220aaattagcca ggcgtggtga tgggtgcctg tagtcccagc tactcgggtg gctgaggcag 62280gagaatggct gaacctggga agcggagctt gcagtgagcc gagatagcac cactgcactc 62340cagcctgggt gacagagcca gactccatct caaaaaaaaa aaaaaaaaaa aaaaaaagaa 62400taccctggaa aagttagcca aaaaatgtct attcaggcgt cagatatgat agtaagataa 62460ttagtttgcg atgcggactt tatatgcagg atatttgggt gtttatggag gaaaagtgaa 62520gtcgatttta ccttcaagag gccaacagcc agctggagag gaagtgcctg ctcccagtag 62580cgtctgctgg tgagaccgac ttccacttga ctagctgagc ccattgacat aatgtgatgg 62640ttctattctc ccttcaggcc tggaaccggg aaccgaatat acaatttatg tcattgccct 62700gaagaataat cagaagagcg agcccctgat tggaaggaaa aagacaggta agagtatctt 62760gcaggtaaca aggagaaaga taggacaaaa ctaataacaa atgagcaatc ttgcaatatg 62820aaaaggttct ccatgttttg atgcatttct tgtgattttt tttatctaac agcatagtgt 62880atatattgta ttctttaata ggagaaataa tttaacatgc actgcagagt ttggttttat 62940tttttttctt tactacagcc actcaatata aagccttgtt attcaccttt taaaaattca 63000aacaagatgt taaaatgtaa aagaagagct atcattgctc ttcttttata ccttctgttg 63060aattttaaaa tgtttccttt tttaaaggag ggagagaaac ctctcacatt tatctttatt 63120tggtttctac aacttagagc taaataatgt cttacttttg catccagttt cagttaattt 63180caagaaaatg tgtattcctg atataggaaa atttcaaaaa tgaacatgtg tgttttatct 63240atttttacca tttcaaacca tgaaaaactg ttgagccaaa cctctgtaat tctcatactt 63300atgacactga tatgattagt ctggattcta cttcctacaa cttgcttctc aaatttaaaa 63360aagaaagaga gaaagagaaa gactgcacat ttcagttcca ttaggtctaa tttgagcaga 63420ggcagcttct acggggctca gcggtttaaa gctgtgtgta tgataaattc atactaacac 63480ttttttcttt ctaaactata aagaaacctt tgagaaaaat cctaaagatt tctttctgga 63540aaaagtgttt tgtgatctca gaactgctca ttttctggtg gcttttatca aattgatgaa 63600cagtcattgt tgcctgaatc gattattatc attgctgcta cttcctggag cttaatgcgc 63660tttgcttttt tggctctaac ctctctcggc tagacgagct tccccaactg gtaacccttc 63720cacaccccaa tcttcatgga ccagagatct tggatgttcc ttccacagtt caaaagaccc 63780ctttcgtcac ccaccctggg tatgacactg gaaatggtat tcagcttcct ggcacttctg 63840gtcagcaacc cagtgttggg caacaaatga tctttgagga acatggtttt aggcggacca 63900caccgcccac aacggccacc cccataaggc ataggccaag accatacccg ccgaatgtag 63960gtgaggaaat ccaaattggt cacatcccca gggaagatgt agactatcac ctgtacccac 64020acggtccggg actcaatcca aatgcctcta caggacaaga agctctctct cagacaacca 64080tctcatgggc cccattccag gacacttctg agtacatcat ttcatgtcat cctgttggca 64140ctgatgaaga acccttacag gtaattaatt gttctcttca cttctcatgg ggcagcacag 64200aaaggaataa gttaggtaac tgaagtgacc agccctcgaa taaaaagtgg cttcatggcc 64260gggtgtgatg gctcacgcct gtaatcccag cactttggga ggccgaggca ggtggatcat 64320ttgaggttag gagttcaaga ccagcctggc caacatggtg aaacctcgtc tcttgaaaaa 64380aaaaaaaaaa aaaaagtggc tccactttta gaacctctta gaagatggca catttaagcc 64440ctgctttttt ttttttttaa atcccaatat ggctctactt tggaggacat accagagagt 64500cactagcttt tatttcatag agaaaatgaa actatttctc ttattctcac acatttgagg 64560ttcctttttg agtaagatag atgattctag aaaagaaaga tattctacct gaatttccat 64620ttgtgtgcag aagtctaaaa cactaccttt acgatttgtc cttgaagaac cccactatct 64680acaacatatc taaagaaaaa aaaaaacagg cgaagctgtg catagcagct gataagtgat 64740tgattctcta aaacgtatat tatttaattt gtgttgacag tatccatttt tttttttccc 64800cgagatggag tcttgctcta tggccctggc tggagtgcag tggcgtgatc tcggctcact 64860gcaacctctg cctcccaggt tcaagcaatt ctcctgcctc agcctcccaa atagctggga 64920ttacaggcat gtgccaccgc acccagctaa tttttgtatt tttagtagag acggggtttc 64980acgatgttgg ccaggatggt ctcgatctcc tgacctcgtg atccgcccgc cttggcctcc 65040caaagtgctg ggactacaag catgagccac ccactacacc cggcccactg acagtatcaa 65100tttttattgt gttgttactt ttagaaagtg gcagaattta aaaactgaca acactgtagg 65160aaatttatga gcttagaaac atgagtttga ggatttgccc aactgtttta aggactccac 65220actggggtca gatgtcacct ggaggagcat gaccgtgtct cccatatagc gcagtgtcca 65280ggttttatgt gaagcaaaca tggccagggc ttccagaggg cttatgcaga cctgcgactg 65340aagcaagatc aatggcaggc cgtctctagt attgtcgagg gctcctgtta actacggagc 65400acgtaggtag attgttggca ggaaaatctg gcaggaacga tggcccctat ccttgttcca 65460tttctctcct cagctggtta ggaccactat accctccctc tttttttttt ttttgttttt 65520tgttttttgt cttcctttgc tttgtttaaa cagtgagggt tattggtaag aggagagccc 65580gtgtcattcc tcactataat gctttctctc tgctttggat gtaccgataa ttgcagttca 65640gggttcctgg aacttctacc agtgccactc tgacaggcct caccagaggt gccacctaca 65700acgtcatagt ggaggcactg aaagaccagc agaggcataa ggttcgggaa gaggttgtta 65760ccgtgggcaa ctctggtatg taaacacgta ctatttagac acaggctccc ctctgctgta 65820caccagagat gggcttttct gttgactgta cctttgttgc cattgtcttt ttatctttgg 65880gatttaatgc aacacatcaa catgaaataa atgagcaact ttatattaaa ttaatctctc 65940ccccacctcc tgccatatcc tgttgtcttc acaaaatgca tacgtaattg acagactctc 66000aaatggtgat atgattatag atctggaagg gatttcaaat attatttagt acaacactct 66060gagtccttac ttctgagtat ctgagttgat atagggcaca ggtttcctga tgtcttttcc 66120cagaccctct ccatctcacc atgctgctgt cctcttgagt gattaaatac tgaaacgatt 66180acctataaag aaaatacccc ttctgcagac atggggacag ttggcttttg ctcctgatat 66240aaaatgctac caacattgtg catttctgtc tgcagagaat gttattccaa tgttatttcc 66300atttttttcc aatgttattt ccattttttt ttctgactat acaggttaaa agcttctata 66360gaggttaaaa gatcattaac tcttctttgt agcacctggg aaatcctttt aaatcaatag 66420cgtgccacct ggctgctcaa tttgcagcag ctgaaaattc accaaggcac atgagatagg 66480ggataatcaa aaccgtgaat ccccaatctt ccaacaggag agttctctac tccacaccaa 66540cagagagtgc taagtcctgt ctatgccaag tgacagattt tattcctaag gccagttgtt 66600taattttagc cccttccctc atctgataga agactgtgct actttacatg tataaattcc 66660tgtgaattaa gcagttgagc atttggctgg agagaggttg ggaggagatt attttgtgtt 66720tgttgtatta catatccaca gtaatgctta tctttgcctt ttgtggtttt actagtagaa 66780tgccacgtga acagaatttt caagagcaaa aaggtctttg tgcttttcta agtcattttt 66840tttttttttt tttaaagatt ccatctcttt aactttagtt aggatggaat ttgaactcct 66900ggctcttttg agtatagaaa cccctagtaa caatttaagt tccttccatt tttcttttaa 66960actccttatt cccagcagca gtattctaca ttctaaccag gttctcccag ctttgagacg 67020tctcagactt accagttctc caaaacgcta ttttctttaa gggtgacacc ttttaaaaat 67080taggcacctc aaatatctac tgcttttgag cttttgagtt ttgcactgta aaaagaaaaa 67140tacacagtgg gattttaagt caaattagtt tatctaattt ttagggaata atttgaagca 67200tgctttgttt gcatagattt ttttaaaata agcttttcca aatcataaag agataagatc 67260ttaggtaaca tgaagagact cccttactta ttcctaaatc atctatattc caagggcatt 67320ttcttatttg gaacagttga cctcactgat aaagctgtct caccactata ataacaatgt 67380ccaaaatcta ggctttctgc actattatgc aaaaattaca ataataaaag tgaaaattac 67440attataatgg tatattaaaa tgctaagact tttgcattat aagcaaaaga cagcctttaa 67500taattattct ttatttagtg aacattttct aagtcttgga aaagggtcaa tgttttgaat 67560tcatggcctt atataatctt cacaagattc cccaggaggt atagatattt ttattattac 67620gctagtattg cagatgaggg aagcaaggca gagtggtatt aaatagctgg cccaaggtca 67680ctcaggtacc aatggagagg catcattagt ctttgcatcc cactaaagtt ctccactagc 67740ttcaattgcc tcaagatctg ttccatgttc tatgaagtag tttcaacaga aatggcaatt 67800atcttagaag caagggaaaa ataaaagatg ggcttcctgt cgggtgcctg tgacaggtgt 67860cacatctaac catggttttt tagagcagtt aatgccttga tagaacagat gaatgcctct 67920taatcctcct ggaattcttg ttttagatta agtcattgta tacagtcatt cgattttctt 67980cttatggtcc aaatcgatta ataagatgtc tctttttgct ttttcttcct tttcttcata 68040gtcaacgaag gcttgaacca acctacggat gactcgtgct ttgaccccta cacagtttcc 68100cattatgccg ttggagatga gtgggaacga atgtctgaat caggctttaa actgttgtgc 68160cagtgcttag gctttggaag tggtcatttc agatgtgatt catctagtga gtagttgctt 68220tgtccatcca cttccgtgtt tgtctcctca agttccatgc atgcactcat gtgccaagga 68280agcatgtttg gaagacacag gttcttccaa acatgaagca aacaagagaa tactgtttga 68340ctcgaagtaa tattttgcat catagaaaaa tgatgggaaa ttttacttgt tggacattgc 68400ttcatttcaa gggttgtatg ccaatacaac tattaattac acataagatt atggtgctaa 68460tttgattttt gaaattttct gtgaaaacaa atggataaag acttttggaa ccaggtctat 68520ttaagagtat tagagacaca gaaaaacctc aaatctcttt taatcttcag tgttgaatga 68580gatcagaggt gaacatttag actcaaaaac agcctccttc aacataaacc aaacatgcac 68640atatcatagt acccatgcac acacttttgc gtcacacaca tagcccaggt agcttgaacg 68700ttgctagaaa tatgaaagaa aaaacagata atctgctttt agatcattaa aaatcaactt 68760gaattgataa atgtttgatt ttcaaattct aatacgtttt aattttcaaa ttttttaagt 68820taaaatgtgc ctaggaaata tctattatgc tttgagatta ggattagaat ttataaacct 68880ttcatttatt ctttgtgttt aggagatgtg atgattattg acaattggtt catttttata 68940ggtgttgacc gttatgccta taaataagcc tcctatagac atacagaaat catatcctgt 69000ggaattagaa tataagactt ggtaaaagag attttcaaag tattttactt aacttgtata 69060cttgaaatca tttaatccag actgaagttg taaaagccag ccagtgtttt caatatagac 69120ttccatgttt gaccatctga aaatgaaaaa cactaaaaac atcacatgct gtttaggagc 69180tggaaatttt aatatttgac ttcaagtaga tggtttttaa ctcctgaaat cgaactacgt 69240ttaagtttgt atgtttatta cctgtttgag cacttaggtg caattgtggg agcggggatg 69300tcaagttcat ttatgtgact ctttggctca acttacataa tctttgtttt gatatcacag 69360ttgtctaatt attttacttt gtagcttaag gcaggctgaa ttgttgataa aatggaaaaa 69420gtagtatatt gttatataag cttctgaggt gtgttttgtt gtataagccc tggaggttaa 69480aaagtcatcc cttatgtata gtagttaaag gcataaaact gtgactttta gatattccac 69540agaaccagac ttatttgatg tggataataa ccaatgattt agcattttgt ttgcttttgt 69600tttattttat ccgggttcat tttttactct tcccatgtac atgaaacagg tggtggcgtg 69660tagagatcag ctgatccttg ttttatggtt aattgaacta ctttgtatcc agggtttctg 69720caaatccaaa agtgattttt catctaggat ctattcctaa cagtctactc caatcccact 69780ttagttttcc acaattttaa atcttaatag tgagaattca aatgaaagtc atttcatttg 69840actattctga tgacatgatt gtggcagaat aaattgggtc ttaaaatgcc ctagaaaatg 69900gtaaatgata aaaaataata ttttaaaatt caaccaaaga aatggcccat tggccaggtg 69960tggtggctca cacctgtaat cccagcactt ttggaggctg aggcgggtgg atcacctgag 70020ctcacgagtt tgagaccagc ctacccaaca tggtaaaacc ccatctctac aaaaaataca 70080aaaaaaaaaa aaaaaaaaaa aatagcactg tggggagtgc ctgtaatccc agctactcag 70140gaggctgagg caggataact gcatgaaccc aggagatgga ggttacagtg agccgagatt 70200gcaccacttc actccagtct gggcgacaga gaaagacttt gtctcaaaaa aaaaaaaaaa 70260aataaaaagt aaataaataa ataaaataaa tggcccatta taggggtttt tatctttaac 70320ttgctatttt tccagatcat ggttctgaag accctgtgac acgtcccagt tcacctactg 70380tcttgtgagt cagaatatac aaataacttt ttggtcctga ctttccccac ccctacagga 70440tggtgccatg acaatggtgt gaactacaag attggagaga agtgggaccg tcagggagaa 70500aatggccaga tgatgagctg cacatgtctt gggaacggaa aaggagaatt caagtgtgac 70560cctcgtatgt catcacagat catttttagt gccttattaa gcattctcac tttcattatc 70620aggctgtaac tctcattcac agaaatgatt ggagacttta ggtctccttg aggagtgaac 70680agtgggtttc ttaatctttt gatttgggaa agtggagaca agcttcaaaa atgagtcatg 70740atttaatgtt attacaggac actttagcac ttgtccaacc tgagtatttt gaccattatc 70800tgcagtaaaa tgctacaaag aagctttatt ggtctgtaga ttcaactttt aaaatatgat 70860ttccatcttc ccgttggacc ctttccagtg tattaggtct aatttttgga agtgccaccc 70920taagatctgt atagcagtac tgctcttagg gatgattcac ataaatatgt ggtgtttgcg 70980ctgtgatgat acaaatttag gacagaaata gaacccaccc ctagatcaag tctgcagtat 71040tgttctcagc ttatgcgtgc atctgtcttg tgtctatatg cagatgaggc aacgtgttat 71100gatgatggga agacatacca cgtaggagaa cagtggcaga aggaatatct cggtgccatt 71160tgctcctgca catgctttgg aggccagcgg gtaagactgg atgtgccagg ctccctacaa 71220gttagataag ataaagggtg ggctcctgca aggatgtgtc gtacacacag gaggggcaga 71280gacccttcgg aagtattaaa ataccacatt tcctgttggc atacaactgc tgacatagag 71340ctctagagca gctctatgtc taccttacat gccattcatt ctttctatta ctcttagtag 71400aaagaatgaa tgaatggcat gtagagtacc aaaaacacaa gtcttgagtc attcttaata 71460gcaacacctg tcatttatat gatgttagaa tcattttcct aagctcccta gcatgtcaga 71520gatactattt acactgaaaa atagtgaagc agagatacta ttcaaattaa ttagtggtaa 71580atagaatgtg tttcatttca gccggttctc cccatcctgg gcagcctgag accctcccct 71640cccctactat tctcaggctg cttctatttt tcagcaaagt gttaagtgca gtgtagctct 71700aggcctccaa ctccattctg atggacaggt gtcccatggc aacgttgtta aatattttga 71760ataatatctc agatgtaaga aaatgccact tcttttaacc tctctcttga ttcagaacag 71820atgcttgtta taggtctagc actgtgctaa gtagtatagg aaaaacagag gaaatgagaa 71880atggcttggc tcttaatgat atagttgaag atgttaaatt agcatacatt tcaaagtcaa 71940gctaattaag ttctaagtgg gtctgacaaa tacagttctg ggtaggctgg aattagcaag 72000aaagagaagc atgaactggc tgaggtttac gatgactaag gtttagttgg gaggggagaa 72060agcagagaga cacaccccct gggatagaaa ggagctggcc caggtgggct ttggtgagcc 72120aaccctctgc ctgctgtctt ctggtaagaa aatagatggg aagaagtggc ttatggaggg 72180ccttggcaac ccattattta agccagtact tctcaaccat ttctaaaata tgcccagtat 72240aacaaaaaat aataagcctt tctctaatat gatttcaaat ttcaaaatga aattatgtgt 72300aactcaaaag caatggaatg tgacagccct ttgttttcaa cgaagacatg ccctcccagc 72360aactccccaa atcctggtgg gtgaggggca tgcttcacac tcaagggtga gactcattgg 72420ttaatgccaa atgcattaac caattacagg tatccaagat gcaaagaaac atgatggaaa 72480atagtctttg ggaaaattaa tctggcagca ggggtgttca ggggcctgtc ttggctccac 72540caggggcagc ccatggaaac tactatgatc ttgtttcacc cccagtgatt acatggggag 72600ggaggtgctc ccaattctga tggaggagaa ttggagattg gaatttagat tgaattcagt 72660atctctctct gtctctctct ctctctctct cccattaaca cttacaacga ctgtgattgt 72720atgaccttag aactcagtca ttctggtatg aaattgtgtg atggagaatg aatttgctgg 72780gaagttgatt ttggtctcac ttcagcatct tctcattatt tatgcacatg aaacctttca 72840tgtgcgacac ttattctatt ctcaagtgct aaatgaaaca tttaagacag gagtggaaac 72900tgttcacttt ctcatatgaa agcaagattc agtgattctg taaggaggta gtcactggta 72960ttgtgttagg tattaagggg catatgtgct taaacagaga aatatgtcta aaatatttaa 73020attctaatat aaaaaagaaa gtgactgtat tatttagggc tgcattttag ttgtaagaaa 73080aaagtccaac tcaagcaaaa atggcccaca caatggaaca gtcccaggac ccaccggctt 73140caggggctgc tccagcaatg gcgcccggac tccctcttgc tccgcgtgcc ttcccatgca 73200ctggcttcgt gcttcagcgg ggtctctgct gatggtgcca ttgatgactg acctccatga 73260gcttgcttta ccccctgcca gcttaagaac agtagtgaaa gagaacatgt gtgtcctccc 73320atttccagta aaaacttcag gcaggagcct cactggctca gcttggtccc gtttccatct 73380cccatgccat ctccggccag gtgacaggct accatgtcac tgcctaggga agtttaggaa 73440gagagtggca aagtggtgca ttagaaagaa catggccagg tcaccccacc tcctgggcgg 73500caggcccaac tccaccagtg gtccactgtg tgacttccct gctccctcta agcaagtcac 73560tcctctcctc tgggtctctg tttccttacc tataaaatga gaacgtttct tcatgtgatc 73620tcaagtccct tttaaaatcg ctaggattct ttgaaaacct tttctatcat ctagtgcaga 73680gaacttgttg aggaagttgg gattggaatg agcctcagca gatgggcaag gtttgaatag 73740gaagagaaga gacatttcag gagaaagaaa caacatagag agacagatgt aggtataaga 73800tatggtaata agccaaaatg tattaagagt tataaatgca tgaaatcatc atcaaagctt 73860gcttagtgat taactgctta tattttgcca gtgcatatga tgtgacattt ttctttaact 73920caaacactaa attacgatgt cctcaggtta tcataaaccc catttgactt catgcctcta 73980ctctctcagg gctggcgctg tgacaactgc cgcagacctg ggggtgaacc cagtcccgaa 74040ggcactactg gccagtccta caaccagtat tctcagagat accatcagag aacaaacact 74100gtaagtgcat tagcagcaca agtgtgttcc ctcatactag acagtctctt tctacaggta 74160tctttcttca gaatgaacca agtgttttaa ttaattaaaa aaaaaaacaa ctcataaatg 74220acttaagtga aacactgtat tccataatat agtttaagtt ataatttatg taactcttga 74280acatctccta ttgcccagta tgctgctagg ttcttgaaac taggaagaaa tattatccta 74340tctataagca gctgtcatga gtccccacct ccccgcattt ttttttctgt acactttaca 74400gtatttgcca ctaatttttt tttccttctt cctttttaac agaatgttaa ttgcccaatt 74460gagtgcttca tgcctttaga tgtacaggct gacagagaag attcccgaga gtaaatcatc 74520tttccaatcc agaggaacaa gcatgtctct ctgccaagat ccatctaaac tggagtgatg 74580ttagcagacc cagcttagag ttcttctttc tttcttaagc cctttgctct ggaggaagtt 74640ctccagcttc agctcaactc acagcttctc caagcatcac cctgggagtt tcctgagggt 74700tttctcataa atgagggctg cacattgcct gttctgcttc gaagtattca ataccgctca 74760gtattttaaa tgaagtgatt ctaagatttg gtttgggatc aataggaaag catatgcagc 74820caaccaagat gcaaatgttt tgaaatgata tgaccaaaat tttaagtagg aaagtcaccc 74880aaacacttct gctttcactt aagtgtctgg cccgcaatac tgtaggaaca agcatgatct 74940tgttactgtg atattttaaa tatccacagt actcactttt tccaaatgat cctagtaatt 75000gcctagaaat atctttctct tacctgttat ttatcaattt ttcccagtat ttttatacgg 75060aaaaaattgt attgaaaaca cttagtatgc agttgataag aggaatttgg tataattatg 75120gtgggtgatt attttttata ctgtatgtgc caaagcttta ctactgtgga aagacaactg 75180ttttaataaa agatttacat tccacaactt gaagttcatc tatttgatat aagacacctt 75240cgggggaaat aattcctgtg aatattcttt ttcaattcag

caaacatttg aaaatctatg 75300atgtgcaagt ctaattgttg atttcagtac aagattttct aaatcagttg ctacaaaaac 75360tgattggttt ttgtcacttc atctcttcac taatggagat agctttacac tttctgcttt 75420aatagattta agtggacccc aatatttatt aaaattgcta gtttaccgtt cagaagtata 75480atagaaataa tctttagttg ctcttttcta accattgtaa ttcttccctt cttccctcca 75540cctttccttc attgaataaa cctctgttca aagagattgc ctgcaaggga aataaaaatg 75600actaagatat taaaa 7561522355PRTHomo sapiensMISC_FEATUREFn1 isoform 3 preproprotein 2Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly Leu 1625 1630 1635 Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu 1640 1645 1650 Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser 1655 1660 1665 Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu 1670 1675 1680 Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr 1685 1690 1695 Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro 1700 1705 1710 Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu 1715 1720 1725 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1730 1735 1740 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1745 1750 1755 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1760 1765 1770 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1775 1780 1785 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1790 1795 1800 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1805 1810 1815 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1820 1825 1830 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1835 1840 1845 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1850 1855 1860 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1865 1870 1875 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1880 1885 1890 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1895 1900 1905 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1910 1915 1920 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1925 1930 1935 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1940 1945 1950 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1955 1960 1965 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1970 1975 1980 Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val 1985 1990 1995 Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val 2000 2005 2010 Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly Tyr 2015 2020 2025 Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln Gln 2030 2035 2040 Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe Arg 2045 2050 2055 Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg Pro 2060 2065 2070 Arg Pro Tyr Pro Pro Asn Val Gly Gln Glu Ala Leu Ser Gln Thr 2075 2080 2085 Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile Ile 2090 2095 2100 Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe Arg 2105 2110 2115 Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr Arg 2120 2125 2130 Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln Gln 2135 2140 2145 Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser Val 2150 2155 2160 Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro 2165 2170 2175 Tyr Thr Val Ser His

Tyr Ala Val Gly Asp Glu Trp Glu Arg Met 2180 2185 2190 Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe Gly 2195 2200 2205 Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp Asn 2210 2215 2220 Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly Glu 2225 2230 2235 Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys Gly 2240 2245 2250 Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp Gly 2255 2260 2265 Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu Gly 2270 2275 2280 Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp Arg 2285 2290 2295 Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu Gly 2300 2305 2310 Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His Gln 2315 2320 2325 Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met Pro 2330 2335 2340 Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2345 2350 2355 3657PRTHomo sapiensMISC_FEATUREFn1 isoform 7 preproprotein 3Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Val Ser Ile Pro Pro Arg Asn Leu Gly 645 650 655 Tyr 42176PRTHomo sapiensMISC_FEATUREFn1 isoform 6 preproprotein 4Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425

Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Thr Ile Pro Ala Pro Thr Asp Leu 1625 1630 1635 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1640 1645 1650 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1655 1660 1665 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1670 1675 1680 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1685 1690 1695 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1700 1705 1710 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1715 1720 1725 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1730 1735 1740 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1745 1750 1755 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1760 1765 1770 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1775 1780 1785 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1790 1795 1800 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1805 1810 1815 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1820 1825 1830 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1835 1840 1845 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1850 1855 1860 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1865 1870 1875 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1880 1885 1890 Pro Leu Ile Gly Arg Lys Lys Thr Gly Gln Glu Ala Leu Ser Gln 1895 1900 1905 Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile 1910 1915 1920 Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe 1925 1930 1935 Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr 1940 1945 1950 Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln 1955 1960 1965 Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser 1970 1975 1980 Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp 1985 1990 1995 Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg 2000 2005 2010 Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe 2015 2020 2025 Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp 2030 2035 2040 Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly 2045 2050 2055 Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys 2060 2065 2070 Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp 2075 2080 2085 Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu 2090 2095 2100 Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp 2105 2110 2115 Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu 2120 2125 2130 Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His 2135 2140 2145 Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met 2150 2155 2160 Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2165 2170 2175 52421PRTHomo sapiensMISC_FEATUREFn1 isoform 2 preproprotein 5Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile 1265 1270 1275 Thr Asp Ser Ser Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser 1280 1285 1290 Thr Ile Ile Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly 1295 1300 1305 Ile Pro Ile Phe Glu Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr 1310 1315 1320 Thr Val Thr Gly Leu Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val 1325 1330 1335 Ile Thr Leu Ile Asn Gly Gly Glu Ser Ala Pro Thr Thr Leu Thr 1340 1345 1350 Gln Gln Thr Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn 1355 1360 1365 Ile Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser 1370 1375 1380 Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn 1385 1390 1395 Glu Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala 1400 1405 1410 Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser 1415 1420 1425 Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly 1430 1435 1440 Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser 1445 1450 1455 Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg 1460 1465 1470 Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe 1475 1480 1485 Ser Gly

Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser 1490 1495 1500 Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser 1505 1510 1515 Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly 1520 1525 1530 Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val 1535 1540 1545 Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala 1550 1555 1560 Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly 1565 1570 1575 Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr 1580 1585 1590 Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr 1595 1600 1605 Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 1610 1615 1620 Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 1625 1630 1635 Met Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp 1640 1645 1650 Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr 1655 1660 1665 Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro 1670 1675 1680 Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu 1685 1690 1695 Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln 1700 1705 1710 Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly 1715 1720 1725 Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp 1730 1735 1740 Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser 1745 1750 1755 Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly 1760 1765 1770 Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu 1775 1780 1785 Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln 1790 1795 1800 Pro Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp 1805 1810 1815 Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp 1820 1825 1830 Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr 1835 1840 1845 Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro 1850 1855 1860 Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys 1865 1870 1875 Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg 1880 1885 1890 Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro 1895 1900 1905 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile 1910 1915 1920 Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp 1925 1930 1935 Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys 1940 1945 1950 Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr 1955 1960 1965 Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser 1970 1975 1980 Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser 1985 1990 1995 Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser 2000 2005 2010 Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr 2015 2020 2025 Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg 2030 2035 2040 Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr 2045 2050 2055 Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser 2060 2065 2070 Glu Pro Leu Ile Gly Arg Lys Lys Thr Val Gln Lys Thr Pro Phe 2075 2080 2085 Val Thr His Pro Gly Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro 2090 2095 2100 Gly Thr Ser Gly Gln Gln Pro Ser Val Gly Gln Gln Met Ile Phe 2105 2110 2115 Glu Glu His Gly Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr 2120 2125 2130 Pro Ile Arg His Arg Pro Arg Pro Tyr Pro Pro Asn Val Gly Gln 2135 2140 2145 Glu Ala Leu Ser Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp 2150 2155 2160 Thr Ser Glu Tyr Ile Ile Ser Cys His Pro Val Gly Thr Asp Glu 2165 2170 2175 Glu Pro Leu Gln Phe Arg Val Pro Gly Thr Ser Thr Ser Ala Thr 2180 2185 2190 Leu Thr Gly Leu Thr Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu 2195 2200 2205 Ala Leu Lys Asp Gln Gln Arg His Lys Val Arg Glu Glu Val Val 2210 2215 2220 Thr Val Gly Asn Ser Val Asn Glu Gly Leu Asn Gln Pro Thr Asp 2225 2230 2235 Asp Ser Cys Phe Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly 2240 2245 2250 Asp Glu Trp Glu Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys 2255 2260 2265 Gln Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser 2270 2275 2280 Arg Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys 2285 2290 2295 Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys 2300 2305 2310 Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala 2315 2320 2325 Thr Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp 2330 2335 2340 Gln Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly 2345 2350 2355 Gly Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly 2360 2365 2370 Glu Pro Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr 2375 2380 2385 Ser Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro 2390 2395 2400 Ile Glu Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp 2405 2410 2415 Ser Arg Glu 2420 62296PRTHomo sapiensMISC_FEATUREFn1 isoform 5 preproprotein 6Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300

1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Thr Ile Pro Ala Pro Thr Asp Leu 1625 1630 1635 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1640 1645 1650 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1655 1660 1665 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1670 1675 1680 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1685 1690 1695 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1700 1705 1710 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1715 1720 1725 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1730 1735 1740 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1745 1750 1755 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1760 1765 1770 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1775 1780 1785 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1790 1795 1800 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1805 1810 1815 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1820 1825 1830 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1835 1840 1845 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1850 1855 1860 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1865 1870 1875 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1880 1885 1890 Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val 1895 1900 1905 Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val 1910 1915 1920 Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly Tyr 1925 1930 1935 Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln Gln 1940 1945 1950 Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe Arg 1955 1960 1965 Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg Pro 1970 1975 1980 Arg Pro Tyr Pro Pro Asn Val Gly Glu Glu Ile Gln Ile Gly His 1985 1990 1995 Ile Pro Arg Glu Asp Val Asp Tyr His Leu Tyr Pro His Gly Pro 2000 2005 2010 Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser Gln 2015 2020 2025 Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile 2030 2035 2040 Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe 2045 2050 2055 Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr 2060 2065 2070 Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln 2075 2080 2085 Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser 2090 2095 2100 Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp 2105 2110 2115 Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg 2120 2125 2130 Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe 2135 2140 2145 Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp 2150 2155 2160 Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly 2165 2170 2175 Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys 2180 2185 2190 Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp 2195 2200 2205 Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu 2210 2215 2220 Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp 2225 2230 2235 Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu 2240 2245 2250 Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His 2255 2260 2265 Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met 2270 2275 2280 Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2285 2290 2295 72330PRTHomo sapiensMISC_FEATUREFn1 isoform 4 preproprotein 7Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240

1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly Leu 1625 1630 1635 Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu 1640 1645 1650 Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser 1655 1660 1665 Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu 1670 1675 1680 Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr 1685 1690 1695 Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro 1700 1705 1710 Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu 1715 1720 1725 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1730 1735 1740 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1745 1750 1755 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1760 1765 1770 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1775 1780 1785 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1790 1795 1800 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1805 1810 1815 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1820 1825 1830 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1835 1840 1845 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1850 1855 1860 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1865 1870 1875 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1880 1885 1890 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1895 1900 1905 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1910 1915 1920 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1925 1930 1935 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1940 1945 1950 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1955 1960 1965 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1970 1975 1980 Pro Leu Ile Gly Arg Lys Lys Thr Val Gln Lys Thr Pro Phe Val 1985 1990 1995 Thr His Pro Gly Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly 2000 2005 2010 Thr Ser Gly Gln Gln Pro Ser Val Gly Gln Gln Met Ile Phe Glu 2015 2020 2025 Glu His Gly Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro 2030 2035 2040 Ile Arg His Arg Pro Arg Pro Tyr Pro Pro Asn Val Gly Gln Glu 2045 2050 2055 Ala Leu Ser Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr 2060 2065 2070 Ser Glu Tyr Ile Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu 2075 2080 2085 Pro Leu Gln Phe Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu 2090 2095 2100 Thr Gly Leu Thr Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala 2105 2110 2115 Leu Lys Asp Gln Gln Arg His Lys Val Arg Glu Glu Val Val Thr 2120 2125 2130 Val Gly Asn Ser Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp 2135 2140 2145 Ser Cys Phe Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp 2150 2155 2160 Glu Trp Glu Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln 2165 2170 2175 Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg 2180 2185 2190 Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp 2195 2200 2205 Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu 2210 2215 2220 Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr 2225 2230 2235 Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln 2240 2245 2250 Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly 2255 2260 2265 Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu 2270 2275 2280 Pro Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser 2285 2290 2295 Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile 2300 2305 2310 Glu Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser 2315 2320 2325 Arg Glu 2330 82477PRTHomo sapiensMISC_FEATUREFn1 isoform 1 preproprotein 8Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp

1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile 1265 1270 1275 Thr Asp Ser Ser Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser 1280 1285 1290 Thr Ile Ile Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly 1295 1300 1305 Ile Pro Ile Phe Glu Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr 1310 1315 1320 Thr Val Thr Gly Leu Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val 1325 1330 1335 Ile Thr Leu Ile Asn Gly Gly Glu Ser Ala Pro Thr Thr Leu Thr 1340 1345 1350 Gln Gln Thr Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn 1355 1360 1365 Ile Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser 1370 1375 1380 Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn 1385 1390 1395 Glu Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala 1400 1405 1410 Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser 1415 1420 1425 Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly 1430 1435 1440 Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser 1445 1450 1455 Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg 1460 1465 1470 Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe 1475 1480 1485 Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser 1490 1495 1500 Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser 1505 1510 1515 Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly 1520 1525 1530 Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val 1535 1540 1545 Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala 1550 1555 1560 Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly 1565 1570 1575 Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr 1580 1585 1590 Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr 1595 1600 1605 Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 1610 1615 1620 Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 1625 1630 1635 Met Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp 1640 1645 1650 Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr 1655 1660 1665 Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro 1670 1675 1680 Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu 1685 1690 1695 Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln 1700 1705 1710 Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly 1715 1720 1725 Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp 1730 1735 1740 Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser 1745 1750 1755 Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly 1760 1765 1770 Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu 1775 1780 1785 Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln 1790 1795 1800 Pro Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp 1805 1810 1815 Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp 1820 1825 1830 Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr 1835 1840 1845 Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro 1850 1855 1860 Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys 1865 1870 1875 Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg 1880 1885 1890 Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro 1895 1900 1905 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile 1910 1915 1920 Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp 1925 1930 1935 Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys 1940 1945 1950 Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr 1955 1960 1965 Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser 1970 1975 1980 Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser 1985 1990 1995 Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser 2000 2005 2010 Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr 2015 2020 2025 Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg 2030 2035 2040 Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr 2045 2050 2055 Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser 2060 2065 2070 Glu Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu 2075 2080 2085 Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp 2090 2095 2100 Val Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly 2105 2110 2115 Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln 2120 2125 2130 Gln Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe 2135 2140 2145 Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg 2150 2155 2160 Pro Arg Pro Tyr Pro Pro Asn Val Gly Glu Glu Ile Gln Ile Gly 2165 2170 2175 His Ile Pro Arg Glu Asp Val Asp Tyr His Leu Tyr Pro His Gly 2180 2185 2190 Pro Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser 2195 2200 2205 Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr 2210 2215 2220 Ile Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln 2225 2230 2235 Phe Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu 2240 2245 2250 Thr Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp 2255 2260 2265 Gln Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn 2270 2275 2280 Ser Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe 2285 2290 2295 Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu 2300 2305 2310 Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly 2315 2320 2325 Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His 2330 2335 2340 Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln 2345 2350 2355 Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly 2360 2365 2370 Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp 2375 2380 2385 Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr 2390 2395 2400 Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly 2405 2410 2415 Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro 2420 2425 2430 Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr 2435 2440 2445 His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe 2450 2455 2460 Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2465 2470 2475 92386PRTHomo sapiensMISC_FEATURElargest Fn splice variant 9Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Gln Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp

900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly Leu 1625 1630 1635 Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu 1640 1645 1650 Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser 1655 1660 1665 Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu 1670 1675 1680 Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr 1685 1690 1695 Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro 1700 1705 1710 Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu 1715 1720 1725 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1730 1735 1740 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1745 1750 1755 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1760 1765 1770 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1775 1780 1785 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1790 1795 1800 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1805 1810 1815 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1820 1825 1830 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1835 1840 1845 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1850 1855 1860 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1865 1870 1875 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1880 1885 1890 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1895 1900 1905 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1910 1915 1920 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1925 1930 1935 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1940 1945 1950 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1955 1960 1965 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1970 1975 1980 Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val 1985 1990 1995 Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val 2000 2005 2010 Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly Tyr 2015 2020 2025 Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln Gln 2030 2035 2040 Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe Arg 2045 2050 2055 Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg Pro 2060 2065 2070 Arg Pro Tyr Pro Pro Asn Val Gly Glu Glu Ile Gln Ile Gly His 2075 2080 2085 Ile Pro Arg Glu Asp Val Asp Tyr His Leu Tyr Pro His Gly Pro 2090 2095 2100 Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser Gln 2105 2110 2115 Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile 2120 2125 2130 Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe 2135 2140 2145 Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr 2150 2155 2160 Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln 2165 2170 2175 Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser 2180 2185 2190 Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp 2195 2200 2205 Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg 2210 2215 2220 Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe 2225 2230 2235 Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp 2240 2245 2250 Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly 2255 2260 2265 Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys 2270 2275 2280 Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp 2285 2290 2295 Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu 2300 2305 2310 Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp 2315 2320 2325 Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu 2330 2335 2340 Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His 2345 2350 2355 Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met 2360 2365 2370 Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2375 2380 2385 1049PRTStreptococcus pyogenesMISC_FEATUREFunctional Upstream Domain (FUD) of F1 adhesion protein 10Lys Asp Gln Ser Pro Leu Ala Gly Glu Ser Gly Glu Thr Glu Tyr Ile 1 5 10 15 Thr Glu Val Tyr Gly Asn Gln Gln Asn Pro Val Asp Ile Asp Lys Lys 20 25 30 Leu Pro Asn Glu Thr Gly Phe Ser Gly Asn Met Val Glu Thr Glu Asp 35 40 45 Thr 117PRTStreptococcus pyogenes 11Met Gly Gly Gln Ser Glu Ser 1 5 1244PRTStreptococcus pyogenes 12Met Gly Gly Gln Ser Glu Ser Val Glu Phe Thr Lys Asp Thr Gln Thr 1 5 10 15 Gly Met Ser Gly Gln Thr Thr Pro Gln Ile Glu Thr Glu Asp Thr Lys 20 25 30 Glu Pro Gly Val Leu Met Gly Gly Gln Ser Glu Ser 35 40 138PRTArtificial SequenceSynthetic polypeptide 13Gly Pro Gln Gly Ile Trp Gly Gln 1 5 148PRTArtificial SequenceSynthetic polypeptide 14Gly Asp Gln Gly Ile Ala Gly Phe 1 5 1510PRTArtificial SequenceSynthetic polypeptide 15Phe Lys Gly Gly Arg Gly Asp Ser Pro Gly 1 5 10 1610PRTArtificial SequenceSynthetic polypeptide 16Phe Lys Gly Gly Gly Lys Phe Ile Thr Cys 1 5 10

Claims

1. A method of inhibiting neovascularization in a subject, the method comprising administering to the subject an agent that interferes with fibronectin (Fn) matrix assembly in an amount effective to inhibit neovascularization.

2. The method of claim 1, wherein the agent does not promote apoptosis.

3. The method of claim 1, wherein the agent does not interfere with binding between integrins and soluble Fn.

4. The method of claim 1, wherein the agent comprises an antibody or fragment thereof that binds Fn.

5. The method of claim 4, wherein the antibody or fragment thereof binds Fn at or near Fn III_1,2 modules.

6. The method of claim 1, wherein the agent comprises an Fn fragment.

7. The method of claim 6, wherein the Fn fragment interferes with polymerization of intact Fn dimers.

8. The method of claim 6, wherein the agent is comprises a nucleic acid sequence encoding an Fn fragment.

9. The method of claim 1, wherein the agent comprises a microbial surface component reorganizing adhesive matrix molecule (MSCRAMM).

10. The method of claim 9, wherein the agent comprises a functional upstream domain (FUD) of Streptococcus pyogenes adhesion F1 protein.

11. The method of claim 10, wherein the agent is a nucleic acid comprising a nucleic acid sequence encoding a FUD of Streptococcus pyogenes adhesion F1 protein.

12. A method of identifying an agent that inhibits neovascularization, the method comprising detecting fibronectin (Fn) matrix assembly by stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent, wherein a decrease in Fn matrix assembly in the presence of the agent compared to Fn matrix assembly in the absence of the agent is indicative of an agent that inhibits neovascularization.

13. A method of identifying an agent that inhibits neovascularization, the method comprising detecting changes in nuclear architecture in stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent, wherein a reduction in nuclear architecture organization identifies an agent that inhibits neovascularization.

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