WO2004089282A2 - Therapeutic polypeptides, nucleic acids encoding same, and methods of use - Google Patents

Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies that immunospecifically bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the novel polypeptide, polynucleotide, or antibody specific to the polypeptide. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.

Description

THERAPEUTIC POLYPEPTIDES, NUCLEIC ACIDS ENCODING SAME, AND METHODS OF USE

TECHNICAL FIELD

The present invention relates to novel polypeptides, and the nucleic acids encoding them, having properties related to stimulation of biochemical or physiological responses in a cell, a tissue, an organ or an organism. More particularly, the novel polypeptides are gene products of novel genes, or are specified biologically active fragments or derivatives thereof. Methods of use encompass diagnostic and prognostic assay procedures as well as methods of treating diverse pathological conditions.

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BACKGROUND OF THE INVENTION

Eukaryotic cells are characterized by biochemical and physiological processes, which under normal conditions are exquisitely balanced to achieve the preservation and propagation of the cells. When such cells are components of multicellular organisms such as vertebrates or, more particularly, organisms such as mammals, the regulation of the biochemical and physiological processes involves intricate signaling pathways. Frequently, such signaling pathways involve extracellular signaling proteins, cellular receptors that bind the signaling proteins and signal transducing components located within the cells.

Signaling proteins may be classified as endocrine effectors, paracrine effectors or autocrine effectors. Endocrine effectors are signaling molecules secreted by a given organ into the circulatory system, which are then transported to a distant target organ or tissue. The target cells include the receptors for the endocrine effector, and when the endocrine effector binds, a signaling cascade is induced. Paracrine effectors involve secreting cells and receptor cells in close proximity to each other, for example, two different classes of cells in the same tissue or organ. One class of cells secretes the paracrine effector, which then reaches the second class of cells, for example by diffusion through the extracellular fluid. The second class of cells contains the receptors for the paracrine effector; binding of the effector results in induction of the signaling cascade that elicits the corresponding biochemical or physiological effect. Autocrine effectors are highly analogous to paracrine effectors, except that the same cell type that secretes the autocrine effector also contains the receptor. Thus the autocrine effector binds to receptors on the same cell, or on identical neighboring cells. The binding process then elicits the characteristic biochemical or physiological effect.

Signaling processes may elicit a variety of effects on cells and tissues including, by way of nonlimiting example, induction of cell or tissue proliferation, suppression of growth or proliferation, induction of differentiation or maturation of a cell or tissue, and suppression of differentiation or maturation of a cell or tissue.

Many pathological conditions involve dysregulation of expression of important effector proteins. In certain classes of pathologies the dysregulation is manifested as diminished or suppressed level of synthesis and secretion of protein effectors. In other classes of pathologies the dysregulation is manifested as increased or up-regulated level of synthesis and secretion of protein effectors. In a clinical setting a subject may be suspected of suffering from a condition brought on by altered or mis-regulated levels of a protein effector of interest. Therefore there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject, and to compare the level with that characteristic of a nonpathological condition. There also is a need to provide the protein effector as a product of manufacture. Administration of the effector to a subject in need thereof is useful in treatment of the pathological condition. Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished or suppressed levels of the protein effector of interest. In addition, there is a need for a method of treatment of a pathological condition brought on by a increased or up-regulated levels of the protein effector of interest.

Antibodies are multi-ham proteins that bind specifically to a given antigen, and bind poorly, or not at all, to substances deemed not to be cognate antigens. Antibodies are comprised of two short chains termed light chains and two long chains termed heavy chains. These chains are constituted of immunoglobulin domains, of which generally there are two classes: one variable domain per chain, one constant domain in light chains, and three or more constant domains in heavy chains. The antigen-specific portion of the immunoglobulin molecules resides in the variable domains; the variable domains of one light chain and one heavy chain associate with each other to generate the antigen-binding moiety. Antibodies that bind immunospecifically to a cognate or target antigen bind with high affinities. Accordingly, they are useful in assaying specifically for the presence of the antigen in a sample. In addition, they have the potential of inactivating the activity of the antigen.

Therefore there is a need to assay for the level of a protein effector of interest in a biological sample from such a subject, and to compare this level with that characteristic of a nonpathological condition. In particular, there is a need for such an assay based on the use of an antibody that binds immunospecifically to the antigen. There further is a need to inhibit the activity of the protein effector in cases where a pathological condition arises from elevated or excessive levels of the effector based on the use of an antibody that binds immunospecifically to the effector. Thus, there is a need for the antibody as a product of manufacture. There further is a need for a method of treatment of a pathological condition brought on by an elevated or excessive level of the protein effector of interest based on administering the antibody to the subject. SUMMARY OF THE INVENTION

The invention is based in part upon the discovery of isolated polypeptides including amino acid sequences selected from mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NON1, ΝOV2, NOV3, etc., nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NONX" nucleic acid or polypeptide sequences.

The invention also is based in part upon variants of a mature form of the amino acid sequence selected from the group consisting of SEQ ID ΝO:2n, wherein n is an integer between 1 and 102, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed. In another embodiment, the invention includes the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102. In another embodiment, the invention also comprises variants of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed. The invention also involves fragments of any of the mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% of the residues are changed.

In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic variants of the sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102. These allelic variants include amino acid sequences that are the translations of nucleic acid sequences differing by a single nucleotide from nucleic acid sequences selected from the group consisting of SEQ ID NOS: 2n-l, wherein n is an integer between 1 and 102. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution. In another embodiment, the invention comprises a pharmaceutical composition involving a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, and a pharmaceutically acceptable carrier. In another embodiment, the invention involves a kit, including, in one or more containers, this pharmaceutical composition.

In another embodiment, the invention includes the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease being selected from a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein said therapeutic is the polypeptide selected from this group.

In another embodiment, the invention comprises a method for determining the presence or amount of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, in a sample, the method involving providing the sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, thereby determining the presence or amount of polypeptide in the sample.

In another embodiment, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, in a first mammalian subject, the method involving measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and comparing the amount of the polypeptide in this sample to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, the disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

In another embodiment, the invention involves a method of identifying an agent that binds to a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, the method including introducing the polypeptide to the agent; and determining whether the agent binds to the polypeptide. The agent could be a cellular receptor or a downstream effector. In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n. wherein n is an integer between 1 and 102, the method including providing a cell expressing the polypeptide of the invention and having a property or function ascribable to the polypeptide; contacting the cell with a composition comprising a candidate substance; and determining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition devoid of the substance, the substance is identified as a potential therapeutic agent.

In another embodiment, the invention involves a method for screening for a modulator of activity or of latency or predisposition to a pathology associated with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, the method including administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of the invention, wherein the test animal recombinantly expresses the polypeptide of the invention; measuring the activity of the polypeptide in the test animal after administering the test compound; and comparing the activity of the protein in the test animal with the activity of the polypeptide in a control animal not administered the polypeptide, wherein a change in the activity of the polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of, or predisposition to, a pathology associated with the polypeptide of the invention. The recombinant test animal could express a test protein transgene or express the transgene under the control of a promoter at an increased level relative to a wild-type test animal The promoter may or may not b the native gene promoter of the transgene.

In another embodiment, the invention involves a method for modulating the activity of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, the method including introducing a cell sample expressing the polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide.

In another embodiment, the invention involves a method of treating or preventing a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, the method including administering the polypeptide to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject. The subject could be human.

In another embodiment, the invention involves a method of treating a pathological state in a mammal, the method including administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, or a biologically active fragment thereof.

In another embodiment, the invention involves an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, a variant of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, or any variant of the polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and the complement of any of the nucleic acid molecules.

In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.

In another embodiment, the invention involves an isolated nucleic acid molecule including a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102. that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.

In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2n-l, wherein n is an integer between 1 and 102.

In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, and a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein the nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, or a complement of the nucleotide sequence.

In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein the nucleic acid molecule has a nucleotide sequence in which any nucleotide specified in the coding sequence of the chosen nucleotide sequence is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides in the chosen coding sequence are so changed, an isolated second polynucleotide that is a complement of the first polynucleotide, or a fragment of any of them.

In another embodiment, the invention includes a vector involving the nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102. This vector can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.

In another embodiment, the invention involves a method for determining the presence or amount of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, in a sample, the method including providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount of the probe bound to the nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in the sample. The presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type. The cell type can be cancerous.

In another embodiment, the invention involves a method for determining the presence of or predisposition for a disease associated with altered levels of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102, in a first mammalian subject, the method including measuring the amount of the nucleic acid in a sample from the first mammalian subject; and comparing the amount of the nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compounds. The sequences are collectively referred to herein as "NOVX nucleic acids" or "NONX polynucleotides" and the corresponding encoded polypeptides are referred to as "ΝONX polypeptides" or "ΝONX proteins." Unless indicated otherwise, "ΝONX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the ΝONX nucleic acids and their encoded polypeptides. TABLE A. Sequences and Corresponding SEQ ID Numbers

Table A indicates the homology of NOVX polypeptides to known protein families. Thus, the nucleic acids and polypeptides, antibodies and related compounds according to the invention corresponding to a NOVX as identified in column 1 of Table A will be useful in therapeutic and diagnostic applications implicated in, for example, pathologies and disorders associated with the known protein families identified in column 5 of Table A.

Pathologies, diseases, disorders, conditions and the like that are associated with NOVX sequences include, but are not limited to, e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, metabolic disturbances associated with obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, diabetes, metabolic disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, as well as conditions such as transplantation and fertility.

NONX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various ΝOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, ΝOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the ΝOVX polypeptides belong.

Consistent with other known members of the family of proteins, identified in column 5 of Table A, the ΝOVX polypeptides of the present invention show homology to, and contain domains that are characteristic of, other members of such protein families. Details of the sequence relatedness and domain analysis for each ΝOVX are presented in Example A.

The ΝOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table A.

The ΝOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of the expression analysis for each ΝOVX are presented in Example C. Accordingly, the ΝOVX nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of a variety of diseases with differential expression in normal vs. diseased tissues, e.g., detection of a variety of cancers.

Additional utilities for ΝOVX nucleic acids and polypeptides according to the invention are disclosed herein. NOVX clones

NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.

The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy. Pathological conditions can be diagnosed by determining the amount of the new protein in a sample or by determining the presence of mutations in the new genes. Specific uses are described for each of the NOVX genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.

The NOVX nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) a biological defense weapon.

In one specific embodiment, the invention includes an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ TD NO:2n, wherein n is an integer between 1 and 102, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; and (e) a fragment of any of (a) through (d).

In another specific embodiment, the invention includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102_. in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, or any variant of said polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and (f) the complement of any of said nucleic acid molecules.

In yet another specific embodiment, the invention includes an isolated nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence selected from the group consisting of SEQ ED NO:2n-l, wherein n is an integer between 1 and 102; (b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; (c) a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

NOYX Nucleic Acids and Polypeptides

One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.

A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide, precursor form, or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell (e.g., host cell) in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a post-translational modification step other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.

The term "probe", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), about 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.

The term "isolated" nucleic acid molecule, as used herein, is a nucleic acid that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3 '-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about 0.5 kb, or about 0.1 kb, of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, or of chemical precursors or other chemicals.

A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS: 2n-l, wherein n is an integer between 1 and 102, or a complement of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, (eds.), CURRENT PROTOCOLS ΓN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993).

A nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template with appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, that it can hydrogen bond with few or no mismatches to a nucleotide sequence of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, thereby forming a stable duplex.

As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

A "fragment" provided herein is defined as a sequence of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to -How for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, and is at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.

A full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking an ATG start codon therefore encodes a truncated C-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 5' direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 3' direction of the disclosed sequence.

A "derivative" is a nucleic acid sequence or amino acid sequence formed from the native compounds either directly, by modification or partial substitution. An "analog" is a nucleic acid sequence or amino acid sequence that has a structure similar to, but not identical to, the native compound, e.g. they differs from it in respect to certain components or side chains. Analogs may be synthetic or derived from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. A "homolog" is a nucleic acid sequence or amino acid sequence of a particular gene that is derived from different species.

Derivatives and analogs may be full length or other than full length. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.

A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.

The nucleotide sequences determined from the cloning of the human NO X genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102; or an anti-sense strand nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102; or of a naturally occurring mutant of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102.

Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe has a detectable label attached, e.g. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.

"A polypeptide having a biologically-active portion of a NOVX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, that encodes a polypeptide having a NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.

NOVX Nucleic Acid and Polypeptide Variants

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID NO:2n- 1, wherein n is an integer between 1 and 102. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO:2/., wherein n is an integer between 1 and 102.

In addition to the human NOVX nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID NO:2/.-l, wherein n is an integer between 1 and 102, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 65% homologous to each other typically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.

As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60 °C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS ΓN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ED NO:2n-l, wherein n is an integer between 1 and 102, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2?.-l, wherein n is an integer between 1 and 102, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Reinhardt's solution, 0.5% SDS and 100 mg ml denatured salmon sperm DNA at 55 °C, followed by one or more washes in IX SSC, 0.1% SDS at 37 °C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ED NO:2;z- 1, wherein n is an integer between 1 and 102, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50 °C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ED NO:2n-l, wherein n is an integer between 1 and 102, thereby leading to changes in the amino acid sequences of the encoded NOVX protein, without altering the functional ability of that NOVX protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:2π, wherein n is an integer between 1 and 102. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.

Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ED NO:2rc-l, wherein n is an integer between 1 and 102, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 40% homologous to the amino acid sequences of SEQ ED NO:2n, wherein n is an integer between 1 and 102. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO:2/_, wherein n is an integer between 1 and 102; more preferably at least about 70% homologous to SEQ ED NO:2n, wherein n is an integer between 1 and 102; still more preferably at least about 80% homologous to SEQ ID NO:27z, wherein n is an integer between 1 and 102; even more preferably at least about 90% homologous to SEQ ED NO:2/z, wherein n is an integer between 1 and 102; and most preferably at least about 95% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 102.

An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID NO:2n, wherein n is an integer between 1 and 102, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ED ϊ^~0:2n-l, wherein n is an integer between 1 and 102, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced any one of SEQ ID NO:27z-l, wherein n is an integer between 1 and 102, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID NO:2.z-l, wherein n is an integer between 1 and 102, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MEN, ME F_. HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STΝK, STPA, SGΝD, SΝDEQK, ΝDEQHK, ΝEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.

In one embodiment, a mutant ΝOVX protein can be assayed for (ϊ) the ability to form protein:protein interactions with other ΝOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant ΝOVX protein and a ΝOVX ligand; or (Hi) the ability of a mutant ΝOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins). In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).

Interfering RNA

In one aspect of the invention, NOVX gene expression can be attenuated by RNA interference. One approach well-known in the art is short interfering RNA (siRNA) mediated gene silencing where expression products of a NOVX gene are targeted by specific double stranded NOVX derived siRNA nucleotide sequences that are complementary to at least a 19-25 nt long segment of the NOVX gene transcript, including the 5' untranslated (UT) region, the ORF, or the 3' UT region. See, e.g., PCT applications WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304, WO02/16620, and WO02/29858, each incorporated by reference herein in their entirety. Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX gene include, e.g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX polypeptide, and polypeptides involved in a NOVX regulatory pathway.

According to the methods of the present invention, NOVX gene expression is silenced using short interfering RNA. A NOVX polynucleotide according to the invention includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a NOVX polynucleotide sequence, for example, by processing the NOVX ribopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded NOVX RNA or by chemical synthesis of nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated herein by reference in its entirety. When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

The most efficient silencing is generally observed with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang makes an additional small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases. In one embodiment, the nucleotides in the 3' overhang are ribonucleotides. In an alternative embodiment, the nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-deoxyribonucleotides in the 3' overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.

A contemplated recombinant expression vector of the invention comprises a NOVX DNA molecule cloned into an expression vector comprising operatively-linked regulatory sequences flanking the NOVX sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands. An RNA molecule that is antisense to NOVX mRNA is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the NOVX mRNA is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands may hybridize in vivo to generate siRNA constructs for silencing of the NOVX gene. Alternatively, two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct. Finally, cloned DNA can encode a construct having secondary structure, wherein a single transcript has both the sense and complementary antisense sequences from the target gene or genes. In an example of this embodiment, a hairpin RNAi product is homologous to all or a portion of the target gene. In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the NOVX sequence may be identical or may be different, such that their expression may be modulated independently, or in a temporal or spatial manner.

In a specific embodiment, siRNAs are transcribed intracellularly by cloning the NOVX gene templates into a vector containing, e.g., a RNA pol III transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA HI. One example of a vector system is the GeneSuppressor™ RNA Interference kit (commercially available from Imgenex). The U6 and HI promoters are members of the type III class of Pol HI promoters. The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for HI promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed siRNA, which is similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript. A siRNA vector appears to have an advantage over synthetic siRNAs where long term knock-down of expression is desired. Cells transfected with a siRNA expression vector would experience steady, long-term mRNA inhibition. In contrast, cells transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division. The long-term gene silencing ability of siRNA expression vectors may provide for applications in gene therapy.

In general, siRNAs are chopped from longer dsRNA by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular proteins into an endonuclease complex. In vitro studies in Drosophila suggest that the siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex, called an RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER. RISC uses the sequence encoded by the antisense siRNA strand to find and destroy mRNAs of complementary sequence. The siRNA thus acts as a guide, restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.

A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 tolOO nt downstream of the start codon. Alternatively, 5' or 3' UTRs and regions nearby the start codon can be used but are generally avoided, as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. An initial BLAST homology search for the selected siRNA sequence is done against an available nucleotide sequence library to ensure that only one gene is targeted. Specificity of target recognition by siRNA duplexes indicate that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA degradation. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88. Hence, consideration should be taken to accommodate SNPs, polymorphisms, allelic variants or species-specific variations when targeting a desired gene.

In one embodiment, a complete NOVX siRNA experiment includes the proper negative control. A negative control siRNA generally has the same nucleotide composition as the NOVX siRNA but lack significant sequence homology to the genome. Typically, one would scramble the nucleotide sequence of the NOVX siRNA and do a homology search to make sure it lacks homology to any other gene. Two independent NOVX siRNA duplexes can be used to knock-down a target NOVX gene. This helps to control for specificity of the silencing effect. In addition, expression of two independent genes can be simultaneously knocked down by using equal concentrations of different NOVX siRNA duplexes, e.g., a NOVX siRNA and an siRNA for a regulator of a NOVX gene or polypeptide. Availability of si-_-- .A-associa_.ng proteins is believed to be more limiting than target mRNA accessibility.

A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g., AA(N19)TT). A desirable spacer region has a G/C-content of approximately 30% to 70%, and more preferably of about 50%. If the sequence AA(N19)TT is not present in the target sequence, an alternative target region would be AA(N21). The sequence of the NOVX sense siRNA corresponds to (N19)TT or N21, respectively. In the latter case, conversion of the 3' end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the NOVX polynucleotide. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. Symmetric 3' overhangs may help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200, incorporated by reference herein in its entirely. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition.

Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21) sequence, one may search for the sequence NA(N21). Further, the sequence of the sense strand and antisense strand may still be synthesized as 5' (N19)TT, as it is believed that the sequence of the 3 '-most nucleotide of the antisense siRNA does not contribute to specificity. Unlike antisense or ribozyme technology, the secondary structure of the target mRNA does not appear to have a strong effect on silencing. See, Harborth, et al. (2001) J. Cell Science 114: 4557-4565, incorporated by reference in its entirety.

Transfection of NOVX siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECT AMINE Reagent (commercially available from Invitrogen). An assay for NOVX gene silencing is generally performed approximately 2 days after transfection. No NOVX gene silencing has been observed in the absence of transfection reagent, allowing for a comparative analysis of the wild-type and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-well plate, approximately 0.84 μg of the siRNA duplex is generally sufficient. Cells are typically seeded the previous day, and are transfected at about 50% confluence. The choice of cell culture media and conditions are routine to those of skill in the art, and will vary with the choice of cell type. The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful NOVX silencing. The efficiency of transfection needs to be carefully examined for each new cell line to be used. Preferred cell are derived from a mammal, more preferably from a rodent such as a rat or mouse, and most preferably from a human. Where used for therapeutic treatment, the cells are preferentially autologous, although non-autologous cell sources are also contemplated as within the scope of the present invention.

For a control experiment, transfection of 0.84 μg single-stranded sense NOVX siRNA will have no effect on NOVX silencing, and 0.84 μg antisense siRNA has a weak silencing effect when compared to 0.84 μ,g of duplex siRNAs. Control experiments again allow for a comparative analysis of the wild-type and silenced NOVX phenotypes. To control for transfection efficiency, targeting of common proteins is typically performed, for example targeting of lamin A/C or transfection of a CMV-driven EGFP-expression plasmid (e.g. commercially available from Clontech). In the above example, a determination of the fraction of lamin A/C knockdown in cells is determined the next day by such techniques as immunofluorescence, Western blot, Northern blot or other similar assays for protein expression or gene expression. Lamin A/C monoclonal antibodies may be obtained from Santa Cruz Biotechnology.

Depending on the abundance and the half life (or turnover) of the targeted NOVX polynucleotide in a cell, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no NOVX knock-down phenotype is observed, depletion of the NOVX polynucleotide may be observed by immunofluorescence or Western blotting. If the NOVX polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the targeted protein is observed, it may be desirable to analyze whether the target mRNA (NOVX or a NOVX upstream or downstream gene) was effectively destroyed by the transfected siRNA duplex. Two days after transfection, total RNA is prepared, reverse transcribed using a target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable NOVX protein may exist in the cell. Multiple transfection in sufficiently long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfection steps are required, cells are split 2 to 3 days after transfection. The cells may be transfected immediately after splitting.

An inventive therapeutic method of the invention contemplates administering a NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX expression or activity. The NOVX ribopolynucleotide is obtained and processed into siRNA fragments, or a NOVX siRNA is synthesized, as described above. The NOVX siRNA is administered to cells or tissues using known nucleic acid transfection techniques, as described above. A NOVX siRNA specific for a NOVX gene will decrease or knockdown NOVX transcription products, which will lead to reduced NOVX polypeptide production, resulting in reduced NOVX polypeptide activity in the cells or tissues.

The present invention also encompasses a method of treating a disease or condition associated with the presence of a NOVX protein in an individual comprising administering to the individual an RNAi construct that targets the mRNA of the protein (the mRNA that encodes the protein) for degradation. A specific RNAi construct includes a siRNA or a double stranded gene transcript that is processed into siRNAs. Upon treatment, the target protein is not produced or is not produced to the extent it would be in the absence of the treatment.

Where the NOVX gene function is not correlated with a known phenotype, a control sample of cells or tissues from healthy individuals provides a reference standard for determining NOVX expression levels. Expression levels are detected using the assays described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the like. A subject sample of cells or tissues is taken from a mammal, preferably a human subject, suffering from a disease state. The NOVX ribopolynucleotide is used to produce siRNA constructs, that are specific for the NOVX gene product. These cells or tissues are treated by administering NOVX siRNA's to the cells or tissues by methods described for the transfection of nucleic acids into a cell or tissue, and a change in NOVX polypeptide or polynucleotide expression is observed in the subject sample relative to the control sample, using the assays described. This NOVX gene knockdown approach provides a rapid method for determination of a NOVX minus (NOVX^") phenotype in the treated subject sample. The NOVX^" phenotype observed in the treated subject sample thus serves as a marker for monitoring the course of a disease state during treatment.

In specific embodiments, a NOVX siRNA is used in therapy. Methods for the generation and use of a NOVX siRNA are known to those skilled in the art. Example techniques are provided below.

Production of RNAs

Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using known methods such as transcription in RNA expression vectors. In the initial experiments, the sense and antisense RNA are about 500 bases in length each. The produced ssRNA and asRNA (0.5 μM) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCl were heated to 95° C for 1 min then cooled and annealed at room temperature for 12 to 16 h. The RNAs are precipitated and resuspended in lysis buffer (below). To monitor annealing, RNAs are electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide. See, e.g., Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989).

Lysate Preparation

Untreated rabbit reticulocyte lysate (Ambion) are assembled according to the manufacturer's directions. dsRNA is incubated in the lysate at 30° C for 10 min prior to the addition of mRNAs. Then NOVX mRNAs are added and the incubation continued for an additional 60 min. The molar ratio of double stranded RNA and mRNA is about 200:1. The NOVX mRNA is radiolabeled (using known techniques) and its stability is monitored by gel electrophoresis.

In a parallel experiment made with the same conditions, the double stranded RNA is internally radiolabeled with a ³²P-ATP. Reactions are stopped by the addition of 2 X proteinase K buffer and deproteinized as described previously (Tuschl et ah, Genes Dev., 13:3191-3197 (1999)). Products are analyzed by electrophoresis in 15% or 18% polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gels for radioactivity, the natural production of 10 to 25 nt RNAs from the double stranded RNA can be determined. The band of double stranded RNA, about 21-23 bps, is eluded. The efficacy of these 21-23 mers for suppressing NOVX transcription is assayed in vitro using the same rabbit reticulocyte assay described above using 50 nanomolar of double stranded 21-23 mer for each assay. The sequence of these 21-23 mers is then determined using standard nucleic acid sequencing techniques.

RNA Preparation

21 nt RNAs, based on the sequence determined above, are chemically synthesized using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany). Synthetic oligonucleotides are deprotected and gel-purified (Elbashir, Lendeckel, & Tuschl, Genes & Dev. 15, 188-200 (2001)), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al., Biochemistry, 32:11658-11668 (1993)).

These RNAs (20 μM) single strands are incubated in annealing buffer (100 M potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C followed by 1 h at 37° C.

Cell Culture

A cell culture known in the art to regularly express NOVX is propagated using standard conditions. 24 hours before transfection, at approximately 80% confluency, the cells are trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105 cells/ml) and transferred to 24-well plates (500 ml/well). Transfection is performed using a commercially available lipofection kit and NOVX expression is monitored using standard techniques with positive and negative control. A positive control is cells that naturally express NOVX while a negative control is cells that do not express NOVX. Base-paired 21 and 22 nt siRNAs with overhanging 3' ends mediate efficient sequence-specific mRNA degradation in lysates and in cell culture. Different concentrations of siRNAs are used. An efficient concentration for suppression in vitro in mammalian culture is between 25 nM to 100 nM final concentration. This indicates that siRNAs are effective at concentrations that are several orders of magnitude below the concentrations applied in conventional antisense or ribozyme gene targeting experiments.

The above method provides a way both for the deduction of NOVX siRNA sequence and the use of such siRNA for in vitro suppression. In vivo suppression may be performed using the same siRNA using well known in vivo transfection or gene therapy transfection techniques.

Antisense Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:27z-l, wherein n is an integer between 1 and 102, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ED NO:27z, wherein n is an integer between 1 and 102, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ED NO:2n-l, wherein n is an integer between 1 and 102, are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a NOVX protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).

Examples of modified nucleotides that can be used to generate the antisense. nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diamiιιopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol HI promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.

Ribozymes and PNA Moieties

Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i.e., SΕQ D NO:2n-l, wherein n is an integer between 1 and 102). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.

In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleotide bases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomer can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.

PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S_ nucleases (See, Hyrup, et al, 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).

In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drag delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleotide bases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et ah, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

NOVX Polypeptides

A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in any one of SEQ ED NO:2τ., wherein n is an integer between 1 and 102. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID NO:27i, wherein n is an integer between 1 and 102, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.

In general, a NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.

One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.

Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence of SEQ ID NO:2τz, wherein n is an integer between 1 and 102) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.

Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.

In an embodiment, the NOVX protein has an amino acid sequence of SEQ ED NO:2/z, wherein n is an integer between 1 and 102. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 102, and retains the functional activity of the protein of SEQ ID NO:2n, wherein n is an integer between 1 and 102, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 102, and retains the functional activity of the NOVX proteins of SEQ ED NO:2?ι, wherein n is an integer between 1 and 102.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").

The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102.

The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) 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 region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

Chimeric and Fusion Proteins

The invention also provides NOVX chimeric or fusion proteins. As used herein, a NOVX "chimeric protein" or "fusion protein" comprises a NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a NOVX protein of SEQ ED NO:2τι, wherein n is an integer between 1 and 102, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within a NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of a NOVX protein. In one embodiment, a NOVX fusion protein comprises at least one biologically-active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically-active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically-active portions of a NOVX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

In one embodiment, the fusion protein is a GST-NO X fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-tr ansferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.

In another embodiment, the fusion protein is a NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.

A NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by 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, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.

NOVX Agonists and Antagonists

The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the NOVX proteins.

Variants of the NOVX proteins that function as either NOVX agonists (z.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et ah, 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries

In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S_\ nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX proteins.

Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include 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 isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein Engineering 6:327-331.

NOVX Antibodies

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_a , F_at_>' and F(_a_.)2 fragments, and an F_ab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG_, IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID NO:2?ι, wherein n is an integer between 1 and 102, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX protein sequence will indicate which regions of a NOVX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. A NOVX polypeptide or a fragment thereof comprises at least one antigenic epitope. An anti-NOVX antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (K_D) is <1 μM, preferably < 100 nM, more preferably < 10 nM, and most preferably < 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.

Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are 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, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

Monoclonal Antibodies

The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dβkker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem.. 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No.4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. Humanized Antibodies

The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature. 332:323-327 (1988); Verhoeyen et al., Science. 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. BioL 2:593-596 (1992)).

Human Antibodies

Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al .( Nature Biotechnology 14. 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).

Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

Fab Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F_a expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F_ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(_{a '})2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F_a fragment generated by reducing the disulfide bridges of an F(_at>')₂ fragment; (iii) an F_ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F_v fragments.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J„ 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytic ally cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the H and V domains of one fragment are forced to pair with the complementary V_L and _H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRDI (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioelher bond. Examples of suitable reagents for this purpose include immothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

Effector Function Engineering

It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.. 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design. 3: 219-230 (1989).

Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹1, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science. 238: 1098 (1987). Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.

Immunoliposomes

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA. 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81_.(19): 1484 (1989).

Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention

Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds (see below).

An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H.

Antibody Therapeutics

Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.

Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.

A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York. If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

ELISA Assay

An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., F_a or F_(a )₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELIS As), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. NOVX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, useful expression vectors in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS ΓNENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculo virus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith and Johnson, 1988. Gene 61: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lid (Studiβr et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986. Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotiexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.

Transgenic NOVX Animals

The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 102, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of any one of SEQ ED NOS:2n-l, wherein n is an integer between 1 and 102), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ED NOS:2n-l, wherein n is an integer between 1 and 102, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).

Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3 '-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.

The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.

Pharmaceutical Compositions

The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppos: itory bases such as cocoa butter and other glycerides) or retention enemas for rectal del: ivery. In one embod: iment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

Il is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Screening and Detection Methods

The isolated nucleic acid molecules of the invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.

The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.

Screening Assays

The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, .e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. The invention also includes compounds identified in the screening assays described herein.

In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a NOVX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; 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 approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.

A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, et al, 1993. Science 261: 1303; Carrell, et al, 1994. Angew . Chem. Int. Ed. Engl 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test 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. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX 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 NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule. As used herein, a "target molecule" is a molecule with which a NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule, a NOVX target molecule can be a non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one embodiment, a NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.

Determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca , diacylglycerol, EP₃, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or delecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.

In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound lo bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX 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 NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.

In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to a NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate a NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.

In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein 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 NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule.

The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, li-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton^® X-100, Triton^® X-114, Thesit^®, Isotridecypoly(ethylene glycol ether)_n, N-dodecyl~N,N-dimethyl-3-ammonio-l-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-chol-ιmidopropyl)dimethylartιminiol-2-hydroxy-l-propane sulfonate (CHAPSO).

In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX 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 that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NO VX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to 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, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX 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 NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.

In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.

In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Cell 72: 223-232; Madura, et al, 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.

The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (Hi) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.

Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of a NOVX sequence, i.e., of SEQ ED NOS:2n-l, wherein n is an integer between 1 and 102, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.

Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787.

Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms. Tissue Typing

The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No. 5,272,057).

Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ED NOS:2n-l, wherein n is an integer between 1 and 102, are used, a more appropriate number of primers for positive individual identification would be 500-2,000. Predictive Medicine

The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.

Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials. These and other agents are described in further detail in the following sections. Diagnostic Assays

An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ED NOS:2n-l, wherein n is an integer between 1 and 102, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELIS As), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).

The methods of the invention can also be used to detect genetic lesions in a NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an addition of one or more nucleotides to a NOVX gene; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (vϋ) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells. In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al, 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NONX-gene (see, Abravaya, et al, 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRΝA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a ΝOVX gene under conditions such that hybridization and amplification of the ΝOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a ΝOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DΝA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DΝA indicates mutations in the sample DΝA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in NOVX can be identified by hybridizing sample and control nucleic acids, e.g., DNA or RNA to high-density arrays containing hundreds or thousands of oligonucleotide probes. See, e.g., Cronin, et al., 1996. Human Mutation 1: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NONX can be identified in two-dimensional arrays containing light-generated DΝA probes as described in Cronin, et al, supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DΝA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the ΝONX gene and detect mutations by comparing the sequence of the sample ΝOVX with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Νaeve, et al, 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al., 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol 38: 147-159).

Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S_\ nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Car ino genesis 15: 1657-1662. According to an exemplary embodiment, a probe based on a NOVX sequence, e.g., a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal Tech. Appl 9: 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 1: 5.

In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al, 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al, 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3 '-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a NONX gene.

Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which ΝONX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect on ΝOVX activity (e.g., ΝOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of ΝOVX protein, expression of ΝOVX nucleic acid, or mutation content of ΝOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome Pregnancy Zone Protein Precursor enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drag effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for

CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drag responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.

By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.

In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.

Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.

These methods of treatment will be discussed more fully, below.

Diseases and Disorders

Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like). Prophylactic Methods

In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating NOVX expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.

Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia). Determination of the Biological Effect of the Therapeutic

In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue. In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.

Prophylactic and Therapeutic Uses of the Compositions of the Invention

The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune , disorders, hematopoietic disorders, and the various dyslipidemias.

Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example A: Polynucleotide And Polypeptide Sequences, And Homology Data

The NOV1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1 A.

KCPGRPWKSVSEINPTTQMKESYYFDLTDG S

NOVlb, CG104903-03 SEQ ID NO: 3 1981 bp DNA Sequence ORF Start: ATG at 50 fORF Stop: end of sequence

AATTCGGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACC-ACTCAATTGTGCAΆACGAATTGTTCCAAAGAGAATT

TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA ACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAΆTAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC GTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACG-GACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCA-GTCCT-GACCATGGACA-AAGCATAAGCATGG-CA-GGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NOVlb, CG104903-03 SEQ ED NO: 4 644 aa MW at71956.8kD Protein Sequence ]

MKLITILFLCSRL LSLTQESQSEEIDC-TOKD F--AV_⁾A-- KKYNSQNQSra!.QFVLYRITEATKTVGS

DTFYSFKYEIKΞGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPW

TAQYDC GCVHPISTQSPDLEPI RHGIQYFN-INTQHSS FM NEVKRAQRQVVAGLNFRITYSIVQT

NCSKENF FLTPDCKS W GDTGECTD-IAYIDIQ RIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP

TNSPE EETLTHTITKLNAENNATFYF ID-JVKKARVQWAGKKYFIDFVARETTCSKESNEELTESC

ETKKLGQS DCNAEVYWP EKKIYPTV CQPLG IS MKRPPGFSPFRSSRIGEIKEETTVSPPHTS APAQDEERDSGKEQGHTRRHDWGHEKQRKHN GHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK

FI-LDDDIiEHQGG--V_₁DHGH--HKHGHGHG-_I-_^GK-α_G-_INGWKTEHLASSSEDSTTPSAQTQEKTE

PTPIPS AKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNG SFNPISDFPDTTSP

KCPGRP KSVSEINPTTQMKΞSYYFDLTDG S

NOVlc, 316514816 SEQ ID NO: 5 1935 bp DNA Sequence ORF Start: at 1 ORF Stop: TAG at 1933

ATGAAACTAATTACCATCCTTTTCCTCTGCTCCAGGCTACTACTAAGTTTAAC

CCAGGAATCACAGTCCGAGGAAATTGACTGCAATGACAAGGATTTATTTAAAGCTGTGGATGCTGCTC

TGAAGAAATATAACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCATAACTGAAGCCACT

AAGACGGTTGGCTCTGACACGTTTTATTCCTTCAAGTACGAAATCAAGGAGGGGGACTGTCCTGTTCA

AAGTGGCAAAACCTGGCAGGACTGTGAGTACAAGGATGCAGCAAAAGCAGCCACTGGAGAATGCACGG

CAACCGTGGGGAAGAGGAGCAGTACGAAATTCTCCGTGGCTACCCAGACCTGCCAGATTACTCCAGCC

GAGGGCCCTGTGGTGACAGCCCAGTACGACTGCCTCGGCTGTGTGCATCCTATATCAACGCAGAGCCC

AGACCTGGAGCCCATTCTGAGACACGGCATTCAGTACTTTAACAACAACACTCAACATTCCTCCCTCT

TCACGCTTAATGAAGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACTTTCGAATTACCTAC

TCAATTGTGCAAACGAATTGTTCCAAAGAGAATTTTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTG

GAATGGTGATACCGGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATTGCTTCCTTCT

CACAGAACTGTGACATTTATCCAGGGAAGGATTTTGTACAACCACCTACCAAGATTTGCGTGGGCTGC

CCCAGAGATATACCCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCACAAAGCTTAA

TGCAGAGAATAACGCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTACAGGTGGTGG CTGGCAAGAAATATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGAG TTGACCGAAAGCTGTGAGACCAAAΆAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTGGT ACCCTGGGAGAAAAAAATTTACCCTACTGTCAACTGTCAACCACTGGGAATGATCTCACTGATGAAAA GGCCTCCAGGTTTTTCACCTTTCCGATCATCACGAATAGGGGAAATAAAAGAAGAAACAACTGTAAGT CCACCCCACACTTCCATGGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCATAC TCGTAGACATGACTGGGGCCATGA--AAACAAAGAAAACATAATCTTGGCCATGGCCATAAACATGAAC GTGACCAAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTCTT GGTCATGGACATAAGTTCAAACTTGATGATGATCTTGAACACCAAGGGGGCCATGTCCTTGACCATGG ACATAAGCATAAGCATGGTCATGGCCACGGAAAACAT-__-AATAAAGGCAAAAAGAATGGAAAGCACA ATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTCTGAAGACAGTACTACACCTTCTGCACAGACACAA GAGAAGACAGAAGGGCCAACACCCATCCCTTCCCTAGCCΑAGCCAGGTGTAACAGTTACCTTTTCTGA CTTTCAGGACTCTGATCTCATTGCAACTATGATGCCTCCTATATCACCAGCTCCCATACAGAGTGATG ACGATTGGATCCCTGATATCCAGATAGACCCAAATGGCCTTTCATTTAACCCAATATCAGATTTTCCA GACACGACCTCCCCAAAATGTCCTGGACGCCCCTGGAAGTCAGTTAGTGAAATTAATCCAACCACACA AATG-__AGAA_CT_ATTAT_TCGA_C-CACTGA_GGCC_T-CTTJ__

NOVlc, 316514816 SEQ ID NO: 6 644 aa MW at 71927.7 kD jProtein Sequence

MKLITILF CSRL LS TQESQSEEIDC-TOKDLFI-AVO---- KKYNSQNQS NQFV YRITEAT

KTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPA

EGPVVTAQYDC GCVΗPISTQSPD EPILRHGIQYF-T-I TQHSSLFTLNEVKRAQRQVVAGLNFRITY

SIVQTNCSKENFLF TPDCKSL NGDTGECTDNAYIDIQ RIASFSQNCDIYPGKDFVQPPTKICVGC

PRDIPTNSPELEETLTHTITK NAE-πSTATFYFKID VKKARVQVVAGK YFIDFVARETTCSKESNEE TESCETKK GQS DCNA__n-VVP E KIYPTV_ICQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVS

PPHTS-__PAQDEERDSGKEQGHTRRHD GHEKQR-_-NLGHGHKHERDQGHGHQRGHG GHGHEQQHG

GHGHKFK DDD EHQGGHVLDHGH_Η-_.GHGHGKH---^GK-_.GKHNGWKTEH ASSSEDSTTPSAQTQ

,EKTEGPTPIPSL--KPGVTVTFSDFQDSD_,IATMMPPISPAPIQSDDD IPDIQIDPNGLSFNPISDFP

DTTSPKCPGRP KSVSEINPTTQMKESYYFDLTDGI.S

NOVld, 308780224 SEQ ED NO: 7 327 bp DNA Sequence ORF Start: at 1 ORF Stop: end of sequence

TGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGAC

ATGACTGGGGCCATGAAAAACAAAGAAAACATAATCTTGGCCATGGCCATA-^ACATGAACGTGACCAA)

GGGCATGGGCACCAAAGAGGACATGGCCT-GGCCATGGACACGAACAACAGCA-GG-CTTGG-CA-GG

ACATAAG-TCAAACTTGATGATGATCTTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGC

ATAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGG

NOVld, 308780224 SEQ ED NO: 8 109 aa MW at 12335.1 kD Protein Sequence

APAQDEERDSGKEQGHTRRHD GHE QRKHNLGHGHKHERDQGHGHQRGHG GHGHEQQHG GHG HKF LDDD EHQGGHVLϋHGHKHKHGHGHGKHKNKGKNGKHNG

NOVle, 308900326 SEQ ID NO: 9 1071 bp DNA Sequence ORF Start: at 1 ORF Stop: end of sequence

ATGAAACTAATTACCATCCTTTTCCTCTGCTCCAGGCTACTAC-AAGTTTAA

CCCAGGAATCACAGTCCGAGGAAATTGACTGCAATGACAAGGATTTA-TTAAAGCTGTGGATGCTGCT

CTGAAGAAATATAACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCATAACTGAAGCCAC

TAAGACGGTTGGCTCTGACACGTTTTATTCCTTCAAGTACGAAATCAAGGAGGGGGATTGTCCTGTTC

AAAG-GGCAAAACCTGGCAGGACTGTGAG-ACAAGGA-GCTGCAAAAGCAGCCAC-GGAGAA-GCACG

GCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTCCGTGGCTACCCAGACCTGCCAGATTACTCCAGC

CGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCCTCGGCTGTGTGCATCCTATATCAACGCAGAGCC

CAGACCTGGAGCCCATTCTGAGACACGGCATTCAGTACTTTAACAACAACACTCAACATTCCTCCCTC

TTCATGCTTAATGAAGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACTTTCGAATTACCTA

CTCAATTGTGCAAACGAATTGTTCCAAAGAGAATTTTCTGTTCTTAACTCCAGACTGCAAGTCCCTTT

GGAATGGTGATACCGGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATTGCTTCCTTC

TCACAGAACTGTGACATTTATCCAGGGAAGGATTTTGTACAACCACCTACCAAGATTTGCGTGGGCTG

CCCCAGGGATATACCCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCACAAAGCTTA

ATGCAGAGAATAACGCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTACAGGTGGTG

GCTGGCAAGAAA-ATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGA

GTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTG

NOVle, 308900326 SEQ ID NO: 10 357 aa MW at 40027.4 kD Protein Sequence

M-_-_- _ __-F__-SM-__LSLTOESOSEΞIDC-TOKDLF_-AVD

AGAAATATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACC GAAAGCTGTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTG GGAGAAAAAAATTTACCCTACTGTCAACTGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTC CAGGTTTTTCACCTTTCCGATCATCACGAATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCC CACACTTCCATGGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAG ACATGACTGGGGCCATGA--AACAAAGAAAACATAATCTTGGCCATGGCCATAAACATGAACGTGACC AAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCAT GGACATAAGTTCAAACTTGATGATGATCTTGΆACACCAAGGGGGCCATGTCCTTGACCATGGACATAA GCAT--AGCΑTGGTCATGGCCACGGA--AΑCATAAAAATAAAGGC--AAAAGAATGGAAAGCACAATGGTT GGAAAACAGAGCATTTGGCAAGCTCTTCTGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAG ACAGAAGGGCCAACACCCATCCCTTCCCTAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCA GGACTCTGATCTCATTGCAACTATGATGCCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATT GGATCCCTGATATCCAGATAGACCCAAATGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACG ACCTCCCCAAAATGTCCTGGACGCCCCTGGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAA AGAATCTTATTATTTCGATCTCACTGATGGCCTTTCT

NOVli, 311750024 SEQ ID NO: 18 622 aa MW at 69424.4kD Protein Sequence

SEEIDC1_DKD FKAVOAALKKYNSQNQS-_.QFV YRITEATKTVGSDTFYSFKYEIKEGDCPVQSG

KTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPWTAQYDC GCVHPISTQSPD

EPILRHGIQYFN__.TQHSS FM NE - _.RAQRQVVAGLNFRITYSIVQTNCSKE_.F FLTPDCKSLW-.G

DTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAE

N-IATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKΞSNEE TESCETKKLGQSLDCNAΞVYVVPW

EKKIYPTVNCQP GMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTS APAQDEERDSGKΞQGHTRR

HD GHEKQRKHN GHGHKHERDQGHGHQRGHGLGHGHEQQHG GHGHKFKLDDD EHQGGHV DHGHK

HKHGHGHGKHK KGKK GKH GWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPS AKPGVTVTFSDFQ

DSDLIATMMPPISPAPIQSDDDWIPDIQIDPNG SFNPISDFPDTTSPKCPGRP KSVSΞINPTTQMK

ESYYFDLTDG S

NOVlj, CG104903-01 SEQ ED NO: 19 1357 bp

DNA Sequence JORF Start: ATG at 1 JORF Stop: TGA at 1195

ATGAAACTAATTACCA-CCTTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTC CGAGGAAATTGATGACTGCAATGACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATA ACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACAGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCACGCT-AATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATG-ACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACT-CGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCTCACTGA-GAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTAGTCACCTAAGGTCCTGCGAGTACAAGGGTCGACCCCCAA AGGCAGGGGCAGAGCCAGCATCTGAGAGGGAGGTCTCTTGACCAATGGGCAGAATCTTCACTCCAGGC

ACATAGCCCCAACCACCTCTGCCAGCAACCTTGAGAGGAAGGACAAGAAGAAAGATGGGATAGAATTT

AAATAGAGAAGAATGCCATTTTATCACTCTGCCTCTGGGTGAAATAAAGATCAGTCTTGATGTTC

NOVlj, CG104903-01 SEQ ED NO: 20 398 aa MW at 44684. ll D

Protein Sequence

M__-,ITI F CSRL SLTQESQSEΞIDDCl roKDLFKA - _)AA KKY SQNQSI QFV YRKTWQDCEYK DAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPWTAQYDC GCVHPISTQSPDLEPI RHGIQ YFN-J-ITQHSSLFT NEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDCKSL GDTGECTDNA YIDIQ RIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKI DNVKI-_RVQ\A AGKKYFIDFVARETTCSKΞSNEELTESCETKKLGQSLDCNAEVYVVPWΞKKIYPTV-I CQPLGMISLMKRPPGFSPFRSSRIGEIKEΞTTSH RSCΞYKGRPPKAGAEPASEREVS

NOVlk, CG104903-02 SEQ ID NO: 21 ^"p48 bp^~

DNA Sequence JORF Start: ATG at 1 RF Stop: TA A at 1846

ATGAAACTAATTACCATCCTTTTCCTCTGC-CCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTC CGAGGAAATTGATGACTGCAATGACAAGGATTTATTT-^GCTGTGGATG^ ACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACAGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CG_GGC_ACCCAGACC-GCCAGAT_AC-CCAGCCGAGGGCCCTGTGG-GACAGCCCAG-ACGAC_GCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCACGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGAT-GAACTTTCGAATTACC-ACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCC-TCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TG-ACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACAC-GACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGA_T GACAATGTGAAAΆAAGCAAGAGTACAGGTGGTGGCTGGCAΆGAAATATTTTATTGACTTCGTGGCCAG GGAAACCΆCATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAA--AAATTTACCCTACTG-CAAC TGTCAACCACTGGGAATGA_C-CACTGA-GAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCA-GGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TGAACACCAAGGGGGCCATGTCCTTGACCATGGACATΆAGCATAΆGCATGGTCATGGCCACGGAAAΆ CATAAAAATA--AGGCAAAAΑGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAΑGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGACAGACCCAAA TGGCCTT_CATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCC_GGACGCCCC_ GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGA-CTCAC-GAT GGCCTTTCTTAA

NOVlk, CG104903-02 SEQ EDNO: 22 615 aa MW at 68746.lkD Protein Sequence

MlITI F CS-i S TQESQSEΞIDDC-roKD FKAVDAA KKY-ISQNQS-INQFVLYRKTWQDCEYK DAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPWTAQYDCLGCVHPISTQSPD EPILRHGIQ YF-J-E.TQHSSLFT NEV -AQRQVVAGLNFRITYSIVQTNCSKENF FIJTPDCKS WNGDTGΞCTDNA YIDIQLRIASFSQNCDIYPG DFVQPPTKICVGCPRDIPTNSPE EETLTHTITKNAEIMNATFYFKI D_T^K-_RVQ-VAGKKYFIDFVARET_CSKESNEE TESCETKK GQSLDC-IAEVYVVP EKKIYPTV3SI CQP GMISLMKRPPGFSPFRSSRIGEI EETTVSPPHTSMAPAQDEERDSGKEQGHTRRHD GHE QR -ϋNπjGHGHKHERDQGHGHQRGHGI-GHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGH HKHGHGHGK HKN G KNGKHNG KTEH ASSSEDSTTPSAQTQEKTEGP IPS AKPGVTVTFSDFQDSDLIATMM PPISPAPIQSDDDWIPDIQTDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFD TD GLS

NOV11, CG104903-04 SEQ ED NO: 23 1380 bp

DNA Sequence ORF Start: ATG at 3 JORF Stop: TGA at 1284 CATGAAACTAATTACCATCCTTTTCCTCTGCTCCAGGCTGCTACTAAG-TTAACCCAGGAATCACAG TCCGAGGAAATTGACTGCAATGACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAA CAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCT CTGACACGTTTTATTCCTTCAAGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACC TGGCAGGACTGTGAG-ACAAGGA-GC-GCAAAAGCAGCCAC-GGAGAATGCACGGCAACCGTGGGGAA GAGGAGCAGTACGAAATTCTCCGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGG TGACAGCCCAGTACGACTGCCTCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCC ATTCTGAGACACGGCATTCAGTACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGA AGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAA CGAATTGTTCCAAAGAGAATT-TCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACC GGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGA CATTTATCCAGGGAAGGATTTTGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATAC CCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAAC GCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATA TTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCT GTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAA AAAATTTACCCTACTGTCAACTGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTT TTCACCT-TCCGATCATCACGAATAGGGGAAATAAAAGAAGAAACAACTAGTCACCTAAGGTCCTGCG AGTACAAGGGTCGACCCCCAAAGGCAGGGGCAGAGCCAGCATCTGAGAGGGAGGTCTCTTGACCAATG

GGCAGAATCTTCACTCCAGGCACATAGCCCCAACCACCTCTGCCAGCAACCTTGAGAGGAAGGACAAG

AAGAAAGATGGGATAGAATT

NOV11, CG104903-04 SEQ ED NO: 24 427 aa MW at 47882.7kD Protein Sequence M-_-,ITILFLCS-i-. SLTQΞSQSEEIDClTOKO F-_ VDAA KKYNSQNQS__.QFV YRITEATKTVGS DTFYSFKYEI ΞGDCPVQSGKT QDCEYKDAAKAATGΞCTATVGKRSSTKFSVATQTCQITPAEGPW TAQYDCLGCVHPISTQSPDLEPIL--HGIQYFKtt_.TQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQT NCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETLTHTITK NAΞ ATFYFKID-IVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESC ETKKLGQS DCNAEVYWP EKKIYPTV-ICQPLGMISLMKRPPGFSPFRSSRIGEIKEETTSH RSCE YKGRPPKAGAEPASEREVS

NOVlm, CG104903-05 jSEQ ID NO: 25 1297 bp DNA Sequence JORF Start: ATG at 50 JORF Stop: TAA at 1295

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACAGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGGTTTTTCACCTTTCCGATCATCACGAATAGGG GAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATGAAGAGCG GGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGAAAACATA ATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGGCCTTGGC CATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATCTTGAACA CCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAACATAAAA ATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTCTGAAGAC AGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCCTAGCCAA GCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATGCCTCCTA TATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAATGGCCTT TCAT-TAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCC-GGAAGTC AGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGATGGCCTTT CTTAA

NOVlm, CG104903-05 SEQ ID NO: 26 415 aa MW at 45897.3kD Protein Sequence

M-_-,ITILFLCS-- LLS TQESQSEEIDCKTOKDLF-_ VD-^ -,KKYNSQNQSNNQFVLYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKT QDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPW TAQYDC GCVHPISTQSPGFSPFRSSRIGEIKEETTVSPPHTS APAQDΞERDSGKEQGHTRRHDWGH EKQRKH-JLGHGHKHERDQGHGHQRGHG GHGHEQQHGLGHGHKFK DDDLEHQGGHVLDHGHKHKHGH GHGKHKNKGKKNGKHNGWKTEH ASSSEDSTTPSAQTQEKTΞGPTPIPSLAKPGVTVTFSDFQDSDLI ATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRP KSVSEINPTTQMKESYYF D TDGLS

NOVln, CG104903-06 SEQ ID NO: 27 1892 bp

DNA Sequence |θRF Start: ATG at 50 JORF Stop: TAA at 458

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGT-TAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GA_GCTGCAAAAGCAGCCACTGGAGAATGCACAGCAACCGTGGGAAGAGGAGCAGTACGAAATTCTCC GTGGCTACCCAGACCTGGAGCCCA-TC-GAGACACGGCAT-CAGTAC---AACAACAACAC-CAΑCAT

TCCTCCCTCTTCACGCTTAATGAAGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACTTTCG lAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATTTTCTGTTCTTAACTCCAGACTGCA

AGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATT

GCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTTTG-ACAACCACCTACCAAGATTTG!

CGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCAI

CAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTAl

CAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAG;

TAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGi

TTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAACTGTCAACCACTGGGAATGATCTCA;

CTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACGAATAGGGGAAATAAAAGAAGAAAC lAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAAC lAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGAAAACATAATCTTGGCCATGGCCAT lAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAl

GCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATCTTGAACACCAAGGGGGCCATGTCC:

TTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAAT;

NOVlq, CG104903-11 SEQ ED NO: 34 16 aa! MW at l862.2kD Protein Sequence | I

HKWKGKK GKHNG KT

NOVlr, CG104903-12|SEQ ID NO: 35J48 bp DNA Sequence ORF Start: at 1 JORF Stop: end of sequence: lAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAAT

NOVlr, CG104903-12 SEQ ID NO: 36 16 aa MW at 1792.1kD| Protein Sequence

KHGHGHGKHKNKG KN

NOVls, CG104903-13JSEQ ID NO: 37J48 bp DNA Sequence ORF Start: at 1 jORF Stop: end of sequence

GTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA

NOVls, CG104903-13 SEQ ID NO: 38 16 aa MW at 1781.0kD [Protein Sequence j

V DHGHKHKHGHGHGK

NOVlt, CG104903-14JSEQ ID NO: 39|48 bp

DNA Sequence ORF Start: at lJORF Stop: end of sequence

GGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCAT

NOVlt, CG104903-14 SEQ ID NO: 40 16 aa MW at 1657.8kD! Protein Sequence

GHGLGHGHEQQHGLGH

NOVlu, CG104903-15JSEQ ID NO: 41|48 bp DNA Sequence ORF Start: at 1 ORF Stop: end of sequence

GACCAAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACAC

NOVlu, CG104903-15 SEQ ED NO: 42i 16 aa MW at 1686.8kDi Protein Sequence

DQGHGHQRGHGGHGH

NOVlv, CG104903-16 SEQ ID NO: 43 1863 bp DNA Sequence ORF Start: at 1 ORF Stop: at 1863

GAGGAAATTGACTGCAATGACAAGGAT-TATTTAAAGCTGTGGATGCTGCTCTGAAGA

AATATAACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACG

GTTGGCTCTGACACGTTT-ATTCCTTCAAGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGG

CAAAACCTGGCAGGACTGTGAGTACAAGGATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCG

TGGGGAAGAGGAGCAGTACGAAATTCTCCGTGGCTACCCAGACC-GCCAGATTACTCCAGCCGAGGGC

CCTGTGGTGACAGCCCAGTACGACTGCCTCGGCTGTG-GCATCCTATATCAACGCAGAGCCCAGACCT

GGAGCCCA-TCTGAGACACGGCATTCAGTACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGC

T_AATGAAGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACT_TCGAATTACC_ACTCAATT

GTGCAAACGAATTGTTCCAAAGAGAATTTTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGG

TGATACCGGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGA

ACTGTGACATTTATCCAGGGAAGGATTTTGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGG

GATATACCCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGA

GAATAACGCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCA

AGAAATATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACC

GAAAGCTGTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTG

GGAGAAAAAAATTTACCCTACTGTCAACTGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTC

CAGGTTTTTCACCTTTCCGATCATCACGAATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCC

CACACTTCCATGGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAG

ACATGACTGGGGCCATGAAAAACAAAGAAAACATAATCTTGGCCATGGCCATAAACATGAACGTGACC

AAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCAT

GGACATAAGTTCAAACTTGATGATGATCTTGSACACCAAGGGGGCCATGTCCTTGACCATGGACAT.AA

GCATAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTT GGAAAACAGAGCATTTGGCAAGCTCTTCTGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAG ACAGAAGGGCCAACACCCATCCCTTCCCTAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCA GGACTCTGATCTCATTGCAACTATGATGCCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATT GGATCCCTGATATCCAGATAGACCCAAATGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACG ACCTCCCCAAAATGTCCTGGACGCCCCTGGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAA AGAA_CT-A_TATT-CGATC-CAC-GA-GGCCTTTCT

NOVlv, CG104903-16 SEQ ID NO: 44 621 aa MW at 69336.6kD Protein Sequence

EEIDC_TDKDLF-_ λ -__LKKY_ISQNQS__.QFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKT QDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPWTAQYDC GCVHPISTQSPD EPI LRHGIQYF__WTQHSS FMLNEVK- _ QRQ -^AGI-NFRITYSIVQTNCSKENFLF TPDCKS WNGDTG ECTDNAYIDIQLRIASFSQNCDIYPG_-DFVQPPTKICVGCPRDIPTNSPELΞΞT THTITKLNAE1_ A FYFKID VKKARVQWAGKKYFIDFVARETTCSKESNEELTESCETKK GQSLDCNAEVΥWP EKK IYPTV_ICQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDΞERDSGKEQGH_RRHDW GHEKQRKHN GHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFK DDDLEHQGGHV DHGHKHKH GHGHGKHKNKGKK GKHNG KTEH ASSSEDSTTPSAQTQΞKTEGPTPIPS AKPGVTVTFSDFQDSD LIATMMPPISPAPIQSDDDWIPDIQIDPMG SFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESY YFDLTDGLS

NOVlw, CG104903-17 SEQ ID NO: 45 |1071 bp DNA Sequence ORF Start: ATG at 1 JORF Stop: at 1071

A_GAAAC-AAT_ACCA_CC--__CC-C-GCTCCAGGCTAC-ACTAAGTT-AA

CCCAGGAATCACAGTCCGAGGAAATTGACTGCAATGACAAGGATTTATTTAAAGC-GTGGATGCTGCT

CTGAAGAAATATAACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCATAACTGAAGCCAC

TAAGACGGTTGGCTCTGACACGTTTTATTCC-TCAAGTACGAAATCAAGGAGGGGGATTGTCCTGTTC

AAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAGGATGCTGCAAAAGCAGCCACTGGAGAATGCACG

GCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTCCGTGGCTACCCAGACCTGCCAGATTACTCCAGC

CGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCCTCGGCTGTGTGCATCCTATATCAACGCAGAGCC

CAGACCTGGAGCCCAT-CTGAGACACGGCAT-CAG-ACT-TAACAACAACACTCAACATTCC-CCCTC

TTCATGCTTAATGAAGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACTTTCGAATTACCTA

CTCAATTGTGCAAACGAATTGTTCCAAAGAGAATTTTCTGTTCTTAACTCCAGACTGCAAGTCCCTTT

GGAATGGTGATACCGGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATTGCTTCCTTC

TCACAGAACTGTGACATTTATCCAGGGAAGGATTTTGTACAACCACCTACCAAGATTTGCGTGGGCTG

CCCCAGGGATATACCCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCACAAAGCTTA

ATGCAGAGAATAACGCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTACAGGTGGTG

GCTGGCAAGAAATATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGA

GTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTG

NOVlw, CG104903-17 SEQ ED NO: 46 357 aa MW at 40026.7kD Protein Sequence

M- ITILFLCSR SLTQESQSEEIDC-TOKDLFKAVDAA KKY-ISQNQS-JNQFV YRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKT QDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPλAt TAQYDC GCVHPISTQSPD EPILRHGIQYFN-J-ITQHSSLFML-TEVKRAQRQVVAGLNFRITYSIVQT NCSKENF FLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQWAGKKYFIDFVARΞTTCSKESNEELTESC ETKKLGQSLDCNAEVYV

NOVlx, CG104903-18 SEQ ED NO: 47 327 bp DNA Sequence ORF Start: at 1 ORF Stop: at 327

GCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGAC

ATGACTGGGGCCATGAAAAACAAAGAAAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAA

GGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGG

ACATAAGTTCAAACTTGATGATGATCTTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGC

ATAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGT

NOVlx, CG104903-18 SEQ ID NO: 48 109 aa MW at 12335.3kD Protein Sequence

APAQDEERDSGKΞQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKF: KLDDDLΞHQGGHVLDHGHI-HKHGHGHGKHK KGKK GKH G

NOVly, CG104903-19 SEQ ID NO: 49 729 bp DNA Sequence ORF Start: at 1 ORF Stop: at 729

GGTT-TTCACCTTTCCGATCATCACGAATAGGGGAAATAAAAGAAGAAACAACTGTAA GTCCACCCCACACTTCCATGGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCAT ACTCGTAGACATGACTGGGGCCATGAAAAACAAAGAAAACATAATCTTGGCCATGGCCATAAACATGA ACGTGACCAAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTC TTGGTCATGGACATAAGTTCAAACTTGATGATGATCTTGAACACCAAGGGGGCCATGTCCTTGACCAT GGACATAAGCATAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAATGGAAAGCA CAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTCTGAAGACAGTACTACACCTTCTGCACAGACAC AAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCCTAGCCAAGCCAGGTGTAACAGTTACCTTTTC- GACTTTCAGGACTCTGATCTCATTGCAACTATGATGCCTCCTATATCACCAGCTCCCATACAGAGTGA TGACGATTGGATCCCTGATATCCAGATAGACCCAAATGGCCTTTCATTTAACCCAATATCAGATTTTC CAGACACGACC_CC_CAAAA_G_CC_GGACGCCCC_GGAAG_CAG__AG_GAAATTAAT

NOVly, CG104903-19 SEQ ID NO: 50 243 aa MW at 26861.2kD Protein Sequence

GFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHD GHΞKQRKH LGHGHKHERDQ GHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHV_.DHGHI-HKHGHGHGKH_-MKGKK-.GKH-.GW KTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDW IPDIQIDPNG SFNPISDFPDTTSPKCPGRPWKSVSEIN

NOVlz, CG104903-20 (SEQ ID NO: 51 J-935 br7

DNA Sequence ORF Start: ATG at 1 lORF Stop: TAG at 1935

ATGAAACTAATTACCATCCTTTTCCTCTGCTCCAGGCTACTACTAAGTTTAAC

CCAGGAATCACAGTCCGAGGAAATTGACTGCAATGACAAGGATTTATTTAAAGCTGTGGATGCTGCTC

TGAAGAAATATAACAGTCAAAACCAAAGTAACAACCAGTTTGTATTGTACCGCATAACTGAAGCCACT

AAGACGGTTGGCTCTGACACGTTTTATTCCTTCAAGTACGAAATCAAGGAGGGGGACTGTCCTGTTCA

AAGTGGCAAAACCTGGCAGGACTGTGAGTACAAGGATGCAGCAAAAGCAGCCACTGGAGAATGCACGG

CAACCGTGGGGAAGAGGAGCAGTACGAAATTCTCCGTGGCTACCCAGACCTGCCAGATTACTCCAGCC

GAGGGCCCTGTGGTGACAGCCCAGTACGACTGCCTCGGCTGTGTGCATCCTATATCAACGCAGAGCCC

AGACCTGGAGCCCATTCTGAGACACGGCATTCAGTACTTTAACAACAACACTCAACATTCCTCCCTCT

TCACGCTTAATGAAGTAAAACGGGCCCAAAGACAGGTGGTGGCTGGATTGAACTTTCGAATTACCTAC

TCAATTGTGCAAACGAATTGTTCCAAAGAGAATTTTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTG

GAATGGTGATACCGGTGAATGTACAGATAATGCATACATCGATATTCAGCTACGAATTGCTTCCTTCT

CACAGAACTGTGACATTTATCCAGGGAAGGATTTTG-ACAACCACCTACCAAGATTTGCGTGGGCTGC

CCCAGAGATATACCCACCAACAGCCCAGAGCTGGAGGAGACACTGACTCACACCATCACAAAGCTTAA

TGCAGAGAATAACGCAACTTTCTATTTCAAGATTGACAATGTGAAAAAAGCAAGAGTACAGGTGGTGG

CTGGCAAGAAATATTTTATTGACTTCGTGGCCAGGGAAACCACATGTTCCAAGGAAAGTAATGAAGAG

TTGACCGAAAGCTGTGAGACCAAAAAACTTGGCCAAAGCCTAGATTGCAACGCTGAAGTTTATGTGGT

ACCCTGGGAGAAAAAAATTTACCCTACTGTCAACTGTCAACCACTGGGAATGATCTCACTGATGAAAA

GGCCTCCAGGTTTTTCACC_T_CCGATCATCACGAATAGGGGAAATAAAAGAAGAAACAACTGTAAGT

CCACCCCACACTTCCATGGCACCTGCACAAGATGAAGAGCGGGATTCAGGAAAAGAACAAGGGCATAC

TCGTAGACATGACTGGGGCCATGAAAAACAAAGAAAACATAATCTTGGCCATGGCCATAAACATGAAC

GTGACCAAGGGCATGGGCACCAAAGAGGACATGGCCTTGGCCATGGACACGAACAACAGCATGGTCTT

GGTCATGGACATAAGTTCAAACTTGATGATGATCTTGAACACCAAGGGGGCCATGTCCTTGACCATGG

ACATAAGCATAAGCATGGTCATGGCCACGGAAAACATAAAAATAAAGGCAAAAAGAATGGAAAGCACA

ATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTCTGAAGACAGTACTACACCTTCTGCACAGACACAA

GAGAAGACAGAAGGGCCAACACCCATCCCTTCCCTAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGA

CTTTCAGGACTCTGATCTCATTGCAACTATGATGCCTCCTATATCACCAGCTCCCATACAGAGTGATG

ACGATTGGATCCCTGATATCCAGATAGACCCAAATGGCCTTTCATTTAACCCAATATCAGATTTTCCA

GACACGACCTCCCCAAAATGTCCTGGACGCCCCTGGAAGTCAGTTAGTGAAATTAATCCAACCACACA

AATGAAAGAATCTTATTATTTCGATCTCACTGATGGCCTTTCTTAG

NOVlz, CG104903-20 SEQ ID NO: 52 644 aa MW at 71926.7kD Protein Sequence

MKLITILFLCSRLLLSLTQΞSQSEEIDCNDKDLFKAVOAALKKY SQNQS__.QFVLYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPW TAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFTLNEVKRAQRQVVAGLNFRITYSIVQT NCSKΞNFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPELΞETLTHTITKLNAEN ATFYFKIDNVKKARVQWAGKKYFIDFVARΞTTCSKESNEELTESC ETKKLGQSLDCNAEVYWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTS MAPAQDEERDSGKΞQGHTRRHDWGHEKQRKHNLGHGHKHΞRDQGHGHQRGHGLGHGHEQQHGLGHGHK F]_LDDDLEHQGGHVLDHGHKHKHGHGHGKHK_KGKKi_GKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEINPT-QMKESYYFDL-DGLS NOVlaa, SNP13381566 of SEQ ID NO: 53 1981 bp CG104903-03, DNA Sequence ORF Start: ATG at 50 ORF Stop: end of sequence

SNP Pos: 105 SNP Change: A to G

AATTCCGGTTGAAACCATGCCTCAGCTCCTAGAGGGAGATTGTTAGATCa.TG-AACTAATTACCATCC T-TTCCTCTGCTCCAGGCTACTACTAAG-TTAACCCGGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCA-IAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAAT-ACCTAC-CAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGG-GAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATC-CACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG _|AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG i-_AGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCA-GGACATAAGT-CAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCT-TTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CC_CC-A_A-CACCAGCTCCCA_ACAGAGTGATGACGAT-GGA-CCC-GA-A-CCAGA-AGACCCAAΑ TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NOVlaa, SNP13381566 of SEQ ID NO: 54 644 aa MW at 71984.8k_D CG104903-03, Protein Sequence SNP Pos: 19 SNP Change: Gin to Arg

M_-LITILFLCS_-LLLSLTRESQSΞEIDC-ROKDLF-_\VDAALKKYNSQNQSN_IQFVLYRITEATKTVGS ;D_FYSFKYΞIKEGDCPVQSGKT QDCEYKDAAKAATGECTATVGKRSSTKFSVATQ_CQITPAEGPW |TAQYDCLGCVHPISTQSPDLEPILPJIGIQYF_]_INTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQ_ :NCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP _!TNSPELEETLTHTIT-__NAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESC ETKKLGQSLDCNAEVYWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTS I APAQDEΞRDSGKΞQGHTRRHD GHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FKLDDDLEHQGGHVLDHGHKHKHGHGHGKHB- KGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSF1.PISDFPDTTSP KCPGRPWKSVSΞINPTTQMKΞSYYFDLTDGLS

NOVlab, SNP13379157 of SEQ ID NO: 55 1981 bp

CG104903-03, DNA Sequence |θRF Start: ATG at 5θ[θRF Stop: end of sequence

SNP Pos: 253 SNP Change: T to C

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATG-VAACTAAT-ACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGC-GTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCCGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCAC-GGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCA-CC-A-ATCAACGCΑGAGCCCAGACC-GGAGCCCAT-C-GAGACACGGCA--CAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGA-TGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGAC-GCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA iAAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NOVlab, SNP13379157 of SEQ ID NO: 56 644 aa MW at 71956.8kD

CG104903-03, Protein Sequence SNP Pos: 68 ISNP Change: Ser to Ser

!M___,ITILFLCSRLLLSLTQESQSEEIDCIrøKDLF-_ VD---__KK_NSQNQS__.QFV_.YRITΞATKTVGS DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPW TAQYDCLGCVHPISTQSPDLEPILRHGIQYF-tt-NTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQT NCSKENFLFLTPDCKSLW GDTGΞCTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKESNΞELTΞSC ETKKLGQSLDCNAEVYWPWEKKIYPTVNCQPLGMISL KRPPGFSPFRSSRIGEIKΞETTVSPPHTS MAPAQDEERDSGKEQGHTRRHDWGHEKQRKH-JLGHGHKHERDQGHGHQRGHGLGHGHΞQQHGLGHGHK FKLDDDLEHQGGHV-jDHGH-ΗKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVlac, SNP13379158 of SEQ ID NO: 57 1981 bp

CG104903-03, DNA Sequence |θϊ_F Su_it ATG at 5θ|θRF Stop: end of sequence

SNP Pos: 295 SNP Change: T to C

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGACTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACA-TCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATT GCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCC-AGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGCCCATGGACACG--AC _ACAGCATGGTCTTGGTCATGGACAT___GTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATG_-__-GCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGT-AGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT iNOVlac, SNP13379158 of ID NO: 58 644 aa MW at 71956.8kD

[CGI 04903-03, Protein Sequence }sϊ?p^"Pos:^"82 SNP Change: Asp to Asp _lMK-DITILFLCSRLLLSLTQESQSEEIDC_ro__-LF__\VDAALKKY_ISQNQS__.QF - __JYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPW TAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQT NCSKE-IFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETI.THTITKLNAE-rNATFYFKID_ KKARVQVVAGKKYFIDFVARETTCSKESNEELTESC ETKKLGQSLDCNAEVYWPWEKKIYPTV CQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTS APAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FBIJDDDLEHQGGHVIJDHGHKH-ΉGHGHGKHK-IKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPP1SPAPIQSDDD IPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEI1.PTTQMKESYYFDLTDGLS iNOVlad, SNP13379159 of SEQ ID NO: 59 1981 bp CG104903-03, DNA Sequence ORF Start: ATG at 50 ORF Stop: end of sequence

SNP Pos: 356 JSNP Change: G to A

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC TTTTCCTCTGCTCCAGGCTAC-ACTAAGTT-AACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAACCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG

GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGAC-AAAAAACTTGGCC

LAAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC GTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAΆGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAΆGCTCTTC

I GAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC

TAGCCAAGCCAGGTGTAΆCAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT 'GGCCTTTCT

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAAC-AATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT IGACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG IGATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGCGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAΆCΆΆCACTCAACATTCCTCCCTCTTCATGCTTAΆTGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG LAGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC :TGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG ;AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG LAAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NOVlaf, SNP13380032 of SEQ ED NO: 64644 aa MW at 71928.7kD

CG104903-03, Protein Sequence _NPPoTl36 i JSNP Change: Val to Ala

MKLITILFLCSRLLLSLTQESQSΞEIDC-JDKDLF__^VD-___KKYNSQ-TQS-1NQFVLYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVA TAQYDCLGCVHPISTQSPDLEPILRHGIQYF_I__.TQHSSLFMLNEVKRAQRQVVAGL-.FRITYSIVQT NCSKENFLFLTPDCKSLW GDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPΞLEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKΞSNEELTESC ETKKLGQSLDCNAEVYWPWEKKIYPTV CQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTS MAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FKLDDDLEHQGGHVLDHGHKHKHGHGHGKHK KGKK GKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEINPTTQMKΞSYYFDLTDGLS

NOVlag, SNP13380035 of JSEQ ID NO: 65 J1981 bp

CG104903-03, DNA Sequence |ORF Start: ATG at 50 RF Stop: end of sequence

SNP Pos: 978 SNP Change: A to G

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG ITACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA JGGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT iTTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA JTACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACGGGTGGTGGCTGGCAAGAAA-AT-TTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCT^ AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTΆGTGAAΆTTAATCCAACCACΆCAAATGAAAGAATCTTATTATTTCGATCTCACTGAT

GGCCTTTCT

NOVlag, SNP13380035 of SEQ ID NO: 66J644 aa MW at 71984.8kD

CG104903-03, Protein Sequence ^

MKLITILFLCSP-DLLSLTQESQSEEIDC-rDKDLF- VO-^ -KKYNSQNQSN-rQF liYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKT QDCEYI-DAAI-AATGECTATVGKRSSTKFSVATQTCQITPAEGPVV TAQYDCLGC\raPISTQSPDLEPILRHGIQYF_r-røTQHSSLFMLNΞVKRAQRQVVAGLNFRITYSIVQT _-CSKENFLFLTPDCKSLW_IGDTGECTD_fAYIDIQLRIASFSQ-.CDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETLTHTITKL.NAE__.ATFYFKIDNVKKARVKWAGKKYFIDFVARETTCSKESNEELTESC ETKKLGQSLDCNAEVYWP EKKIYPTVNCQPLGMISL KRPPGFSPFRSSRIGEIKEETTVSPPHTS MAPAQDEEP_)SGKEQGHTRRHDWGHEKQRKHNLGHGHKHE--DQGHGHQRGHGLGHGHEQQHG_.GHGHK FKLDDDLEHQGG--VLDHGH-_-KHGHGHG--HK-JKGK-_.GKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSP ^{κc P(}_2__-__-_^^

CG104903-03, DNA Sequence . QRF Start: ATG at 50JORF Stop: end of sequence

SNP Pos: 1008 JSNP Change: A to G

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTG -CTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGA-ACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGΆGACACTGACTCACACCATCACAAΆGCT-AATGCAGAGAATAACGCAACTTTC-ATTTCAA-ATT

GACAATGTGAAAAΆAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGGCTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NOVlah, SNP13375317 of SEQ ID NO: 68644 aa MW at 71898.8kD

CG104903-03, Protein Sequence |sNP Pos: 320 j JSNP Change: Asp to Gty

M__-,ITILFLCSRLLLSLTQESQSEEIDC_roKDLF-_\VD-^ALKKY SQNQS NQFVLYRITEATKTVGS DTFYSFKYEIKEGDCPVOSGKTWODCEYKDAAKAATGECTATVGKRSSTKFSVATOTCOITPAEGPW TAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQλA/AGLNFRITYSIVQT NCSKENFLFLTPDCKSLW_JGDTGECTD-.AYIDIQLRIASFSQ_JCDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETLTHTITKLNAEN ATFYFKIDNVKKARVQWAGKKYFIGFVARETTCSKESNEELTΞSC ETKKLGQSLDCNAEVYWPWEKKIYPTV CQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTS MAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEI-.PTTQMI-ESYYFDLTDGLS

NOVlai, SNP13375316 of JSEQ 3D NO: 69 _ jl981_bp_ CG104903-03, DNA Sequence ORF Start: ATG at 50 ORF Stop: end of sequence

SNP Pos: 1023 , SNP Change: A to G

AATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAΆCCCAGGAATCACAGTCCGΆGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAΆGTGGCAAAACCTGGCAGGACTGTGAGTACAΆG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCΆTTCTGAGACACGGCATTCAG TACTTTAACAΆCAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGGAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAAAGGCAAAAAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NONlai, SΝP13375316 of jSEQ ID NO: 70J644 aa |MW at71884.7kD CG104903-03, Protein Sequence SNP Pos: 325 ]SNP Change: Gluto Gly KLITILFLCS- -LLLSLTQESQSΞEIDC_IDKDLF-UWDAALKKYNSQ-IQS1_.QFVLYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPW TAQYDCLGC VHP I STQS PDLEPILRHGI Q YFN-INTQHS SLFML EVKRAQRQ WAGLNFRITYS I VQT NCSKE-IFLFLTPDCKSLW-JGDTGECTDNAYIDIQLRIASFSQ-ICDIYPGKDFVQPPTKICVGCPRDIP TNSPΞLEETLTHTITKLNAΞN ATFYFKIDNVKKARVQWAGKKYFIDFVARGTTCSKΞSNEELTESC ETKKLGQSLDCNAEVYWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEΞTTVSPPHTS MAPAQDEERDSGKEQGHTRFΗDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVlaj, SNP13379639 of |SEQ ID NO: 71 |l981 bp

CG104903-03, DNA Sequence ^RF Start^ ATG at 50 ORF Stop: end of sequence

SNP Pos: 1041 JSNP Change: A to G lAATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAG^GGA^GTCCTGTTCA^GTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGGAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC TGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG |AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG JAAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA IAAACΆTAATCTTGGCCATGGCCATAAACATGAΆCGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAΆCAACΆGCATGGTCTTGGTCΆTGGACΆTAΆGTTCAΆACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTGACCATGGACATAAGCATAAGCATGGTCATGGCCACGGAAAA CATAAAAATAΆAGGCAAAΆAGAATGGAAAGCACAATGGTTGGAAAACAGAGCATTTGGCAAGCTCTTC TGAAGACAGTACTACACCTTCTGCACAGACACAAGAGAAGACAGAAGGGCCAACACCCATCCCTTCCC TAGCCAAGCCAGGTGTAACAGTTACCTTTTCTGACTTTCAGGACTCTGATCTCATTGCAACTATGATG CCTCCTATATCACCAGCTCCCATACAGAGTGATGACGATTGGATCCCTGATATCCAGATAGACCCAAA TGGCCTTTCATTTAACCCAATATCAGATTTTCCAGACACGACCTCCCCAAAATGTCCTGGACGCCCCT GGAAGTCAGTTAGTGAAATTAATCCAACCACACAAATGAAAGAATCTTATTATTTCGATCTCACTGAT GGCCTTTCT

NONlaj, SΝP13379639 of SEQ ID NO: 72644 aa jMW at 71884.7kD

CG104903-03, Protein Sequence JsNp p_os: 331 SNP Change: Glu to Gly

MKLITILFLCS-iLLSLTQESQSEEIDClrøKDLFKAVDAALKKYNSQNQS-_.QFVLYRITEATKTVGS DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAA___ATGECTATVGKRSS _KFSVATQTCQITPAEGPW TAQYDCLGCVHPISTQSPDLΞPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQT NCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIP TNSPELEETLTHTITKLNAE NATFYFKIDNVKKARVQλtVAGKKYFIDFVARETTCSKGSNEELTESC ETKKLGQSLDCNAEVYWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEΞTTVSPPHTS MAPAQDEERDSGKEQGHTRRHDWGHEKQRKH LGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKH GWKTEHLASSSEDSTTPSAQTQΞKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDD IPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVlak, SNP13376277 of SEQ ED NO: 73 1981 bp

CG104903-03, DNA Sequence ORF Start: ATG at 50 ORF Stop: end of sequence

SNP Pos: 1157 SNP Change: T to C lAATTCCGGTTGAAACCATCCCTCAGCTCCTAGAGGGAGATTGTTAGATCATGAAACTAATTACCATCC

TTTTCCTCTGCTCCAGGCTACTACTAAGTTTAACCCAGGAATCACAGTCCGAGGAAATTGACTGCAAT GACAAGGATTTATTTAAAGCTGTGGATGCTGCTCTGAAGAAATATAACAGTCAAAACCAAAGTAACAA CCAGTTTGTATTGTACCGCATAACTGAAGCCACTAAGACGGTTGGCTCTGACACGTTTTATTCCTTCA AGTACGAAATCAAGGAGGGGGATTGTCCTGTTCAAAGTGGCAAAACCTGGCAGGACTGTGAGTACAAG GATGCTGCAAAAGCAGCCACTGGAGAATGCACGGCAACCGTGGGGAAGAGGAGCAGTACGAAATTCTC CGTGGCTACCCAGACCTGCCAGATTACTCCAGCCGAGGGCCCTGTGGTGACAGCCCAGTACGACTGCC TCGGCTGTGTGCATCCTATATCAACGCAGAGCCCAGACCTGGAGCCCATTCTGAGACACGGCATTCAG TACTTTAACAACAACACTCAACATTCCTCCCTCTTCATGCTTAATGAAGTAAAACGGGCCCAAAGACA GGTGGTGGCTGGATTGAACTTTCGAATTACCTACTCAATTGTGCAAACGAATTGTTCCAAAGAGAATT TTCTGTTCTTAACTCCAGACTGCAAGTCCCTTTGGAATGGTGATACCGGTGAATGTACAGATAATGCA TACATCGATATTCAGCTACGAATTGCTTCCTTCTCACAGAACTGTGACATTTATCCAGGGAAGGATTT TGTACAACCACCTACCAAGATTTGCGTGGGCTGCCCCAGAGATATACCCACCAACAGCCCAGAGCTGG AGGAGACACTGACTCACACCATCACAAAGCTTAATGCAGAGAATAACGCAACTTTCTATTTCAAGATT GACAATGTGAAAAAAGCAAGAGTACAGGTGGTGGCTGGCAAGAAATATTTTATTGACTTCGTGGCCAG GGAAACCACATGTTCCAAGGAAAGTAATGAAGAGTTGACCGAAAGCTGTGAGACCAAAAAACTTGGCC AAAGCCTAGATTGCAACGCTGAAGTTTATGTGGTACCCTGGGAGAAAAAAATTTACCCTACTGTCAAC CGTCAACCACTGGGAATGATCTCACTGATGAAAAGGCCTCCAGGTTTTTCACCTTTCCGATCATCACG AATAGGGGAAATAAAAGAAGAAACAACTGTAAGTCCACCCCACACTTCCATGGCACCTGCACAAGATG AAGAGCGGGATTCAGGAAAAGAACAAGGGCATACTCGTAGACATGACTGGGGCCATGAAAAACAAAGA AAACATAATCTTGGCCATGGCCATAAACATGAACGTGACCAAGGGCATGGGCACCAAAGAGGACATGG CCTTGGCCATGGACACGAACAACAGCATGGTCTTGGTCATGGACATAAGTTCAAACTTGATGATGATC TTGAACACCAAGGGGGCCATGTCCTTC

MAPAQDEERDSGKEQGHTRRHDWGHΞKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHK FKLDDDLEHQGGHVLDHGHKHKHGHGHGKHK KGKK-IGKHNGWKTEHLASSSEDSTTPSAQTQEKTEG PTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAP QSDDD IPDIQIDPNGLSFNPISDFPDTTSP KCPGRPWKSVSEINPTTQMKESYYFD TDG S

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table IB.

Table IB. Comparison of the NOY1 protein sequences.

NOVla MKLITILFLCSRLLLSLTQESQSΞEIDCITOKDLF- DAALKKYNSQNQS-INQFV

NOVlb

NOVle HRGRTMKLITILFLCSRLLLSLTQESQSEEIDCND__DLF__VVDAALKKYNSQNQS-INQFV

NOVld

NOVle

NOVlf

NOVlg TRSPTDC_TOI_3LF-_-V--AALKK_-.SQ

NOVlh EEIDCNDKDLFKAVDAALKKYNSQNQSNNQFV

NOVli TRSEEIDCNDKDLFKAVDA

NOVlj

NOVlk

NOV11

NOVlm

NOVln

NOVlo

NOVlp

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOV1v EEIDCNDKDLFKATDAALKKYNSQNQS-INQFV

NOVlw

NOVlx

NOVly

NOVlz MKLITILFLCSRLL S TQESQSEEIDCND--DLFKAVDAALKKYNSQNQSNNQFV

NOVla LYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRS NOVlb ^" -MK ITILFLCSR LSLTQESQSEEIDCNDKDLFKAVDAA KKYNSQNQSNNQFVLYRI

NOVle LYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRS

NOVld

NOVle

NOVlf

NOVlg NQSNNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGEC

NOVlh LYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRS

NOVli ALKKYNSQNQS-INQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDA

NOVlj

NOVlk MK ITILFLCSRL LSLTQESQSEEIDDCNDKDLFKAV

NOVli

NOVlm

NOVln

NOVlo

NOVlp

NOVlq

NOVlr

NOVls

NOVlt

NOVlu NOVlv YRITEATKTVGSDTFYSFKYΞIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRS

NOVlw

NOVlx

NOVly

NOV1z LYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRS

NOVla STKFSVATQTCQITPAEGPWTAQYDCLGCVHPISTQSPDLEPI RHGIQYFNNNTQHSS

NOVlb TEATKTVGSDTFYSFKYΞIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKF

NOVle STKFSVATQTCQITPAEGPλ/VTAQYDCLGCVHPISTQSPD EPI RHGIQYFNNNTQHSS

NOVld

NOVle

NOVlf

NOVlg TATVGKRSSTKFSVATQTCQITPAEGPWTAQYDC GCVHPISTQSPD EPILRHGIQYF

NOVlh STKFSVATQTCQITPAEGPWTAQYDCLGCVHPISTQSPDLEPI RHGIQYFNNNTQHSS

NOVli AKAATGΞCTATVGKRSSTKFSVATQTCQITPAEGPWTAQYDCLGCVHPISTQSPD EPI

NOVlj

NOVlk DAA KKYNSQNQSNNQFVLYRKTWQDCEYKDAAKAATGΞCTATVGKRSSTKFSVATQTCQ

NOVli

NOVlm

NOVln

NOVlo

NOVlp

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv STKFSVATQTCQITPAEGPWTAQYDC GCVHPISTQSPDLΞPILRHGIQYFNNNTQHSS

NOVlw

NOVlx

NOVly

NOVlz STKFSVATQTCQITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSS

NOVla LFMNEVKRAQRQWAGLNFRITYSIVQTNCSKENF F TPDCKS NGDTGECTDNAYI

NOVlb SVATQTCQITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFML

NOVle FTLNEVKRAQRQWAGLNFRITYSIVQTNCSKENF F TPDCKSWNGDTGECTDNAYI

NOVld

NOVle

NOVlf

NOV1g NNNTQHSSLFMLNEVKRAQRQWAGLNFRITYSIVQTNCSKENFLF TPDCKSLWNGDTG

NOVlb. FMNEVKRAQRQWAGLNFRITYSIVQTNCSKENFLF TPDCKSLWNGDTGECTDNAYI

NOVli LRHGIQYFNNNTQHSS FMLNΞVKRAQRQVVAGLNFRITYSIVQTNCSKENFLF TPDCK

NOVlj

NOVlk ITPAΞGPWTAQYDC GCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFTNEVKRAQR

NOVli

NOVlm

NOVln

NOVlo ^■

NOVlp

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv LFMLNEVKRAQRQWAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYI

NOVlw

NOVlx

NOVly

NOVlz FT NEVKRAQRQWAGLNFRITYSIVQTNCSKENF FLTPDCKSLWNGDTGECTDNAYI NOVla DIQ RIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEΞTLTHTITKNAENNA

NOVlb NEVKRAQRQWAGNFRITYSIVQTNCSKENFLFLTPDCKS WNGDTGECTDNAYIDIQL

NOVle DIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPE EΞT THTITKLNAΞNNA

NOVld

NOVle

NOVlf

NOVlg ECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTIT

NOVlh DIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEET THTITKLNAENNA

NOVli SLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPE E

NOVlj

NOVlk QWAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQN

NOVli

NOVlm MKLITILFLC

NOVln

NOVlo

NOVlp

NOVlq

NOVlr

NOVls

_Novit

NOVlu

NOVlv DIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEET THTITKLNAENNA

NOVlw

NOVlx

NOVly

NOVlz DIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEET THTITKLNAENNA

NOVla TFYFKIDNVKKARVQWAGKKYFIDFVARΞTTCSKESNEΞLTESCETKKLGQS DCNAEV

NOVlb RIASFSQNCOIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYF

NOVle TFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKΞSNΞELTESCETKKLGQSLDCNAEV

NOVld

NOVle

NOVlf

NOVlg KLNAENNATFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKESNEELTΞSCETKKLGQ

NOVlh TFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKESNEELTESCΞTKKLGQS DCNAEV

NOVli ETLTHTITKLNAENNATFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKESNEELTES

NOVlj

NOVlk CDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKA

NOVli

NOVlm SR L S TQESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFV YRITEATKTVGSDT

NOVln

NOVlo

NOVlp

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv TFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKESNEELTESCΞTKKLGQSLDCNAEV

NOVlw

NOVlx

NOVly

NOVlz TFYFKIDNVKKARVQWAGKKYFIDFVARETTCSKESNEELTESCETKK GQSLDCNAEV

NOVla YWPWEKKIYPTVNCQP G ISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDE

NOVlb KID_Γ\Π<I ARVQVVAGKKY.FIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVΎVVP

NOVle YWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKΞETTVSPPHTSMAPAQDE

NOVld T NOVle

NOVlf TRSGFSPFRSSRIGEIKEΞTTVSPPHTSMAPAQDΞ

NOVlg SLDCNAEV-WPWEKKIYPTVNCQP GMISLMKRPPGFSPFRSSRIGΞIKEETTVSPPHT

NOVlh YWPWEKKIYPTVNCQPLGMIS MKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDE

NOVli CETKKLGQS DCNAEVYWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEE

NOVlj

NOVlk RVQWAGKKYFIDFVARETTCSKESNEELTESCETI.KLGQSLDCNAΞVYWPWEI.KIYPT

NOVli

NOVlm FYSFKYΞIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITP

NOVln

NOVlo

NOVlp

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv YWPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDE

NOVlw

NOVlx

NOVly GFSPFRSSRIGEIKEETTVSPPHTSMAPAQDΞ

NOVlz YWPWΞKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDΞ

NOVla ERDSGKΞQGHTRRHDWGHEKQRKHNLGHGHKHΞRDQGHGHQRGHG GHGHΞQQHGLGHGH

NOVlb WEKKIYPTVNCQPLGMIS MKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDS

NOVle ERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGH

NOVld RSAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQ

NOVle TRSPTMK ITILF

NOVlf ERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHΞRDQGHGHQRGHGLGHGHEQQHG GHGH

NOVlg SMAPAQDEERDSGKΞQGHTRRHDWGHΞKQRKHNLGHGHKHERDQGHGHQRGHG GHGHEQ

NOVlh ERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHΞQQHGLGHGH

NOVli TTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHΞKQRKHNLGHGHKHERDQGHGHQRGH

NOVlj MKLITILF

NOVlk VNCQPLGMIS MKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTR

NOVli MKLITILF

NOVlm AEGPWTAQYDCLGCVHPISTQSPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEΞRDS

NOVln . MKLITILF

NOVlo MKLITILF

NOVlp MKLITILF

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv ERDSGKEQGHTRRHDWGHΞKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGH

NOVlw MKLITILF

NOVlx APAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGH

NOVly ERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHΞRDQGHGHQRGHGLGHGHEQQHGLGHGH

NOVlz ERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGH

NOVla KFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPS

NOVlb GKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKL

NOVle KFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPS

NOV1d QHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGVDG

NOVle LCSRLLLSLTQESQSE-EIDCNDI-DLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVG

NOVlf KFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPS

NOVlg QHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTΞHLASS

NOVlh KFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPS

NOV1i GLGHGHΞQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGW NOVl LCSRLLLSLTQΞSQSEEIDDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYR

NOVlk RHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQG

NOVli LCSRLLLSLTQESQSE-EIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVG

NOVlm GKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKL

NOVln LCSRLLLSLTQESQSE-EIDCNDKDLFKAVDAALKKYNSQNQSNNQFλ/LYRITEATKTVG

NOVlo LCSRLLLSLTQΞSQSEEIDDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYR

NOVlp LCSRLLLSLTQESQSE-EIDCNDKDLFKAVDi-ALKKYNSQNQSNNQFVLYR

NOVlq HKNKGKKNGKHNGWKT

NOVlr KHGHGHGKHKNKGKKN

NOVls VLDHGHKHKHGHGHGK

NOVlt GHGLGHGHEQQHGLGH

NOVlu DQGHGHQRGHGLGHGH

NOVlv KFKLDDDLEHQGG--VLDHGHKHKHGHGHGKH__NfKGK-_^GKHNGWKTEHLASSSΞDSTTPS

NOVlw LCSRLLLSLTQESQSE-EIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVG

NOVlx GLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHI_NIKGKKNGKHNG-

NOVly KFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPS

NOVlz KF--LDDDLEHQGGHVLDHGHK-_KHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPS

NOVla AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVlb DDDLΞHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQ

NOVle AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVld

NOVle SDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQ

NOVlf AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVlg SEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDD

NOVlh AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVli KTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPA

NOV1j KTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQ

NOVlk GHVLDHGHKHKHGHGHGKHKNKGKKNGKHNG KTEHLASSSEDSTTPSAQTQEKTEGPTP

NOVli SDTFYSFKYEIKEGDCPVQSGKTWQDCΞYKDAAKAATGECTATVGKRSSTKFSVATQTCQ

NOVlm DDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSΞDSTTPSAQTQ

NOVln SDTFYSFKYEIKEGDCPVQSGKT QDCEYKDAAKAATGECTATVGRG AVRNSP

NOV1o KTWQDCEYKDAAKAATGECTA VGKRSSTKFSVATQTCQ

NOVlp IT-EATKTATGECTATVGKRSSTKFSVATQTCQ

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVlw SDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQ

NOVlx

NOVly AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVlz AQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDP

NOVla NGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVlb ΞKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLS

NOVle NGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVld

NOVle ITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQR

NOVlf NGLSFNPISDFPDTTSPKCPGRP KSVSEINLEG

NOVlg IPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTLEG

NOVlh NGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVli PIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSΞINPTTQMKESYYFDL

NOVlj ITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFTLNEVKRAQR

NOVlk IPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQTDPNGLSFNPISDFP

NOVli ITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQR

NOVlm EKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLS

NOVln WLPRPGAHSETRHSVL NOVlo ITPAEGPWTAQYDCLGCVHPISTQS P

NOVlp ITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFTLNEVKRAQR

NOVlq

NOVlr

NOVls

NOVlt — ,

NOVlu

NOVlv NGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVlw ITPAEGPWTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQR

NOVlx

NOVly NGLSFNPISDFPDTTSPKCPGRPWKSVSEIN

NOVlz NGLSFNPISDFPDTTSPKCPGRP KSVSEINPTTQMKESYYFDLTDGLS

NOVla

NOVlb FNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVle

NOVld

NOVle QWAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQN

NOVlf

NOVlg

NOVlh

NOVli TDGLSVDG

NOVlj QWAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQN

NOVlk DTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVli QWAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQN

NOVlm FNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS

NOVln

NOVlo

NOVlp QWAGLNFRITYSIVQTNCSKENFLFLTPDCESLWNGDTGECTDNAYIDIQLRIASFSQN

NOVlq

NOVlr

NOVls

NOVlt

NOVlu

NOVlv

NOVlw QWAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQN

NOVlx

NOVly

NOVlz

NOVla

NOVlb

NOVle

NOVld

NOVle CDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKA

NOVlf

NOVlg

NOVlh

NOVli

NOVlj CDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKA

NOVlk

NOVli CDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKA

NOVlm

NOVln

NOVlo

NOVlp CDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKA

NOVlq

NOVlr

NOVls NOVlt NOVlu NOVlv NOVlw CDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKA NOVlx NOVly NOVlz

NOVla NOVlb NOVle NOVld NOVle RVQWAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVLEG- NOVlf NOVlg NOVlh NOVli NOVlj RVQWAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAΞVYWPWEKKIYPT NOVlk NOVli RVQWAGKKYFIDFVARETTCSKESNEELTESCΞTKKLGQSLDCNAEVYWPWEKKIYPT NOVlm NOVln NOVlo NOVlp RVQWAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVY PWEKKIYPT NOVlq NOVlr NOVls NOVlt NOVlu NOVlv NOVlw RVQWAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYV- NOVlx NOVly NOVlz

NOVla NOVlb NOVle NOVld NOVle NOVlf NOVlg NOVlh NOVli NOVlj VNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTSHLRSCEYKGRPPKAGAΞPASEREVS NOVlk NOVli VNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTSHLRSCEYKGRPPKAGAEPASEREVS NOVlm NOVln NOVlo GFSPFRSSRIGEIKEETTSHLRSCEYKGRPPKAGAEPASEREVS NOVlp VNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTSHLRSCEYKGRPPKAGAEPVSΞREVS NOVlq NOVlr NOVls NOVlt NOVlu NOVlv NOVlw NOVlx NOVly NOVlz

NOVla (SEQ ID NO 2)

NOVlb (SEQ ID NO: 4)

NOVle (SEQ ID NO 6)

NOVld (SEQ ID NO- 8)

NOVle (SEQ ID NO 10)

NOVlf (SEQ ID NO 12)

NOVlg (SEQ ID NO 14)

NOVlh (SEQ ID NO 16)

NOVli (SEQ ID NO 18)

NOVlj (SEQ ID NO- 20)

NOVlk (SEQ ID NO- 22)

NOVli (SEQ ID NO 24)

NOVlm (SEQ ID NO- 26)

NOVln (SEQ ID NO 28)

NOVlo (SEQ ID NO 30)

NOVlp (SEQ ID NO 32)

NOVlq (SEQ ID NO: 34)

NOVlr (SEQ ID NO 36)

NOVls (SEQ ID NO: 38)

NOVlt (SEQ ID NO 40)

NOVlu (SEQ ID NO: 42)

NOVlv (SEQ ID NO 44)

NOVlw (SEQ ID NO: 46)

NOVlx (SEQ ID NO 48)

NOVly (SEQ ID NO: 50)

NOVlz (SEQ ID NO: 52)

Further analysis of the NOVla protein yielded the following properties shown in Table lC.

Table lC. Protein Sequence Properties NOVla

SignalP analysis: I Cleavage site between residues 24 and 25

PSORT π analysis:

PSG: a new signal peptide prediction method

N-region: length 2; pos.chg 1;- neg.chg 0 H-region: length 9; peak value 11.25 PSG score: 6.85

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.04 possible cleavage site: between 18 and 19

>» Seems to have a eleavable signal peptide (1 to 18)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 19

Tentative number of TMS(s) for the threshold 0.5: number of TMS(s) .. fixed PERIPHERAL Likelihood = 7.90 (at 135) ALOM score: 7.90 {number of TMSs : 0)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 9

Charge difference: -4.0 C.-2.0) ~ N( 2.0)

N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 3.52 Hyd Moment (95) : 7.10 G content : 0 D/E content: 1 S/T content: 4 Score: -3.12

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 22 SRL|LL

NUCDISC: discrimination of nuclear localization signals pat4 -. none pat7 : none bipartite : none content of basic residues: 11.2% NLS Score: -0.47

KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals: none

SKL: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal : none

VAC : possible vacuolar targeting motif : none

RNA-binding motif : none

Actinin-type actin-binding motif: type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none

Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail : none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: nuclear Reliability: 89

COIL: Lupas's algorithm to detect coiled-coil regions total : 0 residues Final Results (k = 9/23 ) :

66 .7 % : extracellular, including cell wall 22 .2 % : mitochondrial 11.1 % : nuclear

» prediction for CG104903-09 is exc (k=9 )

A search of the NOVla protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table ID.

In a BLAST search of public sequence databases, the NOVla protein was found to have homology to the proteins shown in the BLASTP data in Table IE.

PFam analysis predicts that the NOVla protein contains the domains shown in the Table IF.

Example 2.

The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.

Table 2A. NOV2 Sequence Analysis

NOV2a, CG120844-02 |SEQ D NO: 77 j689 bp

DNA Sequence JORF Start: at 2 JORF Stop: TAA at 659

CTCCTTCCAGGACCTGGACCTCTGCCCTCTGGATGGCGGCATCCAGCTACGAATCTCCGACCACCACT ACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTGTTGTGGCCATGGACAAGCTGAGGAAGATGCTGGTT CCCTGCCCACAGACCTTCCAGGAGAATGACCTGAGCACCTTCTTTCCCTTCATCTTTGAAGAAGAACC TATCTTCTTCGACACATGGGATAACGAGGCTTATGTGCACGATGCACCTGTACGATCACTGAACTGCA CGCTCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTCCACCTC CAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTC CTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGA AGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCC CAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTTCCTGGGAGGGACCAA AGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCCTAAAGAGAGCTGTACCCAGAGA GTCCTGTGC

NOV2a, CG120844-02 SEQ ED NO: 78 219 aa MW at 25075.3kD Protein Sequence

SFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVWAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEP IFFDT DNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGEESNDKIPVALGLKEKNLYLS CVLKDDKPTLQLΞSVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKj GGQDITDFTMQFVSS

NOV2b, 251426189 SEQ ED NO: 79 EoO bp

DNA Sequence |0-_F Start7at 2 __F Stop: end of sequence

CACCGGATCCCTCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTC TCCACCTCCAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTG TACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTA CCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTG AGTCTGCCCAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTTCCTGGGA GGGACCAAAGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTCTCGAGGGC

NOV2b, 251426189 SEQ ID NO: 80 133 aa MW at 15050.1kD

Protein Sequence I

TGSL-_DSQQKSLVMSGPYELKALHLQGΞESNDKIPV- GLKEKNI-ιYLSCVLKDDKPTLQLESVDPKJ_Y PKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSLEG

NOV2c, CG120844-01 SEQ ID NO: 81 820 bp

DNA Sequence (ORF Start: ATG at 3 JORF Stop: TAA at 810

CCATGGCAGAAGTACCTGAGCTCGCCAGTGAAATGATGGCTTATTACAGTGGCAATGAGGATGACTTG TTCTTTGAAGCTGATGGCCCTAAACAGATGAAGTGCTCCTTCCAGGACCTGGACCTCTGCCCTCTGGA TGGCGGCATCCAGCTACGAATCTCCGACCACCACTACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTG TTGTGGCCATGGACAAGCTGAGGAAGATGCTGGTTCCCTGCCCACAGACCTTCCAGGAGAATGACCTG AGCACCTTCTTTCCCTTCATCTTTGAAGAAGAACCTATCTTCTTCGACACATGGGATAACGAGGCTTA TGTGCACGATGCACCTGTACGATCACTGAACTGCACGCTCCGGGACTCACAGCAAAAAAGCTTGGTGA TGTCTGGTCCATATGAACTGAAAGCTCTCCACCTCCAGGGACAGGATATGGAGCAACAAGTGGTGTTC TCCATGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAA GAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCA AAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTG GAATTTGAGTCTGCCCAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTT CCTGGGAGGGACCAAAGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCTO.AAAGAG AGCT

NOV2c, CG120844-01 SEQ ID NO: 82 269 aa MW at 30747.6kD Protein Sequence

MAEVPELAS^ VAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNΞAYVHDAPVRSLNCTLRDSQQKSLVM SGPYELKALHLQGQDMEQQWFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPK NYPKKKMEKRF VFNKI ΞINNKLEFE S AQF PNWYI STS QAENMPVFLGGTKGGQDITDFTMQFVS S

NOV2d, SNP13377796 of SEQ ID NO: 83 689 bp CG120844-02, DNA Sequence ORF Start: at 2 ORF Stop: TAA at 659

JSNP Pos: 231 JSNP Change: A to G

CTCCTTCCAGGACCTGGACCTCTGCCCTCTGGATGGCGGCATCCAGCTACGAATCTCCGACCACCACT ACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTGTTGTGGCCATGGACAAGCTGAGGAAGATGCTGGTT CCCTGCCCACAGACCTTCCAGGAGAATGACCTGAGCACCTTCTTTCCCTTCATCTTTGAAGAAGAACC TATCTTCTTCGACACATGGGATAACGGGGCTTATGTGCACGATGCACCTGTACGATCACTGAACTGCA CGCTCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTCCACCTC CAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTC CTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGA AGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCC CAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTTCCTGGGAGGGACCAA AGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCC-AAAGAGAGCTGTACCCAGAGA GTCCTGTGC

NOV2d, SNP13377796 of SEQ ED NO: 84 219 aa MW at 25003.2kD

CG120844-02, Protein Sequence |s_^ Posi 77^" SNP Change: Glu to Gly

SFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVWAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEP IFFDT DNGAYVHDAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGEESNDKIPVALGLKEKNLYLS CVLKDDKPTLQLESVDPKNYPKKKMΞKRFVFNKIEINNKLEFESAQFPNYISTSQAENMPVFLGGTK GGQDITDFTMQFVSS

NOV2e, SNP13377795 of SEQ ID NO: 85 689 bp CG120844-02, DNA Sequence ORF Start: at 2 ]ORF Stop: TAA at 659

SNP Pos: 272 JSNP Change: A to G

CTCCTTCCAGGACCTGGACCTCTGCCCTCTGGATGGCGGCATCCAGCTACGAATCTCCGACCACCACT ACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTGTTGTGGCCATGGACAAGCTGAGGAAGATGCTGGTT CCCTGCCCACAGACCTTCCAGGAGAATGACCTGAGCACCTTCTTTCCCTTCATCTTTGAAGAAGAACC TATCTTCTTCGACACATGGGATAACGAGGCTTATGTGCACGATGCACCTGTACGATCACTGAACTGCG CGCTCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTCCACCTC CAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTC CTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGA AGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCC CAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTTCCTGGGAGGGACCAA AGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCCTAAAGAGAGCTGTACCCAGAGA GTCCTGTGC

NOV2e, SNP13377795 of SEQ ED NO: 86 219 aa MW at 25045.2kD

CG120844-02, Protein Sequence SNP Pos: 91 SNP Change: Thr to Ala

SFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVWAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEP IFFDTWDNEAYVHDAPVRSLNC-5-LRDSQQKSLVMSGPYELK--LHLQGEESNDKIPVALGLKEKNLYLS CVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTK GGQDITDFTMQFVS S

NOV2f, SNP13377794 of SEQ ID NO: 87 j689 bp CG120844-02, DNA Sequence ORF Start: at 2 ORF Stop: TAA at 659

SNP Pos: 374 SNP Change: G to A

CTCCTTCCAGGACCTGGACCTCTGCCCTCTGGATGGCGGCATCCAGCTACGAATCTCCGACCACCACT ACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTGTTGTGGCCATGGACAAGCTGAGGAAGATGCTGGTT CCCTGCCCACAGACCTTCCAGGAGAATGACCTGAGCACCTTCTTTCCCTTCATCTTTGAAGAAGAACC TATCTTCTTCGACACATGGGATAACGAGGCTTATGTGCACGATGCACCTGTACGATCACTGAACTGCA CGCTCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTCCACCTC CAAGGAGAAGAAAGTAATGACAAAATACCTGTGACCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTC CTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGA AGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCC CAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTTCCTGGGAGGGACCAA AGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCCTAAAGAGAGCTGTACCCAGAGA

CAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTC CTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGA AGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCC CAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACATGCCCGTCTTCCTGGGAGGGGCCAA AGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCCTAAAGAGAGCTGTACCCAGAGA GTCCTGTGC

NOV2i, SNP13377792 of SEQ ED NO: 94 219 aa |MW at 25045.2kD

CG120844-02, Protein Sequence fsNP Pos: 203 SNP Change: Thr to Ala

SFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQE-JDLSTFFPFIFEEEP IFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGEESNDKIPVALGLKEKNLYLS CVLKDDKPTLQLESVDPK_ΓYPKKKMEKRFVFNKIEI_MI-_EFESAQFP_IWYISTSQAE_Π_PVFLGGAK GGQDITDFTMQFVSS

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 2B.

Table 2B. Comparison of the NOV2 protein sequences.

NOV2a SFQDLDLCPLDGGIQLRISDHHYSKG NOV2b NOV2c MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKG

NOV2a FRQAASVWAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDT DNEAYVHDAPVR NOV2b NOV2c FRQ-_-SVVV--MDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDT DNEAYVHDAPVR

NOV2a SLNCTLRDSQQKSLVMSGPYELKALHLQG- . ΞESNDKIPVALGLKE NOV2b — TGSLRDSQQKSLVMSGPYELKALHLQG EESNDKIPVALGLKE NOV2c SLNCTLRDSQQKSLV-1SGPYELKALHLQGQDMEQQWFSMSFVQGEESNDKIPVALGLKE

NOV2a KNLYLSCVL-_->DKPTLQLESVDPK_JYPKK__-EKR_VFNKIEINN___JEFESAQFPNWYIST NOV2b --NLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEIN_I--LEFESAQFP- re NOV2c iKNLYLSCVLKDDKPTLQLESVDPKϊreP- K-α-EKRFVFNKIEI

NOV2a SQAENMPVFLGGTKGGQDITDF TMQFVSS — NOV2b SQAENMPVFLGGTKGGQDITDFTMQFVSLEG NOV2c SQAENMPVFLGGTKGGQDITDFTMQFVSS - -

NOV2a ( SEQ ID NO 78 ) NOV2b ( SEQ ID NO 80 ) NOV2c ( SEQ ID NO 82 )

Further analysis of the NON2a protein yielded the following properties shown in Table 2C.

Table 2C. Protein Sequence Properties ΝOV2a

Signal? analysis: No Known Signal Sequence Predicted

PSORT π analysis:

PSG: a new signal peptide prediction method

N-region: length 11; pos.chg 0; neg.chg 3 H-region: length 5; peak value 0.00 PSG score: -4.40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -6.91 possible cleavage site: between 52 and 53

>» Seems to have no N-terminal signal peptide

ALOM: Klein et al ' s method for TM region allocation Init position for calculation: 1

Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 5.73 (at 31) ALOM score: 5.73 (number of TMSs: 0)

MITDISC : discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 2.74 Hyd Moment (95) : 5.40 G content: 0 D/E content: 2 S/T content: 1 Score: -7.29

Gavel : prediction of cleavage sites for mitochondrial preseq cleavage site motif not found

NUCDISC: discrimination of nuclear localization signals pat4: PKKK (4) at 157 pat7: PKKKMΞK (5) at 157 bipartite: none content of basic residues: 11.0% NLS Score: 0.21

KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals : none

SKL: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

Actinin-type actin-binding motif: type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none

Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail: none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs : none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs : none

NNCN : Reinhardt ' s method for Cytoplasmic/Nuclear discrimination Prediction : cytoplasmic Reliability: 70 . 6

COIL : Lupas ' s algorithm to detect coiled-coil regions total : 0 residues

3Ul ts (k = 9/23 ) :

43 . 5 % : cytoplasmic

34 8 % : nuclear

17 4 % : mitochondrial

4 3 % : Golgi

» prediction for CG120844-02 is cyt (k=23 )

A search of the NOV2a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 2D.

In a BLAST search of public sequence databases, the NO V2a protein was found to have homology to the proteins shown in the BLASTP data in Table 2E.

PFam analysis predicts that the NOV2a protein contains the domains shown in the Table 2F.

Example 3.

The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.

GCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACG AAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTT CCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCT GCAGGACAGGGGACAGATGACCAG

NOV3a, CG127616-01 |SEQ ID NO: 96 104 aa MW at 11567.4kD

Protein Sequence

M_VHECP-_^- _-LLSLLSLPLGLPV^ RTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

NOV3b, CG127616-02 SEQ ED NO: 97 |324 bp DNA Sequence ORF Start: at 3 ORF Stop: TGA at 318

CCTGGCTATCTGTTCTAGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTG GGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTT GGAGGCCAAGGAGGCCGAGAATATCACGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTC CACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGA AAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGACCAG

NOV3b, CG127616-02 SEQ ED NO: 98 105 aa MW at 11741.6kD Protein Sequence

WLSVLECPAL LLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITKEAISPPDAASAAP LRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

NOV3c, 227803412 SEQ ID NO: 99 1336 bp DNA Sequence ORF Start: at 1 jORF Stop: TGA at 325

CGCGGATCCACCATGGGGGTGCACGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTCGCT CCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTi ACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAj GCTGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCT CCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGACTCGAGCGG

NOV3c, 227803412 SEQ ID NO: 100 108 aa MW at 11968.8kD Protein Sequence

RGS MG .^ECP . _-,._-- LS S P G P . _.G PR ICDS ER LΞAKΞAENI _KE ISPPDAAS AAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

NOV3d, CG127616-03 SEQ ID NO: 101 591 bp DNA Sequence ORF Start: at 3 |θ!gjtopø GA. at 585

CCTGGCTATCTGTTCTAGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTG GGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTT GGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCA CTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAA GTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGATCAACTCTTC CCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTC TGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTC CGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCT GAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGACCAG

NON3d, CG127616-03 SEQ ED NO: 102 194 aa MW at 21494.7kD Protein Sequence

WLSVLECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENIT VPDTKVNFYAKRMEVGQQAVEV QGLALLSEAVLRGQALLINSSQP EPLQLHVDKAVSGLRSLTTL LRALGAQKΞAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

NOV3e, CG127616-04 SEQ ED NO: 103 282 bp DNA Sequence ORF Start: at 7 ORF Stop: at 277

GG -2CTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACG CCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGA AGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTC CGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTG CAGGCTCGAG

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 3B.

Table 3B. Comparison of the NO 3 protein sequences.

NOV3a MGVHECPALWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITK—

NOV3b WLSVLECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLΞAKEAENITK--

NOV3c RGSTMGVHECPAL LLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITK—

NOV3d LSVLECPALWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGC

NOV3e LLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITK--

NOV3f MEVGQQAVEVWQGLALLSEAV LRGQALLVNSSQPWEPLQLHVDKAVSGLRSL

NOV3g MEVGQQAVEV QGLALLSEAV LRGQALLVNSSQPWEPLQLHVDKAVSGLRSL

NOV3h MEVGQQAVEVWQGLALLSEAV LRGQALLVNSSQPWEPLQLHVDKAVSGLRSL

NOV3i MGWECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITK--

NOV3j ECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITK--

NOV3a

NOV3b

NOV3c

NOV3d AEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLINSSQPWE

NOV3f TTLLRALG NOV3g TTLLRALG

NOV3h TTLLRALG

NOV3i

NOV3J

NOV3A EAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3B EAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3C EAISPPDAΆSAAPLRTITADTFRKLFRVΎSNFL

NOV3d PLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3e EAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3f AQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3g AQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3h AQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3i EAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3J EAISPPDAASAAPLRTITADTFRKLFRVYSNFL

NOV3a RGKLKLYTGEACRTGDR

NOV3b RGKLKLYTGEACRTGDR

NOV3c RGKLKLYTGEACRTGDR

NOV3d RGKLKLYTGEACRTGDR

NOV3e RGKLKLYTGΞACR

NOV3f RGKLKLYTGEACRTGDR

NOV3g RGKLKLYTGEACRTGDR

NOV3h RGKLKLYTGEACRTGDR

NOV3i RGKLKLYTGEACRTGDR

NOV3J RGKLKLYTGEACRTGDR

NOV3a (SEQ ID NO 102)

NOV3b (SEQ ID NO 104)

NOV3c (SEQ ID NO 106)

NOV3d (SEQ ID NO 108)

NOV3e (SEQ ID NO 110)

NOV3f (SEQ ID NO 112)

NOV3g (SEQ ID NO 114)

NOV3h (SEQ ID NO 116)

NOV3i (SEQ ID NO 118)

NOV3J (SEQ ID NO 120)

Further analysis of the NOV3a protein yielded the following properties shown in Table 3C.

Table 3C. Protein Sequence Properties NOV3a

SignalP analysis: Cleavage site between residues 28 and 29

PSORT π analysis:

PSG: a new signal peptide prediction method

N-region: length 5; pos.chg 0; neg.chg 1 H-region: length 25; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.97 possible cleavage site: between 22 and 23

>» Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1

Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 1

INTEGRAL Likelihood = -4.88 Transmembrane 10 - 26 PERIPHERAL Likelihood = 11.09 (at 56) ALOM score: -4.88 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 17 Charge difference: -0.5 C( 0.0) - N( 0.5) N >= C : N-terminal side will be inside

>» membrane topology: type 2 (cytoplasmic tail 1 to 10)

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 6.40 Hyd Moment (95): 3.98 G content: 3 D/E content: 2 S/T content: 2 Score: -7.17

Gavel : prediction of cleavage sites for mitochondrial preseq R-2 motif at 41 PRL | IC

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 13.5% NLS Score: -0.47

KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals : none

SKL: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

Actinin-type actin-binding motif: type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none

Prenylation motif : none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail: none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 76.7

COIL: Lupas ' s algorithm to detect coiled-coil regions total : 0 residues

Final Results (k = 9/23)

30 4 mitochondrial

26 1 o, cytoplasmic

13 0 % Golgi

8 7 % vacuolar

8 7 % endoplasmic reticulum

4 3 % extracellular, including cell wall

4 3 % nuclear

4 3 % vesicles of secretory system

» prediction for CG127616-01 is mit (k=23 )

A search of the NOV3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3D.

In a BLAST search of public sequence databases, the NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3E.

PFam analysis predicts that the NOV3a protein contains the domains shown in the Table

3F.

Example 4.

The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.

GGTACGAGGTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCC-AGCTCGAGGGC

NOV4d, 306448506 SEQ ID NO: 122 172 aa MW at 19900.6kD Protein Sequence

THMRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRW RHGQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIEΞNGHNTYASQRWRR RGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS

NOV4e, CG54455-01 SEQ ID NO: 123 1340 bp

DNA Sequence ORF Start: ATG at 130 ORF Stop: TGA at 640

CCATTGGCCGGCGTCCCCGCCCCAGCGAACCCGGCCCCGCCCCCGAGGCGCCCCATTGGCCCCGCCGC

GCGAAGGCAGAGCCGCGGACGCCCGGGAGCGACGAGCGCGCAGCGAACCGGGTGCCGGGTCATCCGCC GCCGCCTGTGGCTGGGCCTGGCCTGGCTGCTGCTGGCGCGGGCGCCGGACGCCGCGGGAACCCCGAGC GCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACGTGCGCTGGCGGCGCCTCTTCTCCTC CACTCACTTCTTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAGGGCACCCGCTGGCGCCACGGCCAGG ACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCATCAAAGCAGTGTCCTCAGGCTTC TACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTCTACACCGTGGACTGCAGGTTCCG GGAGCGCATCGAAGAGAACGGCCACAACACCTACGCCTCACAGCGCTGGCGCCGCCGCGGCCAGCCCA TGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCCGGACGCGGCGGTACCACCTGTCC GCCCACTTCCTGCCCGTCCTGGTCTCCTGAGGCCCTGAGAGGCCGGCGGCTCCCCAAGCCATTGGCCG

GCGTCCCCGCCCCAGCGAACCCGGCCCCGCCCCCGAGGCGCCCCATTGGCCCCGCCGCGCGAAGGCAG lAGCCGCGGACGCCCGGGAGCGACGAGCGCGCAGCGAACCGGGTGCCGGGTCATGCGCCGCCGCCTGTG

GCTGGGCCTGGCCTGGCTGCTGCTGGCGCGGGCGCCGGACGCCGCGGGAACCCCGAGCGCGTCGCGGG

GACCGCGCAGCTACCCGCACCTGGAGGGCGACGTGCGCTGGCGGCGCCTCTTCTCCTCCACTCACTTC

TTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAGGGCACCCGCTGGCGCCACGGCCAGGACAGCATCCT

GGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCATCAAAGCAGTGTCCTCAGGCTTCTACGTGGCCA

TGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTCTACACCGTGGACTGCAGGTTCCGGGAGCGCATC

GAAGAGAACGGCCACAACACCTACGCCTCACAGCGCTGGCGCCGCCGCGGCCAGCCCATGTTCCTGGC

GCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCCGGACGCGGCGGTACCACCTGTCCGCCCACTTCC

TGCCCGTCCTGGTCTCCTGAGGCCCTGAGAGGCCGGCGGCTCCCCAAG

NOV4e, CG54455-01 SEQ ID NO: 124 170 aa MW at 19662.4kD Protein Sequence

MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLΞGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRH GQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIEENGHNTYASQRWRRRG QPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS

NOV4f, CG54455-02 SEQ ED NO: 125 943 bp DNA Sequence ORF Start: at 3 jORF Stop: TGA at 483

TAGGCCGCCTCTGGCTGGGCCTAGCCTGGCTGCTGTTGACGCGGGCACCGGGCGCTCCGGGAGGGTAC CCGCATCTGGAGGGCGACGTGCGCTGGCGCCGCCTCTTCTCCTCCACTCACTTTTTCCTGCGTGTGGA CCTTGGTGGTCGGGTGCAGGGGACGCGTTGGCGGCACGGCCAGGACAGTATAGTGGAGATCCGTTCTG TCCGTGTGGGCACTGTGGTGATCAAAGCTGTGTACTCAGGCTTCTATGTGGCCATGAATCGCAGGGGC CGCCTCTATGGGTCGCGGGTCTACTCTGTGGACTGTAGGTTCCGGGAGCGCATCGAGGAGAACGGCTA CAACACATACGCCTCGCGACGTTGGAGGCACCGCGGCCGACCCATGTTCCTGGCACTTGACAGCCAAG GCATTCCCAGGCAAGGCAGACGGACACGACGGCACCAACTGTCCACACACTTCCTGCCAGTCTTGGTC TCGTCTTGAAGGGCCTGCCAATGGTTCAGGAGGCATGGGCGGCACACAGGGCCTGGAAGATCCGGAGC

TGAACAACCAAGGGCCAGGCCAGAGACCCTGGGCCAACACGAGTCTTTATGTCACAAGCCGGGCGCCC

GCTGGCTGCCGGGCATGGAGACATGGCAGGGTCCCTGCAAGTGAAGCCAGCGCTCAGGGGGATACACA

GAACTGGCAGTTTGCATCCATCTAGTTTGGAGATGAGAACACTCTGGGCACAGCACGGAGAGGTTTGG AGGTGGGAACACACACCAGGTGGATGAGGAAAGCAGGCAGGGAGGACCGGGGAGGGTGGACATGTCAC GGAGGGCAGGGCCCGCGTCAGTGGGGACAGAGACATGTTCGCCCATGTGGCCGAAGCTTGGGGCTGGA

GTTAAGAGCACTTCTTGCTCTTCCAAGGGGCCTGAGTTTAATTCTTCCACATCAGGCTG

NOV4f, CG54455-02 SEQ ID NO: 126 160 aa MW at 18639.2kD Protein Sequence

GRLWLGLAWLLLTRAPGAPGGYPHLEGDVRWRRLFSSTHFFLRVDLGGRVQGTRWRHGQDSIVEIRSV RVGTVVI-^VYSGFYVAMNRRGRLYGSRVYSVDCRFRERIEENGYNTYASRRWRHRGRPMFLALDSQG IPRQGRRTRRHQLSTHFLPVLVSS

NOV4g, CG54455-04 jSEQ ED NO: 127 444 bp jDNA Sequence ORF Start: at 1 ORF Stop: end of sequence

ACCCCGAGCGCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACGTGCGCTGGCGGCGCCT CTTCTCCTCCACTCACTTCTTCCTGCGCGTGGATCCCGGgGGCCGCGTGCAGGGCACCCG ACGGCCAGGACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCATCAAAGCAGTGTCC TCAGGCTTCTACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTCTACACCGTGGACTG CAGGTTCCGGGAGCGCATCGAAGAGAACGGCCACAACACCTACGCCTCACAGCGCTGGCGCCGCCGCG GCCAGCCCATGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCCGGACGCGGCGGTAC CACCTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCC

NOV4g, CG54455-04 SEQ ED NO: 128 148 aa MW at l7173.4kD Protein Sequence JL

TPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRHGQDSILEIRSVHVGV IKAVS SGFYV_-MNRRGRLYGSRLYTVDCRFRERIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRY: HLSAHFLPVLVS

NOV4h, CG54455-05 SEQ ID NO: 129 J550 bp DNA Sequence ORF Start: ATG at 19 ORF Stop: TGA at 529

CGAACCGGGTGCCGGGTCATGCGCCGCCGCCTGTGGCTGGGCCTGGCCTGGCTGCTGCTGGCGCGGGC GCCGGACGCCGCGGGAACCCCGAGCGCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACG TGCGCTGGCGGCGCCTCTTCTCCTCCACTCACTTCTTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAG GGCACCCGCTGGCGCCACGGCCAGGACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGT CATCAAAGCAGTGTCCTCAGGCTTCTACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGAC TCTACACCGTGGACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGGCCACAACACCTACGCCTCACAG CGCTGGCGCCGCCGCGGCCAGCCCATGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGG CCGGACGCGGCGGTACCACCTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCCTGAGGCCCTGAGAGGC CGGCGG

NOV4h, CG54455-05 SEQ ID NO: 130 170 aa MW at 19662.4kD Protein Sequence

MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRH! GQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIEENGHNTYASQRWRRRGJ QPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS

NOV4i, CG54455-06 SEQ ID NO: 131 456 bp DNA Sequence ORF Start: at 7 |ORF Stop: at 451

AGATCTACCCCGAGCGCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACGTGCGCTGGCG GCGTCTCTTCTCCTCCACTCACTTCTTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAGGGCACCCGCT GGCGCCACGGCCAGGACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCATCAAAGCA GTGTCCTCAGGCTTCTACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTCTACACCGT GGACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGGCCACAACACCTACGCCTCACAGCGCTGGCGCC GCCGCGGCCAGCCCATGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCCGGACGCGG CGGTACCACCTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCCCTCGAG

NOV4i, CG54455-06 SEQ ED NO: 132 148 aa MW at 17173.4kD Protein Sequence

TPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRHGQDSILEIRSVHVGVWIKAVS SGFYVAMNRRGRLYGSRLYTVDCRFRERIEΞNGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRY HLSAHFLPVLVS

NOV4J, CG54455-08 SEQ ED NO: 133 538 bp DNA Sequence ORF Start: ATG at 17 ORF Stop: TAG at 527

CACCGAGCTCCCCACCA-GCGCCGCCGCCTGTGGCTGGGCCTGGCCTGGCTGCTGCTGGCGCGGGCGC CGGACGCCGCGGGAACCCCGAGCGCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACGTG CGCTGGCGGCGTCTCTTCTCCTCCACTCACTTCTTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAGGG CACCCGCTGGCGCCACGGCCAGGACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCA TCAAAGCAGTGTCCTCAGGCTTCTACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTC TACACCGTGGACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGGCCACAACACCTACGCCTCACAGCG CTGGCGCCGCCGCGGCCAGCCCATGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCC GGACGCGGCGGTACCACCTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCCTAGGTCGACGGC

NOV4j, CG54455-08 SEQ ID NO: 134 170 aa MW at 19662.4kD Protein Sequence

MRRRLWLGLΆWLLLARΆPDAΆGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRH GQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIΞENGHNTYASQRWRRRG QPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS NOV4k, CG54455-09 SEQ ID NO: 135 530 bp DNA Sequence ORF Start: ATG at 12 ORF Stop: TAG at 528

AGATCTCCACCATGCGCCGCCGCCTGTGGCTGGGCCTGGCCTGGCTGCTGCTGGCGCGGGCGCCGGAC GCCGCGGGAACCCCGAGCGCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACGTGCGCTG GCGGCGTCTCTTCTCCTCCACTCACTTCTTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAGGGCACCC GCTGGCGCCACGGCCAGGACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCATCAAA GCAGTGTCCTCAGGCTTCTACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTCTACAC CGTGGACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGGCTACAACACCTACGCCTCACAGCGCTGGC GCCGCCGCGGCCAGCCCATGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCCGGACG CGGCGGTACCACCTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCCCTCGAGTAG

NOV4k, CG54455-09 SEQ ID NO: 136 172 aa MW at 19930.7kD Protein Sequence

MRRRLWLGLAWLLLARΆPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRH GQDSILEIRSVHVGVVVI--AVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIEΞNGYNTYASQRWRRRG QPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVSLE

NOV41, SNP13379002 of SEQ ID NO: 137 J510 bp

CG54455-03, DNA Sequence ORF Start: ATG at l|ORF Stop: end of sequence

SNP Pos: 358 JSNP Change: G to A

ATGCGCCGCCGCCTGTGGCTGGGCCTGGCCTGGCTGCTGCTGGCGCGGGCGCCGGACGCCGCGGGAAC CCCGAGCGCGTCGCGGGGACCGCGCAGCTACCCGCACCTGGAGGGCGACGTGCGCTGGCGGCGCCTCT TCTCCTCCACTCACTTCTTCCTGCGCGTGGATCCCGGCGGCCGCGTGCAGGGCACCCGCTGGCGCCAC GGCCAGGACAGCATCCTGGAGATCCGCTCTGTACACGTGGGCGTCGTGGTCATCAAAGCAGTGTCCTC AGGCTTCTACGTGGCCATGAACCGCCGGGGCCGCCTCTACGGGTCGCGACTCTACACCGTGGACTGCA GGTTCCGGGAGCGCATCAAAGAGAACGGCCACAACACCTACGCCTCACAGCGCTGGCGCCGCCGCGGC CAGCCCATGTTCCTGGCGCTGGACAGGAGGGGGGGGCCCCGGCCAGGCGGCCGGACGCGGCGGTACCA CCTGTCCGCCCACTTCCTGCCCGTCCTGGTCTCC

NOV41, SNP13379002 of jSEQjD NO: 138 170 aa MW at 19661.4kD CG54455-03, Protein Sequence SNP Pos: 120 SNP Change: Glu to Lys

MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRH GQDSILEIRSVHVGVVVII-AVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIKENGHNTYASQRWRRRG QPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 4B.

Table 4B. Comparison of the NOV4 protein sequences.

NOV4a MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4b ISTMRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLΞGDVRWRRLFSSTHFFLRVDP

NOV4c MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4d -THMRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4e MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4f GRLWLGLAWLLLTRAP GAPGG YPHLEGDVRWRRLFSSTHFFLRVDL

NOV4g TPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4h MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4i TPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4J MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4k MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDP

NOV4a GGRVQGTRWRHGQDSILEIRSVHVGVWIKAVSSGFYV-iMNRRGRLYGSRLYT DCRFRE

NOV4b GGRVQGTRWRHGQDSILEIRSV--VGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE

NOV4c GGRVQGTRWRHGQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE

NOV4d GGRVQGTRWRHGQDSILEIRSVHVGWVIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE

NOV4e GGRVQGTRWRHGQDSILEIRSV /GVVVI-_WSSGFYV-d_NRRGRLYGSRLYTVDCRFRE

NOV4f GGRVQGTRWRHGQDSIVEIRSVRVGTWIKAVYSGFYVAMNRRGRLYGSRVYSVDCRFRE NOV4g GGRVQGTRWRHGQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE NOV4h GGRVQGTRWRHGQDSILEIRSVHVGVVVIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE NOV4i GGRVQGTRWRHGQDSILEIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE NOV4j GGRVQGTRWRHGQDSILEIRSV--VGVVVIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE NOV4k GGRVQGTRWRHGQDSILΞIRSVHVGVWIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRE

NOV a RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4b RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVSLE NOV4c RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS-- NOV4d RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4e RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4f RIEENGYNTYASRRWRHRGRPMFLALDSQGIPRQGRRTRRHQLSTHFLPVLVSS- NOV4g RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4h RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4i RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4J RIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS— NOV4k RIΞENGYNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVSLE

NOV a (SEQ ID NO 116) NOV4b (SEQ ID NO 118) NOV4c (SEQ ID NO 120) NOV4d (SEQ ID NO 122) NOV4e (SEQ ID NO 124) NOV4f (SEQ ID NO 126) NOV4g (SEQ ID NO 128) NOV4h (SEQ ID NO 130) NOV4i (SEQ ID NO 132) NOV4J (SEQ ID NO 134) NOV4k (SEQ ID NO 136)

Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.

Table 4C. Protein Sequence Properties NOV4a

SignalP analysis: Cleavage site between residues 23 and 24

PSORT π analysis:

PSG: a new signal peptide prediction method

N-region : length 4 ; pos . chg 3 ; neg . chg 0 H-region: length 11; peak value 9.66 PSG score: 5.26

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 2.77 possible cleavage site: between 17 and 18

>» Seems to have a eleavable signal peptide (1 to 17)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 18

Tentative number of TMS(s) for the threshold 0.5: Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 12.15 (at 45) ALOM score: -1.86 (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 8

Charge difference: -3.0 C( 1.0) - N( 4.0)

N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 4 Hyd Moment (75) : 12.20 Hyd Moment (95): 7.10 G content: 1 D/E content: 1 S/T content: 0 Score: -0.86

Gavel : prediction of cleavage sites for mitochondrial preseq R-10 motif at 54 RRL FS

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: PGGRTRR (3) at 151 bipartite: none content of basic residues: 18.8% NLS Score: -0.22

KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals :

XXRR-like motif in the N-terminus: RRRL none

SKL: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal : none

VAC : possible vacuolar targeting motif : none

RNA-binding motif: none

Actinin-type actin-binding motif: type 1: none type 2 : none

NMYR: N-myristoylation pattern : none

Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail : none

Dileucine motif in the tail : none checking 63 PROSITE DNA binding motifs : none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt ' s method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 89 COIL : Lupas ' s algorithm to detect coiled-coil regions total : 0 residues

Final Results (k = 9/23 ) :

47 8 _ : mitochondrial 17 4 % : endoplasmic reticulum 8 7 %: extracellular, including cell wall 8 7 %: Golgi 8 7 % : cytoplasmic 4 3 % : vacuolar 4 3 % : nuclear

» prediction for CG54455-03 is mit (k=23 )

A search of the NOV4a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4D.

In a BLAST search of public sequence databases, the NOV4a protein was found to have homology to the proteins shown in the BLASTP data in Table 4E.

PFam analysis predicts that the NOV4a protein contains the domains shown in the Table 4F.

Example 5.

The NOV5 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 5A.

Table 5A. NO 5 Sequence Analysis

NOV5a, CG54611-06 SEQ ID NO: 139 1081 bp DNA Sequence ORF Start: ATG at 19 ORF Stop: TAG at 1075

GCGCCCTCTCGCGCGGCGA-GGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCT GGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCA TCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATC ATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCG

TTCCACTGGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTA GGCACCGGCCGCGGCTCCCCCTGGACGG

NOV5c, CG54611-01 SEQ ID NO: 144 352 aa MW at 39364.3kD Protein Sequence

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDI-ATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTC_WSQPDFRAIGDFLI_DKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLV YYEASPNFCEPNPΞTGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5d, CG54611-02 SEQ ID NO: 145 1060 bp

DNA Sequence JORF Start: ATG at 2 JORF Stop: TAG at 1058

GATCGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCAGCTACCCGATCT GGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTGTGTGCCAGCATC CCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCCCAGCGTGGCCGA GGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGAACTGCACCACCG TCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCGGCCTTTGTCCAC GCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCACGGCCGCCATCTG TGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCTGTAGCGAGGACA TCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGATGCCCGCTCA GCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCACCTCAAGTGCAA GTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCGACTTCCGCGCCA TCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAGCACCGGGAGTCC CGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGAGCGCGACCTGGT CTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCGGCACGCGCGACC GCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGGCCGCGGCCACAAC GCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCTACGTCAGCTGCCA GGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTAG

NOV5d, CG54611-02 SEQ ED NO: 146 352 -_TlMW at 39364.3kD Protein Sequence a

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLV YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5e, CG54611-03 SEQ ID NO: 147 1002 bp DNA Sequence ORF Start: at 1 j ORF Stop: end of sequence

AGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCT GTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGC CCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGG AACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTC GGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCA CGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGC TGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGTC AGATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGC ACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCC GACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAA GCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGG AGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTC GGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGG CCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCT ACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAG

NO V5e, CG54611-03 SEQ ID NO: 148 334 aa MW at 37433.9kD Protein Sequence

SYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAΞGIKIGIQECQHQFRGRRW NCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGG CSEDIEFGGMVSRΞFADARENRSDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQP DFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSF

GATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCA CCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCG ACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAG CACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGA GCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCG GCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGGC CGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCTA CGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGCACCATCACCACCATCACT IGΛCTCGAGCGG

NOV5h, CG54611-07 SEQID NO: 154 362 aa MW at40533.5kD Protein Sequence j 1

TGSTMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMP SVAΞGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGT AAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMH LKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTE RDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCY IVSCQECTRVYDVHTCKHHHHHH

NOV5L CG54611-08 SEQ ID NO: 155 1071 bp

DNA Sequence ORF Start: ATG at 10 JORF Stop: at 1066

GGATCCACCATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCAGCTA

CCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTGTGTG CCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCCCAGC GTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGAACTG CACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCGGCCT TTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCACGGCC GCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCTGTAG CGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGATG CCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCACCTC AAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCGACTT CCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAGCACC GGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGAGCGC GACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCGGCAC GCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGGCCGCG GCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCTACGTC AGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGCTCGAG

NOV5i, CG54611-08 SEQ ID NO: 156 ^352 aa MW at 39364.3kD Protein Sequence iMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC 'GCSSRHQGSPGKGW-MGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLV ^YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5j, CG54611-09 1 S _Eτ.Qr. I TTD. N MOr.:. 115 '.7π 2932 bp

DNA Sequence |₀RF Start: ATG at 79 ORF Stop: TAG at 1135

AGCTCCCAGGGCCCGGCCCCCCCCGGCGCTCACGCTCTCGGGGCGGACTCCCGGCCCTCCGCGCCCTC

CCGCGCGGCGATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCAGCT

ACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTGTGT GCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCCCAG CGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGAACT CCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCGGCC TTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCACGGC CGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCTGTA GCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGAT 'GCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCACCT CAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCGACT ITCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAGCAC iCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGAGCG CGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCGGCA CGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGGCCGC GGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCC^ CAGCTG_CAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAG_AGGCACCGGCCGCGGCTCCCC

CTGGACGGGGCGGGCCCTGCCTGAGGGTGGGCTTTTCCCTGGGTGGAGCAGGACTCCCACCTAAACGGl

GGCAGTACTCCTCCCTGGGGGCGGGACTCCTCCCTGGGGGTGGGGCTCCTACCTGGGGGCAGAACTCC

TACCTGAAGGCAGGGCTCCTCCCTGGAGCCAGTGTCTCCTCTCTGGTGGCTGGGCTGCTCCTGAATGA

GGCGGAGCTCCAGGATGGGGAGGGGCTCTGCGTTGGCTTCTCCCTGGGGACGGGGCTCCCCTGGACAG

AGGCGGGG.TACAGATTGGGCGGGGCTTCTCTTGGGTGGGACAGGGCTTCTCCTGCGGGGGCGAGGCC iCCTCCCAGTAAGGGCGTGGCTCTGGGTGGGCGGGGCACTAGGTAGGCTTCTACCTGCAGGCGGGGCTC

CTCCTGAAGGAGGCGGGGCTCTAGGATGGGGCACGGCTCTGGGGTAGGCTGCTCCCTGAGGGCGGAGC

GCCTCCTTAGGAGTGGGGTTTTATGGTGGATGAGGCTTCTTCCTGGATGGGGCAGAGCTTCTCCTGAC

CAGGGCAAGGCCCCTTCCACGGGGGCTGTGGCTCTGGGTGGGCGTGGCCTGCATAGGCTCCTTCCTGT

GGGTGGGGC.TTCTCTGGGACCAGGCTCCAATGGGGCGGGGCTTCTCTCCGCGGGTGGGACTCTTCCCT

GGGAACCGCCCTCCTGATTAAGGCGTGGCTTCTGCAGGAATCCCGGCTCCAGAGCAGGAAATTCAGCC

CACCAGCCACCTCATCCCCAACCCCCTGTAAGGTTCCATCCACCCCTGCGTCGAGCTGGGAAGGTTCC lATGAAGCGAGTCGGGTCCCCAACCCGTGCCCCTGGGATCCGAGGGCCCCTCTCCAAGCGCCTGGCTTT;

GGAATGCTCCAGGCGCGCCGACGCCTGTGCCACCCCTTCCTCAGCCTGGGGTTTGACCACCCACCTGAi

CCAGGGGCCCTACCTGGGGAAAGCCTGAAGGGCCTCCCAGCCCCCAACCCCAAGACCAAGCTTAGTCC

TGGGAGAGGACAGGGACTTCGCAGAGGCAAGCGACCGAGGCCCTCCCAAAGAGGCCCGCCCTGCCCGG

GCTCCCACACCGTCAGGTACTCCTGCCAGGGAACTGGCCTGCTGCGCCCCAGGCCCCGCCCGTCTCTG

CTCTGCTCAGCTGCGCCCCCTTCTTTGCAGCTGCCCAGCCCCTCCTCCCTGCCCTCGGGTCTCCCCAC

CTGCACTCCATCCAGCTACAGGAGAGATAGAAGCCTCTCGTCCCGTCCCTCCCTTTCCTCCGCCTGTC

CACAGCCCCTTAAGGGAAAGGTAGGAAGAGAGGTCCAGCCCCCCAGGCTGCCCAGAGCTGCTGGTCTC lATTTGGGGGCGTTCGGGAGGTTTGGGGGGCATCAACCCCCCGACTGTGCTGCTCGCGAAGGTCCCACA!

GCCCTGAGATGGGCCGGCCCCCTTCCTGGCCCCTCATGGCGGGACTGGAGAAATGGTCCGCTTTCCTGl

GAGCCAATGGCCCGGCCCCTCCTGACTCATCCGCCTGGCCCGGGAATGAATGGGGAGGCCGCTGAACC

CACCCGGCCCATATCCCTGGTTGCCTCATGGCCAGCGCCCCTCAGCCTCTGCCACTGTGAACCGGCTC

CCACCCTCAAGGTGCGGGGAGAAGAAGCGGCCAGGCGGGGCGCCCCAAGAGCCCAAAAGAGGGCACAC

CGCCATCCTCTGCCTCAAATTCTGCGTTTTTGGTTTTAATGTTATATCTGATGCTGCTATATCCACTG;

TCCAACGG

NOV5j, CG54611-09 SEQ ID NO: 158 352 aa MW at 39364.3kD Protein Sequence

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE CIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWΞKHRESRGWVETLRPRYTYFKVPTERDLV ΥYΞASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV^, CG54611-10 JSEQ ED Nθ7 l59 1014 bp

DNA Sequence |₀RF Start: at 7 ORF Stop: at 1009

GGATCCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCC

CATCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGA TCATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGC CGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAG GGAGTCGGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAG AAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGG GGTGGCTGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAA CCGGCCAGATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCC ACATGCACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCG CAACCCGACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGT GGAGAAGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGC CCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGC TCCTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTG CTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGT GCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGCTCGAG

NOV5k, CG54611-10 SEQ ID NO: 160 334 aa MW at 37444.0kD

Protein Sequence

SYPIWW^A GPQΫSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQΞCQHQFRGRRW NCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGG CSEDIEFGGMVSREFADARΞNRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCW SQP DFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTΞRDLVYYEASPNFCEPNPΞTGSF GTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK NOV51, CG54611-ll SEQ ID NO: 161 1081 bp DNA Sequence ORF Start: ATG at 14 JORF Stop: TAG at 1070

CACCGGATCCACCATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCA GCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTG TGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCC CAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGA ACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCG GCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCAC GGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCT GTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCA GATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCA CCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCG ACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAG CACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGA GCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCG GCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGATCTGCTGTGCTGCGGC CGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCTA CGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTA.GCTCGAGGGC

NOV51. CG546U-11 SEQ ED NO: 162 352 aa MW at 39364.3kD Protein Sequence

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE; GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC^' GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK] CHGLSGSCΞVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVl YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQl ECTRVYDVHTCK

NOV5m, CG54611-12 SEQ ED NO: 163 947 bp DNA Sequence ORF Start: ATG at 5 ORF Stop: TAG at 944

CTTGATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCAGCTACCCGA TCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTGTGTGCCAGC ATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCCCAGCGTGGC CGAGGGCATCAAGATCGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGAACTGCACCA CCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCGGCCTTTGTC CACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCGCGGCCGCCAT CTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCTGTAGCGAGG ACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGATGTCCGC TCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGACAAGTACGACAGCGCCTCGGAGATGGT GGTGGAGAAGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGG TGCCCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACG GGCTCCTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCT GTGCTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACT GGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTAGG

NOV5m, CG54611-12 SEQ ID NO: 164 313 aa MW at 34988.3kD Protein Sequence

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGAAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDVRSAMNRHNNEAGRQDKYDSASEMW EKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLC CGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK

NOV5n, CG54611-13 SEQ ID NO: 165 1194 bp DNA Sequence ORF Start: ATG at 28 jORF Stop: TAG at 1165

GATGGCCCACTCGGATACTTCTTACTGA-GGCCCCACTCGGATACTTCTTACTGATGGCCCCACTCGG ATACTTCTTACTGATGGCCCCACTCGGATACTTCTTACTGATGGCCCCACTCGGATACTTCTTACTCC TCTGCAGCCTGAAGCAGGCTCTGGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTAT TCCTCCCTGGGCTCGCAGCCCATCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTT CTGCAGGAACTACGTGGAGATCATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCC AGCACCAGTTCCGCGGCCGCCGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCC GTGCTGGACAAAGCTACCAGGGAGTCGGCC-TTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGC AGTGACACGCTCATGTGCAGAAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCAC

GGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTAGAAGGGCGAATTCCGCC

NOV5p, CG54611-15 SEQ ID NO: 170 352 aa MW at 39364.3kD Protein Sequence

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRΞSAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIΞFGGMVSREFADARENRPDARS-MNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVITCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLV YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5q, CG54611-16 SEQ ED NO: 171 1194 bp DNA Sequence ORF Start: ATG at 28 ORF Stop: TAG at 1165

GATGGCCCACTCGGA-ACTTCTTACTG__-GGCCCCACTCGGA-ACTTCTTACTGATGGCCCCACTCGG ATACTTCTTACTGATGGCCCCACTCGGATACTTCTTACTGATGGCCCCACTCGGATACTTCTTACTCC TCTGCAGCCTGAAGCAGGCTCTGGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTAT TCCTCCCTGGGCTCGCAGCCCATCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTT CTGCAGGAACTACGTGGAGATCATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCC AGCACCAGTTCCGCGGCCGCCGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCC GTGCTGGACAAAGCTACCAGGGAGTCGGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGC AGTGACACGCTCATGTGCAGAAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCAC CGGGCAAGGGCTGGAAGTGGGGTGGCTGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAG TTCGCCGACGCCCGGGAGAACCGGCCAGATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGG GCGCCAGGCCATCGCCAGCCACATGCACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGG TGAAGACATGCTGGTGGTCGCAACCCGACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGAC AGCGCCTCGGAGATGGTGGTGGAGAAGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCG CTACACCTACTTCAAGGTGCCCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCG AGCCCAACCCTGAGACGGGCTCTTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATC GACGGCTGCGACCTGCTGTGCTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTG CCGCTGCGTGTTCCACTGGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACA CCTGCAAG-AGGCACGTGCACACCTGCAAGTAGGCATC

NOV5q, CG54611-16 SEQ ED NO: 172 379 aa MW at 42383. lkD Protein Sequence

MAPLGYFLLMAPLGYFLLMAPLGYFLLMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPIL CASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRΞS AFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRP DARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEK HRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCG RGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK

NOV5r, CG54611-17 SEQ ID NO: 173 1060 bp DNA Sequence ORF Start: ATG at 2 ORF Stop: TAG at 1058

GATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCAGCTACCCGATCT GGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTGTGTGCCAGCATC CCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCCCAGCGTGGCCGA GGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGAACTGCACCACCG TCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCGGCCTTTGTCCAC GCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCACGGCCGCCATCTG TGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCTGTAGCGAGGACA TCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGATGCCCGCTCA GCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCACCTCAAGTGCAA GTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCGACTTCCGCGCCA TCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAGCACCGGGAGTCC CGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGAGCGCGACCTGGT CTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCGGCACGCGCGACC GCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGGCCGCGGCCACAAC GCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCTACGTCAGCTGCCA GGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAG-AG

NOV5r, CG54611-17 SEQ ID NO: 174 352 aa MW at 39364.3kD

Protein Sequence

MAPLGYFLLLCSLKQALGSYPIW SLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIOECOHOFRGRRWNCTTVHDSLAIFGPVLDKATRΞSAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGG--VSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLV YYΞASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5s, CG54611-18 SEQ ID NO: 175 1060 bp

DNA Sequence ORF Start: ATG at 2 JORF Stop: TAG at 1058

G__-SGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCTGAAGCAGGCTCTGGGCAGCTACCCGATCT GGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGGGCTCGCAGCCCATCCTGTGTGCCAGCATC CCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAACTACGTGGAGATCATGCCCAGCGTGGCCGA GGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTTCCGCGGCCGCCGGTGGAACTGCACCACCG TCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACAAAGCTACCAGGGAGTCGGCCTTTGTCCAC GCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGCTCATGTGCAGAAGGCACGGCCGCCATCTG TGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGGCTGGAAGTGGGGTGGCTGTAGCGAGGACA TCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACGCCCGGGAGAACCGGCCAGATGCCCGCTCA GCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCCATCGCCAGCCACATGCACCTCAAGTGCAA GTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATGCTGGTGGTCGCAACCCGACTTCCGCGCCA TCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGGAGATGGTGGTGGAGAAGCACCGGGAGTCC CGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTACTTCAAGGTGCCCACGGAGCGCGACCTGGT CTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCCTGAGACGGGCTCCTTCGGCACGCGCGACC GCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCGACCTGCTGTGCTGCGGCCGCGGCCACAAC GCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTGTTCCACTGGTGCTGCTACGTCAGCTGCCA GGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTAG

NOV5s, CG54611-18 SEQ ED NO: 176 352 aa MW at 39364.3kD Protein Sequence

MAPLGYFLLLCSLKQALGSYPIW SLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLV YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5t, SNP13378438 of jSEQ ID NO: 177 JΪΪΪ6 bp CG54611-01, DNA Sequence ORF Start: ATG at 31 ORF Stop: TAG at 1087

SNP Pos: 149 SNP Change: T to C

TCCCGGCCCTCCGCGCCCTCTCGCGCGGCGATGGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCT GAAGCAGGCTCTGGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGG GCTCGCAGCCCACCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAAC TACGTGGAGATCATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTT CCGCGGCCGCCGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACA AAGCTACCAGGGAGTCGGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGC TCATGTGCAGAAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGG CTGGAAGTGGGGTGGCTGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACG CCCGGGAGAACCGGCCAGATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCC ATCGCCAGCCACATGCACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATG CTGGTGGTCGCAACCCGACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGG AGATGGTGGTGGAGAAGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTAC TTCAAGGTGCCCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCC TGAGACGGGCTCCTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCG ACCTGCTGTGCTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTG TTCCACTGGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTA GGCACCGGCCGCGGCTCCCCCTGGACGG

NOV5t, SNP13378438 of SEQ ID NO: 178 352 aa MW at 39352.3kD CG54611-01, Protein Sequence SNP Pos: 40 fSNP Change: Ile to Thr

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPTLCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGG_ΓVSREFADAREKKPD_^SAMN___\INEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLV ^!YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

GAAGCAGGCTCTGGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGG GCTCGCAGCCCATCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAAC TACGTGGAGATCATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTT CCGCGGCCGCCGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACA ^!AAGCTACCAGGGAGTCGGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGC TCATGTGCAGAAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGG CTGGAAGTGGGGTGGCTGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACG CCCGGGAGAACCGGCCAGATGCCCGCTCΆGCCATGAACCGCCACΆACAACGΆGGCTGGGCGCCΆGGCC ATCGCCAGCCACATGCACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATG CTGGTGGTCGCAACCCGACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGG AGATGGTGGTGGAGAΆGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTAC TTCAAGGTGCCCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAΆCTTCTGCGAGCCCΆACCC TGAGACGGGCCCCTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCG ACCTGCTGTGCTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTG TTCCACTGGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAG__- GGCACCGGCCGCGGCTCCCCCTGGACGG

NOV6y, SNP13381647 of SEQ ID NO: 188 352 aa |MW at 39374.4kD CG54611-01, Protein Sequence SNP Pos: 289 JSNP Change: Serto Pro _iMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQΞCQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSRΞFADARΞNRPDARSA-_NRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLV .YYEASPNFCEPNPETGPFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5z, SNP13381648 of SEQ ID NO: 189 1116 bp CG54611-01, DNA Sequence ORF Start: ATG at 31 ORF Stop: TAG at 1087

SNP Pos: 961 SNP Change: T to C

TCCCGGCCCTCCGCGCCCTCTCGCGCGGCGA-GGCCCCACTCGGATACTTCTTACTCCTCTGCAGCCT GAAGCAGGCTCTGGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGG gCTCGCAGCCCATCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAAC TACGTGGAGATCATGCCCAGCGTGGCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTT CCGCGGCCGCCGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACA AAGCTACCAGGGAGTCGGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGC TCATGTGCAGAAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGG CTGGAAGTGGGGTGGCTGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACG CCCGGGAGAACCGGCCAGATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCC ,ATCGCCAGCCACATGCACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATG CTGGTGGTCGCAACCCGACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGG AGATGGTGGTGGAGAAGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTAC _|TTCAAGGTGCCCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCC TGAGACGGGCTCCTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCG ACCTGCTGCGCTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTG TTCCACTGGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGTGCACACCTGCAAGTA GGCACCGGCCGCGGCTCCCCCTGGACGG

NOV5z, SNP13381648 of SEQ ED NO: 190J352 aa JMW at 39417.4kD

CG54611-01, Protein Sequence JSNP Pos: 311 SNP Change: Cys to Arg

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARE_RPD-__SAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLV YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLRCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDVHTCK

NOV5aa, SNP13381649 of SEQ ED NO: 191 1116 bp CG54611-01, DNA Sequence ORF Start: ATG at 31 ORF Stop: TAG at 1087

SNP Pos: 1073 SNP Change: T to C

TCCCGGCCCTCCGCGCCCTCTCGCGCGGCGa__GCCCCACTCGGATACTTCTTACTCCTCTGCAGCCT GAAGCAGGCTCTGGGCAGCTACCCGATCTGGTGGTCGCTGGCTGTTGGGCCACAGTATTCCTCCCTGG GCTCGCAGCCCATCCTGTGTGCCAGCATCCCGGGCCTGGTCCCCAAGCAGCTCCGCTTCTGCAGGAAC TACGTGGAGATCATGCCCAGCGTGgCCGAGGGCATCAAGATTGGCATCCAGGAGTGCCAGCACCAGTT CCGCGGCCGCCGGTGGAACTGCACCACCGTCCACGACAGCCTGGCCATCTTCGGGCCCGTGCTGGACA AAGCTACCAGGGAGTCGGCCTTTGTCCACGCCATTGCCTCAGCCGGTGTGGCCTTTGCAGTGACACGC TCATGTGCAGAAGGCACGGCCGCCATCTGTGGCTGCAGCAGCCGCCACCAGGGCTCACCAGGCAAGGG CTGGAAGTGGGGTGGCTGTAGCGAGGACATCGAGTTTGGTGGGATGGTGTCTCGGGAGTTCGCCGACG CCCGGGAGAACCGGCCAGATGCCCGCTCAGCCATGAACCGCCACAACAACGAGGCTGGGCGCCAGGCC ATCGCCAGCCACATGCACCTCAAGTGCAAGTGCCACGGGCTGTCGGGCAGCTGCGAGGTGAAGACATG CTGGTGGTCGCAACCCGACTTCCGCGCCATCGGTGACTTCCTCAAGGACAAGTACGACAGCGCCTCGG AGATGGTGGTGGAGAAGCACCGGGAGTCCCGCGGCTGGGTGGAGACCCTGCGGCCGCGCTACACCTAC TTCAAGGTGCCCACGGAGCGCGACCTGGTCTACTACGAGGCCTCGCCCAACTTCTGCGAGCCCAACCC TGAGACGGGCTCCTTCGGCACGCGCGACCGCACCTGCAACGTCAGCTCGCACGGCATCGACGGCTGCG ACCTGCTGTGCTGCGGCCGCGGCCACAACGCGCGAGCGGAGCGGCGCCGGGAGAAGTGCCGCTGCGTG TTCCACTGGTGCTGCTACGTCAGCTGCCAGGAGTGCACGCGCGTCTACGACGCGCACACCTGCAAG_2_ GGCACCGGCCGCGGCTCCCCCTGGACGG

NOV5aa, SNP13381649 of SEQ ID NO: 192 52 aa |MW at 39336.3kD CG54611-01, Protein Sequence SNP Pos: 348 SNP Change: Val to Ala

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAE GIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAIC GCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCK CHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLV YYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQ ECTRVYDAHTCK

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 5B.

Table 5B. Comparison of the NOV5 protein sequences.

NOV5a MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5b GSSYPIWWSLAVGPQYS

NOV5c MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5d MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5e SYPIWWSLAVGPQYS

NOV5f SYPIWWSLAVGPQYS

NOV5g MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5h TGSTMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5i MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5J MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5k SYPIWWSLAVGPQYS

NOV51 MAPLGYFLLLCSLKQALGSYPIW SLAVGPQYS

NOV5m MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5n MAPLGYFLLMAPLGYFLLMAPLGYFLLMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5o MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5p MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5q MAPLGYFLLMAPLGYFLLMAPLGYFLLMAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5r MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5S MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYS

NOV5a SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5b SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAΞGIKIGIQECQHQFRGRRWNCTTVHD

N0V5c SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5d SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

N0V5e SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5f SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQΞCQHQFRGRRWNCTTVHD

NOV5g SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5h SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5i SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5J SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5k SLGSQPILCASIPGLVPKQLRFCR-JYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV51 SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD N0V5m SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

N0V5n SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5o SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5p SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5q SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAΞGIKIGIQECQHQFRGRRWNCTTVHD

NOV5r SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5s SLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHD

NOV5a SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5b SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5c SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5d SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5e SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5f SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5g SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5h SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5i SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5j SLAIFGPVLDKΆTRESAFVΉAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

N0V5k SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV51 SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5m SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGAAAICGCSSRHQGSPGKGWKWG

NOV5n SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5o SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5p SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5q SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5r SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWG

NOV5s SLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAΞGTAAICGCSSRHQGSPGKGWKWG

NOV5a GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5b GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5c ~ GCSEDIEFGG_-/SREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5d GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5e GCSEDIEFGGMVSREFADARENRSDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5f GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5g GCSEDIEFGG-T^SREFADAREN-IPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5h GCSΞDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5i GCSEDIEFGG_-/SREFADARE_ PDARSA_^RHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5j GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5k GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV51 GCSEDIΞFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5m GCSEDIEFGGMVSREFADARENRPDVRSAMNRHISINEAGRQ

NOV5n GCSEDIΞFGGMVSREFADARΞNRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

N0V5O GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5p GCSEDIΞFGGMVSREFADARΞNRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5q GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5r GCSEDIEFGGMVSREFADARΞNRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5S GCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCE

NOV5a VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5b VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVΞTLRPRYTYFKVPTERDLVY

NOV5c VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTΞRDLVY

NOV5d VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5e VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5f VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5g VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5h VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5i VICTCW SQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5J VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5k VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTΞRDLVY NOV51 VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRΞSRGWVETLRPRYTYFKVPTERDLVY NOV5m DKYDSASEMWΞKHRΞSRGWVETLRPRYTYFKVPTERDLVY NOV5n VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY NOV5o VKTCWWSQPDFRAIGDFL--DKYDSASΞMWEKHRESRGWVETLRPRYTYFKVPTERDLVY NOV5p VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY NOV5q VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY NOV5r VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY NOV5s VKTCWWSQPDFRAIGDFLKDKYDSASEMWEKHRESRGWVETLRPRYTYFKVPTERDLVY

NOV5a YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5b YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5c YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5d YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRRΞKCRCVFHW NOV5e YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5f YEASPNFCEPNPETGSFG NOV5g YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5h YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5i YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFH NOV5J YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRRΞKCRCVFHW NOV5k YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAΞRRRΞKCRCVFHW NOV51 YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5m YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5n YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5o YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5p YEASPNFCΞPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRRΞKCRCVFHW NOV5q YEASPNFCΞPNPΞTGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAΞRRREKCRCVFHW NOV5r YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW NOV5s YEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHW

NOV5a CCYVSCQECTRVYDVHTCK NOV5b CCYVSCQECTRVYDVHTCKLE NOV5c CCYVSCQECTRVYDVHTCK NOV5d CCYVSCQECTRVYDVHTCK NOV5e CCYVSCQECTRVYDVHTCK NOV5f TRVYDVHTCK NOV5g CCYVSCQECTRVYDVHTCK NOV5h CCYVSCQECTRVYDVHTCKHHHHHH NOV5i CCYVSCQECTRVYDVHTCK NOV5J CCYVSCQECTRVYDVHTCK NOV5k CCYVSCQECTRVYDVHTCK NOV51 CCYVSCQECTRVYDVHTCK NOV5m CCYVSCQECTRVYDVHTCK NOV5n CCYVSCQECTRVYDVHTCK NOV5o CCYVSCQECTRVYDVHTCK NOV5p CCYVSCQECTRVYDVHTCK NOV5q CCYVSCQECTRVYDVHTCK NOV5r CCYVSCQECTRVYDVHTCK NOV5s CCYVSCQECTRVYDVHTCK

NOV5a NOV5b NOV5c NOV5d NOV5e NOV5f NOV5g NOV5h NOV5i NOV5J

NOV5k (SEQ ID NO 160)

NOV51 (SEQ ID NO 162)

NOV5m (SEQ ID NO 164)

NOV5n (SEQ ID NO 166)

NOV5o (SEQ ID NO 168)

NOV5p (SEQ ID NO 170)

NOV5q (SEQ ID NO 172)

NOV5r (SEQ ID NO 174)

NOV5s (SEQ ID NO 176)

Further analysis of the NOV5a protein yielded the following properties shown in Table

5C.

Table 5C. Protein Sequence Properties NOV5a

SignalP analysis: Cleavage site between residues 19 and20

PSORT π analysis:

PSG: a new signal peptide prediction method

N-region: length 0; pos.chg 0; neg. chg 0 H-region: length 13; peak value 9.00 PSG score: 4.60

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.73 possible cleavage site: between 18 and 19

>» Seems to have a eleavable signal peptide (1 to 18)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 19

Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 4.61 (at 33) ALOM score: -0.37 (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 9 Charge difference: 0.0 C( 1.0) - N( 1.0) N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 1.56 Hyd Momen (95) : 3.50 G content: 5 D/E content: 1 S/T content: 7 Score: -4.38

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 67 CRN|YV

MJCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 12.5% NLS Score: -0.47 KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals : none

SKL: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

Actinin-type actin-binding motif : type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none

Prenylation motif : none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail : none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt ' s method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 55.5

COIL: Lupas ' s algorithm to detect coiled-coil regions total : 0 residues

Final Results (k = 9/23) :

33. .3 %: extracellular, including cell wall

33. .3 %: mitochondrial

11. .1 %: Golgi

11. .1 %: vacuolar

11. .1 %: endoplasmic reticulum

» prediction for CG54611-06 is exc (k=9)

A search of the NOV5a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 5D.

In a BLAST search of public sequence databases, the NOV5a protein was found to have homology to the proteins shown in the BLASTP data in Table 6E.

PFam analysis predicts that the NOV5a protein contains the domains shown in the Table 5F.

Example 6.

The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.

GCCGGCCTCAAGAACCCCCACGAGGGTTACGAGGTACTCAAGTTTGACGACGTGGTCACCAACCTAGG CAACAACTACGACGCGGCCAGCGGCAAGTTAACGTGCAACATTCCCGGCACCTACTTTTTCACCTACC ATGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGCAAGAATGGCCAGGTGCGG GCCAGTGCTATTGCCCAGGACGCGGACCAGAACTACGACTACGCCAGCAACAGCGTGATCCTGCACCT GGACGCCGGCGACGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACGGCGGCAACAGCAACAAAT ACAGCACGTTCTCTGGCTTCATCATCTACTCCGACTGAGCTCCCCAC

NOV6a, CG92035-02 SEQ ID NO: 194 252 aa MW at 25749.4kD Protein Sequence

IVLIPVLVSSGGPEGHYEMLGTCRMVCDPYPARGPGAGARTDGGDALSEQSGAPPPSTLVQGPQGKPGR GKPGPPGPPGDPGPPGPVGPPGEKGEPGKPGPPGLPGAGGSGAISTATYTTVPRVAFYAGLKNPHEG YEλtLKFDDVVTNLGNNYDAASGKLTCNIPGTYFFTYHVLMRGGDGTSM ADLCKNGQVRASAIAQDAD QNYDYASNSVILHLDAGDΞVFIKLDGGKAHGGNSNKYSTFSGFIIYSD

NOV6b, 214458541 JSEQ ID NO: 195 1387 bp DNA Sequence ORF Start: at 1 ORF Stop: end of sequence

GGATCCGCCTTCTACGCCGGCCTCAAGAACCCCCACGAGGGTTACGAGGTACTCAAGTTTGACGACGT GGTCACCAACCTAGGTAACAACTACGACGCGGCCAGCGGCGAGTTTACGTGCAACATTCCCGGCACCT ACTTTTTCACCTACCATGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGCAAG AATGGCCAGGTGCGGGCCAGTGCTATTGCCCAGGACGCGGACCAGAACTACGACTACGCCAGCAGCAG CGTGATCCTGCACCTGGACGCCGGTGACGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACGGCG GCAACAGCAACAAATACAGCACGTTCTCTGGCTTCATCATCCTCGAG

NOV6b, 214458541 SEQ ID NO: 196 129 aa MW at 13937.2kD Protein Sequence

GSAFYAGLKNPHEGYEVLKFDDVVTNLGN-T_D--ASGEFTCNIPGTYFFTY-WLMRGGDGTSMADLCK NGQVRASAlAQDADQNYDYASSSVILHLDAGDΞVFIKLDGGKAHGGNSNKYSTFSGFIILE

NOV6c, 214458545 SEQ ID NO: 197 387 bp DNA Sequence ORF Start: at 1 lORF Stop: end of sequence

GGATCCGCCTTCTACGCCGGCCTCAAGAACCCCCACGAGGGTTACGAGGTACTCAAGTTTGACGACGT GGTCACCAACCTAGGCAACAACTACGACGCGGCCAGCGGCAAGTTTACGTGCAACATTCCCGGCACCT ACTTTTTCACCTACCATGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGCAAG AATGGCCAGGTGCGGGCCAGTGCTATTGCCCAGGACGCGGACCAGAACTACGACTACGCCAGCAACAG CGTGATCATGCACCTGGACGCCGGCGACGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACGGCG GCAACAGCAACAAATACAGCACGTTCTCTGGCTTCATCATCCTCGAG

NOV6c, 214458545 SEQ ID NO: 198 129 aa MW at l3981.3kD Protein Sequence

GSAFYAGL--NPHEGYEVLKFDDVVTNLGNNYDAASGKFTCNIPGTYFFTY-WLMRGGDGTSMWADLCKI NGQVRASAIAQDADQNYDYASNSVIMHLDAGDΞVFIKLDGGKAHGGNSNKYSTFSGFIILE

NOV6d, 214458564 SEQ ID NO: 199 |387 bp DNA Sequence ORF Start: at 1 JORF Stop: end of sequence

GGATCCGCCTTCTACGCCGGCCTCAAGAACCCCCACGAGGGTTCCGAGGTACTCAAGTTTGACGACGT GGTCACCAACCTAGGCAACAACTACGACGCGGCCAGCGGCAAGTTTACGTGCAACATTCCCGGCACCT ACTTTTTCACCTACCATGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGCAAG AATGGCCAGGTGCGGGCCAGTGCTATTGCCCAGGACGCGGACCAGAACTACGACTACGCCAGCAACAG CGTGATCCTGCACCTGGACGCCGGCGACGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACGGCG GCAACAGCAACAAATACAGCACGTTCTCTGGCTTCATCATCCTCGAG

NOV6d, 214458564 SEQ ID NO: 200 129 aa MW at 13887.2kD Protein Sequence

GSAFYAGL--NPHEGSEVLKFDDVVTNLGNNYDAASGKFTCNIPGTYFFTYHVLMRGGDGTSMWADLCK NGQVRASAIAQDADQNYDYASNSVILHLDAGDEVFIKLDGGKAHGGNSNKYSTFSGFIILΞ

NOV6e, CG92035-01 |SEQ Γ NO: 201 813 bp

DNA Sequence |θl_F Sta-_7ATC at 28^~ fORF Stop: TGA at 802

GCGGCGGCGGCCGCCGCGGGTGTGGTGATGCTGCTGGTGCTGGTGGTGCTCATCCCCGTGCTGGTGAG CTCGGGCGGCCCGGAAGGCCACTATGAGATGCTGGGCACCTGCCGCATGGTGTGCGACCCCTACCCCG CGCGGGGCCCCGGCGCCGGCGCGCGGACCGACGGCGGCGACGCCCTC^ CCGCCTTCCACGCTGGTGCAGGGCCCCCAGGGGAAGCCGGGCCGCACCGGCAAGCCCGGCCCTCCGGG GCCTCCCGGGGACCCAGGTCCTCCCGGCCCTGTGGGGCCGCCGGGGGAGAAGGGTGAGCCAGGCAAGC CGGGCCCTCCGGGGCTGCCGGGCGCGGGGGGCAGCGGCGCCATCAGCACTGCCACCTACACCACGGTG CCGCGCGTGGCCTTCTACGCCGGCCTCAAGAACCCCCACGAGGGTTACGAGGTACTCAAGTTTGACGA CGTGGTCACCAACCTAGGCAACAACTACGACGCGGCCAGCGGCAAGTTAACGTGCAACATTCCCGGCA CCTACTTTTTCACCTACCATGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGC AAGAATGGCCAGGTGCGGGCCAGTGCTATTGCCCAGGACGCGGACCAGAACTACGACTACGCCAGCAA CAGCGTGATCCTGCACCTGGACGCCGGCGACGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACG GCGGCAACAGCAACAAATACAGCACGTTCTCTGGCTTCΑTCATCTACTCCGACΕez-GCTCCCCAC

NOV6e, CG92035-01 SEQ ID NO: 202 258 aa MW at 26418.3kD Protein Sequence

MLLVLWLIPVLVSSGGPEGHYEMLGTCR-_VCDPYPARGPGAGARTDGGDALSEQSGAPPPSTLVQGP QGKPGRTGKPGPPGPPGDPGPPGPVGPPGEKGEPGKPGPPGLPGAGGSGAISTATYTTVPRVAFYAGL I_.PHΞGYEVLKFDDVVTNLG__ r_D--ASGKLTCNIPGTYFFTYHVL RGGDGTSMWADLC--_ IAQDADQNYDYASNSVILHLDAGDEVFIKLDGGKAHGGNSNKYSTFSGFIIYSD

NOV6f, CG9203 )3 ^"~ SEQ ID NO: 203 387 bp DNA Sequence ORF Start: at 7 ORF Stop: at 382

GG-VrCCGCCTTCTACGCCGGCCTCAAGAACCCCCACGAGGGTTACGAGGTACTCAAGTTTGACGACGT GGTCACCAACCTAGGCAACAACTACGACGCGGCCAGCGGCAAGTTTACGTGCAACATTCCCGGCACCT ACTTTTTCACCTACCATGTCCTCATGCGCGGCGGCGACGGCACCAGTATGTGGGCAGACCTCTGCAAG AATGGCCAGGTGCGGGCCAGTGCTATTGCCCAGGACGCGGACCAGAACTACGACTACGCCAGCAACAG CGTGATCCTGCACCTGGACGCCGGTGACGAGGTCTTCATCAAGCTGGATGGAGGCAAAGCACACGGCG GCAACAGCAACAAATACAGCACGTTCTCTGGCTTCATCATCCTCGAG

NOV6f, CG92035-03 SEQ ID NO: 204 125 aa MW at 13576.9kD Protein Sequence

AFYAGL-_.PHEGYEVLKFDDVVTNLGN_rYD- SGKFTCNIPGTYFFTY--VLMRGGDGTSM ADLCKNG QVRASAIAQDADQNYDYASNSVILHLDAGDEVFIKLDGGKAHGGNSNKYSTFSGFII

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 6B.

Table 7B. Comparison of the NOV6 protein sequences.

NOV6a VLIPVLVSSGGPEGHYEMLGTCRMVCDPYPARGPGAGARTDGGDALSEQSGAPP

NOV6b

NOV6c

NOV6d

NOVδe MLLV_,VV_.IPVLVSSGGPEGHYE LGTC-_-VCDPYPARGPGAGARTDGGDALSEQSGAPP

NOV6f

NOV6a PSTLVQGPQGKPGRTGKPGPPGPPGDPGPPGPVGPPGEKGEPGKPGPPGLPGAGGSGAIS

NOV6b

NOV6c

NOV6d

NOV6e PSTLVQGPQGKPGRTGKPGPPGPPGDPGPPGPVGPPGEKGEPGKPGPPGLPGAGGSGAIS

NOV6f

NOV6a TATYTTVPRVAFYAGLKNPHEGYEVLKFDDWTNLGNNYDAASGKLTCNIPGTYFFTYHV

NOV6b GSAFYAGLKNPHEGYEVLKFDDWTNLGNNYDAASGEFTCNIPGTYFFTYHV

NOV6c GSAFYAGLKNPHEGYEVLKFDDWTNLGNNYDAASGKFTCNIPGTYFFTYHV

NOVβd GSAFYAGL NPHEGSEVLKFDDWTNLGNNYDAASGKFTCNIPGTYFFTYHV

NOV6e TATYTTVPRVAFYAGL-_.PHEGYEVLKFDDVVTNLGNNYDAASGKLTCNIPGTYFFTYHV

NOV6f AFYAGLKNPHEGYEVLKFDDWTNLGNNYDAASGKFTCNIPGTYFFTYHV

NOV6a LMRGGDGTSMWADLCKNGQVRASAl QDADQNYDYASNSVILHLDAGDEVFIKLDGGKAH

NOV6b LMRGGDGTSMWADLCKNGQVRASAIAQDADQNYDYASSSVILHLDAGDEVFIKLDGGKAH

NOV6c LMRGGDGTS_IWADLCK_GQVRASAIAQDADQNYDYASNSVI_-HLDAGDEVFIKLDGGKAH NOV6d LMRGGDGTSMWADLC-_.GQVT -ASAIAQDADQNYDYASNSVILHLDAGDEVFIKLDGGKAH NOV6e LMRGGDGTS]_WADLC-_.GQV-_ SAIAQDADQNYDYASNSVILHLDAGDEVFIKLDGG__-H NOV6f LMRGGDGTSM ADLCKNGQV-_ SAIAQDADQNYDYASNSVILHLDAGDEVFIKLDGG__-H

NOV6a GGNSNKYSTFSGFIIYSD NOV6b GGNSNKYSTFSGFIILE-

NOV6c GGNSNKYSTFSGFIILE- NOV6d GGNSNKYSTFSGFIILΞ- NOV6e GGNSNKYSTFSGFIIYSD NOV6 f GGNSNKYSTFSGFII

NOV6a ( SEQ ID NO 194 NOVδb ( SEQ ID NO 196 NOV6c ( SEQ ID NO 198 NOV6d ( SEQ ID NO 200 NOV6e ( SEQ ID NO 202 NOV6f ( SEQ ID NO 204

Further analysis of the NO V6a protein yielded the following properties shown in Table 6C.

Table 6C. Protein Sequence Properties NOV6a

SignalP analysis: No Known Signal Sequence Predicted

PSORT π analysis:

PSG: a new signal peptide prediction method

N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 12; peak value 8.85 PSG score: 4.45

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -5.49 possible cleavage site: between 14 and 15

>» Seems to have no N-terminal signal peptide

ALOM: Klein et al ' s method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: number of TMS(s) .. fixed PERIPHERAL Likelihood = 7.43 (at 160) ALOM score: 7.43 (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 6 Charge difference: -2.5 C(-1.5) - N( 1.0) N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Momen (75) : 3.67 Hyd Moment (95): 2.25 G content: 3 D/E content: 2 S/T content: 2 Score: -8.66

Gavel : prediction of cleavage sites for mitochondrial preseq cleavage site motif not found MJCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite : none content of basic residues: 7.1% NLS Score: -0.47

KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals : none

SKL: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

Actinin-type actin-binding motif: type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none >

Prenylation motif : none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail : none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt ' s method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 70.6

COIL: Lupas ' s algorithm to detect coiled-coil regions total : 0 residues

Final Results (k = 9/23)

43 5 %: cytoplasmic

34 8 nuclear

13 0 -a : mitochondrial

4 3 Ό ' extracellular, including cell wall

4 3 %: vacuolar

» prediction for CG92035-02 is cyt (k=23J A search of the NOV6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6D.

In a BLAST search of public sequence databases, the NOV6a protein was found to have homology to the proteins shown in the BLASTP data in Table 6E.

PFam analysis predicts that the NOV6a protein contains the domains shown in the Table 6F.

Example B: Sequencing Methodology and Identification of NOVX Clones

1. GeneCalling™ Technology: This is a proprietary method of performing differential gene expression profiling between two or more samples developed at CuraGen and described by Shimkets, et al., "Gene expression analysis by transcript profiling coupled to a gene database query" Nature Biotechnology 17:198-803 (1999). cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then digested with up to as many as 120 pairs of restriction enzymes and pairs of linker-adaptors specific for each pair of restriction enzymes were ligated to the appropriate end. The restriction digestion generates a mixture of unique cDNA gene fragments. Limited PCR amplification is performed with primers homologous to the linker adapter sequence where one primer is biotinylated and the other is fluorescently labeled. The doubly labeled material is isolated and the fluorescently labeled single strand is resolved by capillary gel electrophoresis. A computer algorithm compares the electropherograms from an experimental and control group for each of the restriction digestions. This and additional sequence-derived information is used to predict the identity of each differentially expressed gene fragment using a variety of genetic databases. The identity of the gene fragment is confirmed by additional, gene-specific competitive PCR or by isolation and sequencing of the gene fragment.

2. SeqCalling™ Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

3. PathCalling™ Technology: The NOVX nucleic acid sequences are derived by laboratory screening of cDNA library by the two-hybrid approach. cDNA fragments covering either the full length of the DNA sequence, or part of the sequence, or both, are sequenced. In silico prediction was based on sequences available in CuraGen Corporation's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof. The laboratory screening was performed using the methods summarized below: cDNA libraries were derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then directionally cloned into the appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). Such cDNA libraries as well as commercially available cDNA libraries from Clontech (Palo Alto, CA) were then transferred from E.coli into a CuraGen Corporation proprietary yeast strain (disclosed in U. S. Patents 6,057,101 and 6,083,693, incorporated herein by reference in their entireties).

Gal4-binding domain (Gal4-BD) fusions of a CuraGen Corporation proprietary library of human sequences was used to screen multiple Gal4-AD fusion cDNA libraries resulting in the selection of yeast hybrid diploids in each of which the Gal4-AD fusion contains an individual cDNA. Each sample was amplified using the polymerase chain reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such PCR product was sequenced; sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations. Physical clone: the cDNA fragment derived by the screening procedure, covering the entire open reading frame is, as a recombinant DNA, cloned into pACT2 plasmid (Clontech) used to make the cDNA library. The recombinant plasmid is inserted into the host and selected by the yeast hybrid diploid generated during the screening procedure by the mating of both CuraGen Corporation proprietary yeast strains N106' and YULH (U. S. Patents 6,057,101 and 6,083,693).

4. RACE: Techniques based on the polymerase chain reaction such as rapid amplification of cDNA ends (RACE), were used to isolate or complete the predicted sequence of the cDNA of the invention. Usually multiple clones were sequenced from one or more human samples to derive the sequences for fragments. Various human tissue samples from different donors were used for the RACE reaction. The sequences derived from these procedures were included in the SeqCalling Assembly process described in preceding paragraphs. 5. Exon Linking: The NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.

6. Physical Clone: Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clones used for expression and screening purposes.

Example C: Quantitative expression analysis of clones in various cells and tissues

The quantitative expression of various NOV genes was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ-PCR) performed on an Applied Biosystems (Foster City, CA) ABI PRISM® 7700 or an ABI PRISM® 7900 HT Sequence Detection System.

RNA integrity of all samples was determined by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s: 18s) and the absence of low molecular weight RNAs (degradation products). Control samples to detect genomic DNA contamination included RTQ-PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.

RNA samples were normalized in reference to nucleic acids encoding constitutively expressed genes (i.e., β-actin and GAPDH). Alternatively, non-normalized RNA samples were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen Corporation, Carlsbad, CA, Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 μg of total RNA in a volume of 20 μl or were scaled up to contain 50 μg of total RNA in a volume of 100 μl and were incubated for 60 minutes at 42°C. sscDNA samples were then normalized in reference to nucleic acids as described above.

Probes and primers were designed according to Applied Biosystems Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default reaction condition settings and the following parameters were set before selecting primers: 250 nM primer concentration; 58°-60° C primer melting temperature (Tm) range; 59° C primer optimal Tm; 2° C maximum primer difference (if probe does not have 5' G, probe Tm must be 10° C greater than primer Tm; and 75 bp to 100 bp amplicon size. The selected probes and primers were synthesized, by Synthegen (Houston, TX). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: 900 nM forward and reverse primers, and 200nM probe.

Normalized RNA was spotted in individual wells of a 96 or 384- well PCR plate (Applied Biosystems, Foster City, CA). PCR cocktails included a single gene-specific probe and primers set or two multiplexed probe and primers sets. PCR reactions were done using TaqMan® One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No. 4313803) following manufacturer's instructions. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles: 95° C 10 min, then 40 cycles at 95° C for 15 seconds, followed by 60° C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) and plotted using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression was the reciprocal of the RNA difference multiplied by 100. CT values below 28 indicate high expression, between 28 and 32 indicate moderate expression, between 32 and 35 indicate low expression and above 35 reflect levels of expression that were too low to be measured reliably.

Normalized sscDNA was analyzed by RTQ-PCR using IX TaqMan® Universal Master mix (Applied Biosystems; catalog No.4324020), following the manufacturer's instructions. PCR amplification and analysis were done as described above. Panels 1, 1.1, 1.2, and 1.3D

Panels 1, 1.1, 1.2 and 1.3D included 2 control wells (genomic DNA control and chemistry control) and 94 wells of cDNA samples from cultured cell lines and primary normal tissues. Cell lines were derived from carcinomas (ca) including: lung, small cell (s cell var), non small cell (non-s or non-sm); breast; melanoma; colon; prostate; glioma (glio), astrocytoma (astro) and neuroblastoma (neuro); squamous cell (squam); ovarian; liver; renal; gastric and pancreatic from the American Type Culture Collection (ATCC, Bethesda, MD). Normal tissues were obtained from individual adults or fetuses and included: adult and fetal skeletal muscle, adult and fetal heart, adult and fetal kidney, adult and fetal liver, adult and fetal lung, brain, spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose. The following abbreviations are used in reporting the results: metastasis (met); pleural effusion (pi. eff or pi effusion) and * indicates established from metastasis. General_screening_panel_vl.4, vl.5, vl.6 and vl.7 Panels 1.4, 1.5, 1.6 and 1.7 were as described for Panels 1, 1.1, 1.2 and 1.3D, above except that normal tissue samples were pooled from 2 to 5 different adults or fetuses.

Panels 2D, 2.2, 2.3 and 2.4

Panels 2D, 2.2, 2.3 and 2.4 included 2 control wells and 94 wells containing RNA or cDNA from human surgical specimens procured through the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI), Ardais (Lexington, MA) or Clinomics BioSciences (Frederick, MD). Tissues included human malignancies and in some cases matched adjacent normal tissue (NAT). Information regarding histopathological assessment of tumor differentiation grade as well as the clinical stage of the patient from which samples were obtained was generally available. Normal tissue RNA and cDNA samples were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics and Invitrogen (Carlsbad, CA).

HASS Panel v 1.0

The HASS Panel vl.O included 93 cDNA samples and two controls including: 81 samples of cultured human cancer cell lines subjected to serum starvation, acidosis and anoxia according to established procedures for various lengths of time; 3 human primary cells; 9 malignant brain cancers (4 medulloblastomas and 5 glioblastomas); and 2 controls. Cancer cell lines (ATCC) were cultured using recommended conditions and included: breast, prostate, bladder, pancreatic and CNS. Primary human cells were obtained from Clonetics (Walkersville, MD). Malignant brain samples were gifts from the Henry Ford Cancer Center.

ARDAIS Panel vl.O and vl.l

The ARDAIS Panel vl.O and vl.l included 2 controls and 22 test samples including: human lung adenocarcinomas, lung squamous cell carcinomas, and in some cases matched adjacent normal tissues (NAT) obtained from Ardais (Lexington, MA). Unmatched malignant and non-malignant RNA samples from lungs with gross histopathological assessment of tumor differentiation grade and stage and clinical state of the patient were obtained from Ardais. ARDAIS Prostate vl.O

ARDAIS Prostate vl.O panel included 2 controls and 68 test samples of human prostate malignancies and in some cases matched adjacent normal tissues (NAT) obtained from Ardais (Lexington, MA). RNA from unmatched malignant and non-malignant prostate samples with gross histopathological assessment of tumor differentiation grade and stage and clinical state of the patient were also obtained from Ardais. ARDAIS Kidney vl.O

ARDAIS Kidney vl.O panel included 2 control wells and 44 lest samples of human renal cell carcinoma and in some cases matched adjacent normal tissue (NAT) obtained from Ardais (Lexington, MA). RNA from unmatched renal cell carcinoma and normal tissue with gross histopathological assessment of tumor differentiation grade and stage and clinical state of the patient were also obtained from Ardais. ARDAIS Breast vl.O

ARDAIS Breast vl.O panel included 2 control wells and 71 test samples of human breast malignancies and in some cases matched adjacent normal tissue (NAT) obtained from Ardais (Lexington, MA). RNA from unmatched malignant and non-malignant breast samples with gross histopathological assessment of tumor differentiation grade and stage and clinical state of the patient were also obtained from Ardais. Panel 3D, 3.1 and 3.2 Panels 3D, 3.1, and 3.2 included two controls, 92 cDNA samples of cultured human cancer cell lines and 2 samples of human primary cerebellum. Cell lines (ATCC, National Cancer Institute (NCI), German tumor cell bank) were cultured as recommended and were derived from: squamous cell carcinoma of the tongue, melanoma, sarcoma, leukemia, lymphoma, and epidermoid, bladder, pancreas, kidney, breast, prostate, ovary, uterus, cervix, stomach, colon, lung and CNS carcinomas. Panels 4D, 4R, and 4.1D

Panels 4D, 4R, and 4. ID included 2 control wells and 94 test samples of RNA (Panel 4R) or cDNA (Panels 4D and 4. ID) from human cell lines or tissues related to inflammatory conditions. Controls included total RNA from normal tissues such as colon, lung (Stratagene, La JoUa, CA), thymus and kidney (Clontech, Palo Alto, CA). Total RNA from cirrhotic and lupus kidney was obtained from BioChain Institute, Inc., (Hayward, CA). Crohn's intestinal and ulcerative colitis samples were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Cells purchased from Clonetics (Walkersville, MD) included: astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, and human umbilical vein endothelial. These primary cell types were activated by incubating with various cytokines (IL-1 beta -1-5 ng/ml, TNF alpha -5-10 ng/ml, IFN gamma -20-50 ng/ml, IL-4 -5-10 ng/ml, IL-9 -5-10 ng/ml, IL-13 5-10 ng/ml) or combinations of cytokines as indicated. Starved endothelial cells were cultured in the basal media (Clonetics, Walkersville, MD) with 0.1% serum.

Mononuclear cells were prepared from blood donations using Ficoll. LAK cells were cultured in culture media [DMEM, 5% FCS (Hyclone, Logan, UT), 100 mM non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco)] and interleukin 2 for 4-6 days. Cells were activated with 10-20 ng/ml PMA and 1-2 μg/ml ionomycin, 5-10 ng/ml IL-12, 20-50 ng/ml IFN gamma or 5-10 ng/ml D -18 for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in culture media with -5 mg/ml PHA (phytohemagglutinin) or PWM (pokeweed mitogen; Sigma- Aldrich Corp., St. Louis, MO). Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing them 1:1 at a final concentration of -2x10⁶ cells/ml in culture media. The MLR samples were taken at various time points from 1-7 days for RNA preparation.

Monocytes were isolated from mononuclear cells using CD 14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culturing in culture media with 50 ng ml GMCSF and 5 ng/ml IL-4 for 5-7 days.

Macrophages were prepared by culturing monocytes for 5-7 days in culture media with -50 ng/ml 10% type AB Human Serum (Life technologies, Rockville, MD) or MCSF (Macrophage colony stimulating factor; R&D, Minneapolis, MN). Monocytes, macrophages and dendritic cells were stimulated for 6 or 12-14 hours with 100 ng/ml lipopolysaccharide (LPS). Dendritic cells were also stimulated with 10 μg/ml anti-CD40 monoclonal antibody (Pharmingen, San Diego, CA) for 6 or 12-14 hours.

CD4+ lymphocytes, CD8+ lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. CD45+RA and CD45+RO CD4+ lymphocytes were isolated by depleting mononuclear cells of CD8+, CD56+ , CD14+ and CD19+ cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45RO Miltenyi beads were then used to separate the CD45+RO CD4+ lymphocytes from CD45+RA CD4+ lymphocytes. CD45+RA CD4+ , CD45+RO CD4 +and CD8+ lymphocytes were cultured in culture media at 10⁶ cells/ml in culture plates precoated overnight with 0.5 mg/ml anti- CD28 (Pharmingen, San Diego, CA) and 3 μg/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8+ lymphocytes, isolated CD8+ lymphocytes were activated for 4 days on anti-CD28, anti-CD3 coated plates and then harvested and expanded in culture media with IL-2 (1 ng/ml). These CD8+ cells were activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as described above. RNA was isolated 6 and 24 hours after the second activation and after 4 days of the second expansion culture. Isolated NK cells were cultured in culture media with 1 ng ml IL-2 for 4-6 days before RNA was prepared.

B cells were prepared from minced and sieved tonsil tissue (NDRI). Tonsil cells were pelleted and resupended at 10⁶ cells/ml in culture media. Cells were activated using 5 μg/ml PWM (Sigma-Aldrich Corp., St. Louis, MO) or -10 μg/ml anti-CD40 (Pharmingen, San Diego, CA) and 5-10 ng/ml IL-4. Cells were harvested for RNA preparation after 24, 48 and 72 hours.

To prepare primary and secondary Thl/Th2 and Tri cells, umbilical cord blood CD4+ lymphocytes (Poietic Systems, German Town, MD) were cultured at 10⁵- 10⁶cells/ml in culture media with IL-2 (4 ng/ml) in 6-well Falcon plates (precoated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml anti-CD3 (OKT3; ATCC) then washed twice with PBS).

To stimulate Thl phenotype differentiation, IL-12 (5 ng/ml) and anti-_L4 (1 μg/ml) were used; for Th2 phenotype differentiation, IL-4 (5 ng/ml) and anti-IFN gamma (1 μg/ml) were used; and for Tri phenotype differentiation, IL-10 (5 ng/ml) was used. After 4-5 days, the activated Thl, Th2 and Tri lymphocytes were washed once with DMEM and expanded for 4-7 days in culture media with IL-2 (1 ng/ml). Activated Thl, Th2 and Tri lymphocytes were re-stimulated for 5 days with anti-CD28/CD3 and cytokines as described above with the addition of anti-CD95L (1 μg/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Tri lymphocytes were washed and expanded in culture media with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Tri after 6 and 24 hours following the second and third activations with plate-bound anti-CD3 and anti- CD28 mAbs and 4 days into the second and third expansion cultures.

Leukocyte cells lines Ramos, EOL-1, KU-812 were obtained from the ATCC. EOL-1 cells were further differentiated by culturing in culture media at 5 xlO⁵ cells/ml with 0.1 mM dbcAMP for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO cells/ml. RNA was prepared from resting cells or cells activated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 6 and 14 hours. RNA was prepared from resting CCD 1106 keratinocyte cell line (ATCC) or from cells activated with -5 ng/ml TNF alpha and 1 ng/ml IL-1 beta. RNA was prepared from resting NCI-H292, airway epithelial tumor cell line (ATCC) or from cells activated for 6 and 14 hours in culture media with 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13, and 25 ng/ml IFN gamma. RNA was prepared by lysing approximately 10 cells/ml using Trizol (Gibco BRL) then adding 1/10 volume of bromochloropropane (Molecular Research Corporation, Cincinnati, OH), vortexing, incubating for 10 minutes at room temperature and then spinning at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was placed in a 15 ml Falcon Tube and an equal volume of isopropanol was added and left at -20° C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min and washed in 70% ethanol. The pellet was redissolved in 300 μl of RNAse-free water with 35 ml buffer (Promega, Madison, WI) 5 μl DTT, 7 μl RNAsin and 8 μl DNAse and incubated at 37° C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down, placed in RNAse free water and stored at -80° C.

AI_comprehensive panel_vl.O

Autoimmunity (Al) comprehensive panel vl.O included two controls and 89 cDNA test samples isolated from male (M) and female (F) surgical and postmortem human tissues that were obtained from the Backus Hospital and Clinomics (Frederick, MD). Tissue samples included : normal, adjacent (Adj); matched normal adjacent (match control); joint tissues (synovial (Syn) fluid, synovium, bone and cartilage, osteoarthritis (OA), rheumatoid arthritis (RA)); psoriatic; ulcerative colitis colon; Crohns disease colon; and emphysmatic, asthmatic, allergic and chronic obstructive pulmonary disease (COPD) lung.

Pulmonary and General inflammation (PGI) panel vl.O Pulmonary and General inflammation (PGI) panel vl.O included two controls and 39 test samples isolated as surgical or postmortem samples. Tissue samples include: five normal lung samples obtained from Maryland Brain and Tissue Bank, University of Maryland (Baltimore, MD), International Bioresource systems, IBS (Tuscon, AZ), and Asterand (Detroit, MI), five normal adjacent intestine tissues (NAT) from Ardais (Lexington, MA), ulcerative colitis samples (UC) from Ardais (Lexington, MA); Crohns disease colon from NDRI, National Disease Research Interchange (Philadelphia, PA); emphysematous tissue samples from Ardais (Lexington, MA) and Genomic Collaborative Inc. (Cambridge, MA), asthmatic tissue from Maryland Brain and Tissue Bank, University of Maryland (Baltimore, MD) and Genomic Collaborative Ine (Cambridge, MA) and fibrotic tissue from Ardais (Lexinton, MA) and Genomic Collaborative (Cambridge, MA). Cellular OA/RA Panel

Cellular OA.RA panel includes 2 control wells and 35 test samples comprised of cDNA generated from total RNA isolated from human cell lines or primary cells representative of the human joint and its inflammatory condition. Cell types included normal human osteoblasts (Nhost) from Clonetics (Cambrex, East Rutherford, NJ), human chondrosarcoma SW1353 cells from ATCC (Manossas, VA)), human fibroblast-like synoviocytes from Cell Applications, Inc. (San Diego, CA) and MH7A cell line (a rheumatoid fibroblast-like synoviocytes transformed with S V40 T antigen) from Riken Cell bank ( Tsukuba Science City, Japan). These cell types were activated by incubating with various cytokines (IL-1 beta -1-10 ng/ml, TNF alpha -5-50 ng/ml, or prostaglandin E2 for Nhost cells) for 1, 6, 18 or 24 h. All these cells were starved for at least 5 h and cultured in their corresponding basal medium with - 0.1 to 1 % FBS. Minitissue OA RA Panel

The OA/RA mini panel includes two control wells and 31 test samples comprised of cDNA generated from total RNA isolated from surgical and postmortem human tissues obtained from the University of Calgary (Alberta, Canada), NDRI (Philadelphia, PA), and Ardais Corporation (Lexington, MA). Joint tissue samples include synovium, bone and cartilage from osteoarthritic and rheumatoid arthritis patients undergoing reconstructive knee surgery, as well as, normal synovium samples (RNA and tissue). Visceral normal tissues were pooled from 2-5 different adults and included adrenal gland, heart, kidney, brain, colon, lung, stomach, small intestine, skeletal muscle, and ovary. AI.05 chondrosarcoma

AI.05 chondrosarcoma plates included SW1353 cells (ATCC) subjected to serum starvation and treated for 6 and 18 h with cytokines that are known to induce MMP (1, 3 and 13) synthesis (e.g. ILlbeta). These treatments included: IL-lbeta (10 ng/ml), IL-lbeta + TNF-alpha (50 ng/ml), IL-lbeta + Oncostatin (50 ng/ml) and PMA (100 ng/ml). Supernatants were collected and analyzed for MMP 1, 3 and 13 production. RNA was prepared from these samples using standard procedures. Panels 5D and 51

Panel 5D and 51 included two controls and cDNAs isolated from human tissues, human pancreatic islets cells, cell lines, metabolic tissues obtained from patients enrolled in the Gestational Diabetes study (described below), and cells from different stages of adipocyte differentiation, including differentiated (AD), midway differentiated (AM), and undifferentiated (U; human mesenchymal stem cells).

Gestational Diabetes study subjects were young (18 - 40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarean section. Uterine wall smooth muscle (UT), visceral (Vis) adipose, skeletal muscle (SK), placenta (PI) greater omentum adipose (GO Adipose) and subcutaneous (SubQ) adipose samples (less than 1 cc) were collected, rinsed in sterile saline, blotted and flash frozen in liquid nitrogen. Patients included: Patient 2, an overweight diabetic Hispanic not on insulin; Patient 7-9, obese non-diabetic Caucasians with body mass index (BMI) greater than 30; Patient 10, an overweight diabetic Hispanic, on insulin; Patient 11, an overweight nondiabetic African American; and Patient 12, a diabetic Hispanic on insulin. Differentiated adipocytes were obtained from induced donor progenitor cells

(Clonetics, Walkersville, MD). Differentiated human mesenchymal stem cells (HuMSCs) were prepared as described in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr 2 1999: 143-147. mRNA was isolated and sscDNA was produced from Trizol lysates or frozen pellets. Human cell lines (ATCC, NCI or German tumor cell bank) included: kidney proximal convoluted tubule, uterine smooth muscle cells, small intestine, liver HepG2 cancer cells, heart primary stromal cells and adrenal cortical adenoma cells. Cells were cultured, RNA extracted and sscDNA was produced using standard procedures. Panel 51 also contains pancreatic islets (Diabetes Research Institute at the University of Miami School of Medicine).

Human Metabolic RTQ-PCR Panel

Human Metabolic RTQ-PCR Panel included two controls (genomic DNA control and chemistry control) and 211 cDNAs isolated from human tissues and cell lines relevant to metabolic diseases. This panel identifies genes that play a role in the etiology and pathogenesis of obesity and/or diabetes. Metabolic tissues including placenta (PI), uterine wall smooth muscle (Ut), visceral adipose, skeletal muscle (Sk) and subcutaneous (SubQ) adipose were obtained from the Gestational Diabetes study (described above). Included in the panel are: Patients 7 and 8, obese non-diabetic Caucasians; Patient 12 a diabetic Caucasian with unknown BMI, on insulin (treated); Patient 13, an overweight diabetic Caucasian, not on insulin (untreated); Patient 15, an obese, untreated, diabetic Caucasian; Patient 17 and 25, untreated diabetic Caucasians of normal weight; Patient 18, an obese, untreated, diabetic Hispanic; Patient 19, a non-diabetic Caucasian of normal weight; Patient 20, an overweight, treated diabetic Caucasian; Patient 21 and 23, overweight non- diabetic Caucasians; Patient 22, a treated diabetic Caucasian of normal weight; Patient 23, an overweight non-diabetic Caucasian; and Patients 26 and 27, obese, treated, diabetic Caucasians.

Total RNA was isolated from metabolic tissues including: hypothalamus, liver, pancreas, pancreatic islets, small intestine, psoas muscle, diaphragm muscle, visceral (Vis) adipose, subcutaneous (SubQ) adipose and greater omentum (Go) from 12 Type II diabetic (Diab) patients and 12 non diabetic (Norm) at autopsy. Control diabetic and non-diabetic subjects were matched where possible for: age; sex, male (M); female (F); ethnicity, Caucasian (CC); Hispanic (HI); African American (AA); Asian (AS); and BMI, 20-25 (Low BM), 26-30 (Med BM) or overweight (Overwt), BMI greater than 30 (Hi BMI) (obese).

RNA was extracted and ss cDNA was produced from cell lines (ATCC) by standard methods. CNS Panels CNS Panels CNSD .01 , CNS Neurodegeneration V 1.0 and CNS Neurodegeneration

V2.0 included two controls and 46 to 94 test cDNA samples isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital). Brains were removed from calvaria of donors between 4 and 24 hours after death, and frozen at -80° C in liquid nitrogen vapor. Panel CNSD.01

Panel CNSD.01 included two specimens each from: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy (PSP),

Depression, and normal controls. Collected tissues included: cingulate gyrus (Cing Gyr), temporal pole (Temp Pole), globus palladus (Glob palladus), substantia nigra (Sub Nigra), primary motor strip (Brodman Area 4), parietal cortex (Brodman Area 7), prefrontal cortex (Brodman Area 9), and occipital cortex (Brodman area 17). Not all brain regions are represented in all cases.

Panel CNS Neurodegeneration Vl.O

The CNS Neurodegeneration Vl.O panel included: six Alzheimer's disease (AD) brains and eight normals which included no dementia and no Alzheimer's like pathology (control) or no dementia but evidence of severe Alzheimer's like pathology (Control Path), specifically senile plaque load rated as level 3 on a scale of 0-3; 0 no evidence of plaques, 3 severe AD senile plaque load. Tissues collected included: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), occipital cortex (Brodman area 17) superior temporal cortex (Sup Temporal Ctx) and inferior temporal cortex (Inf Temproal Ctx). Gene expression was analyzed after normalization using a scaling factor calculated by subtracting the Well mean (CT average for the specific tissue) from the Grand mean (average CT value for all wells across all runs). The scaled CT value is the result of the raw CT value plus the scaling factor.

Panel CNS Neurodegeneration V2.0 The CNS Neurodegeneration V2.0 panel included sixteen cases of Alzheimer's disease (AD) and twenty-nine normal controls (no evidence of dementia prior to death) including fourteen controls (Control) with no dementia and no Alzheimer's like pathology and fifteen controls with no dementia but evidence of severe Alzheimer's like pathology (AH3), specifically senile plaque load rated as level 3 on a scale of 0-3; 0 no evidence of plaques, 3 severe AD senile plaque load. Tissues from the temporal cortex (Brodman Area 21) included the inferior and superior temporal cortex that was pooled from a given individual (Inf & Sup Temp Ctx Pool). A. NOVl (CG104903-03 and CG104903-09 and CG104903-10): HMW kininogen with 5'utr.

Expression of genes the CG104903-03, CG104903-09 and CG104903-10 was assessed using the primer-probe sets Ag3374 and Ag4269, described in Tables AA and AB. Results of the RTQ-PCR runs are shown in Tables AC and AD.

Table AA. Probe Name Ag3374

Table AB. Probe Name Ag4269

Start SEQ ID

Primers Sequences jLength Position I No

Forward 5 ' -acagagcatttggcaagct-3 ' 19 1610 208

TΞT-5 ' -cagtactacaccttctgcacagacaca-3 ' Probe

..jTAMRA 27 1639 209

Reverse 5 ' -gttggcccttctgtcttctc-3 ' 20 1667 210

Table AC. Ardais Kidney 1.0

Table AD. Panel 4. ID

Ardais Kidney 1.0 Summary: Ag3374 Much higher expression of this gene was detected in normal kidney tissues (CT=19.71-23.21) adjacent to a tumor, while most renal tumor tissues had low to non-detectable levels of its expression. Thus, expression of this gene can be used as a marker of normal kidney. In addition, therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product are useful in the treatment of renal cancer.

Panel 4.1D Summary: Ag5115/Ag4269 Two experiments with two different probe and primer sets were in good agreement with highest expression of the CG104903- 06 in the kidney (CTs=27). Moderate levels of expression of this gene was also seen in thymus, lung and colon. The probe and primer sets for Ag5115 are specific to CG104903- 06. In a second experiment with Ag4269 low levels of expression of this gene was also seen in selected samples, including T cells, neutrophils, and activated dermal fibroblasts, indicating that the protien encoded by this gene play an important role in T cell development. In addition, moderate expression was seen in liver cirrhosis (CT=28.73). Thus, therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product are useful in modulation of liver function, identification and treatment of inflammatory or autoimmune diseases which effect the liver including liver cirrhosis and fibrosis.

B. N V2a (CG 120-44-02): IL-1 Beta-like

Expression of gene CG120844-02 was assessed using the primer-probe sets Agll41, Ag6369, Ag6601, Ag6658 and Ag6701, described in Tables BA, BB, BC, BD and BE. Results of the RTQ-PCR runs are shown in Tables BF, BG and BH.

Table BA. Probe Name Agl 141

Start SEQ ID

Primers Sequences Length Position JNo_

Forward 5 ' -tctcaagcagaaaacatgcc-3 ' 20 572 211

TET-5 ' -aggcggccaggatataactgacttca-3 '

Probe TAMRA 26 613 212

Reverse 5 ' -ggaagacacaaattgcatgg-3 20 639 213

Table BB. Probe Name Ag6369

Table BC. Probe Name Ag6601

Table BD. Probe Name Ag6658

Table BE. Probe Name Ag6701

Table BF. AI_comprehensive panel_ l.O

Table BG. Cellular OA/RA

Table BH. General_screening_panel_vl.4

AI_comprehensive panel_vl.O Summary: Agll41/Ag6369 Highest express on of this gene was detected in matched control sample for ulcerative colitis (CTs=27). Significant expression of this gene was also seen in samples derived from normal and orthoarthitis/rheumatoid arthritis bone, cartilage, synovium and synovial fluid samples, from normal lung, COPD lung, emphysema, atopic asthma, asthma, Crohn's disease (normal matched control and diseased), ulcerative colitis(normal matched control and diseased), and psoriasis (normal matched control and diseased). Therefore, therapeutic modulation of this gene, encoded protein and/or use of antibodies or small molecule targeting this gene or gene product will ameliorate symptoms/conditions associated with autoimmune and inflammatory disorders including psoriasis, allergy, asthma, inflammatory bowel disease, rheumatoid arthritis and osteoarthritis.

Cellular OA/RA Summary: Agll41 Highest expression of this gene was detected in TNF alpha treated MH7A (synoviocyte) cell line (CT=19.7). Expression of this gene was upregulated in activated normal osteoblast (Nhost), chondrocytes (SW1353 cell line) and synoviocyte cell lines. Therefore, modulation of this gene, encoded protein and/or use of antibodies or small molecule targeting this gene or gene product is useful in the treatment of inflammatory and autoimmune diseases such as osteoarthritis and rheumatoid arthritis.

General_screening_panel_vl.4 Summary: Agl 141 Highest expression of this gene was detected in a brain cancer U87-MG cell line (CT=20.9). High to moderate expression of this gene was also seen in number of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression levels of this gene is useful as marker to detect the presence of these cancers. Therapeutic modulation of this gene, encoded protein and/or use of small molecule targeting this gene or gene product is effective in the treatment of pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers.

Among tissues with metabolic or endocrine function, this gene was expressed at moderate levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation of this gene, encoded protein is useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

This gene was expressed at moderate levels in all regions of the central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therapeutic modulation of this gene, encoded protein and/or use of antibodies or small molecule targeting this gene or gene product is useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

C. NOV3 (CG127616-01 and CG127616-02): FL_104 aa (del 54-143aa) and Partial (del l-4aa; 54-143aa) [Erythropoietin precursor-like] Expression of genes CGI 27616-01 and CGI 27616-02 was assessed using the primer-probe sets Ag4746 and Ag6361, described in Tables CA and CB. Results of the RTQ-PCR runs are shown in Tables CC and CD.

Table CA. Probe Name Ag4746

Table CB. Probe Name Ag6361

TET-5 ' -ttccgagtctactccaatttcctccg-3 '

Probe TAMRA 26 243 230

Reverse 15 ' -cctgtgtacagcttcagctttc-3 22 271 231

Table CC. General_screening_panel_vl.4

Table CD. general oncology screening panel_y_2.4

General_screening_panel_vl.4 Summary: Ag4746 Highest expression of this gene was seen in a liver cancer cell line (CT=30.3). Moderate levels of expression were also seen in colon, renal and lung cancer cell lines, with low but significant expression detectable in lymph node and fetal liver. The transcript for this gene encodes a putative variant of erythropoietin (Epo), which is produced in the kidney and liver of normal adults. Human erythropoietin (Epo) is an acidic glycoprotein hormone that mediates the production of red blood cells, promotes erythroid differentiation, initiates hemoglobin synthesis. In addition, Epo has been shown to be a potent growth factor for the development of red blood cells from hematopoetic stem cells. Thus, the expression in hematopoietic tissues in this panel is consistent with the characterization of this novel protein as a novel variant of Epo. Modulation of the expression or function of this gene product is useful in the treatment of hematopoietic disorders. general oncology screening panel_v_2.4 Summary: Ag4746 Moderate expression of this gene was seen in two samples derived from kidney cancers (CTs=32). The expression of this gene is useful as a marker for kidney cancer.

D. NOV4 (CG54455-03 and CG54455-07): FGF-10X

Expression of genes CG54455-03 and CG54455-07 was assessed using the primer- probe sets Ag4346, Ag4347 and Ag7772, described in Tables DA, DB and DC. Results of the RTQ-PCR runs are shown in Tables DD, DE, DF, DG, DH, DI, DJ and DK.

Table DA. Probe Name Ag4346

Table DB. Probe Name Ag4347

Table DC. Probe Name Ag7772

Table DD. AI_comprehensive panel_vl.O

Table DE. Cellular OA/RA

Table DF. General_screening_panel_vl.4

Table DG. Mini tissue OA/RA

Table DH.PGI1.0

Table DI. Panel 3D

Table DJ. Panel 4. ID

Table DK. general oncology screening panel_v_2.4

AI_comprehensive panel_vl.0 Summary: Ag4347 Highest expression of this gene was detected in normal lung (CT=30.8). This gene showed a wide spread low expression in this panel. Moderate to low levels of expression of this gene were detected in samples derived from normal and orthoarthitis/rheumatoid arthritis bone, cartilage, and synovium samples, from normal lung, COPD lung, emphysema, atopic asthma, asthma, allergy, Crohn's disease (normal matched control and diseased), ulcerative colitis (normal matched control and diseased), and psoriasis (normal matched control and diseased). Therefore, therapeutic modulation of this gene, encoded protein and/or use of antibodies or small molecule targeting this gene or gene product is useful in the treatment of autoimmune and inflammatory disorders including psoriasis, allergy, asthma, inflammatory bowel disease, rheumatoid arthritis and osteoarthritis

Cellular OA/RA Summary: Ag4346 Highest expression of this gene was detected in MH7A (synoviocyte) cell line (CT=33.5). Low expression of this gene was also detected in untreated and activated SW1353 (chondrocyte) cell lines, and activated MH7A cells. Therefore, modulation of this gene or encoded protein will be useful in the treatment of orthoarthritis and rheumatoid arthritis.

(General_screening_panel_vl.4 Summary: Ag4347 Highest expression of this gene was detected in ovarian cancer SK-OV-3 cell line (CT=32). Low expression of this gene was detected in number of cancer cell lines derived from ovarian, breast, and colon cancers. Therefore, therapeutic modulaion of this gene, expressed protein and/or use of antibodies or small molecule drug targeting this gene or gene product is useful in the treatment of ovarian, breast and colon cancers. Low levels of expression of this gene were seen in all the regions of the central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Expression analysis of this gene using CuraChip 1.2 (see example 2) showed that this gene was down-regulated in the temporal cortex of Alzheimer's patient but was up-regulated in patients who were found to have serious Alzheimer disease-like pathology with no associated dementia relative to the control patients. Therefore, therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drug targeting this gene or gene product is useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression. Mini tissue OA/RA Summary: Ag4346 Highest expression of this gene was detected in brain (CT=29.9). Significant expression of this gene was also seen in normal synovium, kidney, colon, ovary and small intestine. Expression of this gene was slightly downregulated in synovium from OA and RA patients. Therefore, modulation of this gene and/or encoded protein is useful in the treatment of orthoarthritis and rheumatoid arthritis. PGI1.0 Summary: Ag4346 Highest expression of this gene was detected in lung fibrosis sample (CT=30.8). Significant levels of expression of this gene was also detected in emphyzema and asthma lung. Therefore, therapeutic modulaion of this gene, encoded protein and/or use of expressed protein, antibodies or small molecule drug targeting this gene or gene product is useful in the treatment of emphyzema, asthma and lung fibrosis.

Panel 3D Summary: Ag4347 Low expression of this gene was detected mainly in a small cell lung cancer DMS-79 and DMS-114 cell lines (CTs=33-34). Therefore, therapeutic modulation of this gene, encoded protein, and/or use of small molecule drug targeting this gene or gene product is useful in the treatment of small cell lung cancer.

Panel 4.1D Summary: Ag4346 Moderate levels of expression of this gene was detected mainly in kidney sample (CT=31.8). Therefore, therapeutic modulation of this gene is useful in the treatment of kidney related diseases including lupus erythematosus and glomerulonephritis. general oncology screening panel_v_2.4 Summary: Ag4347 Low expression of this gene was detected in a colon cancer, a metastatic melanoma and a kidney cancer samples. Therefore, therapeutic modulation of this gene, encoded protein,and/or use of antibodies or small molecule drug targeting this gene or gene product is useful in the treatment of these cancers.

E. NOV5a (CG54611-06): WNT-3A PROTEIN PRECURSOR

Expression of gene CG54611-06 was assessed using the primer-probe sets Ag2445 and Ag7111, described in Tables EA and EB. Results of the RTQ-PCR runs are shown in Tables EC and ED.

Table EA. Probe Name Ag2445

Table EB. Probe Name Ag7111

Table EC. Ardais Panel 1.1

Table ED. General_screening_panel_vl.7

Ardais Panel 1.1 Summary: Ag2445 Highest expression of this gene was detected in a matched control sample for lung cancer (CT=28.5). The gene expression was down-regulated in coresponding cancer tissues. This gene encodes wingless-type MMTV integration site family, member 3A (WNT3A). Therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product are useful in the treatment of lung cancer.

General_screening_panel_vl.7 Summary: Ag2445 and Ag7111 Results from two experiments using the two different probe and primer sets that respond to the AL391534_C gene are in very good agreement. Moderate expression was detected in normal lung (CT=28.7, 29.8) but not in any of the 9 lung cancer lines examined. It is consistant with data from patient tissues, see Ardais Panel 1.1. Thus, therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product is useful in the treatment of lung cancer. Moderate expression was also detected in trachea (CT=30.26, 31.89), one (out of 9) colon cancer line (CT=28.9, 30.2) and one (out of 3) Melanoma cell line (CT=29.6, 30.6).

F. NOVδa (CG92035-02): CIQ-EELATED FACTOR PRECURSOR

Expression of gene CG92035-02 was assessed using the primer-probe sets Ag3767, Ag4935 and Ag4902, described in Tables FA, FB and FC. Results ofthe RTQ- PCR runs are shown in Tables FD and FE.

Table FA. Probe Name Ag3767

Table FB. Probe Name Ag4935

Table FC. Probe Name Ag4902

Table FD. General_screening_panel_vl.4

General_screening_panel_vl.4 Summary: Ag3767 High expression of this gene was detected in 2 renal cancer A498 and 786-0 cell lines (CTs=26-28). Moderate expression of this gene was also seen in number of cancer cell lines derived from melanoma, brain, breast, ovary, lung and prostate cancers. Therefore, expression of this gene can be used as diagnostic marker to detect the presence of these cancers. Therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product is useful in the treatment of kidney, melanoma, brain, breast, ovary, lung and prostate cancers.

This gene was expressed at moderate to low levels in all regions of the central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product is useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression. This gene was expressed at much higher level in fetal (CT=32.3) when compared to adult heart(CT=35). This observation indicates that the protein product may enhance heart growth or development in the fetus and thus act in a regenerative capacity in the adult. Therapeutic modulation of this gene, expressed protein and/or use of antibodies or small molecule drugs targeting the gene or gene product is useful in treatment of heart related diseases.

Panel 4.1D Summary: Ag3767/Ag4935 Highest expression of this gene was detected in resting astrocytes and kidney (CTs=30-32). In addition, moderate to low expression of this gene was also seen in keratinocytes, resting and activated mucoepidermoid NCI-H292, resting and activated lung and dermal fibroblasts. Therefore, therapeutic modulation of this gene or its protein product through the use of antibodies or small molecule drug is useful in the threatment of psoriasis, asthma, allergies, chronic obstructive pulmonary disease, emphysema and kidney related diseases including lupus erythematosus.

Example D. Expression of CG54455-06 in stable CHO-K1 cells A 456 bp long Bglll-Xhol fragment containing the CG54455-06 (mature form of

CG54455-01) sequence was subcloned into BamHI-XhoI digested pEE14.4FL2_MSA to generate plasmid 3337. The resulting plasmid 3337 was transfected into CHO-K1 cells using the LipofectaminePlus reagent following the manufacturer's instructions (Invitrogen/Gibco) and stable clones were selected based on resistance against MSX. The culture media was DMEM, 10% FBS, lx nonessential amino acids. The expression and secretion levels of the clone were assessed by Western blot analysis using HRP conjugated V5 antibody. The V5 epitope is fused to the gene of interest at the Cter, in the pEE14.4Sec vector. Fig. 1 shows that CG54455 is expressed, and a 94 kDa protein is secreted by the CHO-K1 cells. OTHER EMBODIMENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims. The claims presented are representative of the inventions disclosed herein. Other, unclaimed inventions are also contemplated. Applicants reserve the right to pursue such inventions in later claims.

Claims

CLAIMSWhat is claimed is:

1. An isolated polypeptide comprising the mature form of an amino acid sequence selected from the group consisting of SEQ ED NO:2n, wherein n is an integer between 1 and 102.

2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO:2n, wherein n is an integer between 1 and 102.

3. An isolated polypeptide comprising an amino acid sequence which is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102.

4. An isolated polypeptide, wherein the polypeptide comprises an amino acid sequence comprising one or more conservative substitutions in the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102.

5. The polypeptide of claim 1 wherein said polypeptide is naturally occurring.

6. A composition comprising the polypeptide of claim 1 and a carrier.

7. A kit comprising, in one or more containers, the composition of claim 6.

8. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a pathology associated with the polypeptide of claim 1, wherein the therapeutic comprises the polypeptide of claim 1.

9. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:

(a) providing said sample;

(b) introducing said sample to an antibody that binds immunospecifically to the polypeptide; and

(c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.

10. A method for determining the presence of or predisposition to a disease associated with altered levels of expression of the polypeptide of claim 1 in a first mammalian subject, the method comprising: a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and b) comparing the expression of said polypeptide in the sample of step (a) to the expression of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease, wherein an alteration in the level of expression of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.

11. A method of identifying an agent that binds to the polypeptide of claim 1 , the method comprising:

(a) introducing said polypeptide to said agent; and

(b) determining whether said agent binds to said polypeptide.

12. The method of claim 11 wherein the agent is a cellular receptor or a downstream effector.

13. A method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of the polypeptide of claim 1, the method comprising:

(a) providing a cell expressing the polypeptide of claim 1 and having a property or function ascribable to the polypeptide;

(b) contacting the cell with a composition comprising a candidate substance; and

(c) determining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition in the absence of the substance, the substance is identified as a potential therapeutic agent.

14. A method for screening for a modulator of activity of or of latency or predisposition to a pathology associated with the polypeptide of claim 1, said method comprising:

(a) administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of claim 1, wherein said test animal recombinantly expresses the polypeptide of claim 1;

(b) measuring the activity of said polypeptide in said test animal after administering the compound of step (a); and

(c) comparing the activity of said polypeptide in said test animal with the activity of said polypeptide in a control animal not administered said polypeptide, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator activity of or latency or predisposition to, a pathology associated with the polypeptide of claim 1.

15. The method of claim 14, wherein said test animal is a recombinant test animal that expresses a test protein transgene or expresses said transgene under the control of a promoter at an increased level relative to a wild-type test animal, and wherein said promoter is not the native gene promoter of said transgene.

16. A method for modulating the activity of the polypeptide of claim 1, the method comprising contacting a cell sample expressing the polypeptide of claim 1 with a compound that binds to said polypeptide in an amount sufficient to modulate the activity of the polypeptide.

17. A method of treating or preventing a pathology associated with the polypeptide of claim 1, the method comprising administering the polypeptide of claim 1 to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject.

18. The method of claim 17, wherein the subject is a human.

19. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, or a biologically active fragment thereof.

20. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ED NO:2n-l, wherein n is an integer between 1 and 102.

21. The nucleic acid molecule of claim 20, wherein the nucleic acid molecule is naturally occurring.

22. A nucleic acid molecule, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 102.

23. An isolated nucleic acid molecule encoding the mature form of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102.

24. A composition comprising an isolated nucleic acid molecule, said molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 102, and a carrier.

25. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ED NO: 2n-l, wherein n is an integer between 1 and 102, or a complement of said nucleotide sequence.

26. A vector comprising the nucleic acid molecule of claim 20.

27. The vector of claim 26, further comprising a promoter operably linked to said nucleic acid molecule.

28. A cell comprising the vector of claim 26.

29. An antibody that immunospecifically binds to the polypeptide of claim 1.

30. The antibody of claim 29, wherein the antibody is a monoclonal antibody.

31. The antibody of claim 29, wherein the antibody is a humanized antibody.

32. A method for determining the presence or amount of the nucleic acid molecule of claim 20 in a sample, the method comprising:

(a) providing said sample;

(b) introducing said sample to a probe that binds to said nucleic acid molecule; and

(c) determining the presence or amount of said probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.

33. The method of claim 32 wherein presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type.

34. The method of claim 33 wherein the cell or tissue type is cancerous.

35. A method for determining the presence of or predisposition to a disease associated with altered levels of expression of the nucleic acid molecule of claim 20 in a first mammalian subject, the method comprising: a) measuring the level of expression of the nucleic acid in a sample from the first mammalian subject; and b) comparing the level of expression of said nucleic acid in the sample of step (a) to the level of expression of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of expression of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

36. A method of producing the polypeptide of claim 1 , the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ED NO:2n-l, wherein n is an integer between 1 and 102.

37. The method of claim 36 wherein the cell is a bacterial cell.

38. The method of claim 36 wherein the cell is an insect cell.

39. The method of claim 36 wherein the cell is a yeast cell.

40. The method of claim 36 wherein the cell is a mammalian cell.

41. A method of producing the polypeptide of claim 2, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102.

42. The method of claim 41 wherein the cell is a bacterial cell.

43. The method of claim 41 wherein the cell is an insect cell.

44. The method of claim 41 wherein the cell is a yeast cell.

45. The method of claim 41 wherein the cell is a mammalian cell.

46. An isolated polypeptide comprising an amino acid sequence at least 95% similar to SEQ ID NO: 4, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 19, 68, 82, 103, 115, 136, 310, 320, 325, 331, 370 and 581 when numbered in accordance with SEQ ED NO: 4.

47. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ED NO: 3, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 105, 253, 295, 356, 392, 456, 978, 1008, 1023, 1041, 1157 and 1791 when numbered in accordance with SEQ ID NO: 3.

48. An isolated polypeptide comprising an amino acid sequence at least 95% similar to SEQ ED NO: 78, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 77, 91 125, 174, 189 and 203 when numbered in accordance with SEQ ID NO: 78.

49. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 77, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 231, 272, 374, 522, 566 and 608 when numbered in accordance with SEQ ID NO: 77.

50. An isolated polypeptide comprising an amino acid sequence at least 95% similar to SEQ ID NO: 116, wherein said amino acid sequence comprises al least one amino acid substitution, wherein said substitution is at amino acid position 120 when numbered in accordance with SEQ ID NO: 116.

51. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 115, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at nucleic acid position 358 when numbered in accordance with SEQ ID NO: 115.

52. An isolated polypeptide comprising an amino acid sequence at least 95% similar to SEQ ID NO: 32, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 40, 44, 134, 212, 234, 289, 311 and 348 when numbered in accordance with SEQ ID NO: 32.

53. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ED NO: 31, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 149, 160, 430, 664, 731, 895, 961 and 1073 when numbered in accordance with SEQ ID NO: 31.