NOD nucleic acids and polypeptides

Abstract

The present invention relates to the NOD proteins and nucleic acids encoding the NOD proteins. The present invention further provides assays for the detection of NOD polymorphisms and mutations associated with disease states, as well as methods of screening for ligands and modulators of NOD proteins.

Description

[0001] This application claims priority to provisional patent application Ser. No. 60/452,274, filed Mar. 05, 2004; which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the NOD proteins and nucleic acids encoding the NOD proteins. The present invention further provides assays for the detection of NOD polymorphisms and mutations associated with disease states, as well as methods of screening for ligands and modulators of NOD proteins.

BACKGROUND OF THE INVENTION

[0004] The removal of infectious agents by the host is fundamental for the survival of multicellular organisms. In animals and plants, the initial detection of microbial agents relies on specialized host receptors that recognize molecules expressed exclusively by microbes (Dang and Jones, Nature 411, 826-833 (2001); Medzhitov, Nature Rev. Immunol. 1, 135-145 (2001)). In animals, detection of microbial agents is mediated by the recognition of pathogen-associated molecular patterns (PAMPs) by specific host pattern-recognition receptors (PRRs) (Medzhitov, supra). Because the structure of each PAMP is highly conserved and invariant in microorganisms of the same class, the animal can recognize most or all microbes with a limited number of PRRs. The identification and characterization of plasma membrane Toll-like receptors (TLRs) as PRRs have provided fundamental insight into the mechanisms of host defense in animals. There is now compelling evidence that TLRs play a pivotal role in mediating immune responses to bacterial pathogens (Medzhitov, supra; Akira et al., Nat. Immunol. 2, 675-680 (2001)) In mammals, TLRs mediate host immune responses by inducing the secretion of several proinflammatory cytokines and co-stimulatory surface molecules through the activation of transcriptional factors including NF-.kappa.B (Medzhitov, supra; Akira et al., supra). The

SUMMARY OF THE INVENTION

[0005] The present invention relates to the NOD proteins and nucleic acids encoding the NOD proteins. The present invention further provides assays for the detection of NOD polymorphisms and mutations associated with disease states, as well as methods of screening for ligands and modulators of NOD proteins.

[0006] Accordingly, in some embodiments, the present invention provides a composition comprising an isolated and purified nucleic acid sequence encoding a protein selected from the group consisting of SEQ ID NOs: 12-22. In some embodiments, the sequence is operably linked to a heterologous promoter. In some embodiments, the sequence is contained within a vector. In some embodiments, the vector is within a host cell. In some embodiments, the nucleic acid comprises one of SEQ ID NOs: 1 and variants thereof that are at least 80%, preferably at least 90%, and even more preferably at least 95% identical to SEQ ID NOs: 12-22. In some embodiments, the nucleic acid comprises one of SEQ ID NOs: 1-11.

[0007] The present invention further provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NOs: 12-22 or variants thereof that are at least 80% identical to SEQ ID NOs: 12-22. In some embodiments, the polypeptide is at least 90%, and preferably at least 95% identical to SEQ ID NOs: 12-22. In some embodiments, the polypeptide comprises one of SEQ ID NOs: 12-22.

[0008] The present invention additionally provides a method of generating an inflammation profile, comprising providing a sample from a subject, wherein the sample comprises nucleic acid; and detecting the presence or absence of expression of at least two NOD genes in the sample, thereby generating an inflammation profile. In some embodiments, the detecting comprises detecting the presence or absence of expression of at least 5, and preferably at least 10 NOD genes in said sample. In some embodiments, the nucleic acid comprises genomic DNA. In other embodiments, the nucleic acid comprises mRNA.

DESCRIPTION OF THE FIGURES

[0009] FIG. 1 shows the domain structures of exemplary NOD nucleic acids and proteins of some embodiments of the present invention. CARD, caspase-recruitment domain; DC, dendritic cell; DT, DEFCAP/TUCAN expanded homology domain; EBD, effector-binding domain; NOD, nucleotide-binding oligomerization domain; PYD, pyrin domain; LRR, leucine-rich repeat; WD40R, WD40 repeat; BIR, baculoviral inhibitor-of-apoptosis repeat; TIR, Toll/interleukin-1 receptor.

[0010] FIG. 2 shows an induced proximity model of NOD protein activation. EBD, effector binding domain; LRD, ligand recognition domain; NOD, Nucleotide-binding oligomerization domain.

[0011] FIG. 3 shows signaling pathways mediated by NOD1, NOD2, IPAF and Cryopyrin.

[0012] FIG. 4 shows a model for the role of NOD 1, NOD2 and related NODs in innate and adaptive immunity. APC, antigen-presenting cell; MHC-II, major histocompatibility complex class II molecules; TCR, T-cell receptor; TLR, Toll-like receptors.

[0013] FIG. 5 shows hypothetical mechanisms of disease in patients with mutations in NOD2, Cryopyrin, CIITA and Pyrin.

[0014] FIG. 6 shows Table 2.

[0015] FIG. 7 shows the nucleic acid sequence of NOD3 (SEQ ID NO: 1).

[0016] FIG. 8 shows the nucleic acid sequence of NOD5 (SEQ ID NO:2).

[0017] FIG. 9 shows the nucleic acid sequence of NOD6 (SEQ ID NO:3).

[0018] FIG. 10 shows the nucleic acid sequence of NOD8 (SEQ ID NO:4).

[0019] FIG. 11 shows the nucleic acid sequence of NOD9 (SEQ ID NO:5).

[0020] FIG. 12 shows the nucleic acid sequence of NOD12 (SEQ ID NO:6).

[0021] FIG. 13 shows the nucleic acid sequence of NOD14 (SEQ ID NO:7).

[0022] FIG. 14 shows the nucleic acid sequence of NOD17 (SEQ ID NO:9).

[0023] FIG. 15 shows the nucleic acid sequence of NOD26 (SEQ ID NO: 10).

[0024] FIG. 16 shows the nucleic acid sequence of NOD27 (SEQ ID NO: 11).

[0025] FIG. 17 shows the amino acid sequence of NOD3 (SEQ ID NO:12).

[0026] FIG. 18 shows the amino acid sequence of NOD5 (SEQ ID NO: 13).

[0027] FIG. 19 shows the amino acid sequence of NOD6 (SEQ ID NO:14).

[0028] FIG. 20 shows the amino acid sequence of NOD8 (SEQ ID NO: 15).

[0029] FIG. 21 shows the amino acid sequence of NOD9 (SEQ ID NO:16).

[0030] FIG. 22 shows the amino acid sequence of NOD12 (SEQ ID NO: 17).

[0031] FIG. 23 shows the amino acid sequence of NOD14 (SEQ ID NO:18).

[0032] FIG. 24 shows the amino acid sequence of NOD17 (SEQ ID NO:20).

[0033] FIG. 25 shows the amino acid sequence of NOD26 (SEQ ID NO:21).

[0034] FIG. 26 shows the amino acid sequence of NOD27 (SEQ ID NO:22).

[0035] FIG. 27 shows the nucleic acid sequence of NOD16 (SEQ ID NO:8).

[0036] FIG. 28 shows the nucleic acid sequence of NOD 16 (SEQ ID NO: 19).

DEFINITIONS

[0037] To facilitate understanding of the invention, a number of terms are defined below.

[0038] As used herein, the term "NOD" when used in reference to a protein or nucleic acid refers to a NOD protein or nucleic acid encoding a NOD protein of the present invention. The term NOD encompasses both proteins that are identical to wild-type NODs and those that are derived from wild type NOD (e.g., variants of NOD polypeptides of the present invention) or chimeric genes constructed with portions of NOD coding regions). In some embodiments, the "NOD" is a wild type NOD nucleic acid (SEQ ID NOs: 1 -11) or amino acid (SEQ ID NOs: 12-22) sequence. In other embodiments, the "NOD" is a variant or mutant.

[0039] As used herein, the term "instructions for using said kit for said detecting the presence or absence of a variant NOD nucleic acid or polypeptide in said biological sample" includes instructions for using the reagents contained in the kit for the detection of variant and wild type NOD nucleic acids or polypeptides. In some embodiments, the instructions further comprise the statement of intended use required by the U.S. Food and Drug Administration (FDA) in labeling in vitro diagnostic products. The FDA classifies in vitro diagnostics as medical devices and requires that they be approved through the 510(k) procedure. Information required in an application under 510(k) includes: 1) The in vitro diagnostic product name, including the trade or proprietary name, the common or usual name, and the classification name of the device; 2) The intended use of the product; 3) The establishment registration number, if applicable, of the owner or operator submitting the 510(k) submission; the class in which the in vitro diagnostic product was placed under section 513 of the FD&C Act, if known, its appropriate panel, or, if the owner or operator determines that the device has not been classified under such section, a statement of that determination and the basis for the determination that the in vitro diagnostic product is not so classified; 4) Proposed labels, labeling and advertisements sufficient to describe the in vitro diagnostic product, its intended use, and directions for use. Where applicable, photographs or engineering drawings should be supplied; 5) A statement indicating that the device is similar to and/or different from other in vitro diagnostic products of comparable type in commercial distribution in the U.S., accompanied by data to support the statement; 6) A 510(k) summary of the safety and effectiveness data upon which the substantial equivalence determination is based; or a statement that the 510(k) safety and effectiveness information supporting the FDA finding of substantial equivalence will be made available to any person within 30 days of a written request; 7) A statement that the submitter believes, to the best of their knowledge, that all data and information submitted in the premarket notification are truthful and accurate and that no material fact has been omitted; 8) Any additional information regarding the in vitro diagnostic product requested that is necessary for the FDA to make a substantial equivalency determination. Additional information is available at the Internet web page of the U.S. FDA.

[0040] As used herein, the term "inflammation profile" refers to the pattern of expression of two or more NOD genes of the present invention (e.g., the NOD genes described by SEQ ID NOs: 1-11). In some embodiments, the pattern of expression comprises the presence or absence of expression. In other embodiments, the pattern of expression comprises the level of expression or localization of expression of the NOD genes. The inflammation profiles of the present invention find use the characterization of inflammatory diseases and in determining a subject's risk of contacting an inflammatory disease. For example, in some embodiments, inflammation profiles from a subject are compared to control profiles associated with disease or predisposition to disease.

[0041] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., including but not limited to, mRNA, tRNA and rRNA) or precursor (e.g., NOD). The polypeptide, RNA, or precursor can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0042] In particular, the term "NOD gene" or "NOD genes" refers to the full-length NOD nucleotide sequence (e.g., contained in SEQ ID NOs: 1-11). However, it is also intended that the term encompass fragments of the NOD sequences, mutants of the NOD sequences, as well as other domains within the full-length NOD nucleotide sequences. Furthermore, the terms "NOD nucleotide sequence" or "NOD polynucleotide sequence" encompasses DNA, cDNA, and RNA (e.g., mRNA) sequences.

[0043] Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms, such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

[0044] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

[0045] The term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the terms "modified," "mutant," "polymorphism," and "variant" refer to a gene or gene product that displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0046] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

[0047] DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides or polynucleotide, referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

[0048] As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene," means a nucleic acid sequence comprising the coding region of a gene or, in other words, the nucleic acid sequence that encodes a gene product. The coding region may be present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

[0049] As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.

[0050] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence 5'-"A-G-T-3'," is complementary to the sequence 3'-"T-C-A-5'." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Complementarity can include the formation of base pairs between any type of nucleotides, including non-natural bases, modified bases, synthetic bases and the like.

[0051] The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is referred to using the functional term "substantially homologous." The term "inhibition of binding," when used in reference to nucleic acid binding, refers to inhibition of binding caused by competition of homologous sequences for binding to a target sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

[0052] The art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).

[0053] When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

[0054] A gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other.

[0055] When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

[0056] As used herein, the term "competes for binding" is used in reference to a first polypeptide with an activity which binds to the same substrate as does a second polypeptide with an activity, where the second polypeptide is a variant of the first polypeptide or a related or dissimilar polypeptide. The efficiency (e.g., kinetics or thermodynamics) of binding by the first polypeptide may be the same as or greater than or less than the efficiency substrate binding by the second polypeptide. For example, the equilibrium binding constant (K.sub.D) for binding to the substrate may be different for the two polypeptides. The term "K.sub.m" as used herein refers to the Michaelis-Menton constant for an enzyme and is defined as the concentration of the specific substrate at which a given enzyme yields one-half its maximum velocity in an enzyme catalyzed reaction.

[0057] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T.sub.m of the formed hybrid, and the G:C ratio within the nucleic acids.

[0058] As used herein, the term "T.sub.m" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T.sub.m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T.sub.m value may be calculated by the equation: T.sub.m=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T.sub.m.

[0059] As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that "stringency" conditions may be altered by varying the parameters just described either individually or in concert. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency" conditions may occur between homologs with about 85-100% identity, preferably about 70-100% identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency" conditions may occur between homologs with about 50-70% identity). Thus, conditions of "weak" or "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.

[0060] "High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times. Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times.SSPE, 1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.

[0061] "Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times. Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0.times.SSPE, 1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed. "Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5.times. Denhardt's reagent [50.times. Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.

[0062] The present invention is not limited to the hybridization of probes of about 500 nucleotides in length. The present invention contemplates the use of probes between approximately 10 nucleotides up to several thousand (e.g., at least 5000) nucleotides in length. One skilled in the relevant understands that stringency conditions may be altered for probes of other sizes (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization [1985] and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY [1989]).

[0063] The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA sequence given in a sequence listing or may comprise a complete gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman [Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)] by the homology alignment algorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol. 48:443 (1970)], by the search for similarity method of Pearson and Lipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988)], by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length sequences of the compositions claimed in the present invention (e.g., NOD).

[0064] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0065] The term "fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to the native protein, but where the remaining amino acid sequence is identical to the corresponding positions in the amino acid sequence deduced from a full-length cDNA sequence. Fragments typically are at least 4 amino acids long, preferably at least 20 amino acids long, usually at least 50 amino acids long or longer, and span the portion of the polypeptide required for intermolecular binding of the compositions (claimed in the present invention) with its various ligands and/or substrates.

[0066] The term "polymorphic locus" is a locus present in a population that shows variation between members of the population (i.e., the most common allele has a frequency of less than 0.95). In contrast, a "monomorphic locus" is a genetic locus at little or no variations seen between members of the population (generally taken to be a locus at which the most common allele exceeds a frequency of 0.95 in the gene pool of the population).

[0067] As used herein, the term "genetic variation information" or "genetic variant information" refers to the presence or absence of one or more variant nucleic acid sequences (e.g., polymorphism or mutations) in a given allele of a particular gene (e.g., a NOD gene of the present invention).

[0068] As used herein, the term "detection assay" refers to an assay for detecting the presence or absence of variant nucleic acid sequences (e.g., polymorphisms or mutations) in a given allele of a particular gene (e.g., a NOD gene). Examples of suitable detection assays include, but are not limited to, those described below in Section III B.

[0069] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

[0070] "Amplification" is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

[0071] Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q.beta. replicase, MDV-1 RNA is the specific template for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]).

[0072] As used herein, the term "amplifiable nucleic acid" is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template."

[0073] As used herein, the term "sample template" refers to nucleic acid originating from a sample that is analyzed for the presence of "target" (defined below). In contrast, "background template" is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

[0074] As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

[0075] As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0076] As used herein, the term "target," refers to a nucleic acid sequence or structure to be detected or characterized. Thus, the "target" is sought to be sorted out from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within the target sequence.

[0077] As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified."

[0078] With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of .sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

[0079] As used herein, the terms "PCR product," "PCR fragment," and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

[0080] As used herein, the term "amplification reagents" refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

[0081] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

[0082] As used herein, the term "recombinant DNA molecule" as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.

[0083] As used herein, the term "antisense" is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). Included within this definition are antisense RNA ("asRNA") molecules involved in gene regulation by bacteria. Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. Once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. The designation (-) (i.e., "negative") is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.

[0084] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding NOD includes, by way of example, such nucleic acid in cells ordinarily expressing NOD where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0085] As used herein, a "portion of a chromosome" refers to a discrete section of the chromosome. Chromosomes are divided into sites or sections by cytogeneticists as follows: the short (relative to the centromere) arm of a chromosome is termed the "p" arm; the long arm is termed the "q" arm. Each arm is then divided into 2 regions termed region 1 and region 2 (region 1 is closest to the centromere). Each region is further divided into bands. The bands may be further divided into sub-bands. For example, the 11p15.5 portion of human chromosome 11 is the portion located on chromosome 11 (11) on the short arm (p) in the first region (1) in the 5th band (5) in sub-band 5 (0.5). A portion of a chromosome may be "altered;" for instance the entire portion may be absent due to a deletion or may be rearranged (e.g., inversions, translocations, expanded or contracted due to changes in repeat regions). In the case of a deletion, an attempt to hybridize (i.e., specifically bind) a probe homologous to a particular portion of a chromosome could result in a negative result (i.e., the probe could not bind to the sample containing genetic material suspected of containing the missing portion of the chromosome). Thus, hybridization of a probe homologous to a particular portion of a chromosome may be used to detect alterations in a portion of a chromosome.

[0086] The term "sequences associated with a chromosome" means preparations of chromosomes (e.g., spreads of metaphase chromosomes), nucleic acid extracted from a sample containing chromosomal DNA (e.g., preparations of genomic DNA); the RNA that is produced by transcription of genes located on a chromosome (e.g., hnRNA and mRNA), and cDNA copies of the RNA transcribed from the DNA located on a chromosome. Sequences associated with a chromosome may be detected by numerous techniques including probing of Southern and Northern blots and in situ hybridization to RNA, DNA, or metaphase chromosomes with probes containing sequences homologous to the nucleic acids in the above listed preparations.

[0087] As used herein the term "portion" when in reference to a nucleotide sequence (as in "a portion of a given nucleotide sequence") refers to fragments of that sequence. The fragments may range in size from four nucleotides to the entire nucleotide sequence minus one nucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).

[0088] As used herein the term "coding region" when used in reference to structural gene refers to the nucleotide sequences that encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by one of the three triplets, which specify stop codons (i.e., TAA, TAG, TGA).

[0089] As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, NOD antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind a NOD polypeptide. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind a NOD polypeptide results in an increase in the percent of NOD-reactive immunoglobulins in the sample. In another example, recombinant NOD polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant NOD polypeptides is thereby increased in the sample.

[0090] The term "recombinant DNA molecule" as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.

[0091] The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule.

[0092] The term "native protein" as used herein, is used to indicate a protein that does not contain amino acid residues encoded by vector sequences; that is the native protein contains only those amino acids found in the protein as it occurs in nature. A native protein may be produced by recombinant means or may be isolated from a naturally occurring source.

[0093] As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four consecutive amino acid residues to the entire amino acid sequence minus one amino acid.

[0094] The term "Southern blot," refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size followed by transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58 [1989]).

[0095] The term "Northern blot," as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52 [1989]).

[0096] The term "Western blot" refers to the analysis of protein(s) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane. The proteins are run on acrylamide gels to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest. The binding of the antibodies may be detected by various methods, including the use of radiolabeled antibodies.

[0097] The term "antigenic determinant" as used herein refers to that portion of an antigen that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies that bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the "immunogen" used to elicit the immune response) for binding to an antibody.

[0098] The term "transgene" as used herein refers to a foreign, heterologous, or autologous gene that is placed into an organism by introducing the gene into newly fertilized eggs or early embryos. The term "foreign gene" refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of an animal by experimental manipulations and may include gene sequences found in that animal so long as the introduced gene does not reside in the same location as does the naturally-occurring gene. The term "autologous gene" is intended to encompass variants (e.g., polymorphisms or mutants) of the naturally occurring gene. The term transgene thus encompasses the replacement of the naturally occurring gene with a variant form of the gene.

[0099] As used herein, the term "vector" is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "vehicle" is sometimes used interchangeably with "vector."

[0100] The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0101] As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo. For example, host cells may be located in a transgenic animal.

[0102] The terms "overexpression" and "overexpressing" and grammatical equivalents, are used in reference to levels of mRNA to indicate a level of expression approximately 3-fold higher than that typically observed in a given tissue in a control or non-transgenic animal. Levels of mRNA are measured using any of a number of techniques known to those skilled in the art including, but not limited to Northern blot analysis (See, Example 10, for a protocol for performing Northern blot analysis). Appropriate controls are included on the Northern blot to control for differences in the amount of RNA loaded from each tissue analyzed (e.g., the amount of 28S rRNA, an abundant RNA transcript present at essentially the same amount in all tissues, present in each sample can be used as a means of normalizing or standardizing the RAD50 mRNA-specific signal observed on Northern blots). The amount of mRNA present in the band corresponding in size to the correctly spliced NOD transgene RNA is quantified; other minor species of RNA which hybridize to the transgene probe are not considered in the quantification of the expression of the transgenic mRNA.

[0103] The term "transfection" as used herein refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0104] The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into the genomic DNA.

[0105] The term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term "transient transfectant" refers to cells that have taken up foreign DNA but have failed to integrate this DNA.

[0106] The term "calcium phosphate co-precipitation" refers to a technique for the introduction of nucleic acids into a cell. The uptake of nucleic acids by cells is enhanced when the nucleic acid is presented as a calcium phosphate-nucleic acid co-precipitate. The original technique of Graham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]), has been modified by several groups to optimize conditions for particular types of cells. The art is well aware of these numerous modifications.

[0107] A "composition comprising a given polynucleotide sequence" as used herein refers broadly to any composition containing the given polynucleotide sequence. The composition may comprise an aqueous solution. Compositions comprising polynucleotide sequences encoding NODs (e.g., SEQ ID NOs:1-11) or fragments thereof may be employed as hybridization probes. In this case, the NOD encoding polynucleotide sequences are typically employed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

[0108] The term "test compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample. Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.

[0109] The term "sample" as used herein is used in its broadest sense. A sample suspected of containing a human chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. A sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.

[0110] As used herein, the term "response," when used in reference to an assay, refers to the generation of a detectable signal (e.g., accumulation of reporter protein, increase in ion concentration, accumulation of a detectable chemical product).

[0111] As used herein, the term "reporter gene" refers to a gene encoding a protein that may be assayed. Examples of reporter genes include, but are not limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol. 7:725 [1987] and U.S. Pat Nos. 6,074,859; 5,976,796; 5,674,713; and 5,618,682; all of which are incorporated herein by reference), green fluorescent protein (e.g., GenBank Accession Number U43284; a number of GFP variants are commercially available from CLONTECH Laboratories, Palo Alto, Calif.), chloramphenicol acetyltransferase, .beta.-galactosidase, alkaline phosphatase, and horse radish peroxidase.

[0112] As used herein, the terms "computer memory" and "computer memory device" refer to any storage media readable by a computer processor. Examples of computer memory include, but are not limited to, RAM, ROM, computer chips, digital video disc (DVDs), compact discs (CDs), hard disk drives (HDD), and magnetic tape.

[0113] As used herein, the term "computer readable medium" refers to any device or system for storing and providing information (e.g., data and instructions) to a computer processor. Examples of computer readable media include, but are not limited to, DVDs, CDs, hard disk drives, magnetic tape and servers for streaming media over networks.

[0114] As used herein, the term "entering" as in "entering said genetic variation information into said computer" refers to transferring information to a "computer readable medium." Information may be transferred by any suitable method, including but not limited to, manually (e.g., by typing into a computer) or automated (e.g., transferred from another "computer readable medium" via a "processor").

[0115] As used herein, the terms "processor" and "central processing unit" or "CPU" are used interchangeably and refer to a device that is able to read a program from a computer memory (e.g., ROM or other computer memory) and perform a set of steps according to the program.

[0116] As used herein, the term "computer implemented method" refers to a method utilizing a "CPU" and "computer readable medium."

GENERAL DESCRIPTION OF THE INVENTION

[0117] The nucleotide-binding oligomerization domain (NOD) was first found in Apaf-1 and its nematode homologue CED-4, two pivotal regulators of developmental and p53-dependent programmed cell death (Lui and Hengartner, supra; Derry et al., supra). Subsequently, two NOD-containing molecules, NOD1 (CARD4) and NOD2, were identified through database searches for Apaf-1/CED-4 homologues. Since then, the NOD protein family has greatly expanded and currently contains a large number of proteins from animals, plants, fungi and bacteria, including >20 human proteins homologous to Apaf-1 and NOD1 (FIG. 1). The majority of NOD family members are comprised of three distinct functional domains, an amino-terminal effector binding domain (EBD), a centrally located NOD and a carboxy-terminal ligand recognition domain (LRD) (Table 2). The NOD mediates self oligomerization, which, in some embodiments, function in the activation of downstream effector molecules. The EBD of mammalian NOD proteins mediates the binding to effector molecules which determines the downstream events activated upon signaling, including apoptosis and NF-.kappa.B activation (Table 2).

[0118] Some NOD proteins share the same type of effector domain (e.g., CARD or PYD). In some embodiments, the NOD proteins activate different signaling cascades as the interaction between these domains and those present in downstream binding partners is highly specific. For example, the PYD of ASC, a downstream adaptor molecule involved in NOD signalling, associates with the PYD of cryopyrin, but not with the PYD present in NALP2, PAN2, PYPAF3, PYPAF4, PYPAF6 or NOD27 (Grenier et al., FEBS Lett. 530, 73-78 (2002)). In other embodiments, certain NOD proteins like NOD1 and NOD2 interact with and use a common downstream molecule, RICK, to activate identical or similar signalling pathways (FIG. 3). Transient expression of NOD1 and NOD2 in mammalian cells induces NF-.kappa.B activation (Bertin et al., J. Biol. Chem. 274, 12955-12958 (1999); Inohara et al., J. Biol. Chem. 274, 14560-14567 (1999); Ogura et al., J. Biol. Chem. 276, 4812-4818 (2001)). Mutational analyses demonstrated that the CARDs and the NODs of NOD 1 and NOD2 were required for the induction of NF-KB whereas its LRRs were dispensable (Inohara et al., J. Biol. Chem. 274, 14560-14567 (1999); Ogura et al., J. Biol. Chem. 276, 4812-4818 (2001)). Thus, in some embodiments, the CARDs act as effector domains for NOD 1 and NOD2 signalling. Both NOD 1 and NOD2 physically associate with RICK, a CARD-containing protein kinase through homophilic CARD-CARD interactions (Inohara et al., J. Biol.. Chem. 274, 14560-14567 (1999); Ogura et al., J. Biol. Chem. 276, 4812-4818 (2001)). A role for RICK in NOD1 and NOD2 signalling is supported by several studies (Inohara et al., supra; Ogura et al., supra).

[0119] Several NOD-LRR proteins, including IPAF, cryopyrin, and DEFCAP, associate with ASC (Manji et al., J. Biol. Chem. 277, 11570-11575 (2002); Geddes et al., Biochem. Biophys. Res. Commun. 284, 77-82 (2001); Martinon et al., Mol. Cell. 10, 417-426 (2002)). ASC (also called TMS1/PYCARD) is an adaptor molecule originally identified in a sub-cytosolic fraction called the "speck" in cells undergoing apoptosis. ASC is composed of an amino-terminal PYD and a carboxy-terminal CARD. Co-expression of ASC with several PYD-containing NOD proteins including cryopyrin, PYPAF5 or PYPAF7, as well as with the CARD-containing IPAF, induces NF-.kappa.B activation (Manji et al., supra). Thus, in some embodiments, PYD-containing NOD proteins use the adaptor ASC for signaling (Grenier et al., supra). NF-.kappa.B activation induced through ASC signalling is inhibited by dominant forms of NEMO/IKK.gamma. (Manji et al., supra; Grenier et al., supra). Thus, ASC signals, as was reported for RICK, through the common IKK signalling pathway of NF-.kappa.B activation (FIG. 3).

[0120] Multiple NOD proteins including NOD 1, NOD2, IPAF and DEFCAP promote activation of pro-inflammatory caspases. For example, NOD1 promotes caspase-1 activation in transient overexpression studies (Yoo et al., Biochem. Biophys. Res. Commun. 299, 652-658 (2002)). IPAF, cryopyrin, DEFCAP, PYPAF5 and PYPAF7 have been found to regulate, in the presence of ASC, the activation of caspase-1, interleukin-1.beta. converting enzyme (Grenier et al., supra; Wang et al., J. Biol. Chem. 277, 29874-29880 (2002)). DEFCAP, the only NOD family member known to possess both a CARD and PYD, can form an endogenous multi-protein complex containing ASC, caspase-1 and caspase-5 dubbed "the inflammasome" which promotes caspase activation and processing of pro-interleukin-1.beta. (Martinon et al., Mol. Cell. 10, 417-426 (2002)).

[0121] In some embodiments, NOD proteins (e.g., Apaf-1, NOD 1, NOD2, DEFCAP, IPAF and cryopyrin) induce or enhance apoptosis (Inohara et al., J. Biol. Chem. 274, 14560-14567 (1999); Ogura et al., J. Biol. Chem. 276, 4812-4818 (2001); Geddes et al., Biochem. Biophys. Res. Commun. 284, 77-82 (2001); Poyet et al., J. Biol. Chem. 276, 28309-28313 (2001); Hlaing et al., J. Biol. Chem. 276, 9230-9238 (2001); Zou et al., Cell 90, 405-413 (1997)). For example, NOD1 and DEFCAP interact with multiple caspases and/or Apaf-1 (Hlaing et al., supra; Inohara and Nunez, Oncogene, 20, 6473-6481 (2001)). Co-expression of IPAF or cryopyrin with ASC or forced oligomerization of IPAF or cryopyrin induces apoptosis in mammalian cells, which requires caspase activity. NOD 1, IPAF, cryopyrin, PYPAF5 and PYPAF7 induce both NF-.kappa.B and caspase-1 activation. Thus, in some embodiments, NOD pro-apoptotic activity results from the activation of inflammatory caspases. In other embodiments, apoptotic caspases contribute to the activation of inflammatory caspases.

[0122] In some embodiments, the induction of both NF-.kappa.B and apoptosis by NOD proteins is similar to that observed with TLRs, PKR and death receptors (DRs), which induce apoptosis through the activation of caspases. Upon DR signalling, the induction of apoptosis is suppressed in vivo by simultaneous activation of NF-KB, which leads to the expression of anti-apoptotic genes (Beg and Baltimore, Science 274, 782-784 (1996); Wang et al., Science 281, 1680-1683 (1998); Micheau et al., Mol. Cell. Biol. 21, 5299-5305 (2001)). Thus, in some embodiments, under physiological conditions, the pro-apoptotic activity induced through NOD proteins is suppressed by simultaneous induction of NF-.kappa.B activity.

[0123] Genetic variation in three human NOD proteins has been implicated in the development of genetic diseases (Hull et al., Curr Opin Rheumatol. 15, 61-69 (2003)). For example, mutations in CIITA are known to cause type II lymphocyte bare syndrome (LBS), a hereditary immunodeficiency disorder characterized by the absence of MHCII expression (Steimle et al., Cell 75, 135-146 (1993); Reith and Mach, Annu Rev Immunol. 19, 331-373. (2001)). More recently, mutations in NOD2 and CIAS1 (the gene encoding cryopyrin) have been implicated in several autoinflammatory diseases. A frameshift mutation, L1007fsinsC, and two missense mutations (G908R and R702W) in NOD2 are associated with Crohn's disease (CD), a common inflammatory disease of the intestinal tract (Ogura et al., Nature 411, 603-606 (2001); Hugot et al., Nature 411, 599-603 (2001); Hampe et al., Lancet 357, 1925-1928 (2001)). Having one copy of the mutated alleles confers a 2-4-fold increased risk of developing CD, whereas homozygocity or compound heterozygocity for NOD2 mutations increases the risk 20-40-fold, indicating that lack of NOD2 function is important for disease development. All three CD-associated mutations result in proteins that are deficient in inducing PGN- and MDP-mediated NF-.kappa.B activation. Activation of NF-.kappa.B induced by MDP is absent in mononuclear cells derived from CD patients homozygous for L1007fsinsC.

[0124] In addition to CD, missense mutations in the coding region of NOD2 have been associated with Blau syndrome, an autosomal dominant trait characterized by arthritis, uveitis and skin rashes (Miceli-Richard et al., Nat. Genet. 29, 19-20 (2001)). NOD2 mutations resulting in Blau syndrome are located in the NOD (Miceli-Richard et al., supra). NOD2 mutant proteins found in patients with Blau syndrome induce increased basal NF-.kappa.B activity, when compared to wild-type NOD2. Thus, variant proteins found in patients with Blau syndrome may represent constitutively active NOD2 mutations. This is in contrast to CD-associated NOD2 variants, which have normal or reduced levels of basal activity but are defective in their response bacterial components (Ogura et al., Nature 411, 603-606 (2001); Bonen et al., Gastroenterology 124, 140-146 (2003)).

[0125] Mutations in the CIAS1 gene, which encodes cryopyrin, are the cause of several autoinflammatory syndromes characterized by recurrent episodes of seemingly unprovoked inflammation (Hoffman et al., Nature Genet. 29, 301-305 (2001); Feldmann et al., Am. J Hum. Genet. 71, 198-203 (2002); Aksentijevich et al., Arthritis Rheum. 46, 3340-3348 (2002); Aganna et al., Arthritis Rheum., 46, 2445-2452 (2002)). These autosomal-dominant diseases include familial cold autoinflammatory syndrome (FACS), Muckle-Wells syndrome (MWS) and neonatal-onset multisystem inflammatory disease (NOMID, also known as chronic infantile neurologic cutaneous articular syndrome or CINCA). Patients with FACS, MWS and NOMID carry missense mutations that localize to the NOD of cryopyrin. The R260W mutation associated with FACS and MWS corresponds to the R334W NOD2 mutation found in Blau syndrome (Miceli-Richard et al., supra). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism of the present invention is not required to practice the present invention. Nonetheless, it is contemplated that this observation suggests that R206W cryopyrin may represent a constitutively active mutation which may lead to a deregulated activation of NF-.kappa.B and inflammatory caspases (FIG. 5).

[0126] Pyrin has been implicated in familial Mediterranean fever (FMF), an autosomal-recessive disease characterized by recurrent episodes of fever and localized inflammation (The International FMF Consortium, Cell 90, 797-807 (1997)). The gene mutated in FMF encodes a protein called pyrin, which is composed of an amino-terminal PYD, a B-type zinc-finger box, a coiled coil, a PRY domain and a Spla and Ryanodine receptor (SPRY) domain (The International FMF Consortium, supra).

[0127] In some embodiments, the present invention provides novel NOD genes (e.g., those described in SEQ ID NOs: 1-22 and Table 1). The novel NOD genes of the present invention were identified by searching public gene databases for proteins with homology to known NOD proteins. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism of the present invention is not necessary to understand the present invention. Nonetheless, it is contemplated that these genes are associated with inflammatory diseases. In particular, analysis conducted during the course of development of the present invention revealed that linkage analysis of NOD27 revealed a locus in the chromosomal region that is associated with psoriasis. Accordingly, it is further contemplated that NOD27 is associated with psoriasis.

[0128] In some embodiments, the present invention provides an "expression profile" of inflammatory diseases. For example, in some embodiments, the expression and or presence of variant alleles of the NOD proteins of the present invention is determined. Such expression profiles can then be correlated with disease states or susceptibility to disease.

DETAILED DESCRIPTION OF THE INVENTION

[0129] The present invention relates to the NOD proteins and nucleic acids encoding the NOD proteins. The present invention further provides assays for the detection of NOD polymorphisms and mutations associated with disease states. Exemplary embodiments of the present invention are described below.

I. NOD Polynucleotides

[0130] As described above, the present invention provides novel NOD family genes. Accordingly, the present invention provides nucleic acids encoding NOD genes, homologs, variants (e.g., polymorphisms and mutants), including but not limited to, those described in SEQ ID NOs: 1-11. Table 1 describes the NOD genes of the present invention. In some embodiments, the present invention provide polynucleotide sequences that are capable of hybridizing to SEQ ID NOs: 1-11 under conditions of low to high stringency as long as the polynucleotide sequence capable of hybridizing encodes a protein that retains a biological activity of the naturally occurring NODs. In some embodiments, the protein that retains a biological activity of naturally occurring NOD is 70% homologous to wild-type NOD, preferably 80% homologous to wild-type NOD, more preferably 90% homologous to wild-type NOD, and most preferably 95% homologous to wild-type NOD. In preferred embodiments, hybridization conditions are based on the melting temperature (T.sub.m) of the nucleic acid binding complex and confer a defined "stringency" as explained above (See e.g., Wahl, et al., Meth. Enzymol., 152:399-407 [1987], incorporated herein by reference).

[0131] In other embodiments of the present invention, additional alleles of NOD genes are provided. In preferred embodiments, alleles result from a polymorphism or mutation (i.e., a change in the nucleic acid sequence) and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes that give rise to alleles are generally ascribed to deletions, additions or substitutions of nucleic acids. Each of these types of changes may occur alone, or in combination with the others, and at the rate of one or more times in a given sequence. Examples of the alleles of the present invention include those encoded by SEQ ID NOs: 1-11 (wild type) and disease alleles thereof.

[0132] In still other embodiments of the present invention, the nucleotide sequences of the present invention may be engineered in order to alter an NOD coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques that are well known in the art (e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.).

[0133] In some embodiments of the present invention, the polynucleotide sequence of NOD may be extended utilizing the nucleotide sequence (e.g., SEQ ID NOs: 1-I1) in various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, it is contemplated that restriction-site polymerase chain reaction (PCR) will find use in the present invention. This is a direct method that uses universal primers to retrieve unknown sequence adjacent to a known locus (Gobinda et al., PCR Methods Applic., 2:318-22 [1993]). First, genomic DNA is amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

[0134] In another embodiment, inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res., 16:8186 [1988]). The primers may be designed using Oligo 4.0 (National Biosciences Inc, Plymouth Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72.degree. C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. In still other embodiments, walking PCR is utilized. Walking PCR is a method for targeted gene walking that permits retrieval of unknown sequence (Parker et al., Nucleic Acids Res., 19:3055-60 [1991]). The PROMOTERFINDER kit (Clontech) uses PCR, nested primers and special libraries to "walk in" genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

[0135] Preferred libraries for screening for full length cDNAs include mammalian libraries that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred, in that they will contain more sequences that contain the 5' and upstream gene regions. A randomly primed library may be particularly useful in case where an oligo d(T) library does not yield full-length cDNA. Genomic mammalian libraries are useful for obtaining introns and extending 5' sequence.

[0136] In other embodiments of the present invention, variants of the disclosed NOD sequences are provided. In preferred embodiments, variants result from polymorphisms or mutations (i.e., a change in the nucleic acid sequence) and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many variant forms. Common mutational changes that give rise to variants are generally ascribed to deletions, additions or substitutions of nucleic acids. Each of these types of changes may occur alone, or in combination with the others, and at the rate of one or more times in a given sequence.

[0137] It is contemplated that it is possible to modify the structure of a peptide having a function (e.g., NOD function) for such purposes as altering the biological activity (e.g., Nod signaling). Such modified peptides are considered functional equivalents of peptides having an activity of a NOD peptide as defined herein. A modified peptide can be produced in which the nucleotide sequence encoding the polypeptide has been altered, such as by substitution, deletion, or addition. In particularly preferred embodiments, these modifications do not significantly reduce the biological activity of the modified NOD genes. In other words, construct "X" can be evaluated in order to determine whether it is a member of the genus of modified or variant NOD's of the present invention as defined functionally, rather than structurally. In preferred embodiments, the activity of variant NOD polypeptides is evaluated by methods described herein (e.g., the generation of transgenic animals or the use of signaling assays).

[0138] Moreover, as described above, variant forms of NOD genes are also contemplated as being equivalent to those peptides and DNA molecules that are set forth in more detail herein. For example, it is contemplated that isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Accordingly, some embodiments of the present invention provide variants of NOD disclosed herein containing conservative replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur -containing (cysteine and methionine) (e.g., Stryer ed., Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether a change in the amino acid sequence of a peptide results in a functional polypeptide can be readily determined by assessing the ability of the variant peptide to function in a fashion similar to the wild-type protein. Peptides having more than one replacement can readily be tested in the same manner.

[0139] More rarely, a variant includes "nonconservative" changes (e.g., replacement of a glycine with a tryptophan). Analogous minor variations can also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs (e.g., LASERGENE software, DNASTAR Inc., Madison, Wis.).

[0140] As described in more detail below, variants may be produced by methods such as directed evolution or other techniques for producing combinatorial libraries of variants, described in more detail below. In still other embodiments of the present invention, the nucleotide sequences of the present invention may be engineered in order to alter a NOD coding sequence including, but not limited to, alterations that modify the cloning, processing, localization, secretion, and/or expression of the gene product. For example, mutations may be introduced using techniques that are well known in the art (e.g., site-directed mutagenesis to insert new restriction sites, alter glycosylation patterns, or change codon preference, etc.). TABLE-US-00001 TABLE 1 Nod Genes Nod Gene SEQ ID NO (Nucleic acid) SEQ ID NO (Polypeptide) Nod3 1 12 Nod5 2 13 Nod6 3 14 Nod8 4 15 Nod9 5 16 Nod12 6 17 Nod14 7 18 Nod16 8 19 Nod17 9 20 Nod26 10 21 Nod27 11 22

II. NOD Polypeptides

[0141] In other embodiments, the present invention provides NOD polynucleotide sequences that encode NOD polypeptide sequences (e.g., the polypeptides of SEQ ID NOs: 12-22). Other embodiments of the present invention provide fragments, fusion proteins or functional equivalents of these NOD proteins. In some embodiments, the present invention provides mutants of NOD polypeptides. In still other embodiments of the present invention, nucleic acid sequences corresponding to NOD variants, homologs, and mutants may be used to generate recombinant DNA molecules that direct the expression of the NOD variants, homologs, and mutants in appropriate host cells. In some embodiments of the present invention, the polypeptide may be a naturally purified product, in other embodiments it may be a product of chemical synthetic procedures, and in still other embodiments it may be produced by recombinant techniques using a prokaryotic or eukaryotic host (e.g., by bacterial, yeast, higher plant, insect and mammalian cells in culture). In some embodiments, depending upon the host employed in a recombinant production procedure, the polypeptide of the present invention may be glycosylated or may be non-glycosylated. In other embodiments, the polypeptides of the invention may also include an initial methionine amino acid residue.

[0142] In one embodiment of the present invention, due to the inherent degeneracy of the genetic code, DNA sequences other than the polynucleotide sequences of SEQ ID NOs: 1-11 that encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express NOD. In general, such polynucleotide sequences hybridize to SEQ ID NOs: 1-11 under conditions of high to medium stringency as described above. As will be understood by those of skill in the art, it may be advantageous to produce NOD-encoding nucleotide sequences possessing non-naturally occurring codons. Therefore, in some preferred embodiments, codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., Nucl. Acids Res., 17 [1989]) are selected, for example, to increase the rate of NOD expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.

[0143] 1. Vectors for Production of NOD

[0144] The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. In some embodiments of the present invention, vectors include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies). It is contemplated that any vector may be used as long as it is replicable and viable in the host.

[0145] In particular, some embodiments of the present invention provide recombinant constructs comprising one or more of the sequences as broadly described above (e.g., SEQ ID NOs: 1-11). In some embodiments of the present invention, the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In still other embodiments, the heterologous structural sequence (e.g., SEQ ID NOs: 1-11) is assembled in appropriate phase with translation initiation and termination sequences. In preferred embodiments of the present invention, the appropriate DNA sequence is inserted into the vector using any of a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.

[0146] Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Such vectors include, but are not limited to, the following vectors: 1) Bacterial--pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); 2) Eukaryotic--pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3) Baculovirus--pPbac and pMbac (Stratagene). Any other plasmid or vector may be used as long as they are replicable and viable in the host. In some preferred embodiments of the present invention, mammalian expression vectors comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. In other embodiments, DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

[0147] In certain embodiments of the present invention, the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) promoter) to direct mRNA synthesis. Promoters useful in the present invention include, but are not limited to, the LTR or SV40 promoter, the E. coli lac or trp, the phage lambda P.sub.L and P.sub.R, T3 and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, and mouse metallothionein-I promoters and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. In other embodiments of the present invention, recombinant expression vectors include origins of replication and selectable markers permitting transformation of the host cell (e.g., dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli).

[0148] In some embodiments of the present invention, transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0149] In other embodiments, the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. In still other embodiments of the present invention, the vector may also include appropriate sequences for amplifying expression.

[0150] 2. Host Cells for Production of NOD Polypeptides

[0151] In a further embodiment, the present invention provides host cells containing the above-described constructs. In some embodiments of the present invention, the host cell is a higher eukaryotic cell (e.g., a mammalian or insect cell). In other embodiments of the present invention, the host cell is a lower eukaryotic cell (e.g., a yeast cell). In still other embodiments of the present invention, the host cell can be a prokaryotic cell (e.g., a bacterial cell). Specific examples of host cells include, but are not limited to, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2 cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7 lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127, 3T3, 293, 293T, HeLa and BHK cell lines.

[0152] The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. In some embodiments, introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (See e.g., Davis et al., Basic Methods in Molecular Biology, [1986]). Alternatively, in some embodiments of the present invention, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

[0153] Proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., [1989].

[0154] In some embodiments of the present invention, following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. In other embodiments of the present invention, cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. In still other embodiments of the present invention, microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0155] 3. Purification of NOD polypeptides

[0156] The present invention also provides methods for recovering and purifying NOD polypeptides from recombinant cell cultures including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. In other embodiments of the present invention, protein-refolding steps can be used as necessary, in completing configuration of the mature protein. In still other embodiments of the present invention, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0157] The present invention further provides polynucleotides having a coding sequence of a NOD gene (e.g., SEQ ID NOs: 1-11) fused in frame to a marker sequence that allows for purification of the polypeptide of the present invention. A non-limiting example of a marker sequence is a hexahistidine tag which may be supplied by a vector, preferably a pQE-9 vector, which provides for purification of the polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host (e.g., COS-7 cells) is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 [1984]).

[0158] 4. Truncation Mutants of NOD Polypeptide

[0159] In addition, the present invention provides fragments of NOD polypeptides (i.e., truncation mutants). In some embodiments of the present invention, when expression of a portion of the NOD protein is desired, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., J. Bacteriol., 169:751 [1987]) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., Proc. Natl. Acad. Sci. USA 84:2718 [1990]). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerivisiae), or in vitro by use of purified MAP.

[0160] 5. Fusion Proteins Containing NOD

[0161] The present invention also provides fusion proteins incorporating all or part of the NOD polypeptides of the present invention. Accordingly, in some embodiments of the present invention, the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. It is contemplated that this type of expression system will find use under conditions where it is desirable to produce an immunogenic fragment of a NOD protein. In some embodiments of the present invention, the VP6 capsid protein of rotavirus is used as an immunologic carrier protein for portions of a NOD polypeptide, either in the monomeric form or in the form of a viral particle. In other embodiments of the present invention, the nucleic acid sequences corresponding to the portion of a NOD polypeptide against which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of NOD as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the hepatitis B surface antigen fusion proteins that recombinant hepatitis B virions can be utilized in this role as well. Similarly, in other embodiments of the present invention, chimeric constructs coding for fusion proteins containing a portion of a NOD polypeptide and the poliovirus capsid protein are created to enhance immunogenicity of the set of polypeptide antigens (See e.g., EP Publication No. 025949; and Evans et al., Nature 339:385 [1989]; Huang et al., J. Virol., 62:3855 [1988]; and Schlienger et al., J. Virol., 66:2 [1992]).

[0162] In still other embodiments of the present invention, the multiple antigen peptide system for peptide-based immunization can be utilized. In this system, a desired portion of NOD is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see e.g., Posnett et al., J. Biol. Chem., 263:1719 [1988]; and Nardelli et al., J. Immunol., 148:914 [1992]). In other embodiments of the present invention, antigenic determinants of the NOD proteins can also be expressed and presented by bacterial cells.

[0163] In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression of proteins, such as a NOD protein of the present invention. Accordingly, in some embodiments of the present invention, NOD polypeptides can be generated as glutathione-S-transferase (i.e., GST fusion proteins). It is contemplated that such GST fusion proteins will enable easy purification of NOD polypeptides, such as by the use of glutathione-derivatized matrices (See e.g., Ausabel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY [1991]). In another embodiment of the present invention, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of a NOD polypeptide, can allow purification of the expressed NOD fusion protein by affinity chromatography using a Ni.sup.2+ metal resin. In still another embodiment of the present invention, the purification leader sequence can then be subsequently removed by treatment with enterokinase (See e.g., Hochuli et al., J. Chromatogr., 411:177 [1987]; and Janknecht et al., Proc. Natl. Acad. Sci. USA 88:8972).

[0164] Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment of the present invention, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, in other embodiments of the present invention, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (See e.g., Current Protocols in Molecular Biology, supra).

[0165] 6. Variants of NOD

[0166] Still other embodiments of the present invention provide mutant or variant forms of NOD polypeptides (i.e., muteins). It is possible to modify the structure of a peptide having an activity of a NOD polypeptide of the present invention for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life, and/or resistance to proteolytic degradation in vivo). Such modified peptides are considered functional equivalents of peptides having an activity of the subject NOD proteins as defined herein. A modified peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition.

[0167] Moreover, as described above, variant forms (e.g., mutants or polymorphic sequences) of the subject NOD proteins are also contemplated as being equivalent to those peptides and DNA molecules that are set forth in more detail. For example, as described above, the present invention encompasses mutant and variant proteins that contain conservative or non-conservative amino acid substitutions.

[0168] This invention further contemplates a method of generating sets of combinatorial mutants of the present NOD proteins, as well as truncation mutants, and is especially useful for identifying potential variant sequences (i.e., mutants or polymorphic sequences) that are involved in inflammatory diseases or resistance to inflammatory diseases. The purpose of screening such combinatorial libraries is to generate, for example, novel NOD variants that can act as either agonists or antagonists, or alternatively, possess novel activities all together.

[0169] Therefore, in some embodiments of the present invention, NOD variants are engineered by the present method to provide altered (e.g., increased or decreased) biological activity. In other embodiments of the present invention, combinatorially-derived variants are generated which have a selective potency relative to a naturally occurring NOD. Such proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols.

[0170] Still other embodiments of the present invention provide NOD variants that have intracellular half-lives dramatically different than the corresponding wild-type protein. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process that result in destruction of, or otherwise inactivate NOD polypeptides. Such variants, and the genes which encode them, can be utilized to alter the location of NOD expression by modulating the half-life of the protein. For instance, a short half-life can give rise to more transient NOD biological effects and, when part of an inducible expression system, can allow tighter control of NOD levels within the cell. As above, such proteins, and particularly their recombinant nucleic acid constructs, can be used in gene therapy protocols.

[0171] In still other embodiments of the present invention, NOD variants are generated by the combinatorial approach to act as antagonists, in that they are able to interfere with the ability of the corresponding wild-type protein to regulate cell function.

[0172] In some embodiments of the combinatorial mutagenesis approach of the present invention, the amino acid sequences for a population of NOD homologs, variants or other related proteins are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example, NOD homologs from one or more species, or NOD variants from the same species but which differ due to mutation or polymorphisms. Amino acids that appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.

[0173] In a preferred embodiment of the present invention, the combinatorial NOD library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential NOD protein sequences. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential NOD sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOD sequences therein.

[0174] There are many ways by which the library of potential NOD homologs and variants can be generated from a degenerate oligonucleotide sequence. In some embodiments, chemical synthesis of a degenerate gene sequence is carried out in an automatic DNA synthesizer, and the synthetic genes are ligated into an appropriate gene for expression. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential NOD sequences. The synthesis of degenerate oligonucleotides is well known in the art (See e.g., Narang, Tetrahedron Lett., 39:39 [1983]; Itakura et al., Recombinant DNA, in Walton (ed.), Proceedings of the 3rd Cleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289 [1981]; Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura et al., Science 198:1056 [1984]; Ike et al., Nucl. Acid Res., 11:477 [1983]). Such techniques have been employed in the directed evolution of other proteins (See e.g., Scott et al., Science 249:386 [1980]; Roberts et al., Proc. Natl. Acad. Sci. USA 89:2429 [1992]; Devlin et al., Science 249: 404 [1990]; Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378 [1990]; each of which is herein incorporated by reference; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815; each of which is incorporated herein by reference).

[0175] It is contemplated that the NOD nucleic acids of the present invention (e.g., SEQ ID NOs: 1-11, and fragments and variants thereof) can be utilized as starting nucleic acids for directed evolution. These techniques can be utilized to develop NOD variants having desirable properties such as increased or decreased biological activity.

[0176] In some embodiments, artificial evolution is performed by random mutagenesis (e.g., by utilizing error-prone PCR to introduce random mutations into a given coding sequence). This method requires that the frequency of mutation be finely tuned. As a general rule, beneficial mutations are rare, while deleterious mutations are common. This is because the combination of a deleterious mutation and a beneficial mutation often results in an inactive enzyme. The ideal number of base substitutions for targeted gene is usually between 1.5 and 5 (Moore and Arnold, Nat. Biotech., 14, 458 [1996]; Leung et al., Technique, 1:11 [1989]; Eckert and Kunkel, PCR Methods Appl., 1: 17-24 [1991]; Caldwell and Joyce, PCR Methods Appl., 2:28 [1992]; and Zhao and Arnold, Nuc. Acids. Res., 25:1307 [1997]). After mutagenesis, the resulting clones are selected for desirable activity (e.g., screened for NOD activity). Successive rounds of mutagenesis and selection are often necessary to develop enzymes with desirable properties. It should be noted that only the useful mutations are carried over to the next round of mutagenesis.

[0177] In other embodiments of the present invention, the polynucleotides of the present invention are used in gene shuffling or sexual PCR procedures (e.g., Smith, Nature, 370:324 [1994]; U.S. Pat. Nos. 5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are herein incorporated by reference). Gene shuffling involves random fragmentation of several mutant DNAs followed by their reassembly by PCR into full length molecules. Examples of various gene shuffling procedures include, but are not limited to, assembly following DNase treatment, the staggered extension process (STEP), and random priming in vitro recombination. In the DNase mediated method, DNA segments isolated from a pool of positive mutants are cleaved into random fragments with DNaseI and subjected to multiple rounds of PCR with no added primer. The lengths of random fragments approach that of the uncleaved segment as the PCR cycles proceed, resulting in mutations in present in different clones becoming mixed and accumulating in some of the resulting sequences. Multiple cycles of selection and shuffling have led to the functional enhancement of several enzymes (Stemmer, Nature, 370:398 [1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91:10747 [1994]; Crameri et al., Nat. Biotech., 14:315 [1996]; Zhang et al., Proc. Natl. Acad. Sci. USA, 94:4504 [1997]; and Crameri et al., Nat. Biotech., 15:436 [1997]). Variants produced by directed evolution can be screened for NOD activity by the methods described herein.

[0178] A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis or recombination of NOD homologs or variants. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.

[0179] 7. Chemical Synthesis of NOD Polypeptides

[0180] In an alternate embodiment of the invention, the coding sequence of NOD is synthesized, whole or in part, using chemical methods well known in the art (See e.g., Caruthers et al., Nucl. Acids Res. Symp. Ser., 7:215 [1980]; Crea and Horn, Nucl. Acids Res., 9:2331 [1980]; Matteucci and Caruthers, Tetrahedron Lett., 21:719 [1980]; and Chow and Kempe, Nucl. Acids Res., 9:2807 [1981]). In other embodiments of the present invention, the protein itself is produced using chemical methods to synthesize either an entire NOD amino acid sequence or a portion thereof. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (See e.g., Creighton, Proteins Structures And Molecular Principles, W H Freeman and Co, New York N.Y. [1983]). In other embodiments of the present invention, the composition of the synthetic peptides is confirmed by amino acid analysis or sequencing (See e.g., Creighton, supra).

[0181] Direct peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202 [1995]) and automated synthesis may be achieved, for example, using ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequence of a NOD polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with other sequences to produce a variant polypeptide.

III. Detection of NOD Alleles

[0182] In some embodiments, the present invention provides methods of detecting the presence of wild type or variant (e.g., mutant or polymorphic) NOD nucleic acids or polypeptides. The detection of mutant NOD polypeptides finds use in the diagnosis of disease (e.g., inflammatory disease).

A. Detection of Variant NOD Alleles

[0183] In some embodiments, the present invention provides alleles of NOD that increase a patient's susceptibility to inflammatory diseases. Any mutation that results in an altered phenotype (e.g., increase in inflammatory disease or resistance to inflammatory disease) is within the scope of the present invention.

[0184] Accordingly, the present invention provides methods for determining whether a patient has an increased susceptibility to an inflammatory disease by determining whether the individual has a variant NOD allele. In other embodiments, the present invention provides methods for providing a prognosis of increased risk for inflammatory disease to an individual based on the presence or absence of one or more variant alleles of NOD.

[0185] A number of methods are available for analysis of variant (e.g., mutant or polymorphic) nucleic acid sequences. Assays for detection variants (e.g., polymorphisms or mutations) fall into several categories including, but not limited to, direct sequencing assays, fragment polymorphism assays, hybridization assays, and computer based data analysis. Protocols and commercially available kits or services for performing multiple variations of these assays are available. In some embodiments, assays are performed in combination or in hybrid (e.g., different reagents or technologies from several assays are combined to yield one assay). The following exemplary assays are useful in the present invention: directs sequencing assays, PCR assays, mutational analysis by dHPLC (e.g., available from Transgenomic, Omaha, Nebr. or Varian, Palo Alto, Calif.), fragment length polymorphism assays (e.g., RFLP or CFLP (See e.g. U.S. Patents U.S. Pat. Nos. 5,843,654; 5,843,669; 5,719,208; and 5,888,780; each of which is herein incorporated by reference)), hybridization assays (e.g., direct detection of hybridization, detection of hybridization using DNA chip assays (See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; 5,858,659; 6,017,696; 6,068,818; 6,051,380; 6,001,311; 5,985,551; 5,474,796; PCT Publications WO 99/67641 and WO 00/39587, each of which is herein incorporated by reference), enzymatic detection of hybridization (See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; 5,994,069; 5,962,233; 5,538,848; 5,952,174 and 5,919,626, each of which is herein incorporated by reference)), and mass spectrometry assays. In addition, assays for the detection of variant NOD proteins find use in the present invention (e.g., cell free translation methods, See e.g., U.S. Pat. No. 6,303,337, herein incorporated by reference) and antibody binding assays.

[0186] B. Kits for Analyzing Risk of Inflammatory Disease

[0187] The present invention also provides kits for determining whether an individual contains a wild-type or variant (e.g., mutant or polymorphic) allele or polypeptide of NOD. In some embodiments, the kits are useful determining whether the subject is at risk of developing an inflammatory disease (e.g., Crohn's disease or psoriasis). The diagnostic kits are produced in a variety of ways. In some embodiments, the kits contain at least one reagent for specifically detecting a mutant NOD allele or protein. In preferred embodiments, the reagent is a nucleic acid that hybridizes to nucleic acids containing the mutation and that does not bind to nucleic acids that do not contain the mutation. In other embodiments, the reagents are primers for amplifying the region of DNA containing the mutation. In still other embodiments, the reagents are antibodies that preferentially bind either the wild-type or mutant NOD proteins.

[0188] In some embodiments, the kit contains instructions for determining whether the subject is at risk for an inflammatory disease. In preferred embodiments, the instructions specify that risk for developing an inflammatory disease is determined by detecting the presence or absence of a mutant NOD allele in the subject, wherein subjects having an mutant allele are at greater risk for developing an inflammatory disease.

[0189] The presence or absence of a disease-associated mutation in a NOD gene can be used to make therapeutic or other medical decisions. For example, couples with a family history of inflammatory diseases may choose to conceive a child via in vitro fertilization and pre-implantation genetic screening. In this case, fertilized embryos are screened for mutant (e.g., disease associated) alleles of a NOD gene and only embryos with wild type alleles are implanted in the uterus.

[0190] In other embodiments, in utero screening is performed on a developing fetus (e.g., amniocentesis or chorionic villi screening). In still other embodiments, genetic screening of newborn babies or very young children is performed. The early detection of a NOD allele known to be associated with an inflammatory disease allows for early intervention (e.g., genetic or pharmaceutical therapies).

[0191] In some embodiments, the kits include ancillary reagents such as buffering agents, nucleic acid stabilizing reagents, protein stabilizing reagents, and signal producing systems (e.g., florescence generating systems as Fret systems). The test kit may be packaged in any suitable manner, typically with the elements in a single container or various containers as necessary along with a sheet of instructions for carrying out the test. In some embodiments, the kits also preferably include a positive control sample.

[0192] C. Bioinformatics

[0193] In some embodiments, the present invention provides methods of determining an individual's risk of developing an inflammatory disease based on the presence of one or more variant alleles of a NOD gene. In some embodiments, the analysis of variant data is processed by a computer using information stored on a computer (e.g., in a database). For example, in some embodiments, the present invention provides a bioinformatics research system comprising a plurality of computers running a multi-platform object oriented programming language (See e.g., U.S. Pat. No. 6,125,383; herein incorporated by reference). In some embodiments, one of the computers stores genetics data (e.g., the risk of contacting an inflammatory disease associated with a given polymorphism, as well as the sequences). In some embodiments, one of the computers stores application programs (e.g., for analyzing the results of detection assays). Results are then delivered to the user (e.g., via one of the computers or via the internet.

[0194] For example, in some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given NOD allele or polypeptide) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

[0195] The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information providers, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., presence of wild type or mutant NOD genes or polypeptides), specific for the diagnostic or prognostic information desired for the subject.

[0196] The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of developing an inflammatory disease) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

[0197] In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

[0198] In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the association of a given NOD allele with inflammatory diseases.

IV. Generation of NOD Antibodies

[0199] The present invention provides isolated antibodies or antibody fragments (e.g., FAB fragments). Antibodies can be generated to allow for the detection of a NOD protein of the present invention. The antibodies may be prepared using various immunogens. In one embodiment, the immunogen is a human NOD peptide to generate antibodies that recognize human NOD. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, Fab expression libraries, or recombinant (e.g., chimeric, humanized, etc.) antibodies, as long as it can recognize the protein. Antibodies can be produced by using a protein of the present invention as the antigen according to a conventional antibody or antiserum preparation process.

[0200] Various procedures known in the art may be used for the production of polyclonal antibodies directed against a NOD polypeptide. For the production of antibody, various host animals can be immunized by injection with the peptide corresponding to the NOD epitope including but not limited to rabbits, mice, rats, sheep, goats, etc. In a preferred embodiment, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).

[0201] For preparation of monoclonal antibodies directed toward NOD, it is contemplated that any technique that provides for the production of antibody molecules by continuous cell lines in culture will find use with the present invention (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Kohler and Milstein, Nature 256:495-497 [1975]), as well as the trioma technique, the human B-cell hybridoma technique (See e.g., Kozbor et al., Immunol. Tod., 4:72 [1983]), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 [1985]).

[0202] In an additional embodiment of the invention, monoclonal antibodies are produced in germ-free animals utilizing technology such as that described in PCT/US90/02545). Furthermore, it is contemplated that human antibodies will be generated by human hybridomas (Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-2030 [1983]) or by transforming human B cells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96 [1985]).

[0203] In addition, it is contemplated that techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated by reference) will find use in producing NOD specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science 246:1275-1281 [1989]) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a NOD polypeptide.

[0204] In other embodiments, the present invention contemplated recombinant antibodies or fragments thereof to the proteins of the present invention. Recombinant antibodies include, but are not limited to, humanized and chimeric antibodies. Methods for generating recombinant antibodies are known in the art (See e.g., U.S. Pat. Nos. 6,180,370 and 6,277,969 and "Monoclonal Antibodies" H. Zola, BIOS Scientific Publishers Limited 2000. Springer-Verlay New York, Inc., New York; each of which is herein incorporated by reference).

[0205] It is contemplated that any technique suitable for producing antibody fragments will find use in generating antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule. For example, such fragments include but are not limited to: F(ab')2 fragment that can be produced by pepsin digestion of the antibody molecule; Fab' fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent.

[0206] In the production of antibodies, it is contemplated that screening for the desired antibody will be accomplished by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.

[0207] In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. As is well known in the art, the immunogenic peptide should be provided free of the carrier molecule used in any immunization protocol. For example, if the peptide was conjugated to KLH, it may be conjugated to BSA, or used directly, in a screening assay.)

[0208] The foregoing antibodies can he used in methods known in the art relating to the localization and structure of NOD (e.g., for Western blotting), measuring levels thereof in appropriate biological samples, etc. The antibodies can be used to detect a NOD in a biological sample from an individual. The biological sample can be a biological fluid, such as, but not limited to, blood, serum, plasma, interstitial fluid, urine, cerebrospinal fluid, and the like, containing cells.

[0209] The biological samples can then be tested directly for the presence of a human NOD using an appropriate strategy (e.g., ELISA or radioimmunoassay) and format (e.g., microwells, dipstick (e.g., as described in International Patent Publication WO 93/03367), etc. Alternatively, proteins in the sample can be size separated (e.g., by polyacrylamide gel electrophoresis (PAGE), in the presence or not of sodium dodecyl sulfate (SDS), and the presence of NOD detected by immunoblotting (Western blotting). Immunoblotting techniques are generally more effective with antibodies generated against a peptide corresponding to an epitope of a protein, and hence, are particularly suited to the present invention.

[0210] Another method uses antibodies as agents to alter signal transduction. Specific antibodies that bind to the binding domains of NOD or other proteins involved in intracellular signaling can be used to inhibit the interaction between the various proteins and their interaction with other ligands. Antibodies that bind to the complex can also be used therapeutically to inhibit interactions of the protein complex in the signal transduction pathways leading to the various physiological and cellular effects of NOD. Such antibodies can also be used diagnostically to measure abnormal expression of NOD, or the aberrant formation of protein complexes, which may be indicative of a disease state.

V. Gene Therapy Using NOD

[0211] The present invention also provides methods and compositions suitable for gene therapy to alter NOD expression, production, or function. As described above, the present invention provides human NOD genes and provides methods of obtaining NOD genes from other species. Thus, the methods described below are generally applicable across many species. In some embodiments, it is contemplated that the gene therapy is performed by providing a subject with a wild-type allele of a NOD gene (i.e., an allele that does not contain a NOD disease allele (e.g., free of disease causing polymorphisms or mutations). Subjects in need of such therapy are identified by the methods described above.

[0212] Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (See e.g., Miller and Rosman, BioTech., 7:980-990 [1992]). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors that are used within the scope of the present invention lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (i.e., on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.

[0213] Preferably, the replication defective virus retains the sequences of its genome that are necessary for encapsidating the viral particles. DNA viral vectors include an attenuated or defective DNA viruses, including, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, that entirely or almost entirely lack viral genes, are preferred, as defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Mol. Cell. Neurosci., 2:320-330 [1991]), defective herpes virus vector lacking a glycoprotein L gene (See e.g., Patent Publication RD 371005 A), or other defective herpes virus vectors (See e.g., WO 94/21807; and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 [1992]; See also, La Salle et al., Science 259:988-990 [1993]); and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61:3096-3101 [1987]; Samulski et al., J. Virol., 63:3822-3828 [1989]; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 [1988]).

[0214] Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector (e.g., adenovirus vector), to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-gamma (IFN-.gamma.), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

[0215] In a preferred embodiment, the vector is an adenovirus vector. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to type 2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animal origin (See e.g., WO 94/26914). Those adenoviruses of animal origin that can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (e.g., Mavl, Beard et al., Virol., 75-81 [1990]), ovine, porcine, avian, and simian (e.g., SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800)).

[0216] Preferably, the replication defective adenoviral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest. Still more preferably, at least the E1 region of the adenoviral vector is non-functional. The deletion in the E1 region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3A fragment). Other regions may also be modified, in particular the E3 region (e.g., WO 95/02697), the E2 region (e.g., WO 94/28938), the E4 region (e.g., WO 94/28152, WO 94/12649 and WO 95/02697), or in any of the late genes L1-L5.

[0217] In a preferred embodiment, the adenoviral vector has a deletion in the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenoviruses are disclosed in WO 95/02697 and WO 96/22378. In still another preferred embodiment, the adenoviral vector has a deletion in the E1 region into which the E4 region and the nucleic acid sequence are inserted.

[0218] The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (See e.g., Levrero et al., Gene 101:195 [1991]; EP 185 573; and Graham, EMBO J., 3:2917 [1984]). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid that carries, inter alia, the DNA sequence of interest. The homologous recombination is accomplished following co-transfection of the adenovirus and plasmid into an appropriate cell line. The cell line that is employed should preferably (i) be transformable by the elements to be used, and (ii) contain the sequences that are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. Examples of cell lines that may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol., 36:59 [1977]), which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines that are able to complement the E1 and E4 functions, as described in applications WO 94/26914 and WO 95/02697. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques that are well known to one of ordinary skill in the art.

[0219] The adeno-associated viruses (AAV) are DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.

[0220] The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368; U.S. Pat. No., 5,139,941; and EP 488 528, all of which are herein incorporated by reference). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques.

[0221] In another embodiment, the gene can be introduced in a retroviral vector (e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289 and 5,124,263; all of which are herein incorporated by reference; Mann et al., Cell 33:153 [1983]; Markowitz et al., J. Virol., 62:1120 [1988]; PCT/US95/14575; EP 453242; EP178220; Bernstein et al. Genet. Eng., 7:235 [1985]; McCormick, BioTechnol., 3:689 [1985]; WO 95/07358; and Kuo et al., Blood 82:845 [1993]). The retroviruses are integrating viruses that infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Defective retroviral vectors are also disclosed in WO 95/02697.

[0222] In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed that contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions that are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719, herein incorporated by reference), the PsiCRIP cell line (See, WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences that may include a part of the gag gene (Bender et al., J. Virol., 61:1639 [1987]). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

[0223] Alternatively, the vector can be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et. al., Proc. Natl. Acad. Sci. USA 84:7413-7417 [1987]; See also, Mackey, et al., Proc. Natl. Acad. Sci. USA 85:8027-8031 [1988]; Ulmer et al., Science 259:1745-1748 [1993]). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold, Science 337:387-388 [1989]). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, herein incorporated by reference.

[0224] Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).

[0225] It is also possible to introduce the vector in vivo as a naked DNA plasmid. Methods for formulating and administering naked DNA to mammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are herein incorporated by reference.

[0226] DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, including but not limited to transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (See e.g., Wu et al., J. Biol. Chem., 267:963 [1992]; Wu and Wu, J. Biol. Chem., 263:14621 [1988]; and Williams et al., Proc. Natl. Acad. Sci. USA 88:2726 [1991]). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther., 3:147 [1992]; and Wu and Wu, J. Biol. Chem., 262:4429 [1987]).

VI. Transgenic Animals Expressing Exogenous NOD Genes and Homologs, Mutants, and Variants Thereof

[0227] The present invention contemplates the generation of transgenic animals comprising an exogenous NOD gene or homologs, mutants, or variants thereof. In preferred embodiments, the transgenic animal displays an altered phenotype as compared to wild-type animals. In some embodiments, the altered phenotype is the overexpression of mRNA for a NOD gene as compared to wild-type levels of NOD expression. In other embodiments, the altered phenotype is the decreased expression of mRNA for an endogenous NOD gene as compared to wild-type levels of endogenous NOD expression. In some preferred embodiments, the transgenic animals comprise mutant alleles of NOD. Methods for analyzing the presence or absence of such phenotypes include Northern blotting, mRNA protection assays, and RT-PCR. In other embodiments, the transgenic mice have a knock out mutation of a NOD gene. In preferred embodiments, the transgenic animals display an altered susceptibility to inflammatory diseases.

[0228] Such animals find use in research applications (e.g., identifying signaling pathways that a NOD protein is involved in), as well as drug screening applications (e.g., to screen for drugs that prevent or treat inflammatory diseases. For example, in some embodiments, test compounds (e.g., a drug that is suspected of being useful to treat an inflammatory disease are administered to the transgenic animals and control animals with a wild type NOD allele and the effects evaluated. The effects of the test and control compounds on disease symptoms are then assessed.

[0229] The transgenic animals can be generated via a variety of methods. In some embodiments, embryonal cells at various developmental stages are used to introduce transgenes for the production of transgenic animals. Different methods are used depending on the stage of development of the embryonal cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter, which allows reproducible injection of 1-2 picoliters (pl) of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host genome before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. U.S. Pat. No. 4,873,191 describes a method for the micro-injection of zygotes; the disclosure of this patent is incorporated herein in its entirety.

[0230] In other embodiments, retroviral infection is used to introduce transgenes into a non-human animal. In some embodiments, the retroviral vector is utilized to transfect oocytes by injecting the retroviral vector into the perivitelline space of the oocyte (U.S. Pat. No. 6,080,912, incorporated herein by reference). In other embodiments, the developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986]). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al., EMBO J., 6:383 [1987]). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of cells that form the transgenic animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et al., supra [1982]). Additional means of using retroviruses or retroviral vectors to create transgenic animals known to the art involves the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilized eggs or early embryos (PCT International Application WO 90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

[0231] In other embodiments, the transgene is introduced into embryonic stem cells and the transfected stem cells are utilized to form an embryo. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley et al., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065 [1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can be efficiently introduced into the ES cells by DNA transfection by a variety of methods known to the art including calcium phosphate co-precipitation, protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated transfection. Transgenes may also be introduced into ES cells by retrovirus-mediated transduction or by micro-injection. Such transfected ES cells can thereafter colonize an embryo following their introduction into the blastocoel of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal (for review, See, Jaenisch, Science 240:1468 [1988]). Prior to the introduction of transfected ES cells into the blastocoel, the transfected ES cells may be subjected to various selection protocols to enrich for ES cells which have integrated the transgene assuming that the transgene provides a means for such selection. Alternatively, the polymerase chain reaction may be used to screen for ES cells that have integrated the transgene. This technique obviates the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.

[0232] In still other embodiments, homologous recombination is utilized to knock-out gene function or create deletion mutants (e.g., mutants in which a particular domain of a NOD is deleted). Methods for homologous recombination are described in U.S. Pat. No. 5,614,396, incorporated herein by reference.

VIII. Drug Screening Using NOD

[0233] In some embodiments, the isolated nucleic acid and polypeptides of NOD genes of the present invention (e.g., SEQ ID NOS: 1-22) and related proteins and nucleic acids are used in drug screening applications for compounds that alter (e.g., enhance or inhibit) NOD activity and signaling. The present invention further provides methods of identifying ligands of the NOD proteins of the present invention.

[0234] As described above, NOD family proteins (e.g., Nod2) have been shown to mediate the host response to bacterial muropeptides. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that the NOD family proteins of the present invention are involved in host responses to microbes (e.g., bacteria, virus, fungi, etc.). It is further contemplated that some NODs recognize endogenous compounds (e.g., derived from host cells) as ligands. For example, some NODs may recognize host cell proteins induced by stress (e.g. heat shock proteins). Accordingly, in some embodiments, the present invention provides methods of screening for ligands of NOD family proteins (e.g., ligands derived from microbes or host factors). For example, in some embodiments, an assay that measures NOD signaling is used to screen libraries of compounds (e.g., microbial or host derived compounds) for their ability to alter NOD family signaling.

[0235] In other embodiments, the present invention provides methods of screening compounds for the ability to alter NOD signaling mediated by natural ligands (e.g., identified using the methods described above). Such compounds find use in the treatment of disease mediated by NOD family members (e.g., inflammatory diseases).

[0236] In one screening method, the two-hybrid system is used to screen for compounds (e.g., proteins) capable of altering NOD function(s) (e.g., interaction with a binding partner) in vitro or in vivo. In one embodiment, a GAL4 binding site, linked to a reporter gene such as lacZ, is contacted in the presence and absence of a candidate compound with a GAL4 binding domain linked to a NOD fragment and a GAL4 transactivation domain II linked to a binding partner fragment. Expression of the reporter gene is monitored and a decrease in the expression is an indication that the candidate compound inhibits the interaction of a NOD with the binding partner. Alternately, the effect of candidate compounds on the interaction of a NOD with other proteins (e.g., proteins known to interact directly or indirectly with the binding partner) can be tested in a similar manner In some embodiments, the present invention provides methods of identifying NOD binding partners or ligands that utilize immunoprecipitation. In some embodiments, antibodies to NOD proteins are utilized to immunoprecipitated NODs and any bound proteins. In other embodiments, NOD fusion proteins are generated with tags and antibodies to the tags are utilized for immunoprecipitation. Potential binding partners that immunoprecipitate with NODs can be identified using any suitable method.

[0237] In another screening method, candidate compounds are evaluated for their ability to alter NOD signaling by contacting NOD, binding partners, binding partner-associated proteins, or fragments thereof, with the candidate compound and determining binding of the candidate compound to the peptide. The protein or protein fragments is/are immobilized using methods known in the art such as binding a GST-NOD fusion protein to a polymeric bead containing glutathione. A chimeric gene encoding a GST fusion protein is constructed by fusing DNA encoding the polypeptide or polypeptide fragment of interest to the DNA encoding the carboxyl terminus of GST (See e.g., Smith et al., Gene 67:31 [1988]). The fusion construct is then transformed into a suitable expression system (e.g., E. coli XA90) in which the expression of the GST fusion protein can be induced with isopropyl-.beta.-D-thiogalactopyranoside (IPTG). Induction with (IPTG should yield the fusion protein as a major constituent of soluble, cellular proteins. The fusion proteins can be purified by methods known to those skilled in the art, including purification by glutathione affinity chromatography. Binding of the candidate compound to the proteins or protein fragments is correlated with the ability of the compound to disrupt the signal transduction pathway and thus regulate NOD physiological effects (e.g., inflammatory disease).

[0238] In another screening method, one of the components of the NOD/binding partner signaling system is immobilized. Polypeptides can be immobilized using methods known in the art, such as adsorption onto a plastic microtiter plate or specific binding of a GST-fusion protein to a polymeric bead containing glutathione. For example, in some embodiments, GST-NOD is bound to glutathione-Sepharose beads. The immobilized peptide is then contacted with another peptide with which it is capable of binding in the presence and absence of a candidate compound. Unbound peptide is then removed and the complex solubilized and analyzed to determine the amount of bound labeled peptide. A decrease in binding is an indication that the candidate compound inhibits the interaction of NOD with the other peptide. A variation of this method allows for the screening of compounds that are capable of disrupting a previously-formed protein/protein complex. For example, in some embodiments a complex comprising a NOD or a NOD fragment bound to another peptide is immobilized as described above and contacted with a candidate compound. The dissolution of the complex by the candidate compound correlates with the ability of the compound to disrupt or inhibit the interaction between NOD and the other peptide.

[0239] Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to NOD peptides and is described in detail in WO 84/03564, incorporated herein by reference. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are then reacted with NOD peptides and washed. Bound NOD peptides are then detected by methods well known in the art.

[0240] Another technique uses NOD antibodies, generated as discussed above. Such antibodies are capable of specifically binding to NOD peptides and compete with a test compound for binding to NOD. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants of a NOD peptide.

[0241] The present invention contemplates many other means of screening compounds. The examples provided above are presented merely to illustrate a range of techniques available. One of ordinary skill in the art will appreciate that many other screening methods can be used.

[0242] In particular, the present invention contemplates the use of cell lines transfected with NOD genes and variants thereof for screening compounds for activity, and in particular to high throughput screening of compounds from combinatorial libraries (e.g., libraries containing greater than 10.sup.4 compounds). The cell lines of the present invention can be used in a variety of screening methods. In some embodiments, the cells can be used in second messenger assays that monitor signal transduction following activation of cell-surface receptors. In other embodiments, the cells can be used in reporter gene assays that monitor cellular responses at the transcription/translation level. In still further embodiments, the cells can be used in cell proliferation assays to monitor the overall growth/no growth response of cells to external stimuli.

[0243] In second messenger assays, the host cells are preferably transfected as described above with vectors encoding NOD or variants or mutants thereof. The host cells are then treated with a compound or plurality of compounds (e.g., from a combinatorial library) and assayed for the presence or absence of a response. It is contemplated that at least some of the compounds in the combinatorial library can serve as agonists, antagonists, activators, or inhibitors of the protein or proteins encoded by the vectors. It is also contemplated that at least some of the compounds in the combinatorial library can serve as agonists, antagonists, activators, or inhibitors of protein acting upstream or downstream of the protein encoded by the vector in a signal transduction pathway.

[0244] In some embodiments, the second messenger assays measure fluorescent signals from reporter molecules that respond to intracellular changes (e.g., Ca.sup.2+ concentration, membrane potential, pH, IP.sub.3, cAMP, arachidonic acid release) due to stimulation of membrane receptors and ion channels (e.g., ligand gated ion channels; see Denyer et al., Drug Discov. Today 3:323 [1998]; and Gonzales et al., Drug. Discov. Today 4:431-39 [1999]). Examples of reporter molecules include, but are not limited to, FRET (florescence resonance energy transfer) systems (e.g., Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators (e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM), chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitive indicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), and pH sensitive indicators (e.g., BCECF).

[0245] In general, the host cells are loaded with the indicator prior to exposure to the compound. Responses of the host cells to treatment with the compounds can be detected by methods known in the art, including, but not limited to, fluorescence microscopy, confocal microscopy (e.g., FCS systems), flow cytometry, microfluidic devices, FLIPR systems (See, e.g., Schroeder and Neagle, J. Biomol. Screening 1:75 [1996]), and plate-reading systems. In some preferred embodiments, the response (e.g., increase in fluorescent intensity) caused by compound of unknown activity is compared to the response generated by a known agonist and expressed as a percentage of the maximal response of the known agonist. The maximum response caused by a known agonist is defined as a 100% response. Likewise, the maximal response recorded after addition of an agonist to a sample containing a known or test antagonist is detectably lower than the 100% response.

[0246] The cells are also useful in reporter gene assays. Reporter gene assays involve the use of host cells transfected with vectors encoding a nucleic acid comprising transcriptional control elements of a target gene (i.e., a gene that controls the biological expression and function of a disease target) spliced to a coding sequence for a reporter gene. Therefore, activation of the target gene results in activation of the reporter gene product. In some embodiments, the reporter gene construct comprises the 5' regulatory region (e.g., promoters and/or enhancers) of a protein whose expression is controlled by NOD in operable association with a reporter gene. Examples of reporter genes finding use in the present invention include, but are not limited to, chloramphenicol transferase, alkaline phosphatase, firefly and bacterial luciferases, .beta.-galactosidase, .beta.-lactamase, and green fluorescent protein. The production of these proteins, with the exception of green fluorescent protein, is detected through the use of chemiluminescent, calorimetric, or bioluminecent products of specific substrates (e.g., X-gal and luciferin). Comparisons between compounds of known and unknown activities may be conducted as described above.

[0247] Specifically, the present invention provides screening methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to a NOD of the present invention, have an inhibitory (or stimulatory) effect on, for example, NOD expression or NOD activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a NOD substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., NOD genes) either directly or indirectly in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions. Compounds, which stimulate the activity of a variant NOD or mimic the activity of a non-functional variant are particularly useful in the treatment of inflammatory diseases.

[0248] In one embodiment, the invention provides assays for screening candidate or test compounds that are substrates of a NOD protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of a NOD protein or polypeptide or a biologically active portion thereof.

[0249] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the `one-bead one-compound` library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[0250] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422 [1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al., Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061 [1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

[0251] Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids (Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage (Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990]; Felici, J. Mol. Biol. 222:301 [1991]).

[0252] In one embodiment, an assay is a cell-based assay in which a cell that expresses a NOD protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate a NOD's activity is determined. Determining the ability of the test compound to modulate NOD activity can be accomplished by monitoring, for example, changes in enzymatic activity. The cell, for example, can be of mammalian origin.

[0253] The ability of the test compound to modulate NOD binding to a compound, e.g., a NOD substrate, can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to a NOD can be determined by detecting the labeled compound, e.g., substrate, in a complex.

[0254] Alternatively, a NOD is coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate NOD binding to a NOD substrate in a complex. For example, compounds (e.g., substrates) can be labeled with .sup.125I, .sup.35S .sup.14C or .sup.3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0255] The ability of a compound (e.g., a NOD substrate) to interact with a NOD with or without the labeling of any of the interactants can be evaluated. For example, a microphysiorneter can be used to detect the interaction of a compound with a NOD without the labeling of either the compound or the NOD (McConnell et al. Science 257:1906-1912 [1992]). As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and a NOD polypeptide.

[0256] In yet another embodiment, a cell-free assay is provided in which a NOD protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the NOD protein or biologically active portion thereof is evaluated. Preferred biologically active portions of NOD proteins to be used in assays of the present invention include fragments that participate in interactions with substrates or other proteins, e.g., fragments with high surface probability scores.

[0257] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[0258] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FRET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,968,103; each of which is herein incorporated by reference). A fluorophore label is selected such that a first donor molecule's emitted fluorescent energy will be absorbed by a fluorescent label on a second, `acceptor` molecule, which in turn is able to fluoresce due to the absorbed energy.

[0259] Alternately, the `donor` protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the `acceptor` molecule label may be differentiated from that of the `donor`. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the `acceptor` molecule label in 1 5 the assay should be maximal. An FRET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0260] In another embodiment, determining the ability of a NOD protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345 [1991] and Szabo et al. Curr. Opin. Struct. Biol. 5:699-705 [1995]). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BlAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

[0261] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[0262] It may be desirable to immobilize a NOD protein, an anti-NOD antibody or its target molecule to facilitate separation of complexed from non-complexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a NOD protein, or interaction of a NOD protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase-NOD fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOD protein, and the mixture incubated under conditions conducive for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.

[0263] Alternatively, the complexes can be dissociated from the matrix, and the level of NOD binding or activity determined using standard techniques. Other techniques for immobilizing either a NOD protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated NOD protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[0264] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-IgG antibody).

[0265] This assay is performed utilizing antibodies reactive with NOD protein or target molecules but which do not interfere with binding of the NOD protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or NOD protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOD protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the NOD protein or target molecule.

[0266] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including, but not limited to: differential centrifugation (see, for example, Rivas and Minton, Trends Biochem Sci 18:284-7 [1993]); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (See e.g., Heegaard J. Mol. Recognit 11: 141-8 [1998]; Hageand Tweed J. Chromatogr. Biomed. Sci. Appl 699:499-525 [1997]). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[0267] The assay can include contacting the NOD protein or biologically active portion thereof with a known compound that binds the NOD to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOD protein, wherein determining the ability of the test compound to interact with a NOD protein includes determining the ability of the test compound to preferentially bind to NOD or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[0268] To the extent that a NOD can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins, inhibitors of such an interaction are useful. A homogeneous assay can be used can be used to identify inhibitors.

[0269] For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared such that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496, herein incorporated by reference, that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified. Alternatively, a NOD protein can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 [1993]; Madura et al., J. Biol. Chem. 268.12046-12054 [1993]; Bartel et al., Biotechniques 14:920-924 [1993]; Iwabuchi et al., Oncogene 8:1693-1696 [1993]; and Brent WO 94/10300; each of which is herein incorporated by reference), to identify other proteins, that bind to or interact with a NOD ("NOD-binding proteins" or "NOD-bp") and are involved in NOD activity. Such NOD-bps can be activators or inhibitors of signals by the NOD proteins or targets as, for example, downstream elements of a NOD-mediated signaling pathway.

[0270] Modulators of NOD expression can also be identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of a NOD mRNA or protein evaluated relative to the level of expression of the NOD mRNA or protein in the absence of the candidate compound. When expression of the NOD mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of a NOD mRNA or protein expression. Alternatively, when expression of NOD mRNA or protein is less (i.e., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOD mRNA or protein expression. The level of NOD mRNA or protein expression can be determined by methods described herein for detecting NOD mRNA or protein.

[0271] A modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a NOD protein can be confirmed in vivo, e.g., in an animal such as an animal model for a disease (e.g., an animal with inflammatory disease).

B. Therapeutic Agents

[0272] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a NOD modulating agent or mimetic, a NOD specific antibody, or a NOD-binding partner) in an appropriate animal model (such as those described herein) to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, as described above, novel agents identified by the above-described screening assays can be, e.g., used for treatments of inflammatory disease (e.g., including, but not limited to, psoriasis or Crohn's disease). In some embodiments, the agents are NOD ligands or ligand analogs (e.g., identified using the drug screening methods described above).

IX. Pharmaceutical Compositions Containing NOD Nucleic Acid, Peptides, and Analogs

[0273] The present invention further provides pharmaceutical compositions which may comprise all or portions of NOD polynucleotide sequences, NOD polypeptides, inhibitors or antagonists of NOD bioactivity, including antibodies, alone or in combination with at least one other agent, such as a stabilizing compound, and may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.

[0274] The methods of the present invention find use in treating diseases or altering physiological states characterized by mutant NOD alleles (e.g., inflammatory disease). Peptides can be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used (e.g., delivery via liposome). Such methods are well known to those of ordinary skill in the art. The formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal. Therapeutic administration of a polypeptide intracellularly can also be accomplished using gene therapy as described above.

[0275] As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and interaction with other drugs being concurrently administered.

[0276] Accordingly, in some embodiments of the present invention, NOD nucleotide and NOD amino acid sequences can be administered to a patient alone, or in combination with other nucleotide sequences, drugs or hormones or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert. In another embodiment of the present invention, NOD polynucleotide sequences or NOD amino acid sequences may be administered alone to individuals subject to or suffering from a disease.

[0277] Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes may, for example, include oral or transmucosal administration; as well as parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

[0278] For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0279] In other embodiments, the pharmaceutical compositions of the present invention can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.

[0280] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. For example, an effective amount of NOD may be that amount that suppresses apoptosis. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.

[0281] In addition to the active ingredients these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0282] The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).

[0283] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0284] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[0285] Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, (i.e., dosage).

[0286] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0287] Compositions comprising a compound of the invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. For polynucleotide or amino acid sequences of NOD, conditions indicated on the label may include treatment of condition related to inflammatory diseases.

[0288] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.

[0289] For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models (particularly murine models) to achieve a desirable circulating concentration range that adjusts NOD levels.

[0290] A therapeutically effective dose refers to that amount of NOD that ameliorates symptoms of the disease state. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0291] The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state; age, weight, and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0292] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212, all of which are herein incorporated by reference). Those skilled in the art will employ different formulations for NOD than for the inhibitors of NOD. Administration to the bone marrow may necessitate delivery in a manner different from intravenous injections.

[0293] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Sequence CWU 1

1

22 1 3414 DNA Homo sapiens 1 tgagaactca ggctggcaca gggattccca gggcatctac caccacgcag ctggagcagg 60 gctgagccca ggagcatgga gatggacgcc cccaggcccc ccagtcttgc tgtccctgga 120 gcagcatcga ggcccgggag gctgctggat ggggggcacg gcaggcagca ggttcaggcc 180 ctctcttcac agctcctgga ggtgatcccc gactccatga ggaagcaaga ggtgcggacg 240 ggcagggagg ccggccaggg ccacggtacg ggctccccag ccgagcaggt gaaagccctc 300 atggatctgc tggctgggaa gggcagtcaa ggctcccacg ccccgcaggc cctggatagg 360 acaccggatg ccccgctggg gccctgcagc aatgactcaa ggatacagag gcaccgcaag 420 gccctgctga gcaaggtggg aggtggcccg gagctgggcg gaccctggca caggctggcc 480 tccctcctgc tggtggaggg cctgacggac ctgcagctga gggaacacga cttcacacag 540 gtggaggcca cccgcggggg cgggcacccc gccaggaccg tcgccctgga ccggctcttc 600 ctgcctctct cccgggtgtc tgtcccaccc cgggtctcca tcactatcgg ggtggccggc 660 atgggcaaga ccaccctggt gaggcacttc gtccgcctct gggcccatgg gcaggtcggc 720 aaggacttct cgctggtgct gcctctgacc ttccgggatc tcaacaccca cgagaagctg 780 tgtgccgacc gactcatctg ctcggtcttc ccgcacgtcg gggagcccag cctggcggtg 840 gcagtcccag ccagggccct cctgatcctg gacggcttgg atgagtgcag gacgcctctg 900 gacttctcca acaccgtggc ctgcacggac ccaaagaagg agatcccggt ggaccacctg 960 atcaccaaca tcatccgtgg caacctcttt ccggaagttt ccatctggat cacctcccgt 1020 cccagtgcat ctggccagat cccagggggc ctggtggacc ggatgacgga gatccggggc 1080 tttaacgagg aggagatcaa ggtgtgtttg gagcagatgt tccccgagga ccaggccctt 1140 ctgggctgga tgctgagcca agtgcaggct gacagggccc tgtacctgat gtgcaccgtc 1200 ccagccttct gcaggctcac ggggatggcg ctaggccacc tgtggcgcag caggacgggg 1260 ccccaggatg cagagctgtg gcccccgagg accctgtgcg agctctactc atggtacttt 1320 aggatggccc tcagcgggga ggggcaggag aagggcaagg caagccctcg catcgagcag 1380 gtggcccatg gtggccgcaa gatggtgggg acattgggcc gtctggcctt ccatgggctg 1440 ctcaagaaga aatacgtgtt ttacgagcaa gacatgaagg cgtttggtgt agacctcgct 1500 ctgctgcagg gcgccccgtg cagctgcttc ctgcagagag aggagacgtt ggcatcgtca 1560 gtggcctact gcttcaccca cctgtccctg caggagtttg tggcagccgc gtattactat 1620 ggcgcatcca ggagggccat cttcgacctc ttcactgaga gcggcgtatc ctggcccagg 1680 ctgggcttcc tcacgcattt caggagcgca gcccagcggg ccatgcaggc agaggacggg 1740 aggctggacg tgttcctgcg cttcctctcc ggcctcttgt ctccgagggt caatgccctc 1800 ctggccggct ccctgctggc ccaaggcgag caccaggcct accggaccca ggtggctgag 1860 ctcctgcagg gctgcctgcg ccccgatgcc gcagtctgtg cacgggccat caacgtgttg 1920 cactgcctgc atgagctgca gcacaccgag ctggcccgca gcgtggagga ggccatggag 1980 agcggggccc tggccaggct gactggtccc gcgcaccgcg ctgccctggc ctacctcctg 2040 caggtgtccg acgcctgtgc ccaggaggcc aacctgtccc tgagcctcag ccagggcgtc 2100 cttcagagcc tgctgcccca gctgctctac tgccggaagc tcaggctgga caccaaccag 2160 ttccaggacc ccgtgatgga gctgctgggc agcgtgctga gtgggaagga ctgtcgcatt 2220 cagaagatca gcttggcgga gaaccagatc agtaacaaag gggccaaagc tctggccaga 2280 tccctcttgg tcaacagaag tctgacctct ctggacctcc gcggtaactc cattggacca 2340 caaggggcca aggcgctggc agacgctttg aagatcaacc gcaccctgac ctccctgagc 2400 ctccagggca acaccgttag ggatgatggt gccaggtcca tggctgaggc cttggcctcc 2460 aaccggaccc tctccatgct gcacctgcag aagaacagca tcgggcccat gggagcccag 2520 cggatggcag atgccttgaa gcagaacagg agtctgaaag agctcatgtt ctccagtaat 2580 agtattggtg atggaggtgc caaggccctg gctgaggccc tgaaggtgaa ccagggcctg 2640 gagagcctgg acctgcagag caattccatc agtgacgcag gagtggcagc actgatgggg 2700 gccctctgca ccaaccagac cctcctcagc ctcagccttc gagaaaactc catcagtccc 2760 gagggagccc aggccatcgc tcatgccctc tgcgccaaca gcaccctgaa gaacctggac 2820 ctgacagcca acctcctcca cgaccagggt gcccgggcca tcgcagtggc agtgagagaa 2880 aaccgcaccc tcacctccct tcacctgcag tggaacttca tccaggccgg cgctgcccag 2940 gccctgggac aagcactaca gctcaacagg agcctcacca gcttagattt acaggagaac 3000 gccatcgggg atgacggagc gtgtgcggtg gcccgtgcac tgaaggtcaa cacagccctc 3060 actgctctct atctccaggt ggcctcaatt ggtgcttcag gcgcccaggt gctaggggaa 3120 gccttggctg tgaacagaac cttggagatt ctcgacttaa gaggaaatgc cattggggtg 3180 gctggagcca aagccctggc aaatgctctg aaggtaaact caagtctccg gagactcaat 3240 cttcaagaga attctctggg gatggacggg gcgatatgca ttgccacagc actgtctgga 3300 aaccacaggc tccagcatat caatctccag ggaaaccaca ttggggactc cggggccagg 3360 atgatctcag aggccatcaa gacaaatgct cccacgtgca ctgttgaaat gtga 3414 2 3521 DNA Homo sapiens 2 aggcctgaat atttggacaa gatggcagat tcatcatcat cttctttctt tcctgatttt 60 gggctgctat tgtatttgga ggagctaaac aaagaggaat taaatacatt caagttattc 120 ctaaaggaga ccatggaacc tgagcatggc ctgacaccct ggaatgaagt gaagaaggcc 180 aggcgggagg acctggccaa tttgatgaag aaatattatc caggagagaa agcctggagt 240 gtgtctctca aaatctttgg caagatgaac ctgaaggatc tgtgtgagag agcgaaagaa 300 gagatcaact ggtcggccca gactatagga ccagatgatg ccaaggctgg agagacacaa 360 gaagatcagg aggcagtgct gggtgatgga acagaataca gaaatagaat aaaggaaaaa 420 ttttgcatca cttgggacaa gaagtctttg gctggaaagc ctgaagattt ccatcatgga 480 attgcagaga aagatagaaa actgttggaa cacttgttcg atgtggatgt caaaaccggt 540 gcacagccac agatcgtggt gcttcaggga gctgctggag ttgggaaaac aaccttggtg 600 agaaaggcaa tgttagattg ggcagagggc agtctctacc agcagaggtt taagtatgtt 660 ttttatctca atgggagaga aattaaccag ctgaaagaga gaagctttgc tcaattgata 720 tcaaaggact ggcccagcac agaaggcccc attgaagaaa tcatgtacca gccaagtagc 780 ctcttgttta ttattgacag tttcgatgaa ctgaactttg cctttgaaga acctgagttt 840 gcactgtgcg aagactggac ccaagaacac ccagtgtcct tcctcatgag tagtttgctg 900 aggaaagtga tgctccctga ggcatcctta ttggtgacaa caagactcac aacttctaag 960 agactaaagc agttgttgaa gaatcaccat tatgtagagc tactaggaat gtctgaggat 1020 gcaagagagg agtatattta ccagtttttt gaagataaga ggtgggccat gaaagtattc 1080 agttcactaa aaagcaatga gatgctgttt agcatgtgcc aagtccccct agtgtgctgg 1140 gccgcttgta cttgtctgaa gcagcaaatg gagaagggtg gtgatgtcac attgacctgc 1200 caaacaacca cagctctgtt tacctgctat atttctagct tgttcacacc agtagatgga 1260 ggctctccta gtctacccaa ccaagcccag ctgagaagac tgtgccaagt cgctgccaaa 1320 ggaatatgga ctatgactta cgtgttttac agagaaaatc tcagaaggct tgggttaact 1380 caatctgatg tctctagttt tatggacagc aatattattc agaaggacgc agagtatgaa 1440 aactgctatg tgttcaccca ccttcatgtt caggagtttt ttgcagctat gttctatatg 1500 ttgaaaggca gttgggaagc tgggaaccct tcctgccagc cttttgaaga tttgaagtca 1560 ttacttcaaa gcacaagtta taaagacccc catttgacac agatgaagtg ctttttgttt 1620 ggccttttga atgaagatcg agtaaaacaa ctggagagga cttttaactg taaaatgtca 1680 ctgaagataa aatcaaagtt acttcagtgt atggaagtat taggaaacag tgactattct 1740 ccatcacagc tgggatttct ggagttgttt cactgtctgt atgagactca agataaagcg 1800 tttataagcc aggcaatgag atgtttccca aaggttgcca ttaatatttg tgagaaaata 1860 catttgcttg tatcttcttt ctgccttaag cactgccggt gtttgcggac catcaggctg 1920 tctgtaactg tggtatttga gaagaagata ttaaaaacaa gcctcccaac taacacttgg 1980 gatggtgatc gcattactca ctgttggcaa gatctctgtt ctgtgcttca tacaaatgaa 2040 cacttgagag aattggacct gtaccatagc aaccttgata aatcagcaat gaatatcctg 2100 catcatgaac taaggcaccc aaactgtaaa ctacaaaagc tactgttgaa atttatcact 2160 ttccctgatg gttgtcagga tatctctact tctttgattc ataacaagaa tctgatgcat 2220 cttgacctaa aagggagtga tataggggat aatggagtaa agtcattgtg tgaggccttg 2280 aaacacccag agtgtaaact acagactctc aggctggaat cttgcaacct aactgtattt 2340 tgttgtctaa atatatctaa tgctctcatc agaagccaga gcctgatatt tctgaatctg 2400 tcaaccaata atctgttgga tgatggagtg cagcttttgt gtgaggcctt aagacatcca 2460 aagtgttatc tagagagact gtccttagaa agctgtggtc tcacagaggc tggctgtgag 2520 tatctttctt tggctctcat cagcaataaa agactgacac atttgtgctt ggcagacaat 2580 gtcttgggtg atggtggagt aaagcttatg agtgatgccc tgcaacatgc acaatgtact 2640 ctgaagagcc ttgtgctgag gcgttgccat ttcacttcac ttagcagtga atatctgtca 2700 acttctcttc tacacaacaa gagcctgacg catctggatc taggatcaaa ctggctacaa 2760 gacaatggag tgaagcttct gtgtgatgtc tttcggcatc caagctgtaa tcttcaggac 2820 ttggaattga tgggctgtgt tctcactaat gcatgttgtc tggatctggc ttctgttatt 2880 ttgaataacc caaacctgag gagcctggac cttgggaaca acgatttgca ggatgatgga 2940 gtgaaaattc tgtgtgatgc tttgagatat ccaaactgta acattcagag gctcgggttg 3000 gaatactgtg gtttgacatc tctctgctgt caagatctct cctctgctct tatctgcaac 3060 aaaagactga taaaaatgaa tctgacacag aataccttag gatatgaagg aattgtgaag 3120 ttatataaag tcttgaagtc tcctaagtgt aaactacaag ttctagggtt gtgcaaagag 3180 gcatttgatg aggaagccca gaagctgctg gaagctgtgg gagttagcaa tccacactta 3240 atcattaagc cagattgtaa ctatcataat gaagaagatg tgtcttggtg gtggtgtttc 3300 tgatttgaag aaactgacat tcctttaaaa atataaatat aaatacatac atacatagat 3360 atatacccag acttgggtgc ttagcttcag atactctatg cccagagata gtgcacttgg 3420 cagctgtcag ataccattca tctacttctc tgtaaaatgt ctgttctact tcacacagtg 3480 gtcgagaggc taaaataaaa tgaaaagcat aaaactctct g 3521 3 3484 DNA Homo sapiens 3 acacctcagt tcacaatcct ggggcgatat ggcagaatct tttttttcgg attttggctt 60 gttgtggtat ctgaaggagc tcagaaagga agagttttgg aaatttaagg agctcctcaa 120 acaacctttg gagaaatttg aactcaagcc aatcccctgg gctgagctga agaaggcctc 180 caaagaagat gtagcaaagc tgctggacaa acattaccca ggaaagcagg catgggaggt 240 aacactgaac ctgtttctac agatcaatag gaaagatctc tggacaaagg ctcaggaaga 300 gatgagaaat aagctaaacc catacagaaa gcatatgaag gaaacatttc aactcatatg 360 ggagaaggaa acctgtcttc acgtccctga gcatttctac aaagaaacca tgaaaaatga 420 gtataaagaa ttgaatgacg catatactgc tgcggctaga cgacacactg tggtcctgga 480 aggtcctgat ggaattggaa aaacaaccct tttaagaaaa gtgatgttgg actgggcaga 540 gggaaactta tggaaggaca ggttcacatt tgtgtttttc ctcaatgtct gtgaaatgaa 600 cggtatcgca gagaccagct tactggagct cctctctagg gactggccgg agtcttcaga 660 gaagatcgaa gacatttttt cccagccaga gagaattctg ttcatcatgg atggctttga 720 gcaactgaag tttaacttac aacttaaggc tgacttgagc gatgattgga ggcagcggca 780 gccaatgcca attatcctga gcagtttgtt gcaaaaaaag atgcttccag aatcctctct 840 ccttattgca ttaggaaaac tggctatgca aaaacactat tttatgttgc ggcatccaaa 900 actcataaag ctcttaggat tcagtgaatc tgaaaagaag tcgtatttct cctacttctt 960 tggtgagaag agcaaagccc tgaaagtctt caattttgtg agagataatg ggccgctgtt 1020 tatcttgtgc cataatccct ttacgtgctg gttggtctgt acttgtgtga aacagaggct 1080 agagagggga gaagaccttg aaataaactc ccaaaacacc acctatttat atgcatcctt 1140 tttaacaact gtattcaaag caggaagtca gagttttcca cctaaggtga acagagcccg 1200 actaaaaagc ctgtgtgctt tggctgcaga gggaatttgg acatatacat ttgtattttc 1260 ccatggggat ctccggagga atgggttatc tgagtctgag ggcgtgatgt gggtgggtat 1320 gagactcctc caaaggagag gggactgttt tgccttcatg catctgtgta tccaagagtt 1380 ttgtgccgcc atgttttatt tgctcaaacg acccaaagac gatcctaacc cggccattgg 1440 aagcataacc cagcttgtaa gagcaagtgt ggttcagcct caaaccctct tgacccaggt 1500 ggggatattc atgtttggaa tttcaacaga agaaatcgtc agcatgctgg agacctcctt 1560 tggttttcca ctgtcaaaag acctaaagca ggaaataacc caatgccttg aaagtttaag 1620 tcaatgtgaa gctgataggg aagccatagc tttccaggaa ctattcattg gtttgtttga 1680 aactcaggaa aaagaatttg taaccaaagt gatgaatttc tttgaagaag ttttcattta 1740 tattggtaac atagaacatt tggtaatagc ttcattctgc ctgaagcatt gtcaacattt 1800 aacgacactt cgcatgtgtg tggagaatat ctttccagat gactcaggat gcatctcaga 1860 ttacaatgag aagctcgtct actggcggga gctttgctca atgttcatta ccaacaagaa 1920 cttccagatt ttagacatgg aaaataccag ccttgatgat ccctccctgg cgattctttg 1980 caaagcgctg gctcagcctg tttgtaaact ccgaaaactc atatttactt ctgtgtactt 2040 tggacatgat tcagaattat ttaaggcagt tcttcacaac cctcatctga aacttctgag 2100 cctgtacggc actagcctct cccagtctga catcagacac ctgtgtgaga cgctgaaaca 2160 tccaatgtgc aagatagaag agctgatact gggaaagtgt gacatctcca gtgaagtttg 2220 tgaagacatc gcctccgtcc tggcctgcaa cagcaagctg aaacacctct ccttggtaga 2280 aaatcccttg agggacgaag gaatgacgtt gctgtgtgaa gccctgaagc actcacactg 2340 tgccctggag aggctgatgt tgatgtactg ctgtctcacc tctgtctcct gtgactccat 2400 ttccgaagtc ctcttgtgca gtaagtccct gtccctcctc gatctgggct caaatgccct 2460 ggaagataat ggagtggcat ctctgtgtgc agcgctgaag cacccaggct gcagcatacg 2520 ggagctgtgg ttgatgggct gtttccttac ttccgattcc tgtaaggaca ttgctgctgt 2580 tcttatttgc aatgggaaac tgaagaccct gaaacttggg cataatgaaa taggagacac 2640 tggtgtcaga cagttatgtg cagctttgca gcatcctcac tgtaaattag agtgtctcgg 2700 gctgcaaacg tgtccgatca cccgtgcctg ctgcgacgac atcgccgcag cactcatcgc 2760 ctgcaaaaca ctgaggagcc tgaacctcga ctggattgcc ttggatgctg atgcagtggt 2820 ggtgctgtgt gaggcattga gccacccgga ctgtgccctg cagatgctgg ggctgcacaa 2880 atctggcttt gatgaagaaa ctcagaagat cctgatgtct gtggaagaaa aaattcccca 2940 tctgaccatt tcacatggac cttggattga cgaggaatac aagatcaggg gtgtgctcct 3000 ctgatgggga acaccctgaa gtagtcgtct cacaaaggct ttccttggcc acagtgggac 3060 cttcacctgg cacctctatc ctgtaattgc acatcatggc agcagggctg tgatttcaga 3120 ggtactccct aagtgttcta gcaatatgat tatggagtgt gattcagtgt acatgctgat 3180 tgtctttgcc tcggtcctat atccccttgt ctttagaaat cccatcctgc cttgtgatat 3240 ttagaagcac aagtacgtta aacaagtgct aaacgctctg gaaagcatgg ctttattttc 3300 ttaatggatg tcttggtgtg taggagcatg catttgtagg caccacaatc cggatacttc 3360 tgacacagaa gtgatgctag aatgtgtcta tagattgtat tgctagcatc cagactttct 3420 agtttgtcca gatttcgatt tgatcaattt tcttgtccaa taaaaaagca tttccaaatc 3480 tcta 3484 4 1974 DNA Homo sapiens 4 atcaccatgg ccatggccaa ggccagaaag ccccgggagg cattgctctg ggccttgagt 60 gaccttgagg agaacgattt caagaagtta aagttctact tacgggatat gaccctgtct 120 gagggccagc ccccactggc cagaggggag ttggagggcc tgattccggt ggacctggca 180 gaattactga tttcaaagta tggagaaaag gaggctgtga aagttgtcct caagggcttg 240 aaggtcatga acctgttgga acttgtggac cagctcagcc atatttgtct gcatgattac 300 agagaagtat accgagagca tgtgcgctgc ctagaggaat ggcaggaagc aggagtcaat 360 ggcagataca accaggtgct cctggtggcc aagcccagct cagagagccc agaatcactt 420 gcctgcccct tcccggagca ggagctggag tctgtcacgg tggaggctct atttgattca 480 ggggaaaagc cctcactggc cccatcctta gttgtgctac aggggtcggc tggcactgga 540 aagacaactc tcgccagaaa aatggtgttg gactgggcca ccggtactct gtacccaggc 600 cggtttgatt atgtctttta tgtaagctgc aaagaagtgg tcctgctgct ggagagcaaa 660 ctggagcagc tccttttctg gtgctgcggg gacaatcaag cccctgtcac agagattctg 720 aggcagccag agcggctcct gttcatcctg gatggctttg atgagctgca gaggcccttt 780 gaagaaaagt tgaagaagag gggtttgagt cccaaggaga gcctgctgca ccttctaatt 840 aggagacata cactccccac gtgctccctt ctcatcacca cccggcccct ggctttgagg 900 aatctggagc ccttgctgaa acaagcacgt catgtccata tcctaggctt ctctgaggag 960 gagagggcga ggtacttcag ctcctatttc acggatgaga agcaagctga ccgtgccttc 1020 gacattgtac agaaaaatga cattctctac aaagcgtgtc aggttccagg catttgctgg 1080 gtggtctgct cctggctgca ggggcagatg gagagaggca aagttgtctt agagacacct 1140 agaaacagca ctgacatctt catggcttac gtctccacct ttctgccgcc cgatgatgat 1200 gggggctgct ccgagctttc ccggcacagg gtcctgagga gtctgtgctc cctagcagct 1260 gaagggattc agcaccagag gttcctattt gaagaagctg agctcaggaa acataattta 1320 gatggcccca ggcttgccgc tttcctgagt agtaacgact accaattggg acttgccatc 1380 aagaagttct acagcttccg ccacatcagc ttccaggact tttttcatgc catgtcttac 1440 ctggtgaaag aggaccaaag ccggctgggg aaggagtccc gcagagaagt gcaaaggctg 1500 ctggaggtaa aggagcagga agggaatgat gagatgaccc tcactatgca gtttttactg 1560 gacatctcga aaaaagacag cttctcgaac ttggagctca agttctgctt cagaatttct 1620 ccctgtttag cgcaggatct gaagcatttt aaagaacaga tggaatctat gaagcacaac 1680 aggacctggg atttggaatt ctccctgtat gaagctaaaa taaagaatct ggtaaaaggt 1740 attcagatga acaatgtatc attcaagata aaacattcaa atgaaaagaa atcacagagc 1800 cagaatttat tttctgtcaa aagcagcttg agtcatggac ctaaggagga gcaaaaatgt 1860 ccttctgtcc atggacagaa ggagggcaaa gataatatag caggaacaca aaaggaagct 1920 tctactggaa aaggcagagg gacagaggaa acaccaaaaa atacttacat ataa 1974 5 3525 DNA Homo sapiens 5 gctctgacct tctttcccag gatgaggtgg ggccaccatt tgcccagggc ctcttggggc 60 tctggtttta gaagagcact ccagcgacca gatgatcgta tccccttcct gatccactgg 120 agttggcccc ttcaagggga gcgtcccttt gggcccccta gggcctttat acgccaccac 180 ggaagctcgg tagatagcgc tcccccatcc gggaggcatg gacggctgtt ccccagcgcc 240 tctgcaactg aagctataca gcggcaccgc cggaacctgg ctgagtggtt cagccggctg 300 cccagggagg agcgccagtt tggcccaacc tttgccctag acacggtcca cgttgaccct 360 gtgatccgcg agagtacccc tgatgagcta cttcgcccac ccgcggagct ggccctggag 420 catcagccac cccaggccgg gctcccccca ctggccttgt ctcagctctt taacccggat 480 gcctgtgggc gccgggtgca gacagtggtg ctgtatggga cagtgggcac aggcaagagc 540 acgctggtgc gcaagatggt tctggactgg tgttatgggc ggctgccggc cttcgagctg 600 ctcatcccct tctcctgtga ggacctgtca tccctgggcc ctgccccagc ctccctgtgc 660 caacttgtgg cccagcgcta cacgcccctg aaggaggttc tgcccctgat ggctgctgct 720 gggtcccacc tcctctttgt gctccatggc ttagagcatc tcaacctcga cttccggctg 780 gcaggcacgg gactttgtag tgacccggag gaaccgcagg aaccagctgc tatcatcgtc 840 aacctgctgc gcaaatacat gctgcctcag gccagcattc tggtgaccac tcggccctct 900 gccattggcc gtatccccag caagtacgtg ggccgctatg gtgagatctg cggtttctct 960 gataccaacc tgcagaagct ctacttccag ctccgcctca accagccgta ctgcgggtat 1020 gccgttggcg gttcaggtgt ctctgccaca ccagctcagc gtgaccacct ggtgcagatg 1080 ctctcccgga acctggaggg gcaccaccag atagccgctg cctgcttcct gccgtcctat 1140 tgctggctcg tttgtgccac cttgcacttc ctgcatgccc ccacgcctgc tgggcagacc 1200 cttacaagca tctataccag cttcctgcgc ctcaacttca gcggggaaac cctggacagc 1260 actgacccct ccaatttgtc cctgatggcc tatgcagccc gaaccatggg caagttggcc 1320 tatgaggggg tgtcctcccg caagacctac ttctctgaag aggatgtctg tggctgcctg 1380 gaggctggca tcaggacgga ggaggagttt cagctgctgc acatcttccg tcgggatgcc 1440 ctgaggtttt tcctggcccc atgtgtggag ccagggcgtg caggcacctt cgtgttcacc 1500 gtgcccgcca tgcaggaata cctggctgcc ctctacattg tgctgggttt gcgcaagacg 1560 accctgcaaa aggtgggcaa ggaagtggct gagctcgtgg gccgtgttgg ggaggacgtc 1620 agcctggtac tgggcatcat ggccaagctg ctgcctctgc gggctctgcc tctgctcttc 1680 aacctgatca aggtggttcc acgagtgttt gggcgcatgg tgggtaaaag ccgggaggcg 1740 gtggctcagg ccatggtgct ggagatgttt cgagaggagg actactacaa cgatgatgtt 1800 ctggaccaga tgggcgccag tatcctgggc gtggagggcc cccggcgcca cccagatgag 1860 ccccctgagg atgaagtctt cgagctcttc cccatgttca tgggggggct tctctctgcc 1920 cacaaccgag ctgtgctagc tcagcttggc tgccccatca agaacctgga tgccctggag 1980 aatgcccagg ccatcaagaa gaagctgggc aagctgggcc ggcaggtgct gcccccatca 2040 gagctccttg accacctctt cttccactat gagttccaga accagcgctt ctccgctgag 2100 gtgctcagct ccctgcgtca gctcaacctg gcaggtgtgc gcatgacacc agtcaagtgc 2160 acagtggtgg cagctgtgct gggcagcgga aggcatgccc tggatgaggt gaacttggcc 2220 tcctgccagc tagatcctgc tgggctgcgc acactcctgc ctgtcttcct gcgtgcccgg 2280 aagctgggct tgcaactcaa cagcctgggc cctgaggcct gcaaggacct ccgagacctg 2340 ttgctgcatg accagtgcca aattaccaca ctgcggctgt ccaacaaccc gctgacggag 2400 gcaggtgttg ccgtgctaat ggaggggctg gcaggaaaca cctcagtgac

gcacctgtcc 2460 ctgctgcaca cgggccttgg ggacgaaggc ctggagctgc tggctgccca gctggaccgc 2520 aaccggcagc tgcaggagct gaacgtggcg tacaacggtg ctggtgacac agcggccctg 2580 gccctggcca gagctgcccg ggagcaccct tccctggaac tgctacacct ctacttcaat 2640 gagctgagct cagagggccg ccaggtcttg cgagacttgg ggggtgctgc tgaaggtggt 2700 gcccgggtgg tggtgtcact gacagagggg acggcggtgt cagaatactg gtcagtgatc 2760 ctcagtgaag tccagcggaa cctcaatagc tgggatcggg cccgggttca gcgacacctt 2820 gagctcctac tgcgggatct ggaagatagc cggggtgcca cccttaatcc ttggcgcaag 2880 gcccagctgc tgcgagtgga gggcgaggtc agggccctcc tggagcagct gggaagctct 2940 ggaagctgag acactggcgg caggcaccta gctatgtgac cactggccct aaaccttttc 3000 cctctgtggc ctcctggctt gcactgctcc ctctagaaag attccttcag gtctggaggc 3060 agaggaatgg gcatagctga gccagttgcc ctcctagggc atgtttgacc aggactgagt 3120 ctggaatctc caagttaaag atggtgaatc aatgcttcgg gcttggagat ggaacatgcc 3180 tcctctccat tcagctagaa ggaccaaagc atgtggcatt tggatggcca gagtgccctg 3240 aagcaccact accaaccttg cctccccctc ctctcaaaga gcctctgatt gtgtcaccaa 3300 ggggctcaca tcttatgtct gccatgccag gggtgtcgcc atccagatgt gttggaagct 3360 tcccctcctg ccttatgctc acctgtggac accgaggatg ccctcacatt ggtgctttct 3420 cctcatcctc atgccccctt tgccacaatg gtatgatggc ttggtagccc ctcgaggcag 3480 atgcacctga cttgctgcta ttaaaaagcc gtgtgccttc tacca 3525 6 3373 DNA Homo sapiens 6 ttcttcagcc ttaacctaag gtctcatact cggagcacta tgacatcgcc ccagctagag 60 tggactctgc agacccttct ggagcagctg aacgaggatg aattaaagag tttcaaatcc 120 cttttatggg cttttcccct cgaagacgtg ctacagaaga ccccatggtc tgaggtggaa 180 gaggctgatg gcgagaaact ggcagaaatt ctggtcaaca cctcctcaga aaattggata 240 aggaatgcga ctgtgaacat cttggaagag atgaatctca cggaattgtg taagatggca 300 aaggctgaga tgatggagga cggacaggtg caagaaatag ataatcctga gctgggagat 360 gcagaagaag actcggagtt agcaaagcca ggtgaaaagg aaggatggag aaattcaatg 420 gagaaacagt ctttggtctg gaagaacacc ttttggcaag gagacattga caatttccat 480 gacgacgtca ctctgagaaa ccaacggttc attccattct tgaatcccag aacacccagg 540 aagctaacac cttacacggt ggtgctgcac ggccccgcag gcgtggggaa aaccacgctg 600 gccaaaaagt gtatgctgga ctggacagac tgcaacctca gcccgacgct cagatacgcg 660 ttctacctca gctgcaagga gctcagccgc atgggcccct gcagttttgc agagctgatc 720 tccaaagact ggcctgaatt gcaggatgac attccaagca tcctagccca agcacagaga 780 atcctgttcg tggtcgatgg ccttgatgag ctgaaagtcc cacctggggc gctgatccag 840 gacatctgcg gggactggga gaagaagaag ccggtgcccg tcctcctggg gagtttgctg 900 aagaggaaga tgttacccag ggcagccttg ctggtcacca cgcggcccag ggcactgagg 960 gacctccagc tcctggcgca gcagccgatc tacgtaaggg tggagggctt cctggaggag 1020 gacaggaggg cctatttcct gagacacttt ggagacgagg accaagccat gcgtgccttt 1080 gagctaatga ggagcaacgc ggccctgttc cagctgggct cggcccccgc ggtgtgctgg 1140 attgtgtgca cgactctgaa gctgcagatg gagaaggggg aggacccggt ccccacctgc 1200 ctcacccgca cggggctgtt cctgcgtttc ctctgcagcc ggttcccgca gggcgcacag 1260 ctgcggggcg cgctgcggac gctgagcctc ctggccgcgc agggcctgtg ggcgcagatg 1320 tccgtgttcc accgagagga cctggaaagg ctcggggtgc aggagtccga cctccgtctg 1380 ttcctggacg gagacatcct ccgccaggac agagtctcca aaggctgcta ctccttcatc 1440 cacctcagct tccagcagtt tctcactgcc ctgttctacg ccctggagaa ggaggagggg 1500 gaggacaggg acggccacgc ctgggacatc ggggacgtac agaagctgct ttccggagaa 1560 gaaagactca agaaccccga cctgattcaa gtaggacact tcttattcgg cctcgctaac 1620 gagaagagag ccaaggagtt ggaggccact tttggctgcc ggatgtcacc ggacatcaaa 1680 caggaattgc tgcaatgcaa agcacatctt catgcaaata agcccttatc cgtgaccgac 1740 ctgaaggagg tcttgggctg cctgtatgag tctcaggagg aggagctggc gaaggtggtg 1800 gtggccccgt tcaaggaaat ttctattcac ctgacaaata cttctgaagt gatgcattgt 1860 tccttcagcc tgaagcattg tcaagacttg cagaaactct cactgcaggt agcaaagggg 1920 gtgttcctgg agaattacat ggattttgaa ctggacattg aatttgaaag ctcaaacagc 1980 aacctcaagt ttctggaagt gaaacaaagc ttcctgagtg actcttctgt gcggattctt 2040 tgtgaccacg taacccgtag cacctgtcat ctgcagaaag tggagattaa aaacgtcacc 2100 cctgacaccg cgtaccggga cttctgtctt gctttcattg ggaagaagac cctcacgcac 2160 ctgaccctgg cagggcacat cgagtgggaa cgcacgatga tgctgatgct gtgtgacctg 2220 ctcagaaatc ataaatgcaa cctgcagtac ctgaggttgg gaggtcactg tgccaccccg 2280 gagcagtggg ctgaattctt ctatgtcctc aaagccaacc agtccctgaa gcacctgcgt 2340 ctctcagcca atgtgctcct ggatgagggt gccatgttgc tgtacaagac catgacacgc 2400 ccaaaacact tcctgcagat gttgtcgttg gaaaactgtc gtcttacaga agccagttgc 2460 aaggaccttg ctgctgtctt ggttgtcagc aagaagctga cacacctgtg cttggccaag 2520 aaccccattg gggatacagg ggtgaagttt ctgtgtgagg gcttgagtta ccctgattgt 2580 aaactgcaga ccttggtgtt acagcaatgc agcataacca agcttggctg tagatatctc 2640 tcagaggcgc tccaagaagc ctgcagcctc acaaacctgg acttgagtat caaccagata 2700 gctcgtggat tgtggattct ctgtcaggcg ttagagaatc caaactgtaa cctaaaacac 2760 ctacgcctct ggagctgctc cctcatgcct ttctattgtc agcatcttgg atctgctctc 2820 ctcagcaatc agaagcttga aactctggac ctgggccaga atcatttgtg gaagagtggc 2880 ataattaagc tctttggggt tctaagacaa agaactggat ccttgaagat actcaggttg 2940 aagacctatg aaactaattt ggaaatcaag aagctgttgg aggaagtgaa agaaaagaat 3000 cccaagctga ctattgattg caatgcttcc ggggcaacgg cacctccgtg ctgtgacttt 3060 ttttgctgag cagcctggga tcgctctacg aattacacag gaagcgggat tcgggtctct 3120 aagatgtctt atgaatgcag gtcagagggt cacatgttaa cactagagtc tgtcgagagg 3180 taggatttga cactggtttt ctcactattt ttgggagatt ctgcacgagt cacgcacccc 3240 cttcacatga cgctatgtac tttctcacag ggataataaa gttagagcac tctcgttgca 3300 gctgcgttta ttgacatgct caggagcaaa cctgcaataa acatggtact ctgtgcttcg 3360 tctaggagga agt 3373 7 3540 DNA Homo sapiens 7 tgagaaactg catgtgttgg gcaagatgaa cttttctgta atcacctgcc ccaacggtgg 60 taccaaccaa gggcttctgc cttacctgat ggccctggat cagtatcagc tggaggaatt 120 caagctttgc ttggaacccc agcagctgat ggacttctgg tcggcccccc aggggcactt 180 cccgcgtatc ccctgggcaa acttgagagc tgccgaccct ttgaatctgt cctttctttt 240 ggatgaacac ttcccaaaag gtcaggcatg gaaagtggtc ctcggcatct tccagacaat 300 gaatctgacc tcactgtgtg agaaagttag agccgagatg aaagagaatg tgcagaccca 360 agagctgcaa gatccaaccc aggaagatct agagatgcta gaagcagcag cagggaatat 420 gcagacccag ggatgccaag atccaaacca agaagaacta gacgagctag aagaagaaac 480 agggaatgta caggcccagg gatgccaaga tccaaaccaa gaagaaccag agatgctaga 540 ggaagcagac cacagaagaa aatacagaga gaacatgaag gctgaactac tggagacatg 600 ggacaacatc agttggccta aagaccacgt atatatccgt aatacatcaa aggacgaaca 660 tgaggaactg cagcgcctac tggatcctaa taggactaga gcccaggccc agacgatagt 720 cttggtgggg agggcagggg ttgggaagac caccttggca atgcaggcta tgctgcactg 780 ggcaaatgga gttctctttc agcaaaggtt ctcctatgtt ttctatctca gctgccataa 840 aataaggtac atgaaggaaa ctacctttgc tgaattgatt tctttggatt ggcccgattt 900 tgatgccccc attgaagagt tcatgtctca accagagaag ctcctgttta ttattgatgg 960 ctttgaggaa ataatcatat ctgagtcacg ctctgagagc ttggatgatg gctcgccatg 1020 tacagactgg taccaggagc tcccagtgac caaaatccta cacagcttgt tgaagaaaga 1080 attggttccc ctggctacct tactgatcac gatcaagacc tggtttgtga gagatcttaa 1140 ggcctcatta gtgaatccat gctttgtaca aattacaggg ttcacagggg acgacctacg 1200 ggtatatttc atgagacact ttgatgactc aagtgaagtt gagaaaatcc tgcagcagct 1260 aagaaaaaac gaaactctct ttcattcctg cagtgccccc atggtgtgtt ggaccgtatg 1320 ttcctgtctg aagcagccga aggtgaggta ttacgatctc cagtcaatca ctcagactac 1380 caccagtctg tatgcctatt ttttctccaa cttgttctcc acagcagagg tagatttggc 1440 agatgacagc tggccaggac aatggagggc cctctgcagt ctggccatag aagggctgtg 1500 gtctatgaac ttcacgttta acaaagaaga cactgagatc gagggcctgg aagtgccttt 1560 cattgattct ctctacgagt tcaatattct tcaaaagatc aatgactgtg ggggttgcac 1620 tactttcacc cacctaagtt tccaggagtt ttttgcagcc atgtcctttg tgctagagga 1680 acctagagaa ttccctcccc attccacaaa gccacaagag atgaagatgt tactgcaaca 1740 cgtcttgctt gacaaagaag cctactggac tccagtggtt ctgttcttct ttggtctttt 1800 aaataaaaac atagcaagag aactggaaga tactttgcat tgtaaaatat ctcccagggt 1860 aatggaggaa ttattaaagt ggggagaaga gttaggtaag gctgaaagtg cctctctcca 1920 atttcacatt ctacgacttt ttcactgcct acacgagtcc caggaggaag acttcacaaa 1980 gaagatgttg ggtcgtatct ttgaagttga ccttaatatt ttggaggacg aagaactcca 2040 agcttcttca ttttgcctaa agcactgtaa aaggttaaat aagctaaggc tttctgttag 2100 cagtcacatc cttgaaaggg acttggaaat tctggagaca agcaagtttg attccaggat 2160 gcacgcatgg aacagcattt gctctacgtt ggtcacaaat gagaatctgc atgagctaga 2220 cctgagtaac agcaaacttc atgcttcctc tgtgaagggt ctctgtcttg cactgaaaaa 2280 tccaagatgc aaagtccaga aactgacgtg caaatcggta actcctgagt gggttctgca 2340 ggacctcatt attgcccttc agggtaacag caagctgacc catctgaact tcagctctaa 2400 caagctggga atgactgtcc ccctgattct taaagctttg agacactcag cttgcaacct 2460 caagtatctg tgcctggaga aatgcaactt gtcggcagcc agctgtcagg acctagcctt 2520 gtttctcacc agcatccaac acgtaactcg attgtgcctg ggatttaatc ggctccaaga 2580 tgatggcata aagctattgt gtgcggccct gactcacccc aagtgtgcct tagagagact 2640 ggagctctgg ttttgccagc tggcagcacc cgcttgcaag cacttgtcag atgctctcct 2700 gcagaacagg agcctgacac acctgaatct gagcaagaac agcctgagag acgagggagt 2760 caagttcctg tgtgaggcct tgggtcgccc agatggtaac ctgcagagcc tgaatttgtc 2820 aggttgttct ttcacaagag agggctgtgg agagctggct aatgccctca gccataatca 2880 taatgtgaaa atcttagatt tgggagaaaa tgatcttcag gatgatggag tgaagctact 2940 gtgtgaggct ctgaaaccac atcgtgcatt gcacacactt gggttggcga aatgcaatct 3000 gacaactgct tgctgccagc atctcttctc tgttctcagc agcagtaaga gcctggtcaa 3060 tctgaacctt ctaggcaatg aattggatac tgatggtgtc aagatgctat gcttcaaaaa 3120 gacctgcaca atgtagtgag agaggagata cagacctcac agaaggagct ctgtctgaaa 3180 ctcaagtgtg cgtgggattt taatgacctt gaagacaagt ggtggtggtg atcccacgga 3240 ttagatgcca cgtggcttga ccatggatct tgggggaaag ccaccaggac atcctggcct 3300 gtgtgtcgct ccaatgtcac catttgtggg gacaaatgag ctgttccctg caggaggctt 3360 tgtcacggtt gttggaggcc gcccattgca cgcccaggtc tggaatccta gtgtaatact 3420 gtgtctggta ccaagatcat aagttggctg tgccttcagt cttgtctatg tcctccttgg 3480 tgtaatgttt ttaattcttg gaggtgttga gagaattcaa taaagcaaag catataaaaa 3540 8 3934 DNA Homo sapiens 8 gtctcgtgtt tctctcttcc aatcggttgt ctttatcgtg gacactgagg tgttctctgc 60 cttgactaaa gatgagtgac gtgaatccac cctctgacac ccccattccc ttttcatcct 120 cctccactca cagttctcat attccgccct ggacattctc ttgctacccc ggctccccat 180 gtgaaaatgg ggtcatgctg tacatgagaa acgtgagcca tgaggagcta caacggttca 240 agcagctctt actgactgag ctcagtactg gcaccatgcc catcacctgg gaccaggtcg 300 agacagccag ctgggcagag gtggttcatc tcttgataga gcgtttccct ggacgacgcg 360 cttgggatgt gacttcgaac atctttgcca ttatgaactg tgataaaatg tgtgttgtag 420 tccgcagaga gataaatgcc attctgccta ccttggaacc agaggacttg aatgtgggag 480 aaacacaggt gaatctggag gaaggagaat ctggtaaaat acggcggtat aaatcgaatg 540 tgatggaaaa gtttttcccc atatgggaca ttacgacttg gcctggaaac cagagggact 600 tcttctacca aggtgtacac aggcacgagg agtacttacc atgtctgctt ctgcccaaaa 660 gaccccaggg tagacagccc aagaccgtgg ccatacaggg agctcctggg atcggaaaaa 720 caatcctggc caaaaaggtg atgtttgagt gggccagaaa caagttctac gcccacaagc 780 gctggtgtgc tttctacttc cattgccaag aggtgaacca gacgacagac cagagcttct 840 ccgagctgat tgagcaaaag tggcctggat ctcaggacct cgtgtcaaag attatgtcca 900 aacccgacca acttctgctg ctcttggatg gctttgagga gctcacatct accctcattg 960 acagactgga ggacctgagt gaagactgga ggcagaaatt gcctgggtct gtcctactga 1020 gcagtttgct gagcaaaacg atgcttccag aggccacgct actgatcatg ataagattta 1080 cctcttggca gacatgcaag cccttgctga aatgtccctc tctcgtaacc cttccggggt 1140 ttaatacgat ggaaaaaatc aagtatttcc agatgtattt tggacacaca gaggagggag 1200 accaagtctt gagtttcgcc atggaaaaca ccattctctt ctccatgtgc cgggtccctg 1260 tggtttgctg gatggtctgc tctggtctga aacagcaaat ggagagagga aacaatctca 1320 cacagtcatg tccaaatgcc acctctgtgt tcgtccggta tatttctagc ttgtttccca 1380 ccagagctga gaacttttcc agaaagatcc accaagcaca actggaaggt ctgtgtcact 1440 tggccgcaga cagcatgtgg cacaggaaat gggtgttagg taaagaagat cttgaggaag 1500 ccaagctgga tcagacggga gtcaccgcct tccttggcat gagtattctt cggagaattg 1560 caggtgagga agaccactat gtctttaccc tcgtgacttt tcaggaattt tttgcggcct 1620 tgttttatgt tctctgtttc ccacaaagac tcaaaaattt tcatgtgttg agccacgtga 1680 atatccagcg cctgatagcg agtcccagag gaagcaaaag ctatctctct cacatgggac 1740 ttttcttatt cggttttctg aacgaggcct gcgcttcggc cgtggaacag tcattccaat 1800 gcaaggtgtc tttcggtaat aagaggaaac tgctgaaagt catacctctg ttgcataaat 1860 gtgacccacc ttctccgggc agtggggtcc cgcagttatt ctactgtctg catgaaatcc 1920 gggaggaagc ctttgtaagc caagccctaa atgattatca taaagttgtc ttgagaattg 1980 gcaacaacaa agaagttcaa gtgtctgctt tttgcctgaa gcggtgtcaa tatttgcatg 2040 aggtggaact gaccgtcacc ctgaacttca tgaacgtgtg gaagctcagc tccagctccc 2100 atcctggctc tgaagcgcca gagagcaatg ggctgcatcg ttggtggcaa gacttatgct 2160 ctgtgtttgc aacgaatgat aagctggaag tcctgactat gaccaacagt gttttggggc 2220 ctcctttttt gaaggctctc gcggccgcac tgaggcaccc tcagtgcaaa ctgcaaaagc 2280 tactcctaag gcgtgtgaat agcaccatgt tgaaccagga cttaatcggt gttttgacgg 2340 ggaaccagca tctgagatac ttggaaatac aacatgtgga agtggagtcc aaagctgtga 2400 agcttctatg cagggtgctg agatcccccc ggtgccgtct gcagtgtctc aggttggaag 2460 actgcttggc cacccctaga atttggactg atcttggcaa taatcttcaa ggtaacgggc 2520 atctaaagac tctcatacta agaaaaaact ccctggagaa ctgtggggcg tattacctgt 2580 ctgtggccca gctggagagg ctgtcgatag agaactgcaa ccttacacag cttacttgtg 2640 aaagccttgc ctcctgtctc aggcagagta agatgctgac ccacctgagc ttggcagaaa 2700 acgccttgaa agatgaaggg gccaagcata tttggaatgc cctgccacac ctgagatgtc 2760 ctctgcagag gctggtactg agaaagtgtg acttgacctt taattgctgt caggatatga 2820 tctctgcgct ctgtaaaaat aaaaccctga aaagtcttga cctaagtttt aatagcctga 2880 aggatgatgg ggtgatcctg ctgtgtgagg ccctgaagaa ccctgactgt acattacaga 2940 tcctggagct ggaaaactgc ctgttcacct ccatctgctg ccaggccatg gcttccatgc 3000 tccgcaaaaa ccaacatctg agacatctgg acttgagcaa gaatgcgatt ggagtctatg 3060 gtattctgac cttgtgcgag gccttctcaa gccaaaagaa gagagaagag gtcattttct 3120 gtattcctgc ctggactcga ataactagct tctccccaac tcctcaccca cccgacttca 3180 cgggaaaaag tgactgccta tcccagatta atccttaggc cgtccagtca tctttctctg 3240 gggcttgatt gatcagttcc cactctgaca actggcaaat accaggcgtt atcatcctgt 3300 atgcattaac gtactttccc ctgaaacaga gcaacccagt caacaccaca gaacctcagc 3360 tttgaaccct ggagtgagga cggtgatgcc ctgtgtgtat taatatgcta tgtaaggctg 3420 ggcgtggtgg ctcacgcctg taacccagca ctatgggagg tcgaggtggg cagattacct 3480 gaggtcagga gttccagacc agcctggcca acatggtgaa accccgcctc tactaaaaaa 3540 aaaaatacaa aaaattaggc gtggtggtgg gctcctgtaa tcccagctgc tcgggaggct 3600 gaggcaggag aatcacttga atctaggagg cagagtttgc agtgagctga gatcacgcca 3660 ttgcactcca gcctgggcga cagagcaaga ctctgtctca agaagaaaaa aaaaatacat 3720 atacacataa atatatatat gtgtgtgtgt atatatatat atatatatat atatgctata 3780 taaagtttaa atgaaatgct ttgagtcacc taagacagga tatagacaaa gtcttcatcg 3840 tcttcttgct tcttctacct ttatttattc tcagctctga atgtatgaac ctgctcaatc 3900 acctcatctt aaaaataaaa tcactgtccc taga 3934 9 3102 DNA Homo sapiens 9 atggcagaat cggattctac tgactttgac ctgctgtggt atctagagaa tctcagtgac 60 aaggaatttc agagttttaa gaagtatctg gcacgcaaga ttcttgattt caaactgcca 120 cagtttccac tgatacagat gacaaaagaa gaactggcta acgtgttgcc aatctcttat 180 gagggacagt atatatggaa tatgctcttc agcatatttt caatgatgcg taaggaagat 240 ctttgtagga agatcattgg cagacgaaac cgcaatcagg aggcatgcaa agctgtcatg 300 aggagaaaat tcatgctgca atgggaaagt cacacttttg gaaaatttca ttataaattt 360 tttcgtgacg tttcgtcaga tgtgttctac atacttcaat tagcctatga ttctaccagc 420 tattattcag caaacaatct caatgtgttc ctgatgggag agagagcatc tggaaaaact 480 attgttataa atctggctgt gttgaggtgg atcaagggtg agatgtggca gaacatgatc 540 tcgtacgtcg ttcacctcac ttctcacgaa ataaaccaga tgaccaacag cagcttggct 600 gagctaatcg ccaaggactg gcctgacggc caggctccca ttgcagacat cctgtctgat 660 cccaagaaac tccttttcat cctcgaggac ttggacaaca taagattcga gttaaatgtc 720 aatgaaagtg ctttgtgtag taacagcacc cagaaagttc ccattccagt tctcctggtc 780 agtttgctga agagaaaaat ggctccaggc tgctggttcc tcatctcctc aaggcccaca 840 cgtgggaata atgtaaaaac gttcttgaaa gaggtagatt gctgcacgac cttgcagctg 900 tcgaatggga agagggagat atattttaac tctttcttta aagaccgcca gagggcgtcg 960 gcagccctcc agcttgtaca tgaggatgaa atactcgtgg gtctgtgccg agtcgccatc 1020 ttatgctgga tcacgtgtac tgtcctgaag cggcagatgg acaaggggcg tgacttccag 1080 ctctgctgcc aaacacccac tgatctacat gcccactttc ttgctgatgc gttgacatca 1140 gaggctggac ttactgccaa tcagtatcac ctaggtctcc taaaacgtct gtgtttgctg 1200 gctgcaggag gactgtttct gagcaccctg aatttcagtg gtgaagacct cagatgtgtt 1260 gggtttactg aggctgatgt ctctgtgttg caggccgcga atattctttt gccgagcaac 1320 actcataaag accgttacaa gttcatacac ttgaacgtcc aggagttttg tacagccatt 1380 gcatttctga tggcagtacc caactatctg atcccctcag gcagcagaga gtataaagag 1440 aagagagaac aatactctga ctttaatcaa gtgtttactt tcatttttgg tcttctaaat 1500 gcaaacagga gaaagattct tgagacatcc tttggatacc agctaccgat ggtagacagc 1560 ttcaagtggt actcggtggg atacatgaaa catttggacc gtgacccgga aaagttgacg 1620 caccatatgc ctttgtttta ctgtctctat gagaatcggg aagaagaatt tgtgaagacg 1680 attgtggatg ctctcatgga ggttacagtt taccttcaat cagacaagga tatgatggtc 1740 tcattatact gtctggatta ctgctgtcac ctgaggacac ttaagttgag cgttcagcgc 1800 atctttcaaa acaaagagcc acttataagg ccaactgcta gtcaaatgaa gagccttgtc 1860 tactggagag agatctgctc tcttttttat acaatggaga gcctccggga gctgcatatc 1920 tttgacaatg accttaatgg tatttcagaa aggattctgt ctaaagccct ggagcattct 1980 agctgtaaac ttcgcacact caagttgtcc tatgtctcga ctgcttctgg ttttgaagac 2040 ttactcaagg ctttggctcg taatcggagc ctgacatacc tgagtatcaa ctgtacgtcc 2100 atttccctaa atatgttttc acttctgcat gacatcctgc acgagcccac atgccaaata 2160 agtcatctga gcttgatgaa atgtgatttg cgagccagcg aatgcgaaga aatcgcctct 2220 ctcctcatca gtggcgggag tctgagaaaa ctgaccttat ccagcaatcc gctgaggagc 2280 gacgggatga acatactgtg tgatgccttg cttcatccca actgcactct tatatcactg 2340 gtgttagtct tctgctgtct cactgaaaat tgctgcagcg cccttggaag agtgcttctg 2400 ttcagcccaa ctctaagaca actagacctg tgtgtgaatc gcttaaaaaa ttacggagtg 2460 ttgcatgtga cgtttccctt gctgtttcca acctgtcagt tagaggagct tcatctgtct 2520 ggctgtttct ttagcagcga tatctgtcaa tatattgcca tagttattgc tactaatgaa 2580 aaactgagga gcctggagat tgggagcaac aaaatagaag atgcaggaat gcagctgcta 2640 tgtggtggtt tgagacatcc caactgcatg ttggtgaata ttgggctaga agagtgcatg 2700 ttaaccagtg cctgctgtcg atctcttgcc tctgttctta ccaccaacaa aacactagaa 2760 agactcaact tgcttcaaaa tcacttgggc aatgatggag ttgcaaaact tcttgagagc 2820 ttgatcagcc cagattgtgt acttaaggta gttgggcttc cattaactgg cctgaacaca 2880 caaacccagc agttgctgat gactgtaaag gaaagaaaac ccagtttgat ctttctgtct 2940

gaaacttggt ctttaaagga aggcagagaa attggtgtga cacctgcttc tcagccaggt 3000 tcaataatac ctaattctaa tttggattac atgtttttca aatttcccag aatgtctgca 3060 gccatgagaa cgtcaaatac agcatctagg caaccccttt ga 3102 10 2928 DNA Homo sapiens 10 atgaggtggg gccaccattt gcccagggcc tcttggggct ctggttttag aagagcactc 60 cagcgaccag atgatcgtat ccccttcctg atccactgga gttggcccct tcaaggggag 120 cgtccctttg ggccccctag ggcctttata cgccaccacg gaagctcggt agatagcgct 180 cccccacccg ggaggcatgg acggctgttc cccagcgcct ctgcaactga agctatacag 240 cggcaccgcc ggaacctggc tgagtggttc agccggctgc ccagggagga gcgccagttt 300 ggcccaacct ttgccctaga cacggtccac gttgaccctg tgatccgcga gagtacccct 360 gatgagctac ttcgcccacc cgcggagctg gccctggagc atcagccacc ccaggccggg 420 ctccccccac tggccttgtc tcagctcttt aacccggatg cctgtgggcg ccgggtgcag 480 acagtggtgc tgtatgggac agtgggcaca ggcaagagca cgctggtgcg caagatggtt 540 ctggactggt gttatgggcg gctgccggcc ttcgagctgc tcatcccctt ctcctgtgag 600 gacctgtcat ccctgggccc tgccccagcc tccctgtgcc aacttgtggc ccagcgctac 660 acgcccctga aggaggttct gcccctgatg gctgctgctg ggtcccacct cctctttgtg 720 ctccatggct tagagcatct caacctcgac ttccggctgg caggcacggg actttgtagt 780 gacccggagg aaccgcagga accagctgct atcatcgtca acctgctgcg caaatacatg 840 ctgcctcagg ccagcattct ggtgaccact cggccctctg ccattggccg tatccccagc 900 aagtacgtgg gccgctatgg tgagatctgc ggtttctctg ataccaacct gcagaagctc 960 tacttccagc tccgcctcaa ccagccgtac tgcgggtatg ccgttggcgg ttcaggtgtc 1020 tctgccacac cagctcagcg tgaccacctg gtgcagatgc tctcccggaa cctggagggg 1080 caccaccaga tagccgctgc ctgcttcctg ccgtcctatt gctggctcgt ttgtgccacc 1140 ttgcacttcc tgcatgcccc cacgcctgct gggcagaccc ttacaagcat ctataccagc 1200 ttcctgcgcc tcaacttcag cggggaaacc ctggacagca ctgacccctc caatttgtcc 1260 ctgatggcct atgcagcccg aaccatgggc aagttggcct atgagggggt gtcctcccgc 1320 aagacctact tctctgaaga ggatgtctgt ggctgcctgg aggctggcat caggacggag 1380 gaggagtttc agctgctgca catcttccgt cgggatgccc tgaggttttt cctggcccca 1440 tgtgtggagc cagggcgtgc aggcaccttc gtgttcaccg tgcccgccat gcaggaatac 1500 ctggctgccc tctacattgt gctgggtttg cgcaagacga ccctgcaaaa ggtgggcaag 1560 gaagtggctg agctcgtggg ccgtgttggg gaggacgtca gcctggtact gggcatcatg 1620 gccaagctgc tgcctctgcg ggctctgcct ctgctcttca acctgatcaa ggtggttcca 1680 cgagtgtttg ggcgcatggt gggtaaaagc cgggaggcgg tggctcaggc catggtgctg 1740 gagatgtttc gagaggagga ctactacaac gatgatgttc tggaccagat gggcgccagt 1800 atcctgggcg tggagggccc ccggcgccac ccagatgagc cccctgagga tgaagtcttc 1860 gagctcttcc ccatgttcat gggggggctt ctctctgccc acaaccgagc tgtgctagct 1920 cagcttggct gccccatcaa gaacctggat gccctggaga atgcccaggc catcaagaag 1980 aagctgggca agctgggccg gcaggtgctg cccccatcag agctccttga ccacctcttc 2040 ttccactatg agttccagaa ccagcgcttc tccgctgagg tgctcagctc cctgcgtcag 2100 ctcaacctgg caggtgtgcg catgacacca gtcaagtgca cagtggtggc agctgtgctg 2160 ggcagcggaa ggcatgccct ggatgaggtg aacttggcct cctgccagct agatcctgct 2220 gggctgcgca cactcctgcc tgtcttcctg cgtgcccgga agctgggctt gcaactcaac 2280 agcctgggcc ctgaggcctg caaggacctc cgagacctgt tgctgcatga ccagtgccaa 2340 attaccacac tgcggctgtc caacaacccg ctgacggcgg caggtgttgc cgtgctaatg 2400 gaggggctgg caggaaacac ctcagtgacg cacctgtccc tgctgcacac gggccttggg 2460 gacgaaggcc tggagctgct ggctgcccag ctggaccgca accggcagct gcaggagctg 2520 aacgtggcgt acaacggtgc tggtgacaca gcggccctgg ccctggccag agctgcccgg 2580 gagcaccctt ccctggaact gctacacctc tacttcaatg agctgagctc agagggccgc 2640 caggtcttgc gagacttggg gggtgctgct gaaggtggtg cccgggtggt ggtgtcactg 2700 acagagggga cggcggtgtc agaatactgg tcagtgatcc tcagtgaagt ccagcggaac 2760 ctcaatagct gggatcgggc ccgggttcag cgacaccttg agctcctact gcgggatctg 2820 gaagatagcc ggggtgccac ccttaatcct tggcgcaagg cccagctgct gcgagtggag 2880 ggcgaggtca gggccctcct ggagcagctg ggaagctctg gaagctga 2928 11 6763 DNA Homo sapiens 11 ggaggagccg cgagcgctga gggtgagtgc cgggagctct gagggagtct gcactatgga 60 aacaacctgt caatccagct caaggcacac atagcccaga cacccatgag accctctccg 120 tggggaccct agagcaccta tcatgaacga ggagaccaag gctggctcct catggacccc 180 gttggcctcc agctcggcaa caagaacctg tggagctgtc ttgtgaggct gctcaccaaa 240 gacccagaat ggctgaacgc caagatgaag ttcttcctcc ccaacacgga cctggattcc 300 aggaacgaga ccttggaccc tgaacagaga gtcatcctgc aactcaacaa gctgcatgtc 360 cagggttcgg acacctggca gtctttcatt cattgcgtgt gcatgcagct ggaggtgcct 420 ctggacctgg aggtgcttct gctaagtact tttggctatg atgatgggtt caccagccag 480 ctgggagctg aggggaaaag ccaacctgaa tctcagctcc accatggcct gaagcgccca 540 catcagagct gtgggtcctc accccgccgg aagcagtgca agaagcagca gctagagttg 600 gccaagaagt acctgcagct cctgcggacc tctgcccagc agcgctacag gagccaaatc 660 cctgggtcag ggcagcccca cgccttccac caggtctatg tccctccaat cctgcgccgg 720 gccacagcat ccttagacac tccggagggg gccattatgg gggacgtcaa ggtggaagat 780 ggtgctgacg tgagcatctc ggacctcttc aacaccaggg ttaacaaggg cccgagggtg 840 accgtgcttt tggggaaggc tggcatgggc aagaccacgc tggcccaccg gctctgccag 900 aagtgggcag agggccatct gaactgtttc caggccctgt tcctttttga attccgccag 960 ctcaacttga tcacgaggtt cctgacaccg tccgagctcc tttttgatct gtacctgagc 1020 cctgaatcgg accacgacac tgtcttccag tacctggaga agaacgctga ccaagtcctg 1080 ctgatctttg atgggctaga tgaggccctc cagcctatgg gtcctgatgg cccaggccca 1140 gtcctcaccc ttttctccca tctctgcaat gggaccctcc tgcctggctg ccgggtgatg 1200 gctacctccc gtccagggaa gctgcctgcc tgcctgcctg cagaggcagc catggtccac 1260 atgttgggct ttgatgggcc acgggtggaa gaatatgtga atcacttctt cagcgcccag 1320 ccatcgcggg agggggccct ggtggagtta cagacaaatg gacgtctccg aagcctgtgt 1380 gcggtgcccg cactgtgcca agtcgcctgt ctctgcctcc accatctgct tcctgaccac 1440 gccccaggcc agtctgtggc cctcctgccc aacatgactc agctctatat gcagatggtg 1500 ctcgccctca gcccccctgg gcacttgccc acctcgtccc tactggacct gggggaggtg 1560 gccctgaggg gcctggagac agggaaggtt atcttctatg caaaagatat tgctccaccc 1620 ttgatagctt ttggggccac tcacagcctg ctgacttcct tctgcgtctg cacaggccct 1680 gggcaccagc agacaggcta tgctttcacc cacctcagcc tgcaggagtt tcttgctgcc 1740 ctgcacctga tggccagccc caaggtgaac aaagacacac ttacccagta tgttaccctc 1800 cattcccgct gggtacagcg gaccaaagct agactgggcc tctcagacca cctccccacc 1860 ttcctggcgg gcctggcatc ctgcacctgc cgccccttcc ttagccacct ggcgcagggc 1920 aatgaggact gtgtgggtgc caagcaggct gctgtagtgc aggtgttgaa gaagttggcc 1980 acccgcaagc tcacagggcc aaaggttgta gagctgtgtc actgtgtgga tgagacacag 2040 gagcctgagc tggccagtct caccgcacaa agcctcccct atcaactgcc cttccacaat 2100 ttcccactga cctgcaccga cctggccacc ctgaccaaca tcctagagca cagggaggcc 2160 cccatccacc tggattttga tggctgtccc ctggagcccc actgccctga ggctctggta 2220 ggctgtgggc agatagagaa tctcagcttt aagagcagga agtgtgggga tgcctttgca 2280 gaagccctct ccaggagctt gccgacaatg gggaggctgc agatgctggg gttagcagga 2340 agtaaaatca ctgcccgagg catcagccac ctggtgaaag ctttgcctct ctgtccacag 2400 ctgaaagaag tcagttttcg ggacaaccag ctcagtgacc aggtggtgct gaacattgtg 2460 gaggttctcc ctcacctacc acggctccgg aagcttgacc tgagcagcaa cagcatctgc 2520 gtgtcaaccc tactctgctt ggcaagggtg gcagtcacgt gtcctaccgt caggatgctt 2580 caggccaggg agcggaccat catcttcctt ctttccccgc ccacagagac aactgcagag 2640 ctacaaagag ctccagacct gcaggaaagt gacggccaga ggaaaggggc tcagagcaga 2700 agcttgacgc tcaggctgca gaagtgtcag ctccaggtcc acgatgcgga ggccctcata 2760 gccctgctcc aggaaggccc tcacctggag gaagtggacc tctcagggaa ccagctggaa 2820 gatgaaggct gtcggctgat ggcagaggct gcatcccagc tgcacatcgc caggaagctg 2880 gacctcagcg acaacgggct ttctgtggcc ggggtgcatt gtgtgctgag ggccgtgagt 2940 gcgtgctgga ccctggcaga gctgcacatc agcctgcagc acaaaactgt gatcttcatg 3000 tttgcccagg agccagagga gcagaagggg ccccaggaga gggctgcatt tcttgacagc 3060 ctcatgctcc agatgccctc tgagctgcct ctgagctccc gaaggatgag gctgacacat 3120 tgtggcctcc aagaaaagca cctagagcag ctctgcaagg ctctgggagg aagctgccac 3180 ctcggtcacc tccacctcga cttctcaggc aatgctctgg gggatgaagg tgcagcccgg 3240 ctggctcagc tgctcccagg gctgggagct ctgcagtcct tgaacctcag tgagaacggt 3300 ttgtccctgg atgccgtgtt gggcttggtt cggtgcttct ccactctgca gtggctcttc 3360 cgcttggaca tcagctttga aagccaacac atcctcctga gaggggacaa gacaagcagg 3420 gatatgtggg ccactggatc tttgccagac ttcccagctg cagccaagtt cttagggttc 3480 cgtcagcgct gcatccccag gagcctctgc ctcagtgagt gtcctctgga gcccccaagc 3540 ctcacccgcc tctgtgccac tctgaaggac tgcccgggac ccctggaact gcaattgtcc 3600 tgtgagttcc tgagtgacca gagcctggag actctactgg actgcttacc tcaactccct 3660 cagctgagcc tgctgcagct gagccagacg ggactgtccc cgaaaagccc cttcctgctg 3720 gccaacacct taagcctgtg tccacgggtt aaaaaggtgg atctcaggtc cctgcaccat 3780 gcaactttgc acttcagatc caacgaggag gaggaaggcg tgtgctgtgg caggttcaca 3840 ggctgcagcc tcagccagga gcacgtagag tcactctgct ggttgctgag caagtgtaaa 3900 gacctcagcc aggtggatct ctcagcaaac ctgctgggcg acagcggact cagatgcctt 3960 ctggaatgtc tgccgcaggt gcccatctcc ggtttgcttg atctgagtca caacagcatt 4020 tctcaggaaa gtgccctgta cctgctggag acactgccct cctgcccacg tgtccgggag 4080 gcctcagtga acctgggctc tgagcagagc ttccggattc acttctccag agaggaccag 4140 gctgggaaga cactcaggct aagtgagtgc agcttccggc cagagcacgt gtccaggctg 4200 gccaccggct tgagcaagtc cctgcagctg acggagctca cgctgaccca gtgctgcctg 4260 ggccagaagc agctggccat cctcctgagc ttggtggggc gacccgcagg gctgttcagc 4320 ctcagggtgc aggagccgtg ggcggacaga gccagggttc tctccctgtt agaagtctgc 4380 gcccaggcct caggcagtgt cactgaaatc agcatctccg agacccagca gcagctctgt 4440 gtccagctgg aatttcctcg ccaggaagag aatccagaag ctgtggcact caggttggct 4500 cactgtgacc ttggagccca ccacagcctt cttgtcgggc agctgatgga gacatgtgcc 4560 aggctgcagc agctcagctt gtctcaggtt aacctctgtg aggacgatga tgccagttcc 4620 ctgctgctgc agagcctcct gctgtccctc tctgagctga agacatttcg gctgacctcc 4680 agctgtgtga gcaccgaggg cctcgcccac ctggcatctg gtctgggcca ctgccaccac 4740 ttggaggagc tggacttgtc taacaatcaa tttgatgagg agggcaccaa ggcgctgatg 4800 agggcccttg aggggaaatg gatgctaaag aggctggacc tcagtcacct tctgctgaac 4860 agctccacct tggccttgct tactcacaga ctaagccaga tgacctgcct gcagagcctc 4920 agactgaaca ggaacagtat cggtgatgtc ggttgctgcc acctttctga ggctctcagg 4980 gctgccacca gcctagagga gctggacttg agccacaacc agattggaga cgctggtgtc 5040 cagcacttag ctaccatcct gcctgggctg ccagagctca ggaagataga cctctcaggg 5100 aatagcatca gctcagccgg gggagtgcag ttggcagagt ctctcgttct ttgcaggcgc 5160 ctggaggagt tgatgcttgg ctgcaatgcc ctgggggatc ccacagccct ggggctggct 5220 caggagctgc cccagcacct gagggtccta cacctaccat tcagccatct gggcccaggt 5280 ggggccctga gcctggccca ggccctggat ggatcccccc atttggaaga gatcagcttg 5340 gcggaaaaca acctggctgg aggggtcctg cgtttctgta tggagctccc gctgctcaga 5400 cagatagacc tggtttcctg taagattgac aaccagactg ccaagctcct cacctccagc 5460 ttcacgagct gccctgccct ggaagtaatc ttgctgtcct ggaatctcct cggggatgag 5520 gcagctgccg agctggccca ggtgctgccg aagatgggcc ggctgaagag agtggacctg 5580 gagaagaatc agatcacagc tttgggggcc tggctcctgg ctgaaggact ggcccagggg 5640 tctagcatcc aagtcatccg cctctggaat aaccccattc cctgcgacat ggcccagcac 5700 ctgaagagcc aggagcccag gctggacttt gccttctttg acaaccagcc ccaggcccct 5760 tggggtactt gatggccccc tcaagacctt tggaatccag ccaagtgatg cacccaaatg 5820 atccaccttt cgcccactgg gataaatgac tcaggaaaga agagcctcgg cagggcgctc 5880 tgcactccac ccaggaggaa ggatacgtgt gtcctgctgc agtcctcagg gagaactttt 5940 ttgggaacca ggagctgggt ctggacaaag gagtaccctg cattacgtgg gatatgtgtg 6000 atcaattggg gacatgcgac acacaatgag ggtgtcatga caatgcatga cacgtacggt 6060 tatatgtggc agtgtgaccc cttgacatgt ggcgttacat gaaagtcagt gtggcacgtg 6120 ttctgtggca tgggtgctgg catcccaagt ggcaggatac atgattgttg gtctatatat 6180 gacacatgac aaatgtccat gtcacaggac tcatggctgg ccagatgacc tcaggctggc 6240 ccaagatcta atttattaat ttttaaagca aatacatatt tatagattgt gtgtatggag 6300 cagctaagtc aggaaaagtc ttccgcccga gctgggaggg gagagtgtcc atgcactgac 6360 cagtccaggg gctcaagggc cagggctctg gaacaagcca gggactcagc cattaagtcc 6420 cctcctgcct caatcctcag cctacccatc tataaacttg atgactcctc ccttacttac 6480 atactagctt ccaaggacag gtggaggtag ggccagcctg gcgggagtgg agaagcccag 6540 tctgtcctat gtaagggaca aagccaggtc taatggtact gggtaggggg cactgccaag 6600 acaataagct aggctactgg gtccagctac tactttggtg ggattcaggt gagtctccat 6660 gcacttcaca tgttacccag tgttcttgtt acttccaagg agaaccaaga atggctctgt 6720 cacactcgaa gccaggtttg atcaataaac acaatggtat tcc 6763 12 1112 PRT Homo sapiens 12 Met Glu Met Asp Ala Pro Arg Pro Pro Ser Leu Ala Val Pro Gly Ala 1 5 10 15 Ala Ser Arg Pro Gly Arg Leu Leu Asp Gly Gly His Gly Arg Gln Gln 20 25 30 Val Gln Ala Leu Ser Ser Gln Leu Leu Glu Val Ile Pro Asp Ser Met 35 40 45 Arg Lys Gln Glu Val Arg Thr Gly Arg Glu Ala Gly Gln Gly His Gly 50 55 60 Thr Gly Ser Pro Ala Glu Gln Val Lys Ala Leu Met Asp Leu Leu Ala 65 70 75 80 Gly Lys Gly Ser Gln Gly Ser His Ala Pro Gln Ala Leu Asp Arg Thr 85 90 95 Pro Asp Ala Pro Leu Gly Pro Cys Ser Asn Asp Ser Arg Ile Gln Arg 100 105 110 His Arg Lys Ala Leu Leu Ser Lys Val Gly Gly Gly Pro Glu Leu Gly 115 120 125 Gly Pro Trp His Arg Leu Ala Ser Leu Leu Leu Val Glu Gly Leu Thr 130 135 140 Asp Leu Gln Leu Arg Glu His Asp Phe Thr Gln Val Glu Ala Thr Arg 145 150 155 160 Gly Gly Gly His Pro Ala Arg Thr Val Ala Leu Asp Arg Leu Phe Leu 165 170 175 Pro Leu Ser Arg Val Ser Val Pro Pro Arg Val Ser Ile Thr Ile Gly 180 185 190 Val Ala Gly Met Gly Lys Thr Thr Leu Val Arg His Phe Val Arg Leu 195 200 205 Trp Ala His Gly Gln Val Gly Lys Asp Phe Ser Leu Val Leu Pro Leu 210 215 220 Thr Phe Arg Asp Leu Asn Thr His Glu Lys Leu Cys Ala Asp Arg Leu 225 230 235 240 Ile Cys Ser Val Phe Pro His Val Gly Glu Pro Ser Leu Ala Val Ala 245 250 255 Val Pro Ala Arg Ala Leu Leu Ile Leu Asp Gly Leu Asp Glu Cys Arg 260 265 270 Thr Pro Leu Asp Phe Ser Asn Thr Val Ala Cys Thr Asp Pro Lys Lys 275 280 285 Glu Ile Pro Val Asp His Leu Ile Thr Asn Ile Ile Arg Gly Asn Leu 290 295 300 Phe Pro Glu Val Ser Ile Trp Ile Thr Ser Arg Pro Ser Ala Ser Gly 305 310 315 320 Gln Ile Pro Gly Gly Leu Val Asp Arg Met Thr Glu Ile Arg Gly Phe 325 330 335 Asn Glu Glu Glu Ile Lys Val Cys Leu Glu Gln Met Phe Pro Glu Asp 340 345 350 Gln Ala Leu Leu Gly Trp Met Leu Ser Gln Val Gln Ala Asp Arg Ala 355 360 365 Leu Tyr Leu Met Cys Thr Val Pro Ala Phe Cys Arg Leu Thr Gly Met 370 375 380 Ala Leu Gly His Leu Trp Arg Ser Arg Thr Gly Pro Gln Asp Ala Glu 385 390 395 400 Leu Trp Pro Pro Arg Thr Leu Cys Glu Leu Tyr Ser Trp Tyr Phe Arg 405 410 415 Met Ala Leu Ser Gly Glu Gly Gln Glu Lys Gly Lys Ala Ser Pro Arg 420 425 430 Ile Glu Gln Val Ala His Gly Gly Arg Lys Met Val Gly Thr Leu Gly 435 440 445 Arg Leu Ala Phe His Gly Leu Leu Lys Lys Lys Tyr Val Phe Tyr Glu 450 455 460 Gln Asp Met Lys Ala Phe Gly Val Asp Leu Ala Leu Leu Gln Gly Ala 465 470 475 480 Pro Cys Ser Cys Phe Leu Gln Arg Glu Glu Thr Leu Ala Ser Ser Val 485 490 495 Ala Tyr Cys Phe Thr His Leu Ser Leu Gln Glu Phe Val Ala Ala Ala 500 505 510 Tyr Tyr Tyr Gly Ala Ser Arg Arg Ala Ile Phe Asp Leu Phe Thr Glu 515 520 525 Ser Gly Val Ser Trp Pro Arg Leu Gly Phe Leu Thr His Phe Arg Ser 530 535 540 Ala Ala Gln Arg Ala Met Gln Ala Glu Asp Gly Arg Leu Asp Val Phe 545 550 555 560 Leu Arg Phe Leu Ser Gly Leu Leu Ser Pro Arg Val Asn Ala Leu Leu 565 570 575 Ala Gly Ser Leu Leu Ala Gln Gly Glu His Gln Ala Tyr Arg Thr Gln 580 585 590 Val Ala Glu Leu Leu Gln Gly Cys Leu Arg Pro Asp Ala Ala Val Cys 595 600 605 Ala Arg Ala Ile Asn Val Leu His Cys Leu His Glu Leu Gln His Thr 610 615 620 Glu Leu Ala Arg Ser Val Glu Glu Ala Met Glu Ser Gly Ala Leu Ala 625 630 635 640 Arg Leu Thr Gly Pro Ala His Arg Ala Ala Leu Ala Tyr Leu Leu Gln 645 650 655 Val Ser Asp Ala Cys Ala Gln Glu Ala Asn Leu Ser Leu Ser Leu Ser 660 665 670 Gln Gly Val Leu Gln Ser Leu Leu Pro Gln Leu Leu Tyr Cys Arg Lys 675 680 685 Leu Arg Leu Asp Thr Asn Gln Phe Gln Asp Pro Val Met Glu Leu Leu 690 695 700 Gly Ser Val Leu Ser Gly Lys Asp Cys Arg Ile Gln Lys Ile Ser Leu 705 710 715 720 Ala Glu Asn Gln Ile Ser Asn Lys Gly Ala Lys Ala Leu Ala Arg Ser 725 730 735 Leu Leu Val Asn Arg Ser Leu Thr Ser Leu Asp Leu Arg Gly Asn Ser 740 745 750 Ile Gly Pro Gln Gly Ala Lys Ala Leu Ala Asp Ala Leu Lys Ile Asn 755 760 765 Arg Thr Leu Thr Ser Leu Ser Leu Gln Gly Asn Thr Val Arg Asp Asp 770 775 780 Gly Ala Arg Ser Met Ala Glu Ala Leu Ala Ser Asn Arg Thr Leu Ser 785 790 795 800 Met Leu His Leu Gln Lys Asn Ser Ile Gly Pro Met Gly Ala Gln Arg 805 810 815 Met Ala Asp Ala Leu Lys Gln Asn Arg Ser Leu Lys Glu Leu Met Phe

820 825 830 Ser Ser Asn Ser Ile Gly Asp Gly Gly Ala Lys Ala Leu Ala Glu Ala 835 840 845 Leu Lys Val Asn Gln Gly Leu Glu Ser Leu Asp Leu Gln Ser Asn Ser 850 855 860 Ile Ser Asp Ala Gly Val Ala Ala Leu Met Gly Ala Leu Cys Thr Asn 865 870 875 880 Gln Thr Leu Leu Ser Leu Ser Leu Arg Glu Asn Ser Ile Ser Pro Glu 885 890 895 Gly Ala Gln Ala Ile Ala His Ala Leu Cys Ala Asn Ser Thr Leu Lys 900 905 910 Asn Leu Asp Leu Thr Ala Asn Leu Leu His Asp Gln Gly Ala Arg Ala 915 920 925 Ile Ala Val Ala Val Arg Glu Asn Arg Thr Leu Thr Ser Leu His Leu 930 935 940 Gln Trp Asn Phe Ile Gln Ala Gly Ala Ala Gln Ala Leu Gly Gln Ala 945 950 955 960 Leu Gln Leu Asn Arg Ser Leu Thr Ser Leu Asp Leu Gln Glu Asn Ala 965 970 975 Ile Gly Asp Asp Gly Ala Cys Ala Val Ala Arg Ala Leu Lys Val Asn 980 985 990 Thr Ala Leu Thr Ala Leu Tyr Leu Gln Val Ala Ser Ile Gly Ala Ser 995 1000 1005 Gly Ala Gln Val Leu Gly Glu Ala Leu Ala Val Asn Arg Thr Leu 1010 1015 1020 Glu Ile Leu Asp Leu Arg Gly Asn Ala Ile Gly Val Ala Gly Ala 1025 1030 1035 Lys Ala Leu Ala Asn Ala Leu Lys Val Asn Ser Ser Leu Arg Arg 1040 1045 1050 Leu Asn Leu Gln Glu Asn Ser Leu Gly Met Asp Gly Ala Ile Cys 1055 1060 1065 Ile Ala Thr Ala Leu Ser Gly Asn His Arg Leu Gln His Ile Asn 1070 1075 1080 Leu Gln Gly Asn His Ile Gly Asp Ser Gly Ala Arg Met Ile Ser 1085 1090 1095 Glu Ala Ile Lys Thr Asn Ala Pro Thr Cys Thr Val Glu Met 1100 1105 1110 13 1093 PRT Homo sapiens 13 Met Ala Asp Ser Ser Ser Ser Ser Phe Phe Pro Asp Phe Gly Leu Leu 1 5 10 15 Leu Tyr Leu Glu Glu Leu Asn Lys Glu Glu Leu Asn Thr Phe Lys Leu 20 25 30 Phe Leu Lys Glu Thr Met Glu Pro Glu His Gly Leu Thr Pro Trp Asn 35 40 45 Glu Val Lys Lys Ala Arg Arg Glu Asp Leu Ala Asn Leu Met Lys Lys 50 55 60 Tyr Tyr Pro Gly Glu Lys Ala Trp Ser Val Ser Leu Lys Ile Phe Gly 65 70 75 80 Lys Met Asn Leu Lys Asp Leu Cys Glu Arg Ala Lys Glu Glu Ile Asn 85 90 95 Trp Ser Ala Gln Thr Ile Gly Pro Asp Asp Ala Lys Ala Gly Glu Thr 100 105 110 Gln Glu Asp Gln Glu Ala Val Leu Gly Asp Gly Thr Glu Tyr Arg Asn 115 120 125 Arg Ile Lys Glu Lys Phe Cys Ile Thr Trp Asp Lys Lys Ser Leu Ala 130 135 140 Gly Lys Pro Glu Asp Phe His His Gly Ile Ala Glu Lys Asp Arg Lys 145 150 155 160 Leu Leu Glu His Leu Phe Asp Val Asp Val Lys Thr Gly Ala Gln Pro 165 170 175 Gln Ile Val Val Leu Gln Gly Ala Ala Gly Val Gly Lys Thr Thr Leu 180 185 190 Val Arg Lys Ala Met Leu Asp Trp Ala Glu Gly Ser Leu Tyr Gln Gln 195 200 205 Arg Phe Lys Tyr Val Phe Tyr Leu Asn Gly Arg Glu Ile Asn Gln Leu 210 215 220 Lys Glu Arg Ser Phe Ala Gln Leu Ile Ser Lys Asp Trp Pro Ser Thr 225 230 235 240 Glu Gly Pro Ile Glu Glu Ile Met Tyr Gln Pro Ser Ser Leu Leu Phe 245 250 255 Ile Ile Asp Ser Phe Asp Glu Leu Asn Phe Ala Phe Glu Glu Pro Glu 260 265 270 Phe Ala Leu Cys Glu Asp Trp Thr Gln Glu His Pro Val Ser Phe Leu 275 280 285 Met Ser Ser Leu Leu Arg Lys Val Met Leu Pro Glu Ala Ser Leu Leu 290 295 300 Val Thr Thr Arg Leu Thr Thr Ser Lys Arg Leu Lys Gln Leu Leu Lys 305 310 315 320 Asn His His Tyr Val Glu Leu Leu Gly Met Ser Glu Asp Ala Arg Glu 325 330 335 Glu Tyr Ile Tyr Gln Phe Phe Glu Asp Lys Arg Trp Ala Met Lys Val 340 345 350 Phe Ser Ser Leu Lys Ser Asn Glu Met Leu Phe Ser Met Cys Gln Val 355 360 365 Pro Leu Val Cys Trp Ala Ala Cys Thr Cys Leu Lys Gln Gln Met Glu 370 375 380 Lys Gly Gly Asp Val Thr Leu Thr Cys Gln Thr Thr Thr Ala Leu Phe 385 390 395 400 Thr Cys Tyr Ile Ser Ser Leu Phe Thr Pro Val Asp Gly Gly Ser Pro 405 410 415 Ser Leu Pro Asn Gln Ala Gln Leu Arg Arg Leu Cys Gln Val Ala Ala 420 425 430 Lys Gly Ile Trp Thr Met Thr Tyr Val Phe Tyr Arg Glu Asn Leu Arg 435 440 445 Arg Leu Gly Leu Thr Gln Ser Asp Val Ser Ser Phe Met Asp Ser Asn 450 455 460 Ile Ile Gln Lys Asp Ala Glu Tyr Glu Asn Cys Tyr Val Phe Thr His 465 470 475 480 Leu His Val Gln Glu Phe Phe Ala Ala Met Phe Tyr Met Leu Lys Gly 485 490 495 Ser Trp Glu Ala Gly Asn Pro Ser Cys Gln Pro Phe Glu Asp Leu Lys 500 505 510 Ser Leu Leu Gln Ser Thr Ser Tyr Lys Asp Pro His Leu Thr Gln Met 515 520 525 Lys Cys Phe Leu Phe Gly Leu Leu Asn Glu Asp Arg Val Lys Gln Leu 530 535 540 Glu Arg Thr Phe Asn Cys Lys Met Ser Leu Lys Ile Lys Ser Lys Leu 545 550 555 560 Leu Gln Cys Met Glu Val Leu Gly Asn Ser Asp Tyr Ser Pro Ser Gln 565 570 575 Leu Gly Phe Leu Glu Leu Phe His Cys Leu Tyr Glu Thr Gln Asp Lys 580 585 590 Ala Phe Ile Ser Gln Ala Met Arg Cys Phe Pro Lys Val Ala Ile Asn 595 600 605 Ile Cys Glu Lys Ile His Leu Leu Val Ser Ser Phe Cys Leu Lys His 610 615 620 Cys Arg Cys Leu Arg Thr Ile Arg Leu Ser Val Thr Val Val Phe Glu 625 630 635 640 Lys Lys Ile Leu Lys Thr Ser Leu Pro Thr Asn Thr Trp Asp Gly Asp 645 650 655 Arg Ile Thr His Cys Trp Gln Asp Leu Cys Ser Val Leu His Thr Asn 660 665 670 Glu His Leu Arg Glu Leu Asp Leu Tyr His Ser Asn Leu Asp Lys Ser 675 680 685 Ala Met Asn Ile Leu His His Glu Leu Arg His Pro Asn Cys Lys Leu 690 695 700 Gln Lys Leu Leu Leu Lys Phe Ile Thr Phe Pro Asp Gly Cys Gln Asp 705 710 715 720 Ile Ser Thr Ser Leu Ile His Asn Lys Asn Leu Met His Leu Asp Leu 725 730 735 Lys Gly Ser Asp Ile Gly Asp Asn Gly Val Lys Ser Leu Cys Glu Ala 740 745 750 Leu Lys His Pro Glu Cys Lys Leu Gln Thr Leu Arg Leu Glu Ser Cys 755 760 765 Asn Leu Thr Val Phe Cys Cys Leu Asn Ile Ser Asn Ala Leu Ile Arg 770 775 780 Ser Gln Ser Leu Ile Phe Leu Asn Leu Ser Thr Asn Asn Leu Leu Asp 785 790 795 800 Asp Gly Val Gln Leu Leu Cys Glu Ala Leu Arg His Pro Lys Cys Tyr 805 810 815 Leu Glu Arg Leu Ser Leu Glu Ser Cys Gly Leu Thr Glu Ala Gly Cys 820 825 830 Glu Tyr Leu Ser Leu Ala Leu Ile Ser Asn Lys Arg Leu Thr His Leu 835 840 845 Cys Leu Ala Asp Asn Val Leu Gly Asp Gly Gly Val Lys Leu Met Ser 850 855 860 Asp Ala Leu Gln His Ala Gln Cys Thr Leu Lys Ser Leu Val Leu Arg 865 870 875 880 Arg Cys His Phe Thr Ser Leu Ser Ser Glu Tyr Leu Ser Thr Ser Leu 885 890 895 Leu His Asn Lys Ser Leu Thr His Leu Asp Leu Gly Ser Asn Trp Leu 900 905 910 Gln Asp Asn Gly Val Lys Leu Leu Cys Asp Val Phe Arg His Pro Ser 915 920 925 Cys Asn Leu Gln Asp Leu Glu Leu Met Gly Cys Val Leu Thr Asn Ala 930 935 940 Cys Cys Leu Asp Leu Ala Ser Val Ile Leu Asn Asn Pro Asn Leu Arg 945 950 955 960 Ser Leu Asp Leu Gly Asn Asn Asp Leu Gln Asp Asp Gly Val Lys Ile 965 970 975 Leu Cys Asp Ala Leu Arg Tyr Pro Asn Cys Asn Ile Gln Arg Leu Gly 980 985 990 Leu Glu Tyr Cys Gly Leu Thr Ser Leu Cys Cys Gln Asp Leu Ser Ser 995 1000 1005 Ala Leu Ile Cys Asn Lys Arg Leu Ile Lys Met Asn Leu Thr Gln 1010 1015 1020 Asn Thr Leu Gly Tyr Glu Gly Ile Val Lys Leu Tyr Lys Val Leu 1025 1030 1035 Lys Ser Pro Lys Cys Lys Leu Gln Val Leu Gly Leu Cys Lys Glu 1040 1045 1050 Ala Phe Asp Glu Glu Ala Gln Lys Leu Leu Glu Ala Val Gly Val 1055 1060 1065 Ser Asn Pro His Leu Ile Ile Lys Pro Asp Cys Asn Tyr His Asn 1070 1075 1080 Glu Glu Asp Val Ser Trp Trp Trp Cys Phe 1085 1090 14 991 PRT Homo sapiens 14 Met Ala Glu Ser Phe Phe Ser Asp Phe Gly Leu Leu Trp Tyr Leu Lys 1 5 10 15 Glu Leu Arg Lys Glu Glu Phe Trp Lys Phe Lys Glu Leu Leu Lys Gln 20 25 30 Pro Leu Glu Lys Phe Glu Leu Lys Pro Ile Pro Trp Ala Glu Leu Lys 35 40 45 Lys Ala Ser Lys Glu Asp Val Ala Lys Leu Leu Asp Lys His Tyr Pro 50 55 60 Gly Lys Gln Ala Trp Glu Val Thr Leu Asn Leu Phe Leu Gln Ile Asn 65 70 75 80 Arg Lys Asp Leu Trp Thr Lys Ala Gln Glu Glu Met Arg Asn Lys Leu 85 90 95 Asn Pro Tyr Arg Lys His Met Lys Glu Thr Phe Gln Leu Ile Trp Glu 100 105 110 Lys Glu Thr Cys Leu His Val Pro Glu His Phe Tyr Lys Glu Thr Met 115 120 125 Lys Asn Glu Tyr Lys Glu Leu Asn Asp Ala Tyr Thr Ala Ala Ala Arg 130 135 140 Arg His Thr Val Val Leu Glu Gly Pro Asp Gly Ile Gly Lys Thr Thr 145 150 155 160 Leu Leu Arg Lys Val Met Leu Asp Trp Ala Glu Gly Asn Leu Trp Lys 165 170 175 Asp Arg Phe Thr Phe Val Phe Phe Leu Asn Val Cys Glu Met Asn Gly 180 185 190 Ile Ala Glu Thr Ser Leu Leu Glu Leu Leu Ser Arg Asp Trp Pro Glu 195 200 205 Ser Ser Glu Lys Ile Glu Asp Ile Phe Ser Gln Pro Glu Arg Ile Leu 210 215 220 Phe Ile Met Asp Gly Phe Glu Gln Leu Lys Phe Asn Leu Gln Leu Lys 225 230 235 240 Ala Asp Leu Ser Asp Asp Trp Arg Gln Arg Gln Pro Met Pro Ile Ile 245 250 255 Leu Ser Ser Leu Leu Gln Lys Lys Met Leu Pro Glu Ser Ser Leu Leu 260 265 270 Ile Ala Leu Gly Lys Leu Ala Met Gln Lys His Tyr Phe Met Leu Arg 275 280 285 His Pro Lys Leu Ile Lys Leu Leu Gly Phe Ser Glu Ser Glu Lys Lys 290 295 300 Ser Tyr Phe Ser Tyr Phe Phe Gly Glu Lys Ser Lys Ala Leu Lys Val 305 310 315 320 Phe Asn Phe Val Arg Asp Asn Gly Pro Leu Phe Ile Leu Cys His Asn 325 330 335 Pro Phe Thr Cys Trp Leu Val Cys Thr Cys Val Lys Gln Arg Leu Glu 340 345 350 Arg Gly Glu Asp Leu Glu Ile Asn Ser Gln Asn Thr Thr Tyr Leu Tyr 355 360 365 Ala Ser Phe Leu Thr Thr Val Phe Lys Ala Gly Ser Gln Ser Phe Pro 370 375 380 Pro Lys Val Asn Arg Ala Arg Leu Lys Ser Leu Cys Ala Leu Ala Ala 385 390 395 400 Glu Gly Ile Trp Thr Tyr Thr Phe Val Phe Ser His Gly Asp Leu Arg 405 410 415 Arg Asn Gly Leu Ser Glu Ser Glu Gly Val Met Trp Val Gly Met Arg 420 425 430 Leu Leu Gln Arg Arg Gly Asp Cys Phe Ala Phe Met His Leu Cys Ile 435 440 445 Gln Glu Phe Cys Ala Ala Met Phe Tyr Leu Leu Lys Arg Pro Lys Asp 450 455 460 Asp Pro Asn Pro Ala Ile Gly Ser Ile Thr Gln Leu Val Arg Ala Ser 465 470 475 480 Val Val Gln Pro Gln Thr Leu Leu Thr Gln Val Gly Ile Phe Met Phe 485 490 495 Gly Ile Ser Thr Glu Glu Ile Val Ser Met Leu Glu Thr Ser Phe Gly 500 505 510 Phe Pro Leu Ser Lys Asp Leu Lys Gln Glu Ile Thr Gln Cys Leu Glu 515 520 525 Ser Leu Ser Gln Cys Glu Ala Asp Arg Glu Ala Ile Ala Phe Gln Glu 530 535 540 Leu Phe Ile Gly Leu Phe Glu Thr Gln Glu Lys Glu Phe Val Thr Lys 545 550 555 560 Val Met Asn Phe Phe Glu Glu Val Phe Ile Tyr Ile Gly Asn Ile Glu 565 570 575 His Leu Val Ile Ala Ser Phe Cys Leu Lys His Cys Gln His Leu Thr 580 585 590 Thr Leu Arg Met Cys Val Glu Asn Ile Phe Pro Asp Asp Ser Gly Cys 595 600 605 Ile Ser Asp Tyr Asn Glu Lys Leu Val Tyr Trp Arg Glu Leu Cys Ser 610 615 620 Met Phe Ile Thr Asn Lys Asn Phe Gln Ile Leu Asp Met Glu Asn Thr 625 630 635 640 Ser Leu Asp Asp Pro Ser Leu Ala Ile Leu Cys Lys Ala Leu Ala Gln 645 650 655 Pro Val Cys Lys Leu Arg Lys Leu Ile Phe Thr Ser Val Tyr Phe Gly 660 665 670 His Asp Ser Glu Leu Phe Lys Ala Val Leu His Asn Pro His Leu Lys 675 680 685 Leu Leu Ser Leu Tyr Gly Thr Ser Leu Ser Gln Ser Asp Ile Arg His 690 695 700 Leu Cys Glu Thr Leu Lys His Pro Met Cys Lys Ile Glu Glu Leu Ile 705 710 715 720 Leu Gly Lys Cys Asp Ile Ser Ser Glu Val Cys Glu Asp Ile Ala Ser 725 730 735 Val Leu Ala Cys Asn Ser Lys Leu Lys His Leu Ser Leu Val Glu Asn 740 745 750 Pro Leu Arg Asp Glu Gly Met Thr Leu Leu Cys Glu Ala Leu Lys His 755 760 765 Ser His Cys Ala Leu Glu Arg Leu Met Leu Met Tyr Cys Cys Leu Thr 770 775 780 Ser Val Ser Cys Asp Ser Ile Ser Glu Val Leu Leu Cys Ser Lys Ser 785 790 795 800 Leu Ser Leu Leu Asp Leu Gly Ser Asn Ala Leu Glu Asp Asn Gly Val 805 810 815 Ala Ser Leu Cys Ala Ala Leu Lys His Pro Gly Cys Ser Ile Arg Glu 820 825 830 Leu Trp Leu Met Gly Cys Phe Leu Thr Ser Asp Ser Cys Lys Asp Ile 835 840 845 Ala Ala Val Leu Ile Cys Asn Gly Lys Leu Lys Thr Leu Lys Leu Gly 850 855 860 His Asn Glu Ile Gly Asp Thr Gly Val Arg Gln Leu Cys Ala Ala Leu 865 870 875 880 Gln His Pro His Cys Lys Leu Glu Cys Leu Gly Leu Gln Thr Cys Pro 885 890 895 Ile Thr Arg Ala Cys Cys Asp Asp Ile Ala Ala Ala Leu Ile Ala Cys 900 905 910 Lys Thr Leu Arg Ser Leu Asn Leu Asp Trp Ile Ala Leu Asp Ala Asp 915 920 925 Ala Val Val Val Leu Cys Glu Ala Leu Ser His Pro Asp Cys Ala Leu 930 935 940 Gln Met Leu Gly Leu His Lys Ser Gly Phe Asp Glu Glu Thr Gln Lys 945 950 955 960 Ile Leu Met Ser Val Glu Glu Lys Ile Pro His Leu Thr Ile Ser His 965 970 975 Gly Pro Trp Ile Asp Glu Glu Tyr Lys Ile Arg Gly Val Leu Leu 980 985 990 15 655 PRT Homo sapiens 15 Met Ala Met Ala Lys Ala Arg Lys Pro Arg Glu Ala Leu Leu Trp Ala 1 5 10 15 Leu Ser Asp Leu Glu Glu Asn Asp Phe Lys Lys Leu Lys Phe Tyr Leu 20 25 30 Arg Asp Met Thr Leu Ser Glu Gly Gln Pro Pro Leu Ala Arg Gly Glu 35 40 45 Leu Glu Gly Leu Ile Pro Val Asp Leu Ala Glu Leu Leu Ile Ser Lys 50 55 60 Tyr Gly Glu Lys

Glu Ala Val Lys Val Val Leu Lys Gly Leu Lys Val 65 70 75 80 Met Asn Leu Leu Glu Leu Val Asp Gln Leu Ser His Ile Cys Leu His 85 90 95 Asp Tyr Arg Glu Val Tyr Arg Glu His Val Arg Cys Leu Glu Glu Trp 100 105 110 Gln Glu Ala Gly Val Asn Gly Arg Tyr Asn Gln Val Leu Leu Val Ala 115 120 125 Lys Pro Ser Ser Glu Ser Pro Glu Ser Leu Ala Cys Pro Phe Pro Glu 130 135 140 Gln Glu Leu Glu Ser Val Thr Val Glu Ala Leu Phe Asp Ser Gly Glu 145 150 155 160 Lys Pro Ser Leu Ala Pro Ser Leu Val Val Leu Gln Gly Ser Ala Gly 165 170 175 Thr Gly Lys Thr Thr Leu Ala Arg Lys Met Val Leu Asp Trp Ala Thr 180 185 190 Gly Thr Leu Tyr Pro Gly Arg Phe Asp Tyr Val Phe Tyr Val Ser Cys 195 200 205 Lys Glu Val Val Leu Leu Leu Glu Ser Lys Leu Glu Gln Leu Leu Phe 210 215 220 Trp Cys Cys Gly Asp Asn Gln Ala Pro Val Thr Glu Ile Leu Arg Gln 225 230 235 240 Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly Phe Asp Glu Leu Gln Arg 245 250 255 Pro Phe Glu Glu Lys Leu Lys Lys Arg Gly Leu Ser Pro Lys Glu Ser 260 265 270 Leu Leu His Leu Leu Ile Arg Arg His Thr Leu Pro Thr Cys Ser Leu 275 280 285 Leu Ile Thr Thr Arg Pro Leu Ala Leu Arg Asn Leu Glu Pro Leu Leu 290 295 300 Lys Gln Ala Arg His Val His Ile Leu Gly Phe Ser Glu Glu Glu Arg 305 310 315 320 Ala Arg Tyr Phe Ser Ser Tyr Phe Thr Asp Glu Lys Gln Ala Asp Arg 325 330 335 Ala Phe Asp Ile Val Gln Lys Asn Asp Ile Leu Tyr Lys Ala Cys Gln 340 345 350 Val Pro Gly Ile Cys Trp Val Val Cys Ser Trp Leu Gln Gly Gln Met 355 360 365 Glu Arg Gly Lys Val Val Leu Glu Thr Pro Arg Asn Ser Thr Asp Ile 370 375 380 Phe Met Ala Tyr Val Ser Thr Phe Leu Pro Pro Asp Asp Asp Gly Gly 385 390 395 400 Cys Ser Glu Leu Ser Arg His Arg Val Leu Arg Ser Leu Cys Ser Leu 405 410 415 Ala Ala Glu Gly Ile Gln His Gln Arg Phe Leu Phe Glu Glu Ala Glu 420 425 430 Leu Arg Lys His Asn Leu Asp Gly Pro Arg Leu Ala Ala Phe Leu Ser 435 440 445 Ser Asn Asp Tyr Gln Leu Gly Leu Ala Ile Lys Lys Phe Tyr Ser Phe 450 455 460 Arg His Ile Ser Phe Gln Asp Phe Phe His Ala Met Ser Tyr Leu Val 465 470 475 480 Lys Glu Asp Gln Ser Arg Leu Gly Lys Glu Ser Arg Arg Glu Val Gln 485 490 495 Arg Leu Leu Glu Val Lys Glu Gln Glu Gly Asn Asp Glu Met Thr Leu 500 505 510 Thr Met Gln Phe Leu Leu Asp Ile Ser Lys Lys Asp Ser Phe Ser Asn 515 520 525 Leu Glu Leu Lys Phe Cys Phe Arg Ile Ser Pro Cys Leu Ala Gln Asp 530 535 540 Leu Lys His Phe Lys Glu Gln Met Glu Ser Met Lys His Asn Arg Thr 545 550 555 560 Trp Asp Leu Glu Phe Ser Leu Tyr Glu Ala Lys Ile Lys Asn Leu Val 565 570 575 Lys Gly Ile Gln Met Asn Asn Val Ser Phe Lys Ile Lys His Ser Asn 580 585 590 Glu Lys Lys Ser Gln Ser Gln Asn Leu Phe Ser Val Lys Ser Ser Leu 595 600 605 Ser His Gly Pro Lys Glu Glu Gln Lys Cys Pro Ser Val His Gly Gln 610 615 620 Lys Glu Gly Lys Asp Asn Ile Ala Gly Thr Gln Lys Glu Ala Ser Thr 625 630 635 640 Gly Lys Gly Arg Gly Thr Glu Glu Thr Pro Lys Asn Thr Tyr Ile 645 650 655 16 975 PRT Homo sapiens 16 Met Arg Trp Gly His His Leu Pro Arg Ala Ser Trp Gly Ser Gly Phe 1 5 10 15 Arg Arg Ala Leu Gln Arg Pro Asp Asp Arg Ile Pro Phe Leu Ile His 20 25 30 Trp Ser Trp Pro Leu Gln Gly Glu Arg Pro Phe Gly Pro Pro Arg Ala 35 40 45 Phe Ile Arg His His Gly Ser Ser Val Asp Ser Ala Pro Pro Ser Gly 50 55 60 Arg His Gly Arg Leu Phe Pro Ser Ala Ser Ala Thr Glu Ala Ile Gln 65 70 75 80 Arg His Arg Arg Asn Leu Ala Glu Trp Phe Ser Arg Leu Pro Arg Glu 85 90 95 Glu Arg Gln Phe Gly Pro Thr Phe Ala Leu Asp Thr Val His Val Asp 100 105 110 Pro Val Ile Arg Glu Ser Thr Pro Asp Glu Leu Leu Arg Pro Pro Ala 115 120 125 Glu Leu Ala Leu Glu His Gln Pro Pro Gln Ala Gly Leu Pro Pro Leu 130 135 140 Ala Leu Ser Gln Leu Phe Asn Pro Asp Ala Cys Gly Arg Arg Val Gln 145 150 155 160 Thr Val Val Leu Tyr Gly Thr Val Gly Thr Gly Lys Ser Thr Leu Val 165 170 175 Arg Lys Met Val Leu Asp Trp Cys Tyr Gly Arg Leu Pro Ala Phe Glu 180 185 190 Leu Leu Ile Pro Phe Ser Cys Glu Asp Leu Ser Ser Leu Gly Pro Ala 195 200 205 Pro Ala Ser Leu Cys Gln Leu Val Ala Gln Arg Tyr Thr Pro Leu Lys 210 215 220 Glu Val Leu Pro Leu Met Ala Ala Ala Gly Ser His Leu Leu Phe Val 225 230 235 240 Leu His Gly Leu Glu His Leu Asn Leu Asp Phe Arg Leu Ala Gly Thr 245 250 255 Gly Leu Cys Ser Asp Pro Glu Glu Pro Gln Glu Pro Ala Ala Ile Ile 260 265 270 Val Asn Leu Leu Arg Lys Tyr Met Leu Pro Gln Ala Ser Ile Leu Val 275 280 285 Thr Thr Arg Pro Ser Ala Ile Gly Arg Ile Pro Ser Lys Tyr Val Gly 290 295 300 Arg Tyr Gly Glu Ile Cys Gly Phe Ser Asp Thr Asn Leu Gln Lys Leu 305 310 315 320 Tyr Phe Gln Leu Arg Leu Asn Gln Pro Tyr Cys Gly Tyr Ala Val Gly 325 330 335 Gly Ser Gly Val Ser Ala Thr Pro Ala Gln Arg Asp His Leu Val Gln 340 345 350 Met Leu Ser Arg Asn Leu Glu Gly His His Gln Ile Ala Ala Ala Cys 355 360 365 Phe Leu Pro Ser Tyr Cys Trp Leu Val Cys Ala Thr Leu His Phe Leu 370 375 380 His Ala Pro Thr Pro Ala Gly Gln Thr Leu Thr Ser Ile Tyr Thr Ser 385 390 395 400 Phe Leu Arg Leu Asn Phe Ser Gly Glu Thr Leu Asp Ser Thr Asp Pro 405 410 415 Ser Asn Leu Ser Leu Met Ala Tyr Ala Ala Arg Thr Met Gly Lys Leu 420 425 430 Ala Tyr Glu Gly Val Ser Ser Arg Lys Thr Tyr Phe Ser Glu Glu Asp 435 440 445 Val Cys Gly Cys Leu Glu Ala Gly Ile Arg Thr Glu Glu Glu Phe Gln 450 455 460 Leu Leu His Ile Phe Arg Arg Asp Ala Leu Arg Phe Phe Leu Ala Pro 465 470 475 480 Cys Val Glu Pro Gly Arg Ala Gly Thr Phe Val Phe Thr Val Pro Ala 485 490 495 Met Gln Glu Tyr Leu Ala Ala Leu Tyr Ile Val Leu Gly Leu Arg Lys 500 505 510 Thr Thr Leu Gln Lys Val Gly Lys Glu Val Ala Glu Leu Val Gly Arg 515 520 525 Val Gly Glu Asp Val Ser Leu Val Leu Gly Ile Met Ala Lys Leu Leu 530 535 540 Pro Leu Arg Ala Leu Pro Leu Leu Phe Asn Leu Ile Lys Val Val Pro 545 550 555 560 Arg Val Phe Gly Arg Met Val Gly Lys Ser Arg Glu Ala Val Ala Gln 565 570 575 Ala Met Val Leu Glu Met Phe Arg Glu Glu Asp Tyr Tyr Asn Asp Asp 580 585 590 Val Leu Asp Gln Met Gly Ala Ser Ile Leu Gly Val Glu Gly Pro Arg 595 600 605 Arg His Pro Asp Glu Pro Pro Glu Asp Glu Val Phe Glu Leu Phe Pro 610 615 620 Met Phe Met Gly Gly Leu Leu Ser Ala His Asn Arg Ala Val Leu Ala 625 630 635 640 Gln Leu Gly Cys Pro Ile Lys Asn Leu Asp Ala Leu Glu Asn Ala Gln 645 650 655 Ala Ile Lys Lys Lys Leu Gly Lys Leu Gly Arg Gln Val Leu Pro Pro 660 665 670 Ser Glu Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn Gln 675 680 685 Arg Phe Ser Ala Glu Val Leu Ser Ser Leu Arg Gln Leu Asn Leu Ala 690 695 700 Gly Val Arg Met Thr Pro Val Lys Cys Thr Val Val Ala Ala Val Leu 705 710 715 720 Gly Ser Gly Arg His Ala Leu Asp Glu Val Asn Leu Ala Ser Cys Gln 725 730 735 Leu Asp Pro Ala Gly Leu Arg Thr Leu Leu Pro Val Phe Leu Arg Ala 740 745 750 Arg Lys Leu Gly Leu Gln Leu Asn Ser Leu Gly Pro Glu Ala Cys Lys 755 760 765 Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr Thr Leu 770 775 780 Arg Leu Ser Asn Asn Pro Leu Thr Glu Ala Gly Val Ala Val Leu Met 785 790 795 800 Glu Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu Leu His 805 810 815 Thr Gly Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln Leu Asp 820 825 830 Arg Asn Arg Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly Ala Gly 835 840 845 Asp Thr Ala Ala Leu Ala Leu Ala Arg Ala Ala Arg Glu His Pro Ser 850 855 860 Leu Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu Gly Arg 865 870 875 880 Gln Val Leu Arg Asp Leu Gly Gly Ala Ala Glu Gly Gly Ala Arg Val 885 890 895 Val Val Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp Ser Val 900 905 910 Ile Leu Ser Glu Val Gln Arg Asn Leu Asn Ser Trp Asp Arg Ala Arg 915 920 925 Val Gln Arg His Leu Glu Leu Leu Leu Arg Asp Leu Glu Asp Ser Arg 930 935 940 Gly Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg Val Glu 945 950 955 960 Gly Glu Val Arg Ala Leu Leu Glu Gln Leu Gly Ser Ser Gly Ser 965 970 975 17 1009 PRT Homo sapiens 17 Met Thr Ser Pro Gln Leu Glu Trp Thr Leu Gln Thr Leu Leu Glu Gln 1 5 10 15 Leu Asn Glu Asp Glu Leu Lys Ser Phe Lys Ser Leu Leu Trp Ala Phe 20 25 30 Pro Leu Glu Asp Val Leu Gln Lys Thr Pro Trp Ser Glu Val Glu Glu 35 40 45 Ala Asp Gly Glu Lys Leu Ala Glu Ile Leu Val Asn Thr Ser Ser Glu 50 55 60 Asn Trp Ile Arg Asn Ala Thr Val Asn Ile Leu Glu Glu Met Asn Leu 65 70 75 80 Thr Glu Leu Cys Lys Met Ala Lys Ala Glu Met Met Glu Asp Gly Gln 85 90 95 Val Gln Glu Ile Asp Asn Pro Glu Leu Gly Asp Ala Glu Glu Asp Ser 100 105 110 Glu Leu Ala Lys Pro Gly Glu Lys Glu Gly Trp Arg Asn Ser Met Glu 115 120 125 Lys Gln Ser Leu Val Trp Lys Asn Thr Phe Trp Gln Gly Asp Ile Asp 130 135 140 Asn Phe His Asp Asp Val Thr Leu Arg Asn Gln Arg Phe Ile Pro Phe 145 150 155 160 Leu Asn Pro Arg Thr Pro Arg Lys Leu Thr Pro Tyr Thr Val Val Leu 165 170 175 His Gly Pro Ala Gly Val Gly Lys Thr Thr Leu Ala Lys Lys Cys Met 180 185 190 Leu Asp Trp Thr Asp Cys Asn Leu Ser Pro Thr Leu Arg Tyr Ala Phe 195 200 205 Tyr Leu Ser Cys Lys Glu Leu Ser Arg Met Gly Pro Cys Ser Phe Ala 210 215 220 Glu Leu Ile Ser Lys Asp Trp Pro Glu Leu Gln Asp Asp Ile Pro Ser 225 230 235 240 Ile Leu Ala Gln Ala Gln Arg Ile Leu Phe Val Val Asp Gly Leu Asp 245 250 255 Glu Leu Lys Val Pro Pro Gly Ala Leu Ile Gln Asp Ile Cys Gly Asp 260 265 270 Trp Glu Lys Lys Lys Pro Val Pro Val Leu Leu Gly Ser Leu Leu Lys 275 280 285 Arg Lys Met Leu Pro Arg Ala Ala Leu Leu Val Thr Thr Arg Pro Arg 290 295 300 Ala Leu Arg Asp Leu Gln Leu Leu Ala Gln Gln Pro Ile Tyr Val Arg 305 310 315 320 Val Glu Gly Phe Leu Glu Glu Asp Arg Arg Ala Tyr Phe Leu Arg His 325 330 335 Phe Gly Asp Glu Asp Gln Ala Met Arg Ala Phe Glu Leu Met Arg Ser 340 345 350 Asn Ala Ala Leu Phe Gln Leu Gly Ser Ala Pro Ala Val Cys Trp Ile 355 360 365 Val Cys Thr Thr Leu Lys Leu Gln Met Glu Lys Gly Glu Asp Pro Val 370 375 380 Pro Thr Cys Leu Thr Arg Thr Gly Leu Phe Leu Arg Phe Leu Cys Ser 385 390 395 400 Arg Phe Pro Gln Gly Ala Gln Leu Arg Gly Ala Leu Arg Thr Leu Ser 405 410 415 Leu Leu Ala Ala Gln Gly Leu Trp Ala Gln Met Ser Val Phe His Arg 420 425 430 Glu Asp Leu Glu Arg Leu Gly Val Gln Glu Ser Asp Leu Arg Leu Phe 435 440 445 Leu Asp Gly Asp Ile Leu Arg Gln Asp Arg Val Ser Lys Gly Cys Tyr 450 455 460 Ser Phe Ile His Leu Ser Phe Gln Gln Phe Leu Thr Ala Leu Phe Tyr 465 470 475 480 Ala Leu Glu Lys Glu Glu Gly Glu Asp Arg Asp Gly His Ala Trp Asp 485 490 495 Ile Gly Asp Val Gln Lys Leu Leu Ser Gly Glu Glu Arg Leu Lys Asn 500 505 510 Pro Asp Leu Ile Gln Val Gly His Phe Leu Phe Gly Leu Ala Asn Glu 515 520 525 Lys Arg Ala Lys Glu Leu Glu Ala Thr Phe Gly Cys Arg Met Ser Pro 530 535 540 Asp Ile Lys Gln Glu Leu Leu Gln Cys Lys Ala His Leu His Ala Asn 545 550 555 560 Lys Pro Leu Ser Val Thr Asp Leu Lys Glu Val Leu Gly Cys Leu Tyr 565 570 575 Glu Ser Gln Glu Glu Glu Leu Ala Lys Val Val Val Ala Pro Phe Lys 580 585 590 Glu Ile Ser Ile His Leu Thr Asn Thr Ser Glu Val Met His Cys Ser 595 600 605 Phe Ser Leu Lys His Cys Gln Asp Leu Gln Lys Leu Ser Leu Gln Val 610 615 620 Ala Lys Gly Val Phe Leu Glu Asn Tyr Met Asp Phe Glu Leu Asp Ile 625 630 635 640 Glu Phe Glu Ser Ser Asn Ser Asn Leu Lys Phe Leu Glu Val Lys Gln 645 650 655 Ser Phe Leu Ser Asp Ser Ser Val Arg Ile Leu Cys Asp His Val Thr 660 665 670 Arg Ser Thr Cys His Leu Gln Lys Val Glu Ile Lys Asn Val Thr Pro 675 680 685 Asp Thr Ala Tyr Arg Asp Phe Cys Leu Ala Phe Ile Gly Lys Lys Thr 690 695 700 Leu Thr His Leu Thr Leu Ala Gly His Ile Glu Trp Glu Arg Thr Met 705 710 715 720 Met Leu Met Leu Cys Asp Leu Leu Arg Asn His Lys Cys Asn Leu Gln 725 730 735 Tyr Leu Arg Leu Gly Gly His Cys Ala Thr Pro Glu Gln Trp Ala Glu 740 745 750 Phe Phe Tyr Val Leu Lys Ala Asn Gln Ser Leu Lys His Leu Arg Leu 755 760 765 Ser Ala Asn Val Leu Leu Asp Glu Gly Ala Met Leu Leu Tyr Lys Thr 770 775 780 Met Thr Arg Pro Lys His Phe Leu Gln Met Leu Ser Leu Glu Asn Cys 785 790 795 800 Arg Leu Thr Glu Ala Ser Cys Lys Asp Leu Ala Ala Val Leu Val Val 805 810 815 Ser Lys Lys Leu Thr His Leu Cys Leu Ala Lys Asn Pro Ile Gly Asp 820 825 830 Thr Gly Val Lys Phe Leu Cys Glu Gly Leu Ser Tyr Pro Asp Cys Lys 835 840 845 Leu Gln Thr Leu Val Leu Gln Gln Cys Ser Ile Thr Lys Leu Gly Cys 850 855 860 Arg Tyr Leu Ser Glu Ala Leu Gln Glu Ala Cys Ser Leu Thr Asn Leu 865 870 875

880 Asp Leu Ser Ile Asn Gln Ile Ala Arg Gly Leu Trp Ile Leu Cys Gln 885 890 895 Ala Leu Glu Asn Pro Asn Cys Asn Leu Lys His Leu Arg Leu Trp Ser 900 905 910 Cys Ser Leu Met Pro Phe Tyr Cys Gln His Leu Gly Ser Ala Leu Leu 915 920 925 Ser Asn Gln Lys Leu Glu Thr Leu Asp Leu Gly Gln Asn His Leu Trp 930 935 940 Lys Ser Gly Ile Ile Lys Leu Phe Gly Val Leu Arg Gln Arg Thr Gly 945 950 955 960 Ser Leu Lys Ile Leu Arg Leu Lys Thr Tyr Glu Thr Asn Leu Glu Ile 965 970 975 Lys Lys Leu Leu Glu Glu Val Lys Glu Lys Asn Pro Lys Leu Thr Ile 980 985 990 Asp Cys Asn Ala Ser Gly Ala Thr Ala Pro Pro Cys Cys Asp Phe Phe 995 1000 1005 Cys 18 1036 PRT Homo sapiens 18 Met Asn Phe Ser Val Ile Thr Cys Pro Asn Gly Gly Thr Asn Gln Gly 1 5 10 15 Leu Leu Pro Tyr Leu Met Ala Leu Asp Gln Tyr Gln Leu Glu Glu Phe 20 25 30 Lys Leu Cys Leu Glu Pro Gln Gln Leu Met Asp Phe Trp Ser Ala Pro 35 40 45 Gln Gly His Phe Pro Arg Ile Pro Trp Ala Asn Leu Arg Ala Ala Asp 50 55 60 Pro Leu Asn Leu Ser Phe Leu Leu Asp Glu His Phe Pro Lys Gly Gln 65 70 75 80 Ala Trp Lys Val Val Leu Gly Ile Phe Gln Thr Met Asn Leu Thr Ser 85 90 95 Leu Cys Glu Lys Val Arg Ala Glu Met Lys Glu Asn Val Gln Thr Gln 100 105 110 Glu Leu Gln Asp Pro Thr Gln Glu Asp Leu Glu Met Leu Glu Ala Ala 115 120 125 Ala Gly Asn Met Gln Thr Gln Gly Cys Gln Asp Pro Asn Gln Glu Glu 130 135 140 Leu Asp Glu Leu Glu Glu Glu Thr Gly Asn Val Gln Ala Gln Gly Cys 145 150 155 160 Gln Asp Pro Asn Gln Glu Glu Pro Glu Met Leu Glu Glu Ala Asp His 165 170 175 Arg Arg Lys Tyr Arg Glu Asn Met Lys Ala Glu Leu Leu Glu Thr Trp 180 185 190 Asp Asn Ile Ser Trp Pro Lys Asp His Val Tyr Ile Arg Asn Thr Ser 195 200 205 Lys Asp Glu His Glu Glu Leu Gln Arg Leu Leu Asp Pro Asn Arg Thr 210 215 220 Arg Ala Gln Ala Gln Thr Ile Val Leu Val Gly Arg Ala Gly Val Gly 225 230 235 240 Lys Thr Thr Leu Ala Met Gln Ala Met Leu His Trp Ala Asn Gly Val 245 250 255 Leu Phe Gln Gln Arg Phe Ser Tyr Val Phe Tyr Leu Ser Cys His Lys 260 265 270 Ile Arg Tyr Met Lys Glu Thr Thr Phe Ala Glu Leu Ile Ser Leu Asp 275 280 285 Trp Pro Asp Phe Asp Ala Pro Ile Glu Glu Phe Met Ser Gln Pro Glu 290 295 300 Lys Leu Leu Phe Ile Ile Asp Gly Phe Glu Glu Ile Ile Ile Ser Glu 305 310 315 320 Ser Arg Ser Glu Ser Leu Asp Asp Gly Ser Pro Cys Thr Asp Trp Tyr 325 330 335 Gln Glu Leu Pro Val Thr Lys Ile Leu His Ser Leu Leu Lys Lys Glu 340 345 350 Leu Val Pro Leu Ala Thr Leu Leu Ile Thr Ile Lys Thr Trp Phe Val 355 360 365 Arg Asp Leu Lys Ala Ser Leu Val Asn Pro Cys Phe Val Gln Ile Thr 370 375 380 Gly Phe Thr Gly Asp Asp Leu Arg Val Tyr Phe Met Arg His Phe Asp 385 390 395 400 Asp Ser Ser Glu Val Glu Lys Ile Leu Gln Gln Leu Arg Lys Asn Glu 405 410 415 Thr Leu Phe His Ser Cys Ser Ala Pro Met Val Cys Trp Thr Val Cys 420 425 430 Ser Cys Leu Lys Gln Pro Lys Val Arg Tyr Tyr Asp Leu Gln Ser Ile 435 440 445 Thr Gln Thr Thr Thr Ser Leu Tyr Ala Tyr Phe Phe Ser Asn Leu Phe 450 455 460 Ser Thr Ala Glu Val Asp Leu Ala Asp Asp Ser Trp Pro Gly Gln Trp 465 470 475 480 Arg Ala Leu Cys Ser Leu Ala Ile Glu Gly Leu Trp Ser Met Asn Phe 485 490 495 Thr Phe Asn Lys Glu Asp Thr Glu Ile Glu Gly Leu Glu Val Pro Phe 500 505 510 Ile Asp Ser Leu Tyr Glu Phe Asn Ile Leu Gln Lys Ile Asn Asp Cys 515 520 525 Gly Gly Cys Thr Thr Phe Thr His Leu Ser Phe Gln Glu Phe Phe Ala 530 535 540 Ala Met Ser Phe Val Leu Glu Glu Pro Arg Glu Phe Pro Pro His Ser 545 550 555 560 Thr Lys Pro Gln Glu Met Lys Met Leu Leu Gln His Val Leu Leu Asp 565 570 575 Lys Glu Ala Tyr Trp Thr Pro Val Val Leu Phe Phe Phe Gly Leu Leu 580 585 590 Asn Lys Asn Ile Ala Arg Glu Leu Glu Asp Thr Leu His Cys Lys Ile 595 600 605 Ser Pro Arg Val Met Glu Glu Leu Leu Lys Trp Gly Glu Glu Leu Gly 610 615 620 Lys Ala Glu Ser Ala Ser Leu Gln Phe His Ile Leu Arg Leu Phe His 625 630 635 640 Cys Leu His Glu Ser Gln Glu Glu Asp Phe Thr Lys Lys Met Leu Gly 645 650 655 Arg Ile Phe Glu Val Asp Leu Asn Ile Leu Glu Asp Glu Glu Leu Gln 660 665 670 Ala Ser Ser Phe Cys Leu Lys His Cys Lys Arg Leu Asn Lys Leu Arg 675 680 685 Leu Ser Val Ser Ser His Ile Leu Glu Arg Asp Leu Glu Ile Leu Glu 690 695 700 Thr Ser Lys Phe Asp Ser Arg Met His Ala Trp Asn Ser Ile Cys Ser 705 710 715 720 Thr Leu Val Thr Asn Glu Asn Leu His Glu Leu Asp Leu Ser Asn Ser 725 730 735 Lys Leu His Ala Ser Ser Val Lys Gly Leu Cys Leu Ala Leu Lys Asn 740 745 750 Pro Arg Cys Lys Val Gln Lys Leu Thr Cys Lys Ser Val Thr Pro Glu 755 760 765 Trp Val Leu Gln Asp Leu Ile Ile Ala Leu Gln Gly Asn Ser Lys Leu 770 775 780 Thr His Leu Asn Phe Ser Ser Asn Lys Leu Gly Met Thr Val Pro Leu 785 790 795 800 Ile Leu Lys Ala Leu Arg His Ser Ala Cys Asn Leu Lys Tyr Leu Cys 805 810 815 Leu Glu Lys Cys Asn Leu Ser Ala Ala Ser Cys Gln Asp Leu Ala Leu 820 825 830 Phe Leu Thr Ser Ile Gln His Val Thr Arg Leu Cys Leu Gly Phe Asn 835 840 845 Arg Leu Gln Asp Asp Gly Ile Lys Leu Leu Cys Ala Ala Leu Thr His 850 855 860 Pro Lys Cys Ala Leu Glu Arg Leu Glu Leu Trp Phe Cys Gln Leu Ala 865 870 875 880 Ala Pro Ala Cys Lys His Leu Ser Asp Ala Leu Leu Gln Asn Arg Ser 885 890 895 Leu Thr His Leu Asn Leu Ser Lys Asn Ser Leu Arg Asp Glu Gly Val 900 905 910 Lys Phe Leu Cys Glu Ala Leu Gly Arg Pro Asp Gly Asn Leu Gln Ser 915 920 925 Leu Asn Leu Ser Gly Cys Ser Phe Thr Arg Glu Gly Cys Gly Glu Leu 930 935 940 Ala Asn Ala Leu Ser His Asn His Asn Val Lys Ile Leu Asp Leu Gly 945 950 955 960 Glu Asn Asp Leu Gln Asp Asp Gly Val Lys Leu Leu Cys Glu Ala Leu 965 970 975 Lys Pro His Arg Ala Leu His Thr Leu Gly Leu Ala Lys Cys Asn Leu 980 985 990 Thr Thr Ala Cys Cys Gln His Leu Phe Ser Val Leu Ser Ser Ser Lys 995 1000 1005 Ser Leu Val Asn Leu Asn Leu Leu Gly Asn Glu Leu Asp Thr Asp 1010 1015 1020 Gly Val Lys Met Leu Cys Phe Lys Lys Thr Cys Thr Met 1025 1030 1035 19 1048 PRT Homo sapiens 19 Met Ser Asp Val Asn Pro Pro Ser Asp Thr Pro Ile Pro Phe Ser Ser 1 5 10 15 Ser Ser Thr His Ser Ser His Ile Pro Pro Trp Thr Phe Ser Cys Tyr 20 25 30 Pro Gly Ser Pro Cys Glu Asn Gly Val Met Leu Tyr Met Arg Asn Val 35 40 45 Ser His Glu Glu Leu Gln Arg Phe Lys Gln Leu Leu Leu Thr Glu Leu 50 55 60 Ser Thr Gly Thr Met Pro Ile Thr Trp Asp Gln Val Glu Thr Ala Ser 65 70 75 80 Trp Ala Glu Val Val His Leu Leu Ile Glu Arg Phe Pro Gly Arg Arg 85 90 95 Ala Trp Asp Val Thr Ser Asn Ile Phe Ala Ile Met Asn Cys Asp Lys 100 105 110 Met Cys Val Val Val Arg Arg Glu Ile Asn Ala Ile Leu Pro Thr Leu 115 120 125 Glu Pro Glu Asp Leu Asn Val Gly Glu Thr Gln Val Asn Leu Glu Glu 130 135 140 Gly Glu Ser Gly Lys Ile Arg Arg Tyr Lys Ser Asn Val Met Glu Lys 145 150 155 160 Phe Phe Pro Ile Trp Asp Ile Thr Thr Trp Pro Gly Asn Gln Arg Asp 165 170 175 Phe Phe Tyr Gln Gly Val His Arg His Glu Glu Tyr Leu Pro Cys Leu 180 185 190 Leu Leu Pro Lys Arg Pro Gln Gly Arg Gln Pro Lys Thr Val Ala Ile 195 200 205 Gln Gly Ala Pro Gly Ile Gly Lys Thr Ile Leu Ala Lys Lys Val Met 210 215 220 Phe Glu Trp Ala Arg Asn Lys Phe Tyr Ala His Lys Arg Trp Cys Ala 225 230 235 240 Phe Tyr Phe His Cys Gln Glu Val Asn Gln Thr Thr Asp Gln Ser Phe 245 250 255 Ser Glu Leu Ile Glu Gln Lys Trp Pro Gly Ser Gln Asp Leu Val Ser 260 265 270 Lys Ile Met Ser Lys Pro Asp Gln Leu Leu Leu Leu Leu Asp Gly Phe 275 280 285 Glu Glu Leu Thr Ser Thr Leu Ile Asp Arg Leu Glu Asp Leu Ser Glu 290 295 300 Asp Trp Arg Gln Lys Leu Pro Gly Ser Val Leu Leu Ser Ser Leu Leu 305 310 315 320 Ser Lys Thr Met Leu Pro Glu Ala Thr Leu Leu Ile Met Ile Arg Phe 325 330 335 Thr Ser Trp Gln Thr Cys Lys Pro Leu Leu Lys Cys Pro Ser Leu Val 340 345 350 Thr Leu Pro Gly Phe Asn Thr Met Glu Lys Ile Lys Tyr Phe Gln Met 355 360 365 Tyr Phe Gly His Thr Glu Glu Gly Asp Gln Val Leu Ser Phe Ala Met 370 375 380 Glu Asn Thr Ile Leu Phe Ser Met Cys Arg Val Pro Val Val Cys Trp 385 390 395 400 Met Val Cys Ser Gly Leu Lys Gln Gln Met Glu Arg Gly Asn Asn Leu 405 410 415 Thr Gln Ser Cys Pro Asn Ala Thr Ser Val Phe Val Arg Tyr Ile Ser 420 425 430 Ser Leu Phe Pro Thr Arg Ala Glu Asn Phe Ser Arg Lys Ile His Gln 435 440 445 Ala Gln Leu Glu Gly Leu Cys His Leu Ala Ala Asp Ser Met Trp His 450 455 460 Arg Lys Trp Val Leu Gly Lys Glu Asp Leu Glu Glu Ala Lys Leu Asp 465 470 475 480 Gln Thr Gly Val Thr Ala Phe Leu Gly Met Ser Ile Leu Arg Arg Ile 485 490 495 Ala Gly Glu Glu Asp His Tyr Val Phe Thr Leu Val Thr Phe Gln Glu 500 505 510 Phe Phe Ala Ala Leu Phe Tyr Val Leu Cys Phe Pro Gln Arg Leu Lys 515 520 525 Asn Phe His Val Leu Ser His Val Asn Ile Gln Arg Leu Ile Ala Ser 530 535 540 Pro Arg Gly Ser Lys Ser Tyr Leu Ser His Met Gly Leu Phe Leu Phe 545 550 555 560 Gly Phe Leu Asn Glu Ala Cys Ala Ser Ala Val Glu Gln Ser Phe Gln 565 570 575 Cys Lys Val Ser Phe Gly Asn Lys Arg Lys Leu Leu Lys Val Ile Pro 580 585 590 Leu Leu His Lys Cys Asp Pro Pro Ser Pro Gly Ser Gly Val Pro Gln 595 600 605 Leu Phe Tyr Cys Leu His Glu Ile Arg Glu Glu Ala Phe Val Ser Gln 610 615 620 Ala Leu Asn Asp Tyr His Lys Val Val Leu Arg Ile Gly Asn Asn Lys 625 630 635 640 Glu Val Gln Val Ser Ala Phe Cys Leu Lys Arg Cys Gln Tyr Leu His 645 650 655 Glu Val Glu Leu Thr Val Thr Leu Asn Phe Met Asn Val Trp Lys Leu 660 665 670 Ser Ser Ser Ser His Pro Gly Ser Glu Ala Pro Glu Ser Asn Gly Leu 675 680 685 His Arg Trp Trp Gln Asp Leu Cys Ser Val Phe Ala Thr Asn Asp Lys 690 695 700 Leu Glu Val Leu Thr Met Thr Asn Ser Val Leu Gly Pro Pro Phe Leu 705 710 715 720 Lys Ala Leu Ala Ala Ala Leu Arg His Pro Gln Cys Lys Leu Gln Lys 725 730 735 Leu Leu Leu Arg Arg Val Asn Ser Thr Met Leu Asn Gln Asp Leu Ile 740 745 750 Gly Val Leu Thr Gly Asn Gln His Leu Arg Tyr Leu Glu Ile Gln His 755 760 765 Val Glu Val Glu Ser Lys Ala Val Lys Leu Leu Cys Arg Val Leu Arg 770 775 780 Ser Pro Arg Cys Arg Leu Gln Cys Leu Arg Leu Glu Asp Cys Leu Ala 785 790 795 800 Thr Pro Arg Ile Trp Thr Asp Leu Gly Asn Asn Leu Gln Gly Asn Gly 805 810 815 His Leu Lys Thr Leu Ile Leu Arg Lys Asn Ser Leu Glu Asn Cys Gly 820 825 830 Ala Tyr Tyr Leu Ser Val Ala Gln Leu Glu Arg Leu Ser Ile Glu Asn 835 840 845 Cys Asn Leu Thr Gln Leu Thr Cys Glu Ser Leu Ala Ser Cys Leu Arg 850 855 860 Gln Ser Lys Met Leu Thr His Leu Ser Leu Ala Glu Asn Ala Leu Lys 865 870 875 880 Asp Glu Gly Ala Lys His Ile Trp Asn Ala Leu Pro His Leu Arg Cys 885 890 895 Pro Leu Gln Arg Leu Val Leu Arg Lys Cys Asp Leu Thr Phe Asn Cys 900 905 910 Cys Gln Asp Met Ile Ser Ala Leu Cys Lys Asn Lys Thr Leu Lys Ser 915 920 925 Leu Asp Leu Ser Phe Asn Ser Leu Lys Asp Asp Gly Val Ile Leu Leu 930 935 940 Cys Glu Ala Leu Lys Asn Pro Asp Cys Thr Leu Gln Ile Leu Glu Leu 945 950 955 960 Glu Asn Cys Leu Phe Thr Ser Ile Cys Cys Gln Ala Met Ala Ser Met 965 970 975 Leu Arg Lys Asn Gln His Leu Arg His Leu Asp Leu Ser Lys Asn Ala 980 985 990 Ile Gly Val Tyr Gly Ile Leu Thr Leu Cys Glu Ala Phe Ser Ser Gln 995 1000 1005 Lys Lys Arg Glu Glu Val Ile Phe Cys Ile Pro Ala Trp Thr Arg 1010 1015 1020 Ile Thr Ser Phe Ser Pro Thr Pro His Pro Pro Asp Phe Thr Gly 1025 1030 1035 Lys Ser Asp Cys Leu Ser Gln Ile Asn Pro 1040 1045 20 1033 PRT Homo sapiens 20 Met Ala Glu Ser Asp Ser Thr Asp Phe Asp Leu Leu Trp Tyr Leu Glu 1 5 10 15 Asn Leu Ser Asp Lys Glu Phe Gln Ser Phe Lys Lys Tyr Leu Ala Arg 20 25 30 Lys Ile Leu Asp Phe Lys Leu Pro Gln Phe Pro Leu Ile Gln Met Thr 35 40 45 Lys Glu Glu Leu Ala Asn Val Leu Pro Ile Ser Tyr Glu Gly Gln Tyr 50 55 60 Ile Trp Asn Met Leu Phe Ser Ile Phe Ser Met Met Arg Lys Glu Asp 65 70 75 80 Leu Cys Arg Lys Ile Ile Gly Arg Arg Asn Arg Asn Gln Glu Ala Cys 85 90 95 Lys Ala Val Met Arg Arg Lys Phe Met Leu Gln Trp Glu Ser His Thr 100 105 110 Phe Gly Lys Phe His Tyr Lys Phe Phe Arg Asp Val Ser Ser Asp Val 115 120 125 Phe Tyr Ile Leu Gln Leu Ala Tyr Asp Ser Thr Ser Tyr Tyr Ser Ala 130 135 140 Asn Asn Leu Asn Val Phe Leu Met Gly Glu Arg Ala Ser Gly Lys Thr 145 150 155 160 Ile Val Ile Asn Leu Ala Val Leu Arg Trp Ile Lys Gly Glu Met Trp 165 170 175 Gln Asn Met Ile Ser Tyr Val Val His Leu Thr Ser His Glu Ile Asn 180 185 190 Gln Met Thr Asn Ser Ser Leu Ala Glu Leu Ile Ala Lys Asp Trp Pro 195 200 205 Asp Gly Gln Ala Pro Ile Ala Asp Ile Leu Ser Asp Pro Lys Lys Leu 210 215

220 Leu Phe Ile Leu Glu Asp Leu Asp Asn Ile Arg Phe Glu Leu Asn Val 225 230 235 240 Asn Glu Ser Ala Leu Cys Ser Asn Ser Thr Gln Lys Val Pro Ile Pro 245 250 255 Val Leu Leu Val Ser Leu Leu Lys Arg Lys Met Ala Pro Gly Cys Trp 260 265 270 Phe Leu Ile Ser Ser Arg Pro Thr Arg Gly Asn Asn Val Lys Thr Phe 275 280 285 Leu Lys Glu Val Asp Cys Cys Thr Thr Leu Gln Leu Ser Asn Gly Lys 290 295 300 Arg Glu Ile Tyr Phe Asn Ser Phe Phe Lys Asp Arg Gln Arg Ala Ser 305 310 315 320 Ala Ala Leu Gln Leu Val His Glu Asp Glu Ile Leu Val Gly Leu Cys 325 330 335 Arg Val Ala Ile Leu Cys Trp Ile Thr Cys Thr Val Leu Lys Arg Gln 340 345 350 Met Asp Lys Gly Arg Asp Phe Gln Leu Cys Cys Gln Thr Pro Thr Asp 355 360 365 Leu His Ala His Phe Leu Ala Asp Ala Leu Thr Ser Glu Ala Gly Leu 370 375 380 Thr Ala Asn Gln Tyr His Leu Gly Leu Leu Lys Arg Leu Cys Leu Leu 385 390 395 400 Ala Ala Gly Gly Leu Phe Leu Ser Thr Leu Asn Phe Ser Gly Glu Asp 405 410 415 Leu Arg Cys Val Gly Phe Thr Glu Ala Asp Val Ser Val Leu Gln Ala 420 425 430 Ala Asn Ile Leu Leu Pro Ser Asn Thr His Lys Asp Arg Tyr Lys Phe 435 440 445 Ile His Leu Asn Val Gln Glu Phe Cys Thr Ala Ile Ala Phe Leu Met 450 455 460 Ala Val Pro Asn Tyr Leu Ile Pro Ser Gly Ser Arg Glu Tyr Lys Glu 465 470 475 480 Lys Arg Glu Gln Tyr Ser Asp Phe Asn Gln Val Phe Thr Phe Ile Phe 485 490 495 Gly Leu Leu Asn Ala Asn Arg Arg Lys Ile Leu Glu Thr Ser Phe Gly 500 505 510 Tyr Gln Leu Pro Met Val Asp Ser Phe Lys Trp Tyr Ser Val Gly Tyr 515 520 525 Met Lys His Leu Asp Arg Asp Pro Glu Lys Leu Thr His His Met Pro 530 535 540 Leu Phe Tyr Cys Leu Tyr Glu Asn Arg Glu Glu Glu Phe Val Lys Thr 545 550 555 560 Ile Val Asp Ala Leu Met Glu Val Thr Val Tyr Leu Gln Ser Asp Lys 565 570 575 Asp Met Met Val Ser Leu Tyr Cys Leu Asp Tyr Cys Cys His Leu Arg 580 585 590 Thr Leu Lys Leu Ser Val Gln Arg Ile Phe Gln Asn Lys Glu Pro Leu 595 600 605 Ile Arg Pro Thr Ala Ser Gln Met Lys Ser Leu Val Tyr Trp Arg Glu 610 615 620 Ile Cys Ser Leu Phe Tyr Thr Met Glu Ser Leu Arg Glu Leu His Ile 625 630 635 640 Phe Asp Asn Asp Leu Asn Gly Ile Ser Glu Arg Ile Leu Ser Lys Ala 645 650 655 Leu Glu His Ser Ser Cys Lys Leu Arg Thr Leu Lys Leu Ser Tyr Val 660 665 670 Ser Thr Ala Ser Gly Phe Glu Asp Leu Leu Lys Ala Leu Ala Arg Asn 675 680 685 Arg Ser Leu Thr Tyr Leu Ser Ile Asn Cys Thr Ser Ile Ser Leu Asn 690 695 700 Met Phe Ser Leu Leu His Asp Ile Leu His Glu Pro Thr Cys Gln Ile 705 710 715 720 Ser His Leu Ser Leu Met Lys Cys Asp Leu Arg Ala Ser Glu Cys Glu 725 730 735 Glu Ile Ala Ser Leu Leu Ile Ser Gly Gly Ser Leu Arg Lys Leu Thr 740 745 750 Leu Ser Ser Asn Pro Leu Arg Ser Asp Gly Met Asn Ile Leu Cys Asp 755 760 765 Ala Leu Leu His Pro Asn Cys Thr Leu Ile Ser Leu Val Leu Val Phe 770 775 780 Cys Cys Leu Thr Glu Asn Cys Cys Ser Ala Leu Gly Arg Val Leu Leu 785 790 795 800 Phe Ser Pro Thr Leu Arg Gln Leu Asp Leu Cys Val Asn Arg Leu Lys 805 810 815 Asn Tyr Gly Val Leu His Val Thr Phe Pro Leu Leu Phe Pro Thr Cys 820 825 830 Gln Leu Glu Glu Leu His Leu Ser Gly Cys Phe Phe Ser Ser Asp Ile 835 840 845 Cys Gln Tyr Ile Ala Ile Val Ile Ala Thr Asn Glu Lys Leu Arg Ser 850 855 860 Leu Glu Ile Gly Ser Asn Lys Ile Glu Asp Ala Gly Met Gln Leu Leu 865 870 875 880 Cys Gly Gly Leu Arg His Pro Asn Cys Met Leu Val Asn Ile Gly Leu 885 890 895 Glu Glu Cys Met Leu Thr Ser Ala Cys Cys Arg Ser Leu Ala Ser Val 900 905 910 Leu Thr Thr Asn Lys Thr Leu Glu Arg Leu Asn Leu Leu Gln Asn His 915 920 925 Leu Gly Asn Asp Gly Val Ala Lys Leu Leu Glu Ser Leu Ile Ser Pro 930 935 940 Asp Cys Val Leu Lys Val Val Gly Leu Pro Leu Thr Gly Leu Asn Thr 945 950 955 960 Gln Thr Gln Gln Leu Leu Met Thr Val Lys Glu Arg Lys Pro Ser Leu 965 970 975 Ile Phe Leu Ser Glu Thr Trp Ser Leu Lys Glu Gly Arg Glu Ile Gly 980 985 990 Val Thr Pro Ala Ser Gln Pro Gly Ser Ile Ile Pro Asn Ser Asn Leu 995 1000 1005 Asp Tyr Met Phe Phe Lys Phe Pro Arg Met Ser Ala Ala Met Arg 1010 1015 1020 Thr Ser Asn Thr Ala Ser Arg Gln Pro Leu 1025 1030 21 975 PRT Homo sapiens 21 Met Arg Trp Gly His His Leu Pro Arg Ala Ser Trp Gly Ser Gly Phe 1 5 10 15 Arg Arg Ala Leu Gln Arg Pro Asp Asp Arg Ile Pro Phe Leu Ile His 20 25 30 Trp Ser Trp Pro Leu Gln Gly Glu Arg Pro Phe Gly Pro Pro Arg Ala 35 40 45 Phe Ile Arg His His Gly Ser Ser Val Asp Ser Ala Pro Pro Pro Gly 50 55 60 Arg His Gly Arg Leu Phe Pro Ser Ala Ser Ala Thr Glu Ala Ile Gln 65 70 75 80 Arg His Arg Arg Asn Leu Ala Glu Trp Phe Ser Arg Leu Pro Arg Glu 85 90 95 Glu Arg Gln Phe Gly Pro Thr Phe Ala Leu Asp Thr Val His Val Asp 100 105 110 Pro Val Ile Arg Glu Ser Thr Pro Asp Glu Leu Leu Arg Pro Pro Ala 115 120 125 Glu Leu Ala Leu Glu His Gln Pro Pro Gln Ala Gly Leu Pro Pro Leu 130 135 140 Ala Leu Ser Gln Leu Phe Asn Pro Asp Ala Cys Gly Arg Arg Val Gln 145 150 155 160 Thr Val Val Leu Tyr Gly Thr Val Gly Thr Gly Lys Ser Thr Leu Val 165 170 175 Arg Lys Met Val Leu Asp Trp Cys Tyr Gly Arg Leu Pro Ala Phe Glu 180 185 190 Leu Leu Ile Pro Phe Ser Cys Glu Asp Leu Ser Ser Leu Gly Pro Ala 195 200 205 Pro Ala Ser Leu Cys Gln Leu Val Ala Gln Arg Tyr Thr Pro Leu Lys 210 215 220 Glu Val Leu Pro Leu Met Ala Ala Ala Gly Ser His Leu Leu Phe Val 225 230 235 240 Leu His Gly Leu Glu His Leu Asn Leu Asp Phe Arg Leu Ala Gly Thr 245 250 255 Gly Leu Cys Ser Asp Pro Glu Glu Pro Gln Glu Pro Ala Ala Ile Ile 260 265 270 Val Asn Leu Leu Arg Lys Tyr Met Leu Pro Gln Ala Ser Ile Leu Val 275 280 285 Thr Thr Arg Pro Ser Ala Ile Gly Arg Ile Pro Ser Lys Tyr Val Gly 290 295 300 Arg Tyr Gly Glu Ile Cys Gly Phe Ser Asp Thr Asn Leu Gln Lys Leu 305 310 315 320 Tyr Phe Gln Leu Arg Leu Asn Gln Pro Tyr Cys Gly Tyr Ala Val Gly 325 330 335 Gly Ser Gly Val Ser Ala Thr Pro Ala Gln Arg Asp His Leu Val Gln 340 345 350 Met Leu Ser Arg Asn Leu Glu Gly His His Gln Ile Ala Ala Ala Cys 355 360 365 Phe Leu Pro Ser Tyr Cys Trp Leu Val Cys Ala Thr Leu His Phe Leu 370 375 380 His Ala Pro Thr Pro Ala Gly Gln Thr Leu Thr Ser Ile Tyr Thr Ser 385 390 395 400 Phe Leu Arg Leu Asn Phe Ser Gly Glu Thr Leu Asp Ser Thr Asp Pro 405 410 415 Ser Asn Leu Ser Leu Met Ala Tyr Ala Ala Arg Thr Met Gly Lys Leu 420 425 430 Ala Tyr Glu Gly Val Ser Ser Arg Lys Thr Tyr Phe Ser Glu Glu Asp 435 440 445 Val Cys Gly Cys Leu Glu Ala Gly Ile Arg Thr Glu Glu Glu Phe Gln 450 455 460 Leu Leu His Ile Phe Arg Arg Asp Ala Leu Arg Phe Phe Leu Ala Pro 465 470 475 480 Cys Val Glu Pro Gly Arg Ala Gly Thr Phe Val Phe Thr Val Pro Ala 485 490 495 Met Gln Glu Tyr Leu Ala Ala Leu Tyr Ile Val Leu Gly Leu Arg Lys 500 505 510 Thr Thr Leu Gln Lys Val Gly Lys Glu Val Ala Glu Leu Val Gly Arg 515 520 525 Val Gly Glu Asp Val Ser Leu Val Leu Gly Ile Met Ala Lys Leu Leu 530 535 540 Pro Leu Arg Ala Leu Pro Leu Leu Phe Asn Leu Ile Lys Val Val Pro 545 550 555 560 Arg Val Phe Gly Arg Met Val Gly Lys Ser Arg Glu Ala Val Ala Gln 565 570 575 Ala Met Val Leu Glu Met Phe Arg Glu Glu Asp Tyr Tyr Asn Asp Asp 580 585 590 Val Leu Asp Gln Met Gly Ala Ser Ile Leu Gly Val Glu Gly Pro Arg 595 600 605 Arg His Pro Asp Glu Pro Pro Glu Asp Glu Val Phe Glu Leu Phe Pro 610 615 620 Met Phe Met Gly Gly Leu Leu Ser Ala His Asn Arg Ala Val Leu Ala 625 630 635 640 Gln Leu Gly Cys Pro Ile Lys Asn Leu Asp Ala Leu Glu Asn Ala Gln 645 650 655 Ala Ile Lys Lys Lys Leu Gly Lys Leu Gly Arg Gln Val Leu Pro Pro 660 665 670 Ser Glu Leu Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn Gln 675 680 685 Arg Phe Ser Ala Glu Val Leu Ser Ser Leu Arg Gln Leu Asn Leu Ala 690 695 700 Gly Val Arg Met Thr Pro Val Lys Cys Thr Val Val Ala Ala Val Leu 705 710 715 720 Gly Ser Gly Arg His Ala Leu Asp Glu Val Asn Leu Ala Ser Cys Gln 725 730 735 Leu Asp Pro Ala Gly Leu Arg Thr Leu Leu Pro Val Phe Leu Arg Ala 740 745 750 Arg Lys Leu Gly Leu Gln Leu Asn Ser Leu Gly Pro Glu Ala Cys Lys 755 760 765 Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln Ile Thr Thr Leu 770 775 780 Arg Leu Ser Asn Asn Pro Leu Thr Ala Ala Gly Val Ala Val Leu Met 785 790 795 800 Glu Gly Leu Ala Gly Asn Thr Ser Val Thr His Leu Ser Leu Leu His 805 810 815 Thr Gly Leu Gly Asp Glu Gly Leu Glu Leu Leu Ala Ala Gln Leu Asp 820 825 830 Arg Asn Arg Gln Leu Gln Glu Leu Asn Val Ala Tyr Asn Gly Ala Gly 835 840 845 Asp Thr Ala Ala Leu Ala Leu Ala Arg Ala Ala Arg Glu His Pro Ser 850 855 860 Leu Glu Leu Leu His Leu Tyr Phe Asn Glu Leu Ser Ser Glu Gly Arg 865 870 875 880 Gln Val Leu Arg Asp Leu Gly Gly Ala Ala Glu Gly Gly Ala Arg Val 885 890 895 Val Val Ser Leu Thr Glu Gly Thr Ala Val Ser Glu Tyr Trp Ser Val 900 905 910 Ile Leu Ser Glu Val Gln Arg Asn Leu Asn Ser Trp Asp Arg Ala Arg 915 920 925 Val Gln Arg His Leu Glu Leu Leu Leu Arg Asp Leu Glu Asp Ser Arg 930 935 940 Gly Ala Thr Leu Asn Pro Trp Arg Lys Ala Gln Leu Leu Arg Val Glu 945 950 955 960 Gly Glu Val Arg Ala Leu Leu Glu Gln Leu Gly Ser Ser Gly Ser 965 970 975 22 1866 PRT Homo sapiens 22 Met Asp Pro Val Gly Leu Gln Leu Gly Asn Lys Asn Leu Trp Ser Cys 1 5 10 15 Leu Val Arg Leu Leu Thr Lys Asp Pro Glu Trp Leu Asn Ala Lys Met 20 25 30 Lys Phe Phe Leu Pro Asn Thr Asp Leu Asp Ser Arg Asn Glu Thr Leu 35 40 45 Asp Pro Glu Gln Arg Val Ile Leu Gln Leu Asn Lys Leu His Val Gln 50 55 60 Gly Ser Asp Thr Trp Gln Ser Phe Ile His Cys Val Cys Met Gln Leu 65 70 75 80 Glu Val Pro Leu Asp Leu Glu Val Leu Leu Leu Ser Thr Phe Gly Tyr 85 90 95 Asp Asp Gly Phe Thr Ser Gln Leu Gly Ala Glu Gly Lys Ser Gln Pro 100 105 110 Glu Ser Gln Leu His His Gly Leu Lys Arg Pro His Gln Ser Cys Gly 115 120 125 Ser Ser Pro Arg Arg Lys Gln Cys Lys Lys Gln Gln Leu Glu Leu Ala 130 135 140 Lys Lys Tyr Leu Gln Leu Leu Arg Thr Ser Ala Gln Gln Arg Tyr Arg 145 150 155 160 Ser Gln Ile Pro Gly Ser Gly Gln Pro His Ala Phe His Gln Val Tyr 165 170 175 Val Pro Pro Ile Leu Arg Arg Ala Thr Ala Ser Leu Asp Thr Pro Glu 180 185 190 Gly Ala Ile Met Gly Asp Val Lys Val Glu Asp Gly Ala Asp Val Ser 195 200 205 Ile Ser Asp Leu Phe Asn Thr Arg Val Asn Lys Gly Pro Arg Val Thr 210 215 220 Val Leu Leu Gly Lys Ala Gly Met Gly Lys Thr Thr Leu Ala His Arg 225 230 235 240 Leu Cys Gln Lys Trp Ala Glu Gly His Leu Asn Cys Phe Gln Ala Leu 245 250 255 Phe Leu Phe Glu Phe Arg Gln Leu Asn Leu Ile Thr Arg Phe Leu Thr 260 265 270 Pro Ser Glu Leu Leu Phe Asp Leu Tyr Leu Ser Pro Glu Ser Asp His 275 280 285 Asp Thr Val Phe Gln Tyr Leu Glu Lys Asn Ala Asp Gln Val Leu Leu 290 295 300 Ile Phe Asp Gly Leu Asp Glu Ala Leu Gln Pro Met Gly Pro Asp Gly 305 310 315 320 Pro Gly Pro Val Leu Thr Leu Phe Ser His Leu Cys Asn Gly Thr Leu 325 330 335 Leu Pro Gly Cys Arg Val Met Ala Thr Ser Arg Pro Gly Lys Leu Pro 340 345 350 Ala Cys Leu Pro Ala Glu Ala Ala Met Val His Met Leu Gly Phe Asp 355 360 365 Gly Pro Arg Val Glu Glu Tyr Val Asn His Phe Phe Ser Ala Gln Pro 370 375 380 Ser Arg Glu Gly Ala Leu Val Glu Leu Gln Thr Asn Gly Arg Leu Arg 385 390 395 400 Ser Leu Cys Ala Val Pro Ala Leu Cys Gln Val Ala Cys Leu Cys Leu 405 410 415 His His Leu Leu Pro Asp His Ala Pro Gly Gln Ser Val Ala Leu Leu 420 425 430 Pro Asn Met Thr Gln Leu Tyr Met Gln Met Val Leu Ala Leu Ser Pro 435 440 445 Pro Gly His Leu Pro Thr Ser Ser Leu Leu Asp Leu Gly Glu Val Ala 450 455 460 Leu Arg Gly Leu Glu Thr Gly Lys Val Ile Phe Tyr Ala Lys Asp Ile 465 470 475 480 Ala Pro Pro Leu Ile Ala Phe Gly Ala Thr His Ser Leu Leu Thr Ser 485 490 495 Phe Cys Val Cys Thr Gly Pro Gly His Gln Gln Thr Gly Tyr Ala Phe 500 505 510 Thr His Leu Ser Leu Gln Glu Phe Leu Ala Ala Leu His Leu Met Ala 515 520 525 Ser Pro Lys Val Asn Lys Asp Thr Leu Thr Gln Tyr Val Thr Leu His 530 535 540 Ser Arg Trp Val Gln Arg Thr Lys Ala Arg Leu Gly Leu Ser Asp His 545 550 555 560 Leu Pro Thr Phe Leu Ala Gly Leu Ala Ser Cys Thr Cys Arg Pro Phe 565 570 575 Leu Ser His Leu Ala Gln Gly Asn Glu Asp Cys Val Gly Ala Lys Gln 580 585 590 Ala Ala Val Val Gln Val Leu Lys Lys Leu Ala Thr Arg Lys Leu Thr 595 600 605 Gly Pro Lys Val Val Glu Leu Cys His Cys Val Asp Glu Thr Gln Glu 610 615 620 Pro Glu Leu Ala Ser Leu Thr Ala Gln Ser Leu Pro Tyr Gln Leu Pro 625 630 635 640 Phe His Asn Phe Pro Leu Thr Cys Thr Asp Leu Ala Thr Leu Thr Asn 645 650 655 Ile

Leu Glu His Arg Glu Ala Pro Ile His Leu Asp Phe Asp Gly Cys 660 665 670 Pro Leu Glu Pro His Cys Pro Glu Ala Leu Val Gly Cys Gly Gln Ile 675 680 685 Glu Asn Leu Ser Phe Lys Ser Arg Lys Cys Gly Asp Ala Phe Ala Glu 690 695 700 Ala Leu Ser Arg Ser Leu Pro Thr Met Gly Arg Leu Gln Met Leu Gly 705 710 715 720 Leu Ala Gly Ser Lys Ile Thr Ala Arg Gly Ile Ser His Leu Val Lys 725 730 735 Ala Leu Pro Leu Cys Pro Gln Leu Lys Glu Val Ser Phe Arg Asp Asn 740 745 750 Gln Leu Ser Asp Gln Val Val Leu Asn Ile Val Glu Val Leu Pro His 755 760 765 Leu Pro Arg Leu Arg Lys Leu Asp Leu Ser Ser Asn Ser Ile Cys Val 770 775 780 Ser Thr Leu Leu Cys Leu Ala Arg Val Ala Val Thr Cys Pro Thr Val 785 790 795 800 Arg Met Leu Gln Ala Arg Glu Arg Thr Ile Ile Phe Leu Leu Ser Pro 805 810 815 Pro Thr Glu Thr Thr Ala Glu Leu Gln Arg Ala Pro Asp Leu Gln Glu 820 825 830 Ser Asp Gly Gln Arg Lys Gly Ala Gln Ser Arg Ser Leu Thr Leu Arg 835 840 845 Leu Gln Lys Cys Gln Leu Gln Val His Asp Ala Glu Ala Leu Ile Ala 850 855 860 Leu Leu Gln Glu Gly Pro His Leu Glu Glu Val Asp Leu Ser Gly Asn 865 870 875 880 Gln Leu Glu Asp Glu Gly Cys Arg Leu Met Ala Glu Ala Ala Ser Gln 885 890 895 Leu His Ile Ala Arg Lys Leu Asp Leu Ser Asp Asn Gly Leu Ser Val 900 905 910 Ala Gly Val His Cys Val Leu Arg Ala Val Ser Ala Cys Trp Thr Leu 915 920 925 Ala Glu Leu His Ile Ser Leu Gln His Lys Thr Val Ile Phe Met Phe 930 935 940 Ala Gln Glu Pro Glu Glu Gln Lys Gly Pro Gln Glu Arg Ala Ala Phe 945 950 955 960 Leu Asp Ser Leu Met Leu Gln Met Pro Ser Glu Leu Pro Leu Ser Ser 965 970 975 Arg Arg Met Arg Leu Thr His Cys Gly Leu Gln Glu Lys His Leu Glu 980 985 990 Gln Leu Cys Lys Ala Leu Gly Gly Ser Cys His Leu Gly His Leu His 995 1000 1005 Leu Asp Phe Ser Gly Asn Ala Leu Gly Asp Glu Gly Ala Ala Arg 1010 1015 1020 Leu Ala Gln Leu Leu Pro Gly Leu Gly Ala Leu Gln Ser Leu Asn 1025 1030 1035 Leu Ser Glu Asn Gly Leu Ser Leu Asp Ala Val Leu Gly Leu Val 1040 1045 1050 Arg Cys Phe Ser Thr Leu Gln Trp Leu Phe Arg Leu Asp Ile Ser 1055 1060 1065 Phe Glu Ser Gln His Ile Leu Leu Arg Gly Asp Lys Thr Ser Arg 1070 1075 1080 Asp Met Trp Ala Thr Gly Ser Leu Pro Asp Phe Pro Ala Ala Ala 1085 1090 1095 Lys Phe Leu Gly Phe Arg Gln Arg Cys Ile Pro Arg Ser Leu Cys 1100 1105 1110 Leu Ser Glu Cys Pro Leu Glu Pro Pro Ser Leu Thr Arg Leu Cys 1115 1120 1125 Ala Thr Leu Lys Asp Cys Pro Gly Pro Leu Glu Leu Gln Leu Ser 1130 1135 1140 Cys Glu Phe Leu Ser Asp Gln Ser Leu Glu Thr Leu Leu Asp Cys 1145 1150 1155 Leu Pro Gln Leu Pro Gln Leu Ser Leu Leu Gln Leu Ser Gln Thr 1160 1165 1170 Gly Leu Ser Pro Lys Ser Pro Phe Leu Leu Ala Asn Thr Leu Ser 1175 1180 1185 Leu Cys Pro Arg Val Lys Lys Val Asp Leu Arg Ser Leu His His 1190 1195 1200 Ala Thr Leu His Phe Arg Ser Asn Glu Glu Glu Glu Gly Val Cys 1205 1210 1215 Cys Gly Arg Phe Thr Gly Cys Ser Leu Ser Gln Glu His Val Glu 1220 1225 1230 Ser Leu Cys Trp Leu Leu Ser Lys Cys Lys Asp Leu Ser Gln Val 1235 1240 1245 Asp Leu Ser Ala Asn Leu Leu Gly Asp Ser Gly Leu Arg Cys Leu 1250 1255 1260 Leu Glu Cys Leu Pro Gln Val Pro Ile Ser Gly Leu Leu Asp Leu 1265 1270 1275 Ser His Asn Ser Ile Ser Gln Glu Ser Ala Leu Tyr Leu Leu Glu 1280 1285 1290 Thr Leu Pro Ser Cys Pro Arg Val Arg Glu Ala Ser Val Asn Leu 1295 1300 1305 Gly Ser Glu Gln Ser Phe Arg Ile His Phe Ser Arg Glu Asp Gln 1310 1315 1320 Ala Gly Lys Thr Leu Arg Leu Ser Glu Cys Ser Phe Arg Pro Glu 1325 1330 1335 His Val Ser Arg Leu Ala Thr Gly Leu Ser Lys Ser Leu Gln Leu 1340 1345 1350 Thr Glu Leu Thr Leu Thr Gln Cys Cys Leu Gly Gln Lys Gln Leu 1355 1360 1365 Ala Ile Leu Leu Ser Leu Val Gly Arg Pro Ala Gly Leu Phe Ser 1370 1375 1380 Leu Arg Val Gln Glu Pro Trp Ala Asp Arg Ala Arg Val Leu Ser 1385 1390 1395 Leu Leu Glu Val Cys Ala Gln Ala Ser Gly Ser Val Thr Glu Ile 1400 1405 1410 Ser Ile Ser Glu Thr Gln Gln Gln Leu Cys Val Gln Leu Glu Phe 1415 1420 1425 Pro Arg Gln Glu Glu Asn Pro Glu Ala Val Ala Leu Arg Leu Ala 1430 1435 1440 His Cys Asp Leu Gly Ala His His Ser Leu Leu Val Gly Gln Leu 1445 1450 1455 Met Glu Thr Cys Ala Arg Leu Gln Gln Leu Ser Leu Ser Gln Val 1460 1465 1470 Asn Leu Cys Glu Asp Asp Asp Ala Ser Ser Leu Leu Leu Gln Ser 1475 1480 1485 Leu Leu Leu Ser Leu Ser Glu Leu Lys Thr Phe Arg Leu Thr Ser 1490 1495 1500 Ser Cys Val Ser Thr Glu Gly Leu Ala His Leu Ala Ser Gly Leu 1505 1510 1515 Gly His Cys His His Leu Glu Glu Leu Asp Leu Ser Asn Asn Gln 1520 1525 1530 Phe Asp Glu Glu Gly Thr Lys Ala Leu Met Arg Ala Leu Glu Gly 1535 1540 1545 Lys Trp Met Leu Lys Arg Leu Asp Leu Ser His Leu Leu Leu Asn 1550 1555 1560 Ser Ser Thr Leu Ala Leu Leu Thr His Arg Leu Ser Gln Met Thr 1565 1570 1575 Cys Leu Gln Ser Leu Arg Leu Asn Arg Asn Ser Ile Gly Asp Val 1580 1585 1590 Gly Cys Cys His Leu Ser Glu Ala Leu Arg Ala Ala Thr Ser Leu 1595 1600 1605 Glu Glu Leu Asp Leu Ser His Asn Gln Ile Gly Asp Ala Gly Val 1610 1615 1620 Gln His Leu Ala Thr Ile Leu Pro Gly Leu Pro Glu Leu Arg Lys 1625 1630 1635 Ile Asp Leu Ser Gly Asn Ser Ile Ser Ser Ala Gly Gly Val Gln 1640 1645 1650 Leu Ala Glu Ser Leu Val Leu Cys Arg Arg Leu Glu Glu Leu Met 1655 1660 1665 Leu Gly Cys Asn Ala Leu Gly Asp Pro Thr Ala Leu Gly Leu Ala 1670 1675 1680 Gln Glu Leu Pro Gln His Leu Arg Val Leu His Leu Pro Phe Ser 1685 1690 1695 His Leu Gly Pro Gly Gly Ala Leu Ser Leu Ala Gln Ala Leu Asp 1700 1705 1710 Gly Ser Pro His Leu Glu Glu Ile Ser Leu Ala Glu Asn Asn Leu 1715 1720 1725 Ala Gly Gly Val Leu Arg Phe Cys Met Glu Leu Pro Leu Leu Arg 1730 1735 1740 Gln Ile Asp Leu Val Ser Cys Lys Ile Asp Asn Gln Thr Ala Lys 1745 1750 1755 Leu Leu Thr Ser Ser Phe Thr Ser Cys Pro Ala Leu Glu Val Ile 1760 1765 1770 Leu Leu Ser Trp Asn Leu Leu Gly Asp Glu Ala Ala Ala Glu Leu 1775 1780 1785 Ala Gln Val Leu Pro Lys Met Gly Arg Leu Lys Arg Val Asp Leu 1790 1795 1800 Glu Lys Asn Gln Ile Thr Ala Leu Gly Ala Trp Leu Leu Ala Glu 1805 1810 1815 Gly Leu Ala Gln Gly Ser Ser Ile Gln Val Ile Arg Leu Trp Asn 1820 1825 1830 Asn Pro Ile Pro Cys Asp Met Ala Gln His Leu Lys Ser Gln Glu 1835 1840 1845 Pro Arg Leu Asp Phe Ala Phe Phe Asp Asn Gln Pro Gln Ala Pro 1850 1855 1860 Trp Gly Thr 1865

Claims

1. A composition comprising an isolated and purified nucleic acid sequence encoding a protein selected from the group consisting of SEQ ID NOs: 12-21.

2. The composition of claim 1, wherein said sequence is operably linked to a heterologous promoter.

3. The composition of claim 1, wherein said sequence is contained within a vector.

4. The composition of claim 3, wherein said vector is within a host cell.

5. The composition of claim 1, wherein said nucleic acid is selected from the group consisting of SEQ ID NOs: 1-10 nd variants thereof that are at least 80% identical to SEQ ID NOs: 1-10.

6. The composition of claim 5, wherein said protein is at least 90% identical to SEQ ID NOs: 12-21.

7. The composition of claim 5, wherein said protein is at least 95% identical to SEQ ID NOs: 12-21.

8. The composition of claim 1, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-10.

9-17. (canceled)