WO2003064629A2

WO2003064629A2 - Therapeutic polypeptides, nucleic acids encoding same, and methods of use

Info

Publication number: WO2003064629A2
Application number: PCT/US2003/003402
Authority: WO
Inventors: Enrique Alvarez; Shlomit R. Edinger; Esha. A. Gangolli; Valerie L. Gerlach; Linda Gorman; Xiaojia Guo; Weizhen Ji; Ramesh Kekuda; Li Li; Charles E. Miller; Muralidhara Padigaru; Meera Patturajan; Luca Rastelli; Daniel K. Rieger; Suresh G. Shenoy; Richard A. Shimkets; Kimberly A. Spytek; Mei Zhong
Original assignee: Curagen Corporation
Priority date: 2002-02-01
Filing date: 2003-02-03
Publication date: 2003-08-07
Also published as: EP1565199A2; CA2474649A1; JP2005527197A; WO2003064629A3

Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies that immunospecifcally bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the novel polypeptide, polynucleotide, or antibody specific to the polypeptide. Vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using same are also included. 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

FIELD OF THE INVENTION

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.

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 ofthe cells. When such cells are components of multicellular organisms such as vertebrates, or more particularly organisms such as mammals, the regulation ofthe 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 ofthe 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 ofthe 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 ofthe effector to a subject in need thereof is useful in treatment ofthe pathological condition. Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished or suppressed levels ofthe 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 multichain 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 ofthe 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 ofthe antigen in a sample. In addition, they have the potential of inactivating the activity ofthe 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 ofthe protein effector in cases where a pathological condition arises from elevated or excessive levels ofthe 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 ofthe amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37. The novel nucleic acids and polypeptides are referred to herein as NOV la, NOV lb, NOV2a, NOV2b, etc. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX" nucleic acid or polypeptide sequences.

The invention also is based in part upon variants of a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% ofthe 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 37. 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 37 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 ofthe amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% ofthe residues are changed.

In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic variants ofthe sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37. 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 37. 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 37 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 37 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 37 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 37 in a first mammalian subject, the method involving measuring the level of expression ofthe polypeptide in a sample from the first mammalian subject; and comparing the amount ofthe polypeptide in this sample to the amount ofthe 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 ofthe 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 37, 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 37, the method including providing a cell expressing the polypeptide ofthe 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 ofthe substance is not observed when the cell is contacted with a composition devoid ofthe 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 37, the method including administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide ofthe invention, wherein the test animal recombinantly expresses the polypeptide ofthe invention; measuring the activity ofthe polypeptide in the test animal after administering the test compound; and comparing the activity ofthe protein in the test animal with the activity ofthe polypeptide in a control animal not administered the polypeptide, wherein a change in the activity ofthe 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 ofthe 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 ofthe 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 37, 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 ofthe 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 37, 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 37 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37; a variant of a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 wherein any amino acid in the mature form ofthe chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence ofthe 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 37; a variant ofthe amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe 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 37 or any variant ofthe polypeptide wherein any amino acid ofthe chosen sequence is changed to a different amino acid, provided that no more than 10% ofthe amino acid residues in the sequence are so changed; and the complement of any ofthe 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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 37. 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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- 1 , wherein n is an integer between 1 and 37; 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 37 is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe nucleotides are so changed; a nucleic acid fragment ofthe sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 37; and a nucleic acid fragment whεiein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-1 , wherein n is an integer between 1 and 37 is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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 37, or a complement ofthe 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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 ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe nucleotides in the chosen coding sequence are so changed, an isolated second polynucleotide that is a complement ofthe 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37. 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37 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 ofthe probe bound to the nucleic acid molecule, thereby determining the presence or amount ofthe nucleic acid molecule in the sample. The presence or amount ofthe 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 ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 37 in a first mammalian subject, the method including measuring the amount ofthe nucleic acid in a sample from the first mammalian subject; and comparing the amount ofthe nucleic acid in the sample of step (a) to the amount ofthe 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 ofthe nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

The invention further provides an antibody that binds immunospecifically to a NOVX polypeptide. The NOVX antibody may be monoclonal, humanized, or a fully human antibody. Preferably, the antibody has a dissociation constant for the binding ofthe NOVX polypeptide to the antibody less than 1 x 10^"9 M. More preferably, the NOVX antibody neutralizes the activity of the NOVX polypeptide.

In a further aspect, the invention provides for the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, associated with a NOVX polypeptide. Preferably the therapeutic is a NOVX antibody.

In yet a further aspect, the invention provides a method of treating or preventing a NOVX-associated disorder, a method of treating a pathological state in a mammal, and a method of treating or preventing a pathology associated with a polypeptide by administering a NOVX antibody to a subject in an amount sufficient to treat or prevent the disorder.

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 ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incoφorated 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 ofthe invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Inhibition of OVCAR-5 cell proliferation by antisense knockdown of CG52414-01

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 "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any ofthe novel sequences disclosed herein. Table A provides a summary ofthe NOVX 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 and condition 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), vascular calcification, fibrosis, 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, osteoarthritis, rheumatoid arthritis, osteochondrodysplasia, adrenoleukodystrophy, congenital adrenal hypeφlasia, prostate cancer, diabetes, metabolic disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, glomerulonephritis, hemophilia, hypercoagulation, idiopathic thrombocytopenic puφura, immunodeficiencies, psoriasis, skin disorders, graft versus host disease, AIDS, bronchial asthma, lupus, Crohn's disease; inflammatory bowel disease, ulcerative colitis, 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,] schizophrenia, depression, asthma, emphysema, allergies, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, as well as conditions such as transplantation, neuroprotection, fertility, or regeneration (in vitro and in vivo).

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 ofthe 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 ofthe family to which the NOVX polypeptides belong.

Consistent with other known members ofthe family of proteins, identified in column 5 of Table A, the NOVX polypeptides ofthe present invention show homology to, and contain domains that are characteristic of, other members of such protein families.

Details ofthe sequence relatedness and domain analysis for each NOVX are presented in

Example A.

The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX 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 NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details ofthe expression analysis for each NOVX are presented in Example C. Accordingly, the NOVX 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 NOVX 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 ofthe 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 ofthe 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 ofthe new protein in a sample or by determining the presence of mutations in the new genes. Specific uses are described for each ofthe 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 ofthe 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 ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 37; (b) a variant of a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 37, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% ofthe 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 37; (d) a variant ofthe amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe 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 ofthe amino acid sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 37; (b) a variant of a mature form ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 37 wherein any amino acid in the mature form ofthe chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence ofthe 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 37; (d) a variant ofthe amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 37, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe 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 37 or any variant of said polypeptide wherein any amino acid ofthe 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 ID NO: 2n-l, wherein n is an integer between 1 and 37; (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 37 is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% ofthe nucleotides are so changed; (c) a nucleic acid fragment ofthe sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 37; 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 37 is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

NOVX Nucleic Acids and Polypeptides

One aspect ofthe 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 ofthe 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 or 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 ofthe 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 step of post-translational modification 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- stranded 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 ofthe nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5¹- and 3'-termini ofthe nucleic acid) in the genomic DNA ofthe 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, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe 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 ofthe invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2n-l , wherein n is an integer between 1 and 37, 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 ofthe nucleic acid sequence of SEQ ID NO:2«-l, wherein n is an integer between 1 and 37, 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 IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)

A nucleic acid ofthe 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 conesponding 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 NO:2n-l, wherein n is an integer between 1 and 37, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes. In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO:2n-l, wherein n is an integer between 1 and 37, 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 NO:2n-l , wherein n is an integer between 1 and 37, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 37, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO:2n-l, wherein n is an integer between 1 and 37, 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 allow 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 ofthe disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment ofthe respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 3' direction ofthe 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 ofthe nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe 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 ofthe 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 ofthe 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 37, as well as a polypeptide possessing NOVX biological activity. Various biological activities ofthe 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 ofthe three "stop" codons, namely, TAA, TAG, or TGA. For the puφoses 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 ofthe human NOVX 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 37; or an anti-sense strand nucleotide sequence of SEQ ID NO:2n-l , wherein n is an integer between 1 and 37; or of a naturally occurring mutant of SEQ ID NO:2n-l, wherein n is an integer between 1 and 37.

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 ofthe 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 37, that encodes a polypeptide having a NOVX biological activity (the biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe 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 37, due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID NO:2n-l , wherein n is an integer between 1 and 37. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

In addition to the human NOVX nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 37, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequences ofthe NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymoφhism 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 ofthe NOVX genes. Any and all such nucleotide variations and resulting amino acid polymoφhisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe NOVX polypeptides, are intended to be within the scope ofthe 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:2n-l, wherein n is an integer between 1 and 37, are intended to be within the scope ofthe invention.

Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe NOVX cDNAs ofthe 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 ofthe 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 37. 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 ofthe 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 ofthe 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% ofthe 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% ofthe 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 IN 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 ofthe invention that hybridizes under stringent conditions to a sequence of SEQ IDNO:2n-l, wherein n is an integer between 1 and 37, 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:2n-l , wherein n is an integer between 1 and 37, 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 ID NO:2n-l , wherein n is an integer between 1 and 37, 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 ID NO:2n-l , wherein n is an integer between 1 and 37, thereby leading to changes in the amino acid sequences ofthe 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:2n, wherein n is an integer between 1 and 37. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences ofthe 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 ofthe 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 ofthe 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 ID NO:2n-l, wherein n is an integer between 1 and 37, 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 ID NO:2n, wherein n is an integer between 1 and 37. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; more preferably at least about 70% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; still more preferably at least about 80% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; even more preferably at least about 90% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; and most preferably at least about 95% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37.

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 37, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 37, 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:2n-l, wherein n is an integer between 1 and 37, 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:2n-l, wherein n is an integer between 1 and 37, the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe 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, MILV, MILF, 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 ofthe following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.

In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and a NOVX ligand; or (iii) the ability of a mutant NOVX 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 ofthe 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 ofthe 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, WO 01/29058, WO01/89304, WO02/16620, and WO02/29858, each incoφorated 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 ofthe 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 Shaφ (1999), Genes & Dev. 13: 3191-3197, incoφorated 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 ofthe 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 ofthe 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 ofthe 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' ofthe 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' ofthe 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 haiφin RNAi product is homologous to all or a portion ofthe target gene. In another example, a haiφin 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 ofthe type III class of Pol III promoters. The +1 nucleotide ofthe 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 ofthe 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 ofthe two siRNA strands.

A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 to 100 nt downstream ofthe 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 ofthe 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, poiymoφhisms, 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 ofthe 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 ofthe 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 siRNA-associating 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 ofthe NOVX sense siRNA conesponds to (N19)TT orN21, respectively. In the latter case, conversion ofthe 3¹ 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 ofthe 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, incoφorated by reference herein in its entirely. The modification ofthe overhang ofthe 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 ofthe sense strand and antisense strand may still be synthesized as 5' (N19)TT, as it is believed that the sequence ofthe 3'-most nucleotide ofthe antisense siRNA does not contribute to specificity. Unlike antisense or ribozyme technology, the secondary structure ofthe target mRNA does not appear to have a strong effect on silencing. See, Harborth, et al. (2001) J. Cell Science 114: 4557-4565, incoφorated by reference in its entirety.

Transfection of NOVX siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECTAMINE 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 ofthe wild-type and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-well plate, approximately 0.84 μg ofthe 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 ofthe 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 ofthe 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 μg of duplex siRNAs. Control experiments again allow for a comparative analysis ofthe 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 ofthe 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) ofthe 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-dcwn of he 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 (asR A) 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 al., 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 mM 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 approx. 80% confluency, the cells are trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105 cells/ml) and transfened 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 ofthe 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:2n-l, wherein n is an integer between 1 and 37, 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 ID NO:2n, wherein n is an integer between 1 and 37, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO:2n- 1 , wherein n is an integer between 1 and 37, are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding a NOVX protein. The term "coding region" refers to the region ofthe 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" ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe molecules or to increase the physical stability ofthe 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-gaϊactosylqueosine, 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-diaminopurine. 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 ofthe 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 ofthe 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 ofthe double helix. An example of a route of administration of antisense nucleic acid molecules ofthe 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 III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-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 ofthe 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 ofthe 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., SEQ ID NO:2n-l , wherein n is an integer between 1 and 37). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe 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 ofthe NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe 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 ofthe molecule. For example, the deoxyribose phosphate backbone ofthe 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; Peπy-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 aπest 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., Sj nucleases (See, Hyrup, et al, \996.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 drug 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 al., 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 ID NO:2n, wherein n is an integer between 1 and 37. The invention also includes a mutant or variant protein any of whose residues may be changed from the coπesponding residues shown in any one of SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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 ofthe 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 ofthe 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 ofthe 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% ofthe volume ofthe 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 ofthe 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 ofthe NOVX proteins (e.g., the amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 37) 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 ofthe 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 ofthe 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 ID NO:2n, wherein n is an integer between 1 and 37. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37, and retains the functional activity ofthe protein of SEQ ID NO:2π, wherein n is an integer between 1 and 37, 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 37, and retains the functional activity ofthe NOVX proteins of SEQ ID NO:2n, wherein n is an integer between 1 and 37. 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 pmposes (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 conesponding 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 conesponding 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. J Mol 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 ofthe analogous nucleic acid sequences refened 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 ofthe DNA sequence of SEQ ID NO:2n-l , wherein n is an integer between 1 and 37.

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 ID NO:2n, wherein n is an integer between 1 and 37, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence conesponding 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 conespond 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 ofthe NOVX polypeptide.

In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus ofthe GST (glutathione S-transferase) 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 ceils), 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 ofthe invention can be incoφorated 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 ofthe 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 ofthe 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 ofthe 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 ofthe NOVX proteins that function as either NOVX agonists (i.e.. mimetics) or as NOVX antagonists. Variants ofthe NOVX protein can be generated by mutagenesis (e.g. , discrete point mutation or truncation of the NOVX protein). An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein. An antagonist ofthe NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe 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 ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.

Variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) ofthe 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 ofthe 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 al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries

In addition, libraries of fragments ofthe 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 Si nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and intemal fragments of various sizes ofthe 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 he 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.

Anti-NOVX Antibodies

Included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. 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_ab, F_ab> and F(_ab-₎₂ fragments, and an F_ab expression library. In general, antibody molecules obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, 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 ofthe 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 ofthe antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein, such as an amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 37, 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. Prefened epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments ofthe invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface ofthe protein, e.g., a hydrophilic region. A hydrophobicity analysis ofthe 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 incoφorated 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 ofthe 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 radiohgand binding assays or similar assays known to those skilled in the art.

A protein ofthe 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 ofthe 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, incoφorated 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 ofthe 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 thyroglobulin, 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 ofthe 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) ofthe monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe 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 ofthe 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 prefened 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 Dekker, 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 ofthe 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 piupose 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 ofthe 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 ofthe invention serve as a prefened 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe human immunoglobulin are replaced by conesponding 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 ofthe CDR regions conespond to those of a non-human immunoglobulin and all or substantially all ofthe 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 ofthe 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 Ban 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 incoφorated, 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 ofthe modifications. The prefened 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 reananged 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 conelative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

F_ab 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 ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F_ab 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(_ab-)₂ fragment produced by pepsin digestion of an antibody molecule; (ii) an F_ab fragment generated by reducing the disulfide bridges of an Ftø_b- fragment; (iii) an F_ab fragment generated by the treatment ofthe 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 ofthe 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 ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the conect bispecific structure. The purification ofthe 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 ofthe hinge, CH2, and CH3 regions. It is prefened to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe 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 prefened interface comprises at least a part ofthe CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface ofthe 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 ofthe 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 ofthe 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 proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence ofthe 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 ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe 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 V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_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 ofthe 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 FcγRIII (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 ofthe 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 thioether bond. Examples of suitable reagents for this puφose 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 ofthe invention with respect to effector function, so as to enhance, e.g., the effectiveness ofthe 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, ¹³¹I, ^I31In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates ofthe 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

1 ,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 ofthe 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 he Invention In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked im unosorbent assay (ELISA) and other immunologically mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

Antibodies directed against a NOVX protein ofthe invention may be used in methods known within the art relating to the localization and/or quantitation of a NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to a NOVX protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as "Therapeutics").

An antibody specific for a NOVX protein ofthe invention (e.g., a monoclonal antibody or a polyclonal antibody) can be used to isolate a NOVX polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. An antibody to a NOVX polypeptide can facilitate the purification of a natural NOVX antigen from cells, or of a recombinantly produced NOVX antigen expressed in host cells. Moreover, such an anti-NOVX antibody can be used to detect the antigenic NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe antigenic NOVX protein. Antibodies directed against a NOVX 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 ¹²⁵1, ¹³¹1, ³⁵S or ³H. Antibody Therapeutics

Antibodies ofthe 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 ofthe interaction between the given antibody molecule and the target antigen in question. In the first instance, administration ofthe antibody may abrogate or inhibit the binding ofthe target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site ofthe 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 sunogate effector ligand, initiating a receptor-based signal transduction event by the receptor.

A therapeutically effective amount of an antibody ofthe 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 ofthe target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity ofthe 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 ofthe 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 ofthe invention, as well as other molecules identified by the screening assays disclosed herein, can be admimstered 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 Absoφtion 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 prefened. 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 ofthe 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 puφose 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_ab or F(_abμ) 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 (/. e. , physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescentiy-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescentiy-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 ofthe 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 ofthe 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 (ELISAs), 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 ofthe 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, expression vectors of utility 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 ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe 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 ofthe 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 IN ENZYMOLOGY 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 ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors ofthe 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 ofthe 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 baculovirus 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 ofthe recombinant protein. Such fusion vectors typically serve three pmposes: (i) to increase expression of recombinant protein; (IΪ) to increase the solubility ofthe 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 ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) 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 1 Id (Studier 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 ofthe 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 ofthe 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. EMBOJ. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Coφoration, San Diego, Calif), and picZ (InVitrogen Coφ, 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 ofthe 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. EMBOJ. 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 ofthe 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 ofthe 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 ofthe 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 viras 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 ofthe 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 ofthe invention pertains to host cells into which a recombinant expression vector ofthe 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 ofthe 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 drags, such as G418, hygromycin and methotrexate. 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 drag selection (e.g., cells that have incoφorated the selectable marker gene will survive, while the other cells die).

A host cell ofthe invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ι.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells ofthe 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 ofthe invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell ofthe 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 ofthe cells ofthe 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 ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe 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 ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.

A transgenic animal ofthe invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retioviral 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 NO:2n-l , wherein n is an integer between 1 and 37, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue ofthe 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 ofthe 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 ofthe 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 ID NO:2n-l, wherein n is an integer between 1 and 37), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NO:2n-l, wherein n is an integer between 1 and 37, 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 ofthe endogenous NOVX protein). In the homologous recombination vector, the altered portion ofthe NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid ofthe 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 ofthe 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 ofthe transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description ofthe 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 ofthe 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 ofthe 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 ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morala or blastocyte and then transfened to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone ofthe 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 refened to herein as "active compounds") ofthe invention, and derivatives, fragments, analogs and homologs thereof, can be incoφorated 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 absoφtion 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 incoφorated herein by reference. Prefened examples of such earners 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 incoφorated into the compositions.

A pharmaceutical composition ofthe 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 ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe 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 absoφtion ofthe injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incoφorating 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 incoφorating 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 ofthe 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 puφose of oral therapeutic administration, the active compound can be incoφorated 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 ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe 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 suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, 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 Coφoration 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. It 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 ofthe 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 ofthe 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, etal, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation ofthe 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 ofthe 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 abenant 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 ofthe 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, absoφtion 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, i.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 ofthe membrane-bound form of a NOVX protein or polypeptide or biologically-active portion thereof. The test compounds ofthe invention can be obtained using any ofthe 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 ofthe assays ofthe 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; Canell, 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 ofthe test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability ofthe 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 ofthe 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 ^,251, 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 ofthe 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 ofthe 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 ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability ofthe 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 ofthe 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 ofthe NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by determining the activity ofthe target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e., intracellular Ca²⁺, diacylglycerol, IP₃, etc.), detecting catalytic/enzymatic activity ofthe 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 detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.

In yet another embodiment, an assay ofthe invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to 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 ofthe test compound to interact with a NOVX protein, wherein determining the ability ofthe test compound to interact with a NOVX protein comprises determining the ability ofthe 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 ofthe test compound to modulate (e.g. stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability ofthe 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 ofthe 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 ofthe 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 ofthe test compound to interact with a NOVX protein, wherein determining the ability ofthe test compound to interact with a NOVX protein comprises determining the ability ofthe NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule. The cell-free assays ofthe 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, n-dodecylglucoside, n-dodecylmaltυside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton^® X-100, Triton^® X-l 14, Thesit^®, Isotridecypoly(ethylene glycol ether)_n, N-dodecyl— N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-choϊamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l -propane sulfonate (CHAPSO).

In more than one embodiment ofthe above assay methods ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe NOVX protein to its target molecule, can be derivatized to the wells ofthe 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 ofthe candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence ofthe 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 ofthe 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 ofthe 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 ofthe 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 involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements ofthe 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 ofthe known transcription factor. If the "bait" and the "prey" proteins are able to interact, n vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains ofthe 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 ofthe 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 ofthe cDNA sequences identified herein (and the conesponding 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 (iii) 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 ofthe 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 ofthe NOVX sequences of SEQ ID NO:2n-l , wherein n is an integer between 1 and 37, or fragments or derivatives thereof, can be used to map the location ofthe NOVX genes, respectively, on a chromosome. The mapping ofthe NOVX sequences to chromosomes is an important first step in conelating 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 ofthe 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 .si^'ti 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 conesponding to noncoding regions of the genes actually are prefened for mapping puφoses. 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 ofthe sequence on the chromosome can be conelated 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 ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for stractural 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 polymoφhisms.

Tissue Typing

The NOVX sequences ofthe 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 ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymoφhisms," described in U.S. Patent No. 5,272,057). Furthermore, the sequences ofthe 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 ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

Panels of conesponding 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 ofthe invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences ofthe invention uniquely represent portions ofthe 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 ofthe allelic variation is due to single nucleotide polymoφhisms (SNPs), which include restriction fragment length polymoφhisms (RFLPs).

Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms 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 ID NO:2n-l , wherein n is an integer between 1 and 37, 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) puφoses to thereby treat an individual prophylactically.

Accordingly, one aspect ofthe 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 puφose 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 ofthe 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 ofthe 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 ofthe invention pertains to monitoring the influence of agents (e.g., drags, 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 ID NO:2n-l , wherein n is an integer between 1 and 37, 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 ofthe 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 ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescentiy-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescentiy-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 ofthe 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 si^'tti hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), 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 prefened 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, mR A 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 abenant 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 drag candidate) to treat a disease or disorder associated with abenant 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 abenant 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 ofthe 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 abenant 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 ofthe NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (f) 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 reanangement 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 ofthe methylation pattern ofthe genomic DNA, (viϊ) 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 prefened 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 ofthe 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 NOVX-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, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a NOVX gene under conditions such that hybridization and amplification ofthe NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe 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 ofthe 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 ofthe 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 NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA 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 DNA indicates mutations in the sample DNA. 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 a sample and control nucleic acids, e.g., DNA or RNA, to high-density anays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in ΝOVX can be identified in two dimensional anays containing light-generated DΝA probes as described in Cronin, et al, supra. Briefly, a first hybridization anay 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 anay that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to ail variants or mutations detected. Each mutation anay 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 NOVX gene and detect mutations by comparing the sequence ofthe sample NOVX with the conesponding 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 ( ee, e.g., Naeve, 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 ofthe duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA DNA hybrids treated with Sj 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 ofthe 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. Carcinogenesis 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 polymoφhism (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 stracture 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 ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary stracture 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. 7: 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, DΝA 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 DΝA 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 DΝA. 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 ofthe 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 ofthe 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 NOVX gene.

Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX 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 NOVX activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

In conjunction with such treatment, the pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drag) ofthe 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 ofthe pharmacologically active drag. Thus, the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, 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. 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 drags (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. 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 polymoφhisms 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 drug effects or show exaggerated drag response and serious toxicity after taking the standard and safe dose of a drag. These polymoφhisms 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 polymoφhic 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 moφhine. 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 ofthe individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drag-metabolizing enzymes to the identification of an individual's drug 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 ofthe 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 pattem) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe 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 ofthe physiological response ofthe 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; (ιϊ) 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; (tv) detecting the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the prc-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration ofthe agent to the subject accordingly. For example, increased administration ofthe agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness ofthe agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i. e. , to decrease the effectiveness ofthe 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 but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

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" (ι^'.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 ofthe invention or antibodies specific to a peptide ofthe 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 ofthe 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, n .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 abenant 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 abenant 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 ofthe NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX abenancy, 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 ofthe invention are further discussed in the following subsections.

Therapeutic Methods

Another aspect ofthe invention pertains to methods of modulating NOVX expression or activity for therapeutic pmposes. The modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe 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 abenant 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 abenant 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 abenant 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 ofthe Biological Effect of the Therapeutic

In various embodiments ofthe 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 ofthe affected tissue.

In various specific embodiments, in vitro assays may be performed with representative cells ofthe 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 ofthe 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 ofthe invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

As an example, a cDNA encoding the NOVX protein ofthe 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 ofthe invention will have efficacy for treatment of patients suffering from diseases, disorders, conditions and the like, including but not limited to those listed herein.

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 ofthe 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 ofthe invention for use in therapeutic or diagnostic methods.

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

EXAMPLES

Example 1.

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

Table IA. NOV1 Sequence Analysis

SEQ ID NO: 1 [2322 bp

NOVla, GGGCGGCCGCAGCCTGAGCCAGGGCCCCCTCCCTCGTCAGGACCGGGGCAGCAAGCAGGCCGGG CGI 26472-01 GGCAGGTCCGGGCACCCACCATGCGAGGCGAGCTCTGGCTCCTGGTGCTGGTGCTCAGGGAGGC

TGCCCGGGCGCTGAGCCCCCAGCCCGGAGCAGGTCACGATGAGGGCCCAGGCTCTGGATGGGCT DNA Sequence GCαυ^GGGACCGTGOMGGCTGGAACσSGAGAGCCCGAGAGAGCCCTGGGCATGTGTCAGAGC CGGACAGGACCCAGCTGAGCCAGGACCTGGGTGGGGGCACCCTGGCCATGGACACGCTGCCAGA TAACAGGACCAGGGTGGTGGAGGACAACCACAGCTATTATGTGTCCCGTCTCTATGGCCCCAGC GAGCCCCACAGCCGGGAACTGTGGGTAGATGTGGCCGAGGCCAACCGGAGCCAAGTGAAGATCC ACACAATACTCTCCAACACCCACCGGCAGGCTTCGAGAGTGGTCT GTCCTTTGATTTCCCTTT CTACGGGCATCCTCTGCGGCAGATCACCATAGCAACTGGAGGCTTCATCTTCATGGGGGACGTG ATCCATCGGATGCTCACAGCTACTCAGTATGTGGCGCCCCTGATGGCCAACTTCAACCCTGGCT ACTCCGACAACTCCACAGTTGTTTACTTTGACAATGGGACAGTCTTTGTGGTTCAGTGGGACCA CGTTTATCTCCAAGGCTGGGAAGACAAGGGCAGTTTCACCTTCCAGGCAGCTCTGCACCATGAC GGCCGCATTGTCTTTGCCTATAAAGAGATCCCTATGTCTGTCCCGGAAATCAGCTCCTCCCAGC ATCCTGTCAAAACCGGCCTATCGGATGCCTTCATGATTCTCAATCCATCCCCGGATGTGCCAGA ATCTCGGCGAAGGAGCATCTTTGAATACCACCGCATAGAGCTGGACCCCAGCAAGGTCArrAGC ATGTCGGCCGTGGAGTTCACCCCATTGCCGACCTGCCTGCAGCATAGGAGCTGTGACGCCTGCA TGTCCTCAGACCTGACCTTCAACTGCAGCTGGTGCCATGTCCTCCAGAGATGCTCCAGTGGCTT TGACCGCTATCGCCAGGAGTGGATGGACTATGGCTGTGCACAGGAGGCAGAGGGCAGGATGTGC GAGGACTTCCAGGATGAGGACCACGACTCAGCCTCCCCTGACACTTCCTTCAGCCCCTATGATG GAGACCTCACCACTACCTCCTCCTCCCTCTTCATCGACAGCCTCACCACAGAAGATGACACCAA GTTGAATCCCTATGCAGGAGGAGACGGCCTTCAGAACAACCTGTCCCCCAAGACAAAGGGCACT CCTGTGCACCTGGGCACCATCGTGGGCATCGTGCTGGCAGTCCTCCTCGTGGCGGCCATCATCC TGGCTGGAATTTACATCΛATGGCCACCCCACΑTCOATGCTGCGCTCTTCTTCATCGAGCGTAG ACCTCACCACTGGCCAGCCATGAAGTTTCGCAGCCACCCTGACCATTCCACCTATGCGGAGGTG GAGCCCTCGGGCCATGAGAAGGAGGGCTTCATGGAGGCTGAGCAGTGCTGAGAACACCAAGTCT CCCCTTTGAAGACTTTGAGGCCACAGAAAAGACAGTTAAAGCAAAGAAGAGAAGTGACTTTTCC

ITGGCCTCTCCCAGCATGCCCTGGGCTGAGATGAGATGGTGGTTTATGGCTCCAGAGCTGCTGTΓ

CGCTTCGTCAGCACACCCCGAATATTGAAGAGGGGGCCAAAAAACAACCACATGGATTTTTTAT lAGGAACAACAACCTAATCTCATCCTGTTTTGATGCAAGGGTTCTCTTCTGTGTCTTGTAACCAT

GAAACAGCAGAAGAACTAACATAACTAACTCCATTTTTGTTTAAGGGGCCTTTACCTATTCCTG

CACCTAGGCTAGGATAACTTTAGAGCACTGACATAAAACGCAAAAACAGGAATCATGCCGTTTG

CΛAAACTAACTCTGGGATTAAAGGGGAAGCATGTAAACAGCTAAC GTTTTTGTTAAAGATTTA

TAGGAATGAGGAGGTTTGGCTATTGTCACATGACAGACTGTTAGCCAAGGACAAAGAAGTTCTG

CAAACCTCCCCTGGACCCTTGCTGGTGTCCAGATGTCTGCGGTTGTCAGCCCCTTCCTTTCCCC

CGACCTAAACATAAAAGACAAGGCAAAGCCCGCATAATTTTAAGACGGTTCTTTAGGACATTAG

TCCACCATCTTCTTGGTTTGCTGGCTCTCCGAAATAAAGTCCCTTTCCTTGCTCCAAAAAAAAA

AAAAAAAAAAAAAAAAAA

ORF Start: ATG at 85 ORF Stop: TGA at 1585

SEQ ID NO: 2 500 aa MW at 55759.7kD

NOVla, MRGE WLLVVLREAARALSPQPGAGHDEGPGSGWAAKGTVRGWNRRARESPGHVSEPDRTQ S CG126472-01 QDLGGGTLAMDT PDNRTRWEDNHSYYVSRLYGPSEPHSRELWVDVAEANRSQVKIHTILSNT HRQASRWLSFDFPFYGHPLRQITIATGGFIFMGDVIHRMLTATQYVAPLMANFNPGYSDNSTV Protein Sequence VYFDNGTVFWQWDHVYLQGWEDKGSFTFQAALHHDGRIVFAYKEIPMSVPEISSSQHPVKTGL SDAF ILNPSPDVPESRRRSIFEYHRIELDPSKVTSMSAVEFTPLPTC QHRSCDACMSSDLTF NCSWCHVLQRCSSGFDRYRQEWMDYGCAQEAEGRMCEDFQDEDHDSASPDTSFSPYDGD TTTS SSLFIDSLTTEDDTKLNPYAGGDG Q NLSPKTKGTPVHLGTIVGIVLAVLLVAAIILAGIYIN GHPTSNAALFFIERRPHHWPAMKFRSHPDHSTYAEVEPSGHEKEGFMEAEQC

SEQ ID NO: 3 2286 bp

NOVlb, GGGCGGCCGCAGCCTGAGCCAGGGCCCCCTCCCTCGTCAGGACCGGGGCAGCAAGCAGGCCGGG CGI 26472-02 GGCAGGTCCGGGCACCCACCATGCGAGGCGAGCTCTGGCTCCTGGTGCTGGTGCTCAGGGAGGC

TGCCCGGGCGCTGAGCCCCCAGCCCGGAGCAGGTCACGATGAGGGCCCAGGCTCTGGATGGGCT DNA Sequence GCCAAAGGGACCGTGCGGGGCTGGAACCGGAGAGCCCGAGAGAGCCCTGGGCATGTGTCAGAGC CGGACAGGACCCAGCTGAGCCAGGACCTGGGTGGGGGCACCCTGGCCATGGACACGCTGCCAGA TAACAGGACCAGGGTGGTGGAGGACAACCACAGCTATTATGTGTCCCGTCTCTATGGCCCCAGC GAGCCCCACAGCCGGGAACTGTGGGTAGATGTGGCCGAGGCCAACCGGAGCCAAGTGAAGATCC ACACAATACTCTCCAACACCCACCGGCAGGCTTCGAGAGTGGTCTTGTCCTTTGATTTCCCTTT CTACGGGCATCCTCTGCGGCAGATCACCATAGCAACTGGAGGCTTCATCTTCATGGGGGACGTG ATCCATCGGATGCTCACAGCTAC CAGTATGTGGCGCCCCTGATGGCCAACTTCAACCCTGGCT ACTCCGACAACTCCACAGTTGTTTACTTTGACAATGGGACAGTCTTTGTGGTTCAGTGGGACCA CGTTTATCTCCAAGGCTGGGAAGACAAGGGCAGTTTCACCTTCCAGGCAGCTCTGCACCATGAC GGCCGCATTGTCTTTGCCTATAAAGAGATCCCTATGTCTGTCCCGGAAATCAGCTCCTCCCAGC ATCCTGTCAAAACCGGCCTATCGGATGCCTTCATGATTCTCAA CCATCCCCGGATGTGCCAGA ATCTCGGCGAAGGAGCATCTT GAATACCACCGCATAGAGCTGGACCCCAGCAAGGTCACCAGC ATGTCGGCCGTGGAGTTCACCCCATTGCCGACCTGCCTGCAGCATAGGAGCTGTGACGCCTGCA TGTCCTCAGACCTGACCTTCAACTGCAGCTGGTGCCATGTCCTCCAGAGATGTTCCAGTGGCTT TGACCGCTATCGCCAGGAGTGGATGGACTATGGCTGTGCACAGGAGGCAGAGGGCAGGATGTGC GAGGACTTCCAGGATGAGGACCACGAC CAGCCTCCCCTGACACTTCCTTCAGCCCCTATGATG GAGACCTCACCACTACCTCCTCCTCCCTCTTCATCGACAGCCTCACCACAGAAGGCCTTCAGAA CAACCTGTCCCCCAAGACAAAGGGCACTCCTGTGCACCTGGGCACCATCGTGGGCATCGTGCTG GCAGTCCTCCTCGTGGCGGCCATCATCCTGGCTGGAATTTACATCAATGGCCACCCCACATCCA ATGCTGCGCTCTTCTTCATCGAGCGTAGACCTCACCACTGGCCAGCCATGAAGTTTCGCAGCCA CCCTAACCATTCCACCTATGCGGAGGTGGAGCCCTCGGGCCATGAGAAGGAGGGCTTCATGGAG GCTGAGCAGTGCTGAGAACACCAAGTCTCCCCTTTGAAGACTTTGAGGCCACAGAAAAGACAGT TAAAGCAAAGAAGAGAAGTGACTTTTCCTGGCCTCTCCCAGCATGCCCTGGGCTGAGATGAGAT

GGTGGTTTATGGCTCCAGAGCTGCTGTTCGCTTCGTCAGCACACCCCGAATATTGAAGAGGGGG

CCAAAAAACAACCACATGGATTTTTTATAGGAACAACAACCTAATCTCATCCTGTTTTGATGCA

AGGGTTCTCTTCTGTGTCTTGTAACCATGAAACAGCAGAAGAACTAACATAACTAACTCCATTT

TTGTTTAAGGGGCCTTTACCTATTCCTGCACCTAGGCTAGGATAACTTTAGAGCACTGACATAA

AACGCAAAAACAGGAATCATGCCGTTTGCAAAACTAACTCTGGGATTAAAGGGGAAGCATGTAA

ACAGCTAACTGTTTTTGTTAAAGATTTATAGGAATGAGGAGGTTTGGCTATTGTCACATGACAG

ACTGTTAGCCAAGGACAAAGAAGTTCTGCAAACCTCCCCTGGACCCTTGCTGGTGTCCAGATGT

CTGCGGTTGTCAGCCCCTTCCTTTCCCCCGACCTAAACATAAAAGACAAGGCAAAGCCCGCATA

LATTTTAAGACGGTTCTTTAGGACATTAGTCCACCATCTTCTTGGTTTGCTGGCTCTCCGAAATA

AAGTCCCTTTCCTTGCTCCAAAAAAAAAAAAAAAAAAAAAAAAAAA

ORF Start: ATG at 85 ORF Stop: TGA at 1549

SEQ ID NO: 4 MWat54511.5kD

NOVlb, MRGELW VLV REAARALSPQPGAGHDEGPGSG AAKGTVRGWNRRARESPGHVSEPDRTQLS CGI26472-02 QDLGGGTLAMDTLPDNRTRWEDNHSYYVSRLYGPSEPHSRELWVDVAEANRSQVKIHTILSNT HRQASRWLSFDFPFYGHP RQITIATGGFIFMGDVIHRMLTATQYVAP MANFNPGYSDNSTV Protein Sequence VYFDNGTVFWQWDHVY QGWEDKGSFTFQAALHHDGRIVFAYKEIPMSVPEISSSQHPVKTG SDAF ILNPSPDVPESRRRSIFEYHRIELDPSKVTSMSAVEFTPLPTCLQHRSCDACMSSDLTF NCSWCHV QRCSSGFDRYRQEWMDYGCAQEAEGRMCEDFQDEDHDSASPDTSFSPYDGD TTTS SSLFIDSLTTEG QNNLSPKTKGTPVHLGTIVGIVLAVLLVAAIILAGIYINGHPTSNAALFFI ERRPHHWPAMKFRSHPNHSTYAEVEPSGHEKEGFMEAEQC

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table IB.

Further analysis ofthe NOVla protein yielded the following properties shown in Table IC.

Table IC. Protein Sequence Properties NOVla

SignalP analysis: Cleavage site between residues 19 and 20

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 4; pos.chg 1; neg.chg 1 H-region: length 8; peak value 11.03 PSG score: 6.62

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.95 possible cleavage site: between 18 and 19

>>> Seems to have a cleavable 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: 1

INTEGRAL Likelihood =-15.87 Transmembrane 427

443

PERIPHERAL Likelihood = 3.87 (at 153) ALOM score: -15.87 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 9 Charge difference: 0.5 C( 1.5) - N( 1.0) C > N: C-terminal side will be inside

>>>Caution: Inconsistent mtop result with signal peptide >>> membrane topology: type la (cytoplasmic tail 444 to 500)

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75): 3.62 Hyd Moment (95): 3.15 G content: 1 D/E conten : 2 S/T content : 0 Score: -6.39

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 12 MRG | EL

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: PESRRRS (4) at 270 bipartite: none content of basic residues: 8.2% NLS Score: -0.13

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

ER Membrane Retention Signals:

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

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

FTS2: 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 : too long tail

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 residue's

Final Results (k = 9/23)

55.6 %: endoplasmic reticulum

22.2 %: Golgi

11.1 %: plasma membrane

11.1 %: extracellular, including cell wall

» prediction for CG126472-01 is end (k=9)

A search ofthe 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

SEQ ID NO: 5 1573 bp

NOV2a, GACTCaGCCTTAGGTACCGGTCAGGCAAAATGCGGTCCTCCCTGGCTCCGGGAGTCTGGTTCTT CG138751-02 CCGGGCCTTCTCCAGGGACAGCTGGTTCCGAGGCCTCATCCTGCTGCTGACCTTCCTAATTTAC GCCTGCTATCACATGTCCAGGAAGCCTATCAGTATCGTCAAGAGCCGTCTGCACCAGAACTGCT DNA Sequence CGGAGCAGATCAAACCCATCAATGATACTCACAGTCTCAATGACACCATGTGGTGCAGCTGGGC CCCATTTGACAAGGACAACTATAAGGAGTTACTAGGGGGCGTGGACAACGCCTTCCTCATCGCC TATGCCaTCGGCATGTTCATCAGTGGGGTTTTTGGGGAGCGGCTTCCGCTCCGTTACTACCTCT CAGCTGGAATGCTGCTCAGTGGCCTTTTCACCTCGCTCTTTGGCCTGGGATAT TCTGGAACAT CCACGAGCTCTGGTACTTTGTGGTCATCCAGGTCTGTAATGGACTCGTCCAGACCACAGGCTGG CCCTCTGTGGTGACCTGTGTTGGCAACTGGTTCGGGAAGGGGAAGCGGGGGTTCATCATGGGCA TCTGGAATTCCCACACATCTGTGGGCAACATCCTGGGCTCCCTGATCGCCGGCATCTGGGTGAA CGGGCAGTGGGGCCTGTCGTTCATCGTGCCTGGCATCATTACTGCCGTCATGGGCGTCATCACC TTCCTCTTCCTCATCGAACACCCAGAAGATGTGGACTGCGCCCCTCCTCAGCACCACGGTGAGC CAGCTGAGAACCAGGACAACCCTGAGGACCCTGGGAACAGTCCCTGCTCTATCAGGGAGAGCGG CCTTGAGACTGTGGCCAAATGCTCCAAGGGGCCATGCGAAGAGCCTGCTGCCATCAGCTTCTTT GGGGCGCTCCGGATCCCAGGCGTGGTCGAGTTCTCTCTGTGTCTGCTGTTTGCCAAGCTGGTCA GTTACACCTTCCTCTACTGGCTGCCCCTCTACATCGCCAATGTGGCTCACTTTAGTGCCAAGGA GGCTGGGGACCTGTCTACACTCTTCGATGTTGGTGGCATCATAGGCGGCATCGTGGCAGGGCTC GTCTCTGACTACACCAATGGCAGGGCαvCCACTTGCTGTGTCATGCTCATCTTGGCTGCCCCCA TGATGTTCCTGTACAACTACATTGGCCAGGACGGGATTGCCAGCTCCATAGTGATGCTGATCAT CTGTGGGGGCCTGGTCAATGGCCCATACGCGCTCATCACCACTGCTGTCTCTGCTGATCTGGGG ACTCΛCAAGAGCCTGAAGGGCAACGCCAAAGCCCTGTCCACGGTCACGGCCATCATTGACGGCA CCGGCTCCATAGGTGCGGCTCTGGGGCCTCTGCTGGCTGGGCTCATCTCCCCCACGGGCTGGAA CAATGTCTTCTACATGCTCATCTCTGCCGACGTCCTAGCCTGCTTGCTCCTTTGCCGGTTAGTA TACAAAGAGATCTTGGCCTGGAAGGTGTCCCTGAGCAGAGGCAGCGGGTGAGTCCGGGGAGCTG AAGCTGCCCCTCTACCAACCTCATTTCTCGTGGGAAT

ORF Start: ATG at 30 ORF Stop: TGA at 1521

SEQ ID NO: 6 497 aa MW at 53902.2kD

NOV2a, MRSSLAPGV FFRAFSRDSWFRGLILLLTFLIYACYHMSRKPISIVKSRLHQNCSEQIKPINDT CGI 38751-02 HSLNDTMWCSWAPFDKDNYKELLGGVDNAFLIAYAIGMFISGVFGERLPLRYYLSAGMLLSGLF TSLFGLGYFWNIHELWYFWIQVCNGLVQTTGWPSWTCVGNWFGKGKRGFIMGI NSHTSVGN Protein Sequence ILGSLIAGIWVNGQWGLSFIVPGIITAVMGVITFLFLIEHPEDVDCAPPQHHGEPAENQDNPED PGNSPCSIRESGLETVAKCSKGPCEEPAAISFFGALRIPGWEFSLCLLFAKLVSYTFLY LPL YIANVAHFSAKEAGDLSTLFDVGGIIGGIVAGLVSDYTNGRATTCCVMLILAAPMMFLYNYIGQ DGIASSIVMLIICGGLVNGPYALITTAVSADLGTHKSLKGNAKALSTVTAIIDGTGSIGAALGP LLAGLISPTGWNNVFYMLISADVLACLLLCRLVYKEILA KVSLSRGSG

SEQ ID NO: 7 1638 bp

NOV2b, ACACGCGCCCAGCTCTGTAGCCTCCTCCGTCGACTCAGCCTTAGGTACCGGTCAGGCAAAATGC CG138751-01 DNA GGTCCTCCCTGGCTCCGGGAGTCTGGTTCTTCCGGGCCTTCTCCAGGGACAGCTGGTTCCGAGG CCTCATCCTGCTGCTGACCTTCCTAATTTACGCCTGCTATCACATGTCCAGGAAGCCTATCAGT Sequence ATCGTCAAGAGCCGTCTGCACCAGAACTGCTCGGAGCAGATCAAACCCATCAATGATACTCACA GTCTCAATGACACCATGTGGTGCAGCTGGGCCCCATTTGACAAGGACAACTATAAGGAGTTACT AGGGGGCGTGGACAACGCCTTCCTCATCGCCTATGCCATCGGCATGTTCATCAGTGGGGTTTTT GGGGAGCGGCTTCCGCTCCGTTACTACCTCTCAGCTGGAATGCTGCTCAGTGGCCTTTTCACCT CGCTCTTTGGCCTGGGATATTTCTGGAACATCCACGAGCTCTGGTACTTTGTGGTCATCCAGGT CTGTAATGGACTCGTCCAGACCACAGGCTGGCCCTCTGTGGTGACCTGTGTTGGCAACTGGTTC GGGAAGGGGAAGCGGGGGTTCATCATGGGCATCTGGAATTCCCACACATCTGTGGGCAACATCC TGGGCTCCCTGATCGCCGGCATCTGGGTGAACGGGCAGTGGGGCCTGTCGTTCATCGTGCCTGG CATCATTACTGCCGTCATGGGCGTCATCACCTTCCTCTTCCTCATCGAACACCCAGAAGATGTG GACTGCGCCCCTCCTCAGCACCACGGTGAGCCAGCTGAGAACCAGGACAACCCTGAGGACCCTG GGAACAGTCCCTGCTCTATCAGGGAGAGCGGCCTTGAGACTGTGGCCAAATGCTCCAAGGGGCC ATGCGAAGAGCCTGCTGCCATCAGCTTCTTTGGGGCGCTCCGGATCCCAGGCGTGGTCGAGTTC TCTCTGTGTCTGCTGTTTGCCAAGCTGGTCAGTTACACCTTCCTCTACTGGCTGCCCCTCTACA TCGCCAATGTGGCTCACTTTAGTGCCAAGGAGGCTGGGGACCTGTCTACACTCTTCGATGTTGG TGGCATCATAGGCGGCATCGTGGCAGGGCTCGTCTCTGACTACACCAATGGCAGGGCCACCACT TGCTGTGTCATGCTCATCTTGGCTGCCCCCATGATGTTCCTGTACAACTACATTGGCCAGGACG GGATTGCCAGCTCCATAGGTGAGGTCCCAGTGATGCTGATCATCTGTGGGGGCCTGGTCAATGG CCCATACGCGCTCATCACCACTGCTGTCTCTGCTGATCTGGGGACTCACAAGAGCCTGAAGGGC ACAGCCAAAGCCCTGTCCACGGTCACGGCCATCATTGACGGCACCGGCTCCATAGGTGCGGCTC TGGGGCCTCTGCTGGCTGGGCTCATCTCCCCCACGGGCTGGAACAATGTCTTCTACATGCTCAT CTCTGCCGACGTCCTAGCCTGCTTGGTCCTTTGCCGGTTAGTATACAAAGAGATCTTGGCCTGG AAGGTGTCCCTGAGCAGAGGCAGCGGGTGAGTCCGGGGAGCTGAAGCTGCCCCTCTACCAACCT CATTTCTCGTGGGAATCAGCCCAGCGCTCAGTTTCTCC

ORF Start: ATG at 61 ORF Stop: TGA at 1564

SEQ ID NO: 8 501 aa MW at 54257.6kD

NOV2b, MRSSLAPGVWFFRAFSRDSWFRGLILLLTFLIYACYHMSRKPISIVKSRLHQNCSEQIKPINDT CGI 38751-01 HSLNDTMWCSWAPFDKDNYKELLGGVDNAFLIAYAIGMFISGVFGERLPLRYYLSAGMLLSGLF TSLFGLGYFWNIHELWYFWIQVCNGLVQTTGWPSWTCVGNWFGKGKRGFIMGIWNSHTSVGN Protein Sequence _ ILGSLIAGIWV GQWGLSFIVPGIITAVMGVITFLFLIEHPEDVDCAPPQHHGEPAENQDNPED PGNSPCSIRESGLETVAKCSKGPCEEPAAISFFGALRIPGWEFSLCLLFAKLVSYTFLYWLPL YIANVAHFSAKEAGDLSTLFDVGGIIGGIVAGLVSDYTNGRATTCCVMLILAAPMMFLYNYIGQ DGIASSIGEVPVMLIICGGLVNGPYALITTAVSADLGTHKSLKGTAKALSTVTAIIDGTGSIGA

ALGiPLLAGLISPTGWNNVFYMLISADVLACLVLCRLVYKEILAWKVSLSRGSG

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 2B.

Further analysis ofthe NOV2a protein yielded the following properties shown in Table 2C.

Table 2C. Protein Sequence Properties NOV2a

SignalP analysis: Cleavage site between residues 37 and 38

PSORT II analysis: PSG: a new signal peptide prediction method N-region: length 2; pos.chg 1; neg.chg 0 H-region: length 10; peak value 0.95 PSG score : 4.55

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -0.87 possible cleavage site: between 34 and 35

>>> Seems to have a cleavable signal peptide (1 to 34)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 35

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

8

INTEGRAL Likelihood = -3.72 Transmembrane 93 -

109

INTEGRAL Likelihood = -0.48 Transmembrane 118 -

134

INTEGRAL Likelihood = -6.90 Transmembrane 211 -

227

INTEGRAL Likelihood = -2.71 Transmembrane 294 -

310

INTEGRAL Likelihood = -2.76 Transmembrane 338 -

354

INTEGRAL Likelihood = -4.83 Transmembrane 362 -

378

INTEGRAL Likelihood = -3.72 Transmembrane 385 -

401

INTEGRAL Likelihood = -6.74 Transmembrane 462 -

478

PERIPHERAL Likelihood = 1.27 (at 438) ALOM score: -6.90 (number of TMSs: 8)

MTOP: Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 17 Charge difference: -1.0 C( 0.0) - N( 1.0) N >= C: N-terminal side will be inside

>>> membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75) : 7.33 Hyd Momen (95) : 6.86 G content: 1 D/E content : 1 S/T content : 3 Score: -1.57

Gavel : prediction of cleavage sites for mitochondrial preseq

R-10 motif at 23 FRA FS

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 6.0% NLS Score: -0.47

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus : RSSL 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 diserimination

Prediction: cytoplasmic

Reliability: 94.1

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

A search ofthe 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 NOV2a 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 3A. Table 3A. NOV3 Sequence Analysis

SEQ ID NO: 9

NOV3a, CGGC<^(^GGTTTCCGCTTGCCTCTGGCCGGGGGTCGGCAACTGCAGGCGTCAGTTTCCCTCAA CGI 70490-01 GATGGCGGACGAGGAGGCTGGAGGTACTGAGAGGATGGAAATCAGCGCGGAGTTACCCCAGACC CCTCAGCGTCTGGCATCTTGGTGGGATCAGCAAGTTGATTTTTATACTGCTTTCTTGCATCATT DNA Sequence TGGCACAATTGGTGCCAGAAATTTACTTTGCTGAAATGGACCCAGACTTGGAAAAGCAGGAGGA AAGTGTACAAATGTCAATATTCACTCCACTGGAATGGTACTTATTTGGAGAAGATCCAGATATT TGCTTAGAGAAATTGAAGCACAGTGGAGCATTTCAGCTTTGTGGGAGGGTTTTCAAAAGTGGAG AGACAACCTATTCTTGCAGGGATTGTGCAATTGATCCAACATGTGTACTCTGTATGGACTGCTT CCAGGACAGTGTTCATAAAAATCATCGTTACAAGATGCATACTTCTACTGGAGGAGGGTTCTGT GACTGTGGAGACACAGAGGCATGGAAAACTGGCCCTTTTTGTGTAAATCATGAACCTGGAAGAG CAGGTACTATAAAAGAGAATTCACGCTGTCCGTTGAATGAAGAGGTAATTGTCCAAGCCAGGAA AATATTTCCTTCAGTGATAAAATATGTCGTAGAAATGACTATATGGGAAGAGGAAAAAGAACTG CCTCCTGAACTCCAGATAAGGGAGAAAAATGAAAGATACTATTGTGTCCTTTTCAATGATGAAC ACCATTCATATGACCACGTCATATACAGCCTACAAAGAGCTCTTGACTGTGAGCTCGCAGAGGC CCAGTTGCATACCACTGCCATTGACAAAGAGGGTCGTCGGGCTGTTAAAGCGGGAGCTTATGCT GCTTGCCAGGAAGCAAAGGAAGATATAAAGAGTCATTCAGAAAATGTCTCTCAACATCCACTTC ATGTAGAAGTATTACACTCAGAGATTATGGCTCATCAGAAATTTGCTTTGCGTCTTGGTTCCTG GATGAACAAAATTATGAGCTATTCAAGTGACTTTAGGCAGATCTTTTGCCAAGCATGCCTTAGA GAAGAACCTGACTCGGAGAATCCCTGTCTCATAAGCAGGTTAATGCTTTGGGATGCAAAGCTTT ATAAAGGTGCCCGTAAGATCCTTCATGAATTGATCTTCAGCAGTTTTTTTATGGAGATGGAATA CAAAAAACTCTTTGCTATGGAATTTGTGAAGTATTATAAACAACTGCAGAAAGAATATATCAGT GATGATCATGACAGAAGTATCTCTATAACTGCACTTTCAGTTCAGATGTTTACTGTTCCTACTC TGGCTCGACATCΓTATTGAAGAGCAGAATGTTATCTCTGTCATTACTGAAACTCTGCTAGAAGT TTTACCTGAGTACTTGGACAGGAACAATAAATTCAACTTCCAGGGTTATAGCCAGGACAAATTG GGAAGAGTATATGCAGTAATATGTGACCTAAAGTATATCCTGATCAGCAAACCCACAATATGGA CAGAAAGATTAAGAATGCAGTTCCTTGAAGGTTTTCGATCTTTTTTGAAGATTCTTACCTGTAT GCAGGGAATGGAAGAAATCCGAAGACAGGTTGGGCAACACATTGAAGTGGATCCTGATTGGGAG GCTGCCATTGCTATACAGATGCAATTGAAGAATATTTTACTCATGTTCCAAGAGTGGTGTGCTT GTGATGAAGAACTCTTACTTGTGGCTTATAAAGAATGTCACAAAGCTGTGATGAGGTGCAGTAC CAGTTTCATATCTAGTAGCAAGACAGTAGTACAATCGTGTGGACATAGTTTGGAAACAAAGTCC TACAGAGTATCTGAGGATCTTGTAAGCATACATCTGCCACTCTCTAGGACCCTTGCTGGTCTTC ATGTACGTTTAAGCAGGCTGGGTGCTGTTTCAAGACTGCATGAATTTGTGTCTTTTGAGGACTT TCAAGTAGAGGTACTAGTGGAATATCCTTTACGTTGTCTGGTGTTGGTTGCCCAGGTTGTTGCT GAGATGTGGCGAAGAAATGGACTGTCTCTTATTAGCCAGGTGTTTTATTACCAAGATGTTAAGT GCAGAGAAGAAATGTATGATAAAGATATCATCATGCTTCAGATTGGTGCATCTTTAATGGATCC CAATAAGTTCTTGTTACTGGTACTTCAGAGGTATGAACTTGCCGAGGCTTTTAACAAGACCATA TCTACAAAAGACCAGGATTTGATTAAACAATATAATACACTAATAGAAGAAATGCTTCAGGTCC TCATCTATATTGTGGGTGAGCGTTATGTACCTGGAGTGGGAAATGTGACCAAAGAAGAGGTCAC AATGAGAGAAATCATTCACTTGCTTTGCATTGAACCCATGCCACACAGTGCCATTGCCAAAAAT TTACCTGAGAATGAAAATAATGAAACTGGCTTAGAGAATGTCATAAACAAAGTGGCCACATTTA AGAAACCAGGTGTATCAGGCCATGGAGTTTATGAACTAAAAGATGAATCACTGAAAGACTTCAA TATGTACTTTTATCATTACTCCAAAACCCAGCATAGCAAGGCTGAACATATGCAGAAGAAAAGG AGAAAACAAGAAAACAAAGATGAAGCATTGCCGCCACCACCACCTCCTGAATTCTGCCCTGCTT TCAGCAAAGTGATTAACCTTCTCAACTGTGATATCATGATGTACATTCTCAGGACCGTATTTGA GCGGGCAATAGACACAGATTCTAACTTGTGGACCGAAGGGATGCTCCAAATGGCTTTTCATATT CTGGCATTGGGTTTACTAGAAGAGAAGCAACAGCTTCAAAAAGCTCCTGAAGAAGAAGTAACAT TTGACTTTTATCATAAGGCTTCAAGATTGGGAAGTTCAGCCATGAATATACAAATGCTTTTGGA AAAACTCAAAGGAATTCCCCAGTTAGAAGGCCAGAAGGACATGATAACGTGGATACTTCAGATG TTTGACACAGTGAAGCGATTAAGAGAAAAATCTTGTTTAATTGTAGCAACCACATCAGGATCGG AATCTATTAAGAATGATGAGATTACTCATGATAAAGAAAAAGCAGAACGAAAAAGAAAAGCTGA

AGCTGCTAGGCTACATCGCCAGAAGATCATGGCTCAGATGTCTGCCTTACAGAAAAACTTCATT

GAAACTCATAAACTCATGTATGACAATACATCAGAAATGCCTGGGAAAGAAGATTCCATTATGG AGGAAGAGAGCACCCCAGCAGTCAGTGACTACTCTAGAATTGCTTTGGGTCCTAAACGGGGTCC ATCTGTTACTGAAAAGGAGGTGCTGACGTGCATCCTTTGCCAAGAAGAACAGGAGGTGAAAATA GAAAATAATGCCATGGTATTATCGGCCTGTGTCCAGAAATCTACTGCCTTAACCCAGCACAGGG GAAAACCCATAGAACTCTCAGGAGAAGCCCTAGACCCACTTTTCATGGATCCAGACTTGGCATA TGGAACTTATACAGGAAGCTGTGGTCATGTAATGCACGCAGTGTGCTGGCAGAAGTATTTTGAA GCTGTACAGCTGAGCTCTCAGCAGCGCATTCATGTTGACCTTTTTGACTTGGAAAGTGGAGAAT

ATCTTTGCCCTCTTTGCAAATCTCTGTGCAATACTGTGA'ΓCCCCATTATTCCTTTGCAACCTCA

AAAGATAAACAGTGAGAATGCAGATGCTCTTGCTCAACTTTTGACCCTGGCACGGTGGATACAG ACTGTTCTGGCCAGAATATCAGGTTATAATATAAGACATGCTAAAGGAGAAAACCCAATTCCTA

Further analysis ofthe NOV3a protein yielded the following properties shown in Table 3B. Table 3B. Protein Sequence Properties NOV3a

SignalP analysis: No Known Signal Sequence Predicted

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 11; pos.chg 1; neg.chg 4 H-region: length 1; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -7.16 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: 5

INTEGRAL Likelihood -1. .81 Transmembrane 644

660

INTEGRAL Likelihood = -5. .57 Transmembrane 1397

-1413

INTEGRAL Likelihood = -1. .28 Transmembrane 1438

-1454

INTEGRAL Likelihood = -3. .50 Transmembrane 1625

-1641

INTEGRAL Likelihood = -2. .76 Transmembrane 1652

-1668

PERIPHERAL Likelihood = 1.27 (at 412) ALOM score: -5.57 (number of TMSs: 5)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 651 Charge difference: 4.0 C( 1.0) - N(-3.0) C > N: C-terminal side will be inside

>>> membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 9.30 Hyd Moment (95): 7.89 G content: 0 D/E content: 2 S/T content: 0 Score: -6.40

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

NUCDISC: discrimination of nuclear localization signals pat4: KKRR (5) at 851 pat4: KRRK (5) at 852 pat4: RKRK (5) at 1019 pat4: PRKR (4) at 1588 pat7: PRKRNSL (5) at 1588 bipartite: none content of basic residues: 10.2% NLS Score: 1.15

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

ER Membrane Retention Signals: none

1028 H 0.56

1029 R 0.56

1030 Q 0.56

1031 K 0.56

1032 I 0.56 total : 29 residues

Final Results (k = 9/23 ) :

56.5 % : endoplasmic reticulum

13 .0 % : vacuolar

8.7 % : mitochondrial

8.7 % : nuclear

4.3 % : Golgi

4.3 % : vesicles of secretory system

4.3 % : cytoplasmic

» prediction for CG170490-0 is end (k=23 )

A search ofthe 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 3C.

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 3D.

PFam analysis predicts that the NOV3a protein contains the domains shown in the Table 3E.

Example 4.

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

SEQ ID NO: 11 2748 bp

NOV4a, CTGTGAGCCGCGAGAGGCCCGGGAGCCGCGCGTCGCCGAGCCGAGCTGACCGAGAGCCCCATGG CG170667-01 CTGTGCAGCGCGCCGCGTCTCCGCGCCGCCCGCCCGCCCCGCTCTGGCCCCGGCTCCTGCTGCC GCTGCTGTTGCTGCTGCTGCCCGCGCCGAGCGAGGGTCTTGGCCACTCTGCTGAACTGGCATTT DNA Sequence GCTGTGGAGCCAAGTGATGATGTTGCCGTCCCCGGGCAGCCTATAGTGCTGGACTGCAGGGTGG AGGGGACCCCTCCAGTGCGAATCACCTGGAGGAAGAATGGGGTAGAGCTGCCAGAGAGTACCCA CTCCACCTTGCTGGCCAATGGGTCCTTGATGATCCGTCACTTCAGGCTGGAGCCGGGAGGCAGC CCTTCGGATGAAGGTGACTATGAGTGTGTGGCCCAGAACCGCTTTGGGCTGGTGGTCAGCCGGA AGGCTCGCATCCAAGCTGCAACCATGTCGGACTTCCACGTGCATCCCCAGGCCACCGTGGGTGA GGAGGGTGGTGTGGCCCGCTTCCAGTGCCAAATCCATGGGCTTCCCAAACCCCTGATCACTTGG GAGAAGAACAGAGTCCCAATTGACACGGACAATGAGAGGTACACATTGCTGCCCAAGGGGGTCC TGCAGATCACAGGACTTCGAGCTGAGGACGGTGGCATCTTCCACTGTGTGGCCTCAAACATCGC (^GTATCCGGATCAGCCΛCGGGGCCAGGCTCACTGTGTCAGGCTCGGGCTCTGGGGCCTACAAG GAGCCAGCCATCCTCGTGGGGCCTGAGAACCTCACCCTGACAGTGCACCAGACCGCGGTGCTTG AGTGTGTCGCCACGGGCAACCCGCGCCCCATTGTGTCCTGGAGCCGCCTGGATGGTCGCCCTAT CGGGGTGGAGGGCATCCAGGTGCTGGGCACAGGAAACCTCATCATCTCAGACGTGACGGTCCAG CACTCTGGCGTCTACGTCTGTGCAGCCAACAGACCTGGCACCCGGGTGAGGAGAACGGCACAGG GCCGGCTGGTGGTGCAAGCCCCAGCTGAGTTTGTGCAGCATCCCCAGTCCATCTCCAGGCCAGC TGGGACCACAGCCATGTTCACCTGCCAAGCCCAGGGTGAGCCACCGCCTCATGTCACGTGGCTG AAAAATGGACAGGTGCTGGGGCCAGGAGGCCACGTCAGGCTCAAGAATAACAACAGCACACTGA CCATTTCTGGAATCGGTCCTGAGGATGAAGCCATTTATCAGTGTGTGGCCGAGAACAGTGCGGG CTCATCACAGGCCAGTGCCAGGCTGACCGTACTGTGGGCTGAGGGGCTCCCCGGGCCTCCCCGC AATGTGCGGGCAGTCTCTGTGTCTTCCACTGAGGTGCGTGTGTCCTGGAGTGAGCCGCTGGCCA ACACCAAGGAGATCATCGGCTACGTCCTGCACATCAGGAAGGCTGCTGACCCACCGGAGCTGGA GTATCAGGAGGCAGTCAGCAAGAGCACCTTTCAGCACCTGGTCAGCGACCTGGAGCCCTCCACA GCCTACAGTTTCTACATCAAGGCCTACACACCAAGGGGGGCCAGCTCAGCCTCTGTGCCCACCC TAGCTAGCACCCTGGGTGAAGCCCCTGCCCCACCCCCACTGTCAGTGCGAGTCCTGGGCAGCTC CTCCTTGCAGCTGCTGTGGGAGCCTTGGCCCCGGCTGGCCCAGCACGAGGGCGGCTTCAAGCTG TTTTACCGCCCAGCAAGCAAGACCTCCTTCACCGGCCCCATCCTGCTGCCTGGAACCGTCTCCT CCTACAACCTCAGCCAGCTCGACCCCACTGCAGTGTATGAGGTGAAGCTGCTCGCCTACAGCCA GCATGGAGATGGCAATGCCACAGTCCGCTTTGTGTCTTTGAGGGGAGCATCTGAGAGGACAGGC ATCGTCATCGGCATCCACATCGGGGTCACTTGCATCATCTTCTGTGTCCTCTTCCTCCTGTTCG GCCAAAGGGGCAGGGTCCTCCTGTGTAAAGATGTGGAAAACCAGCTGTCCCCTCCACAGGGTCC CCGGAGCCΛGAGGGACCCTGGCΛTTCTGGCCCTAAATGGGGCGAGACGGGGACAGCGGGGCCAG CTGGGCCGAGACGAGAAACGTGTGGATATGAAGGAGCTGGAGCAGCTGTTCCCCCCGGCCAGCG CAGCAGGGCAGCCGGACCCCAGACCCACACAGGATCCTGCAGCCCCCGCTCCGTGTGAGGAGAC CCAGCTCTCCGTGCTGCCACTTCAGGGGTGCGGCCTGATGGAGGGGAAGACGACGGAGGCGAAG ACCACAGAGGCCACGGCTCCCTGCGCCGGCCTGGCGGCTGCCCCACCACCCCCAGATGGAGGCC CTGGCCTCCTCAGTGAAGGCCAGGCTTCCAGGCCTGCAGCGGCCCGGGTTACCCAGCCAGCTCA CTCGGAACAGTAGCCAGTGTCTGGCAGGCTCCAGAGGGTGGACGGAGCGGGGCCCATTCTCAGG TCAAAAGCAAGATTTCTACTGTCATGTGGGATTTGGATGGTCCTGGGGGCTCCCCAGCATTTCT ftTCCTGACTGCCTCTTGGGTTGTCAAAACCCAAGGCAGCCTTGACAGGGACCCCCCGGCCCTAA

CACCCATCAGGAGTTGGAGCAGTTCCTGCAGGAGCCTGTTCCTTCCCTGGGCTGACGCCCCCTT jGCCTCTGCCTGGTACCCACATGACTTGGAACTGAACTAACATTTTTCTTTAAAAAGCAAA

ORF Start: ATG at 61 I ORF Stop: TAG at 2443

SEQ ID NO: 12 J794 aa |MW at 84591 ,3kD

NOV4a, MAVQRAASPRRPPAPLWPRLLLPLLLLLLPAPSEGLGHSAELAFAVEPSDDVAVPGQPIVLDCR CGI 70667-01 VEGTPPVRITWRKNGVELPESTHSTLLANGSLMIRHFRLEPGGSPSDEGDYECVAQNRFGLWS RKARIQAATMSDFHVHPQATVGEEGGVARFQCQIHGLPKPLITWEKNRVPIDTDNERYTLLPKG Protein Sequence VLQITGLRAEDGGIFHCVASNIASIRISHGARLTVSGSGSGAYKEPAILVGPENLTLTVHQTAV LECVATGNPRPIVSWSRLDGRPIGVEGIQVLGTGNLIISDVTVQHSGVYVCAANRPGTRVRRTA QGRLWQAPAEFVQHPQSISRPAGTTAMFTCQAQGEPPPHVTWLKNGQλ^GPGGHVRLKNNNST LTISGIGPEDEAIYQCVAENSAGSSQASARLTVLWAEGLPGPPRNVRAVSVSSTEVRVSWSEPL ANTKEIIGYVLHIRKAADPPELEYQEAVSKSTFQHLVSDLEPSTAYSFYIKAYTPRGASSASVP TLASTLGEAPAPPPLSVRVLGSSSLQLLWEPWPRLAQHEGGFKLFYRPASKTSFTGPILLPGTV SSYNLSQLDPTAVYEVKLLAYSQHGDGNATVRFVSLRGASERTGIVIGIHIGVTCIIFCVLFLL FGQRGRVLLCKDVENQLSPPQGPRSQRDPGILALNGARRGQRGQLGRDEKRVDMKELEQLFPPA SAAGQPDPRPTQDPAAPAPCEETQLSVLPLQGCGLMEGKTTEAKTTEATAPCAGLAAAPPPPDG GPGLLSEGQASRPAAARVTQPAHSEQ SEQ ID NO: 13 2805 bp

NOV4b, CTGTGAGCCGCGAGAGGCCCGGGAGCCGCGCGTCGCCGAGCCGAGCTGACCGAGAGCCCCATGG CGI 70667-02 CTGTGCAGCGCGCCGCGTCTCCGCGCCGCCCGCCCGCCCCGCTCTGGCCCCGGCTCCTGCTGCC GCTGCTGTTGCTGCTGCTGCCCGCGCCGAGCGAGGGTCTTGGCCACTCTGCTGAACTGGCATTT DNA Sequence GCTGTGGAGCCAAGTGATGATGTTGCCGTCCCCGGGCAGCCTATAGTGCTGGACTGCAGGGTGG AGGGGACCCCTCCAGTGCGAATCACCTGGAGGAAGAATGGGGTAGAGCTGCCAGAGAGTACCCA CTCCACCTTGCTGGCCAATGGGTCCTTGATGATCCGTCACTTCAGGCTGGAGCCGGGAGGCAGC CCTTCGGATGAAGGTGACTATGAGTGTGTGGCCCAGAACCGCTTTGGGCTGGTGGTCAGCCGGA AGGCTCGCATCCAAGCTGCAACCATGTCGGACTTCCACGTGCATCCCCAGGCCACCGTGGGTGA GGAGGGTGGTGTGGCCCGCTTCCΛGTGCCΛAATCCATGGGCTTCCCΛAACCCCTGATCACTTGG GAGAAGAACAGAGTCCCAATTGACACGGACAATGAGAGGTACACATTGCTGCCCAAGGGGGTCC TGCAGATCACAGGACTTCGAGCTGAGGACGGTGGCATCTTCCACTGTGTGGCCTCAAACATCGC CAGTATCCGGATCAGCCACGGGGCCAGGCTCACTGTGTCAGGCTCGGGCTCTGGGGCCTACAAG GAGCCAGCCATCCTCGTGGGGCCTGAGAACCTCACCCTGACAGTGCACCAGACCGCGGTGCTTG AGTGTGTCGCCACGGGCAACCCGCGCCCCATTGTGTCCTGGAGCCGCCTGGATGGTCGCCCTAT CGGGGTGGAGGGCATCCAGGTGCTGGGCACAGGAAACCTCATCATCTCAGACGTGACGGTCCAG CACTCTGGCGTCTACGTCTGTGCAGCCAACAGACCTGGCACCCGGGTGAGGAGAACGGCACAGG GCCGGCTGGTGGTGCAAGCCCCAGCTGAGTTTGTGCAGCATCCCCAGTCCATCTCCAGGCCAGC TGGGACCACAGCCATGTTCACCTGCCAAGCCCAGGGTGAGCCACCGCCTCATGTCACGTGGCTG AAAAATGGACAGGTGCTGGGGCC^GGAGGCαv.CGTCAGGCTCAAGAATAACAACAGCACACTGA CCATTTCTGGAATCGGTCCTGAGGATGAAGCCATTTATCAGTGTGTGGCCGAGAACAGTGCGGG CTCATCACAGGCCAGTGCCAGGCTGACCGTACTGTGGGCTGAGGGGCTCCCCGGGCCTCCCCGC AATGTGCGGGCAGTCTCTGTGTCTTCCACTGAGGTGCGTGTGTCCTGGAGTGAGCCGCTGGCCA ACACCAAGGAGATCATCGGCTACGTCCTGCACATCAGGAAGGCTGCTGACCCACCGGAGCTGGA GTATCAGGAGGCAGTCAGCAAGAGCACCTTTCAGt^CCTGGTCAGCGACCTGGAGCCCTCCACA GCCTACAGTTTCTACATCAAGGCCTACACACCAAGGGGGGCCAGCTCAGCCTCTGTGCCCACCC TAGCTAGCACCCTGGGTGAAGCCCCTGCCCCACCCCCACTGTCAGTGCGAGTCCTGGGCAGCTC CTCCTTGCAGCTGCTGTGGGAGCCTTGGCCCCGGCTGGCCCAGCACGAGGGCGGCTTCAAGCTG TTTTACCGCCCAGCAAGCAAGACCTCCTTCACCGGCCCCATCCTGCTGCCTGGAACCGTCTCCT CCTACAACCTCAGCCAGCTCGACCCCACTGCAGTGTATGAGGTGAAGCTGCTCGCCTACAGCCA GCATGGAGATGGCAATGCCACAGTCCGCTTTGTGTCTTTGAGGGGAGCATCTGAGAGGACAGCC TTGAGCCCACCATGTGACTGCCGGAAGGAGGAGGCCGCCAACCAGACGTCCACCACAGGCATCG TCATCGGCATCCACATCGGGGTCGCTTGCATCATCTTCTGTGTCCTCTTCCTCCTGTTCGGCCA AAGGGGCAGGGTCCTCCTGTGTAAAGATGTGGAAAACCAGCTGTCCCCTCCACAGGGTCCCCGG AGC(^GAGGGACCCTGGCATTCTGGCCCTAAATGGGGCGAGACGGGGACAGCGGGGCCAGCTGG GCCGAGACGAGAAACGTGTGGATATGAAGGAGCTGGAGCAGCTGTTCCCCCCGGCCAGCGCAGC AGGGCGGCCGGACCCCAGACCCACACAGGATCCTGCAGCCCCCGCTCCGTGTGAGGAGACCCAG CTCTCCTTGCTGCCACTTCAGGGGTGCGGCCTGATGGAGGGGAAGACGACGGAGGCGAAGACCA CAGAGGCCACGGCTCCCTGCGCCGGCCTGGCGGCTGCCCCACCACCCCCAGATGGAGGCCCTGG CCTCCTCAGTGAAGGCCAGGCTTCCAGGCCTGCAGCGGCCCGGGTTACCCAGCCAGCTCACTCG GAACAGTAGCCAGTGTCTGGCAGGCTCCAGAGGGTGGACGGAGCGGGGCCCATTCTCAGGTCAA AAGCAAGATTTCTACTGTCATGTGGGATTTGGATGGTCCTGGGGGCTCCCCAGCATTTCTATCC

ITGACTGCCTCTTGGGTTGTCAAAACCCAAGGCAGCCTTGACAGGGACCCCCCGGCCCTAACACC

CATCAGGAGTTGGAGCAGTTCCTGCAGGAGCCTGTTCCTTCCCTGGGCTGACGCCCCCTTGCCT

CTGCCTGGTACCCACATGACTTGGAACTGAACTAACATTTTTCTTTAAAAAGC

ORF Start: ATG at 61 ORF Stop: TAG at 2503

SEQ ID NO: 14 J814 aa JMW at 86707.6kD

NOV4b, MAVQRAASPRRPPAPL PRLLLPLLLLLLPAPSEGLGHSAELAFAVEPSDDVAVPGQPIVLDCR CGI 70667-02 VEGTPP\'RITWRKNGVELPESTHSTLLANGSLMIRHFRLEPGGSPSDEGDYECVAQNRFGLWS RKARIQAATMSDFHVHPQATVGEEGGVARFQCQIHGLPKPLITWEKNRVPIDTDNERYTLLPKG Protein Sequence VLQITGLRAEDGGIFHCVASNIASIRISHGARLTVSGSGSGAYKEPAILVGPENLTLTVHQTAV LECVATGNPRPIVSWSRLDGRPIGVEGIQVLGTGNLIISDVTVQHSGVYVCAANRPGTRVRRTA QGRLWQAPAEFVQHPQSISRPAGTTAMFTCQAQGEPPPHVTWLKNGQVLGPGGHVRLKNNNST LTISGIGPEDEAIYQCVAENSAGSSQASARLTVLWAEGLPGPPRNVRAVSVSSTEVRVSWSEPL ANTKEIIGYVLHIRKAADPPELEYQEAVSKSTFQHLVSDLEPSTAYSFYIKAYTPRGASSASVP TLASTLGEAPAPPPLSVRVLGSSSLQLLWEPWPRLAQHEGGFKLFYRPASKTSFTGPILLPGTV SSYNLSQLDPTAVYEVKLLAYSQHGDGNATVRFVSLRGASERTALSPPCDCRKEEAANQTSTTG IVIGIHIGVACIIFCVLFLLFGQRGRVLLCKDVENQLSPPQGPRSQRDPGILALNGARRGQRGQ LGRDEKRVDMKELEQLFPPASAAGRPDPRPTQDPAAPAPCEETQLSLLPLQGCGLMEGKTTEAK TTEATAPCAGLAAAPPPPDGGPGLLSEGQASRPAAARVTQPAHSEQ Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 4B.

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 36 and 37

PSORT II analysis: PSG: a new signal peptide prediction method

N-region : length 11 ; pos . chg 3 ; neg . chg 0 H-region: length 7 ; peak value -3 .55 PSG score : -7 .95

GvH: von Heijne ' s method for signal seq . recognition GvH score (threshold: -2.1) : 3 .23 possible cleavage site : between 35 and 36

>>> 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 : 2

Number of TMS (s) for threshold 0.5 : 1

INTEGRAL Likelihood =- 10 .61 Transmembrane 625

641

PERIPHERAL Likelihood = 3.45 (at 279) ALOM score: -10.61 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 632 Charge difference: -0.5 C( 1.0) - N( 1.5) N >= C: N-terminal side will be inside

>>> membrane topology: type 2 (cytoplasmic tail 1 to 625)

MITDISC: discrimination of mitochondrial targeting seq R content: 4 Hyd Moment (75): 9.73 Hyd Moment (95): 7.17 G content: 0 D/E content: 1 S/T content: 2 Score: -0.12

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 29 PRL|LL

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: PGTRVRR (3) at 312 bipartite: none content of basic residues: 9.1% NLS Score: -0.22

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus : AVQR 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

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 20 LL at 21 LL at 24 LL at 25 LL at 26 LL at 27 LL at 28 LL at 90 LL at 188 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):

43.5 %: mitochondrial

26.1 %: cytoplasmic

8.7 %: vacuolar

8.7 %: endoplasmic reticulum

4.3 %: Golgi

4.3 %: vesicles of secretory system

A search ofthe 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.

SEQ ID NO: 16 448 aa |MW at 49630.9kD

NOV5a, MSPTISHKDSSRQRRPGNFSHSLDMKSGPLPPGGWDDSHLDSAGREGDREALLGDTGTGDFLKA CG170791-01 PQSFRAELSSILLLLFLYVLQGIPLGLAGSIPLILQSKNVSYTDQAFFSFVF PFSLKLLWAPL VDAVYVKNFGRRKSWLVPTQYILGLFMIYLSTQVDRIiLGNTDDRTPDVIALTVAFFLFEFLAAT Protein Sequence QDIAVDGWALTMLSRENVGYASTCNSVGQTAGYFLGNVLFLALESADFCNKYLRFQPQPRGIVT LSDFLFFWGTVFLITTTLVALLKKENEVSWKEETQGITDTYKLLFAIIKMPAVLTFCLLILTA KVTVYSMYVSIMAFNAKVSDPLIGGTYMTLLNTVSNLGGNWPSTVALWLVDPLTVKECVGASNQ NCRTPDAVELCKKLGGSCVTALDGYYVESIICVFIGFG FFLGPKFKKLQDEGSSSWKCKRNN

Further analysis ofthe NOV5a protein yielded the following properties shown in Table 5B.

Table 5B. Protein Sequence Properties NOVSa

SignalP analysis: No Known Signal Sequence Predicted

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 9; pos.chg 1; neg.chg 1 H-region: length 2; peak value -20.41 PSG score: -24.81

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -13.86 possible cleavage site: between 32 and 33

>>> 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:

5

INTEGRAL Likelihood = -6 53 Transmembrane 75

91

INTEGRAL Likelihood = -5 79 Transmembrane 176

192

INTEGRAL Likelihood = -3 88 Transmembrane 261

277

INTEGRAL Likelihood = -7 11 Transmembrane 301

317

INTEGRAL Likelihood -2 97 Transmembrane 411

427

PERIPHERAL Likelihood = 1.38 (at 110) ALOM score: -7.11 (number of TMSs: 5)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 82 Charge difference: 0.0 C( 0.0) - N( 0.0) N >= C: N-terminal side will be inside

>>> membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75) : 8.26 Hyd Moment (95) : 5.54 G content : 1 D/E content : 2 S/T content : 7 Score: -2.44

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 25 RRP|GN NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: PKFKKLQ (5) at 429 bipartite: none content of basic residues: 8.5% NLS Score: -0.04

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

ER Membrane Retention Signals:

KKXX-like motif in the C-terminus: CKRN

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

Tyrosines in the tail: none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs:

Leucine zipper pattern (PS00029) : *** found *** LVPTQYILGLFMIYLSTQVDRL at 144 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: 94.1

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

Final Results (k = 9/23)

39.1 %: mitochondrial

39.1 %: endoplasmic reticulum

13.0 %: nuclear

4.3 % -. plasma membrane

4.3 %: vesicles of secretory system

>> prediction for CG170791-01 is mit (k=23) A search ofthe 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 5C.

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 5D.

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

Table 5E. Domain Analysis of NOV5a

Identities/

Pfam Domain NOV5a Match Region Similarities Expect Value for the Matched Region

Example 6.

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

GACTTTGAAGCGATTCAGAACAACATTGCGCTGAAGTACAAGAAGAAAGGCAAGCCAGCAAGCA TGGTGAAGAACAAGGAGCAGGTCCGCCACTTCTACTACCGCACCTGGCACAAGATCACCAAGTA CATCGACTTTGATCATGTGTTCTCTCGAGGCCTGAAGAAGTCATCCCAGGAACTGTATGGCCTG ATCTGCTATGGCGAGCTGCGCAAGAAGATTGGGGGCTGTATGGATGACAAGAATGCAACAAAGC TGAATGAACTCATTCAGGTTGGGATCCATACTTGGGGCAAATCTTACTTCACCTTTTATTTCAT TTCCTCCATGATTGATGGAATGAAGCCAGAGTTCCAGACTCTTTGCTGTATGCTTGAGGACGGT GCACAGAGTGGCCTGTCCGATGAGCGTTCCTTTTGCCAAAACACAGATGTGCTGCCCAGCGGGG GCGTGGTGGGCACCTGCAGCGCCATCCGCGGGAGAACTTATGCCTCAGCGTTCCTTCAAAACTC TTTTCCCAGGGCCACCACTGTACGTTACAAAGGGCGGAACCTGCGGATCAAAGCGCCCATGTGC CGGGCCCTGAAGAAGCTGTGCGATCCAGATGGCTTGAGTGATGAAGAGGACCAGAAGCCAGTGC GCCTGCCTCTGAAAGTCCCTATAGAGCTACAGCCGCGGAACAACCACGCCTGGGCCCGTGTGCA GAGCCTTGCCCAGAACCCACGCCTCAGGAACTTCCAGGAGAAGCAGGTCCACCCCTATGCTCTG TCATCACACGAGGACGCAGCAGTGTGGAGGCGACTGGAGTCCAGGGAGCACTGGGCTGCAGTCC TGTATCTGGGCAGGGATCGCCCAACCTGTGTTCAGGCCGTGGAGGGGATGTCGCGGATGATCGT GGAGCTACATCGAAAGGTCTCCAGCCTCATCGAATTCTTGAAGCAGAAGTGGGCGCTCCATGAG CATCCCGACCTCAGTGCTAGCCAGTGTGGGCCTTCCTTGACGGGCACTCAGCGGAAGACACTCG AGGAGCGGCAGCTGCAGGACTCATGCTCCGCACCGATGCAGGAGAAGGTGACACTGCACTTGTT CCCAGGCGAGAACTGTACACTGACACCGCTGCCGGGCGTGGCTCGCGTGGTGCACTCCAAGGCC TTCTGCACAGTGCACTGGCAGGAGGGCGGCCGGTGCAAGCAGAGTGCCAAGGACGCCCACGTGC TGCCCCCAGCCCAGATCCTGGGCATCCAGAGTGGGCAGGGCACGGCCCGGGGCCAGGTGAAATG CCCGCGGAGCGGAGCTGAGGGCAAGGGTGTGGGGCGGCCCCCTCCTGCGGCTGACGCCTTGCAG AGCTCCGGAGAGAGTTCCCCCGAAAGCGCCCCCGGGGAGGGGGCTGCCCTAAGCTTGAGCAGCC CGGACGCTCCTGACAGGCCTCCTCCCAGGCACCAGGACACTGGGCCATGTCTTGAGAAGACCCC TGCAGAAGGCAGGGACAGTCCCACCCGGGAGCCAGGGGCCTTGCCGTGTGCCTGTGGCCAGCTC CCAGACCTGGAGGACGAGCTCTCGCTTCTAGACCCCTTGCCCCGCTACCTAAAGTCCTGTCAGG ACCTCATTGTCCCCGAGCAGTGCCGCTGTGCGGACACACGGCCTGGGAGCGAGCAGCCCCCTCT GGGCGGGGCGGCCTCCCCAGAGGTGCTGGCTCCTGTCAGCAAGGAGGCTGCTGACCTTGCTCCC ACTGGCCCATCCCCGAGGCCCGGCCCCGGGCTCCTGCTGGATGTTTGCACTAAAGACTTGGCAG ATGCACCTGCGGAGGAGCTCCAGGAGAAGGGGAGCCCCGCGGGGCCTCCGCCGTCTCAGGGACA GCCTGCCGCCAGGCCCCCGAAGGAGGTCCCCGCCAGCCGGCTGGCTCAGCAGCTCCGTGAGGAG GGCTGGAACCTGCAGACCTCCGAAAGCCTCACGCTGGCCGAAGTCTACCTCATGATGGGCAAGC CCAGCAAGCTGCAGCTGGAGTACGACTGGCTGGGGCCCGGCCGCCAGGACCCCCGCCCCGGCTC CCTACCCACCGCCCTCCACAAGCAGCGCCTCCTCAGCTGCCTCCTGAAGCTCATTTCCACCGAG GTCAACCCCAAGCTGGCTCTGGAAGCAAACACCATCTCTACAGCCTCAGTAAGGCCCGCCCAGG AGGAGCAGTCGATGACGCCCCCAGGGAAGGTGGTGACCGTCAGCTCTCGCAGCCCCCGCTGCCC TCGGAACCAGGCCTCCCTCCGCAGCAGCAAGACCTTCCCGCCCAGCTCTGCACCCTGCTCCTCA GGTTTGAGAAACCCTCCAAGACCCCTCTTGGTGCCTGGTCCCTCCAGCACAGGAAGCAATGACT CAGATGGAGGCCTTTTTGCTGTCCCGACAACCTTGCCACCCAACAGCCGACACGGGAAGCTCTT CTCTCCCAGTAAAGAAGCAGAGCTGACTTTCCGCCAGCATCTGAACTCCATCAGTATGCAGTCG GATTTCTTCCTGCCAAAGCCCCGGAAGCTGCGGAACCGGCACCTGCGGAAGCCACTGGTGGTCC AGAGAACACTGCTCCCTAGACCATCGGAAAACCAGTCCCACAACGTTTGTTCCTTCTCCATCCT GTCTAACTCTTCCGTAACTGGGAGAGGTTCGTTCCGGCCCATCCAGTCTTCTCTGACCAAAGCA GCTCTGTCTCGGCCGATCGTGCCCAAGGTCCTTCCACCCCAGGCCACGAGTCACCTGGCCAGTG CTATCGACTTAGCAGCTACAAGTGCCGGCATCCTTTCCGGGAACCCCCTCCCTGCCTTGGACAC CGAGGGCTTGTCTGGCATCTCTCCACTGTCTTCAGACGAGGTGACGGGTGCCATCTCGGGGCAG GACTCTACTGGAACTCACCAGGATGGAGACACCCTCCCCACCGTGGGGGGCTCCGACCCATTTG TCAGCATCCCTTCGAGGCCTGAGCAGGAGCCAGTGGCAGACAGTTTCCAGGGCTCATCTGTTCT CTCCTTATCTGAGCTGCCCAAGGCCCCTCTCCAGAATGGCCTCTCCATACCGCTGTCCTCGTCA GAGAGCTCCAGCACCCGGCTGTCTCCACCAGACGTCTCTGCTCTGCTCGACATCTCCCTGCCCG GCCCACCTGAGGATGCGCTGTCACAGGGCGAGCCTGCCACACACATTAGCGACTCCATCATTGA GATCGCCATCAGCTCCGGTCAGTACGGTGAAGGAGTCCCTCTTTCCCCAGCAAAACTGAATGGC AGTGACAGTTCCAAGAGCCTTCCCTCCCCGTCCAGCAGCCCCCAGCCACACTGGATCGCCTCTC CCACCCACGACCCCCAGTGGTACCCCAGTGACTCCACCGACTCCTCGCTCAGCAGCCTGTTTGC AAGCTTCATCTCCCCAGAGAAGAGCCGGAAGATGTTGCCGACTCCCATTGGGACCAACAGTGGC ACTTCCTTGCTTGGCCCCAGCTTGTTGGATGGAAACTCGCGGGACTCATTTGTGTCCAGGTCCC TGGCTGACGTTGCAGAGGTTGTGGATTCCCAGCTGGTGTGCATGATGAACGAAAACAGCATTGA TTACATTTCTCGGTTCAATGACCTGGCCCAAGAGCTGTCCATCGCTGAGCCTGGCCGCCGAGAA GCTCTGTTTGATGGTGGTGGAGGCGGCCCCGCTGTCAGTGACCTGTCCCAGTGACCACACGTCC TGGTGGCGGATGAAGCCCTCTTCGAGCTAGA ΛAAAATAGATAAGCCCAGCAGCCCCAGAAGAT

GGTCTGAACΆGAGGCATCTCCGCACCCAAGACTGTGCAACGGGCAGGAACGTGGTCACAGAGCT

GCTTCCCCACGAGCAGCAGGCAACGGCGTCCAAGGAGACTAGGATGAGTTCTTGGCAAGGGCCA

GCGTTAGAAATCACTGTGGTACTAGAGCCGTTCTTCACCACGCCTGGGCCCATGTTAGGGTCTG CATAATGATCCCATTTCAGCCTGTGCTCTGCCTCGATTGTTGTGTTGGACATTCCGGTGGCATT TCCTTCTGAGACAAGGGAGTATGTGTGCCTTGGTGTAGTTGCTGTGCACTAGGAGCTGTGATCT CCCTCTCTGCAGGGAGGCCCCAGCCCCTGCTGCTTGCTTTCTGCCAAACCTGTGCTATGCATCA IGCTGTGCCCTCTGTGGACTGTAACGGGCAGGACAGTTGGGTGTGGCCTGGGCTCATGCCTGGTG

1GTGTCACATCCCAAGGCAGCU^V.GAGCATGGATACCGATCACAGGGCTGCTGCGGAGTCGTGGGG

CCCTGGGCTGGTGCCTCCCCTCCCTAGAGGTTTTGTTCGTACTCTTAACAGGGAGTGGGGGCAG

GAAGAGTCCTGTACTATGCAGGTTGTGTGGACTTTACATGGGACCCTGCTAAGCTGGTTGAAAA

TGTTTTTCTTGTGTTTTAAGAATTAGGAGACATGGAAGAGGAAGAACAAAGTCCCCTCTGTAGT iTGGTTTCCTTCCTGTGTCCCTTTGCAAGCTTCCAGGCGATCTAAGGTGTCATTTCTCCCTCCTG

GGGTGACCCTTAGGCGCTAATATGATTACAGCGAAGACTTTCCTGATAAGTTCTCAAACTCGAT jGTGTGACTGTTTGGCACTTGAGACAAACCTGCCTTTGCAGGGAAAGTGTCTCTCACGGGCATTG

GTGTGGGCGTGCCTGACATACGTGTTCAGTCCCTTGCATACCTTTGCCTTGAGACTTCTGTGTC

TCCTTCCCATTTGGGACACCCAGGTGAGGGCCCAGACATCTGGATGTGGTCAGACCTCACCAAA iTATATGCCTTCGTGGTGGTCTCCCTCCTTGCGCCCTCTTGGGTGGCCAGCGTTCCTACTGCAGA iCGGCCCAACATCCAGTCTTTCCCCAGGACAGAGCTAACAAGGGCCCCTTTGCCTTCTCATCCTC

ΛGGAGTTCCAGGCACATGAGTCACCGTCCATCCACATCCAGTGTGGCCTGGAGCTGCTACAGAG

IGTGTTGGGCAGGCCATGCCTGTGCCGCCATCTCTCCCTTCCTGCCTCATTTCATCCCCCGCAGC lAGCCGGGATTGATTGTGCTTTCCTAACCCCCTTGGACCTACTCTCGCTCCτCCCCACCATTCCT

CTTCCCCCACATGTGTGGCACGCTGCAGCCCTCAAGGCCAGCCCTGGCCCCTCCACTGCTTCTC

TCCCCATCCACAATGGAGAAGGTGAAAAGAGGAGGGAAAGGCCTTTGGTGTGGACAAGCATGTG

GACGCCCTCCGTCCTGCAGTCTTGCCAGCCCACCACAGCCACTGTAGACCACAGGCAGGCCGTG

TACTGCACCACTGGGAGGACGTGGAGAGGACAGTGAACTTCCAGGCAAGAGCTTCCTTCTTTTG iTCTCACGAGTTTTTCTTAGAGCTCTTGCCTGAGCTGGCTTCCCTCCTTCAGACATTGACATGAG lATCTTAAGCAAACAGTCCCAAACCTCTTAGGGGTGAAAAAAGAAACATGCCACTTGATTAGGAG

AGAGACAGCAGTGTTTGAACTACAGCATCTTTACACTAGCTTGTGTTTTGTGCTACGTATACCA

GCTTCCAAAATTAGCATCTCATTGAGCCAGAGAAGACAAGGAGATCTCCCTCTGGGCATCTGGC

TTTGCTGCGTCTCTAGAGGGTTAGGATACCAGGCCGAGTTCAGGCCACTGCTAGCTTTCTCATA

CTCCCACAGGCTAGACCAGAGATGCCAAGTCCCAACAGCACTGAGCTGTGTGCACTGTGCCAGG

GACAGGAGGGTTTGTGAACTGCCTGTCAGGGTACCTGTTAGCCCCTGACAACTCAGTGGGGTGA

ΛGTTTTGGAGGTCAGAGTCTGCTTTCGTAGGCTCTTTAGACAGCACCTACCACTTGGTTCTCCA

GCGTAGACTCCTGGGAGCAGCCAACTGCAGCCATTGCCATCCAGTGGGGAGATGGGTTAGGGAG

GAGGACGGGCTGACTCCTCTCCTGTAATAAAGCTGACAAGAGTTCTAGAGGATTCTGCTTCTCT ftGTAACTAGACAGGTGATACGCATTTGCTTGCCACATTAAGGGAAAATGGTGTCATTTGTTGCA

GAAAAACAATGGATACATTTTCTTCTGGCCTAAATGAATATTTATGTGCAAACATAGGCAACTG

TTAAAGGCTGGAATTTTCAAAAGATCCAAACAGAGACTTCCTGCATCTTCTGCCTTTCCAACAG

AAGCGGTGATCGTCTAAGTATGAGCCTGTGGCTTCCTTTGTGCATTTGAGCATGCTGTAATTAA

GATGAGATCAGTTTCTTAGAAAAAGCTTTCCTGAATCCCTCTGACGTTGCCTGGGATCTTTCTG

TTGATTCGTCTTTTCTGGAGATTGGGACAGAGCATCTGTGGTCCAGGGAAGTTAGTCCTCTGGC

CTCAATTCTGTTGTGGATGTGCAGTGATAAGCGGGCATTGCGTGCCTCGGGGGATGCCTAGTTC

GTGGCTTCCTGGCTGTTTTGTCCTTCTGTGTCTTGTAGCTGTAGGGTGCCAGCTCAGGGAGTGG

GGTGTTGGCGGCGTTTCCGCGGTTGGCCTCCTTGCTTTGCCGCACCTCCAGGTTCTGGGCATGA

GAGGCCGTGGCCTCATTTCTGGTGGATAACCTTTTTAGTTTAATAGCATCTTTAATTAGATCAC

ΛGCATTGAATTCAAAATTTCTTCTGCAAAGAAAGTTGTGGGGCATAAGACACCGGGAATGAGGG

AGGAGGAAGACAGTTGTGTTTTCTCTTTAAACCTTGAGCTCTAGCCGATGCATTTGTCAGGAAA iTACAGCACTTTGTCTTAAGAAAACAAGGAAGGAGGCCGGGCGCAGTGGCTCACGCCTGTAATCC iCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACCTGAGGTGGGGAGTATGAGACCACCCTGACT lAACATGGAGAGACCCTGTCTCTACTAAAAGTACAGAATTAGCCGGGCGTGGTGGCGCATGCCCA

TAATCCCAGCTACTGAGGAGACTTGAGGTAGGAGAATCACTTGAACCTCAGCGGCGGAGGTTGC

AGTGAGTCGAGATCGCGCCAGTGCACTCCAGCCTGGGCAAGAAGAGCGAAACTGGGTCTCAAGT

TAAAAAAAGAAAGCAAGGAAAGAGTAATTTACAACGAAGGAAAAAAACCCACAGCACACCCTTC

GCGGCTGTCAGCGCTCTCCTGATGTCACAGTGGCTGCGTGTCCTTGGGGTGGGTGAGGTGTGGG

GAGCCCAGCCCCTGGCCCTGCCTCCCGCGCCCCGCTCCCCTTCTCTCTCTTACTCGGTTAAGCC lATAGCGAGGCCTCCGCTCGTTTCAGATATGAATTTGTTTTATAGATTATAAATATGCATATACA

GTGTATGTATAAAGCAGAATGCCTGCCTTTCCTGGTTATTTTTTGTACCATATTGTAAATTATA

ITTATTTATTCTTTACCAATTTTGGGAATAAAAGGTGTTTTGGTTATTTAATATAATAAGAGCTG

TTAAACTTCTGTTTAAATTTCCAGTTCAACTTGTAAATGTTTTTATTGTGCATAAATACATACT

|AATGTTGATCTAAAAAAAAAAAAAAAAAAAAAAGGGCGGCCGCT

ORF Start: ATG at 1 ORF Stop: TGA at 4276

SEQ ID NO: 18 jl425 aa |MW at 153017Λ1 D

NOV6a, MARPGRGVLSGGAGERGGGVASTAPERSSPASLYFWGVGASIVCSFEVEMPPFSTVELNAGAS CG171174-01 SGGRRVGQRAAAEQEAQEGSSERCGERQRRWLGAPRKRFWHGSEALDLESSRHSSPMSLASNL ALPLHPLGDAFLSGVLTWGSRSSSRNLGSSGGEKEEGKKVRRQWESWSTEDKNTFFEGLYEHGK Protein Sequence DFEAIQNNIALKYKKKGKPASMVKNKEQVRHFYYRTWHKITKYIDFDHVFSRGLKKSSQELYGL ICYGELRKKIGGCMDDKNATKLNELIQVGIHTWGKSYFTFYFISSMIDGMKPEFQTLCCMLEDG AQSGLSDERSFCQNTDVLPSGGWGTCSAIRGRTYASAFLQNSFPRATTVRYKGRNLRIKAPMC RALKKLCDPDGLSDEEDQKPVRLPLKVPIELQPRNNHAWARVQSLAQNPRLRNFQEKQVHPYAL SSHEDAAVWRRLESREHWAAVLYLGRDRPTCVQAVEGMSRMIVELHRKVSSLIEFLKQKWALHE HPDLSASQCGPSLTGTQRKTLEERQLQDSCSAPMQEKVTLHLFPGENCTLTPLPGVARWHSKA FCTVHWQEGGRCKQSAKDAHVLPPAQILGIQSGQGTARGQVKCPRSGAEGKGVGRPPPAADALQ SSGESSPESAPGEGAALSLSSPDAPDRPPPRHQDTGPCLEKTPAEGRDSPTREPGALPCACGQL PDLEDELSLLDPLPRYLKSCQDLIVPEQCRCADTRPGSEQPPLGGAASPEVLAPVSKEAADLAP TGPSPRPGPGLLLDVCTKDLADAPAEELQEKGSPAGPPPSQGQPAARPPKEVPASRLAQQLREE GWNLQTSESLTLAEVYLMMGKPSKLQLEYDWLGPGRQDPRPGSLPTALHKQRLLSCLLKLISTE VNPKLALEANTISTASVRPAQEEQSMTPPGKWTVSSRSPRCPRNQASLRSSKTFPPSSAPCSS GLRNPPRPLLVPGPSSTGSNDSDGGLFAVPTTLPPNSRHGKLFSPSKEAELTFRQHLNSISMQS DFFLPKPRKLRNRHLRKPLWQRTLLPRPSENQSHNVCSFSILSNSSVTGRGSFRPIQSSLTKA ALSRPIVPKVLPPQATSHLASAIDLAATSAGILSGNPLPALDTEGLSGISPLSSDEVTGAISGQ DSTGTHQDGDTLPTVGGSDPFVSIPSRPEQEPVADSFQGSSVLSLSELPKAPLQNGLSIPLSSS ESSSTRLSPPDVSALLDISLPGPPEDALSQGEPATHISDSIIEIAISSGQYGEGVPLSPAKLNG SDSSKSLPSPSSSPQPHWIASPTHDPQWYPSDSTDSSLSSLFASFISPEKSRKMLPTPIGTNSG TrSsLiLGPSLLDGNSRDSFVSRSLADVAEWDSQLVCMMNENSIDYISRFNDLAQELSIAEPGRRE ALFFDGGGGGPAVSDLSQ

Further analysis ofthe NOV6a protein yielded the following properties shown in Table 6B.

Table 6B. Protein Sequence Properties NOV6a

SignalP analysis: No Known Signal Sequence Predicted

PSORT II analysis: PSG: a new signal peptide prediction method

N- region: length 6; pos .chg 2; neg. chg 0 H-region: length 8 ; peak value 6.08 PSG score : 1 .68

GvH: von Heijne ' s method for signal seq. recognition GvH score (threshold: -2.1) : -7.17 possible cleavage site : between 41 and 42

>>> 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 = -2.87 Transmembrane 31

47

PERIPHERAL Likelihood = 2.60 (at 1107) ALOM score: -2.87 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 38 Charge difference: -3.0 C(-3.0) - N( 0.0) N >= C: N-terminal side will be inside

>>> membrane topology: type 2 (cytoplasmic tail 1 to 31)

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75): 3.60 Hyd Momen (95) : 12.59 G content: 8 D/E content: 2 S/T content: 3 Score: -6.80

Gavel : prediction of cleavage sites for mitochondrial preseq

R-2 motif at 16 GRG|VL

NUCDISC: discrimination of nuclear localization signals pat4: PRKR (4) at 99 pat4: KPRK (4) at 1030 pat7: PRKRFW (5) at 99 pat7: PKPRKLR (5) at 1029 pat7: PRKLRNR (5) at 1031 pat7: PEKSRKM (4) at 1328 bipartite: none content of basic residues: 10.8%

NLS Score: 1.67

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus : ARPG 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

Tyrosines in the tail : none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination

Prediction: nuclear

Reliability: 94.1

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

Final Results (k = 9/23):

47.8 %: nuclear

26.1 %: mitochondrial

8.7 %: cytoplasmic

4.3 %: extracellular, including cell wall

4.3 %: Golgi

4 .3 % : plasma membrane

4 .3 % : peroxisomal >> prediction for CG171174-01 is nuc (k=23 )

A search ofthe 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 6C.

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 6D.

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

Example 7.

The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.

Further analysis ofthe NOV7a protein yielded the following properties shown in Table 7B.

Table 7B. Protein Sequence Properties NOV7a

SignalP analysis: Cleavage site between residues 26 and 27

PSORT II analysis: PSG : a new signal peptide prediction method

N-region : length 3 ; pos . chg 0 ; neg . chg 1 H-region : length 31 ; peak value 0.00 PSG score : -4 .40

GvH : von Heijne ' s method for signal seq. recognition GvH score (threshold : -2.1) : 6.68 possible cleavage site : between 25 and 26

>>> 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 = -8 .92 Transmembrane 4

20

PERIPHERAL Likelihood = 1.43 (at 21) ALOM score: -8.92 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 11 Charge difference: 0.0 C( 0.0) - N( 0.0) N >= C: N-terminal side will be inside

>>> membrane topology: type 2 (cytoplasmic tail 1 to 4)

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 7.06 Hyd Moment (95): 6.67 G content: 6 D/E content : 2 S/T content : 4 Score: -8.50

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 54 IRN|VT

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite : none content of basic residues : 8.2%

NLS Score: -0.47

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

ER Membrane Retention Signals:

KKXX-like motif in the C-terminus: EKGK

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

Tyrosines in the tail: 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):

34.8 %: mitochondrial

30.4 %: cytoplasmic

8.7 %: Golgi

8.7 % : vacuolar

8.7 %: endoplasmic reticulum

4.3 %: extracellular, including cell wall

4.3 %: vesicles of secretory system

>> prediction for CG172318-01 is mit (k=23)

A search ofthe NOV7a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7C.

In a BLAST search of public sequence databases, the NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7D.

PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7E.

Table 7E. Domain Analysis of NOV7a

Identities/

Pfam Domain NOV7a Match Region Similarities Expect Value for the Matched Region

Example 8.

The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.

TCCTGGAACCTCAATTGTGAATTTAACTTGCACCACAAACACTACAGAAGACAATTATTCACGT TTAAGGTCATACCAAGTTTCCCTTCACTGCACCTGGCTTGTTGGCACAGATGCCCCTGAGGACA CGCAGTATTTTCTCTACTATAGGTATGGCTCTTGGACTGAAGAATGCCAAGAATACAGCAAAGA CACACTGGGGAGAAATATCGCATGCTGGTTTCCCAGGACTTTTATCCTCAGCAAAGGGCGTGAC TGGCTTGCGGTGCTTGTTAACGGCTCCAGCAAGCACTCTGCTATCAGGCCCTTTGATCAGCTGT TTGCCCTTCACGCCATTGATCAAATAAATCCTCCACTGAATGTCACAGCAGAGATTGAAGGAAC TCGTCTCTCTATCCAATGGGAGAAACCAGTGTCTGCTTTTCCAATCCATTGCTTTGATTATGAA GTAAAAATACACAATACAAGGAATGGATATTTGCAGATAGAAAAATTGATGACCAATGCATTCA TCTCAATAATTGATGATCTTTCTAAGTACGATGTTCAAGTGAGAGCAGCAGTGAGCTCCATGTG CAGAGAGGCAGGGCTCTGGAGTGAGTGGAGCCAACCTATTTATGTGGGAAATGATGAACACAAG CCCTTGAGAGAGTGGTTTGTCATTGTGATTATGGCAACCATCTGCTTCATCTTGTTAATTCTCT CGCTTATCTGTAAAATATGTCATTTATGGATCAAGTTGTTTCCACCAATTCCAGCACCAAAAAG TAATATCAAAGATCTCTTTGTAACCACTAACTATGAGGTCCTCTGCATTTTCATATACATCTTA GATTCGGCTGACϊΛTTTTCrA ΛAAAAAGAAAGCTGGGTCCAGTGAGACGGAAATTGAAGTCA TCTGTTATATAGAGAAGCCTGGAGTTGAGACCCTGGAGGATTCTGTGTTTTGACTGTCACTTTG GCATCCTCTGATGAACTCACACATGCCT

ORF Start: ATG at 14 ORF Stop: TGA at 1331

SEQ ID NO: 22 439 aa

NOV8a, MIIVAHVLLILLGATEILQADLLPDEKISLLPPVNFTIKVTGLAQALLQWKPNPDQEQRNVNLE CGI 72921-01 YQVKINAPKEDDYETRITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGTS IVNLTCTTNTTEDNYSRLRSYQVSLHCT LVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTLGR Protein Sequence NIACWFPRTFILSKGRDWLAλπiVNGSSKHSAIRPFDQLFALHAIDQINPPLNVTAEIEGTRLSI QWEKPVSAFPIHCFDYEVKIHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAG LWSEWSQPIYVGNDEHKPLREWFVIVIMATICFILLILSLICKICHLWIKLFPPIPAPKSNIKD LFVTTNYEVLCIFIYILDSADNFLQKKKAGSSETEIEVICYIEKPGVETLEDSVF

Further analysis ofthe NOV8a protein yielded the following properties shown in Table 8B.

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75): 3.37 Hyd Moment (95) : 3.35 G content: 1 D/E conten : 1 S/T content : 1 Score: -5.87

NUCDISC: discrimination of nuclear localization signals pat4 -. none pat7: none bipartite: none content of basic residues: 8.7% 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

Tyrosines in the tail: too long tail

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)

44.4 %: endoplasmic reticulum

22.2 %: Golgi

22.2 %: extracellular, including cell wall

A search ofthe NOV8a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 8C.

In a BLAST search of public sequence databases, the NOV8a protein was found to have homology to the proteins shown in the BLASTP data in Table 8D.

PFam analysis predicts that the NOV8a protein contains the domains shown in the Table 8E.

Table 8E. Domain Analysis of NOV8a

Identities/

Pfam Domain NOV8a Match Region Similarities Expect Value for the Matched Region

Example 9.

The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.

TGGAAACCAGAGGGACTTCTTCTACCAAGGTGTACACAGGCACGAGGAGTACTTACCATGTCTG CTTCTGCCCAAAAGACCCCAGGGTAGACAGCCCAAGACCGTGGCCATACAGGGAGCTCCTGGGA TCGGAAAAACAATCCTGGCCAAAAAGGTGATGTTTGAGTGGGCCAGAAACAAGTTCTACGCCCA CAAGCGCTGGTGTGCTTTCTACTTCCATTGCCAAGAGGTGAACCAGACGACAGACCAGAGCTTC TCCGAGCTGATTGAGCAAAAGTGGCCTGGATCTCAGGACCTCGTGTCAAAGATTATGTCCAAAC CCGACOΛCTTCTGCTGCTCTTGGATGGC TTGAGGAGCT'CΛCATCTACCCTCATTGACAGACT GGAGGACCΓGAGTGAAGACTGGAGGCAGAAATTGCCTGGGTCTGTCCTACTGAGCAGTTTGCTG AGCAAAACGATGCTTCCAGAGGCCACGCTACTGATCATGATAAGATTTACCTCTTGGCAGACAT GCAAGCCCTTGCTGAAATGTCCCTCTCTCGTAACCCTTCCGGGGTTTAATACGATGGAAAAAAT OΛGTATTTCCAGATGTATTTTGGA⁽^CACAGAGGAGGGAGACCAAGTCTTGAGTTTCGCCATG GAAAACACCATTCTCTTCTCCATGTGCCGGGTCCCTGTGGTTTGCTGGATGGTCTGCTCTGGTC TGAAACAGCAAATGGAGAGAGGAAACAATCTCACACAGTCATGTCCAAATGCCACCTCTGTGTT CGTCCGGTATATTTCTAGCTTGTTTCCCACCAGAGCTGAGAACTTTTCCAGAAAGATCCACCAA GCACAACTGGAAGGTCTGTGTCACTTGGCCGCAGACAGCATGTGGCACAGGAAATGGGTGTTAG GTAAAGAAGATCTTGAGGAAGCCAAGCTGGATCAGACGGGAGTCACCGCCTTCCTTGGCATGAG TATTCTTCGGAGAATTGCAGGTGAGGAAGACCACTATGTCTTTACCCTCGTGACTTTTCAGGAA TTTTTTGCGGCCTTGTTTTATGTTCTCTGTTTCCCAΑWΥVGACTCAAAAATTTTCATGTGTTGA GCCACGTGAATATCCAGCGCCTGATAGCGAGTCCCAGAGGAAGCAAAAGCTATCTCTCTCACAT GGGACTTTTCTTATTCGGTTTTCTGAACGAGGCCTGCGCTTCGGCCGTGGAACAGTCATTCCAA TGCAAGGTGTCTTTCGGTAATAAGAGGAAACTGCTGAAAGTCATACCTCTGTTGCATAAATGTG ACCCACCTTCTCCGGGCAGTGGGGTCCCGCAGTTATTCTACTGTCTGCATGAAATCCGGGAGGA AGCCTTTGTAAGCCAAGCCCTAAATGATTATCΛTAAAGTTGTCTTGAGAATTGGCAACAACAAA GAAGTTCAAGTGTCTGCTTTTTGCCTGAAGCGGTGTCAATATTTGCATGAGGTGGAACTGACCG TCACCCTGAACTTCATGAACGTGTGGAAGCTCAGCTCCAGCTCCCATCCTGGCTCTGACCTAAG GCGTGTGAATAGCACCATGTTGAACCAGGACTTAATCGGTGTTTTGACGGGGAACCAGCATCTG AGATACTTGGAAATACAACATGTGGAAGTGGAGTCCAAAGCTGTGAAGCTTCTATGCAGGGTGC TGAGATCCCCCCGGTGCCGTCTGCAGTGTCTCAGGTTGGAAGACTGCTTGGCCACCCCTAGAAT TTGGACTGATCTTGGCAATAATCTTCAAGGTAACGGGCATCTAAAGACTCTCATACTAAGAAAA AACTCCCTGGAGAACTGTGGGGCGTATTACCTGTCTGTGGCCCAGCTGGAGAGGCTGTCGCAGA GTAAGATGCTGACCCACCTGAGCTTGGCAGAAAACGCCTTGAAAGATGAAGGGGCCAAGCATAT TTGGAATGCCCTGCCACACCTGAGATGTCCTCTGCAGAGGCTGGTACTGAGAAAGTGTGACTTG ACCTTTAATTGCTGTCAGGATATGATCTCTGCGCTCTGTAAAAATAAAACCCTGAAAAGTCTTG ACCTAAGTTTTAATAGCCTGAAGGATGATGGGGTGATCCTGCTGTGTGAGGCCCTGAAGAACCC TGACTGTACATTACAGATCCTGGAGCTGGAAAACTGCCTGTTCACCTCCATCTGCTGCCAGGCC ATGGCTTCCATGCTCCGCAAAAACCAACATCTGAGACATCTGGACTTGAGCAAGAATGCGATTG GAGTCTATGGTATTCTGACCTTGTGCGAGGCCTTCTCAAGCCAAAAGAAGAGAGAAGAGGTCAT TTTCTGTATTCCTGCCTGGACTCGAATAACTAGCTTCTCCCCAACTCCTCACCCACCCGACTTC ACGGGAAAAAGTGACTGCCTATCCCAGATTAATCCTTAΒ

ORF Start: ATG at 1 ORF Stop: TAG at 2917

SEQ ID NO: 24 972 aa MW at ll0966.4kD

NOV9a, MSDVNPPSDTPIPFSSSSTHSSHIPPWTFSCYPGSPCENGVMLYMRNVSHEELQRFKQLLLTEL CGI 73919-01 STGTMPITWDQVETASWAEWHLLIERFPGRRAWDVTSNIFAIMNCDKMCVWRREINAILPTL EPEDLNVGETQVNLEEGESGKIRRYKSNVMEKFFPIWDITTWPGNQRDFFYQGVHRHEEYLPCL Protein Sequence LLPKRPQGRQPKTVAIQGAPGIGKTILAKKVMFE ARNKFYAHKR CAFYFHCQEV QTTDQSF SELIEQKWPGSQDLVSKIMSKPDQLLLLLDGFEELTSTLIDRLEDLSED RQKLPGSVLLSSLL SKTMLPEATLLIMIRFTS QTCKPLLKCPSLVTLPGFNTMEKIKYFQMYFGHTEEGDQVLSFAM ENTILFSMCRVPWCWMVCSGLKQQMERGNNLTQSCPNATSVFVRYISSLFPTRAENFSRKIHQ AQLEGLCHLAADSM HRKWVLGKEDLEEAKLDQTGVTAFLGMSILRRIAGEEDHYVFTLVTFQE FFAALFYVLCFPQRLKNFHVLSHVNIQRLIASPRGSKSYLSHMGLFLFGFLNEACASAVEQSFQ CKVSFGNKRKLLKVIPLLHKCDPPSPGSGVPQLFYCLHEIREEAFVSQALNDYHKWLRIGNNK EVQVSAFCLKRCQYLHEVELTVTLNFMNVWKLSSSSHPGSDLRRVNSTMLNQDLIGVLTGNQHL RYLEIQHVEVESKAVKLLCRVLRSPRCRLQCLRLEDCLATPRIWTDLGNNLQGNGHLKTLILRK NSLENCGAYYLSVAQLERLSQSKMLTHLSLAEJMALKDEGAKHIWNALPHLRCPLQRLVLRKCDL TFNCCQDMISALCKNKTLKSLDLSFNSLKDDGVILLCEALKNPDCTLQILELENCLFTSICCQA MASMLRKNQHLRHLDLSKNAIGVYGILTLCEAFSSQKKREEVIFCIPAWTRITSFSPTPHPPDF TGKSDCLSQINP

Further analysis ofthe NOV9a protein yielded the following properties shown in Table 9B. Table 9B. Protein Sequence Properties NOV9a

SignalP analysis: No Known Signal Sequence Predicted

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 9; pos.chg 0; neg.chg 2 H-region: length 28; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -6.94 possible cleavage site: between 30 and 31

>>> 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:

2

INTEGRAL Likelihood = -2.02 Transmembrane 387

403 INTEGRAL Likelihood = -2.13 Transmembrane 507 523

PERIPHERAL Likelihood = 0.90 (at 555) ALOM score: -2.13 (number of TMSs: 2)

MTOP: Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 394 Charge difference: 1.0 C( 1.0) - N( 0.0) C > N: C-terminal side will be inside

>>> membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 3.53 Hyd Momen (95): 4.97 G content: 0 D/E content : 2 S/T content : 2 Score: -6.93

NUCDISC: discrimination of nuclear localization signals pa 4 : none pat7: none bipartite: none content of basic residues: 10.9% 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: found KIMSKPDQL at 273

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

Tyrosines in the tail : 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):

22 2 % : vacuolar

22 2 %: nuclear

22 2 % : endoplasmic reticulum

11 1 %: Golgi

11 1 % : cytoplasmic

11 1 % : mitochondrial

>> prediction for CG173919-01 is vac (k=9)

A search ofthe NOV9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9C.

In a BLAST search of public sequence databases, the NOV9a protein was found to have homology to the proteins shown in the BLASTP data in Table 9D.

PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9E.

Example 10.

The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 10B.

Further analysis ofthe NOVlOa protein yielded the following properties shown in Table IOC.

Score: -3.26

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 40 LRG|AA

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: PRMRRPP (5) at 80 bipartite: none content of basic residues: 9.9% NLS Score: -0.04

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

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: 94.1

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

Final Results (k = 9/23):

43.5 %: nuclear

26.1 %: mitochondrial

8.7 %: Golgi

8.7 %: cytoplasmic

4.3 %: extracellular, including cell wall

4.3 %: plasma membrane

4.3 %: peroxisomal >> prediction for CG174858-02 is nuc (k=23 )

A search ofthe NOVlOa protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 10D.

In a BLAST search of public sequence databases, the NOVlOa protein was found to have homology to the proteins shown in the BLASTP data in Table 10E.

PFam analysis predicts that the NOVlOa protein contains the domains shown in the Table 10F.

Table 10F. Domain Analysis of NOVlOa

Identities/

Pfa Domain NOVlOa Match Region Similarities Expect Value for the Matched Region

Example 11.

The NOVl 1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11 A.

AATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGAT CTCCCAGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCT GCCGCCCCGCCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTC AATTATGGAGGAGGAAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTG ACCTO3TGTTCC03TGGTTTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTAT ACTGαυvσKWGGTGTTCAAAATTCAGATTGCrGGGGAAGATTGCΛTGGAGGAGTTTCCTGTTA ATCTGCATAGGAATTATCATTGTGAATCCCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGG TGOΪCΛTTCCTGGTGTGCTGCAGCGATTGGGAGTGACATACTTTGTGGTTAAGACTGTGTTGGA GCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAGCTGCCTTTCTCTTCGA GACATCΛCGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCTGTGGCTGGGCT TGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCATTGGAGA TTTCTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGGG AGACGATCACCTTTACCAGCACCCATCTTCTAAGCTGTACTTTACCACACCGAGGTGGCCTATG ACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGG

JAAAAATACTATTGTTCATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGT

GTTGTATTCTTGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCC lAGTAAACaAAAATCTCTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATC

CTGCTGGTCTGTACCAGTTGTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCA

GGAATGAATTCCATTCTGGTATACGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGT

GGAAGCTGAAGGACAACCAGTCCCACAAGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCT

CTGGGTGCTCATTGCCTACaTCCrCTATAGAAAGAAGATTTTTTGGAAAATCTGATGGCTCCCA

CTGAGATGTGCΓGCTGGAAGACTCTAGTAGGCCTGCAGGGAGGACTGAAGCAGCCTTTGTTAAA

IGGGAAGCATTCATTAGGAAATTGACTGGCTGCGTGTTTACAGACTCTGGGGGAAGACACTGATG

TCCTCAAACTGGTTAACTGTGACACGGCTCGCCAGAACTCTGCCTGTCTATTTGTGACTTACAG

ATTTGAAATGTAATTGTCTTTTTTCCTCCATCTTCTGTGGAAATGGATGTCTTTGGAACTTCAT

TCCGAGGAGATAAGCTTTAACTTTCCAAAAGGGAATTGCCATGGGTGTTTTTCTTCTGTGGTGA

GTGAAACAATCTGAGGTCTGGTTCTTGCTGACCTTGTTGCCCTGCAAACTTCCTTTCCACGTGT ftCGCGCACACCAACACGAAATGCCATCACTCCTACTGCGGCTGCTATGAAGCTTACTGGTTGTG

ATGTGTTATAATTTAGTCTGTTTTTTTGATTGAATGCAGTTTAATGTTTCCAGAAAGCCAAAGT

IAATTTTCTTTTCAGATATGCAAGGCTTTGGTGGGTCCAAAAAATGTCTATCACAAGCCATTTTT

TCCTTTTCCTCTCTCGAAAAGTTAAAATATCTATGTGTTATTCCCAAACCCTCTTACCTATGTA

TCTGCCTGTCTGTCCATCATCTTCCTTCCTCCCTATCTCTGTGTATCTGGATGGCAGCCGCTGC

CCAGGGGAGTGGCTGTGGGGAGGGCAGGTACTGTCTTTGCCTGTGGGTCCAGCTGAGCCATCCC

TGCTGGGTGATGCTGGGCAAGACCCTTGGCCCGTCTGGGCCTTGGCTTCCTCACTTGTGAAATG lAGCGGGAAGATGACTCTCAGTTCCTTCCACCTCTTAGACATGGTGAGGTAACAGACATCAAAAG

CTTTTCTGAAATCTTCAGAAGAAATAGTTCCATTACAGAAAACTCTTCAAAATAAATAGTAGTG

UUiAACTTTTAAAAACrCTCATTGGAGTAAGTCTTTTCAAGATGATCCTCCACAATGGAGGCAGC

GTTCCTACTTGTCATCACACAGCTGAAGACATTGTTTCTTAGGTGTGAAATCGGGGACAAAGGA

CAAACAGAGACACACGGCATTGTTCATGGGAGGCATCGTCACCCTCCTGGGTGTTCTGTGGGAA

ITTTCCTGTGTGAGGAAAACGTGGCCACAGGGTTGTGCTGTACCCACCCTTCCCCGGCGAGATGG

CCCTCGGCCTGTGCCGCTGCTTCCACCCTCGCCACTCCATGGCAGCTTTTGGTCTGTTTCCGGC

TCTGCCCTCTGCCCTGAACTCTCATCCGGCTTGTACCTGCCTGCTGGACCCCTCCACCTGGAGG

CCAGCCCATGTCTCAGGCCCAGCCCTAGCCTCTTCTCCTCAAATTCTAAGTGTTTTCTCTTTAG

GTTTCCCTGGCTTTGTGAATGGATCATGTGTCTCTAGGTATAAACCTGACATCATCTTTCCACC

CGGCTTACCTCCACCAGATCTCCCCAGTTCTGTCTCCATCTTCTACCTGCAGCTGCTCTGTTCT

CATGGTCACTGCTGCATCACTGAGTCTGGACCCTTGTTATCATTTTCAAACTGGCCTCCTTCCC

TCGTTCCCCACTTCTTAAAGTCACCTGTCCATTGCCACCAGATTAAGCTTTCTCCAGCCAGATC

ACCTCTCTCTGAGAAACCTCCATTGACATGGAAACACCATTGTCTGGCACACATACTCACATAC

TCACCTTCCCGTCTTGATCCCCACACATCTTTCCAGCCTCCCCTCCCACTCCACTCCCTGCTCC

CTCCTCCACCTCCCCATCCTCTTGTCTCCCCTCCCCTCT

ORF Start: ATG at 1 ORF Stop: TAA at 1375

SEQ ID NO: 30 458 aa MW at 50588.4kD

NOVl la, MGVGRALAALLLAASVLSAALLAPGGSSGRDAQAAPPRDLDKKRHAELKMDQALLLIHNELLWT CGI 76203-01 NLTVYWKSECCYHCLFQVLVNVPQSPKAGKPSAAAASVSTQHGSILQLNDTLEEKEVCRLEYRF GEFGNYSLLVKNIHNGVSEIACDLAVNEDPVDSNLPVSIAFLIGLAVIIVISFLRLLLSLDDFN Protein Sequence N ISKAISSRETDRLINSELGSPSRTDPLDGDVQPATWRLSALPPRLRSVDTFRGIALILMVFV NYGGGKYWYFKHAS NGLTVADLVFPWFVFIMGSSIFLSMTSILQRGCSKFRLLGKIAWRSFLL ICIGIIIVNPNYCLGPLSWDKVRIPGVLQRLGVTYFWKTVLELLFAKPVPEHCASERSCLSLR DITSS PQWLLILVI.EGLWLGLTFLLPVPGCPTGYLGPGGIGDFWQVSKLHWRSCRLHRPPAAG RRSPLPAPIF Further analysis ofthe NOVl la protein yielded the following properties shown in Table 11B.

Table HB. Protein Sequence Properties NOVlla

SignalP analysis: Cleavage site between residues 30 and 31

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 5; pcs.chg 1; neg.chg 0 H-region: length 24; peak value 9.20 PSG score: 4.80

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.28 possible cleavage site: between 18 and 19

>>> Seems to have a cleavable 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:

5

INTEGRAL Likelihood =-12. .31 Transmembrane 163

179

INTEGRAL Likelihood = -3. .35 Transmembrane 284

300

INTEGRAL Likelihood = -4, .73 Transmembrane 313

329

INTEGRAL Likelihood = -1. .06 Transmembrane 351

367

INTEGRAL Likelihood = -6, .85 Transmembrane 394

410

PERIPHERAL Likelihood = 0.95 (at 241) ALOM score: -12.31 (number of TMSs: 5)

MTOP: Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 9 Charge difference: 0.0 C( 2.0) - N( 2.0) N >= C: N-terminal side will be inside

>>> membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75): 8.77 Hyd Moment(95): 7.34 G content: 5 D E content: 1 S/T content: 4 Score: -3.83

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 15 GRA|LA

NUCDISC: discrimination of nuclear localization signals pat4: KKRH (3) at 42 pa 7 : none bipartite: none content of basic residues: 9.4% NLS Score: -0.29

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: GVGR 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 : MGVGRAL

Tyrosines in the tail : none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination

Prediction: cytoplasmic

Reliability: 94.1

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

Final Results (k = 9/23):

77.8 %: endoplasmic reticulum 22.2 % : mitochondrial

» prediction for CG176203-01 is end (k=9)

A search ofthe NOVl la protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 1 IC.

In a BLAST search of public sequence databases, the NOVl la protein was found to have homology to the proteins shown in the BLASTP data in Table 1 ID.

PFam analysis predicts that the NOVl la protein contains the domains shown in the Table HE.

Table HE. Domain Analysis of NOVlla

Identities/

Pfam Domain NOVlla Match Region Similarities Expect Value for the Matched Region

Example 12.

The NOVl 2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 12A.

Table 12A. NOV12 Sequence Analysis

SEQ ID NO: 31 1760 bp

NOVl 2a, GCGGCCGCGGGGGCCTTGCCTTCCGCACTCGGGCGCAGCCGGGTGGATCTCGAGCAGGTGCGGA CG176213-01 GCCCCGGGCGGCGGGCGCGGGTGCGAGGGATCCCTGACGCCTCTGTCCCTGTTTCTTTGTCGCT

CCCAGCCTGTCTGTCGTCGTTTTGGCGCCCCCGCCTCCCCGCGGTGCGGGGTTGCACACCGATC DNA Sequence CTGGGCTTCGCTCGATTTGCCGCCGAGGCGCCTCCCAGACCTAGAGGGGCGCTGGCCTGGAGCA

GCGGGTCGTCTGTGTCCTCTCTCCTCTGCGCCGCGCCCGGGGATCCGAAGGGTGCGGGGCTCTG lAGGAGGTGACGCGCGGGGCCTCCCGCaCCCTGGCCTTGCCCGCATTCTCCCTCTCTCCCAGGTG TGAGCAGCCTATCAGTCACCATGTCCGCAGCCTGGATCCCGGCTCTCGGCCTCGGTGTGTGTCT

GCTGCTGCTGCCGGGGCCCGCGGGCAGCGAGGGAGCCGGTAAACGACTAAAGAAAACACCCGAG AAGAAAACTGGCAATAAAGATTGTAAAGCAGACATTGCATTTCTGATTGATGGAAGCTTTAATA TTGGGCAGCGCCGATTTAATTTACAGAAGAATTTTGTTGGAAAAGTGGCTCTAATGTTGGGAAT TGGAACAGAAGGACCAOiTGTGGGCCTTGTTCAAGCCAGTGAACATCCCAAAATAGAATTTTAC TTGAAAAACTTTACATCAGCα^AAGATGTTTTGTTTGCCATAAAGGAAGTAGGTTTCAGAGGGG GTAATTCOATACΛGGAAAAGCCTTGAAGCATAerGCTCAGAAATTCTTCACGGTAGATGCTGG AGTAAGAAAAGGGATCCCCAAAGTGGTGGTGGTATTTATTGATGGTTGGCCTTCTGATGACATC GAGGAAGCAGG<^TTGTGGCCΛGAGAGTTTGGTGTCAATGTATTTATAGTTTCTGTGGCCAAGC CTATCCCTGAAGAACTGGGGATGGTTCAGGATGTCACATTTGTTGACAAGGCTGTCTGTCGGAA TAATGGCTTCTTCTCTTACCACATGCCCAACTGGTTTGGCACCACAAAATACGTAAAGCCTCTG GTACAGAAGCTGTGCACTCATGAACAAATGATGTGCAGCAAGACCTGTTATAACTCAGTGAACA TTGCCTTTCTAATTGATGGCTCCAGCAGTGTTGGAGATAGCAATTTCCGCCTCATGCTTGAATT TGTTTCCAACΛTAGCα^AGACTTTTGAAATCTCGGACATTGGTGCCAAGATAGCTGCTGTACAG TTTACTTATGATCΛGCGCΛCGGAGTTCAGTTTCACTGACTATAGCACCAAAGAGAATGTCCTAG CTGTCATCAGAAACATCCGCTATATGAGTGGTGGAACAGCTACTGGTGATGCCATTTCCTTCAC TGTTAGAAATGTGTTTGGCCCTATAAGGGAGAGCCCCAACAAGAACTTCCTAGTAATTGTCACA GATGGGCAGTCCTATGATGATGTCCAAGGCCCTGCAGCTGCTGCACATGATGCAGGAATCACTA TCH CT'CTGTTGGTGTGGCTTGGGCACCTCTGGATGACCTGAAAGATATGGCTTCTAAACCGAA GGAGTCTCΛTGCTTTCTTCACAAGAGAGTTCACAGGATTAGAACCAATTGTTTCTGATGTCATC AGAGGCATTTGTAGAGATTTCI AGAATCCCAGaATAATGGTAACATTTTGACAACTGAAAGA AAAAGTACAAGGGGATCCAGTGTGTAAATTGT

ORF Start: ATG at 405 lORF Stop: TAA at 1701

SEQ ID NO: 32 432 I aa |MW at 47177.7kD

NOV12a, MSAAWIPALGLGVCLLLLPGPAGSEGAGKRLKKTPEKKTGNKDCKADIAFLIDGSFNIGQRRFN CGI 76213-01 LQKNFVGKVALMLGIGTEGPHVGLVQASEHPKIEFYLKNFTSAKDVLFAIKEVGFRGGNSNTGK ALKHTAQKFFTVDAGVRKGIPKWWFIDGWPSDDIEEAGIVAREFGVNVFIVSVAKPIPEELG Protein Sequence MVQDVTFVDKAVCRNNGFFSYHMPNWFGTTKYVKPLVQKLCTHEQMMCSKTCYNSVNIAFLIDG SSSVGDSNFRLMLEFVSNIAKTFEISDIGAKIAAVQFTYDQRTEFSFTDYSTKENVLAVIRNIR YMSGGTATGDAISFTVRNVFGPIRESPNKNFLVIVTDGQSYDDVQGPAAAAHDAGITIFSVGVA WAPLDDLKDMASKPKESHAFFTREFTGLEPIVSDVIRGICRDFLESQQ

Further analysis ofthe NOV 12a protein yielded the following properties shown in Table 12B.

C > N: C-terminal side will be inside

>>>Caution: Inconsistent mtop result with signal peptide MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 1.80 Hyd Moment (95) : 2.02 G content: 4 D/E content: 1 S/T content: 2 Score: -7.37

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 10.9% 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

Tyrosines in the tail : none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination

Prediction: cytoplasmic

Reliability: 94.1

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

Final Results (k = 9/23)

A search ofthe NOV 12a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 12C.

In a BLAST search of public sequence databases, the NOV 12a protein was found to have homology to the proteins shown in the BLASTP data in Table 12D.

PFam analysis predicts that the NOV 12a protein contains the domains shown in the Table 12E.

Example 13.

The NOV 13 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 13A.

Table 13A. NOV13 Sequence Analysis

SEQ ID NO: 33 2689 bp

NOVl 3a, CACCAAGCTTCTβGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGCCCG CG50691-05 GGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGAGCT GCGGCGGCACCTTCGGGATTTACGGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCCCCC DNA Sequence GCTCAATGGCX^CTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACTGAT GACCΑACTGCTTGGTTTTAAAC(aTGα^TGAVAACCTTATTGCTGGCTGCAATATAATCAATG GGAAATGTGAATGTAAΑCCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGATΛT GTGCCTTTCAGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGTGAA GTCCAGTTCTCTCCACGTTGTCCTGAAGATTCT'GTTCTGATCGAGGGTTATGCTCCTCCTGGGG AGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCA GCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTC TATGAGTGCΛAACCAGTTTTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGCAGA CCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTACTTT GCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCCACT CCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTA ATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCGAAT GGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGT GAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATCCAG TGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCG GTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACC GTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCGAAG AACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGACAGG GAAGGACTΌCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAAC ACΣ»GGAACTATGTT<^GAACGTAAAΑΥVGGCTGCACCTTGAACTGTCCCTTCGGTTTCCTTA CTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCATAAT CTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGT AAGAAATGTCCΛGAGCTCTCATGCAGTAAGATCTGCCCCTΓGGGTTTCCAGCAGGACAGTCACG GCTGTCTTATCTGCAAGTGCAGAGAGGCCRCRRGCTTCAGCTGGGCCACCCATCCTGTCGGGCAC TTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGGGAA TGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTGGCA ACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCC AGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAAACG TGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGG TGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTAC AGATCAACCTTTTCGGCCTTCCTTGTCCCGCAATAACAGCGTACCTAATTACTGCAAAAATGAT GAAGGGGATATATTCCTGGCAGCTGAGTCCTGGAAGCCTGACGTTTGTACCAGCTGCATCTGCA TTGATAGCGTAATRAGCTGTTTCTCTGAGTCCTGCCCTTCTGTATCCTGTGAAAGACCTGTCTT GAGAAAAGGCCAGTGTTGTCCCTACTGCATAGAAGACACAATTCCAAAGAAGGTGGTGTGCCAC TTCΛGTGGGAAGGCCTATGCCGACGAGGAGX^TGGGACCTTGACAGCTGCACCCACTGCTACT GCCTGCAGGGCCAGACCCTCTGCTCGACCGTCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCAT CAACGTGGAAGGAAGTTGCTGCCCAATGTGTCCAGAAATGTATGTCCCAGAACCAACCAATATA CCCATTGAGAAGACAAACCATCGAGGAGAGGTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTA GTGAAAATGATATCGTCCATCTCCCTAGAGATATGGGTCACCTCCAGGTAGATTACAGACTCGA

G

ORF Start: at 11 ORF Stop: at 2684

SEQ ID NO: 34 891 aa MW at 97577.0kD

NOVl 3a, LVCLPCDESKCEEPRNCPGSIVQGVCGCCYTCASQRNESCGGTFGIYGTCDRGLRCVIRPPLNG CG50691-05 DSLTEYEAGVCEDEN TDDQLLGFKPCNENLIAGCNIINGKCECNTIRTCSNPFEFPSQDMCLS ALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVCQPGN Protein Sequence LNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCTLPTR CECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFRMDNC RFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGDRWRE DDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLTGKDC INGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPIICDK YCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSHGCLICKCREASASAGPPILSGTCLT VDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFWQKPELS TPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQCTDQP FRPSLSRNNSVPNYCKNDEGDIFLAAESWKPDVCTSCICIDSVISCFSESCPSVSCERPVLRKG QCCPYCIEDTIPKKWCHFSGKAYADEERWDLDSCTHCYCLQGQTLCSTVSCPPLPCVEPINVE GSCCPMCPEMYVPEPTNIPIEKTNHRGEVDLEVPLWPTPSENDIVHLPRDMGHLQVDYR

SEQ ID NO: 35 5379 bp

NOVl 3b, GGCCCGGCTGCGAGGAGGAGGCGGCGGCGGCGCAGGAGGATGTACTTGGTGGCGGGGGACAGGG CG50691-04 GGTTGGCCGGCTGCGGGCACCTCCTGGTCTCGCTGCTGGGGCTGCTGCTGCTGCTGGCGCGCTC CGGCACCCGGGCGCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGC DNA Sequence CCGGGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGA GCTGCGGCGGCACCTTCGGGATTTACGGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCC CCCGCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACT GATGACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCaATATAATCA ATGGGAAATGTGAATGTAACACCATTQ3AACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGA TATGTGCCTTTCAGCΓTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAA GCCCGCTGT GAAGTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTG GGGAGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTG CCAGCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGAC CTCTATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGC AGACCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTAC TTTGCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCC A<^CCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTG TTAATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCG AATGGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGT GGTGAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATC CΛGTGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGA CCGGTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCG ACCGTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCG AAGAACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGAC AGGGAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATA AACACCGAGGAACTATGTTCAGAACGTAAACAAGGCTGCACCTTGAACTGTCCCTTCGGTTTCC TTACTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCAT AATC GTGACAAGTATTGTCCΛCTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGC TGTAAGAAATGTCCΛGAGCTCΓCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTC ACGGCTGTCTTATCTGCΛAGTGCAGAGAGGCCTCRGCRTCΛGCTGGGCCACCCATCCTGTCCKSG CACTTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGG GAATGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTG GCAACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAA GCCAGAGCTCAGTACTCCCTCCATΓTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAA A∞TGGAACΛTTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAG AGGTGTGCCCACCGCIΏCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTG TACAGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTATGCCGACGAG GAGCGGTGGGACCTTGACAGCTGCACCCACTGCTACTGCCTGCAGGGCCAGACCCTCTGCTCGA CCGTCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTGCTGCCCAAT GTGTCCAGAAATGTATGTCCCAGAACCAACCAATATACCCATTGAGAAGACAAACCATCGAGGA GAGGTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTAGTGAAAATGATATCGTCCATCTCCCTA GAGATATGGGTCACCTCCAGGTAGATTACAGAGATAACAGGCTGCACCCAAGTGAAGATTCTTC ACTGGACTCCATTGCCTCAGTTGTGGTTCCCATAATTATATGCCTCTCTATTATAATAGCATTC CTATTCATCAATCAGAAGAAACAGTGGATACCACTGCTTTGCTGGTATCGAACACCAACTAAGC CTTCTTCCTTAAATAATCAGCTAGTATCTGTGGACTGCAAGAAAGGAACCAGAGTCCAGGTGGA CAGTTCCCAGAGAATGCTAAGAATTGCAGAACCAGATGCAAGATTCAGTGGCTTCTACAGCATG CAAAAACAGAACCATCTACAGGCAGACAATTTCTACCAAACAGTGTGAAGAAAGGCAACTAGGA TGAGGTTTCAAAAGACGGAAGACGACTAAATCTGCTCTAAAAAGTAAACTAGAATTTGTGCACT

TGCTTAGTGGATTGTATTGGATTGTGACTTGATGTACAGCGCTAAGACCTTACTGGGATGGGCT

CTGTCTACAGCAATGTGCAGAACAAGCATTCCCACTTTTCCTCAAGATAACTGACCAAGTGTTT

TCTTAGAACCAAAGTTTTTAAAGTTGCTAAGATATATTTGCCTGTAAGATAGCTGTAGAGATAT

TTGGGGTGGGGACAGTGAGTTTGGATGGGGAAATGGGTGGGAGGGTGGTGTTGGGAAGAAAAAT

TGGTCAGCTTGGCTCGGGGAGAAACCTGGTAACATAAAAGCAGTTCAGTGGCCCAGAGGTTATT

TTTTTCCTATTGCTCTGAAGACTGCACTGGTTGCTGCAAAGCTCAGGCCTGAATGAGCAGGAAA

CAAAAAAGGCCTTGCGACCCAGCTGCCATAACCACCTTAGAACTACCAGACGAGCACATCAGAA

CCCTTTGACAGCCATCCCAGGTCTAAAGCCACAAGTTTCTTTTCTATACAGTCACAACTGCAGT

AGGCAGTGAGGAAGCCAGAGAAATGCGATAGCGGCATTTCTCTAAAGCGGGTTATTAAGGATAT

ATACAGTTACACTTTTTGCTGCTTTTATTTTCTTCCAAGCCAATCAATCAGCCAGTTCCTAGCA

GAGTCAGCACATGAACAAGATCTAAGTCATTTCTTGATGTGAGCACTGGAGCTTTTTTTTTTTT

IACAACGTGACAGGAAGAGGAGGGAGAGGGTGACGAACACCAGGCATTTCCAGGGGCTATATTTC

ΛCTGTTTGTTGTTGCTTTGTTCTGTTATATTGTTGGTTGTTCATAGTTTTTGTTGAAGCTCTAG

CTTAAGAAGAAACTTTTTTTAAAAAGACTGTTTGGGGATTCTTTTTCCTTATTATATACTGATT

CTAα^AAATAGAAACTACTTCATTTTAATTGTATATTATTCAAGCACCTTTGTTGAAGCTCAAA

ΛAAAATGATGCCTCTTTAAACTTTAGCAATTATAGGAGTATTTATGTAACTATCTTATGCTTCA

LAAAAACAAAAGTATTTGTGTGCATGTGTATATAATATATATATATACATATATATTTATACACA

TACAATTTATGTTTTCCTGTTGAATGTATTTTTATGAGATTTTAACCAGAACAAAGGCAGATAA

AC^GGCΛTTCCATAGCΛGTGCTTTTGATCΛCTTACΛAATTTTTTGAATAACACAAAATCTCATT

CTACCTGCAGTTTAATTGGAAAGATGTGTGTGTGAGAGTATGTATGTGTGTGTGTGTGTGTGTG

TGTGTGCGCGCGCACGCACGCCTTGAGCAGTCAGCATTGCACCTGCTATGGAGAAGGGTATTCC

TTTATTAAAATCTTCCTCATTTGGATTTGCTTTCAGTTGGTTTTCAATTTGCTCACTGGCCAGA piAACAATGGTTTGAAACAACTACTGGAATATTGTCCACAATAAGCTGGAAGTTTGTTGTAGTAT

GCCTCAAATATAACTGaCTGTATACTATAGTGGTAACTTTTCAAACAGCCCTTAGCACTTTTAT

JACTAATTAACCCATTTGTGCATTGAGTTTTCTTTTAAAAATGCTTGTTGTGAAAGACACAGATA

CTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGT AAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCACG GCTGTCTTATCTGCΛAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCAC TTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGGGAA TGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTGGCA ACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCC AGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAAACG TGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGG TGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTAC AGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTATGCCGACGAGGAG CGGTGGGACCTTGACAGCTGCACCCACTGCTACTGCCTGCAGGGCCAGACCCTCTGCTCGACCG TCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTGCTGCCCAATGTG TCCAGAAATGTATGTCCCAGAACCAACCAATATACCCATTGAGAAGACAAACCATCGAGGAGAG GTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTAGTGAAAATGATATCGTCCATCTCCCTAGAG ATATGGGTCACCTCCAGGTAGATTACAGACTCGAGGGC

ORF Start: at 2 ORF Stop: end of sequence

SEQ ID NO: 42 823 aa MW at 90023.6kD

NOV13e, TKLLVCLPCDESKCEEPRNCPGSIVQGVCGCCYTCASQRNESCGGTFGIYGTCDRGLRCVIRPP 308482339 Protein LNGDSLTEYEAGVCEDENWTDDQLLGFKPCNENLIAGCNIINGKCECNTIRTCSNPFEFPSQDM CLSALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVCQ Sequence PGNLNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCTL PTRCECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFRM DNCRFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGDR WREDDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLTG KDCINGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPII CDKYCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSHGCLICKCREASASAGPPILSGT CLTVDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFWQKP ELSTPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQCT EDTIPKKWCHFSGKAYADEERWDLDSCTHCYCLQGQTLCSTVSCPPLPCVEPINVEGSCCPMC PEMYVPEPTNIPIEKTNHRGEVDLEVPLWPTPSENDIVHLPRDMGHLQVDYRLEG

SEQ ID NO: 43 3361 bp

NOV13f, ftAAGAGAGTCTCACCCTGTTTCCCAGACCGGAATGCAGTGGCGTGATCAACCTCGTGGGCTCAA

CG50691-01 DNA IGTGATCCTCCCACCTCAAACTCCTGAGTGCTGGGACCACAGGCATGCACAACCATTCCCAGCTA

ΛTTTTTTGTTTTGTTTTTGTAGAGACTGGGTCTCACTGTGTTGCCCAGGCTGGTCATGAACTCC Sequence TGGGCTCAAGTAATCCCCGTGCCTTGGTCTCTGAAAGTGTTGGGATTACAGGCATGAGCCACTG

ITGCCTGGCCAAAAAAGAGCTCTTTAAAAAATAATTTTGTAGATTGACAAATGTGACTCTTGTAA

TTTTATTGAACATGAAAAAACCCAGGAATCTTTATTTGATATTAAACATTTTTAAAGGCATCTC

ΛGTTGTTGTTGTAATAACACATTAAGAGAAGTAGTGGTTTTTTATTTCCAACCTTTGTGCATAT lAGCTATTTAATGCCTACATGGATGGCTATTATTTCACTTTTTTCAGTTATTATGAAGAGATTGG

GTTTCATTCΛTTTGTAAAGTTTCAGC(^GACTGCCTTTCACAAATTGATTTGTCAAAATTGAAT

GTTAATCTTGACATCCCAGTGCGTTTTTGCCCGCGAACAGGCCTTTGAATGAAGCTGCAAACAC lACATTATCTGGTTGTTAATTGTTTTACAGATGAGAACTGGACTGATGACCAACTGCTTGGTTTT

ΛAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCAATGGGAAATGTGAATGTAACA

Cf^TTCGAACCTGCAGOΛTCCCTTTGAGTTTCCAAGTCAGGATATGTGCCTTTCAGCTTTAAA

GAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGTGAAGTCCAGTTCTCTCCACGT TGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTGGGGAGTGCTGTCCCTTACCCA GCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCAGCCGGGAAACCTGAACAT ACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTCTATGAGTGCAAACCAGTT TTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGCAGACCGCGTGTCCCCCGGACA GCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTACTTTGCCAACAAGATGCGAGTG TCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGαATCCACTCCCCGCATAGTCTCTCGT GGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTAATGATACAAAGCCAGCCT GCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCGAATGGACAACTGTCGGTTCTG TCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGTGAGATAAACTGCGAGAGG TACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATCCAGTGTATCCTTTTAATAATC CCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCGGTGGCGGGAAGACGACTG CACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACCGTCTGCGGACAGACCTGC ACΛAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCGAAGAACCAACCATCATCACAG TTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGACAGGGAAGGACTGCATTAATGG TT OiAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAACACCGAGGAACTATGTTCA GAACGTAAACAAGGCTGC^CCTTGAACTGTCCCTTCGGTTTCCTTACTGATGCCCAΛAACTGTG AGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCATAATCTGTGACAAGTATTGTCC ACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGTAAGAAATGTCCAGAGCTC TCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCGCGGCTGTCTTATCTGCAAGT GCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCACTTGTCTCACCGTGGATGG T(^TCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGGGAATGCTACTGTCTCAATGGA CGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTGGCAACCCCACCATTCACCCTG GACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCCAGAGCTCAGTACTCCCTC CATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAAACGTGGAACATTGACTCCTGT ACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGGTGTGCCCACCGCTGCTCT GCCΛGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTACAGATCAACCTTTTCGGCC TTCCTTGTCCCGOiATAACAGCGTACCTAATTACTGCAAAAATGATGAAGGGGATATATTCCTG GCAGCTGAGTCCTGGAAGCCTGACGTTTGTACCAGCTGCATCTGCATTGATAGCGTAATTAGCT GTTTCTCTGAGTCCTGCCCTTCTGTATCCTGTGAAAGACCTGTCTTGAGAAAAGGCCAGTGTTG TCCCTACTGCATAGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTAT GCCGACGAGGAGCGGTGGGACCTTGACAGCTGCACCCACTACTACTGCCTGCAGGGCCAGACCC TCTGCTCGACCGTCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTG CTGCCCAATGTGTCCAGTTTCACCTTTACCATCTTTGGATATGAGTACAGAACCTATGAGCTGT TAGGTGATTAGCACCTGTCTCTTTACAGAAGAAACTGAGGCTCAGGAAAGAGCCCCTGTGGGAA GAGGACTCACTGTCATGCCTCAGCTTGGTGGAGTTTCACCGGAAATCTACCCATATGCAGGGTC

AAGGCAAAAGAATTCCAAAGTTACGTCTCTCCCTCTCACTCAGGAAAAAACCTGAGGTGGAACT

GAATCAATCCCΑGCTCTGGGGCCTCTGCΑGAAACTTTTACTACTTAGCCATTGACATTTACAGT

ATAATACCTATCTGATCAAACTGGATAATGTAAATATATTTACTGAAGATCAGCTTCTAATCTA

AATGGTTCCAGTGGTAACATAATGGACATCTGA

ORF Start: ATG at 813 |ORF Stop: TAG at 3009

SEQ ID NO: 44 |732 aa JMW at 79910.4kD

NOV13f, MCLSALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVC CG50691-01 QPGNLNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCT LPTRCECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFR Protein Sequence MDNCRFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGD RWREDDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLT GKDCINGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPI ICDKYCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSRGCLICKCREASASAGPPILSG TCLTVDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFWQK PELSTPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQC TDQPFRPSLSRNNSVPNYCKNDEGDIFLAAESWKPDVCTSCICIDSVISCFSESCPSVSCERPV LRKGQCCPYCIEDTIPKKWCHFSGKAYADEERWDLDSCTHYYCLQGQTLCSTVSCPPLPCVEP INVEGSCCPMCPVSPLPSLDMSTEPMSC

SEQ ID NO: 45 2470 bp

NOV13g, CACCAAGCTTCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGCCCG CG50691-06 DNA GGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGAGCT GCGGCGGCACCTTCGGGATTTACGGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCCCCC Sequence GCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACTGAT GACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCAATG GGAAATGTGAATGTAACΛCCATT SAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGATAT GTGCCTTTCGGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGTGAA GTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTGGGG AGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCA GCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTC TATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGCAGA CCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTACTTT GCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCCACT CCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTA ATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCGAAT GGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGT GAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATCCAG TGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCG GTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACC GTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCGAAG AACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGACAGG GAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAAC ACCGAGGAACTATGTTCAGAACGTAAACAAGGCTGCACCTTGAACTGTCCCTTCGGTTTCCTTA CTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCATAAT CTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGT AAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCACG GCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCAC

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 13B.

Further analysis ofthe NOV 13a protein yielded the following properties shown in Table 13C. Table 13C. Protein Sequence Properties NOV13a

SignalP analysis: No Known Signal Sequence Predicted

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 10; pos.chg 1; neg.chg 2 H-region: length 1; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -6.37 possible cleavage site: between 33 and 34

>>> Seems to have no N-terminal signal peptide

0 number of TMS(s) .. fixed

PERIPHERAL Likelihood = 1.64 (at 736)

ALOM score: 1.64 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 6.88 Hyd Moment (95): 6.86 G content: 0 D/E content: 2 S/T content: 0 Score: -6.85

NUCDISC: discrimination of nuclear localization signals pat4: RPKK (4) at 501 pat7: PRPKKCR (5) at 500 pat7: PKKCRPI (5) at 502 bipartite: none content of basic residues : 8.6% NLS Score: 0.64

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

A search ofthe NOVl 3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 13D.

In a BLAST search of public sequence databases, the NOV 13a protein was found to have homology to the proteins shown in the BLASTP data in Table 13E.

PFam analysis predicts that the NOVl 3a protein contains the domains shown in the Table 13F.

Example 14.

The NOV 14 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 14A.

Table 14A. NOV14 Sequence Analysis

SEQ ID NO: 47 |499 bp

NOV14a, TATGGAATAAAGAACCATGACGGAGTCCCATGCGCAGCCAGAGAAGAGACCACCACCCGAGAGA CG51905-03 GGTTTCATCCTACCATGTAACTCTGCTTACAGCCTACTTGCTTCTCACCGGCGTGCTGGGGACA GCAAAGTCTGAGGACTCTGGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAA DNA Sequence GGCCTCTGCACAGCTCΓAGAAGCΓTCAATCCCATTTCCACCCATACATCTCΓTTGTGCTCTCAC ACCCCCACAGCCCTTCTGGAATAAGACCATCACAGCACAGGGTTTGCAAGATGTCTAATGCCAG TCATTCACAGGGCAGCTCAGACCCTGGCCTGCGGTGCATACTAGGTGACTCCACATGAGGTGTC

ATGCTAGATCCTGCAGGGAGAATAAGCACACACAGGCCCGTGACCCATGCTGTGGACTTCATGT

TCTAGGAGGTAGAGGGAGACAGACAAGAATCAAATGACTGTACTAGGCCGG

ORF Start: ATG at 30 [ORF Stop: TAA at 312

SEQ ED NO: 48 94 aa MW at 10354.6kD

NOV14a, MRSQRRDHHPREVSSYHVTLLTAYLLLTGVLGTAKSEDSGWCGPVCKESSGHGIRPLHSSRSFN CG51905-03 PISTHTSLCALTPPQPF NKTITAQGLQDV Protein Sequence

SEQ IDNO: 49 |579bp

NOV14b, TATGGAATAAAGAACCATGACGGAGTCCCATGCGCAGCCAGAGAAGAGACCACCACCCGAGAGA CG51905-01 GGTTTCATCCTACCATGTAACTCTGCTTACAGCCTACTTGCTTCTCACCGGCGTGCTGGGGACA GCΛAAGTCTGAGGACTCTGGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAA DNA Sequence GGCCTCTGCACAGCTCTAGAAGCTTCAATCCCATTTCCACCCATACATCTCTTTGTGCTCTCAC ACCCCCACAGCCCTTCTGGAATAAGACCATCACAGCACAGGGTTTGCAAGATGTCTAATGCCAG TCATTCACAGGGCAGCTCAGACCCTGGCCTGCGGTGCATACTAGGTGACTCCACATGAGGTGTC

ATGCTAGATCCTGCAGGGAGAATAAGCACACACAGGCCCGTGACCCATGCTGTGGACTTCATGT

TCTAGGAGGTAGAGGGAGACAGACAAGAATCAAATGACTGfACTAGGCCGGGCGCACTGGCTCA

CGCCTGTAATCCCAGCACTTTGGGGAGGCCGAGGCAGGTGGATCACGAGGCCAGGCGTTCGAGA

CCA

ORF Start: ATG at 30 ORF Stop: TAA at 312

SEQ ID NO: 50 94 aa MW at 10354.6kD

NOV14b, MRSQRRDHHPREVSSYHVTLLTAYLLLTGVLGTAKSEDSGWCGPVCKESSGHGIRPLHSSRSFN CG51905-01 PISTHTSLCALTPPQPFNKTITAQGLQDV Protein Sequence

SEQ ID NO: 51 j!93 bp

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 14B.

Further analysis of ihe NOV14a protein yielded the following properties shown in Table 14C.

Table 14C. Protein Sequence Properties NOV14a

SignalP analysis: Cleavage site between residues 37 and 38

PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 11 ; pos .chg 4 ; neg . chg 1 H- region: length 0 ; peak value -2.25 PSG score : -6.65

GvH: von Heijne ' s method for signal seq. recognition GvH score (threshold: -2.1) : -0.05 possible cleavage site : between 32 and 33

>>> 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: 0

PERIPHERAL Likelihood = 12.57 (at 62)

ALOM score: -1.59 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 4 Hyd Moment (75): 8.79 Hyd Momen (95): 8.84 G content: 0 D/E content : 2 S/T content : 1 Score: -2.40

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 15 QRR|DH

NUCDISC: discrimination of nuclear localization signals pat : none pat7 : none bipartite: none content of basic residues: 9.6% NLS Score: -0.47

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: RSQR 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

A search ofthe NOV 14a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 14D.

In a BLAST search of public sequence databases, the NOV 14a protein was found to have homology to the proteins shown in the BLASTP data in Table 14E.

PFam analysis predicts that the NOV 14a protein contains the domains shown in the Table 14F.

Table 14F. Domain Analysis of NOV14a

Identities/

Pfam Domain NOV14a Match Region Similarities Expect Value for the Matched Region

Example 15.

The NOV 15 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 15 A.

CATCCAGCAGGAGAACTTCTGGGTTGGCCCCAGCTCGATTGACCTGATCCACCTGGGGGCCAA GTTCTCACCCTGCATCCGGAAGGACGGGCAGATCGAGCAGCTGGTGCTGCGCGAGCGAGACCTG GAGCGGGACTCAGGCTGCTGTGTCCAGAATGACCACTCCGGATGCATCCAGACCCAGCGGAAGG ACTGCTCGGAGACTTTGGCCACTTTTGTCAAGTGGCAGGATGACACTGGGCCCCCCATGGACAA GTCTGATCΓGGGCCAGAAGCGGACTTCGGGGGCTGTCTGCCACCAGGACCCCAGGACCTGCGAG GAGCCAGCCTCCAGCGGTGCCCACATCTGGCCCGATGACATCACTAAGTGGCCGATCTGCACAG

AGCAGGCCAGGAGCAACCACACAGGCTTCCTGCACATGGACTGCGAGATCAAGGGCCGCCCCTG

CTGCATCGGCACCAAGGGCAGCTGTGAGATCACCACCCGGGAATACTGTGAGTTCATGCACGGC TATTTCCATGAGGAAGCAACACTCTGCTCCCAGGTGCACTGCTTGGACAAGGTGTGTGGGCTGC TGCCCTTCCTCAACCCΓGAGGTCC^GATCAGTTCTACAGGCΓCTGGCTGTCTCTCTTCCTACA TGCTGGCGTGGTGCACTGCCTCGTGTCTGTGGTCTTTCAAATGACCATCCTGAGGGACCTGGAG AAGCTGGCCGGCTGGCACCGTATCGCCATCATCTTCATCCTCAGTGGCATCACAGGCAACCTCG CCAGTGCCATCTTTCTCCCATACCGGGCAGAGGTGGGCCCGGCCGGCTCACAGTTCGGCCTCCT CGCCTGCCTCTTCGTGGAGCTCTTCCAGAGCTGGCCGCTGCTGGAGAGGCCCTGGAAGGCCTTC CTCAACCTCTCGGCCATCGTGCTCTTCCTGTTCATCTGTGGCCTCCTGCCCTGGATCGACAACA TCGCCCACATCTTCGGCTTCCTCAGTGGCCTGCTGCTGGCCTTCGCCTTCCTGCCCTACATCAC CTTCGGCACCAGCGACAAGTACCGCAAGCGGGCACTCATCCTGGTGTCACTGCTGGCCTTTGCC GGCCTCTTCGCCGCCCTCGTGCTGTGGCTGTACATCTACCCCATTAACTGGCCCTGGATCGAGC ACCTCACCTGCTTCCCCTTCACCAGCCGCTTCTGCGAGAAGTATGAGCTGGACCAGGTGCTGCA CCTCGAGGGCAAGGGTTTAA

ORF Start: ATG at 17 |ORF Stop: at 2498

SEQ ID NO: 58 827 aa MW at 93378.2kD

NOVl 5a, MASADKNGGSVSSVSSSRLQSRKPPNLSITIPPPEKETQAPGEQDSMLPERKNPAYLKSVSLQE CG52414-03 PRSRWQESSEKRPGFRRQASLSQSIRKGAAQWFGVSGDWEGQRQQ QRRSLHHCSMRYGRLKAS CQRDLELPSQEAPSFQGTESPKPCKMPKIVDPLARGRAFRHPEEMDRPHAPHPPLTPGVLSLTS Protein Sequence FTSVRSGYSHLPRRKRMSVAHMSLQAAAALLKGRSVLDATGQRCRλ/VKRSFAFPSFLEEDWDG ADTFDSSFFSKEEMSSMPDDVFESPPLSASYFRGIPHSASPVSPDGVQIPLKEYGRAPVPGPRR GKRIASKVKHFAFDRKKRHYGLGVVGNVπjNRSYRRSISSTVQRQLESFDSHRPYFTY LTFVHV IITLLVICTYGIAPVGFAQHVTTQLVLRNKGVYESVKYIQQENFWVGPSSIDLIHLGAKFSPCI RKDGQIEQLVLRERDLERDSGCCVQNDHSGCIQTQRKDCSETLATFVKWQDDTGPPMDKSDLGQ KRTSGAVCHQDPRTCEEPASSGAHIWPDDITKWPICTEQARSNHTGFLHMDCEIKGRPCCIGTK GSCEITTREYCEFMHGYFHEEATLCSQVHCLDKVCGLLPFLNPEVPDQFYRLWLSLFLHAGWH CLVSWFQMTILRDLEKLAGWHRIAIIFILSGITGNLASAIFLPYRAEVGPAGSQFGLLACLFV ELFQSWPLLERPWKAFLNLSAIVLFLFICGLLPWIDNIAHIFGFLSGLLLAFAFLPYITFGTSD KYRKRALILVSLLAFAGLFAALVLWLYIYPIN PWIEHLTCFPFTSRFCEKYELDQVLH

SEQ ID NO: 59 |2596 bp

NOVl 5b, TCAATTGACTTGATATGATTTATTATTTTTACTACTTATAAGAATGGAAATAAGTTCTCCTTAG CG52414-01 TTTTTTTCTTGGAGAAAGTCTGACATGTGAGGCACAGATGAGTTATTAAAGGCAGATGACTTTC

CAGCCTTGTCTTAAATGTTCCATTCTTTACCTTAGAAATTATTTAAATTTGTGTCCTGTCCCAG DNA Sequence AGCATCCGCAAGGGCGCAGCCCAGTGGTTTGGAGTCAGCGGCGACTGGGAGGGGCAGCGGCAGC

AGTGGCAGCGCCGCAGCCTGCACCACTGCAGCATGCGCTACGGCCGCCTGAAGGCCTCGTGCCA

GCGTGACCTGGAGCTCCCCAGCCAGGAGGCACCGTCCTTCCAGGGCACTGAGTCCCCAAAGCCC TGCAAGATGCCCAAGATTGTGGATCCGCTGGCCCGGGGCCGGGCCTTCCGCCACCCGGAGGAGA TGGACAGGCCCCACGCCCTGCACCCACCGCTGACCCCCGGAGTCCTGTCCCTCACCTCCTTCAC CAGTGTCCGTTCTGGCTACTCCCACCTGCCACGCCGCAAGAGAATGTCTGTGGCCCACATGAGC TTGCAAGCTGCCGCTGCCCTCCTCAAGGGGCGCTCGGTGCTGGATGCCACCGGACAGCGGTGCC GGGTGGTCAAGCGCAGCTTTGCCTTCCCGAGCTTCCTGGAGGAGGATGTGGTCGATGGGGCAGA CACGTTTGACTCCTCCTTTTTTAGTAAGGAAGAAATGAGCTCCATGCCTGATGATGTCTTTGAG TCCCCCCCACTCTCTGCCAGCTACTTCCGAGGGATCCCACACTCAGCCTCCCCTGTCTCCCCCG ATGGGGTGCAAATCCCTCTGAAGGAGTATGGCCGAGCCCCAGTCCCCGGGCCCCGGCGCGGCAA GCGCATCGCCTCCAAGGTGAAGCACTTTGCCTTTGATCGGAAGAAGCGGCACTACGGCCTCGGC GTGGTGGGCAACTGGCTGAACCGCAGCTACCGCCGCAGCATCAGCAGCACTGTGCAGCGGCAGC TGGAGAGCTTCGACAGCCACCGGCCCTACTTCACCTACTGGCTGACCTTCGTCCATGTCATCAT CACGCTGCTGGTGATTTGCACGTATGGCATCGCACCCGTGGGCTTTGCCCAGCACGTCACCACC CAGCTGGTGCTGCGGAACAAAGGTGTGTACGAGAGCGTGAAGTACATCCAGCAGGAGAACTTCT GGGTTGGCCCCAGCTCGATTGACCTGATCCACCTGGGGGCCAAGTTCTCACCCTGCATCCGGAA GGACGGGCΛGATCGAGCAGCTGGTGCTGCGCGAGCGAGACCTGGAGCGGGACTCAGGCTGCTGT GTCCAGAATGACCACTCCGGCTGCATCCAGACCCAGCGGAAGGACTGCTCGGAGACTTTGGCCA CTTTTGTCAAGTGGCAGGATGACACTGGGCCCCCCATGGACAAGTCTGATCTGGGCCAGAAGCG GACTTCGGGGGCTGTCTGCCACCAGGACCCCAGGACCTGCGAGGAGCCAGCCTCCAGCGGTGCC CACATCTGGCCCGATGACATCACTAAGTGGCCGATCTGCACAGAGCAGGCCAGGAGCAACCACA CAGGCΓTCCTGCACATGGACTGCGAGATCAAGGGCCGCCCCTGCTGCATCGGCACCAAGGGCAG CTGTGAGATCACCACCCGGGAATACTGTGAGTTCATGCACGGCTATTTCCATGAGGAAGCAACA

CTGCCCTTCCTCAACCCTGAGGTCCCAGATCAGTTCTACAGGCTCTGGCTGTCTCTCTTCCTAC ATGCTGGGGTGGTGCACTGCCTCGTGTCTGTGGTCTTTCAAATGACCATCCTGAGGGACCTGGA

GAAGCTGGCCGGCTGGCACCGTATCGCCATCATCTTCATCCTCAGTGGCATCACAGGCAACCTC GCCAGTGCCATCTTTCTCCCATACCGGGCAGAGGTAGGCCCGGCCGGCTCACAGTTCGGCCTCC TCGCCΓGCCTCTTCGTGGAGCTCTTCCAGAGCTGGCCGCTGCTGGAGAGGCCCTGGAAGGCCTT CCTCΛACCTCTCGGCCATCGTGCTCTTCCTGTTCATCTGTGGCCTCCTGCCCTGGATCGACAAC ATCGCCCACATCTTCGGCTTCCTCΛGTGGCCTGCΓGCTGGCCTTCGCCTTCCTGCCCTACATCA CCTTCGGCACCAGCGACAAGTACCGCAAGCGGGCACTCATCCTGGTGTCACTGCTGGCCTTTGC CGGCCTCΓTCGCCGCCCTCGTGCTGTGGCTGTACATCTACCCCATTAACTGGCCCTGGATCGAG CΛCCTCACCTGCTTCCCCTTCACCAGCCGCTTCTGCGAGAAGTATGAGCTGGACCAGGTGCTGC ACTGACCGCTGGGCCACACGGCTGCCCCTCAGCCCTGCTGGAACAGGGTCTGCCTGCGAGGGCT GCCCTCTGCAGAGCGCTCTCTGTGTGCCAGAGAGCCAGAGACCCAAGACAGGGCCCGGGCTCTG

GACCTGGGTGCCCCCCTGCCAGGCGAGGCTGACTCCGCGTGAGATAGATGGTTGGTTAAGGCGG GGTTTTTCCGGGCCGCGCCCCCCCCCTCTAAA

ORF Start: ATG at 338 |ORF Stop: TGA at 2819

SEQ IDNO: 62 |827 aa JMW at 93378.2kD

NOVl5c, MASADKNGGSVSSVSSSRLQSRKPPNLSITIPPPEKETQAPGEQDSMLPERKNPAYLKSVSLQE CG52414-02 PRSRWQESSEKRPGFRRQASLSQSIRKGAAQ FGVSGD EGQRQQWQRRSLHHCSMRYGRLKAS CQRDLELPSQEAPSFQGTESPKPCKMPKIVDPLARGRAFRHPEEMDRPHAPHPPLTPGVLSLTS Protein Sequence FTSVRSGYSHLPRRKRMSVAHMSLQAAAALLKGRSVLDATGQRCRWKRSFAFPSFLEEDWDG ADTFDSSFFSKEEMSSMPDDVFESPPLSASYFRGIPHSASPVSPDGVQIPLKEYGRAPVPGPRR GKRIASKVKHFAFDRKKRHYGLGWGNWLNRSYRRSISSTVQRQLESFDSHRPYFTYWLTFVHV IITLLVICTYGIAPVGFAQHVTTQLVLRNKGVYESVKYIQQENFWVGPSSIDLIHLGAKFSPCI RKDGQIEQLVLRERDLERDSGCCVQNDHSGCIQTQRKDCSETLATFVKWQDDTGPPMDKSDLGQ KRTSGAVCHQDPRTCEEPASSGAHI PDDITKWPICTEQARSNHTGFLHMDCEIKGRPCCIGTK GSCEITTREYCEFMHGYFHEEATLCSQVHCLDKVCGLLPFLNPEVPDQFYRLWLSLFLHAGWH CLVSWFQMTILRDLEKLAGWHRIAIIFILSGITGNLASAIFLPYRAEVGPAGSQFGLLACLFV ELFQSWPLLERP KAFLNLSAIVLFLFICGLLPWIDNIAHIFGFLSGLLLAFAFLPYITFGTSD KYRKRALILVSLLAFAGLFAALVL LYIYPINWPWIEHLTCFPFTSRFCEKYELDQVLH

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 15B.

Further analysis ofthe NOVl 5a protein yielded the following properties shown in Table 15C.

GvH score (threshold: -2.1): -7.64 possible cleavage site: between 21 and 22

>>> Seems to have no N-terminal signal peptide

INTEGRAL Likelihood = -6. .42 Transmembrane 381

397

INTEGRAL Likelihood = -4. .25 Transmembrane 630

646

INTEGRAL Likelihood = -3. .08 Transmembrane 666

682

INTEGRAL Likelihood = 0. .37 Transmembrane 697

713

INTEGRAL Likelihood = -9. .08 Transmembrane 720

736

INTEGRAL Likelihood = -4. .83 Transmembrane 742

758

INTEGRAL Likelihood =-10. .83 Transmembrane 775

791

PERIPHERAL Likelihood = 5.25 (at 600) ALOM score: -10.83 (number of TMSs: 7)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 388 Charge difference: 0.5 C( 1.5) - N( 1.0) C > N: C-terminal side will be inside

>>>Caution: Inconsistent mtop result with signal peptide >>> membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75): 6.30 Hyd Moment (95): 5.24 G content: 2 D/E content: 2 S/T content: 10 Score: -3.23

Gavel: prediction of cleavage sites for mitochondrial preseq

R-2 motif at 32 SRK|PP

NUCDISC: discrimination of nuclear localization signals pat4: PRRK (4) at 204 pat4: RRKR (5) at 205 pat4: RKKR (5) at 335 pat4: KKRH (3) at 336 pat7: PRRKRMS (5) at 204 pat7: PGPRRGK (3) at 316 pat7: PRRGKRI (5) at 318 bipartite: KRIASKVKHFAFDRKKR at 322 content of basic residues: 11.6% NLS Score: 2.37

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

Tyrosines in the tail: 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):

55.6 %: endoplasmic reticulum

11.1 %: vacuolar

11.1 %: mitochondrial

11.1 %: vesicles of secretory system

11.1 %: Golgi

>> prediction for CG52414-03 is end (k=9)

A search ofthe NOVl 5a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 15D.

In a BLAST search of public sequence databases, the NOV 15a protein was found to have homology to the proteins shown in the BLASTP data in Table 15E.

PFam analysis predicts that the NOVl 5a protein contains the domains shown in the Table 15F.

Example 16.

The NOV 16 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 16A.

Table 16A. NOVl 6 Sequence Analysis

SEQ ID NO: 63 1778 bp

NOVl 6a, GAGGCCAGGAGCGCTCCGTCTGGAACGGCGCAGGTCCCAACAGCTGGGGTTCCCCCTCAGCCCG CG52552-06 TGAGCAGCCATGTCCAACCCCAACGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAG

GCCCTCTGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCC DNA Sequence TGGCTACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCACCCAC CCGATGCCCATGAACTACGGCCCAGGCCATGGCTATGATGGGGAGGAGAGAGCGGTGAGTGATA GCTTCGGGCCTGGAGAGTGGGATGACCGGAAAGTGCGACACACTTTTATCCGAAAGGTTTACTC <^TCATCTCCGTGCΛGCTGCT'CATCACTGTGGC(^TCATTGCTATCTTCACCTTTGTGGAACCT GTCAGCGCCTTTGTGAGGAGAAATGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCGTTGTCA CCTACCTGATCCTTGCCTGCTGCCAGGGACCCAGACGCCGTTTCCCATGGAACATCATTCTGCT GACCCTTTTTACTTTTGCCATGGGCTTCATGACGGGCACCATTTCCAGTATGTACCAAACCAAA GCCGTCATCATTGCAATGATCATCACTGCGGTGGTATCCATTTCAGTCACCATCTTCTGCTTTC AGACCAAGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTCCTGGT GACTGGGATTGTCΛCTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCTCCACATGCTCTAT GCTGCTCTGGGGGCΛTTTGTTTCACCCTGTTCCTGGCTTACAACACACAGCTGGTCCTGGGGA ACCGGAAGCACACCATCAGCCCCGAGGACTACATCACTGGCGCCCTGCAGATTTACACAGACAT CATCTACATCTTCACCTTTGTGCTGCAGCTGATGGGGGATCGCAATTAAGGAGCAAGCCCCCAT TTTCACCCGATCCTGGGCTCTCCCTTCCAAGCTAGAGGGCTGGGCCCTATGACTGTGGTCTGGG

CTTTAGGCCCCTTTCCTTCCCCTTGAGTAACATGCCCAGTTTCCTTTCTGTCCTGGAGACAGGT

GGCCTCTCTGGCTATGGATGTGTGGGTACTTGGTGGGGCACGGAGGAGCTAGGGACTAACTGTT

GCTCTTGGTGGGCTTGGCAGGGACTAGGCTGAA ATGTGTCTTCTCCCCGCCACCTACTGTATG

ACACCACATTCTTCCTAACAGCTGGGGTTGTGAGGAATATGAAAAGAGCCTATTCGATAGCTAG

ΛAGGGAATATGAAAGGTAGAAGTGACTTCAAGGTCACGAGGTTCCCCTCCCACCTCTGTCACAG

GCTTCTTGACTACGTAGTTGGAGCTATTTCTTCCCCCAGCAAAGCCAGAGAGCTTTGTCCCCGG

CCTCCTGGACACATAGGCCATTATCCTGTATTCCTTTGGCTTGGCATCTTTTAGCTCAGGAAGG

TAGAAGAGATCTGTGCCCATGGGTC CCTTGCTTCAATCCCTTCTTGTTTCAGTGACATATGTA

TTGTTTATCTGGGTTAGGGATGGGGGACAGATAATAGAACGAGCAAAGTAACCTATACAGGCCA

GCATGGAACAGCATCTCCCCTGGGCTTGCTCCTGGCTTGTGACGCTATAAGACAGAGCAGGCCA

CATGTGGCCATCTGCTCCCCATTCTTGAAAGCTGCTGGGGCCTCCTTGCA

ORF Start: ATG at 74 ORF Stop: TAA at 1007

SEQ ID NO: 64 311 aa MW at 34649.2kD

NOVl 6a, MSNPNAPPPYEDRNPLYPGPLPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP CG52552-06 MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSA FVRRNVAVYYVSYAVFWTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVI Protein Sequence IAMIITAWSISVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAAL GAICFTLFLAYNTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTΓVLQLMGDRN

SEQ ID NO: 65 908 bp

NOVl6b, CGGCCATQTCCAACCCCAGCGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAGGCCC CG52552-04 TCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCCTGGC TACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCCATGGCTATG DNA Sequence ATGGGGAGGAGAGAGCAGTGAGTGATAGCTTCGGGCCTGGAGAGTGGGATGACCGGAAAGTGCG ACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCGTGCAGCTGCTCATCACTGTGGCCATC ATTGCΓATCTTCACCTTTGTGGAACCTGTCAGCGCCTTTGTGAGGAGAAATGTGGCTGTCTACT ACGTGTCCTATGCTGTCTTCGTTGTCACCTACCTGATCCTTGCCTGCTGCCAGGGACCCAGACG CCGTTTCC<^TGGAACATCATTC GCTGACCCTTTTTACΓTTTGCCATGGGCTTCATGACGGCC ACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCATTGCAATGATCATCACTGCGGTGGTAT CCATTTCΆGTCACCATCTTCTGCTTTCAGACCAAGGTGGACTTCACCTCGTGCACAGGCCTCTT CTGTGTCCTGGGAATTGTGCTCCTGGTGACTGGGATTGTCACTAGCATTGTGCTCTACTTCCAA TACGTTTACTGGCRCCAΩTGCTCRATGCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGG CTTACGACACACAGCTGGTCCTGGGGAACCGGAAGCACACCATCAGCCCCGAGGACTACATCAC TGGCGCCCTGCAGATTTACACAGACATCATCTACATCTTCACCTTTGTGCTGCAGCTGATGGGG GATCGCAATTAA

ORF Start: ATG at 6 ORF Stop: TAA at 906

SEQ ID NO: 66 300 aa MW at 33423.7kD

NOVl 6b, MSNPSAPPPYEDRNPLYPGPPPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPHGYDG CG52552-04 EERAVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSAFVRRNVAVYYV SYAVFWTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVIIAMIITAWSI Protein Sequence SVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAALGAICFTLFLAY DTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN

SEQ ID NO: 67 1767 bp

NOV16c, AACGGCGCAGGTCCCAGCAGCTGGGGTTCCCCCTCAGCCCGTGAGCAGCCATGTCCAACCCCAG CG52552-01 CGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAGGCCCTCTGCCCCCTGGGGGCTAT GGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCCTGGCTACCCGCAGCCTGGCTACG DNA Sequence GTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCACCCACCCGATGCCCATGAACTACGGCCC AGGCCATGGCTATGATGGGGAGGAGAGAGCGGTGAGTGATAGCTTCGGGCCTGGAGAATGGGAT GACCGGAAAGTGCGACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCGGGCAGCTGCTCA TCACTGGGGCCATCΛTTGCTATCTTCACCTTTGGGGAACCTGTCAGCGCCTTTGGCAGGAGAAA TGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCAGTGTCACCTACCTGATCCTTGCCTGCTGC CAGGGACCCAGACGCCGTTTCCCATGGAACATCATTCTGCTGACCCTTTTTACTTTTGCCATGG GCTTCATGACGGGCACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCATTGCAATGATCAT CACTGCGGTGGTATCCATTTCAGTCACCATCTTCTGCTTTCAGACCAAGGTGGACTTCACCTCG TGCACΛGGCC^CTTCΓGTGTCCTGGGAATTGTGCTCCTGGTGACTGGGATTGTCACTAGCATTG TGCTCTACTTCCAATACGTTTACTGGCTCCACATGCTCTATGCTGCTCTGGGGGCCATTTGTTT CΛCCCTGTTCCTGGCTTACGACACACAGCTGGTCCTGGGGAACCGGAAGCACACCATCAGCCCC GAGGACTACΛTCΛCTGGCGCCCTGCAGATTTACACAGACATCATCTACATCTTCACCTTTGTGC TGCAGCTGATGGGGGATCGCAATTAAGGAGCAAGCCCCCATTTTCACCCGATCCTGGGCTCTCC CTTCCAAGCTAGAGGGCTGGGCCCTATGACTGTGGTCTGGGCTTTAGGCCCCTTTCCTTCCCCT

TGAGTAACATGCCCAGTTTCCTTTCTGTCCTGGAGACAGGTGGCCTCTCTGGCTATGGATGTGT

GGGTACTTGGTGGGGACGGAGGAGCTAGGGACTAACTGTTGCTCTTGGTGGGCTTGGCAGGGAC

TAGGCTGAAGATGTGTCTTCTCCCCGCCACCTACTGTATGACACCACATTCTTCCTAACAGCTG

GGGTTGTGAGGAATATGAAAAGAGCCTATTCGATAGCTAGAAGGGAATATGAAAGGTAGAAGTG

ACTTCAAGGTCΛ SAGGTTCCCCrCCCACCTCTGTCACAGGCTTCTTGACTACGTAGTTGGAGC

TATTTCTTCCCCCAGCAAAGCCAGAGAGCTTTGTCCCCGGCCTCCTGGACACATAGGCCATTAT

CCΓGTATTCCTTTGGCTTGGCATCTTTTAGCTCAGGAAGGTAGAAGAGATCTGTGCCCATGGGT

CTCCTTGCTTCAATCCCTTCTTGTTTCAGTGACATATGTATTGTTTATCTGGGTTAGGGATGGG

IGGACAGATAATAGAACGAGCAAAGTAACCTATACAGGCCAGCATGGAACAGCATCTCCCCTGGG

CTTGCTCCTGGCTTGTGACGCTATAAGACAGAGCAGGCCACATGTGGCCATCTGCTCCCCATTC

TTGAAAGCTGCTGGGGCCTCCTTGCAGGCTTCTGGATCC

ORF Start: ATG at 51 ORF Stop: TAA at 984

SEQ IDNO: 68 311 aa MWat34442.7kD

NOVl6c, MSNPSAPPPYEDRNPLYPGPLPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP CG52552-01 MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFIRKVYS11SGQLLITGAIIAIFTFGEPVSA FGRRNVAVYYVSYAVFSVTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVI Protein Sequence IAMIITAVVSISVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAAL GAICFTLFLAYDTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN

SEQ IDNO: 69 2059 bp

NOV16d, CGCTCCGTCTGGAACGGCGCAGGTCCCAGCAGCTGGGGTTCCCCCTCAGCCCGTGAGCAGCCAT CG52552-02 GTCCAACCCCAGCGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAGGCCCTCTGCCC CCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCCTGGCTACCCGC DNA Sequence AGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCACCCACCCGATGCCCAT GAACTACGGCCCAGGCCATGGCTATGATGGGGAGGAGAGAGCGGTGAGTGATAGCTTCGGGCCT GGAGAGTGGGATGACCGGAAAGTGCGACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCG TGCAGCTGCTCATCACTGTGGCCATCATTGCTATCTTCACCTTTGTGGAACCTGTCAGCGCCTT TGTGAGGAGAAATGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCGTTGTCACCTACCTGATC CTTGCCTGCTGCCAGGGACCCΛGACGCCGTTTCCCATGGAACATCATTCTGCTGACCCTTTTTA CTTTTGCCATGGGCTTCATGACGGGCACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCAT TGCAATGATCATCACTGCGGTGGTATCCATTTCAGTCACCATCTTCTGCTTTCAGACCAAGGTG AGGGCATGGAGGGCCCTTCCCTGGCCCCCCGACTCCCCTTTCTTATCAGGCCCGGACCCCGGTA CACTAGGGATGTTCCCTAGAGACCTGATCCCCTTCTCCTCATCCGCACCTACAAAACTGTGTCC TGTTTCTGTCCTTAGAATGTTGTGGACATTCCCATACCCCCTAGGAGGCAGCACTGGGACTCCC TGGCΛGGGCCAGTCTGACTGGGCTGGTTGTCACAGCCATCTGACAGGTGCCTCTTTCTTGCTTC CTGGCΛGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTCCTGGTG ACTGGGATTGTCACΓAGCΆTTGTGCTCTTAGCATTGTGCΓCTACTTCCAATACGTTTACTGGCT C<^(^TGCTCTATGCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGGCTTACGACACACAG

CTGGTCCTGGGGAACCGGAAGCACACCATCAGCCCCGAGGACTACATCACTGGCGCCCTGCAGA

TTTACACAGACATCATCTACATCTTCACCTTTGTGCTGCAGCTGATGGGGGATCGCAATTAAGG

AGCAAGCCCCCATTTTCACCCGATCCTGGGCTCTCCCTTCCAAGCTAGAGGGCTGGGCCCTATG

ACTGTGGTCTGGGCTTTAGGCCCCTTTCCTTCCCCTTGAGTAACATGCCCAGTTTCCTTTCTGT

CCTGGAGACAGGTGGCCTCTCTGGCTATGGATGTGTGGGTACTTGGTGGGGACGGAGGAGCTAG

GGACTAACTGTTGCTCTTGGTGGGCTTGGCAGGGACTAGGCTGAAGATGTGTCTTCTCCCCGCC

ACCTACTGTATGACACCACATTCTTCCTAACAGCTGGGGTTGTGAGGAATATGAAAAGAGCCTA

TTCGATAGCTAGAAGGGAATATGAAAGGTAGAAGTGACTTCAAGGTCACGAGGTTCCCCTCCCA

CCTCΓGTCACΆGGCTTCTTGACTACGTAGTTGGAGCTATTTCTTCCCCCAGCAAAGCCAGAGAG

CTTTGTCCCCGGCCΓCCTGGACACATAGGCCATTATCCTGTATTCCTTTGGCTTGGCATCTTTT AGCTCAGGAAGGTAGAAGAGATCTGTGCCCATGGGTCTCCTTGCTTCAATCCCTTCTTGTTTCA GTGACATATGTATTGTTTATCTGGGTTAGGGATGGGGGACAGATAATAGAACGAGCAAAGTAAC CTATACAGGCCAGCATGGAACAGCATCTCCCCTGGGCTTGCTCCTGGCTTGTGACGCTATAAGA (^GAGCAGGCCA<ATGTGGCCATCTGCTCCCCATTCTTGAAAGCTGCTGGGGCCTCCTTGCAGG CTTCTGGATCC

ORF Start: ATG at 63 ORF Stop: TGA at 1023

SEQ ID NO: 70 320 aa MW at 35204.3kD

NOV16d, MSNPSAPPPYEDRNPLYPGPLPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP CG52552-02 MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFI RKVYS I ISVQLLITVAI IAI FTFVEPVSA FVRRNVAVYYVSYAVFWTYLILACCQGPRRRFP NIILLTLFTFAMGFMTGTISSMYQTKAVI Protein Sequence IAMIITAWSISVTIFCFQTKVRAWRALPWPPDSPFLSGPDPGTLGMFPRDLIPFSSSAPTKLC PVSVLRMLWTFPYPLGGSTGTPWQGQSDWAGCHSHLTGASFLLPGRWTSPRAQASSVSWELCSW

SEQ ID NO: 71 2437 bp

NOV16e, ATGCCAGCCCCAAACCTCATCCCTAGTGGAGGCCTTGCTGATGTGGAAGTGGCCAGGGCCCTCA CG52552-03 TGGTAGGCTGGGCAGAAGCCCAAGAACAGGCTCTAAAGCTGCTAAACCCGGCAGTCCTGGTCCC

CGGAGGCTCTTGCCAGTCTGACAGTGTTCTTGGCACTGCTCAAAGGTCCCAGCAGCTGGGGTTC DNA Sequence CCCGTCAGCCCGTGAGCGGCCATGTCCAACCCCAGCGCCCCACCACCATATGAAGACCGCAACC

CCCTGTACCCAGGCCCTCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTA TCCTGCCTACCCTGGCTACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATG CCCCCCACCCACCCGATGCCCATGAACTACGGCCCAGGCCATGGCTATGATGGGGAGGAGAGAG CGGTGAGTGATAGCTTCGGGCCTGGAGAGTGGGATGAC03GAAAGTGCGACΛCACTTTTATCCG AAAGGTTTACTCCATCATCTCCGTGCAGCTGCTCATCACTGTGGCCATCATTGCTATCTTCACC TTTGTGGAACCTGTCΛGO.CCTTTGTGAGGAGAAATGTGGCTGTCTACTACGTGTCCTATGCTG TCTTCGTTGTCACCTACCTGATCCTTGCCTGCTGCCAGGGACCCAGACGCCGTTTCCCATGGAA CATCΛTTCTGCTGACCCTTTTTACTTTTGCCATGGGCRT⁽^TGACGGGCACΑ^V.TTTCCAGTATG TACCAAACCAAAGCCGTCATCATTGCAATGATCATCACTGCGGTGGTATCCATTTCAGTCACCA TCTTCTGCTTTCAGACCAAGGTGGACTTC^CCRRCGTGCACAGGCCTCTTCTGTGTCCTGGGAAT TGTGCTCCTGGTGACΓGGGATTGT^CTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCTC CACΛTGCTCTATGCTGCTCΓGGGGGCCΛTTTGTTTCACCCTGTTCCTGGCTTACGACACACAGC TGGTCCTGGGGAACCGGAAGCACACCATCAGCCCCGAGGACTACATCACTGGCGCCCTGCAGAT TTACACAGACΑT(ATCTACATCTT(^CCTTTGTGCTGCAGCTGATGGGGGATCGCAATTAAGGA GCAAGCCCCCATTTTCACCCGATCCTGGGCTCTCCCTTCCAAGCTAGAGGGCTGGGCCCTATGA

CTGTGGTCTGGGCTTTAGGCCCCTTTCCTTCCCCTTGAGTAACATGCCCAGTTTCCTTTCTGTC

CTGGAGACAGGTGGCCTCTCTGGCTATGGATGTGTGGGTACTTGGTGGGGACGGAGGAGCTAGG

'GACTAACTGTTGCTCTTGGTGGGCTTGGCAGGGACTAGGCTGAAGATGTGTCTTCTCCCCGCCA

CCTACTGTATGACACCACATTCTTCCTAACAGCTGGGGTTGTGAGGAATATGAAAAGAGCCTAT

TCGATAGCTAGAAGGGAATATGAAAGGTAGAAGTGACTTCAAGGTCACGAGGTTCCCCTCCCAC

CTCTGTCACAGGCTTCTTGACTACGTAGTTGGAGCTATTTCTTCCCCCAGCAAAGCCAGAGAGC

TTTGTCCCCGGCCTCCTGGACACATAGGCCATTATCCTGTATTCCTTTGGCTTGGCATCTTTTA

GCTCAGGAAGGTAGAAGAGATCTGTGCCCATGGGTCTCCTTGCTTCAATCCCTTCTTGTTTCAG

TGACATATGTATTGTTTATCTGGGTTAGGGATGGGGGACAGATAATAGAACGAGCAAAGTAACC

TATACAGGCCAGCATGGAACAGCATCTCCCCTGGGCTTGCTCCTGGCTTGTGACGCTATAAGAC

AGAGCAGGCCACATGTGGCCATCTGCTCCCCATTCTTGAAAGCTGCTGGGGCCTCCTTGCAGGC

TTCTGGATCTCTGGTCAGAGTGAACTCTTGCTTCCTGTATTCAGGCAGCTCAGAGCAGAAAGTA AGGGGCAGAGTCATACGTGTGGCCAGGAAGTAGCCAGGGTGAAGAGAGACTCGGTGCGGGCAGG

GAGAATGCCTGGGGGTCCCTCACCTGGCTAGGGAGATACCGAAGCCTACTGTGGTACTGAAGAC

TTCTGGGTTCTTTCCTTCTGCTAACCCAGGGAGGGTCCTAAGAGGAAGGTGACTTCTCTCTGTT

TGTCTTAAGTTGCACTGGGGGATTTCTGACTTGAGGCCCATCTCTCCAGCCAGCCACTGCCTTC

TTTGTAATATTAAGTGCCTTGAGCTGGAATGGGGAAGGGGGACAAGGGTCAGTCTGTCGGGTGG

GGGCAGAAATCAAATCAGCCCAAGGATATAGTTAGGATTAATTACTTAATAGAGAAATCCTAAC

TATATCACACAAAGGGATACAACTATAAATGTAATAAAATTTATGTCTAGAAGTTAAAAAAAAA lAAAAA

ORF Start: ATG at 214 JORF Stop: TAA at 1147

SEQ ID NO: 72 311 aa MW at 34607. lkD

NOV16e, MSNPSAPPPYEDRNPLYPGPPPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP CG52552-03 MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFI RKVYS I I SVQLLI TVAI IAI FTFVEPVSA FVRRNVAVYYVSYAVFWTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVI Protein Sequence IAMIITAWSISVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAAL GAICFTLFLAYDTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN

SEQ ID NO: 73 719 bp

NOVlδf, CAGCTGGGGTTCCCCGTCAGCCCGTGAGCGGCCATGTCCAACCCCAGCGCCCCACCACCATATG CG52552-05 AAGACCGCAACCCCCTGTACCCAGGCCCTCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCT GCCAGGAGGGTATCCTGCCTACCCTGGCTACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTAC DNA Sequence CCACAGCCCATGCCCCCCACCCACCCGATGCCCATGAACTACGGCCCAGGCCATGGCTATGATG

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 16B.

Further analysis ofthe NOVlόa protein yielded the following properties shown in Table 16C.

ALOM: Klein et al ' s method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.

6

INTEGRAL Likelihood = -8.92 Transmembrane 105

121 INTEGRAL Likelihood = -4.25 Transmembrane 138

154 INTEGRAL Likelihood = -2.07 Transmembrane 165

181 INTEGRAL Likelihood 10.30 Transmembrane 191

207 INTEGRAL Likelihood 10.51 Transmembrane 225

241 INTEGRAL Likelihood -3.40 Transmembrane 249 -

265

PERIPHERAL Likelihood = 0.53 (at 292) ALOM score: -10.51 (number of TMSs: 6)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 112 Charge difference: -1.5 C( 1.0) - N( 2.5) N >= C: N-terminal side will be inside

>>> membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75): 4.00 Hyd Moment (95) : 1.75 G content: 0 D/E content: 2 S/T content: 1 Score: -7.55

NUCDISC: discrimination of nuclear localization signals pat4: PRRR (4) at 157 pat7: PRRRFPW (5) at 157 bipartite: none content of basic residues: 5.5% 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

A search ofthe NOVl 6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 16D.

In a BLAST search of public sequence databases, the NOVlόa protein was found to have homology to the proteins shown in the BLASTP data in Table 16E.

PFam analysis predicts that the NOVlόa protein contains the domains shown in the Table 16F.

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 fluorescentiy labeled. The doubly labeled material is isolated and the fluorescentiy labeled single strand is resolved by capillary gel electrophoresis. A computer algorithm compares the electropherograms from an experimental and control group for each ofthe 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 ofthe gene fragment is confirmed by additional, gene-specific competitive PCR or by isolation and sequencing ofthe 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 polymoφhisms (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 ofthe DNA sequence, or part ofthe sequence, or both, are sequenced. In silico prediction was based on sequences available in CuraGen Coφoration'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 iβa\4- 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 Coφoration proprietary yeast strain (disclosed in U. S. Patents 6,057,101 and 6,083,693, incoφorated herein by reference in their entireties).

Gal4-binding domain (Gal4-BD) fusions of a CuraGen Coφoration 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 Coφoration'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 polymoφhisms (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 Coφoration 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 ofthe cDNA ofthe 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 ofthe 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) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe 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 Coφoration'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 ofthe 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 puφoses. Example C. Quantitative expression analysis of clones in various cells and tissues

The quantitative expression of various clones 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). RTQ PCR was performed on an Applied Biosystems ABI PRISM® 7700 or an ABI PRISM® 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and Alzheimer's diseased brains). RNA integrity from all samples is controlled for quality 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 that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by 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.

First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions.

In other cases, non-normalized RNA samples were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen Coφoration; Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 μg of total RNA were performed in a volume of 20 μl and incubated for 60 minutes at 42°C. This reaction can be scaled up to 50 μg of total RNA in a final volume of 100 μl. sscDNA samples are then normalized to reference nucleic acids as described previously, using IX TaqMan® Universal Master mix (Applied Biosystems; catalog No.4324020), following the manufacturer's instructions.

Probes and primers were designed for each assay 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 settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (Tm) range = 58°-60°C, primer optimal Tm = 59°C, maximum primer difference = 2°C, probe does not have 5'G, probe Tm must be 10°C greater than primer Tm, amplicon size 75bp to lOObp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). 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 ofthe probe, respectively. Their final concentrations were: forward and reverse primers, 900nM each, and probe, 200nM.

PCR conditions: When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone and another gene-specific set multiplexed with the target probe). PCR reactions were set up 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 as follows: 95°C 10 min, then 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) 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 is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.

When working with sscDNA samples, normalized sscDNA was used as described previously for RNA samples. PCR reactions containing one or two sets of probe and primers were set up as described previously, using IX TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions. PCR amplification was performed as follows: 95°C 10 min, then 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Results were analyzed and processed as described previously.

Panels 1, 1.1, 1.2, and 1.3D

The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers ofthe following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions ofthe brain, the 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.

In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used: ca. = carcinoma, * = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, pi. eff = pi effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.

General screening panel vl .4, vl.5, vl.6 and 1.7 The plates for Panels 1.4, 1.5, 1.6 and 1.7 include 2 control wells (genomic DNA control and chemistry control) and 88 to 94 wells containing cDNA from various samples. The samples in Panels 1.4, 1.5, 1.6 and 1.7 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers ofthe following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panels 1.4, 1.5, 1.6 and 1.7 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panels 1.4, 1.5, 1.6 and 1.7 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions ofthe brain, the 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. Abbreviations are as described for Panels 1, 1.1, 1.2, and 1.3D. Panels 2D, 2.2, 2.3 and 2.4

The plates for Panels 2D, 2.2, 2.3 and 2.4 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI) or from Ardais or Clinomics). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologist at NDRI/ CHTN/Ardais/Clinomics). Unmatched RNA samples from tissues without malignancy (normal tissues) were also obtained from Ardais or Clinomics. This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage ofthe patient. These matched margins are taken from the tissue surrounding (i.e., immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.

HASS Panel v 1.0

The HASS panel v 1.0 plates are comprised of 93 cDNA samples and two controls. Specifically, 81 of these samples are derived from cultured human cancer cell lines that had been subjected to serum starvation, acidosis and anoxia for different time periods as well as controls for these treatments, 3 samples of human primary cells, 9 samples of malignant brain cancer (4 medulloblastomas and 5 glioblastomas) and 2 controls. The human cancer cell lines are obtained from ATCC (American Type Culture Collection) and fall into the following tissue groups: breast cancer, prostate cancer, bladder carcinomas, pancreatic cancers and CNS cancer cell lines. These cancer cells are all cultured under standard recommended conditions. The treatments used (serum starvation, acidosis and anoxia) have been previously published in the scientific literature. The primary human cells were obtained from Clonetics (Walkersville, MD) and were grown in the media and conditions recommended by Clonetics. The malignant brain cancer samples are obtained as part of a collaboration (Henry Ford Cancer Center) and are evaluated by a pathologist prior to CuraGen receiving the samples . RNA was prepared from these samples using the standard procedures. The genomic and chemistry control wells have been described previously.

ARDAIS Panel v 1.0

The plates for ARDAIS panel v 1.0 generally include 2 control wells and 22 test samples composed of RNA isolated from human tissue procured by surgeons working in close cooperation with Ardais Coφoration. The tissues are derived from human lung malignancies (lung adenocarcinoma or lung squamous cell carcinoma) and in cases where indicated many malignant samples have "matched margins" obtained from noncancerous lung tissue just adjacent to the tumor. These matched margins are taken from the tissue suπounding (i.e., immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue) in the results below. The tumor tissue and the "matched margins" are evaluated by independent pathologists (the surgical pathologists and again by a pathologist at Ardais). Unmatched malignant and non-malignant RNA samples from lungs were also obtained from Ardais. Additional information from Ardais provides a gross histopathological assessment of tumor differentiation grade and stage. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical state of the patient.

ARDAIS Prostate v 1.0

The plates for ARDAIS prostate 1.0 generally include 2 control wells and 68 test samples composed of RNA isolated from human tissue procured by surgeons working in close cooperation with Ardais Coφoration. The tissues are derived from human prostate malignancies and in cases where indicated malignant samples have "matched margins" obtained from noncancerous prostate tissue just adjacent to the tumor. These matched margins are taken from the tissue surrounding (i.e., immediately proximal) to the zone of surgery (designated "NAP', for normal adjacent tissue) in the results below. The tumor tissue and the "matched margins" are evaluated by independent pathologists (the surgical pathologists and again by a pathologist at Ardais). RNA from unmatched malignant and non-malignant prostate samples were also obtained from Ardais. Additional information from Ardais provides a gross histopathological assessment of tumor differentiation grade and stage. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical state ofthe patient.

Panel 3D, 3.1 and 3.2

The plates of Panel 3D, 3.1, and 3.2 are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma ofthe tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D, 3.1, 3.2, 1, 1.1., 1.2, 1.3D, 1.4, 1.5, and 1.6 are of the most common cell lines used in the scientific literature.

Panels 4D, 4R, and 4.1D

Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney (Clontech) was employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, CA). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).

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, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or

12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately l-5ng/ml, TNF alpha at approximately 5-10ng/ml, IFN gamma at approximately 20-50ng/ml, IL-4 at approximately 5-10ng/ml, IL-9 at approximately 5-10ng/ml, IL-13 at approximately 5-10ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum. Mononuclear cells were prepared from blood of employees at CuraGen

Coφoration, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco/Life Technologies, Rockville, MD), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20ng/ml PMA and l-2μg/ml ionomycin, IL-12 at 5-10ng/ml, IFN gamma at 20-50ng/ml and IL-18 at 5-10ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5μg/ml. 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 the isolated mononuclear cells 1:1 at a final concentration of approximately 2xl0⁶cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol (5.5xl0^"5M) (Gibco), and lOmM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging 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 according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^"5M (Gibco), and lOmM Hepes (Gibco), 50ng/ml GMCSF and 5ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^"5M (Gibco), lOmM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50ng/ml.

Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at lOOng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at lOμg/ml for 6 and 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 according to the manufacturer's instructions. CD45RA and CD45RO 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 beads were then used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xlO^"5M (Gibco), and lOmM Hepes (Gibco) and plated at 10⁶cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5μg/ml anti-CD28 (Pharmingen) and 3ug/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, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days ofthe second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^"5M (Gibco), and lOmM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared. To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 10⁶cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco). To activate the cells, we used PWM at 5μg/ml or anti-CD40 (Pharmingen) at approximately lOμg/ml and IL-4 at 5-i0ng ml. Cells were harvested for RNA preparation at 24,48 and 72 hours. To prepare the primary and secondary Thl/Th2 and Tri cells, six-well Falcon plates were coated overnight with lOμg/ml anti-CD28 (Pharmingen) and 2μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CEM lymphocytes (Poietic Systems, German Town, MD) were cultured at 10⁵-10⁶cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^'5M (Gibco), lOmM Hepes (Gibco) and IL-2 (4ng/ml). IL-12 (5ng/ml) and anti-IL4 (1 μg/ml) were used to direct to Thl, while IL-4 (5ng/ml) and anti-IFN gamma (1 μg/ml) were used to direct to Th2 and IL-10 at 5ng/ml was used to direct to Tri . After 4-5 days, the activated Thl, Th2 and Tri lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 1 OOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), lOmM Hepes (Gibco) and IL-2 (lng/ml). Following this, the activated Thl, Th2 and Tri lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but 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 then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way 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 in Interleukin 2.

The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in O.lmM dbcAMP at 5xl0⁵cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5xl0⁵cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 1 OOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^'5M (Gibco), lOmM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at lOng/ml and ionomycin at 1 μg/ml for 6 and 14 hours. Keratinocyte line CCD 106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^"5M (Gibco), and lOmM Hepes (Gibco). CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5ng/ml IL-4, 5ng/ml IL-9, 5ng/ml IL-13 and 25ng/ml IFN gamma.

For these cell lines and blood cells, RNA was prepared by lysing approximately 10⁷cells ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Coφoration) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 φm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15ml Falcon Tube. An equal volume of isopropanol was added and left at -20°C overnight. The precipitated RNA was spun down at 9,000 φm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300μl of RNAse-free water and 35μl buffer (Promega) 5μl DTT, 7μl RNAsin and 8μl DNAse were added. The tube was 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 3M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80°C.

AI_comprehensive panel_vl.O

The plates for AI_comprehensive panel_vl .0 include two control wells and 89 test samples comprised of cDNA isolated from surgical and postmortem human tissues obtained from the Backus Hospital and Clinomics (Frederick, MD). Total RNA was extracted from tissue samples from the Backus Hospital in the Facility at CuraGen. Total RNA from other tissues was obtained from Clinomics.

Joint tissues including synovial fluid, synovium, bone and cartilage were obtained from patients undergoing total knee or hip replacement surgery at the Backus Hospital. Tissue samples were immediately snap frozen in liquid nitrogen to ensure that isolated RNA was of optimal quality and not degraded. Additional samples of osteoarthritis and rheumatoid arthritis joint tissues were obtained from Clinomics. Normal control tissues were supplied by Clinomics and were obtained during autopsy of trauma victims.

Surgical specimens of psoriatic tissues and adjacent matched tissues were provided as total RNA by Clinomics. Two male and two female patients were selected between the ages of 25 and 47. None ofthe patients were taking prescription drugs at the time samples were isolated. Surgical specimens of diseased colon from patients with ulcerative colitis and Crohns disease and adjacent matched tissues were obtained from Clinomics. Bowel tissue from three female and three, male Crohn's patients between the ages of 41-69 were used. Two patients were not on prescription medication while the others were taking dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from three male and four female patients. Four ofthe patients were taking lebvid and two were on phenobarbital.

Total RNA from post mortem lung tissue from trauma victims with no disease or with emphysema, asthma or COPD was purchased from Clinomics. Emphysema patients ranged in age from 40-70 and all were smokers, this age range was chosen to focus on patients with cigarette-linked emphysema and to avoid those patients with alpha- lanti-trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded smokers to prevent those patients that could also have COPD. COPD patients ranged in age from 35-80 and included both smokers and non-smokers. Most patients were taking corticosteroids, and bronchodilators.

In the labels employed to identify tissues in the AI_comprehensive panel_vl .0 panel, the following abbreviations are used:

Al = Autoimmunity

Syn = Synovial Normal = No apparent disease

Rep22 /Rep20 = individual patients

RA = Rheumatoid arthritis

Backus = From Backus Hospital

OA = Osteoarthritis (SS) (BA) (MF) = Individual patients

Adj = Adjacent tissue

Match control = adjacent tissues

-M = Male

-F = Female COPD = Chronic obstructive pulmonary disease

AI.05 chondrosarcoma The AI.05 chondrosarcoma plates are comprised of SW 1353 cells that had been subjected to serum starvation and treatment with cytokines that are known to induce MMP (1, 3 and 13) synthesis (e.g. ILlbeta). These treatment? include: IL-lbeta (10 ng/ml), IL-lbeta + TNF-alpha (50 ng/ml), IL-lbeta + Oncostatin (50 ng/ml) and PMA (100 ng/ml). The SW1353 cells were obtained from the ATCC (American Type Culture Collection) and were all cultured under standard recommended conditions. The SW1353 cells were plated at 3 xlO⁵ cells/ml (in DMEM medium- 10 % FBS) in 6- well plates. The treatment was done in triplicate, for 6 and 18 h. The supernatants were collected for analysis of MMP 1, 3 and 13 production and for RNA extraction. RNA was prepared from these samples using the standard procedures.

Panels 5D and 51

The plates for Panel 5D and 51 include two control wells and a variety of cDNAs isolated from human tissues and cell lines with an emphasis on metabolic diseases. Metabolic tissues were obtained from patients enrolled in the Gestational Diabetes study. Cells were obtained during different stages in the differentiation of adipocytes from human mesenchymal stem cells. Human pancreatic islets were also obtained.

In the Gestational Diabetes study subjects are young (18 - 40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarean section. After delivery ofthe infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) ofthe exposed metabolic tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted and fast frozen within 5 minutes from the time of removal. The tissue was then flash frozen in liquid nitrogen and stored, individually, in sterile screw-top tubes and kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectus) and subcutaneous adipose. Patient descriptions are as follows:

Patient 2: Diabetic Hispanic, overweight, not on insulin Patient 7-9: Nondiabetic Caucasian and obese (BMI>30) Patient 10: Diabetic Hispanic, overweight, on insulin Patient 11 : Nondiabetic African American and overweight

Patient 12: Diabetic Hispanic on insulin Adiocyte differentiation was induced in donor progenitor cells obtained from Osirus (a division of Clonetics/BioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr 2 1999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows:

Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated

Donor 2 and 3 AD: Adipose, Adipose Differentiated

Human cell lines were generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: kidney proximal convoluted tubule, uterine smooth muscle cells, small intestine, liver HepG2 cancer cells, heart primary stromal cells, and adrenal cortical adenoma cells. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. All samples were processed at CuraGen to produce single stranded cDNA.

Panel 51 contains all samples previously described with the addition of pancreatic islets from a 58 year old female patient obtained from the Diabetes Research Institute at the University of Miami School of Medicine. Islet tissue was processed to total RNA at an outside source and delivered to CuraGen for addition to panel 51.

In the labels employed to identify tissues in the 5D and 51 panels, the following abbreviations are used: GO Adipose = Greater Omentum Adipose

SK = Skeletal Muscle

UT = Uterus

PL = Placenta

AD = Adipose Differentiated AM = Adipose Midway Differentiated

U = Undifferentiated Stem Cells Panel CNSD.01

The plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

Disease diagnoses are taken from patient records. The panel contains two brains from each ofthe following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supemuclear Palsy, Depression, and "Normal controls". Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration ofthe substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration. In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:

PSP = Progressive supranuclear palsy

Sub Nigra = Substantia nigra

Glob Palladus= Globus palladus Temp Pole = Temporal pole

Cing Gyr = Cingulate gyrus

BA 4 = Brodman Area 4

Panel CNS_Neurodegeneration_V1.0

The plates for Panel CNS_Neurodegeneration_V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology. Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) patients, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (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).^' Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages ofthe disease; the occipital cortex is spared in AD and therefore acts as a "control" region within AD patients. Not all brain regions are represented in all cases. In the labels employed to identify tissues in the CNS_Neurodegeneration_Vl .0 panel, the following abbreviations are used:

AD = Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy

Control = Control brains; patient not demented, showing no neuropathology Control (Path) = Control brains; patient not demented but showing sever AD-like pathology

SupTemporal Ctx = Superior Temporal Cortex Inf Temporal Ctx = Inferior Temporal Cortex Panel CNS_Neurodegeneration_V2.0

The plates for Panel CNS_Neurodegeneration_V2.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology. Disease diagnoses are taken from patient records. The panel contains sixteen brains from Alzheimer's disease (AD) patients, and twenty-nine brains from "Normal controls" who showed no evidence of dementia prior to death. The twenty-nine normal control brains are divided into two categories: Fourteen controls with no dementia and no Alzheimer's like pathology (Controls) and fifteen controls with no dementia but evidence of severe Alzheimer's like pathology, (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). Tissue from the temporal cotex (Broddmann Area 21) was selected for all samples from the Harvard Brain Tissue Resource Center; from the two sample from the Human Brain and Spinal Fluid Resource Center (samples 1 and 2) tissue from the inferior and superior temporal cortex was used; each sample on the panel represents a pool of inferior and superior temporal cortex from an individual patient. The temporal cortex was chosen as it shows a loss of neurons in the intermediate stages ofthe disease. Selection of a region which is affected in the early stages of Alzheimer's disease (e.g., hippocampus or entorhinal cortex) could potentially result in the examination of gene expression after vulnerable neurons are lost, and missing genes involved in the actual neurodegeneration process. In the labels employed to identify tissues in the CNS_Neurodegeneration_V2.0 panel, the following abbreviations are used:

Control = Control brains; patient not demented, showing no neuropathology AH3 = Control brains; patient not demented but showing sever AD-like pathology

Inf & Sup Temp Ctx Pool = Pool of inferior and superior temporal cortex for a given individual A. CG126472-02: TEM7.

Expression of gene CGI 26472-02 was assessed using the primer-probe set Ag4727, described in Table AA. Results ofthe RTQ-PCR runs are shown in Tables AB, AC, AD, AE and AF. Table AA. Probe Name Ag4727

Table AB. CNS neurodegeneration yl.O

Table AC. General screening panel yl.4

Table AD. Panel 3D

Table AE. Panel 4.1D

Table AF. general oncology screening panel v 2.4

CNS_neurodegeneration_vl.0 Summary: Ag4727 Two experiments with the same probe and primer produce results that are in excellent agreement. This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene appears to be slightly down-regulated in the temporal cortex of Alzheimer's disease patients. Therefore, up-regulation of this gene or its protein product, or treatment with specific agonists for this receptor may be of use in reversing the dementia, memory loss, and neuronal death associated with this disease.

General_screening _panel_vl.4 Summary: Ag4727 Highest expression of this gene is seen in a brain cancer cell line (CT=28). This gene is widely expressed in this panel, with moderate expression seen in a colon and gastric cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

Among tissues with metabolic function, this gene is expressed at moderate to low levels in pituitary, adipose, adrenal gland, pancreas, thyroid, fetal liver and adult and fetal skeletal muscle and heart. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

Interestingly, this gene is expressed at much higher levels in the fetal kidney (CT=29) when compared to the level of expression in the adult kidney (CT=32). This observation suggests that expression of this gene can be used to distinguish between the fetal and adult source of this tissue. In addition, the relative overexpression of this gene in fetal kidney suggests that the protein product may enhance kidney growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of kidney related diseases.

This gene is also expressed at moderate to low significant levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation ofthe expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

Panel 3D Summary: Ag4727 Highest expression of this gene is seen in cerebellum (CT=30). Moderate levels of expression are seen in several cancer cell lines on this panel, in agreement with Panel 1.4. Please see that panel for discussion of this.

Panel 4.1D Summary: Ag4727 Highest expression of this gene is seen in thymus (CT=27.8), with moderate levels of expression seen in normal lung. The protein encoded for by this gene could therefore play an important role in T cell development. Therefore, therapeutic modulation ofthe expression or function of this gene could be utilized to modulate immune function (T cell development) and be important for organ transplant, AIDS treatment or post chemotherapy immune reconstitution. General oncology screening panel_v_2.4 Summary: Ag4727 This gene is widely expressed in this panel, with highest expression in lung cancer (GT=32.6). In addition, this gene is more highly expressed in lung and kidney cancer than in the corresponding normal adjacent tissue. Thus, expression of this gene could be used as a marker of these cancers. Furthermore, therapeutic modulation ofthe expression or function of this gene product may be useful in the treatment of lung and kidney cancer. B. CG170490-01: UBIQUITIN-PROTEIN LIGASE E3 COMPONEN N-RECOGNIN.

Expression of gene CGI 70490-01 was assessed using the primer-probe set Ag6130, described in Table BA. Results ofthe RTQ-PCR runs are shown in Tables BB, BC and BD.

Table BA. Probe Name Ag6130

Table BB. CNS neurodegeneration yl.O

Control (Path) 1 Temporal Ctx |57.0 |Control (Path) 3 Parietal Ctx |6.1

Control (Path) 2 Temporal Ctx |37.9 jControl (Path) 4 Parietal Ctx |34.2

Table BC. General screening panel yl.5

Table BD. Panel 4.1D

CNS_neurodegeneration_vl.0 Summary: Ag6130 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals.

General_screening_panel_vl.5 Summary: Ag6130 Highest expression of this gene is detected in a brain cancer U-118-MG cell line (CT=27.6). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation ofthe expression or function of this gene may be 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 is expressed at moderate levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation ofthe activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes. In addition, this gene is 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. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Interestingly, this gene is expressed at much higher levels in fetal (CT=28.7) when compared to adult liver (CT=33). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of liver related diseases.

Panel 4.1D Summary: Ag6130 Highest expression of this gene is detected in activated secondary Tri cells (CT=29.9). This gene is expressed at moderate to low levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members ofthe T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_vl .5 and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation ofthe gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

C. CG170667-02: PUTATIVE NEURONAL CELL ADHESION MOLECULE.

Expression of gene CGI 70667-02 was assessed using the primer-probe sets Ag6137 and Ag6161 , described in Tables CA and CB. Results of the RTQ-PCR runs are shown in Tables CC and CD. Table CA. Probe Name Ag6137

Table CB. Probe Name Ag6161

Table CC. CNS neurodegeneration yl.O

Table CD. General screening panel yl.5

CNS_neurodegeneration_vl.O Summary: Ag6137 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals.

General screening_panel_vl.5 Summary: Ag6137 Highest expression of this gene is detected in liver cancer cell line (CT=28.2). Moderate to low expression of this gene is detected in number of cancer cell lines derived from pancreatic, brain, colon, renal, breast, and ovarian cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation ofthe expression or function of this gene may be effective in the treatment of pancreatic, brain, colon, renal, breast, and ovarian cancers. Among tissues with metabolic or endocrine function, this gene is expressed at low levels in pancreas, thyroid, pituitary gland, fetal heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation ofthe activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, this gene is expressed at low levels in all regions ofthe central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

D. CG170791-01: acetyl-CoA transporter (ACATN).

Expression of gene CGI 70791-01 was assessed using the primer-probe set Ag6214, described in Table DA. Results ofthe RTQ-PCR runs are shown in Tables DB and DC.

Table DA. Probe Name Ag6214

Table DB. CNS neurodegeneration yl.O

Table DC. Panel 4.1D

CNS_neurodegeneration_vl.O Summary: Ag6214 Low expression of this gene is seen in temporal cortex of a control patient (CT=34.2). Therefore, therapeutic modulation of this gene or its protein product may be useful in the treatment of neurological disorder such as seizure, Alzheimer's, schizophrenia, and forgetfulness.

Panel 4.1D Summary: Ag6214 Low expression of this gene is detected mainly in TNF alpha + IL-1 beta activated HPAEC and bronchial epithelium and TNF alpha activated dermal fibroblasts. Therefore, therapeutic modulation of this gene or its protein product may be useful in the treatment of autoimmune and inflammatory disorders that include psoriasis, chronic obstructive pulmonary disease, asthma, allergy and emphysema.

E. CG171174-01: Novel Plasma Membrane Protein.

Expression of gene CG171174-01 was assessed using the primer-probe set Ag6166, described in Table EA. Results ofthe RTQ-PCR runs are shown in Tables EB, EC and ED.

Table EA. Probe Name Ag6166

Table EB. CNS neurodegeneration yl.O

Table EC. General screening panel yl.5

Table ED. Panel 4.1D

CNS_neurodegeneration_vl.0 Summary: Ag6166 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals.

This gene is found to be slightly upregulated in the temporal cortex of Alzheimer's disease patients. Blockade of this receptor may be of use in the treatment of this disease and decrease neuronal death. Gener ai_screening_panel vl .5 Summary: Ag6166 Highest expression of this gene is detected in cerebellum (CT=27). This gene is expressed at moderate levels in all regions ofthe central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation ofthe expression or function of this gene may be 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 is expressed at moderate to low levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation ofthe activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes. Interestingly, this gene is expressed at much higher levels in fetal (CT=30) when compared to adult liver (CT=34.8). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of liver related diseases.

Panel 4.1D Summary: Ago 166 Highest expression of this gene is detected in TNF alpha treated dermal fibroblasts (CT=33.3). Low expression of this gene is seen in wide range of cell types of significance in the immune response in health and disease. These cells include members ofthe T-cell, B-cell, endothelial cell, activated monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin. Expression of this gene is upregulated in LPS stimulated monocytes, activated Ramos B cells, activated naive and memory T cells and activated polarized T cells. Therefore, modulation ofthe gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

F. CG172921-01: Interleukin-5 receptor alpha chain.

Expression of gene CGI 72921-01 was assessed using the primer-probe sets Ag5894 and Ag6187, described in Tables FA and FB. Results ofthe RTQ-PCR runs are shown in Tables FC and FD. Please note that CG 172921 -01 represents a full length physical clone.

Table FA. Probe Name Ag5894

Table FB. Probe Name Ag6187

Table FC. Al comprehensive panel yl.O

Table FD. Panel 4.1D

AI_comprehensive panel_vl.O Summary: Ag5894 Highest expression is seen in a sample from RA bone (CT=30.9). Furthermore, moderate levels of expression are seen in a cluster of RA derived samples. Thus, expression of this gene could be used to differentiate RA derived samples from other samples on this panel and as a marker of RA. Furthermore, therapeutic modulation ofthe expression or function of this gene may be effective in the treatment of RA.

Panel 4.1D Summary: Ag5894/Ag6187 Two experiments with two different probe and primer sets produce results that are in excellent agreement. Detectable expression is limited to resting neutrophils (CTs=31-33). This expression is reduced to background level (CT=40) in neutrophils activated by TNF-alpha+LPS. This expression profile suggest that this gene is produced by resting neutrophils but not by activated neutrophils. Therefore, the gene product may reduce activation of these inflammatory cells and be useful as a protein therapeutic to reduce or eliminate the symptoms in patients with Crohn's disease, ulcerative colitis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, emphysema, rheumatoid arthritis, lupus erythematosus, or psoriasis. G. CG176203-01: Novel membrane protein.

Expression of gene CGI 76203-01 was assessed using the primer-probe set Ag6370, described in Table GA. Results ofthe RTQ-PCR runs are shown in Tables GB and GC.

Table GA. Probe Name Ag6370

Table GB. General screening panel yl.6

Table GC. Panel 4.1D

General_screening_panel_vl.6 Summary: Ag6370 Highest expression of this gene is detected in ovarian cancer SK-OV-3 cell line (CT=27.7). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation ofthe expression or function of this gene may be 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 is expressed at moderate levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, fetal liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, this gene is expressed at moderate levels in all regions ofthe central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Interestingly, this gene is expressed at much higher levels in fetal (CT=29.3) when compared to adult liver (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of liver related diseases.

Panel 4.1D Summary: Ag6370 Highest expression of this gene is detected in lung microvascular endothelial cells (CT=29.3). This gene is expressed at moderate to low levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members ofthe T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by lung and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_yl .6 and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation ofthe gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

H. CG50691-02: CRIMl Homolog

Expression of gene CG50691-02 was assessed using the primer-probe sets Agl55 and Ag7290, described in Tables HA and HB. Results ofthe RTQ-PCR runs are shown in Tables HC, HD, HE, HF and HG.

Table HA. Probe Name Agl55

Table HB. Probe Name Ag7290

Table HC. Ardais Prostate 1.0

Table HD. CNS neurodegeneration yl.O

Table HE. General screening panel yl.7

Table HF. Panel 1

Table HG. Panel 4.1D

Ardais Prostate 1.0 Summary: Ag7290 Highest expression of this gene_: which encodes a putative CRIMl protein, is seen in normal tissue adjacent to a prostate carcinoma (CT=29.9). In addition, this gene is widely expressed at low levels in most ofthe samples on this panel, suggesting a role for this protein in cell survival and/or proliferation.

CNS_neurodegeneration_vl.0 Summary: Ag7290 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at low levels in the brain. Please see Panel 1.7 for discussion of this gene in the central nervous system.

General_screening_panel_vl .7 Summary: Ag7290 Highest expression of this gene is seen in an ovarian cancer cell line (CT=26.2). This gene is widely expressed in this panel, with moderate to high expression seen in brain, colon, gastric, lung, breast, ovarian, prostate and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. This gene encodes a novel protein with homology to Cysteine-Rich Motor Neuron Protein 1 (CRIMl )which may interact with growth factors implicated in motor neuron differentiation and survival (Kolle, G.; Georgas, K.; Holmes, G. P.; Little, M. H.; Yamada, T. : CRIMl, a novel gene encoding a cysteine-rich repeat protein, is developmentally regulated and implicated in vertebrate CNS development and organogenesis. Mech. Dev. 90: 181-193, 2000. PubMed ID : 10642437 ).

The Criml gene encodes a putative transmembrane protein with an IGF-binding protein motif and multiple VWFC domain, analogous to those of chordin and short gastrulation (sog) proteins that associate with TGFbeta superfamily members, namely Bone Moφhogenic Protein (BMP). In chordin, such repeats are responsible for its dorsalising activity and for binding to bone moφhogenic proteins (BMPs). Chordin is a BMP antagonist that dorsalizes early vertebrate embryonic tissues by binding to ventralizing TGF-beta-like bone moφhogenetic proteins and sequestering them in latent complexes. Scott et al. (Scott, I. C; Blitz, I. L.; Pappano, W. N.; Imamura, Y.; Clark, T. G.; Steiglitz, B. M.; Thomas, C. L.; Maas, S. A.; Takahara, K.; Cho, K. W. Y.; Greenspan, D. S. :

Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 213: 283-300, 1999.) showed that BMP1 and TLL1 counteracted the dorsalizing effects of chordin upon overexpression in Xenopus embryos. They suggested that BMP1 is the major chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis. It also directly binds BMP-4 and BMP-2, and interferes with the binding to the receptors. Bone metastases are a frequent clinical problem in patients with breast, prostate, and other cancers. Formation of these lesions is a site-specific process determined by multiple cellular and molecular interactions between the cancer cells and the bone microenvironment. Bone moφhogenetic protein (BMP) has been shown to be one ofthe significant factors in the prognosis of bone tumors. The overexpression of BMP2, BMP4, and BMP6 were found in most of osteosarcoma or prostate cancer with metastases (Guo, W., Gorlick, R., Ladanyi, M., Meyers, PA., Huvos, AG., Bertino, JR., and Healey, JH.,1999, Expression of bone moφhogenetic proteins and receptors in sarcomas. Clinical Orthopaedics and Related Research 365: 175-183; Hamdy, F., Autzen, P., Robinson, MC, Wilson Home, CH., Neal, DE. and Robson CN., 1997, Immunolocalization and messenger RNA expression of bone moφhogenetic protein-6 in human benign and malignant prostatic tissue. Cancer Research 57: 4427-4431) suggesting a close association between BMPs and skeletal metastases. BMP-2, -4, -6 may be responsible, in part, for osteoblastic changes in metastatic lesions secondary to prostate cancer. Kolle et al. (2000) proposed that CRIMl might be involved in motor neuron survival by interacting with various growth factors. Georgas K et al. (Georgas K, Bowles J, Yamada T, Koopman P, Little MH, 2000, Characterisation of Criml expression in the developing mouse urogenital tract reveals a sexually dimoφhic gonadal expression pattern. Dev Dyn 219(4):582-7) have demonstrated that Criml also displays a striking male-specific expression pattem in the fetal gonads, its expression strongest in the Sertoli cells ofthe developing testis.

CG50691-02 and CG50691-03 (see below) represent two novel splice variants of CRIMl with deletion of internal 58 aa (exon 2) or 65 aa (exon 14). The encoded protein either contains all the characteristic domains as the parent CRIMl or only lacks one ofthe VWFC cysteine-rich repeat domains. Therefore, it is anticipated the splice variants may have similar or altered biological functions as the parent protein that could be involved in the BMP pathway during organogenesis or cancer development. Modulation of this gene product may therefore be useful in the treatment of cancer and in particular the cancers listed above, including prostate cancer. Among tissues with metabolic function, this gene is expressed at moderate levels in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

This gene is also expressed at moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex.

Therefore, therapeutic modulation ofthe expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

In addition, this gene is expressed at much higher levels in kidney (CT=27) when compared to expression in the fetal counteφart (CT=30). Thus, expression of this gene may be used to differentiate between the fetal and adult source of this tissue.

Panel 1 Summary: Agl55 Highest expression is seen in the placenta (CT=20.74). Overall, the expression profile agrees with the results for Panel 1.7. Please see that panel for discussion of this gene. Panel 4.1D Summary: Ag7290 Highest expression is seen in an ILl-b treated sample of HUVECs (CT=30.2). Overall, this transcript is expressed at moderate levels in endothelial cells, including samples derived from HPAEC, HUVEC and lung and dermal microvascular EC. Fibroblasts also express this transcript. Therapies designed with the protein encoded by this transcript could be important in regulating endothelium function including leukocyte extravasation, a major component of inflammation during asthma, IBD, and psoriasis.

I. CG50691-03: CRIMl Homolog.

Expression of gene CG50691-03 was assessed using the primer-probe sets Agl55 and Ag7303, described in Tables IA and IB. Results ofthe RTQ-PCR runs are shown in Tables IC, ID, IE and IF. Table IA. Probe Name Agl55

Table IB. Probe Name Ag7303

Table IC. CNS neurodegeneration yl.O

Table ID. General screening panel yl.7

Table IE. Panel 1

Table IF. Panel 4.1D

CNS_neurodegeneration_vl.0 Summary: Ag7303 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene is appears to be slightly upregulated in the temporal cortex of Alzheimer's disease patients. Therefore, therapeutic modulation ofthe expression or function of this gene may decrease neuronal death and be of use in the treatment of this disease.

General_screening_panel_vl.7 Summary: Ag7303 Highest expression of this gene is seen in a renal cancer cell line (CT=23.5). This gene is widely expressed in this panel, with high to moderate expression seen in brain, colon, gastric, lung, prostate, breast, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

Among tissues with metabolic function, this gene is expressed at high to moderate levels in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

This gene is also expressed at high to moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex.

This gene encodes a novel splice variant of CRIMl. Please see CG50691-02 for further discussion of this gene.

Panel 1 Summary: Agl55 Highest expression is seen in the placenta (CT=20.74). Overall, the expression profile agrees with the results for Panel 1.7. Please see that panel for discussion of this gene. Panel 4.1D Summary: Ag7303 Highest expression of this gene is seen in untreated lung microvascular endothelial cells (CT=30). This transcript is also expressed in clusters of samples derived from HPAEC, HUVEC, lung, and dermal microvascular EC, and lung and dermal fibroblasts. Therefore, therapies designed with the protein encoded by this transcript could be important in regulating endothelium function including leukocyte extravasation, a major component of inflammation during asthma, IBD, and psoriasis.

J. CG50691-04: Cysteine-rich repeat-containing protein S52 precursor.

Expression of gene CG50691-04 was assessed using the primer-probe sets Agl55 and Ag8164, described in Tables JA and JB. Results of the RTQ-PCR runs are shown in Tables JC, JD, JE and JF.

Table JA. Probe Name Agl55

Table JB. Probe Name Ag8164

Table JC. CNS neurodegeneration yl.O

Table JD. Oncology cell line screening panel v3.2

Table JE. Panel 1

Table JF. Panel 4.1D

CNS_neurodegeneration_vl.0 Summary: Ag8164, This profile confirms the expression of this gene at low levels in the brain. Therefore, therapeutic modulation ofthe expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

Oncology cell line _screening_panel v3.2 Summary: Ag8164 Highest expression is seen in a colon cancer cell line (CT=30). In addition, this gene is expressed at moderate to low levels in many samples on this panel, suggesting a role for this gene in cell survival and proliferation. Panel 1 Summary: Agl55 Highest expression is seen in the placenta (CT=20.74). Overall, the expression profile agrees with the results for Panel 1.7. Please see that panel for discussion of this gene.

Panel 4.1D Summary: Ag8164 Highest expression of this gene is seen in untreated lung microvascular endothelial cells (CT=31.1). This transcript is also expressed in clusters of samples derived from HPAEC, HUVEC, lung, and dermal microvascular EC, and lung and dermal fibroblasts. Therefore, therapies designed with the protein encoded by this transcript could be important in regulating endothelium function including leukocyte extravasation, a major component of inflammation during asthma, IBD, and psoriasis.

K CG51905-01 and CG51905-03: 28804279.0.7.

Expression of gene CG51905-01 and CG51905-03 was assessed using the primer-probe sets Ag2814 and Ag203, described in Tables KA and KB. Results ofthe RTQ-PCR runs are shown in Tables KC, KD and KE. Please note that CG51905-03 represents a full-length physical clone ofthe CG51905-01 gene, validating the prediction of the gene sequence.

Table KA. Probe Name Ag2814

Table KB. Probe Name Ag203

Table KC. Panel 1

Table KD. Panel 1.3D

Table KE. Panel 4D

Panel 1 Summary: Ag203 Highest expression of this gene is detected in thymus (CT=29.9). Thus, this gene or its protein product could play an important role in T cell development. Small molecule therapeutics, or antibody therapeutics designed against the protein encoded for by this gene could be utilized to modulate immune function (T cell development) and be important for organ transplant, AIDS treatment or post chemotherapy immune reconstitution.

Moderate to low expression of this gene is also detected in a few cancer cell lines derived from ovarian, lung, renal and brain cancers. Therefore, therapeutic modulation of this gene or its protein product may be useful in the treatment of ovarian, lung, renal and brain cancers.

Low expression of this gene is also seen in lymph node, pancreas, and colon. Please see panel 1.3D for further discussion of this gene.

Panel 1.3D Summary: Ag2814 Highest expression of this gene in mainly seen in fetal skeletal muscle (CT=32.6). Interestingly, this gene is expressed at much higher levels in fetal when compared to adult skeletal muscle (CT = 40). This observation suggests that expression of this gene can be used to distinguish fetal from adult skeletal muscle. In addition, the relative overexpression of this gene in fetal skeletal muscle suggests that the protein product may enhance muscular growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe GPCR encoded by this gene could be useful in treatment of muscle related diseases. More specifically, treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function.

Low expression of this gene is also seen in some ofthe tissues with metabolic or endocrine function including placenta, fetal liver, colorectal region, and stomach. Thus, therapeutic modulation of this gene or its protein product may be useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, this gene is expressed at much higher levels in fetal (CT=34.6) when compared to adult liver (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of liver related diseases. Panel 4D Summary: Ag203/Ag2814 Two experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in activated primary Th2 cells and activated Ramos B cells (CTs-32-32.6). Low expression of this gene is also seen in activated primary TH1 and Tri, activated secondary Thl, Th2 and Tri cells, activated memory T cells, activated PBMC cells, activated B lymphocytes, activated small airway epithelium, basophils, mucoepidermoid cells, dermal fibroblasts, thymus and kidney. Therefore, modulation ofthe gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

L. CG52414-01: Rhomboid (3541612.0.88).

Expression of gene CG52414-01 was assessed using the primer-probe sets Ag2648, Ag2786, Ag2787 and Ag913, described in Tables LA, LB, LC and LD. Results ofthe RTQ-PCR runs are shown in Tables LE, LF, LG, LH, LI, LJ and LK.

Table LA. Probe Name Ag2648

Table LB. Probe Name Ag2786

Table LC. Probe Name Ag2787

Table LD. Probe Name Ag913

Table LE. AI comprehensive panel yl.O

Table LF. CNS neurodegeneration yl.O

Table LG. General screening panel yl.6

Table LH. Oncology cell line screening panel v3.2

Table LI. Panel 1.3D

Table LJ. Panel 2D

Table LK. Panel 4D

AI_comprehensive panel_vl.0 Summary: Ag2787 Highest expression of this gene is detected in a matched control sample for ulcerative colitis (CT=28). Moderate to low levels of expression of this gene is also detected in samples derived from normal and orthoarthitis/ rheumatoid arthritis bone and adjacent bone, cartilage, synovium and synovial fluid 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 product may ameliorate symptoms/conditions associated with autoimmune and inflammatory disorders including psoriasis, allergy, asthma, inflammatory bowel disease, rheumatoid arthritis and osteoarthritis.

CNS_neurodegeneration_vl.O Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals. However, no differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment. Please see Panel 1.3D for a discussion ofthe potential role of this gene in treatment of central nervous system disorders.

General screening panel vl.6 Summary: Ag913 Highest expression of this gene is detected in renal cancer TK-10 cell line (CT=26.9). Moderate to high expression of this gene is 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. In addition, this gene shows widespread expression in this panel which correlates with expression seen in panel 1.3D. Please see panel 1 D for further discussion of this gene.

Interestingly, this gene is expressed at much higher levels in fetal (CTs=30-32.5) when compared to adult lung and liver (CTs=35-40). This observation suggests that expression of this gene can be used to distinguish fetal from adult lung and liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance lung and liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of lung and liver related diseases.

Oncology cell line screening panel_v3.2 Summary: Ag2648 Highest expression of this gene is detected in pancreatic cancer SU86.86 cell line (CT=30). Moderate expression of this gene is seen in number of cancer cell lines derived from lung, bone marrow, epidermoid, vulva, bone, bladder, pancreatic, renal, B cells and T cells, leukemia, lymphoma, cervical, gastric, colon, lung and brain. This expression pattern correlates with that seen in panel 1.3D. Please see panel 1.3D for further discussion of this gene.

Panel 1.3D Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in renal cancer A498 cell line (CTs=28-28.9). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation ofthe expression or function of this gene may be 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 is expressed at moderate to low levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation ofthe activity of this gene may prove useful in the tieatment of endocrine/metabolically related diseases, such as obesity and diabetes. In addition, this gene is expressed at moderate to low levels in all regions ofthe central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Panel 2D Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in bladder and kidney cancers (CTs=26.4-28). High to moderate expression of this gene is also detected in cancer and normal samples derived from colon, prostate, liver, lung, kidney, breast, thyroid, ovary and stomach. Interestingly, expression of this gene is higher in cancer samples especially gastric, bladder, breast, kidney and colon cancer compared to adjacent normal tissues. Therefore, expression of this gene may be used as diagnostic marker to detect the presence of these cancers. In addition, this gene codes for a putative protease belonging to Rhomboid family that activates growth factors ligands (Urban et al. Cell 2001 Oct 19; 107(2): 173-82). Therefore this gene may play a role in tumor cell proliferation and invasion, by activating growth factors like TGFalpha and EGF that mediates cell growth and invasion. Targeting of protease encoded by this gene with a human monoclonal antibody that results in an inhibition ofthe activity of this protein, preferably its putative protease activity, will have therapeutic effect on tumors, preferably on colon, gastric, kidney, ovarian and bladder tumors and will result in reduced tumor cell growth, proliferation and invasion.

Panel 4D Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in LPS activated macrophages and monocytes (CTs=27-28.5). This gene is expressed at high to moderate levels in a wide range of cell types of significance in. the immune response in health and disease. These cells include members ofthe T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. Expression of this gene is stimulated in activated endothelial cells, small airway epithelium and fibroblasts. The ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. Therefore, modulation ofthe gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

Example D: Identification of Single Nucleotide Polymorphisms in NOVX nucleic acid sequences

Variant sequences are also included in this application. A variant sequence can include a single nucleotide polymoφhism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymoφhic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymoφhic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration ofthe amino acid encoded by the gene at the position ofthe SNP. Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result ofthe redundancy ofthe genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation ofthe expression pattern. Examples include alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, and stability of transcribed message.

SeqCalling assemblies produced by the exon linking process were selected and extended using the following criteria. Genomic clones having regions with 98% identity to all or part ofthe initial or extended sequence were identified by BLASTN searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCalling assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs. Some additional genomic regions may have also been identified because selected SeqCalling assemblies map to those regions. Such SeqCalling sequences may have overlapped with regions defined by homology or exon prediction. They may also be included because the location ofthe fragment was in the vicinity of genomic regions identified by similarity or exon prediction that had been included in the original predicted sequence. The sequence so identified was manually assembled and then may have been extended using one or more additional sequences taken from CuraGen Coφoration's human SeqCalling database. SeqCalling fragments suitable for inclusion were identified by the CuraTools™ program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.

The regions defined by the procedures described above were then manually integrated and corrected for apparent inconsistencies that may have arisen, for example, from miscalled bases in the original fragments or from discrepancies between predicted exon junctions, EST locations and regions of sequence similarity, to derive the final sequence disclosed herein. When necessary, the process to identify and analyze SeqCalling assemblies and genomic clones was reiterated to derive the full length sequence (Alderbom et al., Determination of Single Nucleotide Polymoφhisms by Real-time Pyrophosphate DNA Sequencing. Genome Research. 10 (8) 1249-1265, 2000).

Variants are reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments ofthe invention.

Table SNP1: SNP Variants for CG172318-01 (NOV 7a).

Table SNP2: SNP Variants for CG170791-01 (NOV 5a).

Table SNP3: SNP Variant for CG176203-01 (NOV 11a).

Table SNP4: SNP Variants for CG176213-01 (NOV 12a).

Table SNP5: SNP Variant for CG50691-02 (NOV13c).

Table SNP6: SNP Variants for CG52414-01 (NOV 15b).

Table SNP7: SNP Variants for CG52552-04 (NOV 16b).

Example E: Inhibition of expression of NOV15 CG52414-01 using antisense in an ovarian tumor-derived cell line.

Five oligonucleotides were designed and synthesized as mixed-backbone oligonucleotides containing modified phosphorothioate segments at 5' and 3' ends and 2'-O-methyl RNA oligoribonucleotide segments located in the middle. The purity ofthe oligonucleotides was confirmed by mass spectiophometry. The oligonucleotide sequences for CG52414-01 are:

SEQ ID 138: ASI : 5' CCCTCCCAGUCGCCGCTGAC 3' (complementary to the sequence located in 5 'UTR of LIV-1) SEQ ID 139: AS2: 5' TTCAGGCGGCCGAGCGCAT 3' (complementary to the sequence surrounding ATG start codon)

SEQ ID 140: AS3: 5' AGGTCACGCUGGCACGAGGC 3' (complementary to the sequence 3' next to AS2)

SEQ ID 141 : AS4: 5' GCCCUUGAUCUCGCAGTCCA 3* (complementary to the sequence in the middle of SLPI ORF)

SEQ ID 142: AS5: 5' AGCGGUCAGUGCAGCACCTG 3' (complementary to the sequence flanking the 3' stop codon)

OVCAR-5 cells were seeded 10,000 cells/well in a 96 well plate in complete medium 24 hr before transfection and cultured to reach 50% confluency on the day of transfection. Oligonucleotides were diluted with Optimen to 100 and 400 nM, and mixed with Oligofectamine (Invitrogen) according to manufacturer's instructions. Cells were washed with serum-free medium. The oligo and liposome mixture were then added to cells. After 4 hr incubation period, serum were added back to cells. CellTiter 96Aqueous Non-Radioactive Cell Proliferation Assay Kit from Promega was used to determine the number of viable cells 72 hrs after transfection. Briefly, 20 μl of combined MTS solution was diluted with 100 μl complete medium, and added to each well ofthe 96 well plate. After 1 hr incubation at 37°C, the absorbance at 490 nm was recorded using an ELISA plate reader.

Inhibition of expression of CG52414-01 by antisense compounds AS 1 , AS2, AS3 ,

AS4, AS5, alone or in combination, resulted in inhibition of OVCAR-5 cell proliferation (Figure 1). Example F. Relevant Pathways of NOV15 CG52414.

Materials and Methods

PathCalling™ Technology: The sequence of Ace. No CG52414-01 was derived by laboratory screening of cDNA library by the two-hybrid approach. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were sequenced. In silico prediction was based on sequences available in CuraGen Coφoration'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 that follow. 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 Coφoration proprietary yeast strain (disclosed in U. S. Patents 6,057,101 and 6,083,693, incoφorated herein by reference in their entireties).

Gal4-binding domain (Gal4-BD) fusions of a CuraGen Coφortion 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 Coφoration'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 polymoφhisms (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 Coφoration proprietary yeast strains N106' and YULH (U. S. Patents 6,057,101 and 6,083,693).

Interaction protein pairs are added to CuraGen's PathCalling™ Protein Interaction Database. This database allows for the discovery of novel pharmaceutical drug targets by virtue of their interactions and/or presence in pathologically related signaling pathways. Protein interactions are subsequently analyzed using bioinformatic tools within GeneScape™, which provides a means of visualization of binary protein interactions, protein complex formation, as well as complete cellular signaling pathways. The specific interactions, which constitute the specific complexes, may also be useful for therapeutic intervention through the use of recombinant protein or antibody therapies, small molecule drugs, or gene therapy approaches. Protein interactions, which are identified through the mining ofthe PathCalling™ database, can be screened in vitro and in vivo to provide expression, functional, biochemical, and phenotypic information. Assays may be used alone or in conjunction and include, but are not limited to the following technologies; RTQ-PCR, Transfection of recombinant proteins, Co-immunoprecipitation and mass spectrometry, FRET, Affinity Chromatography, Immunohistochemisty or Immunocytochemistey, gene CHIP hybridizations, antisense (i.e., knock-down, knock-up), GeneCalling experiments, and/or biochemical assays (phosphorylation, dephosphorylation, protease, etc.).

Proteins CG52414-01 (Rhomboid like), tousled like kinase 1 and zinc finger ZNF263 protein were found to interact and may result in the formation of a protein complex, or may constitute a series of complexes, which form in order to propagate a cellular signal, which is physiologically relevant to a disease pathology. OTHER EMBODIMENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for puφoses of illustration only, and is not intended to be limiting with respect to the scope ofthe 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 ofthe 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 ofthe embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope ofthe following claims. The claims presented are representative ofthe 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 sequenced selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

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 37.

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 37.

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 ofthe polypeptide of claim 1 in a first mammalian subject, the method comprising: a) measuring the level of expression ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe substance is not observed when the cell is contacted with a composition in the absence ofthe 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 ihe 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 contiol 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 tieat 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 37 or a biologically active fragment thereof.

20. 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 37.

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 37.

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 37.

24. An isolated nucleic acid molecule comprising a nucleic acid selected from the group consisting of 2n-l, wherein n is an integer between 1 and 37.

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 ID NO: 2n-l, wherein n is an integer between 1 and 37, 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 ofthe nucleic acid molecule in said sample.

33. The method of claim 32 wherein presence or amount ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 37.

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 ofthe 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 37.

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.