WO2003064589A2

WO2003064589A2 - Therapeutic polypeptides, nucleic acids encoding same, and methods of use

Info

Publication number: WO2003064589A2
Application number: PCT/US2002/024483
Authority: WO
Inventors: Ramesh Kekuda; Charles E. Miller; Meera Patturajan; Carol E. A. Pena; Daniel K. Rieger; Richard A. Shimkets; Bryan D. Zerhusen; Li Li; Weizhen Ji; Muralidhara Padigaru; Stacie J. Casman; Edward Z. Voss; Ferenc L. Boldog; Linda Gorman; Mario W. Leite; Corine A. M. Vernet; David W. Anderson; Xiaojia Guo; Mei Zhong; Valerie L. Gerlach
Original assignee: Curagen Corporation
Priority date: 2001-01-23
Filing date: 2002-08-02
Publication date: 2003-08-07
Also published as: AU2002365216A8; AU2002365216A1; WO2003064589A3

Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies that immunospecifically bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the novel polypeptide, polynucleotide, or antibody specific to the polypeptide. 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 biochemicalOr 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 of the biochemical and physiological processes involves intricate signaling pathways. Frequently, such signaling pathways involve extracellular signaling proteins, cellular receptors that bind the signaling proteins, and signal transducing components located within the cells. Signaling proteins may be classified as endocrine effectors, paracrine effectors or autocrine effectors. Endocrine effectors are signaling molecules secreted by a given organ into the circulatory system, which are then transported to a distant target organ or tissue. The target cells include the receptors for the endocrine effector, and when the endocrine effector binds, a signaling cascade is induced. Paracrine effectors involve secreting cells and receptor cells in close proximity to each other, for example two different classes of cells in the same tissue or organ. One class of cells secretes the paracrine effector, which then reaches the second class of cells, for example by diffusion through the extracellular fluid. The second class of cells contains the receptors for the paracrine effector; binding of the effector results in induction 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 Kekuda et al. 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 of the 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. κeκιιαa et al.

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 102. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOVl,

NOV2, NOV3, etc., nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "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 102, 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 102. In another embodiment, the invention also comprises variants of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed. The invention also involves fragments of any of the mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% ofthe residues are changed.

In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic variants of the sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102. These allelic variants include amino acid sequences that are the translations of nucleic acid sequences differing by a single nucleotide from nucleic acid sequences selected from the group consisting of SEQ ID NOS: 2n-l, wherein n is an integer between 1 and 102. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution.

In another embodiment, the invention comprises a pharmaceutical composition involving a polypeptide with an amino acid sequence selected from the group consisting of Kekuda et al.

SEQ ID NO:2n, wherein n is an integer between 1 and 102 and a pharmaceutically acceptable carrier. In another embodiment, the invention involves a kit, including, in one or more containers, this pharmaceutical composition.

In another embodiment, the invention includes the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease being selected from a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102 wherein said therapeutic is the polypeptide selected from this group. In another embodiment, the invention comprises a method for determining the presence or amount of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102 in a sample, the method involving providing the sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, thereby determining the presence or amount of polypeptide in the sample.

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

In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer Kekuda et ul. between 1 and 102, 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 of the substance is not observed when the cell is contacted with a composition devoid of the substance, the substance is identified as a potential therapeutic agent.

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

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

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

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

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

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

In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ

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

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

ID NO:2n, wherein n is an integer between 1 and 102, wherein the nucleic acid molecule has a nucleotide sequence in which any nucleotide specified in the coding sequence of the chosen nucleotide sequence is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% 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 102. This vector can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.

In another embodiment, the invention involves a method for determining the presence or amount of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form ofthe amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 102 in a sample, the method including providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount 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 102 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 of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level ofthe nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease. Kekuda et al.

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 ofthe 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 of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compounds. The sequences are collectively referred to herein as "NOVX nucleic acids" or "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 Kekuda et al. sequences disclosed herein. Table A provides a summary ofthe NOVX nucleic acids and their encoded polypeptides.

TABLE A. Sequences and Corresponding SEQ ID Numbers

Kekuda et al.

Kekuda et al.

Table A and B indicate the homology of NOVX polypeptides to known protein families. Thus, the nucleic acids and polypeptides, antibodies and related compounds Kekuda et al. 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), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, metabolic disturbances associated with obesity, transplantation, adrenoleukodystrophy, congenital adrenal hypeφlasia, prostate cancer, diabetes, metabolic disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic puφura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias,] the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, as well as conditions such as transplantation, neuroprotection, fertility, or regeneration. 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 of the 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 Kekuda et al. 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 of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members 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 ofthe invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene Kekuda et al. 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 102; (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 102, 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 102; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 102 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% 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 102; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 102 wherein any amino acid in the mature form ofthe chosen sequence is changed to a different amino acid, provided that no more than 15% of the 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 102; (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 102, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 102 or any variant of said polypeptide wherein any amino acid 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. Kekuda et al.

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 102; (b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 102 is changed from that selected from the group consisting 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 102; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 102 is changed from that selected from the group consisting 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 of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.

A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide 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 Kekuda et al. 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 ofthe 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 of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini ofthe nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 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 of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, or of chemical precursors or other chemicals. Kekuda et al.

A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102, or a complement of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al, (eds.), MOLECULAR CLONING: A

LABORATORY MANUAL 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS 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 corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NO:2«-l , wherein n is an integer between 1 and 102, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

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

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

A "fragment" provided herein is defined as a sequence of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to 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. Kekuda et al.

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

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues 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 of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID

NO:2«-l , wherein n is an integer between 1 and 102, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.

A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the puφoses of this invention, an ORF may be Kekuda et al. 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 bonaftde cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more. The nucleotide sequences determined from the cloning of the human 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:2«- 1 , wherein n is an integer between 1 and 102; or an anti-sense strand nucleotide sequence of SEQ ID NO:2«-l , wherein n is an integer between 1 and 102; or of a naturally occurring mutant of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102. Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe has a detectable label attached, e.g. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.

"A polypeptide having a biologically-active portion of a NOVX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically- active portion of NOVX" can be prepared by isolating a portion of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102, 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. Kekuda et al.

NOVX Nucleic Acid and Polypeptide Variants

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 102, due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102. 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:2«, wherein n is an integer between 1 and 102. In addition to the human NOVX nucleotide sequences of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic 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 of the 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:2«-l , wherein n is an integer between 1 and 102, 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:2«-l, wherein n Kekuda et al. is an integer between 1 and 102. In another embodiment, the nucleic acid is at least 10, 25,

50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 65% homologous to each other typically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion 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% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% 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 Kekuda et al. isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO:2«-l , wherein n is an integer between 1 and 102, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g. , encodes a natural protein).

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

In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102, thereby leading to changes in the amino acid sequences of the Kekuda et al. 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:2«, wherein n is an integer between 1 and 102. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.

For example, amino acid residues that are conserved among the NOVX proteins 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:2«-l, wherein n is an integer between 1 and 102, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 40% homologous to the amino acid sequences of SEQ ID NO:2«, wherein n is an integer between 1 and 102. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO:2«, wherein n is an integer between 1 and 102; more preferably at least about 70% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 102; still more preferably at least about 80% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 102; even more preferably at least about 90% homologous to SEQ ID NO:2«, wherein n is an integer between 1 and 102; and most preferably at least about 95% homologous to SEQ ID NO:2«, wherein n is an integer between 1 and 102.

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

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:2«-l, wherein n is an integer between 1 and 102, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO:2«, wherein n is an integer between 1 and 102, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO :2«-l, wherein n is an integer between 1 and 102, are additionally provided. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" 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 of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion 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 of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically Kekuda et al. synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 -carboxymethylaminomethyl-2-thiouridine,

5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methyl guanine,

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 of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules 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 Kekuαa et at. 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 of the 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:2/7-l, wherein n is an integer between 1 and 102). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence 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 5,1 16,742 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 :141 1-1418.

Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX nucleic acid (e.g., the Kekuda et al.

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; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.

PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S| nucleases (See, Hyrup, et al, I996.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 Kekuda et al. 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: 11 19-11124. 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:2«, wherein n is an integer between 1 and 102. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID NO:2«, wherein n is an integer between 1 and 102, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.

In general, a NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or Kekuda et al. 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 of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly- produced, it is also preferably substantially free of culture medium, /^'. e. , culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.

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

Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence of SEQ ID NO:2«, wherein n is an integer between 1 and 102) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity 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:2«, wherein n is an integer between 1 and 102. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO:2«, wherein n is an integer between 1 and 102, and retains the functional activity ofthe protein of SEQ ID NO:2«, wherein n is an integer between 1 and 102, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2«, wherein n is an integer between 1 and 102, and retains the functional activity of the NOVX proteins of SEQ ID NO:2«, wherein n is an integer between 1 and 102.

Determining Homology Between Two or More Sequences

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

The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs Kekuda et al. 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 of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%,

80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence of SEQ ID NO:2«-l, wherein n is an integer between 1 and 102.

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

Chimeric and Fusion Proteins

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

In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the 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 cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be 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 of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.

A NOVX chimeric or fusion protein 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 Kekuda et al. 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 of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the 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 of the biological activities ofthe naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the NOVX proteins.

Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be Kekuda et al. 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. 1 1 : 477.

Polypeptide Libraries

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

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

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_at>, F_ab- and F(_ab')2 fragments, and an F_ab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGj, IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species. An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID NO:2«, wherein n is an integer between 1 and 102, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments 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 Kekuda et at. 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 of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (K_D) is <1 μM, preferably < 100 nM, more preferably < 10 nM, and most preferably < 100 pM to about 1 pM, as measured by assays such as radiohgand binding assays or similar assays known to those skilled in the art.

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

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

Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic Kekuda et at. 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 of the antigen characterized by a unique binding affinity for it. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies Kekuda et al. that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

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

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell

Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.. Monoclonal Antibody Production Techniques and Applications, Marcel 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 Kekuda et ul. 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 puφose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

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

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

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

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

In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Kekuda et at.

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

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

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

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

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

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 F_(ab')₂ fragment; (iii) an F_ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F_v fragments.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding Kekuda et al. 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 correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

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

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface 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, Kekuda et al. 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 of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One 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 VL 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). Kekuda et al.

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

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CDl 6) 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 purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. Effector Function Engineering

It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Kekuda et al.

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 (/^'. 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 Bi, I, ^J In, Y, and Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14- labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/1 1026.

In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody- receptor conjugate Kekuda et al. 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 ofthe 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 the Invention In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent 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 of the 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 ofthe 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 Kekuda et al. 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 % ^{l j l}I, ^JJS 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 of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target Kekuda et al. 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 surrogate 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 of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug 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 preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain ofthe target Kekuda et al. 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 Kekuda et al. polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof

(e.g., F_ab 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. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method 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 Thory 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 Kekuda et al.

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 of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably- linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g.. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS 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 ofthe 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 of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic Kekuda et al. 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 puφoses: (ϊ) to increase expression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification ofthe 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 of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech 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) 1 19-128. Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector Kekuda et al. 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: 21 1 1-21 18). 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: 1 13-123), 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 Kekuda et al.

Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 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 virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion 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 of the 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 Kekuda et al. foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and 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 d g 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 (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.

Transgenic NOVX Animals

The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell 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-hurnan 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 Kekuda et al. 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, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ ID NO:2«-l , wherein n is an integer between 1 and 102, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue 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 of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene- Kekuda et al. 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:2«-l , wherein n is an integer between 1 and 102), but more preferably, is a non-human homologue of a human

NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NO:2n-

1 , wherein n is an integer between 1 and 102, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).

Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g. , the upstream regulatory region can be altered to thereby alter the expression ofthe endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid 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. 1 13-152. 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 Kekuda et al. animal contain the homologously-recombined DNA by germline transmission ofthe 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 90/1 1354; WO 91/01 140; 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 T351-1355. 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 morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.

Pharmaceutical Compositions

The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be 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 Kekuda et al. 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. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be 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, N.J.) 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, Kekuda et ul. glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention 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 absorption, 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. Kekuda et al.

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. 4,522.81 1. 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 of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Kekuda et al.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (.see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91 : 3054-3057). The pharmaceutical preparation 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 aberrant activity compared to NOVX wild-type protein (e.g. ; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, 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 decon volution; 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 : 1 1422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, et al, 1993. Science 261: 1303; Carrell, et al, 1994. Angew. Chem. Int. Ed Engl. 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061 ; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, Kekuda et al.

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 ¹²T, ^3?S, ^l4C, 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 ofthe test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity of the 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 of the NOVX protein to bind to or interact with a NOVX target molecule. As used herein, a "target molecule" is a molecule with which a NOVX protein binds or interacts in nature, for example, a molecule on the Kekuda et al. 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, IP , 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 of the test compound to bind to the NOVX protein or biologically- active portion thereof. Binding ofthe 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 of the 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 Kekuda et al. the ability of the 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 ofthe 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 of the 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-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton^® X-100, Triton* X-l 14. ThesitX Isotridecypoly(ethylene glycol ether)_n, N-dodecyl— N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-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 Kekuda et al. domain that allows one or both ofthe proteins to be bound to a matrix. For example, GST-

NOVX 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 of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater ( e , statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of Kekuda et al.

NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.

In yet another aspect 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 of the NOVX pathway. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains 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 of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide Kekuda et al. reagents. By way of example, and not of limitation, these sequences can be used to: (/^') 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 ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe NOVX sequences of SEQ ID NO:2«- 1 , wherein n is an integer between 1 and 102, 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 correlating these sequences with genes associated with disease.

Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis 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. Kekuda et al.

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

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

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions ofthe genes actually are preferred 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 correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787.

Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Kekuda et al.

Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence.

Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from 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 corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences 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 polymorphisms (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 purposes. 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, Kekuda et ul. such as those of SEQ ID NO:2«-l, wherein A? is an integer between 1 and 102, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) pmposes 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.. drugs, compounds) on the expression or activity of NOVX in clinical trials.

These and other agents are described in further detail in the following sections. Kekuda et ul.

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:2«-l, wherein n is an integer between 1 and 102, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays 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 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. 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 situ 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 Kekuda et al. antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

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

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

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

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

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

The methods 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 aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression ofthe NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: ( ) a deletion of one or more nucleotides from a NOVX gene; (/ ) an addition of one or more nucleotides to a NOVX gene; (//^"/) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as ofthe methylation pattern ofthe genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells. Kekuda et al.

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 of the 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: 1 173-1 177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1 197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a 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 arrays containing Kekuda et al. 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 NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence ofthe sample NOVX with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., 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 basepair 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 Kekuda et al. 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 polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl. Acad. Sci. USA : 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 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, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of Kekuda et al. high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753. Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986.- Notwre 324: 163; Saiki, et al., 1989. Proc. Natl Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DΝA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DΝA.

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. 1 1 : 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 of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

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

Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which ΝOVX is expressed may be utilized in the prognostic assays described herein. Kekuda et ul.

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 drug) 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 drug. Thus, the pharmacogenomics ofthe individual permits the selection of effective agents (e.g. , drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of 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.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as 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. Kekuda et al.

As an illustrative embodiment, the activity of drug 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 drug response and serious toxicity after taking the standard and safe dose of a drug. 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 CYP2C 19 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 C YP2D6-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 of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug-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, Kekuda et al. 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 pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative ofthe physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe 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 of the agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration 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 Kekuda et ul. 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: (/) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; ( ) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that Kekuda et al. 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, in situ hybridization, and the like).

Prophylactic Methods

In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods 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 puφoses. 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 Kekuda et al. protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.

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

Determination of the Biological Effect of the Therapeutic

In various embodiments 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. Kekuda et ul.

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 nclude 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 of the 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 antibacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.

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

Kekuda et al.

EXAMPLES

Example A: Polynucleotide and Polypeptide Sequences, and Homology Data

Example 1.

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

Table 1 A. NOVl Sequence Analysis

SEQ ID NO: 1 3163 bp

NOVla, CTCCCCACGGCGCCAGGAGGAGGGGCGAGGGCCGGCAGCCCCCTCTCCCGCGCGCGGC

GCAGGAGCCGAGCCCAGCCCGGGGGACCCGCCGCCGCCGGTCATGTGGGCCGGACTGC CGI 13254-01 TCCTTCGGGCCGCCTGTGTCGCGCTCCTGCTGCCGGGGGCACCAGCCCGAGGCTACAC CGGGAGGAAGCCGCCCGGGCACTTCGCGGCCGAGAGACGCCGACTGGGCCCCCACGTC DNA Sequence TGCCTCTCTGGGTTTGGGAGTGGCTGCTGCCCTGGCTGGGCGCCCTCTATGGGTGGTG GGCACTGCACCCTACCCCTCTACTCCTTCGGCTGTGGGAGTGGCATCTGCATCGCTCC CAATGTCTGCTCCTGCCAGGATGGAGAGCAAGGGGCCACCTGCCCAGAAACCCATGGA CCATGTGGGGAGTACGGCTGTGACCTTACCTGCAACCATGGAGGCTGTCAGGAGGTGG CCCGAGTGTGCCCCGTGGGCTTCTCGATGACGGAGACAGCTGTTGGCATCAGGTGTAC AGACATTGACGAATGTGTAACCTCCTCCTGCGAGGGCCACTGTGTGAACACAGAAGGT GGGTTTGTGTGCGAGTGTGGGCCGGGCATGCAGCTGTCTGCCGACCGCCACAGCTGCC AAGACACTGACGAATGCCTAGGGACTCCCTGTCAGCAGAGATGTAAAAACAGCATTGG CAGCTACAAGTGTTCCTGTCGAACTGGCTTCCACCTTCATGGCAACCGGCACTCCTGT GTAGATGTAAACGAGTGTCGGAGGCCATTGGAGAGGCGAGTCTGTCACCATTCCTGCC ACAACACCGTGGGCAGCTTCCTATGCACATGCCGACCTGGCTTCAGGCTCCGAGCTGA CCGCGTGTCCTGTGAAGCTTTCCCGAAAGCCGTGCTGGCCCCATCTGCCATCCTGCAA CCCCGGCAACACCCGTCCAAGATGCTTCTGTTGCTTCCTGAGGCCGGCCGGCCTGCCC TGTCCCCAGGACATAGCCCTCCTTCTGGGGCTCCAGGGCCCCCAGCCGGAGTCAGGAC CACCCGCCTGCCATCTCCCACCCCACGACTACCCACATCCTCCCCTTCTGCCCCTGTG TGGCTGCTGTCCACCCTGCTGGCCACCCCAGTGCCTACTGCCTCCCTGCTGGGGAACC TCAGACCCCCCTCACTCCTTCAGGGGGAGGTGATGGGGACCCCTTCCTCACCCAGGGG CCCTGAGTCCCCCCGACTGGCAGCAGGGCCCTCTCCCTGCTGGCACCTGGGAGCCATG CATGAATCAAGGAGTCGCTGGACAGAGCCTGGGTGTTCCCAGTGCTGGTGCGAGGACG GGAAGGTGACCTGTGAAAAGGTGAGGTGTGAAGCTGCTTGTTCCCACCCAATTCCCTC CAGAGATGGTGGGTGCTGCCCATCGTGCACAGGCTGTTTTCACAGTGGTGTCGTCCGA GCTGAAGGGGATGTGTTTTCACCTCCCAATGAGAACTGCACCGTCTGTGTCTGTCTGG CTGGAAACGTGTCCTGCATCTCTCCTGAGTGTCCTTCTGGCCCCTGTCAGACCCCCCC ACAGACGGATTGCTGTACTTGTGTTCCAGTGAGATGCTATTTCCACGGCCGGTGGTAC GCAGACGGGGCTGTGTTCAGTGGGGGTGGTGACGAGTGTACCACCTGTGTTTGCCAGA ATGGGGAGGTGGAGTGCTCCTTCATGCCCTGCCCTGAGCTGGCCTGCCCCCGAGAAGA GTGGCGGCTGGGCCCTGGGCAGTGTTGCTTCACCTGCCAGGAGCCCACACCCTCGACA GGTTGCTCTCTTGACGACAACGGGGTTGAGTTTCCGATTGGACAGATCTGGTCGCCTG GTGACCCCTGTAGATGGCTCGGTGAGCTGCAAGAGGACAGACTGTGTGGACTCCTGCC CTCACCCGATCCGGATCCCTGGACAGTGCTGCCCAGACTGTTCAGCAGGTTGCACCTA CACAGGCAGAATCTTCTATAACAACGAGACCTTCCCGTCTGTGCTGGACCCATGTCTG AGCTGCATCTGCCTGACAGACTGCAACTACGAGGGAAGGAAGGTGGCGAATGGCCAGG TGTTCACCTTGGATGATGAACCCTGCACCCGGTGCACGTGCCAGCTAGATTCCCTGTC TCCTCTGGAAGAAAAGCAGGGGCTCTCCCCTCACGGAAATGTGGCATTCAGCAAAGCT GGTCGGAGCCTGCATGGAGACACTGAGGCCCCTGTCAACTGTAGCTCCTGTCCTGGGC CCCCGACAGCATCACCCTCGAGGCCGGTGCTTCATCTCCTCCAGCTCCTTTTAAGAAC GAACTTGATGAAAACACAGACTTTACCTACAAGCCCGGCAGGAGCTCATGGTCCACAC TCACTCGCTTTGGGGCTGACAGCCACTTTCCCAGGGGAGCCTGGGGCCTCCCCTCGAC TCTCACCAGGGCCTTCGACCCCTCCAGGAGCCCCCACTCTACCTCTAGCTTCCCCAGG GGCTCCTCAGCCACCTCCTGTGACTCCAGAGCGCTCGTTCTCAGCCTCTGGGGCCCAG ATAGTGTCCAGGTGGCCTCCTCTGCCTGGCACCCTCCTGACGGAAGCTTCAGCACTTT CCATGATGGACCCCAGCCCCTCGAAGACCCCCATCACCCTCCTCGGGCCTCGCGTGCT Kekuda et ul.

TTCTCCCACCACCTCTAGACTCTCCACAGCCCTTGCAGCCACCACCCACCCTGGCCCC

CAGCAGCCCCCAGTGGGGGCTTCTCGGGGGGAAGAGTCCACCATGTAAGGAGGTCACT

GTGTCCGGGAGACTCTGGAGAGAGGACCTCTGCCAGTGGCCCAGGGTGTGTGCAGGGC

AGCTCCAAGGATGAACCTGGTGGGGATGCCTGGGCTCCCTCCTGCAGGGGCCCTGGTG

AGGATGGAAGACCCCCAAGGCTGGATGTAACCTTGTTCCCAAGAAGTGTTTGGAATGT

GCTGTAAGAATGGAGGAAGTCGTTTCCACTGTCAGCATCCTCCCTGGACCGCGTGGCT

GGCTCATCTTTTGAGAAGGGTTGGGACTGCCAAGTTCTCCTGGAGGAAGAGTTGCGTC

CGGCTGGGATTCCACTCACTGGGACTGTACCGCCAGGTGTCATGCGTCTCTCTGAGGT

TTCCTGATTAAAGGTTGTCTCGGTTTCAAAA

ORF Start: ATG at 101 ORF Stop: TAA at 1991

SEQ ID NO: 2 630 aa MW at 66952.5kD jNOVla, M AGLLLRAACVA LLPGAPARGYTGRKPPGHFAAERRRLGPHVC SGFGSGCCPGWA PSMGGGHCTLPLYSFGCGSGICIAPNVCSCQDGEQGATCPETHGPCGEYGCDLTCNHG

I CGI 13254-01 GCQEVARVCPVGFSMTETAVGIRCTDIDECVTSSCEGHCV TEGGFVCECGPGMQLSA DRHSCQDTDECLGTPCQQRCKNSIGSYKCSCRTGFHLHGNRHSCVDVNECRRPLERRV j Protein Sequence CHHSCHNTVGSF CTCRPGFR RADRVSCEAFPKAVLAPSAILQPRQHPSKM LLPE AGRPALSPGHSPPSGAPGPPAGVRTTRLPSPTPRLPTSSPSAPVWLLSTLLATPVPTA S LGN RPPSL QGEVMGTPSSPRGPESPRLAAGPSPC HLGAMHESRSRWTEPGCSQ CWCEDGKVTCEKVRCEAACSHPIPSRDGGCCPSCTGCFHSGWRAEGDVFSPPNENCT VCVCLAGNVSCISPECPSGPCQTPPQTDCCTCVPVRCYFHGR YADGAVFSGGGDECT TCVCQNGEVECSFMPCPELACPREE RLGPGQCCFTCQEPTPSTGCSLDDNGVEFPIG QIWSPGDPCRW GELQEDRLCG LPSPDPDPWTVLPRLFSR HLHRQNLL

SEQ ID NO: 1830 bp iNOVlb, GGTCATGTGGGCCGGACTGCTCCTTCGGGCCGCCTGTGTCGCGCTCCTGCTGCCGGGG i GCACCAGCCCGAGGCTACACCGGGAGGAAGCCGCCCGGGCACTTCGCGGCCGAGAGAC

^CGl 13254-02 GCCGACTGGGCCCCCACGTCTGCCTCTCTGGGTTTGGGAGTGGCTGCTGCCCTGGCTG GGCGCCCTCTATGGGTGGTGGGCACTGCACCCTGCCCCTCTGCTCCTTCGGCTGTGGG

■ DNA Sequence AGTGGCATCTGCATCGCTCCCAATGTCTGCTCCTGCCAGGATGGAGAGCAAGGGGCCA CCTGCCCAGAAACCCATGGACCATGTGGGGAGTACGGCTGTGACCTTACCTGCAGCCA TGGAGGCTGTCAGGAGGTGGCCCGAGTGTGCCCCGTGGGCTTCTCGATGACGGAGACA GCTGTTGGCATCAGGTGTACAGACATTGACGAATGTGTAACCTCCTCCTGCGAGGGCC ACTGTGTGAACACAGAAGGTGGGTTTGTGTGCGAGTGTGGGCCGGGCATGCAGCTGTC TGCCGACCGCCACAGCTGCCAAGACACTGACGAATGCCTAGGGACTCCCTGTCAGCAG AGATGTAAAAACAGCATTGGCAGCTACAAGTGTTCCTGTCGAACTGGCTTCCACCTTC ATGGCAACCGGCACTCCTGTGTAGCTTTCCCGAAAGCCGTGCTGGCCCCATCTGCCAT CCTGCAACCCCGGCAACACCCGTCCAAGATGCTTCTGTTGCTTCCTGAGGCCGGCCGG CCTGCCCTGTCCCCAGGACATAGCCCTCCTTCTGGGGCTCCAGGGCCCCCAGCCGGAG TCAGGACCACCCGCCTGCCATCTCCCACCCCACGACTACCCACATCCTCCCCTTCTGC CCCTGTGTGGCTGCTGTCCACCCTGCTGGCCACCCCAGTGCCTACTGCCTCCCTGCTG GGGAACCTCAGACCCCCCTCACTCCTTCAGGGGGAGGTGATGGGGACCCCTTCCTCAC CCAGGGGCCCTGAGTCCCCCCGACTGGCAGCAGGGCCCTCTCCCTGCTGGCACCTGGG AGCCATGCATGAATCAAGGAGTCGCTGGACAGAGCCTGGGTGTTCCCAGTGCTGGTGC GAGGACGGGAAGGTGACCTGTGAAAAGGTGAGGTGTGAAGCTGCTTGTTCCCACCCAA TTCCCTCCAGAGATGGTGGGTGCTGCCCATCGTGCACAGGCTGTTTTCACAGTGGTGT CGTCCGAGCTGAAGGGGATGTGTTTTCACCTCCCAATGAGAACTGCACCGTCTGTGTC TGTCTGGCTGGAAACGTGTCCTGCATCTCTCCTGAGTGTCCTTCTGGCCCCTGTCAGA CCCCCCCACAGACGGATTGCTGTACTTGTGTTCCAGTGAGATGCTATTTCCACGGCCG GTGGTACGCAGACGGAGCTGTGTTCAGTGGGGGTGGTGACGAGTGTACCACCTGTGTT TGCCAGAATGGGGAGGTGGAGTGCTCCTTCATGCCCTGCCCTGAGCTGGCCTGCCCCC GAGAAGAGTGGCGGCTGGGCCCTGGGCAGTGTTGCTTCACCTGCCAGGAGCCCACACC CTCGACAGGCTGCTCTCTTGACGACAACGGGGTTGAGTTTCCGATTGGACAGATCTGG TCGCCTGGTGACCCCTGTGAGTTATGCATCTGCCAGGCAGATGGCTCGGTGAGCTGCA AGAGGACAGACTGTGTGGACTCCTGCCCTCACCCGATCCGGATCCCTGGACAGTGCTG CCCAGACTGTTCAGCAGGTAATCCCCTGCCTCTGCCCCAAGCCCCCAGGGCAGGGCAT CTCAGGCATCGGGCTCCTTAAGCCCTATACAG Kekuda et al.

Kekuda et al.

NOV Id, GTCWHLGAMHESRSRWTEPGCSQCWCEDGKVTCEKVRCEAACSHPIPSRDGGCCPSCT GCFHSGWRAEGDVFSPPNENCTVCVCLAGNVSCISPECPSGPCQAPPQTDCCTCVPV 212170920 RCYFHGR YADGAVFSGGGDECTTCVCQNGEVECSFMPYPE ACPREEWRLGPGQCCF TCQEPTPSTGCS DDNGVEFPIGVD Protein Sequence

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

Kekuda et al.

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

Table IC. Protein Sequence Properties NOVla

PSort ] 0.5947 probability located in outside; 0.1900 probability located in lysosome analysis: ] (lumen); 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 22 and 23 analysis:

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 I D. Kekuda et ul.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOVla protein was found to have homology to the proteins shown in the BLASTP data in Table 1 E.

Table IE. Public BLASTP Results for NOVla

NOVla Identities/

Protein Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number Residues Portion

Q96DN2 CDNA FLJ32009 fis, clone 1..589 587/589 (99%) 1 0.0 NT2RP7009498, weakly similar to 1..589 587/589 (99%) i J fibulin- 1, isoform A precursor - Homo sapiens (Human), 955 aa.

Q9DBE2 1300015B04Rik protein - Mus 1..615 517/615 (84%) j 0.0 musculus (Mouse), 608 aa. 1..607 547/615 (88%)

Q9IBG7 i Kielin - Xenopus laevis (African 368..589 79/227 (34%) ' 2e-32 I clawed frog), 2327 aa. 1483..1695 109/227 (47%) ^:

Q91 V88 ; POEM (NEPHRONECTIN short 44..373 103/364 (28%) ; le-31 j _' ' isoform) - Mus musculus (Mouse), 35..383 153/364 (41%) \

: 561 aa.

Q9CXD8 ' 6130401 L20Rik protein - Mus 53..261 79/221 (35%) ; 7e-31 ] musculus (Mouse), 528 aa. 96..308 101/221 (44%) j i . _.

PFam analysis indicates that the NOVla protein contains the domains shown in the Table I F.

Kekuda et al.

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: 9 4036 bp

NOV2a, TCCTGGATGAGGCAGCTCAGTCACAGAGGGTGGGCCCCCAGAGAAGGGAAAATTGTGA

GCAGCCCACACTGCTGGCAGATGCGGCATAAGTGTCCCAGCCAGGCTAGGGAGGCGGT CGI 22729-01 GGGCACTGGGTGCACACGATGGCCCTGTGGTTGCTGTCTCAGTCCCGGGCTGTGCTTC CAGGCTTCTCCAGACCACGCCACCAGCCAACAGAAGCGAGACTTCCAGTCCGAGGTCC DNA Sequence TGCTTTCTGCTATGGAACTATTCCACATGACAAGTGGAGGTGATGCAGCGATGTTCAG AGACGGCAAAGAGCCTCAGCCAAGTGCAGAAGCTGCTGCTGCCCCTTCTCTTGCCAAC ATCTCCTGCTTCACCCAGAAGCTGGTGGAGAAGCTGTACAGTGGGATGTTCTCGGCAG ACCCCAGGCATATCCTCCTCTTCATCCTGGAGCACATCATGGTGGTCATTGAGACTGC CTCTTCTCAAAGGGACACTGTCCTCAGCACTTTATACAGCAGTTTAAATAAAGTCATT CTTTATTGCCTATCCAAGCCCCAGCAGTCCCTCTCCGAATGCCTCGGCCTTCTCAGCA TCCTGGGCTTTCTGCAGGAGCACTGGGATGTTGTCTTTGCCACCTACAATTCCAACAT CAGCTTCCTCCTGTGTCTCATGCATTGCCTTTTGCTACTCAATGAGAGAAGTTACCCA GAAGGATTTGGATTGGAGCCCAAGCCTAGAATGTCTACTTATCATCAAGTCTTCCTTT CCCCAAATGAAGACGTGAAAGAAAAAAGAGAAGACTTACCAAGTTTGAGTGATGTCCA ACACAACATCCAGAAGACAGTGCAGACTCTCTGGCAGCAGCTGGTGGCACAAAGGCAG CAGACCCTGGAGGATGCCTTCAAGATCGATCTCTCTGTGAAACCTGGAGAGAGGGAAG TGAAGATTGAAGAGGTCACACCGCTCTGGGAGGAGACGATGCTCAAGGCCTGGCAGCA TTACTTAGCATCTGAGAAGAAGTCACTGGCAAGTCGTTCAAATGTTGCACACCACAGC AAAGTCACTTTGTGGAGTGGAAGCCTGTCCTCAGCCATGAAGCTGATGCCCGGGCGGC AGGCCAAGGACCCTGAGTGCAAGACAGAGGATTTTGTGTCATGTATAGAGAACTACAG AAGAAGAGGACAAGAGCTATATGCATCTTTATACAAAGACCATGTGCAAAGGCGAAAA TGTGGCAACATCAAGGCAGCCAACGCCTGGGCCAGGATCCAGGAGCAGCTTTTTGGGG AGCTGGGCTTGTGGAGCCAGGGGGAAGAAACCAAGCCCTGTTCCCCATGGGAACTCGA Kekuda et ul.

CTGGAGAGAAGGACCAGCTCGAATGAGGAAACGCATCAAACGCTTGTCTCCTTTGGAG GCCCTGAGCTCAGGAAGGCACAAGGAAAGCCAAGACAAAAATGATCATATTTCTCAAA CAAATGCTGAAAACCAAGATGAACTGACACTGAGGGAGGCTGAGGGCGAGCCGGACGA GGTGGGGGTGGACTGCACCCAGCTGACCTTCTTCCCAGCCTTACACGAAAGTCTGCAC TCAGAAGACTTCTTGGAACTGTGTCGGGAAAGACAAGTTATTTTACAAGAGCTTCTTG ATAAAGAAAAGGTGACGCAGAAGTTCTCCCTGGTGATTGTGCAGGGCCACCTGGTGTC AGAAGGGGTCCTGCTTTTTGGCCACCAACACTTCTACATCTGCGAGAACTTCACACTG TCTCCCACGGGTGATGTCTACTGTACCCGTCACTGCTTATCCAACATCAGCGATCCGT TCATTTTCAACCTGTGCAGCAAAGACAGGTCCACTGACCATTACTCGTGCCAGTGCCA CAGCTACGCTGACATGCGGGAGCTACGGCAGGCTCGCTTCCTCCTGCAGGACATCGCC CTGGAGATCTTCTTCCACAATGGATATTCCAAGTTTCTTGTCTTCTACAACAATGATC GGAGTAAGGCCTTTAAAAGCTTCTGCTCTTTCCAACCCAGCCTGAAGGGGAAAGCCAC CTCGGAGGACACCCTCAATCTAAGGAGATACCCCGGCTCTGACAGGATCATGCTGCAG AAGTGGCAGAAAAGGGACATCAGCAATTTTGAGTATCTCATGTACCTCAACACCGCGG CTGGGAGAACCTGCAATGACTACATGCAGTACCCAGTGTTCCCCTGGGTCCTCGCAGA CTACACCTCAGAGACATTGAACTTGGCAAATCCGAAGATTTTCCGGGATCTTTCAAAG CCCATGGGGGCTCAGACCAAGGAAAGGAAGCTGAAATTTATCCAGAGGTTTAAAGAAG TTGAGAAAACTGAAGGAGACATGACTGTCCAGTGCCACTACTACACCCACTACTCCTC GGCCATCATCGTGGCCTCCTACCTGGTCCGGATGCCACCCTTCACCCAGGCCTTCTGC GCTCTGCAGGGCGGAAGCTTCGACGTGGCAGACAGAATGTTCCACAGTGTGAAGAGCA CGTGGGAGTCGGCCTCCAGAGAGAACATGAGTGACGTCAGGGAGCTGACCCCAGAGTT CTTCTACCTGCCTGAGTTCTTAACCAACTGCAACGGGGTAGAGTTCGGCTGCGTGCAG GACGGGACTGTGCTAGGAGACGTGCAGCTCCCTCCCTGGGCTGATGGGGACCCTCGGA AATTCATCAGCCTGCACAGAAAGGCCCTGGAAAGTGACTTTGTCAGTGCCAACCTCCA CCATTGGATAGACCTTATTTTTGGGTACAAGCAGCAGGGGCCAGCCGCAGTGGATGCT GTTAATATCTTCCACCCCTACTTCTACGGTGACAGAATGGACCTCAGCAGCATCACTG ACCCCCTCATCAAAAGCACCATCCTGGGGTTTGTCAGCAACTTTGGACAGGTGCCCAA ACAGCTCTTTACCAAACCTCACCCAGCCAGGACTGCAGCAGGGAAGCCTCTGCCTGGA AAGGATATCTCCACCCCCGTGAGCCTGCCTGGCCACCCACAGCCCTTTTTCTACAGCC TGCAGTCGCTGAGGCCCTCCCAGGTCACGGTCAAAGATATGTACCTCTTTTCTCTAGG CTCAGAGTCCCCCAAAGGGGCCATTGGCCACATTGTCTCTACTGAGAAGACCATTCTG GCTGTAGAGAGGAACAAAGTGCTGCTGCCTCCTCTCTGGAACAGGACCTTCAGCTGGG GCTTTGATGACTTCAGCTGCTGCTTGGGGAGCTACGGCTCCGACAAGGTCCTGATGAC ATTCGAGAACCTGGCTGCCTGGGGCCGCTGTCTGTGCGCCGTGTGCCCATCCCCAACA ACGATTGTCACCTCTGGGACCAGCACTGTGGTGTGTGTGTGGGAGCTCAGCATGACCA AAGGCCGCCCGAGGGGCTTGCGCCTCCGGCAGGCCTTGTATGGACACACACAGGCTGT CACGTGCCTGGCAGCGTCAGTCACCTTCAGCCTCCTGGTGAGCGGCTCCCAGGACTGC iACCTGTATCCTGTGGGATCTGGACCACCTCACCCACGTGACCCGCCTGCCCGCCCATC IGGGAAGGCATCTCAGCCATCACCATCAGTGACGTCTCAGGCACCATTGTCTCCTGTGC JGGGAGCACACTTGTCCCTGTGGAATGTCAATGGACAGCCCCTGGCCAGCATCACCACA IGCCTGGGGCCCAGAAGGAGCCATAACCTGTTGCTGCCTGATGGAGGGCCCAGCATGGG ΑCACAAGCCAGATCATCATCACCGGGAGTCAAGACGGCATGGTCCGGGTTTGGAAGAC TGAGGATGTGAAGATGTCTGTTCCTGGACGGCCAGCAGGAGAGGAGCCCCTGGCTCAG CCTCCAAGCCCAAGAGGCCACAAGTGGGAGAAGAACCTGGCCTTGAGTCGAGAGCTGG ACGTTAGCATTGCTTTGACAGGGAAGCCCAGCAAAACCAGCCCCGCAGTGACTGCTCT GGCCGTGTCCAGAAACCACACCAAACTCCTGGTTGGTGATGAGAGGGGGAGAATATTC TGCTGGTCTGCAGATGGGTAGGAAGAGAGAGGCA

ORF Start: ATG at 7 ORF Stop: TAG at 4021 j SEQ ID NO: 10 1338 aa MW at 150546. lkD

|NOV2a, MRQ SHRG APREGKIVSSPHCWQMRHKCPSQAREAVGTGCTR PCGCCLSPGLCFQA SPDHATSQQKRDFQSEVLLSAMELFHMTSGGDAAMFRDGKEPQPSAEAAAAPSLANIS

I CGI 22729-01 CFTQKLVEKLYSGMFSADPRHILLFILEHIMWIETASSQRDTVLSTLYSS NKVI Y CLSKPQQS SECLGLLSILGFLQEH DWFATYNSNISFLLCLMHCL LNERSYPEG

(Protein Sequence FGLEPKPRMSTYHQVFLSPNEDVKEKREDLPSLSDVQHNIQKTVQTLWQQLVAQRQQT EDAFKIDLSVKPGEREVKIEEVTPLWEETM KAWQHYLASEKKS ASRSNVAHHSKV T WSGSLSSAMKLMPGRQAKDPECKTEDFVSCIENYRRRGQELYASLYKDHVQRRKCG NIKAANAWARIQEQLFGELGL SQGEETKPCSPWELD REGPARMRKRIKRLSPLEAL SSGRHKESQDKNDHISQTNAENQDELTLREAEGEPDEVGVDCTQLTFFPALHESLHSE Kekuda et ul.

DFLELCRERQVILQELLDKE VTQKFSLVIVQGHLVSEGVLLFGHQHFYICENFTLSP TGDVYCTRHCLSNISDPFIFNLCSKDRSTDHYSCQCHSYADMRELRQARFLLQDIALE IFFHNGYSKFLVFY NDRSKAFKSFCSFQPSLKGKATSEDTLNLRRYPGSDRIMLQK QKKDISNFEYLMYLNTAAGRTCNDYMQYPVFPWVLADYTSETLNLANPKIFRDLSKPM GAQTKERKLKFIQRFKEVEKTEGDMTVQCHYYTHYSSAIIVASYLVRMPPFTQAFCAL QGGSFDVADRMFHSVKST ESASRENMSDVRELTPEFFYLPEFLTNCNGVEFGCVQDG TVLGDVQLPPWADGDPRKFISLHRKALESDFVSANLHHWIDLIFGYKQQGPAAVDAVN IFHPYFYGDRMDLSSITDPLIKSTILGFVSNFGQVPKQLFTKPHPARTAAGKP PGKD ISTPVSLPGHPQPFFYSLQSLRPSQVTVKDMYLFSLGSESPKGAIGHIVSTEKTILAV ERNKVLLPPLWNRTFS GFDDFSCCLGSYGSDKVLMTFENLAA GRCLCAVCPSPTTI VTSGTSTWCVWELSMTKGRPRGLRLRQALYGHTQAVTCLAASVTFSLLVSGSQDCTC ILWDLDHLTHVTRLPAHREGISAITISDVSGTIVSCAGAHLSL NVNGQPLASITTAW GPEGAITCCCLMEGPA DTSQIIITGSQDGMVRV KTEDVKMSVPGRPAGEEPLAQPP SPRGHK EKNLALSRELDVSIALTGKPSKTSPAVTALAVSRNHTKLLVGDERGRIFCWi SADG

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

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

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV2a protein was found to have homology to the proteins shown in the BLASTP data in Table 2D. Kekuda et al.

PFam analysis indicates that the NOV2a protein contains the domains shown in the Table 2E.

102 Kekuda et al.

Example 3.

The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.

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

Table 3B. Protein Sequence Properties NOV3a

PSort 0.4600 probability located in plasma membrane; 0.1000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in outside

SignalP Cleavage site between residues 22 and 23 analysis:

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.

I f Kekuda et al.

Kekuda et al.

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

Table 3E. Domain Analysis of NOV3a

Identities/

Pfa Domain | NOV3a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 4.

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

Kekuda et al.

NOV4a, MQRARPTLWAAALTLLVLLRGPPVARAGASSGGLGPWRCEPCDARALAQCAPPPAVC AELVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVN CGI 24229-01 ASAVSRLRAYLLPAPPAPGEPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPL HSKIIIIKKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKF Protein Sequence LNVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVH CYSMQSK

Further analysis ofthe NOV4a protein yielded the following properties shown in Table 4B.

Table 4B. Protein Sequence Properties NOV4a

PSort 0.3703 probability located in outside; 0.1900 probability located in lysosome analysis: (lumen); 0.1080 probability located in nucleus; 0.1000 probability located in endoplasmic reticulum (membrane)

SignalP Cleavage site between residues 28 and 29 analysis:

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 4C.

Kekuda et al.

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

Kekuda et al.

PFam analysis indicates that the NOV4a protein contains the domains shown in the Table 4E.

Example 5.

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

Kekuda et al.

CACGAAGTTGGACATAACTTTGGATCCCCACATGATTCTGGAACAGAGTGCACACCAG GAGAATCTAAGAATTTGGGTCAAAAAGAAAATGGCAATTACATCATGTATGCAAGAGC AACATCTGGGGACAAACTTAACAACAATAAATTCTCACTCTGTAGTATTAGAAATATA AGCCAAGTTCTTGAGAAGAAGAGAAACAACTGTTTTGTTGAATCTGGCCAACCTATTT GTGGAAATGGAATGGTAGAACAAGGTGAAGAATGTGATTGTGGCTATAGTGACCAGTG TAAAGATGAATGCTGCTTCGATGCAAATCAACCAGAGGGAAGAAAATGCAAACTGAAA CCTGGGAAACAGTGCAGTCCAAGTCAAGGTCCTTGTTGTACAGCACAGTGTGCATTCA AGTCAAAGTCTGAGAAGTGTCGGGATGATTCAGACTGTGCAAGGGAAGGAATATGTAA TGGCTTCACAGCTCTCTGCCCAGCATCTGACCCTAAACCAAACTTCACAGACTGTAAT AGGCATACACAAGTGTGCATTAATGGGCAATGTGCAGGTTCTATCTGTGAGAAATATG GCTTAGAGGAGTGTACGTGTGCCAGTTCTGATGGCAAAGATGATAAAGAATTATGCCA TGTATGCTGTATGAAGAAAATGGACCCATCAACTTGTGCCAGTACAGGGTCTGTGCAG TGGAGTAGGCACTTCAGTGGTCGAACCATCACCCTGCAACCTGGATCCCCTTGCAACG ATTTTAGAGGTTACTGTGATGTTTTCATGCGGTGCAGATTAGTAGATGCTGATGGTCC TCTAGCTAGGCTTAAAAAAGCAATTTTTAGTCCAGAGCTCTATGAAAACATTGCTGAA TGGATTGTGGCTCATTGGTGGGCAGTATTACTTATGGGAATTGCTCTGATCATGCTAA TGGCTGGATTTATTAAGATATGCAGTGTTCATACTCCAAGTAGTAATCCAAAGTTGCC TCCTCCTAAACCACTTCCAGGCACTTTAAAGAGGAGGAGACCTCCACAGCCCATTCAG CAACCCCAGCGTCAGCGGCCCCGAGAGAGTTATCAAATGGGACACATGAGACGCTAAC TGCAGCTTTTGCCTTGGTTCTTCCTAGTGCCTACAATGGGAAAACTTCACTCCAAAGA

GAAACCTATTAAGTCATCATCTCCAAACTAAACCCTCACAAGTAACAGTTGAAGAAAA

AATGGCAAGAGATCATATCCTCAGACCAGGTGGAATTACTTAAATTTTAAAGCCTGAA

AATTCCAATTTGGGGGTGGGAGGTGGAAAAGGAACCCAATTTTCTTATGAACAGATAT

TTTTAACTTAATGGCACAAAGTCTTAGAATATTATTATGTGCCCCGTGTTCCCTGTTC

TTCGTTGCTGCATTTTCTTCACTTGCAGGCAAACTTGGCTCTCAATAAACTTTTCG

ORF Start: ATG at 230 ORF Stop: TAA at 1505

SEQ ID NO: 16 425 aa MW at 47237.5kD

NOV5a, MVLLRVLILLLSWAAGMGGQYGNPLNKYIRHYEGLSYNVDSLHQKHQRAKRAVSHITF

! AHEVGHNFGSPHDSGTECTPGES NLGQKENGNYIMYARATSGDKLNWNKFSLCSIRN i CGI 24445-02 ISQVLEKKRNNCFVESGQPICGNGMVEQGEECDCGYSDQCKDECCFDANQPEGRKCKL i KPGKQCSPSQGPCCTAQCAFKSKSEKCRDDSDCAREGICNGFTALCPASDPKPNFTDC

[Protein Sequence NRHTQVCINGQCAGSICEKYGLEECTCASSDGKDDKELCHVCCMKKMDPSTCASTGSV QWSRHFSGRTITLQPGSPC DFRGYCDVFMRCRLVDADGPLARLKKAIFSPELYENIA EWIVAHWWAVLLMGIALIMLMAGFIKICSVHTPSSNPKLPPPKPLPGTLKRRRPPQPI QQPQRQRPRESYQMGHMRR

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

Table 5B. Protein Sequence Properties NOV5a

PSort J 0.4600 probability located in plasma membrane; 0.1800 probability located in analysis: nucleus; 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP j Cleavage site between residues 20 and 21 analysis:

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.

1 1 Kekuda et al.

In a BLAST search of public sequence datbases, the NOV5a protein was found to have homology to the proteins shown in the BLASTP data in Table 5D.

12 Kekuda et al.

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

Table 5E. Domain Analysis of NOV5a

Identities/

Pfa Domain j NOV5a Match Region Similarities Expect Value for the Matched Region squash 200..221 8/22 (36%) 0.25 12/22 (55%)

disintegrin 143..226 33/85 (39%) 2.2e-08 54/85 (64%)

13 Kekuda et al.

Example 6.

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

Further analysis of the NOVόa protein yielded the following properties shown in Table 6B.

r Table 6B. Protein Sequence Properties NOVόa

PSort J 0.5135 probability located in outside; 0.1000 probability located in analysis: : endoplasmic reticulum (membrane); 0.1000 probability located in

' endoplasmic reticulum (lumen); 0.1000 probability located in microbody j (peroxisome)

1 SignalP i Cleavage site between residues 28 and 29 i analysis: :

Kekuda et al.

A search ofthe NOVόa 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.

15 Kekuda et al.

In a BLAST search of public sequence datbases, the NOVόa protein was found to have homology to the proteins shown in the BLASTP data in Table 6D.

PFam analysis indicates that the NOVόa protein contains the domains shown in the Table 6E.

Table 6E. Domain Analysis of NOVόa

j Identities/

J

Pfa Domain NOVόa Match Region ] Similarities Expect Value

! j for the Matched Region

integrin_B 37..165 65/143 (45%) 2.3e-89 Kekuda et al.

Example 7.

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

Table 7A. NOV7 Sequence Analysis

SEQ ID NO: 19 1140 bp

NOV7a, AGGACAACCCCAGCAATGTGGAGAAGCCTGGGGCTTGCCCTGGCTCTCTGTCTCCTCC

CATCGGGAGGAACAGAGAGCCAGGACCAAAGCTCCTTATGTAAGCAACCCCCAGCCTG CGI 24916-01 GAGCATAAGAGATCAAGATCCAATGCTAAACTCCAATGGTTCAGTGACTGTGGTTGCT CTTCTTCAAGCCTCATTTTATGTATTTCTTCCCAAATATTTTAGATTAGAAGACCTGC DNA Sequence GAGTAAAACTGAAGAAAGAAGGATATTCTAATATTTCTTATATTGTTGTTAATCATCA AGGAATCTCTTCTCGATTAAAATACACACATCTTAAGAATAAGGTTTCAGAGCATATT CCTGTTTATCAACAAGAAGAAAACCAAACAGATGTCTGGACTCTTTTAAATGGAAGCA AAGATGACTTCCTCATATATGATAGGTGTGGCCGTCTTGTATATCATCTTGGTTTGCC TTTTTCCTTCCTAACTTTCCCATATGTAGAAGAAGCCATTAAGATTGCTTACTGTGAA AAGAAATGTGGAAACTGCTCTCTCACGACTCTCAAAGATGAAGACTTTTGTAAACGTG TATCTTTGGCTACTGTGGATAAAACAGTTGAAACTCCATCGCCTCATTACCATCATGA GCATCATCACAATCATGGACATCAGCACCTTGGCAGCAGTGAGCTTTCAGAGAATCAG CAACCAGGAGCACCAAATGCTCCTACTCATCCTGCTCCTCCAGGCCTTCATCACCACC ATAAGCACAAGGGTCAGCATAGGCAGGGTCACCCAGAGAACCGAGATATGCCAGCAAG JTGAAGATTTACAAGATTTACAAAAGAAGCTCTGTCGAAAGAGATGTATAAATCAATTA icTCTGTAAATTGCCCACAGATTCAGAGTTGGCTCCTAGGAGCTGATGCTGCCATTGTC

GACATCTGATATTTGAAAAAACAGGGTCTGCAATCACCTGACAGTGTAAAGAAAACCT

CCCATCTTTATGTAGCTGACAGGGACTTCGGGCAGAGGAGAACATAACTGAATCTTGT

CAGTGACGTTTGCCTCCAGCTGCCTGACAAATAAGTCAGCAGCTTATACCCACAGAAG

!CCAGTGCCAGTTGACGCTGAAAGAATCAGGCAAAAAAG iORF Start: ATG at 16 ORF Stop: TGA at 913

SEQ ID NO: 20 1299 aa IMW at 34008.2kD NOV7a, MWRS GLALALCLLPSGGTESQDQSS CKQPPAWSIRDQDPMLNSNGSVTWALLQAS FYVFLPKYFRLEDLRVKLKKEGYSNISYIWNHQGISSRLKYTH KNKVSEHIPVYQQ . JCG124916-01 EENQTDV TLLNGSKDDF IYDRCGR VYHLGLPFSFLTFPYVEEAIKIAYCEKKCGN CS TT KDEDFCKRVS ATVDKTVETPSPHYHHEHHHNHGHQHLGSSE SENQQPGAP j Protein Sequence NAPTHPAPPG HHHHKHKGQHRQGHPENRDMPASEDLQDLQKKLCRKRCINQ LCKLP TDSE APRS Further analysis ofthe NOV7a protein yielded the following properties shown in

Table 7B.

Table 7B. Protein Sequence Properties NOV7a

PSort j 0.5135 probability located in outside; 0.1900 probability located in lysosome analysis: : (lumen); 0.1000 probability located in endoplasmic reticulum (membrane);

] 0.1000 probability located in endoplasmic reticulum (lumen)

17 Kekuda et al.

A searc o t e N V7a prote n aga nst t e eneseq ata ase, a propr etary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7C.

Table 7C. Geneseq Results for NOV7a

NOV7a Identities/

Geneseq j Protein/Organism/Length [Patent Residues/ Similarities for Expect Identifier j #, Date] Match the Matched Value Residues Region

AAU84306 j Human endometrial cancer related 1..299 290/299 (96%) e-176 protein, SEPP1 - Homo sapiens, 381 1..299 294/299 (97%) aa. [WO200209573-A2, 07-FEB- 2002]

AAB03188 Human selenoprotein P - Homo 1..299 290/299 (96%) e-176 sapiens, 381 aa. [WO200031131- 1..299 294/299 (97%) j Al , 02-JUN-2000]

AAB57080 Human prostate cancer antigen 60..299 232/240 (96%) e-142 protein sequence SEQ ID NO: 1658 • 1..240 236/240 (97%) Homo sapiens, 240 aa. [WO200055174-A 1 , 21 -SEP-2000]

AAG03755 j Human secreted protein, SEQ ID 219..299 81/81 (100%) 8e-45 NO: 7836 - Homo sapiens, 1 10 aa. 30..1 10 81/81 (100%) I [EP1033401 -A2, 06-SEP-2000]

AAO06297 Human polypeptide SEQ ID NO 70..147 64/1 13 (56%) 8e-24 20189 - Homo sapiens, 1 13 aa. 1..113 69/1 13 (60%) i [WO200164835-A2, 07-SEP-2001]

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

Table 7D. Public BLASTP Results for NOV7a Kekuda et al.

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

Table 7E. Domain Analysis of NOV7a

Identities/

Pfa Domain NOV7a Match Region ] Similarities Expect Value i for the Matched Region 1

No Significant Matches Found Kekuda et al.

Example 8.

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

Table 8A. NOV8 Sequence Analysis

SEQ ID NO: 21 3123 bp

NOV8a, GATTCCAAGTCGCTGCTGTGCAGAGCAGCAAGTGCTCCGTGCAGGGCTGTTGCTATCA

CTTGGAGGTGAACAGCCTCTTTGCCGGTATTCAGTGAAGAAAGCAAGTCTAAATATGC CGI 26224-01 AGTTCTCTCACTGGAGTGAAAGATGTTTGTTCATTTCTAATCAACTATGCTAGACAGC

TGCAAGCTGAAAAGTGCCTGCAATTTGCCATTTATTTGTAATAAGAAAATAATAAACA DNA Sequence CTGCTGGAACCAGTAATGCAGAAGTCCCCTTGGCTGATCCCGGAATGTACCAGCTGGA

CATTACATTAAGAAGGGGTCAAAGTTTAGCTGCTCGAGATCGAGGAGGGACGAGTGAT

CCATATGTGAAGTTTAAAATCGGAGGAAAAGAAGTTTTTAGAAGTAAGATAATACACA

AGAACCTCAACCCTGTGTGGGAAGAAAAAGCTTGTATTCTGGTTGATCATCTTAGGGA

GCCATTGTATATAAAGGTATTTGACTATGATTTTGGACTACAGGATGACTTTATGGGC

TCAGCCTTTCTGGATCTGACACAATTGGAGTTAAACAGGCCCACAGATGTGACCCTTA

CTCTGAAAGATCCTCATTATCCTGACCATGATCTTGGAATCATTTTGCTCTCAGTCAT

CCTTACCCCTAAAGAAGGAGAGTCCAGGGAGTTTCAGACCCAAAGTTTACGCCTATCA

GACCTACACAGAAAATCGCATCTTTGGAGAGGAATAGTCAGCATCACCTTGATTGAAG

GGAGAGACCTCAAGGCCATGGATTCCAACGGGTTGAGCGATCCCTACGTGAAGTTCCG

GCTTGGGCATCAGAAGTACAAGAGCAAGATTATGCCAAAAACGTTGAATCCTCAGTGG

AGGGAACAATTTGATTTTCACCTTTATGAAGAAAGAGGAGGAGTCATTGATATCACTG

CATGGGACAAAGATGCTGGGAAAAGGGATGATTTCATTGGCAGGTGCCAGGTCGACCT

GTCAGCCCTCAGTAGGGAACAGACGCACAAGCTGGAGTTGCAGCTGGAAGAGGGTGAG

GGACACCTGGTGCTGCTGGTCACTCTGACAGCATCAGCCACAGTCAGCATCTCTGACC

TGTCTGTCAACTCCCTGGAGGACCAGAAGGAACGAGAGGAGATATTAAAGAGATATAG

CCCATTGAGGATATTTCACAACCTGAGAGATGTGGGATTTCTCCAGGTGAAAGTCATC

AGAGCGGAAGGGTTAATGGCTGCCGACGTCACTGGAAAAAGTGACCCATTTTGTGTGG_,

TAGAACTGAACAAAGATAGACTGCTAACACATACTGTCTACAAAAATCTCAATCCTGAJ

GTGGAATAAAGTCTTCACGTTCAACATTAAAGATATCCATTCAGTTCTTGAAGTGACAI

GTTTATGATGAAGATCGGGATCGAAGTGCTGACTTTCTGGGCAAAGTTGCTATACCATJ

TGCTGTCTATTCAAAATGGTGAACAGAAAGCCTACGTCTTGAAAAACAGGCAGCTGAC

AGGGCCAACAAAGGGGGTCATCTATCTTGAAATAGATGTGATTTTTAATGCTGTGAAA

GCCAGCTTACGAACATTAATACCCAAAGAACAGAAGTACATTGAAGAGGAAAACAGAC

TCTCTAAACAGCTGCTACTAAGAAACTTTATCAGAATGAAACGTTGTGTCATGGTGCT

GGTAAATGCTGCATACTACGTTAATAGTTGCTTTGATTGGGATTCACCCCCAAGGAGT

CTCGCTGCTTTTGTGGTAGTGGAGGACATGCTAGAGGACGAGGAAGAAGAAGATGACA

AAGATGACAAGGACAGTGAAAAAAAGGGATTTATAAATAAAATCTATGCCATCCAGGA

GGTATGTGTCAGTGTCCAGAACATCCTAGATGAAGTGGCTTCCTTTGGCGAAAGGATA

AAGAGTACTTTCAACTGGACTGTCCCATTCTTAAGCTGGCTGGCCATTGTAGCCCTCT

GTGTGTTCACAGCCATCCTGTACTGCATTCCGCTGAGATACATTGTCCTTGTCTGGGG

CATCAATAAATTTACAAAAAAGCTTCGGAGTCCATATGCAATTGATAACAATGAACTA

CTTGACTTCCTTTCCAGAGTCCCTTCAGATGTACAAGTGGTGCAATACCAAGAACTGA

AACCAGATCCTTCTCATAGCCCATATAAAAGAAAGAAAAACAATCTTGGCTAGCCAGC

TCCCAGCACTGAGGAGACCAGCATCTGTTTGGGAAGATAAAAGAAAAAGCCCTCAGCC

TCAGCAGCATTTCCTTTCTTTCTGCTTTTTATTTATTTTGCCTTTTTATCATGATCGA

GAGAATCTGTAAATAGTGTACAAAGGCATATGTCTTTGAATATATACTTCTATTGTAC

AGACTCAACTTGATAAAGGTTTTGCTACTGCTGTGTCAAAACCTTGTTAGCTGTGGAT

AATAATATAACACACTGAAAGAACAAATATAAGAATGATAACACTGGAAGATATATTC

TTATCTAATTACAAGTGGATTAAATACTCACCTGTGCTCTGATTAAATCTACATCAAT

TGTAAATGTCGATTTGATTTTAAAGTTTTTTTTTAATGCGACTATTTTTTATCTGAAA

AGTAATCCATTACACTTTTCTATGTTTTATACATTTCAAAAGGGAGGGAAATTCCAAA

GCCTGAATAATGGAATGGATACATTTCAATTTAACATATATTCTGGCTTTAGATCCCG

ACATTCACTCCTGTGCAAATTACTTAGGTATGACTTAGGCTAATTTTAAGCTAATAAG Kekuda et al.

TGAAGGTACATTCACTCCCTCAAGAGAATCAATACTCAGAAGGTTACAAAGTTTTCTT

TATAGAATTTCAATCAATCATTCCATCTAAAACCTTAAAATCTCTACAGGACTACATA

ACATAAATACTGCCAGTTTATAAACGATTGCCTATCTGAATTTTTATACCTACCACTA

CTTTAATTTATACAGTTAGTTAGCAAATTAGCAACCCAGTAAGTACAGTTATCAAAAA

TACTAGGAAACTATATCCATATCGCTTTTGGTGTCAGATTGTATCTGTGCATCTAAAA

ATATTTTAATAAATACTCAAGTGCTCTCAGAGAAAAAAAAAAAAAAAAA

ORF Start: ATG at 163 ORF Stop: TAG at 2197

SEQ ID NO: 22 678 aa MW at 77717.4kD

NOV8a, M DSCKLKSACNLPFICNKKIINTAGTSNAEVPLADPGMYQ DIT RRGQSLAARDRG GTSDPYVKFKIGGKEVFRSKIIHKN NPV EEKACILVDH REPLYIKVFDYDFG QD CGI 26224-01 DFMGSAFLDLTQ ELNRPTDVT TLKDPHYPDHDLGII LSVILTPKEGESREFQTQS RLSDLHRKSHL RGIVSITLIEGRD KAMDSNGLSDPYVKFR GHQKYKSKI PKT Protein Sequence NPQ REQFDFHLYEERGGVIDITA DKDAGKRDDFIGRCQVDLSALSREQTHK ELQL EEGEGH V LVT TASATVSISD SVNSLEDQKEREEILKRYSP RIFHNLRDVGFLQ VKVIRAEGLMAADVTGKSDPFCVVELNKDRLLTHTVYKNLNPEW KVFTFNIKDIHSV LEVTVYDEDRDRSADF GKVAIPL SIQNGEQKAYVLKNRQLTGPTKGVIYLEIDVIF NAVKASLRTLIPKEQKYIEEENRLSKQLLLRNFIRMKRCVMVLVNAAYYVNSCFD DS PPRSLAAFWVEDMLEDEEEEDDKDDKDSEKKGFINKIYAIQEVCVSVQNI DEVASF GERIKSTFNWTVPF S LAIVALCVFTAI YCIPLRYIVLVWGINKFTKKLRSPYAID NNEL DFLSRVPSDVQWQYQELKPDPSHSPYKRKKNNLG

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

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.

Table 8C. Geneseq Results for NOV8a

Geneseq Protein/Organism/Length [Patent NOV8a Identities/ Expect Identifier #, Date] Residues/ Similarities for Value Kekuda et ul.

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

Kekuda et al.

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

Kekuda et al.

Example 9.

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

Table 9A. NOV9 Sequence Analysis

SEQ ID NO: 23 2376 bp

NOV9a, ATGAATGACACAGAAAAACCAGCAGATACTCCCTCTGAGGAAGAGGACTTTGGTGATC CAAGGACATATGACCCAGATTTCAAGGGGCCTGTTGCCAACAGGAGTTGTACAGATGT CG126233-01 TCTGTGCTGTATGATCTTCCTACTGTGTATTATTGGCTACATTGTTTTAGGACTTGTG GCCTGGGTACATGGGGACCCCAGAAGAGCAGCCTATCCTACAGACAGCCAGGGCCACT DNA Sequence TTTGTGGCCAGAAGGGCACTCCCAATGAGAACAAGACCATTTCGTTTTACTTTAACCT GTTACGCTGTACCAGTCCCTCCGTATTGCTAAACCTACAGTGCCCTACCACACAGATC TGTGTCTCCAAGTGCCCAGAAAAATTTTTAACCTATGTGGAAATGCAACTTTTGTACA CAAAAGACAAAAGCTACTGGGAAGACTACCGTCAGTTCTGTAAGACCACTGCTAAGCC TGTGAAGTCTCTCACACAGCTTTTACTGGATGATGATTGTCCAACAGCGATTTTTCCC AGCAAACCTTGTCTCCAGAGATGTTTCCCTGACTTCTCTACCAAAAATGGCACTTTAA CAATAGGAAGTAAGATGATGTTCCAAGATGGAAATGGACGGACAAGAAGTGTTGTAGA ACTCGGGATTGCTGCAAATGGTATCAATAAACTTCTTGATGCAAAGTCACTTGGATTG AAAGTGTTTGAAGACTATGCAAGAACTTGGTATTGGATTCTCATTGGCCTGACGATTG CCATGGTCCTTAGTTGGATATTTTTGATACTTCTGAGGTTCATAGCTGGATGCCTCTT CTGGGTCTTCATGATTGGTGTGATTGGAATTATAGGTTATGGAATATGGCACTGTTAC CAGCAGTACACCAATCTTCAGGAACGCCCAAGTTCTGTATTAACTATCTATGACATCG iGGATTCAGACTAACATAAGCATGTACTTTGAACTGCAACAAACATGGTTCACATTTAT jGATAATACTCTGCATCATTGAAGTGATTGTCATCCTCATGCTGATCTTCCTCAGGAAT JCGAATCCGAGTCGCCATTATCCTGCTGAAGGAAGGAAGCAAAGCCATTGGATATGTTC JCTAGTACATTAGTCTATCCAGCTTTAACTTTCATTTTGCTCTCAATCTGCATTTGCTA icTGGGTCGTGACAGCAGTGTATCAGATTTTTAATACAACTGAAATTGCCAAAGCTTGC JCCTGGGGCTCTGTGTAACTTTGCTTTCTATGGTGGAAAGAGCTTGTACCATCAGTACA TCCCTACCTTCCATGTATACAACTTATTTGTCTTTCTCTGGCTTATAAACTTCGTCAT TGCATTAGGTCAGTGCGCCCTTGCTGGTGCATTCGCTACTTATTACTGGGCCATGAAA AAACCTGATGACATCCCACGATATCCACTTTTTACTGCATTTGGACGAGCCATACGAT ATCACACAGGATCCCTAGCATTTGGATCTTTAATTATTGCATTAATTCAAATGTTTAA AATTGTACTAGAATACTTGGACCACCGTCTTAAACGTACCCAGAACACATTGTCTAAA TTCCTACAATGCTGCCTGAGATGCTGCTTCTGGTGTTTGGAAAATGCAATAAAGTTTT TAAACAGAAATGCCTATATTATGATTGCAATATATGGCAGAAACTTCTGCAGGTCAGC AAAAGATGCTTTCAATCTGCTGATGAGAAATATACTAAAAGTTGCAGTTACAGATGAA GTTACATACTTTGTATTATTCCTGGGGAAACTTCTAGTTGCTGGAAGTATAGGTGTTC TGGCCTTCCTATTCTTCACACAAAGACTGCCAGTGATTGCACAAGGACCAGCATCTTT AAATTACTACTGGGTACCTTTGCTGACAGTCATTTTTGGGTCTTACCTGATTGCACAT GGGTTCTTCAGCGTCTATGCAATGTGTGTTGAAACAATTTTCATCTGCTTCTTGGAAG ATTTAGAAAGAAATGATGGTTCTACTGCAAGACCTTATTATGTGAGTCAACCTTTGCT GAAGATTTTCCAGGAGGAAAATCCACAAACTAGGAAGCAGTAGAAGAGCAAACTGGTC GTCCTACAGCTGTGTGTTACCTTTTCTCCATCTGCTGTGTCTGTGCAACATTTGTTTC ATAAGTGCTTTGTGTTTAGCAACACTGTATTCACGACCTTGTTGGCTTGCATTTGCAT GTTTTATACCAAAGCTTATACTGTACTATGTGAAGCCATCAGAAGTCGCAAGGGAATT GTTAATAACATAAAACATTTTTATACTAAGATCATTTGTTTTGTAATTCGTTTTTAAA GAGTGGCTTGGATGTTTTGAAAATACTACTGAATATGTTAATATTCTTTTAAATCT Kekuda et al.

Further analysis of the NOV9a protein yielded the following properties shown in Table 9B.

Table 9B. Protein Sequence Properties NOV9a

PSort ' 0.6000 probability located in plasma membrane; 0.4000 probability located in analysis: , Golgi body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.0300 probability located in mitochondrial inner membrane

SignalP i Cleavage site between residues 64 and 65 analysis:

A search of the 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.

Kekuda et ul.

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

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

Table 9E. Domain Analysis of NOV9a

Identities/

Pfam Domain NOV9a Match Region Similarities Expect Value for the Matched Region j

No Significant Matches Found Kekuda et ul.

Example 10.

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

Table 10A. NOV10 Sequence Analysis

SEQ ID NO: 25 6065 bp

NOVlOa, CCAGAGGAGCGCCTTCTGCCTCAGAACGGCGTGACTCGGAGAATTGGAGCGTTATTCA

GTATATTAATGTCTTATTGATAATGGCAGAACATCCACCACTACTGGATACAACTCAG CGI 26600-01 ATCTTAAGTAGTGATATTTCTCTTTTGTCTGCCCCTATTGTAAGTGCAGATGGAACAC AACAGGTTATTCTGGTACAAGTTAACCCAGGAGAAGCATTTACAATAAGAAGAGAAGA DNA Sequence TGGACAGTTTCAGTGCATTACAGGTCCTGCTCAGGTTCCAATGATGTCCCCAAATGGT TCTGTGCCTCCTATCTATGTGCCTCCTGGATATGCCCCACAGGTTATTGAAGACAATG GTGTTCGAAGAGTTGTCGTGGTCCCTCAGGCACCAGAGTTTCACCCTGGTAGTCACAC AGTTCTCCACCGTTCTCCACATCCTCCTCTACCTGGTTTCATTTCTGTCCCAACTATG ATGCCGCCTCCACCACGTCATATGTACTCACCCGTGACTGGAGCTGGAGACATGACAA CACAGTATATGCCACAGTATCAGTCTTCACAAGTCTATGGAGATGTAGATGCTCACTC TACACATGGAAGGTCCAACTTTAGAGATGAACGATCTAGTAAAACATATGAACGTTTG CAGAAAAAATTGAAGGATCGCCAAGGAACACAGAAAGATAAAATGAGCAGTCCACCAT CATCACCCCAGAAATGCCCTTCTCCCATTAATGAACATAATGGACTTATAAAAGGACA AATTGCTGGTGGTATAAACACAGGATCAGCAAAAATCAAGTCTGGGAAGGGGAAAGGT GGTACACAAGTTGATACAGAAATTGAAGAAAAAGATGAAGAAACTAAAGCATTTGAAG CACTTCTTTCCAACATTGTCAAACCAGTGGCCTCCGACATCCAGGCAAGGACAGTAGT ACTTACCTGGTCACCACCTTCCAGCCTCATTAATGGTGAAACAGATGAAAGTAGTGTA CCAGAGCTCTATGGTTATGAAGTTCTGATCTCAAGTACTGGAAAAGATGGGAAATACA AAAGTGTATATGTAGGAGAAGAAACAAATATCACTTTAAATGATCTCAAGCCAGCCAT GGATTACCATGCAAAAGTCCAGGCAGAATATAATTCTATAAAGGGAACTCCTTCAGAG GCTGAAATCTTTACCACCTTGAGCTGTGAACCTGATATACCTAATCCACCAAGGATAG CCAATCGGACCAAAAATTCACTCACTTTGCAATGGAAGGCACCTAGTGACAATGGTTC TAAAATCCAAAACTTTGTATTAGAATGGGATGAAGGAAAAGGAAATGGAGAATTTTGT CAGTGTTACATGGGCTCACAGAAACAATTTAAAATTACTAAACTTTCACCAGCAATGG GCTGTAAATTCAGACTATCGGCCAGAAATGACTATGGTACAAGTGGTTTTAGTGAAGA AGTCTTATATTACACCTCAGGCTGTGCTCCTTCTATGCCAGCAAGTCCTGTATTAACC AAGGCTGGAATTACTTGGTTATCCTTACAATGGAGTAAGCCCTCAGGAACACCATCAG ATGAAGGAATTTCTTACATTTTAGAGATGGAGGAAGAAACTTCAGGATATGGTTTTAA GCCTAAATATGATGGAGAAGATCTTGCTTACACAGTGAAAAATCTCAGACGTAGTACT AAGTATAAATTTAAGGTTATTGCTTACAACTCAGAAGGTAAAAGTAATCCAAGTGAAG TAGTAGAATTTACTACTTGCCCTGATAAACCAGGCATACCTGTAAAGCCTTCAGTGAA AGGAAAGATACATTCACACAGTTTTAAAATAACCTGGGATCCACCAAAAGACAATGGC GGAGCAACCATCAATAAATATGTAGTGGAGATGGCAGAAGGTTCTAACGGAAACAAAT GGGAAATGATATACAGTGGTGCTACCAGGGAACATCTTTGTGATCGACTGAATCCAGG CTGTTTCTATCGTTTACGAGTTTACTGCATCAGTGATGGAGGACAGAGTGCGGTCTCT GAATCTTTACTTGTGCAGACTCCAGCTGTGCCTCCTGGCCCATGCCTCCCTCCCAGAT TACAGGGTAGACCCAAAGCAAAAGAAATACAGTTACGATGGGGACCCCCTCTGGTTGA TGGTGGATCACCCATTTCCTGTTACAGTGTGGAAATGTCTCCTATAGAAAAAGATGAA CCTAGAGAAGTTTACCAAGGTTCTGAAGTAGAATGTACAGTGAGCAGCCTTCTTCCTG GAAAGACATACAGCTTCAGACTACGTGCAGCTAACAAAATGGGGTTTGGACCATTTTC AGAAAAATGTGATATTACTACAGCCCCTGGGCCACCAGATCAGTGCAAGCCCCCTCAA GTGACATGTAGATCTGCAACTTGTGCACAAGTGAATTGGGAGGTTCCTTTGAGTAATG GAACAGATGTCACTGAATATCGACTGGAGTGGGGAGGAGTTGAAGGAAGTATGCAGAT ATGTTACTGTGGGCCTGGTCTCAGTTATGAAATAAAAGGACTTTCACCAGCAACTACC TATTATTGCAGGGTCCAGGCTCTGAGTGTTGTGGGTGCAGGCCCTTTCAGTGAAGTAG TAGCCTGTGTGACTCCACCATCAGTTCCTGGCATTGTGACCTGTCTTCAAGAAATAAG CGATGATGAGATAGAAAATCCCCATTATTCACCTTCTACATGCCTTGCAATAAGCTGG GAAAAGCCTTGTGATCATGGTTCGGAAATCCTTGCCTACAGCATAGACTTTGGAGATA AACAATCCCTAACAGTGGGAAAGGTTACAAGCTATATTATCAACAATTTGCAACCAGA TACAACATACAGAATACGAATTCAAGCCTTGAATAGCCTTGGAGCTGGTCCTTTCAGC Kekuda et al.

CATATGATAAAATTAAAAACTAAGCCTCTCCCTCCTGATCCACCTCGTCTGGAATGTG TTGCCTTTAGCCACCAGAACCTTAAGCTGAAATGGGGAGAAGGAACTCCAAAGACATT GTCAACCGATTCTATTCAGTACCACCTTCAGATGGAGGATAAGAATGGACGGTTTGTA TCCCTATACAGAGGACCATGTCATACATACAAAGTACAAAGACTTAATGAGTCAACAT CCTATAAATTCTGTATTCAAGCTTGTAATGAAGCTGGGGAAGGTCCCCTCTCCCAAGA ATATATTTTCACTACTCCAAAATCTGTCCCAGCTGCCTTGAAAGCCCCCAAAATAGAG AAAGTAAATGATCACATTTGTGAAATTACATGGGAGTGTTTACAGCCAATGAAAGGTG ATCCAGTTATTTACAGTCTTCAAGTTATGTTGGGAAAAGATTCAGAATTCAAACAGAT TTACAAGGGTCCCGACTCTTCCTTCCGGTATTCCAGCCTTCAGCTGAACTGTGAATAT CGCTTCCGTGTATGTGCCATTCGCCAGTGCCAAGACTCTCTGGGACACCAGGACCTCG TAGGTCCCTACAGCACCACAGTGCTCTTCATCTCTCAGAGGACTGAACCACCAGCCAG CACCAACAGAGACACTGTGGAAAGCACAAGGACCCGACGGGCACTGAGTGACGAGCAG TGTGCTGCCGTCATCCTTGTGCTGTTTGCTTTCTTTTCCATTTTGATTGCCTTTATCA TTCAGTACTTTGTAATCAAGTGAAAATATAACTTTATTTTTTAACTCTATTACATTTT

ATTTTGTCATGTACTAAAATTATTTCTGTATTGCTTTTATAAAAAACAGTGGCATTTA

GCACTGGCATTGAGACTATAGCACATCATTTTTGCCATTTTCAGTGCTTATATTGTTA

GGTAGAGGCTGGCACTTTATTAGAATGCAAGCCACAAAAATATCAATTTTGTTTTTTT

TTGTTAGGGTGGGTCTTCTTTTTTTCTTTCCCTCTCTCTTTTTTTAACAAATGCCTTC

TTATAGAAAAACTTTCTAAGAGGCAACAATTTAGAATGGATATTTTGACGAATCGGCA

TGAGTGTAACAGTGATAACCTGATCTGTTTGTTTTAAAGATTATTACCAAGTGAAAAA

TTCAGAATGAATAGAATTTACACTAACATGCTATATAAAATGTTAAAGTCTGATGCTG

TGAAAGCAATCTAGTGCTATATTTCTACCTCCTCATTTGTCTTAATTATTTGGTAAGT

GGGATTATGATGAGTAACTGGAGGGGCTTAGAAACAAAAACTGGATGAAAGAGTATGC

ATGAAGAAAAGCTTCTTTGATAAATGTGGAGTTCTTCATTATAAATATATATTCATGA

ATTCACAGATAAGTACTTAAAGAACAGACAGTTTACTTGGCCTAAAAATATTTTGATG

TTTACTCAAAAAGTACCTCTTCAGGTCTTGAGAACATGGAAAAGAATTGAGTGCTTTT

AAATACTTTTTAGAAAGTAATCATAAAAGTAAATTGAATTTCAAACCTATTTGGCTTC

TGTTTTGTGAACCTTTGAACTATATGTATGTGTATAAGGGTATACACATACATATATG

GCATATAACAAGTGTACACATATACACATAACAAGTGTAGAAGTATATATTACATACA

TACACTCACTCTGTCTGGTATAGGCTAATTTTGAAGAACTCCCATAAGTTTCTGCTGC

TTCTCCCATAACTGCTGCCACCACCATCAGAATTCATAATCAAACCTAACCTTTTTGT

TTGGGGCACCAAATCTGAAGACAAAATTAATTTGCACCAGTAAACTTCAAGCTGCTTT

CTTTCTTGAAAACTAAACGTTTAACGTATAATGTCTGTTTGGATACTGTTCCAAATTG

TTGATTGCATGTGGTTAATGTTGCATTAGAGCACTTTGCAATTGCATAATTCATTAAT

GTTTTGTGAGCTTGCATTTGTGAGTTATTGGATGATCAGACTGAATTTTGTCAAGTAT

CACATTGTACATCTTGCCTAGATGTCGATGACTGCAAGTAATAATACAGTTTATAATG

AAACTATCTACAATTCTTGTTTTAGCACATCTGTTATCCGTAAAACACCTGTAACTAG

CTTTTTTAATTTATTATTTGAATTTTAGGATAGCGAATCACTAATTTTTAGTTGCTGA

GGTTGGCATTTTAGTGATTATTAAGCACTTCTGTCAGTCTTTGAAAAAAAGAACGTAT

TTTTTGTGCTTTGAAGATCTCTGAAGAATTTCTTTTATAATAGAATGGGCATGTATTG

TAACAGTTTTATGTCAAATGATCTGTGCTGTAGAAAAACATTAACCCTTGTTCAAAAA

AGAAATGGATAAACTTGGCCTTTCTAAGTGGTAAGAATGACCTGTCACTATAATATAC

TGTATGTTTACATTTTATTTAAATTTAATCTCTTATGTATAGGGTGATAACCTTCCCC

AGAAACAACAGTGATTGCGATTGTTTTCTAGAAACTTCTTTAAAGTGCCACATTTGGC

AGTACAAATGAGTCTGAGTGTAATAGCCCAGAGATTTATATATAGTTGAATGTCTAAA

ATGGTAAAATGTGCCACTGTGTCAAGTTACAGTGGCTTATGTTTTTCATAGTAATTCA

AATGAACTTCCTATTTTTGATAGTAAATGTCATTTAATAGTATACTTGCCATTTGAGC

CTCACTGCAAAATTAGTGCAGAGGAGAAAACAATTTTTAATGTAATCTTGATTTTACC

TCATATACTGTACATTCCAAAAACTCTAAACTTTTTAAAGATTATAGATACACTACCA

AACATATCACCTTAAAATTGTATAAGGCTGAATGAACTTCATACAAATGAAAAAAATC

TCATAAAAATACATAAACTATGTAGCAAAAGTATCTGTAAAATCCATGGAAAATAAAA

GTTGTATCATTCTTTTTGAGATACGTTTATTGTATTCATATATATTCATTATTTGCTA

CCTGTTTAAGAAAGTGAAATGTTATGGTCTCCCCTCTTCCAATGAGCTTAAAACATTT

TTCCCAACAGTATATAAATCTTCAACATGAGAGGATGTATATTTATTATATAAAGCCC

AGTAAAGAATAAAATTAGAAGTTTTATCCTAGG

ORF Start: ATG at 81 ORF Stop: TGA at 3675

SEQ ID NO: 26 1 198 aa MW at 131840.2kD Kekuda et ul.

NOVlOa, MAEHPPLLDTTQI SSDISLLSAPIVSADGTQQVILVQVNPGEAFTIRREDGQFQCIT GPAQVPMMSPNGSVPPIYVPPGYAPQVIEDNGVRRWWPQAPEFHPGSHTV HRSPH CGI 26600-01 PPLPGFISVPTM PPPPRHMYSPVTGAGDMTTQYMPQYQSSQVYGDVDAHSTHGRSNF RDERSSKTYER QKKLKDRQGTQKDKMSSPPSSPQKCPSPINEHNGLIKGQIAGGINT Protein Sequence GSAKIKSGKGKGGTQVDTEIEEKDEETKAFEALLSNIVKPVASDIQARTWLTWSPPS SLINGETDESSVPELYGYEVLISSTGKDGKYKSVYVGEETNIT NDLKPAMDYHAKVQ AEYNSIKGTPSEAEIFTT SCEPDIPNPPRIANRTKNSLTLQWKAPSDNGSKIQNFVL EWDEGKGNGEFCQCYMGSQKQFKITK SPAMGCKFR SARNDYGTSGFSEEV YYTSG CAPSMPASPVLTKAGITWLSLQWSKPSGTPSDEGISYILEMEEETSGYGFKPKYDGED AYTVKNLRRSTKYKFKVIAYNSEGKSNPSEWEFTTCPDKPGIPVKPSVKGKIHSHS FKITWDPPKDNGGATINKYWEMAEGSNGNKWEMIYSGATREHLCDRLNPGCFYR RV YCISDGGQSAVSESLLVQTPAVPPGPCLPPR QGRPKAKEIQLRWGPP VDGGSPISC YSVEMSPIEKDEPREVYQGSEVECTVSSLLPGKTYSFRLRAANKMGFGPFSEKCDITT APGPPDQCKPPQVTCRSATCAQVNWEVPLSNGTDVTEYRLEWGGVEGSMQICYCGPGL SYEIKGLSPATTYYCRVQALSWGAGPFSEWACVTPPSVPGIVTCLQEISDDEIENP HYSPSTC AIS EKPCDHGSEILAYSIDFGDKQSLTVGKVTSYIINNLQPDTTYRIRI QALNSLGAGPFSHMIKLKTKPLPPDPPRLECVAFSHQNLKLKWGEGTPKTLSTDSIQY H QMEDKNGRFVS YRGPCHTYKVQR NESTSYKFCIQACNEAGEGP SQEYIFTTPK SVPAA KAPKIEKVNDHICEITWECLQPMKGDPVIYSLQVM GKDSEFKQIYKGPDSS FRYSSLQLNCEYRFRVCAIRQCQDSLGHQD VGPYSTTVLFISQRTEPPASTNRDTVE STRTRRA SDEQCAAVILV FAFFSI IAFIIQYFVIK

Further analysis ofthe NOVlOa protein yielded the following properties shown in Table 10B.

Table 10B. Protein Sequence Properties NOVlOa

PSort ^~ 0.8500 probability located in endoplasmic reticulum (membrane); 0.6640 analysis: probability located in plasma membrane; 0.1000 probability located in mitochondrial inner membrane; 0.1000 probability located in Golgi body

SignalP No Known Signal Sequence Indicated analysis:

A search of the 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 IOC.

Kekuda et al.

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

Table 10D. Public BLASTP Results for NOVlOa

Kekuda et al.

PFam analysis indicates that the NOVlOa protein contains the domains shown in the Table 10E.

Kekuda et al. fh3 952..1035 23/88 (26%) 0.032 52/88 (59%)

Example 11.

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

Table 11 A. NOV11 Sequence Analysis

SEQ ID NO: 27 1175 bp

±.

NOVl la, ATGGCCACTGCCCAGTTGCAGAGGACTTCCATGACTGCACTGGTATTTCTCAATAAGA TACCACCTGAACACCAGTCTTTGGTGTTAGTGAAGAGTTTCCTCACAGTTTCAGTATC CG127888-01 CTGTATCATGTATTTGAGAGGAATATTTCCAGCATGTGCTTATGGAACCAGATATCTA GATGATCTTTGTGTCAAAATACTGAGAGAAGATAAAAATTGCCCAGGATCTACACAGT DNA Sequence TAGTGAAATGGATACTAGGATGTTACGATGCTTTACAGAAAAAAATATACACAAACCC AGAAGATCCTCAGACAATTTCAGAATGTTACCAATTCAAATTCAAATACACCAATAAT GGACCACTTATGGACTTCATAAGTGAAAGCCAAAGCAATGAGTCTAGCATGTTATGTA CTGACACCGAGAAAGCAAGCACTCTCCTAATTCGCAAGATTTATACCCTAATGCAAAA TCTGGGGCCTTTACCTAATGTTTGTTTGAGCATGAAACGTTTTTACTATGATGAAGTT ACACCCCCAGATTACCAGCCTCCTGGTTTTAAGGATGGTGATTGTGAAGGAGTTATAT TTGAAGGGGAACTTATGTATTTAAATGTGGGCGAAGTCTCAACACCTTTTCCCACCTT CAAAGTAAAAGTGACCACTGAGAGAGAACGAATGGAAAATATTTATTCAACTATACTA TCACTAAAACAAATAAAAACAAAACTTCACAAAATCCTGAGGGACAAAGATGCAGAAG ATGACCAGGCGCATTATACAAGTGATGATTTGGACATTGAAACTAAAATGGAAGAGCA GGAAAAAAACCCTCGATTTTCTGAACTTGGAGAACCAAGTTTAGTTTGTGAGGATGAT GAAATTGTGAGGTATAAAAAAAGTTCAGATCTTTCCATTTCTCATTCTCAGGTTGAGC AGTTAGTCAATAAAACATCGGAACTTGATATGTCTGAAAGCAAAACAAGAAGTGGAAA GTCTTTCAGAATAATGGCAAATGGAAATCAACCAGTAACATCTTCCAAAGAAATTCGG AAGAGAAGTCAACATGAATCTGGGAGAATAGTGCTCCATCACTCGCATTCTTCTAGTC AAGAGTCAGTACCAAAAAGGAGAAAGTTTAGTGAACCAAAGGAACATATATAAAAATT ATTTTTCTTCTGTAT

ORF Start: ATG at 1 ORF Stop: TAA at 1 153

SEQ ID NO: 28 384 aa MW at 43970.6kD

NOVl la, ATAQLQRTSMTALVFLNKIPPEHQSLV VKSFLTVSVSCIMYLRGIFPACAYGTRYL DDLCVKILREDKNCPGSTQ VKWILGCYDALQKKIYTNPEDPQTISECYQFKFKYTNN CG127888-01 GPLMDFISESQSNESSMLCTDTEKASTLLIRKIYTLMQNLGPLPNVC SMKRFYYDEV TPPDYQPPGFKDGDCEGVIFEGEL YLNVGEVSTPFPTFKVKVTTERERMENIYSTIL Protein Sequence S KQIKTK HKILRDKDAEDDQAHYTSDDLDIETK EEQEKNPRFSELGEPSLVCEDD EIVRYKKSSDLSISHSQVEQLVNKTSELDMSESKTRSGKSFRIMANGNQPVTSSKEIR KRSQHESGRIVLHHSHSSSQESVPKRRKFSEPKEHI

Further analysis of the NOVl la protein yielded the following properties shown in Table 1 IB.

Table 11B. Protein Sequence Properties NOVlla Kekuda et al.

PSort 0.6186 probability located in outside; 0.1900 probability located in lysosome analysis: (lumen); 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 53 and 54 analysis:

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.

Kekuda et al.

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

PFam analysis indicates that the NOV 11 a protein contains the domains shown in the Table HE.

Table HE. Domain Analysis of NOVlla

Pfam Domain NOVlla Match Region Identities/ 1 Expect Value Kekuda et al.

Example 12.

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

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

Table 12B. Protein Sequence Properties NOV12a

PSort 0.9190 probability located in plasma membrane; 0.3000 probability located in analysis: lysosome (membrane); 0.2133 probability located in microbody (peroxisome); 0.1000 probability located in endoplasmic reticulum (membrane)

SignalP Cleavage site between residues 23 and 24 analysis:

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. Kekuda et al.

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

Kekuda et al.

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

Kekuda et al.

Example 13.

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

Further analysis ofthe NOVl 3a protein yielded the following properties shown in Table 13B.

Table 13B. Protein Sequence Properties NOV13a

PSort ^* 0.6854 probability located in outside; 0.1000 probability located in analysis: . endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in microbody

(peroxisome)

SignalP ^J, Cleavage site between residues 32 and 33 analysis: =

A search ofthe NOV13a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 13C.

Table 13C. Geneseq Results for NOV13a

Geneseq Protein/Organism/Length [Patent NOVl 3a Identities/ Expect Identifier #, Date] Residues/ » Similarities for Value Kekuda et al.

Match the Matched Residues Region

AAB98325 Human ortholog of r0v0-176.7A 1..59 59/59 (100%) le-27 (PA27) protein sequence - Homo 1..59 59/59 (100%) sapiens, 120 aa. [WO200132926-A2, 10-MAY-2001]

AAY94866 Human protein clone HP 10557 - 1..59 59/59 (100%) le-27 Homo sapiens, 172 aa. 1..59 59/59 (100%) [WO200005367-A2, 03-FEB-2000]

AAB98322 Human PA27 protein (r0v0-176.7A) 1..59 58/59 (98%) le-25 SEQ ID NO:72 - Homo sapiens, 171 1..58 58/59 (98%) aa. [WO200132926- A2, 10-MAY- 2001]

ABB72158 Rat protein isolated from skin cells 1..59 46/59 (77%) 4e-17 SEQ ID NO: 197 - Rattus sp, 171 aa. 1 1..58 48/59 (80%)

[WO200T90357-A1 , 29-NOV-2001 ]

AAB55958 Skin cell protein, SEQ ID NO: 197 - 1..59 46/59 (77%) 4e-17 Rattus sp, 171 aa. [WO200069884- 1..58 48/59 (80%) A2, 23-NOV-2000]

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

Kekuda et ul.

PFam analysis indicates that the NOV 13a protein contains the domains shown in the Table 13E.

Table 13E. Domain Analysis of NOV13a

Identities/ j

Pfam Domain j NOV13a Match Region i Similarities Expect Value i for the Matched Region

No Significant Matches Found

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: 33 ITs 1 bp

NOV 14a, CGAGCGTCGCGGCTATGGCTTATCACTCGGGCTACGGAGCCCACGGCTCCAAGCACAG

GGCCCGGGCAGCCCCGGATCCCCCTCCCCTCTTCGATGACACAAGCGGTGGTTATTCC CG129005-01 AGCCAGCCCGGGGGATACCCAGCCACAGGAGCAGACGTGGCCTTCAGTGTCAACCACT TGCTTGGGGACCCAATGGCCAATGTGGCTATGGCCTATGGCAGCTCCATCGCATCCCA DNA Sequence TGGGAAGGACATGGTGCACAAGGAGCTGCACCGTTTTGTGTCTGTGAGCAAACTCAAG TATTTTTTTGCTGTGGACACAGCCTACGTGGCCAAGAAGCTAGGGCTGCTGGTCTTCC CCTACACACACCAGAACTGGGAAGTGCAGTACAGTCGTGATGCTCCTCTGCCCCCCCG GCAAGACCTCAACGCCCCTGACCTCTATATCCCCACGATGGCCTTCATTACTTACGTG CTCCTGGCTGGGATGGCACTGGGCATTCAGAAAATGATCCTCAGTGTGCTCACGGGGC TGCTGTTCGGCAGCGATGGCTACTACGTGGCGCTGGCCTGGACCTCATCGGCGCTCAT GTACTTCATTGTGCGCTCTTTGCGGACAGCAGCCCTGGGCCCCGACAGCATGGGGGGC Kekuda et al.

Further analysis ofthe NOV 14a protein yielded the following properties shown in Table 14B.

Table 14B. Protein Sequence Properties NOV14a

PSort j 0.7480 probability located in microbody (peroxisome); 0.7000 probability analysis: located in plasma membrane; 0.2000 probability located in endoplasmic

I reticulum (membrane); 0.1000 probability located in mitochondrial inner

_* membrane

SignalP JNo Known Signal Sequence Indicated analysis:

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 14C.

Kekuda et al.

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

Kekuda et al.

PFam analysis indicates that the NOV 14a protein contains the domains shown in the Table 14E.

Kekuda et ul.

Example 15.

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

Table 15A. NOV15 Sequence Analysis

SEQ ID NO: 35 9508 bp

NOV 15a, TACTGCCACCATTGGAACTTTTGATGTTGATGGGGAAGAGTTGCAACACCTCCAGGGT sTGTCCTGCTGATGGTGGCTGCGAATGATTTGCCTTGACAATAGCTGAAAAACCACCAT CG132086-01 ;CTGCAACACGTGGGAGTAAGACTTCTCCTGCTCTTTGCCAGTGGTCTGAGGTGATGAA JCCACCCTGGCTTGGTGTGCTGTGTCCAGCAAACTACAGGGGTGCCGCTGGTAGTTATG DNA Sequence |GTGAAACCAGACACTTTTCTTATCCACGAGATTAAGACTCTTCCTGCTAAAGCGAAGA iTCCAAGACATGGTTGCTATTAGGCACACGGCCTGCAATGAGCAGCAGCGGACAACAAT JGATTCTGCTGTGTGAGGATGGCAGCCTGCGCATTTACATGGCCAACGTGGAGAACACC JTCCTACTGGCTGCAGCCATCCCTGCAGCCCAGCAGTGTCATCAGCATCATGAAGCCTG JTTCGAAAGCGCAAAACAGCTACAATCACAACCCGCACGTCTAGCCAGGTGACTTTCCC 'CATTGACTTTTTTGAACACAACCAGCAGCTGACAGATGTGGAGTTTGGTGGTAACGAC jCTCCTACAGGTCTATAATGCACAACAGATAAAACACCGGCTGAATTCCACTGGCATGT JATGTGGCCAACACCAAGCCCGGAGGCTTCACCATTGAGATTAGTAACAACAATAGCAC JTATGGTGATGACAGGCATGCGGATCCAGATTGGGACTCAAGCAATAGAACGGGCCCCG ^•TCATATATCGAGATCTTCGGCAGAACTATGCAGCTCAACCTGAGTCGCTCACGCTGGT ITTGACTTCCCCTTCACCAGAGAAGAAGCCCTGCAGGCTGATAAGAAGCTGAACCTCTT jCATTGGGGCCTCGGTGGATCCAGCAGGTGTCGCCATGATAGATGCTGTAAAAATTTAT JGGCAAGACTAAGGAGCAGTTTGGCTGGCCTGATGAGCCCCCAGAAGAATTCCCTTCTG jCCTCTGTCAGCAACATCTGCCCTTCAAATCTGAACCAGAGCAACGGCACTGGAGATAG jCGACTCAGCTGCCCCCACTACGACCAGTGGAACTGTCCTGGAGAGGCTGGTTGTGAGT !TCTTTAGAAGCCCTGGAAAGCTGCTTTGCCGTTGGCCCAATCATCGAGAAGGAGAGAA ^'ACAAGAATGCTGCTCAGGAGCTGGCCACTTTGCTGTTGTCCCTGCCAGCACCTGCCAG TGTCCAGCAGCAGTCCAAGAGCCTTCTGGCCAGCCTGCACACCAGCCGCTCGGCCTAC CACAGCCACAAGGATCAGGCCTTGCTGAGCAAAGCTGTGCAGTGTCTCAACACATCTA GCAAAGAGGGCAAGGATTTGGACCCTGAGGTGTTCCAGAGGCTAGTGATCACAGCTCG CTCCATTGCCATCATGCGCCCCAACAACCTTGTCCACTTTACGGAGTCAAAGCTGCCC CAGATGGAAACAGACTGTTTTTTTCCTAGATGTGCCTGCTGGAGTCTAGGGATAGTTG GCATATTGATTGGGGCCCCACTTGAAACTCCCTCCCCAGAAGGAATGGATGAAGGGAA GGAACCGCAGAAGCAGTTGGAAGGAGATTGCTGTAGTTTCATCACCCAGCTTGTGAAC CACTTCTGGAAACTCCATGCATCCAAACCCAAGAATGCCTTCTTGGCACCTGCCTGCC TTCCAGGACTAACTCATATTGAAGCTACTGTCAATGCTCTGGTGGACATCATCCATGG CTACTGTACCTGTGAGCTGGATTGTATTAACACAGCATCCAAGATCTACATGCAGATG CTCTTGTGTCCTGATCCTGCTGTGAGCTTCTCTTGTAAACAAGCTCTAATTCGAGTCC TAAGGCCCAGGAACAAACGGAGACATGTGACTTTACCCTCTTCCCCTCGAAGCAACAC TCCAATGGGAGACAAGGATGATGATGACGATGATGATGCAGATGAGAAAATGCAGTCA Kekuda et nl.

TCAGGGATCCCGAATGGTGGTCACATCCGTCAGGAAAGCCAGGAACAGAGTGAGGTGG

ACCATGGAGATTTTGAGATGGTGTCTGAGTCGATGGTCCTGGAGACAGCTGAAAATGT

CAACAATGGCAACCCCTCTCCCCTGGAGGCCCTGCTGGCAGGCGCAGAGGGCTTCCCC

CCCATGCTGGACATCCCACCTGATGCAGATGACGAGACCATGGTTGAACTAGCCATTG

CCCTGAGCCTGCAGCAGGACCAACAAGCTCCAGCCTCAGACGACGAGGGCAGTACAGC

AGCGACAGATGGTTCTACCCTTCGGACCTCTCCTGCTGACCACGGTGGTAGTGTGGGC

TCGGAGAGCGGGGGCAGTGCAGTGGACTCAGTGGCTGGCGAGCACAGTGTATCTGGCC

GGAGCAGTGCTTATGGCGATGCTACAGCTGAGGGGCATCCGGCTGGACCAGGAAGTGT

CAGCTCAAGCACTGGAGCCATCAGCACCACCACTGGGCACCAGGAGGGAGATGGCTCC

GAGGGAGAAGGAGAAGGAGAAACTGAAGGAGATGTCCACACTAGCAACAGGCTGCACA

TGGTCCGTCTAATGCTGTTGGAGAGATTACTGCAGACCCTGCCTCAATTACGAAACGT

TGGCGGTGTCCGGGCCATCCCATACATGCAGGTCATTCTAATGCTCACTACAGATCTG

GATGGAGAAGATGAGAAAGACAAGGGGGCCCTAGACAACCTGCTCTCCCAGCTTATTG

CTGAGTTGGGTATGGATAAAAAGGATGTCTCCAAGAAGAATGAGCGCAGCGCCCTGAA

TGAAGTCCATCTGGTAGTAATGAGACTCCTGAGTGTCTTCATGTCCCGCACCAAATCT

GGATCCAAGTCTTCCATATGTGAGTCATCTTCCCTCATCTCCAGTGCCACAGCAGCAG

CTCTACTGAGCTCTGGGGCTGTGGACTACTGCCTGCACGTGCTCAAATCACTGCTGGA

ATATTGGAAGAGCCAACAGAATGACGAGGAGCCTGTGGCTACCAGCCAGTTGCTGAAA

CCACATACTACCTCCTCCCCACCTGACATGAGCCCATTCTTTCTCCGCCAGTATGTGA

AGGGTCATGCTGCTGATGTGTTTGAGGCCTATACTCAGCTTCTAACAGAAATGGTACT

GAGGCTTCCTTACCAAATCAAAAAGATTACTGACACCAATTCTCGAATCCCACCTCCT

GTCTTTGACCACTCGTGGTTTTACTTTCTCTCCGAGTACCTCATGATCCAGCAGACTC

CATTTGTGCGCCGTCAAGTCCGCAAACTTCTGCTCTTCATCTGTGGATCCAAAGAGAA

GTACCGCCAGCTCCGGGATTTGCACACCCTGGACTCTCACGTGCGTGGGATCAAGAAG

CTGCTAGAAGAGCAGGGGATATTCCTCCGGGCAAGTGTGGTTACAGCCAGCTCAGGCT

CCGCCTTGCAATATGACACACTCATCAGCCTGATGGAGCACCTGAAAGCCTGTGCAGA

GATTGCCGCCCAGCGAACCATCAACTGGCAGAAATTCTGCATCAAAGATGACTCCGTC

CTGTACTTCCTCCTCCAAGTCAGTTTCCTTGTGGATGAGGGCGTGTCCCCAGTGCTGC

TGCAACTGCTCTCCTGTGCTCTGTGCGGCAGCAAGGTGCTCGCTGCACTGGCAGCCTC

TTCGGGATCCTCCAGTGCTTCTTCCTCCTCAGCCCCTGTGGCTGCCAGTTCTGGACAA

GCCACAACACAGTCCAAGTCTTCCACTAAAAAGAGCAAGAAAGAAGAAAAAGAAAAGG

AGAAAGATGGTGAGACCTCTGGCAGCCAGGAGGACCAGCTGTGCACAGCTCTGGTGAA

CCAGCTGAACAAATTTGCCGATAAGGAAACCCTGATCCAGTTCCTGCGTTGTTTCCTG

TTAGAGTCCAATTCTTCCTCGGTGCGCTGGCAGGCCCACTGTCTGACACTGCACATCT

ACAGAAATTCCAGCAAATCTCAACAGGAGCTCCTGCTAGATCTGATGTGGTCCATCTG

GCCAGAACTCCCAGCCTATGGTCGTAAGGCTGCCCAGTTTGTGGACCTACTAGGATAT

TTCTCCCTGAAAACTCCACAAACAGAGAAGAAGTTGAAGGAGTATTCACAGAAGGCTG

TGGAGATTCTGCGGACTCAAAACCATATTCTTACCAACCACCCCAACTCGAACATTTA

TAACACTTTGTCTGGCTTAGTGGAGTTTGATGGCTATTACCTGGAGAGCGATCCCTGC

CTGGTGTGTAATAACCCGGAAGTACCGTTCTGTTATATCAAGCTGTCTTCCATTAAAG

TGGACACGCGGTACACCACCACCCAGCAGGTTGTGAAGCTCATTGGCAGTCACACCAT

CAGCAAAGTGACAGTGAAAATCGGGGATCTGAAACGGACCAAGATGGTGCGGACCATC

AACCTGTATTATAACAACCGAACCGTGCAGGCCATCGTGGAGTTGAAAAACAAGCCAG

CTCGCTGGCACAAAGCCAAGAAGGTTCAGCTGACCCCTGGACAGACAGAGGTGAAGAT

TGACCTGCCGTTGCCCATTGTGGCCTCCAATCTGATGATTGAGTTTGCAGACTTCTAT

GAAAACTACCAGGCCTCCACAGAGACCCTGCAGTGCCCTCGCTGTAGTGCCTCGGTCC

CTGCCAACCCAGGAGTCTGTGGCAACTGTGGAGAGAATGTGTACCAGTGTCACAAATG

CAGATCCATCAACTACGATGAAAAGGATCCCTTCCTCTGCAATGCCTGTGGCTTCTGT

AAATATGCCCGCTTCGACTTCATGCTCTATGCCAAGCCTTGCTGTGCAGTGGATCCCA

TTGAGAATGAAGAAGACCGGAAGAAGGCTGTATCCAACATCAATACACTTTTGGACAA

AGCTGATCGAGTGTATCATCAGCTGATGGGACACCGGCCACAGCTGGAGAACCTGCTC

TGCAAAGTGAATGAGGCAGCTCCAGAAAAGCCACAGGATGACTCAGGAACAGCAGGGG

GCATCAGCTCCACTTCTGCCAGTGTGAATCGTTACATCCTGCAGTTGGCTCAGGAGTA

TTGTGGAGACTGCAAGAACTCTTTTGATGAACTCTCCAAAATCATCCAGAAAGTCTTT

GCTTCGCGCAAAGAGTTGTTGGAATATGACCTACAGCAGAGGGAAGCAGCCACTAAAT

CATCCCGGACCTCCGTGCAGCCCACATTCACTGCCAGCCAGTACCGTGCCTTATCCGT

CCTGGGCTGTGGCCACACATCCTCCACCAAGTGCTATGGCTGCGCCTCGGCTGTCACA

GAACATTGTATCACACTACTTCGGGCCCTGGCCACCAACCCAGCCTTGAGGCACATCC

TTGTCTCCCAGGGCCTTATCCGGGAGCTCTTTGATTATAATCTTCGCCGAGGGGCTGC

GGCCATGCGGGAGGAGGTCCGCCAGCTCATGTGCCTCCTAACTCGAGACAACCCAGAA

GCCACCCAACAGATGAATGACCTGATTATTGGCAAGGTCTCCACAGCCCTGAAGGGCC Kekuda et nl.

ACTGGGCCAACCCCGATCTGGCAAGTAGCCTGCAGTATGAAATGCTGCTGCTGACGGA TTCTATCTCCAAGGAGGACAGCTGCTGGGAGCTCCGGTTACGCTGTGCTCTCAGCCTT TTCCTCATGGCTGTGAACATTAAGACTCCTGTGGTGGTTGAAAACATTACCCTCATGT GCCTGAGGATCTTGCAGAAGCTGATAAAACCACCTGCTCCCACTAGCAAGAAGAACAA GGATGTCCCCGTTGAGGCCCTCACCACGGTGAAGCCATACTGCAATGAGATCCATGCC CAGGCTCAACTGTGGCTCAAGAGAGACCCCAAGGCATCCTATGATGCCTGGAAGAAGT GTCTTCCTATCAGAGGGATAGATGGCAATGGGAAAGCCCCCAGCAAATCAGAGCTCCG CCATCTCTATTTGACTGAGAAGTATGTGTGGAGGTGGAAACAGTTCCTGAGTCGTCGG GGGAAGAGGACCTCCCCCTTGGATCTCAAACTGGGGCATAACAACTGGCTGCGACAAG TGCTTTTCACTCCAGCAACGCAGGCCGCACGGCAGGCAGCCTGTACCATTGTGGAAGC TCTAGCCACCATTCCCAGCCGCAAGCAGCAGGTCCTGGACCTGCTTACCAGTTACCTG GATGAGCTGAGCATAGCTGGGGAGTGTGCAGCTGAGTACCTGGCTCTCTACCAGAAGC TCATCACTTCTGCGCACTGGAAAGTCTACTTGGCAGCTCGGGGAGTCCTACCCTATGT GGGCAACCTCATCACCAAGGAAATAGCTCGTCTGCTGGCCCTGGAGGAGGCTACCCTG AGTACCGATCTGCAGCAGGGTTATGCCCTTAAAAGTCTCACAGGCCTTCTCTCCTCCT TTGTTGAGGTGGAATCCATCAAAAGACATTTTAAAAGTCGCTTGGTGGGTACTGTGCT GAATGGATACCTGTGCTTGCGGAAGCTGGTGGTGCAGAGGACCAAGCTGATCGATGAG ACGCAGGACATGCTGCTGGAGATGCTGGAGGACATGACCACAGGTACAGAATCAGAAA CCAAGGCCTTCATGGCTGTGTGCATTGAGACAGCCAAGCGCTACAATCTGGATGACTA CCGGACCCCGGTGTTCATCTTCGAGAGGCTCTGCAGCATCATTTATCCTGAGGAGAAT GAAGTCACTGAGTTCTTTGTGACCCTGGAGAAGGATCCCCAACAAGAAGACTTCTTAC AGGGCAGGATGCCTGGGAACCCGTATAGCAGCAATGAGCCAGGCATCGGGCCGCTGAT GAGGGATATAAAGAACAAGATTTGCCAGGACTGTGACTTAGTGGCCCTCCTGGAAGAT GACAGTGGCATGGAGCTTCTAGTGAACAATAAAATCATTAGTTTGGACCTTCCTGTGG CTGAAGTTTACAAGAAAGTCTGGTGTACCACGAATGAGGGAGAGCCCATGAGGATTGT TTATCGTATGCGGGGGCTGCTGGGCGATGCCACAGAGGAGTTCATTGAGTCCCTGGAC TCTACTACAGATGAAGAAGAAGATGAAGAAGAAGTGTATAAAATGGCTGGTGTGATGG CCCAGTGTGGGGGCCTGGAATGCATGCTTAACAGACTCGCAGGGATCAGAGATTTCAA GCAGGGACGCCACCTTCTAACAGTGCTACTGAAATTGTTCAGTTACTGCGTGAAGGTG AAAGTCAACCGGCAGCAACTGGTCAAACTGGAAATGAACACCTTGAACGTCATGCTGG GGACCCTAAACCTGGCCCTTGTAGCTGAACAAGAAAGCAAGGACAGTGGGGGTGCAGC TGTGGCTGAGCAGGTGCTTAGCATCATGGAGATCATTCTAGATGAGTCCAATGCTGAG CCCCTGAGTGAGGACAAGGGCAACCTCCTCCTGACAGGTGACAAGGATCAACTGGTGA ^TGCTCTTGGACCAGATCAACAGCACCTTTGTTCGCTCCAACCCCAGTGTGCTCCAGGG CCTGCTTCGCATCATCCCGTACCTTTCCTTTGGAGAGGTGGAGAAAATGCAGATCTTG GTGGAGCGATTCAAACCATACTGCAACTTTGATAAATATGATGAAGATCACAGTGGTG iATGATAAAGTCTTCCTGGACTGCTTCTGTAAAATAGCTGCTGGCATCAAGAACAACAG CAATGGGCACCAGCTGAAGGATCTGATTCTCCAGAAGGGGATCACCCAGAATGCACTT GACTACATGAAAAAGCACATCCCTAGCGCCAAGAATTTGGATGCCGACATCTGGAAAA AGTTTTTGTCTCGCCCAGCCTTGCCATTTATCCTAAGGCTGCTTCGGGGCCTGGCCAT CCAGCACCCTGGCACCCAGGTTCTGATTGGAACTGATTCCATCCCGAACCTGCATAAG CTGGAGCAGGTGTCCAGTGATGAGGGCATTGGGACCTTGGCAGAGAACCTGCTGGAAG CCCTGCGGGAACACCCTGACGTAAACAAGAAGATTGACGCAGCCCGCAGGGAGACCCG GGCAGAGAAGAAGCGCATGGCCATGGCAATGAGGCAGAAGGCCCTGGGCACCCTGGGC ATGACGACAAATGAAAAGGGCCAGGTCGTGACCAAGACAGCACTCCTGAAGCAGATGG AAGAGCTGATCGAGGAGCCTGGCCTCACGTGCTGCATCTGCAGGGAGGGATACAAGTT CCAGCCCACAAAGGTCCTGGGCATTTATACCTTCACGAAGCGGGTAGCCTTGGAGGAG ATGGAGAATAAGCCCCGGAAACAGCAGGGCTACAGCACCGTGTCCCACTTCAACATTG TGCACTACGACTGCCATCTGGCTGCCGTCAGGTTGGCTCGAGGCCGGGAAGAGTGGGA GAGTGCCGCCCTGCAGAATGCCAACACCAAGTGCAACGGGCTCCTTCCGGTCTGGGGA CCTCATGTCCCTGAATCAGCTTTTGCCACTTGCTTGGCAAGACACAACACTTACCTCC AGGAATGTACAGGCCAGCGGGAGCCCACGTATCAGCTCAACATCCATGACATCAAACT GCTCTTCCTGCGCTTCGCCATGGAGCAGTCGTTCAGCGCAGACACTGGCGGGGGCGGC CGGGAGAGCAACATCCACCTGATCCCGTACATCATTCACACTGTGCTTTACGTCCTGA ACACAACCCGAGCAACTTCCCGAGAAGAGAAGAACCTCCAAGGCTTTCTGGAACAGCC CAAGGAGAAGTGGGTGGAGAGTGCCTTTGAAGTGGACGGGCCCTACTATTTCACAGTC TTGGCCCTTCACATCCTGCCCCCTGAGCAGTGGAGAGCCACACGTGTGGAAATCTTGC GGAGGCTGTTGGTGACCTCGCAGGCTCGGGCAGTGGCTCCAGGTGGAGCCACCAGGCT GACAGATAAGGCAGTGAAGGACTATTCCGCTTACCGTTCTTCCCTTCTCTTTTGGGCC CTCGTCGATCTCATTTACAACATGTTTAAGAAGGTGCCTACCAGTAACACAGAGGGAG GCTGGTCCTGCTCTCTCGCTGAGTACATCCGCCACAACGACATGCCCATCTACGAAGC Kekuda et ul.

TGCCGACAAAGCCCTGAAAACCTTCCAGGAGGAGTTCATGCCAGTGGAGACCTTCTCA GAGTTCCTCGATGTGGCCGGTCTTTTATCAGAAATCACCGATCCAGAGAGCTTCCTGA AGGACCTGTTGAACTCAGTCCCCTGACCACCACACAGCAGCTGCGGCGGCGAAGACGA

AGCTGGCTTGCCTTCCACCCTCTGTTCTCCCTCCTTGTGCATTAAGTTCCCTCCGCGG

GATGCTGCATTGTTACCCCGCCCTCCCCTCTCTCATTTTTCTTGGTGTGGCTTGGGGT

TTTTAGGCTTCCTGTTTTATCTCGTGTGTGTGGTGCACCAGCTATGAGGTTGTCTGTA

ACCCAAGCCATCAAAGGGCCTGTACATACCTAGGAGCCATGAGTTGTCCCGGCCAGCT

TCATACTTGAGTGTGCACATCTTGAGAAATAAACAAGTGACTTAACACACATTG

ORF Start: ATG at 170 ORF Stop: TGA at 9188

SEQ ID NO: 36 3006 aa MW at 334825.2kD

NOVl 5a, MNHPGLVCCVQQTTGVPLWMVKPDTFLIHEIKTLPAKAKIQDMVAIRHTACNEQQRT

TMILLCEDGSLRIYMANVENTSYWLQPSLQPSSVISIMKPVRKRKTATITTRTSSQVT CG132086-01 FPIDFFEHNQQLTDVEFGGNDLLQVYNAQQIKHRLNSTGMYVANTKPGGFTIEISNNN

STMVMTGMRIQIGTQAIERAPSYIEIFGRTMQLNLSRSRWFDFPFTREEALQADKKLN Protein Sequence LFIGASVDPAGVAMIDAVKIYGKTKEQFGWPDEPPEEFPSASVSNICPSNLNQSNGTG

DSDSAAPTTTSGTVLERLWSSLEALESCFAVGPIIEKERNKNAAQELATLLLSLPAP

ASVQQQSKSLLASLHTSRSAYHSHKDQALLSKAVQCLNTSSKEGKDLDPEVFQRLVIT

ARSIAIMRPNNLVHFTESKLPQMETDCFFPRCACWSLGIVGILIGAPLETPSPEG DE

GKEPQKQLEGDCCSFITQLVNHFWKLHASKPKNAFLAPACLPGLTHIEATVNALVDII

HGYCTCELDCINTASKIYMQMLLCPDPAVSFSCKQALIRVLRPRNKRRHVTLPSSPRS

NTPMGDKDDDDDDDADEKMQSSGIPNGGHIRQESQEQSEVDHGDFE VSESMVLETAE

NVNNGNPSPLEALLAGAEGFPPMLDIPPDADDETMVELAIALSLQQDQQAPASDDEGS

TAATDGSTLRTSPADHGGSVGSESGGSAVDSVAGEHSVSGRSSAYGDATAEGHPAGPG

SVSSSTGAISTTTGHQEGDGSEGEGEGETEGDVHTSNRLHMVRLMLLERLLQTLPQLR

NVGGVRAIPYMQVILMLTTDLDGEDEKDKGALDNLLSQLIAELGMDKKDVSKK ERSA

LNEVHLWMRLLSVFMSRTKSGSKSSICESSSLISSATAAALLSSGAVDYCLHVLKSL_ι

LEYWKSQQNDEEPVATSQLLKPHTTSSPPDMSPFFLRQYVKGHAADVFEAYTQLLTEMj

VLRLPYQIKKITDTNSRIPPPVFDHSWFYFLSEYL IQQTPFVRRQVRKLLLFICGSKJ

EKYRQLRDLHTLDSHVRGIKKLLEEQGIFLRASWTASSGSALQYDTLISL EHLKACJ

AEIAAQRTINWQKFCIKDDSVLYFLLQVSFLVDEGVSPVLLQLLSCALCGSKVLAALA

ASSGSSSASSSSAPVAASSGQATTQSKSSTKKSKKEEKEKEKDGETSGSQEDQLCTAL

VNQLNKFADKETLIQFLRCFLLESNSSSVRWQAHCLTLHIYRNSSKSQQELLLDLMWS

IWPELPAYGRKAAQFVDLLGYFSLKTPQTEKKLKEYSQKAVEILRTQNHILTNHPNSN

IYNTLSGLVEFDGYYLESDPCLVCNNPEVPFCYIKLSSIKVDTRYTTTQQWKLIGSH

TISKVTVKIGDLKRTKMVRTINLYYNNRTVQAIVELKNKPARWHKAKKVQLTPGQTEV

KIDLPLPIVASNLMIEFADFYENYQASTETLQCPRCSASVPANPGVCGNCGENVYQCH

KCRSINYDEKDPFLCNACGFCKYARFDFMLYAKPCCAVDPIENEEDRKIAVSNINTLL

DKADRVYHQLMGHRPQLENLLCKVNEAAPEKPQDDSGTAGGISSTSASV RYILQLAQ

EYCGDCKNSFDELSKIIQKVFASRKELLEYDLQQREAATKSSRTSVQPTFTASQYRAL

SVLGCGHTSSTKCYGCASAVTEHCITLLRALATNPALRHI VSQG IRELFDYNLRRG

AAAMREEVRQLMCLLTRDNPEATQQMNDLIIGKVSTALKGHWANPDLASSLQYEMLLL

TDSISKEDSCWELRLRCALSLFLMAV IKTPVWENITLMCLRILQKLIKPPAPTSKK,

NKDVPVEALTTVKPYCNEIHAQAQLWLKRDPKASYDAWKKCLPIRGIDGNGKAPSKSEj

LRHLYLTEKYVWRWKQFLSRRGKRTSPLDLKLGHNNWLRQVLFTPATQAARQAACTIV

EALATIPSRKQQVLDLLTSYLDELSIAGECAAEYLALYQKLITSAHWKVYLAARGVLP

YVGNLITKEIARLLALEEATLSTDLQQGYALKS TGLLSSFVEVESIKRHFKSRLVGT

VLNGYLCLRKLWQRTKLIDETQDMLLEMLEDMTTGTESETKAFMAVCIETAKRYNLD

DYRTPVFIFERLCSIIYPEENEVTEFFVTLEKDPQQEDFLQGRMPGNPYSSNEPGIGP

LMRDIKNKICQDCDLVALLEDDSGMELLVNNKIISLDLPVAEVYKKVWCTTNEGEPMR

IVYRMRGLLGDATEEFIESLDSTTDEEEDEEEVYKMAGVMAQCGGLECMLNRLAGIRD

FKQGRHLLTVLLKLFSYCVKVKVNRQQLVKLE NTLNVMLGTLNLALVAEQESKDSGG

AAVAEQVLSIMEIILDESNAEPLSEDKGNLLLTGDKDQLVMLLDQINSTFVRSNPSVL

QGLLRIIPYLSFGEVEKMQILVERFKPYCNFDKYDEDHSGDDKVFLDCFCKIAAGIKN

NSNGHQLKDLILQKGITQNALDYMKKHIPSAKNLDADIWKKFLSRPALPFILRLLRGL

AIQHPGTQVLIGTDSIPNLHKLEQVSSDEGIGTLAENLLEALREHPDV KKIDAARRE

TRAEKKRMAMAMRQKALGTLGMTTNEKGQWTKTALLKQMEELIEEPGLTCCICREGY

KFQPTKVLGIYTFTKRVALEEMENKPRKQQGYSTVSHFNIVHYDCHLAAVRLARGREE

WESAALQNANTKCNGLLPVWGPHVPESAFATCLARHNTYLQECTGQREPTYQLNIHDI Kekuda et ul.

KLLFLRFAMEQSFSADTGGGGRESNIHLIPYIIHTVLYVLNTTRATSREEKNLQGFLE QPKEKWVESAFEVDGPYYFTVLALHILPPEQWRATRVEILRRLLVTSQARAVAPGGAT RLTDKAVKDYSAYRSSLLFWALVDLIYNMFKKVPTSNTEGGWSCSLAEYIRHNDMPIY EAADKALKTFQEEFMPVETFSEFLDVAGLLSEITDPESFLKDLLNSVP

Further analysis ofthe NOV 15a protein yielded the following properties shown in Table 15B.

Table 15B. Protein Sequence Properties NOV15a

PSort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400 analysis: probability located in plasma membrane; 0.4600 probability located in Golgi body; 0.1800 probability located in nucleus

SignalP No Known Signal Sequence Indicated analysis:

A search of the 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 15C.

Table 15C. Geneseq Results for NOVl 5a h- \ NOV15a Identities/

Geneseq Protein/Organism/Length Residues/ Similarities for Expect Identifier [Patent #, Date] Match the Matched Value Residues Region

AAY53675 Mechanical stress induced protein ..2938 '834/2974 (95%) 0.0 274 amino acid sequence - Rattus 318..3262 : 2881/2974 (96%) sp, 3262 aa. [WO9960164-A1, 25- NOV-1999]

AAU28088 Novel human secretory protein, 584..3006 ] 2423/2458 (98%) 0.0 Seq ID No 257 - Homo sapiens, 1..2458 ] 2423/2458 (98%) 2458 aa. [WO200166689-A2, 13- SEP-2001]

AAM39071 Human polypeptide SEQ ID NO 584..3006 j 2421/2458 (98%) 0.0 Kekuda et nl.

In a BLAST search of public sequence datbases, the NOVl 5a protein was found to have homology to the proteins shown in the BLASTP data in Table 15D.

Kekuda et al.

PFam analysis indicates that the NOVl 5a protein contains the domains shown in the Table 15E.

Example 16.

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

Kekuda et al.

Kekuda et al.

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

Kekuda et al.

Table 16B. Comparison of NOVlόa against NOVl 6b.

NOVlόa Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV 16b 686-711 26/26 (100%) 667-692 26/26 (100%)

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

Table 16C. Protein Sequence Properties NOVlόa

PSort 0.4323 probability located in outside; 0.1376 probability located in microbody j analysis: (peroxisome); 0.1000 probability located in endoplasmic reticulum

\

! (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

! SignalP Cleavage site between residues 27 and 28

I ! analysis:

A search ofthe NOVlόa 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.

Kekuda el al. aa. [WO200050068-A2, 31-AUG- 2000]

AAY69069 Amino acid sequence of a human 27..711 679/703 (96%) 0.0 reduced tropoelastin derivative - 1..698 680/703 (96%) Synthetic, 698 aa. [WO200004043- A1. 27-JAN-2000]

AAY01302 Human tropoelastin variant 27..711 679/703 (96%) 0.0 SHELdelta26A - Homo sapiens, 1..698 680/703 (96%) 698 aa. [WO9903886-A1, 28-JAN- 1999]

AAW46315 Human elastin containing non- 27..711 679/735 (92%) 0.0 natural polypeptide MFU-1 1..730 680/735 (92%) sequence - Homo sapiens, 730 aa. [WO9805685-A2, 12-FEB-1998]

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

Kekuda et al.

PFam analysis indicates that the NOVl 6a protein contains the domains shown in the Table 16F.

Table 16F. Domain Analysis of NOVlόa

i Identities/ ]

Pfa Domain NOVlόa Match Region Similarities j Expect Value j for the Matched Region

No Significant Matches Found

Example 17.

The NOVl 7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 17A.

Table 17A. NOV17 Sequence Analysis

'SEQ ID NO: 41 11072 bp

:NOV17a, lATCCGAGTCACCTGCAGGACCGAAATGGAGGAGAGAGCACAGCACTGCCTGTCCAGAT

I TACTAGACAACTCTGCCCTGAAGCAGCAGGAGTTACCCATACACCGGCTATATTTCAC

JCG132343-01 GGCCAGGAGAGTCCTCTTTGTCTTTTTCGCAACAGGAATATTCTGCCTTTGTATGGGC i ATCATCCTTATATTGTCTGCAAGGAGCACTCAGGAAATAGAGGTTAATTACACAAGAA iDNA Sequence TATGTGCAAATTGTGCAAAACTGCGAGAAAATGCCTCTAATTTTGACAAGGAATGCAC CTGCTCTATTCCCTTTTACCTTTCAGGAAAAATGCAGGGTAATGTTTATATGTACTAC AAATTGTATGGCTTCTATCAGAACCTGTATCTATATATTCGATCCAGAAGTAATAGAC AACTGGTGGGCAAAGATGTAAAAGTAGTTGAGGATTGTGCCCCATTTAAAATGTCCGA CAATAAGACCCCCATCGTTCCTTGTGGTGCTATTGCCAACAGCATGTTCAATGACACC ATAATTCTTTCACACAACATTAATTCATCTGTACAAATCAAAGTGCCAATGTTAAAGA GTAGACTTACGTGGTGGACAGATAAGTATGTCAAATTTCAGAATCTAAGTTTCAAGAA TCTTGCTGATGAATTTAGAGGTACCACAAAGCCCCCAAACTGGCCCAAGCCTATCTAT GACTTGGATAAAAAGGATCCAAGAAACAATGGCTTCCTCAATGATGACTTCATTGTGT GGATGCGGGCAGCTGCCTTTCCCACTTTCAAAAAACTGTATGGTCGACTCAGTCGAAC ACACCATTTTATAGAAGGCTTGCCTGCTGGTAATTATAGTTTCAACATAACCTATAGT TTCCCAGTAACCAGGTTCCACGGAGAAAAATCAGTTGTTCTCTCCACCCTGACATGGT GTGGGGGTAATAGCCTTTTCTTAGGTCTTGCCTACACAGTGACAGGAGCTATGACATG GTTGGCCTCCTTTGCCATGATGGCAATTCACATCATGCTGAAAAACAAGAAAATGTCC TTCTTCCATCAATAAAGTCAAGCTTTAA

ORF Start: ATG at 25 ORF Stop: TAA at 1057

SEQ ID NO: 42 344 aa MW at 39698.8kD Kekuda et al.

|NOV17a, MEERAQHCLSRLLDNSALKQQELPIHRLYFTARRVLFVFFATGIFCLCMGIILILSAR i STQEIEVNYTRICANCAKLRENASNFDKECTCSIPFYLSGKMQGNλTmYYKLYGFYQN

|CG132343-01 LYLYIRSRSNRQLVGKDVKWEDCAPFKMSDNKTPIVPCGAIANSMFNDTIILSHNIN SSVQIKVPMLKSRLTWWTDKYVKFQNLSFKNLADEFRGTTKPPNWPKPIYDLDKKDPR j Protein Sequence NNGFLNDDFIVWMRAAAFPTFKKLYGRLSRTHHFIEGLPAGNYSFNITYSFPVTRFHG EKSWLSTLTWCGGNSLFLGLAYTVTGAMTWLASFAMMAIHIMLKNKK SFFHQ

Further analysis ofthe NOV 17a protein yielded the following properties shown in Table 17B.

Table 17B. Protein Sequence Properties NOVl 7a

PSort 0.7900 probability located in plasma membrane; 0.7294 probability located in analysis: microbody (peroxisome); 0.3000 probability located in Golgi body; 0.2000 ] probability located in endoplasmic reticulum (membrane)

SignalP I Cleavage site between residues 60 and 61 analysis:

A search of the NOV 17a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 17C.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOVl 7a protein was found to have homology to the proteins shown in the BLASTP data in Table 17D.

Kekuda et al.

PFam analysis indicates that the NOV 17a protein contains the domains shown in the Table 17E.

Table 17E. Domain Analysis of NOV17a

Identities/

Pfam Domain NOVl 7a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 18.

The NOV 18 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 18A.

Table 18A. NOV18 Sequence Analysis

SEQ ID NO: 43 1084 bp

|NOV18a, GAAGCTTCTGGATCCTACGCTCATCTCTACAGAGGAGAACATGCACGCAGCAGAGATC

ATGGGGCCCCTCTCAGCCCCTCCCTGCACAGAGCACATCAAATGGAAGGGGCTCCTGC CG132423-01 TCACAGCATTACTTTTAAACTTCTGGAACTTGCCTACCACTGCCCAAGTCATGATTGA AGCCCAGCCACCCAAAGTGTCCGAGGGGAAGGATGTTCTTCTACTTGTCCAAATCAGG DNA Sequence GACCTCTACCATTACATTACATCATATGTAGTAGACGGTCAAATAATTATATATGGAC CGGCATACAGTGGACGAGAAACAGTATATTCCAATGCATCCCTGCTGATCCAGAATGT CACCCGGGAGGACGCAGGATCCTACACCTTACACATCATAAAGCGAGGTGATGGGACT AGAGGAGTAACTGGATATTTCACCTTCACCTTATACCTGGAGACTCCCAAGCCCTCCA ITCTCCAGCAGCAACTTAAACCCCAGGGAGGCCATGGAGACTGTGATCTTAACCTGTAA TCCTGAGACTCCGGACGCAAGCTACCTGTGGTGGATGAATGGTCAGAGCCTCCCTATG ACTCATAGGATGCAGCTGTCTGAAACCAACAGGACCCTCTTTCTATTTGGTGTCACAA AGTATACTGCAGGACCCTATGAATGTGAAATATGGAACTCAGGGAGTGCCAGCCGCAG TGACCCAGTCACCCTGAATCTCCTCCATGGTCCAGACCTCCCCAGAATTTTCCCTTCA GTCACCTCTTACTATTCAGGAGAGAACCTCGACTTGTCCTGCTTCGCAGACTCTAACC CACCAGCACAGTATTCTTGGACAATTAATGGGAAGTTTCAGCTATCAGGACAAAAGCT CTTTATCCCTCAAATTACTCCAAAGCATAATGGGCTCTATGCTTGCTCTGCTCGTAAC TCAGCCACTGGCGAGGAAAGCTCCACATCCTTGACAATCAGAGTCATTGCTCCTCCAG GATTAGGAACTTTTGCTTTCAATAATCCAACGTAGCAGCCGTGATGTCATTTTTGTAT

TTCAGGAAGACTGGCAGGAGATTTATGGAAAAGACTATGA

ORF Start: ATG at 41 ORF Stop: TAG at 1019

SEQ ID NO: 44 326 aa MWat36013.5kD

NOV18a, MHAAEIMGPLSAPPCTEHIKWKGLLLTALLLNFWNLPTTAQVMIEAQPPKVSEGKDVL LLVQIRDLYHYITSYWDGQIIIYGPAYSGRETVYSNASLLIQNVTREDAGSYTLHII CGI32423-01 KRGDGTRGVTGYFTFTLYLETPKPSISSSNLNPREAMETVILTCNPETPDASYLWWMN GQSLPMTHRMQLSETNRTLFLFGVTKYTAGPYECEIWNSGSAΞRSDPVTLNLLHGPDL Protein Sequence PRIFPSVTSYYSGENLDLSCFADSNPPAQYSWTINGKFQLSGQKLFIPQITPKHNGLY ACSARNSATGEESSTSLTIRVIAPPGLGTFAFNNPT Kekuda et al.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 18B.

Table 18B. Comparison of NOV18a against NOV18b.

NOV18a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV18b 1..326 317/326 (97%) 3-328 317/326 (97%)

Further analysis ofthe NOVl 8a protein yielded the following properties shown in Table 18C.

Table 18C. Protein Sequence Properties NOVl 8a

PSort 0.4500 probability located in cytoplasm; 0.2390 probability located in analysis: lysosome (lumen); 0.21 13 probability located in microbody (peroxisome); ] 0.1000 probability located in mitochondrial matrix space

SignalP Cleavage site between residues 41 and 42 Kekuda et ul.

A search of the NOV 18a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 18D.

Kekuda et ul.

In a BLAST search of public sequence datbases, the NOV 18a protein was found to have homology to the proteins shown in the BLASTP data in Table 18E.

Table 18E. Public BLASTP Results for NOVl 8a

NOV18a Identities/

Protein Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number Residues Portion

Q 15242 I Pregnancy-specific beta- 1 - 7..322 315/331 (95%) 0.0 glycoprotein precursor - Homo 1..331 315/331 (95%) I sapiens (Human), 332 aa.

Q8TCD9 j Pregnancy specific beta- 1 - 7..326 287/335 (85%) e-165 glycoprotein 2 - Homo sapiens 1..335 295/335 (87%) ; (Human), 335 aa.

P 1 1465 I Pregnancy-specific beta- 1 - 7..326 285/335 (85%) ^", e-164 ϊ

, glycoprotein 2 precursor (PSBG-2) 1..335 295/335 (87%) ; i (Pregnancy-specific beta-1 glycoprotein E) (PS-beta-E) - Homo sapiens (Human), 335 aa.

C27658 : pregnancy-specific beta-1 7..326 285/336 (84%) i e-162

! glycoprotein E precursor - human, I ..336 295/336 (86%) \ ^: 336 aa.

075237 3 PSGIIA-c - Homo sapiens (Human), 7..3L 261/322 (81%) : e-147 i

^, 5 aa. 1..322 274/322 (85%) |

PFam analysis indicates that the NOVl 8a protein contains the domains shown in the Table 18F.

Table 18F. Domain Analysis of NOV18a

Identities/

Pfa Domain NOV18a Match Region i Similarities Expect Value for the Matched Region Kekuda et al. ig 245-294 16/53 (30%) 7.9e-08 34/53 (64%)

Example 19.

The NOV 19 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 19A.

Table 19A. NOV19 Sequence Analysis

SEQ ID NO: 47 7347 bp

NOV 19a, ATGCAGAAGGAGCTGGGCATTGTGCCTTCCTGCCCTGGCATGAAGAGCCCCAGGCCCC ACCTCCTGCTACCATTGCTGCTGCTGCTGCTGCTGCTGCTGGGGGCTGGGGTGCCAGG CG132541-01 TGCCTGGGGTCAGGCTGGGAGCCTGGACTTGCAGATTGATGAGGAGCAGCCAGCGGGT ACACTGATTGGCGACATCAGTGCGGGGCTTCCGGCAGGCACGGCAGCTCCTCTCATGT DNA Sequence ACTTCATCTCTGCCCAAGAGGGCAGCGGCGTGGGCACAGACCTGGCCATTGACGAACA CAGTGGGGTCGTCCGTACAGCCCGTGTCTTGGACCGTGAGCAGCGGGACCGCTACCGC TTCACTGCAGTCACTCCTGATGGTGCCACCGTAGAAGTTACAGTGCGAGTGGCTGACA TCAACGACCATGCTCCAGCCTTCCCACAGGCTCGGGCTGCCCTGCAGGTACCTGAGCA TACAGCTTTTGGCACCCGCTACCCACTGGAGCCTGCTCGTGATGCAGATGCTGGGCGT CTGGGAACCCAGGGCTATGCGCTATCTGGTGATGGGGCTGGAGAGACCTTCCGGCTGG AGACACGCCCCGGTCCAGATGGGACTCCAGTACCTGAGCTGGTAGTTACTGGGGAACT GGACCGAGAGAACCGCTCACACTATATGCTACAGCTGGAGGCCTATGATGGTGGTTCA CCCCCCCGGAGGGCCCAGGCCCTGCTGGACGTGACACTGCTGGACATCAATGACCATG CCCCGGCTTTCAATCAGAGCCGCTACCATGCTGTGGTGTCTGAGAGCCTGGCCCCTGG CAGTCCTGTCTTGCAGGTGTTCGCATCTGATGCCGATGCTGGTGTCAATGGGGCTGTG ACTTACGAGATCAACCGGAGGCAGAGCGAGGGTGATGGACCCTTCTCCATCGACGCAC ACACGGGGCTGCTGCAGTTAGAGCGGCCACTGGACTTTGAGCAGCGGCGGGTCCATGA ACTGGTGGTGCAAGCACGAGATGGTGGGGCTCACCCTGAGCTGGGCTCGGCCTTTGTG ACTGTGCATGTGCGAGATGCCAATGACAATCAGCCCTCCATGACTGTCATCTTTCTCA: GTGCAGATGGCTCCCCCCAAGTGTCTGAGGCCGCCCCACCTGGACAGCTCGTTGCTCG CATCTCTGTGTCAGACCCAGATGATGGTGACTTTGCCCATGTCAATGTGTCCCTGGAA 13GTGGAGAGGGCCACTTTGCCCTAAGCACCCAAGACAGCGTCATCTATCTGGTGTGTG TGGCTCGGCGGCTGGATCGAGAGGAGAGGGATGCCTATAACTTGAGGGTTACAGCCAC AGACTCAGGCTCACCTCCACTGCGGGCTGAGGCTGCCTTTGTGCTGCACGTCACTGAT GTCAACGACAATGCACCTGCCTTTGACCGCCAGCTCTACCGACCTGAGCCCCTGCCTG AGGTTGCGCTGCCTGGCAGCTTTGTAGTGCGGGTGACTGCTCGGGATCCTGACCAAGG CACCAATGGTCAGGTCACTTATAGCCTAGCCCCTGGCGCCCACACCCACTGGTTCTCC ATTGACCCCACCTCAGGCATTATCACTACGGCTGCCTCACTGGACTATGAGTTGGAAC CTCAGCCACAGCTGATTGTGGTGGCCACAGATGGTGGCCTGCCCCCTCTAGCCTCCTC TGCCACAGTTAGCGTGGCCCTGCAAGATGTGAATGATAATGAGCCCCAATTCCAGAGG ACTTTCTACAATGCCTCACTGCCTGAGGGCACCCAGCCTGGAACTTGCTTCCTGCAGG TGACAGCCACAGACGCGGATAGTGGCCCATTTGGCCTCCTCTCCTATTCCTTGGGTGC TGGACTTGGGTCCTCCGGATCTCCCCCATTCCGCATTGATGCCCACAGCGGTGATGTG TGCACAACCCGGACCCTGGACCGTGACCAGGGGCCCTCAAGCTTTGACTTCACAGTGA CAGCTGTGGATGGGGGAGGCCTCAAGTCCATGGTATATGTGAAGGTGTTTCTGTCAGA CGAGAATGACAACCCTCCTCAGTTTTATCCACGGGAGTATGCTGCCAGTATAAGTGCC CAGAGTCCACCAGGCACAGCTGTGCTGAGGTTGCGTGCCCATGACCCTGACCAGGGAT CCCATGGGCGACTCTCCTACCATATCCTGGCTGGCAACAGCCCCCCACTTTTTACCTT GGATGAGCAATCAGGTCTGTTGACAGTAGCCTGGCCCTTGGCCAGACGGGCCAATTCT GTGGTGCAGCTGGAGATCGGGGCTGAGGACGGAGGTGGCCTACAGGCAGAACCCAGTG CCCGAGTGGACATCAGCATTGTGCCTGGAACCCCCACACCACCCATATTTGAGCAACT ACAGTATGTTTTTTCTGTGCCAGAGGATGTGGCACCAGGCACCAGTGTGGGCATAGTC CAGGCACACAACCCACCAGGTCGCTTGGCACCTGTGACCCTTTCCCTATCAGGTGGGG Kekuda et al.

ATCCCCGAGGACTCTTCTCCCTAGATGCGGTATCAGGACTGTTGCAAACACTTCGCCC

TCTGGACCGGGAGCTACTGGGACCAGTGTTGGAGCTGGAGGTGCGAGCAGGCAGTGGA

GTGCCCCCAGCTTTCGCTGTAGCTCGGGTGCGTGTGCTGCTGGATGATGTGAATGACA

ACTCCCCTGCCTTTCCTGCACCTGAAGACACGGTATTGCTACCACCAAACACTGCCCC

AGGGACTCCCATCTATACACTGCGGGCTCTTGACCCCGACTCAGGTGTTAACAGTCGA

GTCACCTTTACCCTGCTTGCTGGGGGTGGTGGAGCCTTCACCGTGGACCCCACCACAG

GCCATGTACGGCTTATGAGGCCTCTGGGGCCCTCAGGAGGGCCAGCCCATGAGCTGGA

GCTGGAGGCCCGGGATGGGGGCTCCCCACCACGCACCAGCCACTTTCGACTACGGGTG

GTGGTACAGGATGTGGGAACCCGTGGGCTGGCTCCCCGATTCAACAGCCCTACCTACC

GTGTGGACCTGCCCTCAGGCACCACTGCTGGAACTCAGGTCCTGCAAGTGCAGGCCCA

AGCACCAGATGGGGGCCCTATCACCTATCACCTTGCAGCAGAGGGAGCAAGTAGCCCC

TTTGGCCTGGAGCCACAGAGTGGGTGGCTATGGGTGCGGGCAGCACTAGACCGTGAGG

CCCAGGAATTGTACATACTGAAGGTAATGGCAGTGTCTGGGTCCAAAGCTGAGTTGGG

GCAGCAGACAGGCACAGCCACCGTGAGGGTCAGCATCCTCAACCAGAATGAACACAGT

CCCCGCTTGTCTGAGGATCCCACCTTCCTGGCTGTGGCTGAGAACCAGCCCCCAGGGA

CCAGCGTGGGCCGAGTCTTTGCCACTGACCGAGACTCAGGACCCAATGGACGTCTGAC

CTACAGCCTGCAACAGCTGTCTGAAGACAGCAAGGCCTTCCGCATCCACCCCCAGACT

GGTGAGGTGACCACACTCCAAACCCTGGACCGTGAGCAGCAGAGCAGCTATCAGCTCC

TGGTGCAGGTGCAGGATGGAGGGAGCCCACCCCGCAGCACCACAGGCACTGTGCATGT

TGCAGTGCTTGACCTCAACGACAACAGCCCCACGTTCCTGCAGGCTTCAGGAGCTGCT

GGTGGGGGCCTCCCTATACAGGTACCAGACCGCGTGCCTCCAGGAACACTGGTGACGA

CTCTGCAGGCGAAGGATCCAGATGAGGGGGAGAATGGGACCATCTTGTACACGCTAAC

TGGTCCTGGCTCAGAGCTTTTCTCTCTGCACCCTCACTCAGGGGAGCTGCTCACTGCA

GCTCCCCTGATCCGAGCAGAGCGGCCCCACTATGTGCTGACACTGAGTGCTCATGACC

AAGGCAGCCCTCCTCGAAGTGCCAGCCTCCAGCTGCTGGTGCAGGTACTTCCCTCAGC

TCGCTTGGCCGAGCCGCCCCCAGATCTCGCAGAGCGGGACCCAGCGGCACCAGTGCCT

GTCGTGCTGACGGTGACAGCAGCTGAGGGACTGCGGCCCGGCTCTCTGTTGGGCTCGG

TGGCAGCGCCAGAGCCCGCGGGTGTGGGTGCACTCACCTACACACTGGTGGGCGGTGC

CGATCCCGAGGGCACCTTCGCGCTGGATGCGGCCTCAGGGCGCTTGTACCTGGCGCGG

CCCCTGGACTTCGAAGCTGGCCCGCCGTGGCGCGCGCTCACGGTACGCGCTGAGGGGC

CGGGAGGCGCGGGCGCGCGGCTGCTGCGAGTGCAGGTGCAAGTGCAGGACGAGAATGA

GCATGCGCCCGCCTTTGCGCGCGACCCGCTGGCGCTGGCGCTGCCAGAGAACCCGGAG

CCCGGCGCAGCGCTGTACACTTTCCGCGCGTCGGACGCCGACGGCCCCGGCCCCAATA^'

GCGACGTGCGCTACCGCCTGCTGCGCCAGGAGCCGCCCGTGCCGGCGCTTCGCCTGGAi

CGCGCGCACCGGGGCGCTCAGCGCTCCGCGCGGCCTGGACCGAGAGACCACTCCCGCG

CTGCTGCTGCTGGTGGAAGCCACCGACCGGCCCGCCAACGCCAGCCGCCGTCGTGCAGi

CGCGCGTTTCAGCGCGCGTCTTCGTCACGGATGAGAATGACAACGCGCCTGTCTTCGci

CTCGCCGTCACGCGTGCGCCTCCCAGAGGACCAGCCGCCTGGGCCCGCGGCCCTGCAC

GTGGTAGCCCGGGACCCGGATCTGGGCGAGGCTGCACGCGTGTCCTATCGGCTGGCAT

CTGGCGGGGACGGCCACTTCCGGCTGCACTCAAGCACTGGTGCGCTGTCCGTGGTGCG

GCCGTTGGACCGCGAACAACGAGCTGAGCACGTACTGACAGTGGTGGCCTCAGACCAC

GGCTCCCCGCCGCGCTCGGCCACGCAGGTCCTGACCGTCAGTGTCGCTGACGTCAACG

ACGAGGCGCCTACTTTCCAGCAGCAGGAGTACAGCGTCCTCTTGCGTGAGAACAACCC

TCCTGGCACATCTCTGCTCACCCTGCGAGCAACCGACCCCGACGTGGGTGCCAACGGG

CAAGTGACTTATGGAGGCGTCTCTAGCGAAAGCTTTTCTCTGGATCCTGACACTGGTG

TTCTCACGACTCTTCGGGCCCTGGATCGAGAGGAACAGGAGGAGATCAACCTGACAGT

GTATGCCCAGGACAGGGGCTCACCTCCTCAGTTAACGCATGTCACTGTTCGAGTGGCT

GTGGAGGATGAGAATGACCATGCACCAACCTTTGGGAGTGCCCATCTCTCTCTGGAGG

TGCCTGAGGGCCAGGACCCCCAGACCCTTACCATGCTTCGGGCCTCTGATCCAGATGT

GGGAGCCAATGGGCAGTTGCAGTACCGCATCCTAGATGGGGACCCATCAGGAGCCTTT

GTCCTAGACCTTGCTTCTGGAGAGTTTGGCACCATGCGGCCACTAGACAGAGAAGTGG

AGCCAGCTTTCCAGCTGAGGATAGAGGCCCGGGATGGAGGCCAGCCAGCTCTCAGTGC

CACGCTGCTTTTGACAGTGACAGTGCTGGATGCCAATGACCATGCTCCAGCCTTTCCT

GTGCCTGCCTACTCGGTGGAGGTGCCGGAGGATGTGCCTGCAGGGACCCTGCTGCTGC

AGCTACAGGCTCATGACCCTGATGCTGGAGCTAATGGCCATGTGACCTACTACCTGGG

CGCCGGTACAGCAGGAGCCTTCCTGCTGGAGCCCAGCTCTGGAGAACTGCGCACAGCT

GCAGCCTTGGACAGAGAACAGTGTCCCAGCTACACCTTTTCTGTGAGTGCAGTGGATG

GTGCAGCTGCTGGGCCCCTAAGCACCACAGTGTCTGTCACCATCACGGTGCGCGATGT

CAATGACCATGCACCCACCTTCCCCACCAGTCCTCTGCGCCTACGTCTGCCCCGCCCA

GGCCCCAGCTTCAGTACCCCAACCCTGGCTCTGGCCACACTGAGAGCTGAAGATCGTG

ATGCTGGTGCCAATGCTTCCATTCTGTACCGGCTGGCAGGCACACCACCTCCTGGCAC TACTGTGGACTCTTACACTGGTGAAATCCGCGTGGCCCGCTCTCCTGTAGCTCTAGGC CCCCGAGATCGTGTCCTCTTCATTGTGGCCACTGATCTTGGCCGTCCAGCTCGCTCTG CCACTGGTGTGATCATTGTTGGACTGCAGGGGGAAGCTGAGCGTGGACCCCGCTTTCC CCGGGCTAGCAGTGAGGCTACGATTCGTGAGAATGCGCCCCCAGGTACTCCTATTGTC TCCCCCAGGGCCGTCCATGCAGGAGGCACAAATGGACCCATCACCTACAGCATTCTCA GTGGGAATGAGAAAGGGACATTCTCCATCCAGCCTAGTACAGGTGCCATCACAGTTCG CTCAGCAGAGGGGCTAGACTTCGAGGTGAGTCCACGGCTGCGACTGGTGCTGCAGGCA GAGAGTGGAGGAGCCTTTGCCTTCACTGTGCTGACCCTGACCCTGCAAGATGCCAACG ACAATGCTCCCCGTTTCCTGCGGCCCCATTATGTGGCCTTCCTTCCTGAGTCCCGGCC CTTGGAGGGGCCCCTGCTGCAGGTGGAGGCGGATGACCTGGATCAAGGCTCTGGAGGA CAGATTTCCTACAGTCTGGCTGCATCCCAGCCGGCACGTGGATTGTTCCACGTAGACC CAACCACAGGCACTATCACTACCACAGCCATCCTGGACCGTGAGATCTGGGCTGAAAC ACGGTTGGTGCTGATGGCCACAGACAGAGGGAGCCCAGCCCTGGTGGGCTCAGCTACC TTGACGGTGATGGTCATCGACACCAATGACAATCGCCCCACCATCCCCCAACCCTGGG AGCTCCGAGTGTCAGAAGATGGCAAGCCATGTGTGGCAGGTGCGCTGACAGCCATTGT GGCCGGCGAGGAGGAGCTCCGTGGCAGCTATAACTGGGACTACCTGCTGAGCTGGTGC CATCAGCACCAACCACTGGCCAGTGTCTTCACAGAGATCGCTCGGCTCAAGGATGAAG CTCGGCCATGTCCCCCAGCTCCCCGTATCGACCCACCACCCCTCATCACTGCCGTGGC CCACCCAGGAGCCAAGTCTGTGCCCCCCAAGCCAGCAAACACAGCTGCAGCCCGGGCC ATCTTCCCACCAGCTTCTCACCGCTCCCCCATCAGCCGTGAAGGCTCCCTGTCCTCAG CTGCCATGTCCCCCAGCTTCTCACCCTCTCTGTCTCCTCTGGCTGCTCGCTCACCCGT TGTCTCACCAATTGGGGTGGCCCAGGGTCCCTCAGCCTCAGCACTCAGCGCAGAGTCT GGCCTGGAGCCACCTGATGACACGGAGCTGCACATCTAG

ORF Start: ATG at 1 ORF Stop: TAG at 7345

SEQ ID NO: 48 2448 aa MW at 2581 15.8kD

|NOV19a, MQKELGIVPSCPGMKSPRPHLLLPLLLLLLLLLGAGVPGAWGQAGSLDLQIDEEQPAG TLIGDISAGLPAGTAAPLMYFISAQEGSGVGTDLAIDEHSGWRTARVLDREQRDRYR ICG132541 -01 FTAVTPDGATVEVTVRVADINDHAPAFPQARAALQVPEHTAFGTRYPLEPARDADAGR LGTQGYALSGDGAGETFRLETRPGPDGTPVPELWTGELDRENRSHYMLQLEAYDGGS j Protein Sequence PPRRAQALLDVTLLDINDHAPAFNQSRYHAWSESLAPGSPVLQVFASDADAGVNGAV TYEINRRQSEGDGPFSIDAHTGLLQLERPLDFEQRRVHELWQARDGGAHPELGSAFV TVHVRDANDNQPSMTVIFLSADGSPQVSEAAPPGQLVARISVSDPDDGDFAHVNVS E GGEGHFALSTQDSVIYLVCVARRLDREERDAYNLRVTATDSGSPPLRAEAAFVLHVTD VNDNAPAFDRQLYRPEPLPEVALPGSFWRVTARDPDQGTNGQVTYSLAPGAHTHWFS IDPTSGIITTAASLDYELEPQPQLIWATDGGLPPLASSATVSVALQDVNDNEPQFQR TFYNAS PEGTQPGTCFLQVTATDADSGPFGLLSYSLGAGLGSSGSPPFRIDAHSGDV CTTRTLDRDQGPSSFDFTVTAVDGGGLKSMVYVKVFLSDENDNPPQFYPREYAASISA QSPPGTAVLRLRAHDPDQGSHGRLSYHILAGNSPPLFTLDEQSGLLTVAWPLARRANS WQLEIGAEDGGGLQAEPSARVDISIVPGTPTPPIFEQLQYVFSVPEDVAPGTSVGIV QAHNPPGRLAPVTLSLSGGDPRGLFSLDAVSGLLQTLRPLDRELLGPVLELEVRAGSG VPPAFAVARVRVLLDDVNDNSPAFPAPEDTVLLPPNTAPGTPIYTLRALDPDSGVNSR VTFTLLAGGGGAFTVDPTTGHVRLMRPLGPSGGPAHELELEARDGGSPPRTSHFRLRV WQDVGTRGLAPRFNSPTYRVDLPSGTTAGTQVLQVQAQAPDGGPITYHLAAEGASSP FGLEPQSGWLWVRAALDREAQELYILKVMAVSGSKAELGQQTGTATVRVSILNQNEHS PRLSEDPTFLAVAENQPPGTSVGRVFATDRDSGPNGRLTYSLQQLSEDSKAFRIHPQT GEVTTLQTLDREQQSSYQLLVQVQDGGSPPRSTTGTVHVAVLDLNDNSPTFLQASGAA GGGLPIQVPDRVPPGTLVTTLQAKDPDEGENGTILYTLTGPGSELFSLHPHSGELLTA APLIRAERPHYVLTLSAHDQGSPPRSASLQLLVQVLPSARLAEPPPDLAERDPAAPVP WLTVTAAEGLRPGSLLGSVAAPEPAGVGALTYTLVGGADPEGTFALDAASGRLYLAR PLDFEAGPPWRALTVRAEGPGGAGARLLRVQVQVQDENEHAPAFARDPLALALPENPE PGAALYTFRASDADGPGPNSDVRYRLLRQEPPVPALRLDARTGALSAPRGLDRETTPA LLLLVEATDRPANASRRRAARVSARVFVTDENDNAPVFASPSRVRLPEDQPPGPAALH WARDPDLGEAARVSYRLASGGDGHFRLHSSTGALSWRPLDREQRAEHVLTWASDH GSPPRSATQVLTVSVADVNDEAPTFQQQEYSVLLRENNPPGTSLLTLRATDPDVGANG QVTYGGVSSESFSLDPDTGVLTTLRALDREEQEEINLTVYAQDRGSPPQLTHVTVRVA VEDENDHAPTFGSAHLSLEVPEGQDPQTLTMLRASDPDVGA GQLQYRILDGDPSGAF VLDLASGEFGTMRPLDREVEPAFQLRIEARDGGQPALSATLLLTVTVLDANDHAPAFP VPAYSVEVPEDVPAGTLLLQLQAHDPDAGANGHVTYYLGAGTAGAFLLEPSSGELRTA Kekuda et al.

AALDREQCPSYTFSVSAVDGAAAGPLSTTVSVTITVRDVNDHAPTFPTSPLRLRLPRP GPSFSTPTLALATLRAEDRDAGANASILYRLAGTPPPGTTVDSYTGEIRVARSPVALG PRDRVLFIVATDLGRPARSATGVIIVGLQGEAERGPRFPRASSEATIRENAPPGTPIV SPRAVHAGGTNGPITYSILSGNEKGTFSIQPSTGAITVRSAEGLDFEVSPRLRLVLQA ESGGAFAFTVLTLTLQDANDNAPRFLRPHYVAFLPESRPLEGPLLQVEADDLDQGSGG QISYSLAASQPARGLFHVDPTTGTITTTAILDREIWAETRLVLMATDRGSPALVGSAT LTVMVIDTNDNRPTIPQPWELRVSEDGKPCVAGALTAIVAGEEELRGSYNWDYLLSWC HQHQPLASVFTEIARLKDEARPCPPAPRIDPPPLITAVAHPGAKSVPPKPANTAAARA IFPPASHRSPISREGSLSSAAMSPSFSPSLSPLAARSPWSPIGVAQGPSASALSAES GLEPPDDTELHI

SEQ ID NO: 49 | 10759 bp

NOV 19b, GCGGGGGGAGGGGAGGGGAGGGGAGGGGGCGCGGGGCCGCGGCAGCGGAGCTCGCATC

CTCGGCGGGGCGGCTGTGCAGGAGGCGGCGCCCGGGCGTCAGCGGACGGACCGATCGA 1CG132541-02 CGGCCAAGGGCGCGCGGACCGACGGCGGCTGCCCGGAGGGGATCGCGGGCCTCGGAGA

CAGCGACTGCGGACGATGCGCGGCCCCAGGCCCCGCGCGAGCGGGCGCTGCCCGGGGG j DNA Sequence GCTGACCGCGGCCGGACGGCGCCCCAGCACCGGGCGAGGGAGCCCGCGTCGCGCGGAG

GTCAGGGAGCCTGAGCTGGAGCCAGGGCCCCAGTGGGACCTGACCCAAAGTCTGAGGT

CAAGCTGGGCCCAGAGCCTGGCCTGGAGCTGGAGCCCAAGGCACAGCTGGACTACCCT

TGTCATGCAGAAGGAGCTGGGCATTGTGCCTTCCTGCCCTGGCATGAAGAGCCCCAGG

CCCCACCTCCTGCTACCATTGCTGCTGCTGCTGCTGCTGCTGCTGGGGGCTGGGGTGC CAGGTGCCTGGGGTCAGGCTGGGAGCCTGGACTTGCAGATTGATGAGGAGCAGCCAGC GGGTACACTGATTGGCGACATCAGTGCGGGGCTTCCGGCAGGCACGGCAGCTCCTCTC ATGTACTTCATCTCTGCCCAAGAGGGCAGCGGCGTGGGCACAGACCTGGCCATTGACG AACACAGTGGGGTCGTCCGTACAGCCCGTGTCTTGGACCGTGAGCAGCGGGACCGCTA CCGCTTCACTGCAGTCACTCCTGATGGTGCCACCGTAGAAGTTACAGTGCGAGTGGCT GACATCAACGACCATGCTCCAGCCTTCCCACAGGCTCGGGCTGCCCTGCAGGTACCTG AGCATACAGCTTTTGGCACCCGCTACCCACTGGAGCCTGCTCGTGATGCAGATGCTGG GCGTCTGGGAACCCAGGGCTATGCGCTATCTGGTGATGGGGCTGGAGAGACCTTCCGG CTGGAGACACGCCCCGGTCCAGATGGGACTCCAGTACCTGAGCTGGTAGTTACTGGGG lAACTGGACCGAGAGAACCGCTCACACTATATGCTACAGCTGGAGGCCTATGATGGTGG TTCACCCCCCCGGAGGGCCCAGGCCCTGCTGGACGTGACACTGCTGGACATCAATGAC CATGCCCCGGCTTTCAATCAGAGCCGCTACCATGCTGTGGTGTCTGAGAGCCTGGCCC CTGGCAGTCCTGTCTTGCAGGTGTTCGCATCTGATGCCGATGCTGGTGTCAATGGGGC TGTGACTTACGAGATCAACCGGAGGCAGAGCGAGGGTGATGGACCCTTCTCCATCGAC GCACACACGGGGCTGCTGCAGTTAGAGCGGCCACTGGACTTTGAGCAGCGGCGGGTCC ATGAACTGGTGGTGCAAGCACGAGATGGTGGGGCTCACCCTGAGCTGGGCTCGGCCTT TGTGACTGTGCATGTGCGAGATGCCAATGACAATCAGCCCTCCATGACTGTCATCTTT CTCAGTGCAGATGGCTCCCCCCAAGTGTCTGAGGCCGCCCCACCTGGACAGCTCGTTG CTCGCATCTCTGTGTCAGACCCAGATGATGGTGACTTTGCCCATGTCAATGTGTCCCT GGAAGGTGGAGAGGGCCACTTTGCCCTAAGCACCCAAGACAGCGTCATCTATCTGGTG TGTGTGGCTCGGCGGCTGGATCGAGAGGAGAGGGATGCCTATAACTTGAGGGTTACAG CCACAGACTCAGGCTCACCTCCACTGCGGGCTGAGGCTGCCTTTGTGCTGCACGTCAC TGATGTCAACGACAATGCACCTGCCTTTGACCGCCAGCTCTACCGACCTGAGCCCCTG CCTGAGGTTGCGCTGCCTGGCAGCTTTGTAGTGCGGGTGACTGCTCGGGATCCTGACC AAGGCACCAATGGTCAGGTCACTTATAGCCTAGCCCCTGGCGCCCACACCCACTGGTT CTCCATTGACCCCACCTCAGGCATTATCACTACGGCTGCCTCACTGGACTATGAGTTG GAACCTCAGCCACAGCTGATTGTGGTGGCCACAGATGGTGGCCTGCCCCCTCTAGCCT CCTCTGCCACAGTTAGCGTGGCCCTGCAAGATGTGAATGATAATGAGCCCCAATTCCA GAGGACTTTCTACAATGCCTCACTGCCTGAGGGCACCCAGCCTGGAACTTGCTTCCTG CAGGTGACAGCCACAGACGCGGATAGTGGCCCATTTGGCCTCCTCTCCTATTCCTTGG GTGCTGGACTTGGGTCCTCCGGATCTCCCCCATTCCGCATTGATGCCCATAGCGGTGA TGTGTGCACAACCCGGACCCTGGACCGTGACCAGGGGCCCTCAAGCTTTGACTTCACA GTGACAGCTGTGGATGGGGGAGGCCTCAAGTCCATGGTATATGTGAAGGTGTTTCTGT CAGACGAGAATGACAACCCTCCTCAGTTTTATCCACGGGAGTATGCTGCCAGTATAAG TGCCCAGAGTCCACCAGGCACAGCTGTGCTGAGGTTGCGTGCCCATGACCCTGACCAG GGATCCCATGGGCGACTCTCCTACCATATCCTGGCTGGCAACAGCCCCCCACTTTTTA CCTTGGATGAGCAATCAGGGCTGTTGACAGTAGCCTGGCCCTTGGCCAGACGGGCCAA TTCTGTGGTGCAGCTGGAGATCGGGGCTGAGGACGGAGGTGGCCTACAGGCAGAACCC AGTGCCCGAGTGGACATCAGCATTGTGCCTGGAACCCCCACACCACCCATATTTGAGC Kekuda et al.

AACTACAGTATGTTTTTTCTGTGCCAGAGGATGTGGCACCAGGCACCAGTGTGGGCAT

AGTCCAGGCACACAACCCACCAGGTCGCTTGGCACCTGTGACCCTTTCCCTATCAGGT

GGGGATCCCCGAGGACTCTTCTCCCTAGATGCGGTATCAGGACTGTTGCAAACACTTC

GCCCTCTGGACCGGGAGCTACTGGGACCAGTGTTGGAGCTGGAGGTGCGAGCAGGCAG

TGGAGTGCCCCCAGCTTTCGCTGTAGCTCGGGTGCGTGTGCTGCTGGATGATGTGAAT

GACAACTCCCCTGCCTTTCCTGCACCTGAAGACACGGTATTGCTACCACCAAACACTG

CCCCAGGGACTCCCATCTATACACTGCGGGCTCTTGACCCCGACTCAGGTGTTAACAG

TCGAGTCACCTTTACCCTGCTTGCTGGGGGTGGTGGAGCCTTCACCGTGGACCCCACC

ACAGGCCATGTACGGCTTATGAGGCCTCTGGGGCCCTCAGGAGGGCCAGCCCATGAGC

TGGAGCTGGAGGCCCGGGATGGGGGCTCCCCACCACGCACCAGCCACTTTCGACTACG

GGTGGTGGTACAGGATGTGGGAACCCGTGGGCTGGCTCCCCGATTCAACAGCCCTACC

TACCGTGTGGACCTGCCCTCAGGCACCACTGCTGGAACTCAGGTCCTGCAAGTGCAGG

CCCAAGCACCAGATGGGGGCCCTATCACCTATCACCTTGCAGCAGAGGGAGCAAGTAG

CCCCTTTGGCCTGGAGCCACAGAGTGGGTGGCTATGGGTGCGGGCAGCACTAGACCGT

GAGGCCCAGGAATTGTACATACTGAAGGTAATGGCAGTGTCTGGGTCCAAAGCTGAGT

TGGGGCAGCAGACAGGCACAGCCACCGTGAGGGTCAGCATCCTCAACCAGAATGAACA

CAGTCCCCGCTTGTCTGAGGATCCCACCTTCCTGGCTGTGGCTGAGAACCAGCCCCCA

GGGACCAGCGTGGGCCGAGTCTTTGCCACTGACCGAGACTCAGGACCCAATGGACGTC

TGACCTACAGCCTGCAACAGCTGTCTGAAGACAGCAAGGCCTTCCGCATCCACCCCCA

GACTGGAGAAGTGACCACACTCCAAACCCTGGACCGTGAGCAGCAGAGCAGCTATCAG

CTCCTGGTGCAGGTGCAGGATGGAGGGAGCCCACCCCGCAGCACCACAGGCACTGTGC

ATGTTGCAGTGCTTGACCTCAACGACAACAGCCCCACGTTCCTGCAGGCTTCAGGAGC

TGCTGGTGGGGGCCTCCCTATACAGGTACCAGACCGCGTGCCTCCAGGAACACTGGTG

ACGACTCTGCAGGCGAAGGATCCAGATGAGGGGGAGAATGGGACCATCTTGTACACGC

TAACTGGTCCTGGCTCAGAGCTTTTCTCTCTGCACCCTCACTCAGGGGAGCTGCTCAC

TGCAGCTCCCCTGATCCGAGCAGAGCGGCCCCACTATGTGCTGACACTGAGTGCTCATj

GACCAAGGCAGCCCTCCTCGAAGTGCCAGCCTCCAGCTGCTGGTGCAGGTGCTTCCCTJ

CAGCTCGCTTGGCCGAGCCGCCCCCAGATCTCGCAGAGCGGGACCCAGCGGCACCAGTJ

GCCTGTCGTGCTGACGGTGACAGCAGCTGAGGGACTGCGGCCCGGCTCTCTGTTGGGC¹

TCGGTGGCAGCGCCAGAGCCCGCGGGTGTGGGTGCACTCACCTACACACTGGTGGGCG;

GTGCCGATCCCGAGGGCACCTTCGCGCTGGATGCGGCCTCAGGGCGCTTGTACCTGGC,^'

GCGGCCCCTGGACTTCGAAGCTGGCCCGCCGTGGCGCGCGCTCACGGTACGCGCTGAGJ

GGGCCGGGAGGCGCGGGCGCGCGGCTGCTGCGAGTGCAGGTGCAAGTGCAGGACGAGAJ

ATGAGCATGCGCCCGCCTTTGCGCGCGACCCGCTGGCGCTGGCGCTGCCAGAGAACCC!

GGAGCCCGGCGCAGCGCTGTACACTTTCCGCGCGTCGGACGCCGACGGCCCCGGCCCCj

AATAGCGACGTGCGCTACCGCCTGCTGCGCCAGGAGCCGCCCGTGCCGGCGCTTCGCC

TGGACGCGCGCACCGGGGCGCTCAGCGCTCCGCGCGGCCTGGACCGAGAGACCACTCC

CGCGCTGCTGCTGCTGGTGGAAGCCACCGACCGGCCCGCCAACGCCAGCCGCCGTCGT

GCAGCGCGCGTTTCAGCGCGCGTCTTCGTCACGGATGAGAATGACAACGCGCCTGTCT

TCGCCTCGCCGTCACGCGTGCGCCTCCCAGAGGACCAGCCGCCTGGGCCCGCGGCCCT

GCACGTGGTAGCCCGGGACCCGGATCTGGGCGAGGCTGCACGCGTGTCCTATCGGCTG

GCATCTGGCGGGGACGGCCACTTCCGGCTGCACTCAAGCACTGGAGCGCTGTCCGTGGJ

TGCGGCCGTTGGACCGCGAACAACGAGCTGAGCACGTACTGACAGTGGTGGCCTCAGAJ

CCACGGCTCCCCGCCGCGCTCGGCCACGCAGGTCCTGACCGTCAGTGTCGCTGACGTC;

AACGACGAGGCGCCTACTTTCCAGCAGCAGGAGTACAGCGTCCTCTTGCGTGAGAACA

ACCCTCCTGGCACATCTCTGCTCACCCTGCGAGCAACCGACCCCGACGTGGGGGCCAA

CGGGCAAGTGACTTATGGAGGCGTCTCTAGCGAAAGCTTTTCTCTGGATCCTGACACT

GGTGTTCTCACGACTCTTCGGGCCCTGGATCGAGAGGAACAGGAGGAGATCAACCTGA

CAGTGTATGCCCAGGACAGGGGCTCACCTCCTCAGTTAACGCATGTCACTGTTCGAGT

GGCTGTGGAGGATGAGAATGACCATGCACCAACCTTTGGGAGTGCCCATCTCTCTCTG

GAGGTGCCTGAGGGCCAGGACCCCCAGACCCTTACCATGCTTCGGGCCTCTGATCCAG

ATGTGGGAGCCAATGGGCAGTTGCAGTACCGCATCCTAGATGGGGACCCATCAGGAGC

CTTTGTCCTAGACCTTGCTTCTGGAGAGTTTGGCACCATGCGGCCACTAGACAGAGAA

GTGGAGCCAGCTTTCCAGCTGAGGATAGAGGCCCGGGATGGAGGCCAGCCAGCTCTCA

GTGCCACGCTGCTTTTGACAGTGACAGTGCTGGATGCCAATGACCATGCTCCAGCCTT

TCCTGTGCCTGCCTACTCGGTGGAGGTGCCGGAGGATGTGCCTGCAGGGACCCTGCTG

CTGCAGCTACAGGCTCATGACCCTGATGCTGGAGCTAATGGCCATGTGACCTACTACC

TGGGCGCCGGTACAGCAGGAGCCTTCCTGCTGGAGCCCAGCTCTGGAGAACTGCGCAC

AGCTGCAGCCTTGGACAGAGAACAGTGTCCCAGCTACACCTTTTCTGTGAGTGCAGTG

GATGGTGCAGCTGCTGGGCCCCTAAGCACCACAGTGTCTGTCACCATCACGGTGCGCG

ATGTCAATGACCATGCACCCACCTTCCCCACCAGTCCTCTGCGCCTACGTCTGCCCCG Kekuda et al.

CCCAGGCCCCAGCTTCAGTACCCCAACCCTGGCTCTGGCCACACTGAGAGCTGAAGAT

CGTGATGCTGGTGCCAATGCTTCCATTCTGTACCGGCTGGCAGGCACACCACCTCCTG

GCACTACTGTGGACTCTTACACTGGTGAAATCCGCGTGGCCCGCTCTCCTGTAGCTCT

AGGCCCCCGAGATCGTGTCCTCTTCATTGTGGCCACTGATCTTGGCCGTCCAGCTCGC

TCTGCCACTGGTGTGATCATTGTTGGACTGCAGGGGGAAGCTGAGCGTGGACCCCGCT

TTCCCCGGGCTAGCAGTGAGGCTACGATTCGTGAGAATGCGCCCCCAGGGACTCCTAT

TGTCTCCCCCAGGGCCGTCCATGCAGGAGGCACAAATGGACCCATCACCTACAGCATT

CTCAGTGGGAATGAGAAAGGGACATTCTCCATCCAGCCTAGTACAGGTGCCATCACAG

TTCGCTCAGCAGAGGGGCTAGACTTCGAGGTGAGTCCACGGCTGCGACTGGTGCTGCA

GGCAGAGAGTGGAGGAGCCTTTGCCTTCACTGTGCTGACCCTGACCCTGCAAGATGCC

AACGACAATGCTCCCCGTTTCCTGCGGCCCCATTATGTGGCCTTCCTTCCTGAGTCCC

GGCCCTTGGAGGGGCCCCTGCTGCAGGTGGAGGCGGATGACCTGGATCAAGGCTCTGG

AGGACAGATTTCCTACAGTCTGGCTGCATCCCAGCCGGCACGTGGATTGTTCCACGTA

GACCCAACCACAGGCACTATCACTACCACAGCCATCCTGGACCGTGAGATCTGGGCTG

AAACACGGTTGGTGCTGATGGCCACAGACAGAGGGAGCCCAGCCCTGGTGGGCTCAGC

TACCTTGACGGTGATGGTCATCGACACCAATGACAATCGCCCCACCATCCCCCAACCC

TGGGAGCTCCGAGTGTCAGAAGATGCGTTATTGGGCTCAGAGATTGCACAGGTAACAG

GGAATGATGTGGACTCAGGACCCGTGCTGTGGTATGTGCTAAGCCCATCTGGGCCCCA

GGATCCCTTCAGTGTTGGCCGCTATGGAGGCCGTGTCTCCCTCACGGGGCCCCTGGAC

TTTGAGCAGTGTGACCGCTACCAGCTGCAGCTGCTGGCACATGATGGGCCTCATGAGG

GCCGTGCCAACCTCACAGTGCTTGTGGAGGATGTCAATGACAATGCACCTGCCTTCTC

ACAGAGCCTCTACCAGGTAATGCTGCTTGAGCACACACCCCCAGGCAGTGCCATTCTC

TCCGTCTCTGCCACTGATCGGGACTCAGGTGCCAACGGTCACATTTCCTACCACCTGG

CTTCCCCTGCCGATGGCTTCAGTGTTGACCCCAACAATGGGACCCTGTTCACAATAGT

GGGAACAGTGGCCTTGGGCCATGACGGGTCAGGAGCAGTGGATGTGGTGCTGGAAGCA

CGAGACCACGGGGCTCCAGGCCGGGCAGCACGAGCCACAGTGCACGTGCAGCTGCAGG

ACCAGAACGACCACGCCCCGAGCTTCACATTGTCACACTACCGTGTGGCTGTGACTGA

AGACCTGCCCCCTGGCTCCACTCTGCTCACCCTGGAGGCTACAGATGCTGATGGAAGC

CGCAGCCATGCCGCTGTGGACTACAGCATCATCAGTGGCAACTGGGGCCGAGTCTTCC

AGCTGGAACCCAGGCTGGCTGAGGCTGGGGAGAGTGCTGGACCAGGCCCCCGGGCACT

GGGCTGCCTGGTGTTGCTTGAACCTCTAGACTTTGAAAGCCTGACACAGTACAATCTA

ACAGTGGCTGCAGCTGACCGTGGGCAGCCACCCCAAAGCTCAGTCGTGCCAGTCACTG

TCACTGTACTAGATGTCAATGACAACCCACCTGTCTTTACCCGAGCATCCTACCGTGT

GACAGTACCTGAGGACACACCTGTTGGAGCTGAGCTGCTGCATGTAGAGGCCTCTGAC

GCTGACCCTGGCCCTCATGGCCTCGTGCGTTTCACTGTCAGCTCAGGCGACCCATCAG

GGCTCTTTGAGCTGGATGAGAGCTCAGGCACCTTGCGACTGGCCCATGCCCTGGACTG

TGAGACCCAGGCTCGACATCAGCTTGTAGTACAGGCTGCTGACCCTGCTGGTGCACAC

TTTGCTTTGGCACCAGTGACAATTGAGGTCCAGGATGTGAATGATCATGGCCCAGCCT

TCCCACTGAACTTACTCAGCACCAGCGTGGCCGAGAATCAGCCTCCAGGCACTCTCGT

GACCACTCTGCATGCAATCGACGGGGATGCTGGGGCTTTTGGGAGGCTCCGTTACAGC

CTGTTGGAGGCTGGGCCAGGACCTGAGGGCCGTGAGGCATTTGCACTGAACAGCTCAA

CAGGGGAGTTGCGTGCGCGAGTGCCCTTTGACTATGAGCACACAGAAAGCTTCCGGCT

GCTGGTGGGTGCTGCTGATGCTGGGAATCTCTCAGCCTCTGTCACTGTGTCGGTGCTA

GTGACTGGAGAGGATGAGTATGACCCTGTATTTCTGGCACCAGCTTTCCACTTCCAAG

TGCCCGAAGGTGCCCGGCGTGGCCACAGCTTGGGTCACGTGCAGGCCACAGATGAGGA

TGGGGGTGCCGATGGCCTGGTTCTGTATTCCCTTGCCACCTCTTCCCCCTATTTTGGT

ATTAACCAGACTACAGGAGCCCTGTACCTGCGGGTGGACAGTCGGGCACCAGGCAGCG

GAACAGCCACCTCTGGGGGTGGGGGCCGGACCCGGCGGGAAGCACCACGGGAGCTGAG

GCTGGAGGTGATAGCACGGGGGCCTCTGCCTGGTTCCCGGAGTGCCACAGTGCCTGTG

ACCGTGGATATCACCCACACCGCACTGGGCCTGGCACCTGACCTCAACCTGCTATTAG

TAGGGGCCGTGGCAGCCTCCTTGGGAGTTGTGGTGGTGCTTGCACTGGCAGCCCTGGT

CCTAGGACTTGTTCGGGCCCGTAGCCGCAAGGCTGAGGCAGCCCCTGGCCCAATGTCA

CAGGCAGCACCCCTAGCCAGTGACTCACTGCAGAAACTGGGCCGGGAGCCACCTAGTC

CACCACCCTCTGAGCACCTCTATCACCAGACTCTTCCCAGCTATGGTGGGCCAGGAGC

TGGAGGACCCTACCCCCGTGGTGGCTCCTTGGACCCTTCACATTCAAGTGGCCGAGGA

TCAGCAGAGGCTGCAGAGGATGATGAGATCCGCATGATCAATGAGTTCCCCCGTGTGG

CCAGTGTGGCCTCCTCTCTGGCTGCCCGTGGCCCTGACTCAGGCATCCAGCAGGATGC

AGATGGTCTGAGTGACACATCCTGCGAACCACCTGCCCCTGACACCTGGTATAAGGGC

CGAAAGGCAGGGCTGCTGCTGCCAGGTGCAGGAGCCACTCTCTACAGAGAGGAGGGGC

CCCCAGCCACTGCCACAGCCTTCCTGGGGGGCTGTGGCCTGAGCCCTGCACCCACTGG

GGACTATGGCTTCCCAGCAGATGGCAAGCCATGTGTGGCAGGTGCGCTGACAGCCATT Kekuda et al.

GTGGCCGGCGAGGAGGAGCTCCGTGGCAGCTATAACTGGGACTACCTGCTGAGCTGGT GCCCTCAGTTCCAACCACTGGCCAGTGTCTTCACAGAGATCGCTCGGCTCAAGGATGA AGCTCGGCCATGTCCCCCAGCTCCCCGTATCGACCCACCACCCCTCATCACTGCCGTG GCCCACCCAGGAGCCAAGTCTGTGCCCCCCAAGCCAGCAAACACAGCTGCAGCCCGGG CCATCTTCCCACCAGCTTCTCACCGCTCCCCCATCAGCCATGAAGGCTCCCTGTCCTC AGCTGCCATGTCCCCCAGCTTCTCACCCTCTCTGTCTCCTCTGGCTGCTCGCTCACCC GTTGTCTCACCATTTGGGGTGGCCCAGGGTCCCTCAGCCTCAGCACTCAGCGCAGAGT CTGGCCTGGAGCCACCTGATGACACGGAGCTGCACATCTAGCTGTGGCCCAGGCTGGG

CCCCGACCTGGGATGCGCACAGTGTCCCCAACGCAGGCCCCACTCTGAGCCTGCCCTG

GGCAGCCTCGGACTATGACTGGCTACGGGGAGGCCACCACCAGGCCCCAGCTCTCCAC

CCTGAACTCCCCAGCCCCCTCAGAGTACTAGGACCACAGAAGCCCTGTTGCTCACTGA

CCTGTGACCAGGTCCAATGTGGGGAGAAATATGAAGGAGGTAGCAGCCCTGGGTTCTC

CTCAGTGAGGGATCCCTGCCCTGCACCAGCACCCTGAGATGGAGCTGAGACTTTATTT

ATTGGGGGTAGGGGGATGGAGGAGGTCCCTCCAACATGTTTGGACCCAGCTCCTTTGG

GTTCCACTGACACCCCTGCCCCTGCCCCTGCCCAGAACCAAGTGCCATTTCTCACTCT

GGAGCCTTAATAAACTGCAATTTGTATCC

ORF Start: ATG at 41 1 ORF Stop: TAG at 10305

SEQ ID NO: 50 3298 aa MW at 346176.3kD

NOV 19b, MQKELGIVPSCPGMKSPRPHLLLPLLLLLLLLLGAGVPGAWGQAGSLDLQIDEEQPAG TLIGDISAGLPAGTAAPLMYFISAQEGSGVGTDLAIDEHSGWRTARVLDREQRDRYR CG132541-02 FTAVTPDGATVEVTVRVADINDHAPAFPQARAALQVPEHTAFGTRYPLEPARDADAGR LGTQGYALSGDGAGETFRLETRPGPDGTPVPELWTGELDRENRSHYMLQLEAYDGGS I Protein Sequence PPRRAQALLDVTLLDINDHAPAFNQSRYHAWSESLAPGSPVLQVFASDADAGVNGAV TYEINRRQSEGDGPFSIDAHTGLLQLERPLDFEQRRVHELWQARDGGAHPELGSAFV TVHVRDANDNQPSMTVIFLSADGSPQVSEAAPPGQLVARISVSDPDDGDFAHVNVSLE GGEGHFALSTQDSVIYLVCVARRLDREERDAYNLRVTATDSGSPPLRAEAAFVLHVTD VNDNAPAFDRQLYRPEPLPEVALPGSFWRVTARDPDQGTNGQVTYSLAPGAHTHWFS IDPTSGIITTAASLDYELEPQPQLIWATDGGLPPLASSATVSVALQDVNDNEPQFQR TFYNASLPEGTQPGTCFLQVTATDADSGPFGLLSYSLGAGLGSSGSPPFRIDAHSGDV CTTRTLDRDQGPSSFDFTVTAVDGGGLKSMVYVKVFLSDENDNPPQFYPREYAASISA QSPPGTAVLRLRAHDPDQGSHGRLSYHILAGNSPPLFTLDEQSGLLTVAWPLARRANS WQLEIGAEDGGGLQAEPSARVDISIVPGTPTPPIFEQLQYVFSVPEDVAPGTSVGIV QAHNPPGRLAPVTLSLSGGDPRGLFSLDAVSGLLQTLRPLDRELLGPVLELEVRAGSG VPPAFAVARVRVLLDDVNDNSPAFPAPEDTVLLPPNTAPGTPIYTLRALDPDSGVNSR VTFTLLAGGGGAFTVDPTTGHVRLMRPLGPSGGPAHELELEARDGGSPPRTSHFRLRV WQDVGTRGLAPRFNSPTYRVDLPSGTTAGTQVLQVQAQAPDGGPITYHLAAEGASSP FGLEPQSGWLWVRAALDREAQELYILKVMAVSGSKAELGQQTGTATVRVSILNQNEHS PRLSEDPTFLAVAENQPPGTSVGRVFATDRDSGPNGRLTYSLQQLSEDSKAFRIHPQT GEVTTLQTLDREQQSSYQLLVQVQDGGSPPRSTTGTVHVAVLDLNDNSPTFLQASGAA GGGLPIQVPDRVPPGTLVTTLQAKDPDEGENGTILYTLTGPGSELFSLHPHSGELLTA APLIRAERPHYVLTLSAHDQGSPPRSASLQLLVQVLPSARLAEPPPDLAERDPAAPVP WLTVTAAEGLRPGSLLGSVAAPEPAGVGALTYTLVGGADPEGTFALDAASGRLYLAR PLDFEAGPPWRALTVRAEGPGGAGARLLRVQVQVQDENEHAPAFARDPLALALPENPE PGAALYTFRASDADGPGPNSDVRYRLLRQEPPVPALRLDARTGALSAPRGLDRETTPA LLLLVEATDRPANASRRRAARVSARVFVTDENDNAPVFASPSRVRLPEDQPPGPAALH WARDPDLGEAARVSYRLASGGDGHFRLHSSTGALSWRPLDREQRAEHVLTWASDH GSPPRSATQVLTVSVADVNDEAPTFQQQEYSVLLRENNPPGTSLLTLRATDPDVGANG QVTYGGVSSESFSLDPDTGVLTTLRALDREEQEEINLTVYAQDRGSPPQLTHVTVRVA VEDENDHAPTFGSAHLSLEVPEGQDPQTLTMLRASDPDVGANGQLQYRILDGDPSGAF VLDLASGEFGTMRPLDREVEPAFQLRIEARDGGQPALSATLLLTVTVLDANDHAPAFP VPAYSVEVPEDVPAGTLLLQLQAHDPDAGANGHVTYYLGAGTAGAFLLEPSSGELRTA AALDREQCPSYTFSVSAVDGAAAGPLSTTVSVTITVRDVNDHAPTFPTSPLRLRLPRP GPSFSTPTLALATLRAEDRDAGANASILYRLAGTPPPGTTVDSYTGEIRVARSPVALG PRDRVLFIVATDLGRPARSATGVIIVGLQGEAERGPRFPRASSEATIRENAPPGTPIV SPRAVHAGGTNGPITYSILSGNEKGTFSIQPSTGAITVRSAEGLDFEVSPRLRLVLQA ESGGAFAFTVLTLTLQDANDNAPRFLRPHYVAFLPESRPLEGPLLQVEADDLDQGSGG QISYSLAASQPARGLFHVDPTTGTITTTAILDREIWAETRLVLMATDRGSPALVGSAT LTVMVIDTNDNRPTIPQPWELRVSEDALLGSEIAQVTGNDVDSGPVLWYVLSPSGPQD Kekuda et al.

PFSVGRYGGRVSLTGPLDFEQCDRYQLQLLAHDGPHEGRANLTVLVEDVNDNAPAFSQ SLYQV LLEHTPPGSAILSVSATDRDSGANGHISYHLASPADGFSVDPNNGTLFTIVG TVALGHDGSGAVDWLEARDHGAPGRAARATVHVQLQDQNDHAPSFTLSHYRVAVTED LPPGSTLLTLEATDADGSRSHAAVDYSIISGNWGRVFQLEPRLAEAGESAGPGPRALG CLVLLEPLDFESLTQYNLTVAAADRGQPPQSSWPVTVTVLDVNDNPPVFTRASYRVT VPEDTPVGAELLHVEASDADPGPHGLVRFTVSSGDPSGLFELDESSGTLRLAHALDCE TQARHQLWQAADPAGAHFALAPVTIEVQDVNDHGPAFPLNLLSTSVAENQPPGTLVT TLHAIDGDAGAFGRLRYSLLEAGPGPEGREAFALNSSTGELRARVPFDYEHTESFRLL VGAADAGNLSASVTVSVLVTGEDEYDPVFLAPAFHFQVPEGARRGHSLGHVQATDEDG GADGLVLYSLATSSPYFGINQTTGALYLRVDSRAPGSGTATSGGGGRTRREAPRELRL EVIARGPLPGSRSATVPVTVDITHTALGLAPDLNLLLVGAVAASLGWWLALAALVL GLVRARSRKAEAAPGPMSQAAPLASDSLQKLGREPPSPPPSEHLYHQTLPSYGGPGAG GPYPRGGSLDPSHSSGRGSAEAAEDDEIRMINEFPRVASVASSLAARGPDSGIQQDAD GLSDTSCEPPAPDTWYKGRKAGLLLPGAGATLYREEGPPATATAFLGGCGLSPAPTGD YGFPADGKPCVAGALTAIVAGEEELRGSYNWDYLLSWCPQFQPLASVFTEIARLKDEA RPCPPAPRIDPPPLITAVAHPGAKSVPPKPANTAAARAIFPPASHRSPISHEGSLSSA AMSPSFSPSLSPLAARSPWSPFGVAQGPSASALSAESGLEPPDDTELHI

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 19B.

Table 19B. Comparison of NOV19a against NOV19b.

NOV19a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV 19b .2318 2162/2318 (93%) .2314 2166/2318 (93%)

Further ana ysis ofthe NOVl 9a protein yielded the following properties shown in Table 19C.

Table 19C. Protein Sequence Properties NOV19a

I PSort 1 0.7900 probability located in plasma membrane; 0.3000 probability located in i analysis: microbody (peroxisome); 0.3000 probability located in Golgi body; 0.2000 j probability located in endoplasmic reticulum (membrane)

SignalP j Cleavage site between residues 43 and 44 analysis:

A search ofthe NOV 19a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 19D.

Table 19D. Geneseq Results for NOVl 9a

Geneseq Protein/Organism/Length NOVl 9a Identities/ Expect Kekuda et al.

In a BLAST search of public sequence datbases, the NOV 19a protein was found to have homology to the proteins shown in the BLASTP data in Table 19E.

Table 19E. Public BLASTP Results for NOV19a

Protein NOV19a Identities/ Expect

Protein/Organism/Length Accession Residues/ Similarities for Value Kekuda et ul.

PFam analysis indicates that the NOV 19a protein contains the domains shown in the Table 19F.

Kekuda et al.

17: Kekuda et al.

Example 20.

The NOV20 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 20A.

Kekuda et al.

TTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCCCCTGGATGGGCTAATTCCAG

TGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGTCGTGGGAATGAGTCAGCT

CTTTGGGATTGCAAACATGATGGATGGGGAAAGCATAGTAACTGTACTCACCAACAAG

ATGCTGGAGTGACCTGCTCAGATGGATCCAATTTGGAAATGAGGCTGACGCGTGGAGG

GAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACGGTGGGGAACAGTGTGT

GATGATAACTTCAACATAGATCATGCATCTGTCATTTGTAGACAACTTGAATGTGGAA

GTGCTGTCAGTTTCTCTGGTTCATCTAATTTTGGAGAAGGCTCTGGACCAATCTGGTT

TGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACATCAAGGA

TGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAGGGAG

CAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAAGT

GAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTTACGATGCT

GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCCGTCACAGCCATTGGTCGAGTTA

ACGCCAGTAAGGGATTTGGACACATCTGGCTTGACAGCGTTTCTTGCCAGGGACATGA

ACCTGCTGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAAT

GAAGATGCTGGCGTGACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTG

GAGGCAGCCGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGT

GTGTGACAGAGGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGT

GGATCTGCACTCAAAACATCTTATCAAGTGTACTCCAAAATCCAGGCAACAAACACAT

GGCTGTTTCTAAGTAGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTG

GCAATGGGGTGGACTTACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCC

CACAGGGAACCCAGACTGGTTGGAGGGGACATTCCCTGTTCTGGACGTGTTGAAGTGA

AGCATGGTGACACGTGGGGCTCCATCTGTGATTCGGACTTCTCTCTGGAAGCTGCCAG

CGTTCTATGCAGGGAATTACAGTGTGGCACAGTTGTCTCTATCCTGGGGGGAGCTCAC

TTTGGAGAGGGAAATGGACAGATCTGGGCTGAAGAATTCCAGTGTGAGGGACATGAGT

CCCATCTTTCACTCTGCCCAGTAGCACCCCGCCCAGAAGGAACTTGTAGCCACAGCAG

GGATGTTGGAGTAGTCTGCTCAAGATACACAGAAATTCGCTTGGTGAATGGCAAGACC

CCGTGTGAGGGCAGAGTGGAGCTCAAAACGCTTGGTGCCTGGGGATCCCTCTGTAACT

CTCACTGGGACATAGAAGATGCCCATGTTCTTTGCCAGCAGCTTAAATGTGGAGTTGC

CCTTTCTACCCCAGGAGGAGCACGTTTTGGAAAAGGAAATGGTCAGATCTGGAGGCAT

ATGTTTCACTGCACTGGGACTGAGCAGCACATGGGAGATTGTCCTGTAACTGCTCTAG

GTGCTTCATTATGTCCTTCAGAGCAAGTGGCCTCTGTAATCTGCTCAGGAAACCAGTC

CCAAACACTGTCCTCGTGCAATTCATCGTCTTTGGGCCCAACAAGGCCTACCATTCCA

GAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGCCTGGTAAATGGAGGAG

GTCGCTGTGCTGGGAGAGTAGAGATCTATCATGAGGGCTCCTGGGGCACCATCTGTGA

TGACAGCTGGGACCTGAGTGATGCCCACGTGGTTTGCAGACAGCTGGGCTGTGGAGAG

GCCATTAATGCCACTGGTTCTGCTCATTTTGGGGAAGGAACAGGGCCCATCTGGCTGG

ATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCACACGGCTG

GGGGCAGCAAAATTGCAGGCACAAGGAGGATGCGGGAGTTATCTGCTCAGAATTCATG

TCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTGGAAGTTT

TTTACAATGGAGCTTGGGGCACTGTTGGCAAGAGTAGCATGTCTGAAACCACTGTGGG

TGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACCCTGCATCTTTA

GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTG

ACACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGCCCCTCGGA

GGAGACCTGGATCACATGTGACAACAAGATAAGACTTCAGGAAGGACCCACTTCCTGT

TCTGGACGTGTGGAGATCTGGCATGGAGGTTCCTGGGGGACAGTGTGTGATGACTCTT

GGGACTTGGACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAA

AGCATTCAAAGAAGCAGAGTTTGGTCAGGGGACTGGACCGATATGGCTCAATGAAGTG

AAGTGCAAAGGGAATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCATA

GTGAGTGTGGGCACAAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAGTGCAGAA

AACCCCACAAAAAGCCACAACAGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATT

CAATACCGGGAGATGAATTCTTGCCTGAATGCAGATGATCTGGACCTAATGAATTCCT

CAGGAGGCCATTCTGAGCCACACTGAAAAGGAAAATGGGAATTTATAACCCAGTGAGT

TCAGCCTTTAAGATACCTTGATGAAGACCTGGAGTA

ORF Start: ATG at 87 iORF Stop: TGA at 3330

SEQ ID NO: 52 1081 aa MW at 117107.8kD

NOV20a, MSKLRMVLLEDSGSADFRRHFVNLSPFTITWLLLSACFVTSSLGGTDKELRLVDGEN KCSGRVEVKVQEEWGTVCNNGWSMEAVSVICNQLGCPTAIKAPGWANSSAGSGRIWMD CG132888-02 HVSCRGNESALWDCKHDGWGKHSNCTHQQDAGVTCSDGSNLEMRLTRGGNMCSGRIEI Kekuda et ul.

CG132888-02 KFQGRWGTVCDDNFNIDHASVICRQLECGSAVSFSGSSNFGEGSGPIWFDDLICNGNE SA WNCKHQGWGKHNCDHAEDAGVICSKGADLSLRLVDGVTECSGRLEVRFQGEWGTI Protein Sequence CDDGWDSYDAAVACKQLGCPTAVTAIGRVNASKGFGHIWLDSVSCQGHEPAVWQCKHH EWGKHYCNHNEDAGVTCSDGSDLELRLRGGGSRCAGTVEVEIQRLLGKVCDRGWGLKE ADWCRQLGCGSALKTSYQVYSKIQATNTWLFLSSCNGNETSLWDCKNWQWGGLTCDH YEEAKITCSAHREPRLVGGDIPCSGRVEVKHGDTWGSICDSDFSLEAASVLCRELQCG TWSILGGAHFGEGNGQIWAEEFQCEGHESHLSLCPVAPRPEGTCSHSRDVGWCSRY TEIRLVNGKTPCEGRVELKTLGAWGSLCNSHWDIEDAHVLCQQLKCGVALSTPGGARF GKGNGQIWRH FHCTGTEQHMGDCPVTALGASLCPSEQVASVICSGNQSQTLSSCNSS SLGPTRPTIPEESAVACIESGQLRLVNGGGRCAGRVEIYHEGSWGTICDDSWDLSDAH WCRQLGCGEAINATGSAHFGEGTGPIWLDEMKCNGKESRIWQCHSHGWGQQNCRHKE DAGVICSEFMSLRLTSEASREACAGRLEVFYNGAWGTVGKSSMSETTVGWCRQLGCA DKGKINPASLDKAMSIPMWVDNVQCPKGPDTLWQCPSSPWEKRLASPSEETWITCDNK IRLQEGPTSCSGRVEIWHGGSWGTVCDDSWDLDDAQWCQQLGCGPALKAFKEAEFGQ GTGPIWLNEVKCKGNESSLWDCPARRWGHSECGHKEDAAVNCTDISVQKTPQKATTVS SRGENLVHQIQYREMNSCLNADDLDLMNSSGGHSEPH

Further analysis ofthe NOV20a protein yielded the following properties shown in Table 20B.

Table 20B. Protein Sequence Properties NOV20a

PSort 0.6500 probability located in plasma membrane; 0.5658 probability located in \ analysis: mitochondrial inner membrane; 0.3635 probability located in microbody (peroxisome); 0.3000 probability located in Golgi body

! SignalP Cleavage site between residues 46 and 47 analysis:

A search ofthe NOV20a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 20C.

Kekuda et ul.

In a BLAST search of public sequence datbases, the NOV20a protein was found to have homology to the proteins shown in the BLASTP data in Table 20D.

Kekuda et ul.

PFam analysis indicates that the NOV20a protein contains the domains shown in the Table 20E.

Kekuda et ul.

Example 21.

The NOV21 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 21 A.

Table 21A. NOV21 Sequence Analysis

SEQ ID NO: 53 ]4308 bp

NOV21a, ATGGGGAAGAGAGGCATGATGAGAGATCTCTGTGGCTTGTGTGTGCCAAGGTCACCAG TGGAAACTCTCAAGGACAATACCTGTGTCTCCTCCAAGGCCCATCCCTCGTGCCTAAC CG133159-01 ACAGTTCCTGGCAGAGACCAGAAACTCCTTTGACTGTTGTGAACCTGATGAGGTCCCT GATCACTGTCCAGGGCCGCCAGGCTCCAAGCACAGGGCCCGGGCAGCCCCGGATCCCC DNA Sequence CTCCCCTCTTCGATGACACAAGCGGTGGTTATTCCAGCCAGCCCGGGGGATACCCAGC CACAGGAGCAGACGTGGCCTTCAGTGTCAACCACTTGCTTGGGGACCCAATGGCCAAT GTGGCTATGGCCTATGGCAGCTCCATCGCATCCCATGGGAAGGACATGGTGCACAAGG AGCTGCACCGTTTTGTGTCTGTGAGCAAACTCAAGTATTTTTTTGCTGTGGACACAGC CTACGTGGCCAAGAAGCTAGGGCTGCTGGTCTTCCCCTACACACACCAGAACTGGGAA GTGCAGTACAGTCGTGATGCTCCTCTGCCCCCCCGGCAAGACCTCAACGCCCCTGACC TCTATATCCCCAGCGTGCTCTGTTATCCCTTCTTCCAAGAAGCCTTTCCTGACCCCCT GAGCAAGTGGTGGCTCCCTTCTGGGTTCCCACAACTGCCTGTCCACATGGCATTTTTC |AGGCTGCCCACACATACAGCTGACTCTTCTCTGTCCTGTTGGCTGCACAGGGCCAGGC jCCATCGTGGACACCCAGGCGATGGCCTTCATTACTTACGTGCTCCTGGCTGGGATGGC iACTGGGCATTCAGAAAAGGTTCTCCCCGGAGGTGCTGGGCCTGTGTGCAAGCACAGCG CTGGTGTGGGTGGTGATGGAGGTGCTGGCCCTGCTCCTGGGCCTCTACCTGGCCACCG TGCGCAGTGACCTGAGCACCTTTCACCTGCTGGCCTACAGTGGCTACAAATACGTGGG AATGATCCTCAGTGTGCTCACGGGGCTGCTGTTCGGCAGCGATGGCTACTACGTGGCG CTGGCCTGGACCTCATCGGCGCTCATGTACTTCATTGTGCGCTCTTTGCGGACAGCAG CCCTGGGCCCCGACAGCATGGGGGGCCCCGTCCCCCGGCAGCGTCTCCAGCTCTACCT GACTCTGGGAGCTGCAGCCTTCCAGCCCCTCATCATATACTGGCTGACTTTCCACCTG GTCCGGCAGCTGCTACCCTCACCTCCAGAGGTGGTAGAAGAGGAGGGGGATGTTGAGG jCCCAGGGTCACCCACTCTGCTGCACACAGAAACATCAGACAGAAGAGGCCGTGGATGG AGTATTCTGGGACCACCAGCTGGGGGATGACTACCTGTTTAAGCTGCTTTTGATTGGC jGACTCAGGCGTGGGCAAGTCATGCCTGCTCCTGCGGTTTGCTGATGACACGTACACAG (AGAGCTACATCAGCACCATCGGGGTGGACTTCAAGATCCGAACCATCGAGCTGGATGG ^'CAAAACTATCAAACTTCAGATCTGGGACACAGCGGGCCAGGAACGGTTCCGGACCATC ACTTCCAGCTACTACCGGGGGGCTCATGGCATCATCGTGGTGTATGACGTCACTGACC AGATTCACAAGTGCCAGTTCCGGCCCGGCCATTGTTCAAGGCCCTTGAGATTTAACTG CGAACAAGGTGGGGGTGGCTCTGGCATTCTAGTGACGGAAACAGACAATAAACTTGCA TACAGAACCACCGTGACTTTAGGAGTGATAAGGTCAATGCTTCCAATAGAGTTGGAGC AAGTGCGCCAGAAGCTGCTGCAGCTGCTCCGCACCTACTCACCCAGCGCCCAGGTCAA GCGGCTCCTGCAGGCCTGCAAGCTGCTCTACATGGCCCTGAGGACCCAGGAAGGGGAG GGCGCGGGTGCCGACGAGTTCCTGCCTCTGCTGAGCCTCGTCTTGGCCCACTGTGACC TTCCTGAGCTGCTGCTGGAGGCCGAGTACATGTCGGAGCTGCTGGAGCCCAGCCTGCT TACTGGAGAGGGTGGCTACTACCTGACCAGCCTCTCTGCCAGCCTGGCCCTGCTGAGT GGCCTGGGTCAGGCCCACACCCTCCCACTGAGCCCCGTGCAGGAGCTACGGCGCTCCC TCAGCCTCTGGGAGCAGCGCCGCCTGCCTGCCACCCACTGCTTCCAGCACCTCCTCCG AGTAGCCTATCAGGATCCCAGCAGTGGCTGCACCTCCAAGACCCTGGCCGTGCCCCCA GAGGCCTCGATTGCCACCCTGAACCAGCTCTGTGCCACCAAGTTCCGAGTGACCCAGC CCAACACTTTTGGCCTCTTCCTGTACAAGGAGCAGGGCTACCACCGCCTGCCCCCTGG GGCCCTGGCCCACAGGCTGCCCACCACTGGCTACCTCGTCTACCGCCGGGCAGAGTGG CCTGAGACCCAGGGGGCTGTGACAGAGGAGGAGGGCAGTGGGCAGTCAGAGGCAAGAA GCAGAGGGGAGGAGCAAGGGTGCCAGGGAGATGGGGATGCTGGGGTCAAAGCCAGCCC CAGGGACATTCGGGAACAGTCTGAGACAACTGCTGAAGGGGGCCAGGAGTTTGAGTGG Kekuda et al.

Kekuda et al.

Further analysis ofthe NOV21a protein yielded the following properties shown in

Table 2 IB.

Table 21B. Protein Sequence Properties NOV21a

PSort 0.6000 probability located in plasma membrane; 0.4000 probability located in analysis: Golgi body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.3000 probability located in microbody (peroxisome)

SignalP No Known Signal Sequence Indicated analysis:

A search ofthe NOV21a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 21C.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV21 a protein was found to have homology to the proteins shown in the BLASTP data in Table 2 ID.

Kekuda et al.

Homo sapiens (Human), 783 aa. 489..760 272/272 (99%)

PFam analysis indicates that the NOV2 la protein contains the domains shown in the Table 2 IE.

Example 22.

The NOV22 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 22A.

Kekuda et al.

ICG133508-01 CGCCTCGCTGTGCCGGGCCCGGCCGCCCCCTCTCGGGCTGGACGTGGAGACTTGTCGG AGCTTCGAGCTGCAGCCCCCAGAGCGGAGTCCCAGCGCGGCAGGCGCAGGCACCTCTG DNA Sequence TCAGCCTCCTCGCAGTTGTAGTTATTGTGTGTGGCGTGGCCCTGGTGGCAGTTTTTCT CTTTCTCTTTTGGAAGCTGTGCTGGATGCCCTGGAGGAACAAGGAGGCCTCCAGTCCC TCTTCTGCTAATCCCCCCTTGGAAGCCCTCCAGAGCCCCAGCTTCAGAGGCAACATGG CGGACAAGCTGAAGGACCCCAGCACCCTGGGCTTCCTGGAGGCGGCCGTGAAGATCAG CCACACGTCCCCAGATATCCCAGCTGAGGTGCAGATGTCGGTCAAGGAGCACATCATG CGTCACACCCGGCTGCAGCGGCAAACTACAGAGCCAGCGTCATCCACCAGGCACACGT CCTTCAAGCGCCACCTGCCAAGGCAGATGCATGTCTCCAGTGTAGACTATGGCAATGA GCTTCCACCAGCAGCAGAGCAGCCCACCAGCATTGGCCGCATCAAGCCTGAGCTCTAC AAGCAGAAGTCGGTGGATGGGGAGGATGCCAAGTCTGAGGCCACCAAGAGCTGCGGGA AGATCAACTTCAGCCTACGCTACGATTACGAGACCGAGACCCTGATTGTGCGTATCCT GAAGGCTTTTGACCTCCCTGCCAAGGACTTTTGTGGAAGCTCTGACCCTTATGTCAAG ATCTACCTCCTGCCTGACCGCAAATGCAAGCTGCAGACCCGGGTGCACCGCAAGACCC TGAACCCCACCTTTGATGAGAACTTCCACTTCCCTGTGCCCTATGAGGAGCTGGCTGA CCGCAAGCTGCATCTCAGTGTCTTCGACTTTGACCGCTTCTCCCGCCATGACATGATT GGCGAGGTCATCCTGGACAACCTCTTTGAGGCCTCTGACCTGTCTCGGGAAACCTCCA TCTGGAAGGATATCCAATATGCCACAAGTGAAAGCGTGGACTTGGGAGAGATCATGTT CTCCCTTTGCTACCTGCCCACTGCAGGCAGGCTCACCCTCACAGTGATTAAGTGTCGG AACCTCAAGGCGATGGACATCACAGGCTATTCAGATCCCTATGTGAAAGTGTCCTTGC TCTGTGATGGGCGGAGGCTGAAGAAGAAGAAAACAACCATAAAGAAAAACACTCTCAA TCCTGTCTACAATGAGGCCATCATCTTTGACATTCCCCCGGAAAACATGGATCAAGTC AGCCTGCTCATCTCAGTCATGGACTATGATCGAGTGGGCCACAATGAGATCATAGGAG TCTGTCGTGTGGGGATCACTGCTGAAGGCCTGGGCAGGGACCACTGGAACGAGATGCT GGCATACCCCCGGAAGCCCATCGCACACTGGCACTCCTTGGTGGAGGTAAAGAAATCC TTCAAAGAGGGAAACCCTCGGTTGTGATTTCATTCACGTGGATGCCGCAAGCAGAGAG

ACTGCCACCTGGAGTTAGGATGGCAGGGCCGAGCTGCTAGCTTCGACAGTGAGAGCTC

GTGCCCATCTCCGAAACCACCTCCAACACCATGAGATGTGCAGCCAAATAACACAAAT

GGGACTCAGCAATGTTCTCTTTGCACTTGTTCAACCGTCTAAACAGTGTTGTGCAGTC

GCAGTGGCGGCAGCAGCGGCAGCCGTCCGTCACTCCAGAGTCTTACCTGCTCCTGTGT

AGGTCAAAGCTGAGACACTTGTCATGTGGTCAGATCTGTCTTAGTC

ORF Start: ATG at 61 ORF Stop: TGA at 1591

SEQ ID NO: 56 510 aa MW at 57324.3kD

NOV22a, MSGVWGAGGPRCQEALAVLASLCRARPPPLGLDVETCRSFELQPPERSPSAAGAGTSV SLLAVWIVCGVALVAVFLFLFWKLCWMPWRNKEASSPSSANPPLEALQSPSFRGNMA CG133508-01 DKLKDPSTLGFLEAAVKISHTSPDIPAEVQMSVKEHIMRHTRLQRQTTEPASSTRHTS FKRHLPRQMHVSSVDYGNELPPAAEQPTSIGRIKPELYKQKSVDGEDAKSEATKSCGK Protein Sequence INFSLRYDYETETLIVRILKAFDLPAKDFCGSSDPYVKIYLLPDRKCKLQTRVHRKTL NPTFDENFHFPVPYEELADRKLHLSVFDFDRFSRHDMIGEVILDNLFEASDLSRETSI WKDIQYATSESVDLGEIMFSLCYLPTAGRLTLTVIKCRNLKA DITGYSDPYVKVSLL CDGRRLKKKKTTIKKNTLNPVYNEAlIFDIPPENMDQVSLLISVMDYDRVGHNEIIGV CRVGITAEGLGRDHWNEMLAYPRKPIAHWHSLVEVKKSFKEGNPRL

SEQ ID NO: 57 675 bp

!NOV22b, GGATCCCTGATTGTGCGTATCCTGAAGGCTTTTGACCTCCCTGCCAAGGACTTTTGTG GAAGCTCTGACCCTTATGTCAAGATCTACCTCCTGCCTGACCGCAAATGCAAGCTGCA GACCCGGGTGCACCGCAAGACCCTGAACCCCACCTTTGATGAGAACTTCCACTTCCCT GTGCCCTATGAGGAGCTGGCTGACCGCAAGCTGCATCTCAGTGTCTTCGACTTTGACC GCTTCTCCCGCCATGACATGATTGGCGAGGTCATCCTGGACAACCTCTTTGAGGCCTC

TGACCTGTCTCGGGAAACCTCCATCTGGAAGGATATCCAATATGCCACAAGTGAAAGC GTGGACTTGGGAGAGATCATGTTCTCCCTTTGCTACCTGCCCACTGCAGGCAGGCTCA CCCTCACAGTGATTAAGTGTCGGAACCTCAAGGCGATGGACATCACAGGCTATTCAGA TCCCTATGTGAAAGTGTCCTTGCTCTGTGATGGGCGGAGGCTGAAGAAGAAGAAAACA ACCATAAAGAAAAACACTCTCAATCCTGTCTACAATGAGGCCATCATCTTTGACATTC CCCCGGAAAACATGGATCAAGTCAGCCTGCTCATCTCAGTCATGGACTATGATCGAGT GGGCCACAATGAGATCATAGGAGTCTGTCGTCTCGAG Kekuda et al.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 22B.

Table 22B. Comparison of NOV22a against NOV22b.

NOV22a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV22b 245-467 210/223 (94%) 2..224 212/223 (94%)

Further analysis ofthe NOV22a protein yielded the following properties shown in Table 22C.

A search ofthe NOV22a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 22D.

Kekuda et ul.

In a BLAST search of public sequence datbases, the NOV22a protein was found to have homology to the proteins shown in the BLASTP data in Table 22E.

Table 22E. Public BLASTP Results for NOV22a

NOV22a 1 Identities/

Protein Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number Residues \ Portion

Q9R0N8 ι Synaptotagmin VI - Mus 1..510 j 493/511 (96%) 0.0 I musculus (Mouse), 51 1 aa. 1..51 1 498/51 1 (96%) Kekuda et nl.

PFam analysis indicates that the NOV22a protein contains the domains shown in the Table 22F.

Kekuda et al.

Example 23.

The NOV23 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 23A.

Table 23A. NOV23 Sequence Analysis

SEQ ID NO: 59 J 1751 bp

NOV23a, CGGGAGCCTCTCCCTGAGGGGCACCGCGTTCTTCAGGAGCTGGGCCTCCAGTGCGGCG

CGATGTCAGGCGCGGTGACAGCTCTGTGAGTCCGAGGCCGCGGCGTGTGGCTGGGCGG CG133548-01 CTGCGGGGCCTGACCGGTCCGCTCATGGTGCCGCCACGACGCCATCGCGGGGCAGGAA

GGCCAGGGGTGCTGAGTTCTTCACCTCCTTTTAGACTGAGATCTGCCAAGTTTTCCGG DNA Sequence CATTGCTCTTGAGGATCTCAGAAGGGCTCTTAAGACAAGACTGCAAATGGTGTGTGTA TTTGTCATGAACCGAATGAATTCCCAGAACAGTGGTTTCACTCAGCGCAGGCGAATGG CTCTTGGGATTGTTATTCTTCTGCTTGTTGATGTGATATGGGTTGCTTCCTCTGAACT TACTTCGTATGTTTTTACCCAGTACAACAAACCATTCTTCAGCACCTTTGCAAAAACA TCTATGTTTGTTTTGTACCTTTTGGGCTTTATTATTTGGAAGCCATGGAGACAACAGT GTACAAGAGGACTTCGCGGAAAGCATGCTGCTTTTTTTGCAGATGCTGAAGGTTACTT TGCTGCTTGCACAACAGATACAACTATGAATAGTTCTTTGAGTGAACCTCTGTATGTG CCTGTGAAATTCCATGATCTTCCAAGTGAAAAACCTGAGAGCACAAACATTGATACTG AAAAAAGTCCCAAAAAGTCTCGTGTGAGGTTCAGTAATATCATGGAGATTCGACAGCT TCCGTCAAGTCATGCATTGGAAGCAAAGTTGTCTCGCATGTCATATCCTGTGAAAGAA CAAGAATCCATACTGAAAACTGTGGGGAAACTTACTGCAACTCAAGTAGCGAAAATTA GCTTTTTTTTTTGCTTTGTGTGGTTTTTGGCAAATTTGTCATATCAAGAAGCACTTTC AGACACACAAGTTGCTATAGTTAATATTTTATCTTCAACTTCCGGTCTTTTTACCTTA ATCCTTGCTGCAGTATTTCCAAGTAACAGTGGAGATAGATTTACCCTTTCTAAACTAT TAGCTGTAATTTTAAGCATTGGAGGCGTTGTACTGGTAAACCTGGCAGGGTCTGAAAA ACCTGCTGGAAGAGACACAGTAGGTTCCATTTGGTCTCTTGCTGGAGCCATGCTCTAT GCTGTCTATATTGTTATGATTAAGAGAAAAGTAGATAGAGAAGACAAGTTGGATATTC CAATGTTCTTTGGTTTTGTAGGTTTGTTTAATCTGCTGCTCTTATGGCCAGGTTTCTT TTTACTTCATTATACTGGATTTGAGGACTTCGAGTTTCCCAATAAAGTAGTATTAATG TGCATTATCATTAATGGCCTTATTGGAACAGTACTCTCAGAGTTCCTGTGGTTGTGGG GCTGCTTTCTTACCTCATCATTGATAGGCACACTTGCACTAAGCCTTACAATACCTCT GTCCATAATAGCTGACATGTGTATGCAAAAGGTACAGTTTTCTTGGTTATTTTTTGCA GGAGCTATCCCTGTATTTTTTTCATTTTTTATTGTAACTCTCCTATGCCATTATAATA ATTGGGATCCTGTGATGGTGGGAATCAGAAGAATATTTGCTTTTATATGCAGAAAACA Kekuda et al.

TCGAATTCAGAGGGTTCCAGAAGACAGCGAACAGTGTGAGAGTCTCATTTCTATGCAC AGTGTTTCTCAGGAGGATGGAGCTAGTTAGCTGTCTGTTGTCTGTAGCCCAGGTTTGT ATGTGAGCTGG

ORF Start: ATG at 141 ORF Stop: TAG at 1710

SEQ ID NO: 60 523 aa MW at 58872.3kD

NOV23a, MVPPRRHRGAGRPGVLSSSPPFRLRSAKFSGIALEDLRRALKTRLQ VCVFVMNRMNS QNSGFTQRRRMALGIVILLLVDVIWVASSELTSYVFTQYNKPFFSTFAKTSMFVLYLL CG133548-01 GFIIWKPWRQQCTRGLRGKHAAFFADAEGYFAACTTDTTMNSSLSEPLYVPVKFHDLP SEKPESTNIDTEKSPKKSRVRFSNIMEIRQLPSSHALEA LSRMSYPVKEQESILKTV Protein Sequence GKLTATQVAKISFFFCFVWFLANLSYQEALSDTQVAIVNILSSTSGLFTLILAAVFPS NSGDRFTLSKLLAVILSIGGWLVNLAGSEKPAGRDTVGSIWSLAGAMLYAVYIV IK RKVDREDKLDIPMFFGFVGLFNLLLLWPGFFLLHYTGFEDFEFPNKWLMC11ING I GTVLSEFLWLWGCFLTSSLIGTLALSLTIPLSIIADMCMQKVQFSWLFFAGAIPVFFS FFIVTLLCHYNNWDPVMVGIRRIFAFICRKHRIQRVPEDSEQCESLISMHSVSQEDGA S

SEQ ID NO: 61 1607 bp

NOV23b, CGGGAGCCTCTCCCTGAGGGGCACCGCGTTCTTCAGGAGCTGGGCCTCCAGTGCGGCG

CGATGTCAGGCGCGGTGACAGCTCTGTGAGTCCGAGGCCGCGGCGTGTGGCTGGGCGG CG133548-02 CTGCGGGGCCTGACCGGTCCGCTCATGGTGCCGCCACGACGCCATCGCGGGGCAGGAA

GGCCAGGGGTGCTGAGTTCTTCACCTCCTTTTAGACTGAGATCTGCCAAGTTTTCCGG DNA Sequence CATTGCTCTTGAGGATCTCAGAAGGGCTCTTAAGACAAGACTGCAAATGGTGTGTGTA

TTTGTCATGAACCGAATGAATTCCCAGAACAGTGGTTTCACTCAGCGCAGGCGAATGG

CTCTTGGGATTGTTATTCTTCTGCTTGTTGATGTGATATGGGTTGCTTCCTCTGAACT

TACTTCGTTTGCAGATGCTGAAGGTTACTTTGCTGCTTGCACAACAGATACAACTATG

AATAGTTCTTTGAGTGAACCTCTGTATGTGCCTGTGAAATTCCATGATCTTCCAAGTG

AAAAACCTGAGAGCACAAACATTGATACTGAAAAAAGTCCCAAAAAGTCTCGTGTGAG

GTTCAGTAATATCATGGAGATTCGACAGCTTCCGTCAAGTCATGCATTGGAAGCAAAG

TTGTCTCGCATGTCATATCCTGTGAAAGAACAAGAATCCATACTGAAAACTGTGGGGA

AACTTACTGCAACTCAAGTAGCGAAAATTAGCTTTTTTTTTTGCTTTGTGTGGTTTTT

GGCAAATTTGTCATATCAAGAAGCACTTTCAGACACACAAGTTGCTATAGTTAATATT

TTATCTTCAACTTCCGGTCTTTTTACCTTAATCCTTGCTGCAGTATTTCCAAGTAACAJ

GTGGAGATAGATTTACCCTTTCTAAACTATTAGCTGTAATTTTAAGCATTGGAGGCGT!

TGTACTGGTAAACCTGGCAGGGTCTGAAAAACCTGCTGGAAGAGACACAGTAGGTTCCJ

ATTTGGTCTCTTGCTGGAGCCATGCTCTATGCTGTCTATATTGTTATGATTAAGAGAA;

AAGTAGATAGAGAAGACAAGTTGGATATTCCAATGTTCTTTGGTTTTGTAGGTTTGTT

TAATCTGCTGCTCTTATGGCCAGGTTTCTTTTTACTTCATTATACTGGATTTGAGGAC

TTCGAGTTTCCCAATAAAGTAGTATTAATGTGCATTATCATTAATGGCCTTATTGGAA

CAGTACTCTCAGAGTTCCTGTGGTTGTGGGGCTGCTTTCTTACCTCATCATTGATAGG

CACACTTGCACTAAGCCTTACAATACCTCTGTCCATAATAGCTGACATGTGTATGCAA

AAGGTACAGTTTTCTTGGTTATTTTTTGCAGGAGCTATCCCTGTATTTTTTTCATTTT

TTATTGTAACTCTCCTATGCCATTATAATAATTGGGATCCTGTGATGGTGGGAATCAG

AAGAATATTTGCTTTTATATGCAGAAAACATCGAATTCAGAGGGTTCCAGAAGACAGC

GAACAGTGTGAGAGTCTCATTTCTATGCACAGTGTTTCTCAGGAGGATGGAGCTAGTT

AGCTGTCTGTTGTCTGTAGCCCAGGTTTGTATGTGAGCTGG

ORF Start: ATG at 141 ORF Stop: TAG at 1566

SEQ ID NO: 62 475 aa MW at 53094.6kD

NOV23b, MVPPRRHRGAGRPGVLSSSPPFRLRSAKFSGIALEDLRRALKTRLQMVCVFVMNRMNS QNSGFTQRRRMALGIVILLLVDVIWVASSELTSFADAEGYFAACTTDTTMNSSLSEPL CGI 33548-02 YVPVKFHDLPSEKPESTNIDTEKSPKKSRVRFSNIMEIRQLPSSHALEAKLSRMSYPV KEQESILKTVGKLTATQVAKISFFFCFV FLANLSYQEALSDTQVAIVNILSSTSGLF Protein Sequence TLILAAVFPSNSGDRFTLSKLLAVILSIGGWLVNLAGSEKPAGRDTVGSIWSLAGAM LYAVYIVMIKRKVDREDKLDIPMFFGFVGLFNLLLLWPGFFLLHYTGFEDFEFPNKW LMCIIINGLIGTVLSEFLWLWGCFLTSSLIGTLALSLTIPLSIIADMCMQKVQFSWLF Kekuda et al.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 23B.

Table 23B. Comparison of NOV23a against NOV23b.

NOV23a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV23b 15..523 431/509 (84%) 15..475 431/509 (84%)

Further analysis ofthe NOV23a protein yielded the following properties shown in Table 23C.

A searc o the N V a prote n aga nst t e Geneseq atabase, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 23D.

Kekuda et ul.

In a BLAST search of public sequence datbases, the NOV23a protein was found to have homology to the proteins shown in the BLASTP data in Table 23E.

Table 23E. Public BLASTP Results for NOV23a

^■ NOV23a Identities/

Protein Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number Residues Portion

Q8WV83 J Similar to RIKEN cDNA 1..523 522/523 (99%) 0.0

1300003P13 gene - Homo sapiens 1..523 523/523 (99%)

(Human), 523 aa.

Q8R314 I RIKEN cDNA 1300003P13 gene 1..523 492/524 (93%) 0.0 j - Mus musculus (Mouse), 524 aa. 1..524 508/524 (96%) Kekuda et nl.

PFam analysis indicates that the NOV23a protein contains the domains shown in the Table 23F.

Example 24.

The NOV24 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 24A.

Kekuda et ul.

TCTTTTGAAGAAATGCTGCAAGATAAACTAAAAGTGCCAGAAAGTGAAAACAACAAAA CCAGCAATAGTTCTCAGGTCTCAAATGAACAGGATAAGATTGATGCCTATAAACTTTT GAAAAAAGAAATGACTCTAGACTTGAAAACCAAATTTGGCTCAACAGCTGATGCACTT GTATCTGATGATGAGACAACCAGACTCGTTACTTCATTAGAAGATGATTTTGATGAGG AATTGGATACTGAGTATTATGCAGTTGGAAAGGAAGATGAGGAGAACCAAGAAGACTT TGATGAGTTGCCATTACTTACCTTTACAGATGGGGAAGATATGAAAACTCCAGCAAAG TCTGGCGTTGAGAAATATCCAACAGATAAAGAGCAGAATTCAAATGAAGAGGACAAGG TTCAGCTAACTGTGCCCCCTGGCATCAAAAATGATGATAAAAATATACTAACAACCTG GGGGGACACTATCTTCTCTATTGTCACAGGAGGTGAAGAAACAAGAGATACGATGGAT TTAGAGAGCTCTAGTTCAGAGGAAGAAAAAGAAGATGATGATGATGCATTAGTCCCAG ATAGCAAACAGGGGAAACCACAGTCAGCAACAGATTATAGTGACCCTGACAATGTAGA TGATGGTCTTTTTATTGTAGACATTCCTAAAACAAATAATGACAAAGAAGTAAACGCA GAACATCACATTAAAGGAAAAGGGAGGGGAGTTCAGGAATCCAAGAGGGGCCTGGTAC AAGATAAGACAGAATTAGAGGATGAAAATCAAGAAGGCATGACTGTGCACAGTTCTGT TCACAGCAATAACCTCAACTCTATGCCAGCTGCTGAAAAGGGTAAAGACACATTAAAA TCAGCTTATGATGATACAGAAAATGACCTAAAAGGAGCAGCTATTCATATCTCAAAAG GAATGCTCCACGAAGAAAAGCCTGGAGAGCAGATTTTGGAAGGTGGCTCAGAGAGTGA ATCTGCACAGAAAGCTGCAGGGAATCAAATGAATGACAGAAAGATTCAACAGGAATCC CTGGGTAGTGCACCACTCATGGGAGATGACCACCCTAACGCATCCAGAGACAGTGTGG AGGGAGACGCTTTGGTAAATGGGGCCAAACTGCACACGCTTTCAGTGGAGCATCAACG TGAGGAATTGAAAGAGGAATTAGTTCTTAAAACTCAAAACCAACCTAGATTCTCCTCT CCAGATGAGATTGATTTGCCCAGAGAACTGGAAGACGAGGTTCCCATTCTGGGAAGAA ATCTTCCCTGGCAACAAGAAAGAGATGTGGCTGCCACAGCCAGTAAGCAAATGAGTGA GAAGATAAGGCTCTCTGAGGGAGAAGCCAAAGAGGACTCCTTGGATGAAGAGTTTTTT CATCACAAGGCAATGCAGGGCACAGAGGTAGGACAGACAGACCAAACTGACAGCACAG GAGGACCAGCTTTCCTTTCTAAAGTAGAAGAGGATGATTATCCCTCTGAAGAACTACT AGAGGATGAAAACGCTATAAATGCAAAACGGTCTAAAGAAAAAAACCCTGGGAATCAG GGCAGGCAGTTTGATGTTAATCTGCAAGTCCCTGACAGAGCAGTTTTAGGGACCATTC ATCCAGATCCAGAAATTGAAGAAAGCAAGCAAGAAACTAGTATGATTTTGGATAGCGA JAAAAACAAGTGAGACTGCTGCCAAAGGGGTCAACACAGGAGGCAGGGAACCAAATACA 'ATGGTGGAAAAAGAACGCCCTCTGGCAGATAAGAAAGCACAGAGACCATTTGAACGAA GTGACTTTTCTGACAGCATAAAAATTCAGACTCCAGAATTAGGTGAAGTGTTTCAGAA TAAAGATTCTGATTATCTGAAGAACGACAACCCTGAGGAACATCTGAAGACCTCAGGG CTTGCAGGGGAGCCTGAGGGAGAACTCTCAAAAGAGGACCATGAGAACACAGAGAAGT ACATGGGCACAGAAAGCCAGGGGTCTGCTGCTGCAGAACCTGAAGATGACTCGTTCCA CTGGACTCCACATACAAGTGTAGAGCCAGGGCATAGTGACAAGAGGGAGGACTTACTT ATCATAAGCAGCTTCTTTAAAGAACAACAGTCTTTGCAGCGGTTCCAGAAGTACTTTA ATGTCCATGAGCTGGAAGCCTTGCTACAAGAAATGTCATCAAAACTGAAGTCAGCGCA GCAGGAGAGCCTGCCCTATAATATGGAAAAAGTCCTAGATAAGGTCTTCCGTGCTTCT GAGTCACAAATTCTGAGCATAGCAGAAAAAATGCTTGATACTCGTGTGGCTGAAAATA GAGATCTGGGAATGAACGAAAATAACATATTTGAAGAGGCTGCAGTGCTTGATGACAT TCAAGACCTCATCTATTTTGTCAGGTACAAGCACTCCACAGCAGAGGAGACAGCCACA CTGGTGATGGCACCACCTCTAGAGGAAGGCTTGGGTGGAGCAATGGAAGAGATGCAAC CACTGCATGAAGATAATTTCTCACGAGAGAAGACAGCAGAACTTAATGTGCAGGTTCC TGAAGAACCCACCCACTTGGACCAACGTGTGATTGGGGACACTCATGCCTCAGAAGTG TCACAGAAGCCAAATACTGAGAAAGACCTGGACCCAGGGCCAGTTACAACAGAAGACA CTCCTATGGATGCTATTGATGCAAACAAGCAACCAGAGACAGCCGCCGAAGAGCCGGC AAGTGTCACACCTTTGGAAAACGCAATCCTTCTAATATATTCATTCATGTTTTATTTA ACTAAGTCGCTAGTTGCTACATTGCCTGATGATGTTCAGCCTGGGCCTGATTTTTATG GACTGCCATGGAAACCTGTATTTATCACTGCCTTCTTGGGAATTGCTTCGTTTGCCAT TTTCTTATGGAGAACTGTCCTTGTTGTGAAGGATAGAGTATATCAAGTCACGGAACAG CAAATTTCTGAGAAGTTGAAGACTATCATGAAAGAAAATACAGAACTTGTACAAAAAT TGTCAAATTATGAACAGAAGATCAAGGAATCAAAGAAACATGTTCAGGAAACCAGGAA ACAAAATATGATTCTCTCTGATGAAGCAATTAAATATAAGGATAAAATCAAGACACTT GAAAAAAATCAGGAAATTCTGGATGACACAGCTAAAAATCTTCGTGTTATGCTAGAAT CTGAGAGAGAACAGAATGTCAAGAATCAGGACTTGATATCAGAAAACAAGAAATCTAT AGAGAAGTTAAAGGATGTTATTTCAATGAATGCCTCAGAGTTTTCAGAGGTTCAGATT GCACTTAATGAAGCTAAGCTTAGTGAAGAGAAGGTGAAGTCTGAATGCCATCGGGTTC AAGAAGAAAATGCTAGGCTTAAGAAGAAAAAAGAGCAGTTGCAGCAGGAAATCGAAGA CTGGAGTAAATTACATGCTGAGCTCAGTGAGCAAATCAAATCATTTGAGAAGTCTCAG AAAGATTTGGAAGTAGCTCTTACTCACAAGGATGATAATATTAATGCTTTGACTAACT Kekuda et al.

GCATTACACAGTTGAATCTGTTAGAGTGTGAATCTGAATCTGAGGGTCAAAATAAAGG TGGAAATGATTCAGATGAATTAGCAAATGGAGAAGTGGGAGGTGACCGGAATGAGAAG ATGAAAAATCAAATTAAGCAGATGATGGATGTCTCTCGGACACAGACTGCAATATCGG TAGTTGAAGAGGATCTAAAGCTTTTACAGCTTAAGCTAAGAGCCTCCGTGTCCACTAA ATGTAACCTGGAAGACCAGGTAAAGAAATTGGAAGATGACCGCAACTCACTACAAGCT GCCAAAGCTGGACTGGAAGATGAATGCAAAACCTTGAGGCAGAAAGTGGAGATTCTGA ATGAGCTCTATCAGCAGAAGGAGATGGCTTTGCAAAAGAAGCTGAGTCAAGAAGAGTA TGAACGGCAAGAAAGAGAGCACAGGCTGTCAGCTGCAGATGAAAAGGCAGTTTCGGCT GCAGAGGAAGTAAAAACTTACAAGCGGAGAATTGAAGAAATGGAGGATGAATTACAGA AGACAGAGCGGTCATTTAAAAACCAGATCGCTACCCATGAGAAGAAAGCTCATGAAAA CTGGCTCAAAGCTCGTGCTGCAGAAAGAGCTATAGCTGAAGAGAAAAGGGAAGCTGCC AATTTGAGACACAAATTATTAGAATTAACACAAAAGATGGCAATGCTGCAAGAAGAAC CTGTGATTGTAAAACCAATGCCAGGAAAACCAAATACACAAAACCCTCCACGGAGAGG TCCTCTGAGCCAGAATGGCTCTTTTGGCCCATCCCCTGTGAGTGGTGGAGAATGCTCC CCTCCATTGACAGTGGAGCCACCCGTGAGACCTCTCTCTGCTACTCTCAATCGAAGAG ATATGCCTAGAAGTGAATTTGGATCAGTGGACGGGCCTCTACCTCATCCTCGATGGTC AGCTGAGGCATCTGGGAAACCCTCTCCTTCTGATCCAGGATCTGGTACAGCTACCATG ATGAACAGCAGCTCAAGAGGCTCTTCCCCTACCAGGGTACTCGATGAAGGCAAGGTTA ATATGGCTCCAAAAGGGCCCCCTCCTTTCCCAGGAGTCCCTCTCATGAGCACCCCCAT GGGAGGCCCTGTACCACCACCCATTCGATATGGACCACCACCTCAGCTCTGCGGACCT TTTGGGCCTCGGCCACTTCCTCCACCCTTTGGCCCTGGTATGCGTCCACCACTAGGCT TAAGAGAATTTGCACCAGGCGTTCCACCAGGAAGACGGGACCTGCCTCTCCACCCTCG GGGATTTTTACCTGGACACGCACCATTTAGACCTTTAGGTTCACTTGGCCCAAGAGAG TACTTTATTCCTGGTACCCGATTACCACCCCCAACCCATGGTCCCCAGGAATACCCAC CACCACCTGCTGTAAGAGACTTACTGCCGTCAGGCTCTAGAGATGAGCCTCCACCTGC CTCTCAGAGCACTAGCCAGGACTGTTCACAGGCTTTAAAACAGAGCCCATAAAACTAT

GACCTCTGAGGTTTCATTGGAAAGAAAGTGTACTGTGCATTATCCATTACAGTAAAGG

ATTTCATTGGCTTCAAAATCCAAAAGTTTATTTTAAAAGGTTTGTTGTTAGAACTAAG

CTGCCTTGGCAGTGTGCATTTTTGAGCCAAACAATTCAAAAATGTCATTTCTTCCCTA

AATAAAAATCACCTTTTAAGCTAAAAAGAAAAAAAAAAAAAAAAAAAA iORF Start: ATG at 13 jORF Stop: TAA at 5734

|SEQ ID NO: 64 1907 aa MW at 213668.2kD

NOV24a, iMAAAPGLLVWLLVLRLPWRVPGQLDPSTGRRFSEHKLCADDECSVL YRGEALEDFTG PDCRFVNFKKGDPVYVYYKLARGWPEVWAGSVGRTFGYFPKDLIQWHEYTKEELQVP CGI 33569-01 TDETDFVCFDGGRDDFHNYNVEELLGFLELYNSAATDSEKAVEKTLQDMEKNPELSKE REPEPEPVEANSEESDSVFSENTEDLQEQFTTQKHHSHANSQANHAQGEQASFESFEE Protein Sequence MLQDKLKVPESENNKTSNSSQVSNEQDKIDAYKLLKKEMTLDLKTKFGSTADALVSDD ETTRLVTSLEDDFDEELDTEYYAVGKEDEENQEDFDELPLLTFTDGEDMKTPAKSGVE KYPTDKEQNSNEEDKVQLTVPPGIKNDDKNILTTWGDTIFSIVTGGEETRDTMDLESS SSEEEKEDDDDALVPDSKQGKPQSATDYSDPDNVDDGLFIVDIPKTNNDKEVNAEHHI KGKGRGVQESKRGLVQDKTELEDENQEGMTVHSSVHSNNLNSMPAAEKGKDTLKSAYD DTENDLKGAAIHISKGMLHEEKPGEQILEGGSESESAQKAAGNQMNDRKIQQESLGSA PLMGDDHPNASRDSVEGDALVNGAKLHTLSVEHQREELKEELVLKTQNQPRFSSPDEI DLPRELEDEVPILGRNLPWQQERDVAATASKQMSEKIRLSEGEAKEDSLDEEFFHHKA MQGTEVGQTDQTDSTGGPAFLSKVEEDDYPSEELLEDENAINAKRSKEKNPGNQGRQF DVNLQVPDRAVLGTIHPDPEIEESKQETSMILDSEKTSETAAKGVNTGGREPNTMVEK ERPLADKKAQRPFERSDFSDSIKIQTPELGEVFQNKDSDYLKNDNPEEHLKTSGLAGE PEGELSKEDHENTEKYMGTESQGSAAAEPEDDSFHWTPHTSVEPGHSDKREDLLIISS FFKEQQSLQRFQKYFNVHELEALLQEMSSKLKSAQQESLPYNMEKVLDKVFRASESQI LSIAEKMLDTRVAENRDLGMNENNIFEEAAVLDDIQDLIYFVRYKHSTAEETATLVMA PPLEEGLGGAMEEMQPLHEDNFSREKTAELNVQVPEEPTHLDQRVIGDTHASEVSQKP NTEKDLDPGPVTTEDTPMDAIDANKQPETAAEEPASVTPLENAILLIYSFMFYLTKSL VATLPDDVQPGPDFYGLPWKPVFITAFLGIASFAIFLWRTVLWKDRVYQVTEQQISE KLKTIMKENTELVQKLSNYEQKIKESKKHVQETRKQNMILSDEAIKYKDKIKTLEKNQ EILDDTAKNLRVMLESEREQNVKNQDLISENKKSIEKLKDVISMNASEFSEVQIALNE AKLSEEKVKSECHRVQEENARLKKKKEQLQQEIEDWSKLHAELSEQIKSFEKSQKDLE VALTHKDDNINALTNCITQLNLLECESESEGQNKGGNDSDELANGEVGGDRNEKMKNQ IKQMMDVSRTQTAISWEEDLKLLQLKLRASVSTKCNLEDQVKKLEDDRNSLQAAKAG Kekuda et al.

LEDECKTLRQKVEILNELYQQKEMALQKKLSQEEYERQEREHRLSAADEKAVSAAEEV KTYKRRIEEMEDELQKTERSFKNQIATHEKKAHENWLKARAAERAIAEEKREAANLRH KLLELTQKMAMLQEEPVIVKPMPGKPNTQNPPRRGPLSQNGSFGPSPVSGGECSPPLT VEPPVRPLSATLNRRDMPRSEFGSVDGPLPHPRWSAEASGKPSPSDPGSGTATMMNSS SRGSSPTRVLDEGKVNMAPKGPPPFPGVPLMSTPMGGPVPPPIRYGPPPQLCGPFGPR PLPPPFGPGMRPPLGLREFAPGVPPGRRDLPLHPRGFLPGHAPFRPLGSLGPREYFIP GTRLPPPTHGPQEYPPPPAVRDLLPSGSRDEPPPASQSTSQDCSQALKQSP

SEQ ID NO: 65 4985 bp

NOV24b, GCTGACCACAACATGGCTGCGGCGCCTGGGCTGCTCGTCTGGCTGCTCGTGCTCCGGC

TGCCCTGGCGGGTGCCGGGCCAGCTGGACCCCAGCACTGGCCGGCGGTTCTCGGAGCA CGI 33569-02 CAAACTCTGCGCGGACGACGAATGCAGCGTGTTAATGTACCGCGGTGAGGCTCTTGAA GATTTCACAGGCCCGGATTGTCGTTTTGTGAATTTTAAAAAAGGTGATCCTGTATATG DNA Sequence TTTACTATAAACTGGCAAGAGGATGGCCTGAAGTTTGGGCTGGAAGTGTAGGACGCAC TTTTGGATATTTTCCAAAAGATTTAATCCAGGTAGTTCATGAATATACCAAAGAAGAG CTACAAGTTCCAACAGATGAGACGGATTTTGTTTGTTTTGATGGAGGAAGAGATGATT TTCATAATTATAATGTAGAAGAACTTTTAGGGTTTTTGGAACTGTACAATTCTGCAGC TACAGATTCTGAGAAAGCTGTAGAAAAAACTTTACAGGATATGGAAAAAAACCCTGAA TTATCTAAGGAAAGGGAACCTGAACCTGAACCAGTAGAAGCCAACTCAGAGGAAAGTG ATAGTGTATTCTCAGAAAACACTGAGGATCTTCAGGAACAGTTTACAACTCAGAAGCA CCACTCCCATGCAAACAGCCAAGCAAATCATGCTCAGGGAGAGCAGGCTTCATTTGAA, TCTTTTGAAGAAATGCTGCAAGATAAACTAAAAGTGCCAGAAAGTGAAAACAACAAAA¹ CCAGCAATAGTTCTCAGGTCTCAAATGAACAGGATAAGATTGATGCCTATAAACTTTT GAAAAAAGAAATGACTCTAGACTTGAAAACCAAATTTGGCTCAACAGCTGATGCACTT GTATCTGATGATGAGACAACCAGACTCGTTACTTCATTAGAAGATGATTTTGATGAGG! AATTGGATACTGAGTATTATGCAGTTGGAAAGGAAGATGAGGAGAACCAAGAAGACTT TGATGAGTTGCCATTACTTACCTTTACAGATGGGGAAGATATGAAAACTCCAGCAAAG TCTGGCGTTGAGAAATATCCAACAGATAAAGAGCAGAATTCAAATGAAGAGGACAAGG TTCAGCTAACTGTGCCCCCTGGCATCAAAAATGATGATAAAAATATACTAACAACCTGj GGGGGACACTATCTTCTCTATTGTCACAGGAGGTGAAGAAACAAGAGATACGATGGATI TTAGAGAGCTCTAGTTCAGAGGAAGAAAAAGAAGATGATGATGATGCATTAGTCCCAG ATAGCAAACAGGGGAAACCACAGTCAGCAACAGATTATAGTGACCCTGACAATGTAGA TGATGGTCTTTTTATTGTAGACATTCCTAAAACAAATAATGACAAAGAAGTAAACGCA GAACATCACATTAAAGGAAAAGGGAGGGGAGTTCAGGAATCCAAGAGGGGCCTGGTAC lAAGATAAGACAGAATTAGAGGATGAAAATCAAGAAGGCATGACTGTGCACAGTTCTGT TCACAGCAATAACCTCAACTCTATGCCAGCTGCTGAAAAGGGTAAAGACACATTAAAA TCAGCTTATGATGATACAGAAAATGACCTAAAAGGAGCAGCTATTCATATCTCAAAAG GAATGCTCCACGAAGAAAAGCCTGGAGAGCAGATTTTGGAAGGTGGCTCAGAGAGTGA ATCTGCACAGAAAGCTGCAGGGAATCAAATGAATGACAGAAAGATTCAACAGGAATCC CTGGGTAGTGCACCACTCATGGGAGATGACCACCCTAACGCATCCAGAGACAGTGTGG AGGGAGACGCTTTGGTAAATGGGGCCAAACTGCACACGCTTTCAGTGGAGCATCAACG TGAGGAATTGAAAGAGGAATTAGTTCTTAAAACTCAAAACCAACCTAGATTCTCCTCT CCAGATGAGATTGATTTGCCCAGAGAACTGGAAGACGAGGTTCCCATTCTGGGAAGAA ATCTTCCCTGGCAACAAGAAAGAGATGTGGCTGCCACAGCCAGTAAGCAAATGAGTGA GAAGATAAGGCTCTCTGAGGGAGAAGCCAAAGAGGACTCCTTGGATGAAGAGTTTTTT CATCACAAGGCAATGCAGGGCACAGAGGTAGGACAGACAGACCAAACTGACAGCACAG GAGGACCAGCTTTCCTTTCTAAAGTAGAAGAGGATGATTATCCCTCTGAAGAACTACT AGAGGATGAAAACGCTATAAATGCAAAACGGTCTAAAGAAAAAAACCCTGGGAATCAG GGCAGGCAGTTTGATGTTAATCTGCAAGTCCCTGACAGAGCAGTTTTAGGGACCATTC ATCCAGATCCAGAAATTGAAGAAAGCAAGCAAGAAACTAGTATGATTTTGGATAGCGA AAAAACAAGTGAGACTGCTGCCAAAGGGGTCAACACAGGAGGCAGGGAACCAAATACA ATGGTGGAAAAAGAACGCCCTCTGGCAGATAAGAAAGCACAGAGACCATTTGAACGAA GTGACTTTTCTGACAGCATAAAAATTCAGACTCCAGAATTAGGTGAAGTGTTTCAGAA TAAAGATTCTGATTATCTGAAGAACGACAACCCTGAGGAACATCTGAAGACCTCAGGG CTTGCAGGGGAGCCTGAGGGAGAACTCTCAAAAGAGGACCATGAGAACACAGAGAAGT ACATGGGCACAGAAAGCCAGGGGTCTGCTGCTGCAGAACCTGAAGATGACTCGTTCCA CTGGACTCCACATACAAGTGTAGAGCCAGGGCATAGTGACAAGAGGGAGGACTTACTT ATCATAAGCAGCTTCTTTAAAGAACAACAGTCTTTGCAGCGGTTCCAGAAGTACTTTA ATGTCCATGAGCTGGAAGCCTTGCTACAAGAAATGTCATCAAAACTGAAGTCAGCGCA GCAGGAGAGCCTGCCCTATAATATGGAAAAAGTCCTAGATAAGGTCTTCCGTGCTTCT Kekuda et al.

GAGTCACAAATTCTGAGCATAGCAGAAAAAATGCTTGATACTCGTGTGGCTGAAAATA GAGATCTGGGAATGAACGAAAATAACATATTTGAAGAGGCTGCAGTGCTTGATGACAT TCAAGACCTCATCTATTTTGTCAGGTACAAGCACTCCACAGCAGAGGAGACAGCCACA CTGGTGATGGCACCACCTCTAGAGGAAGGCTTGGGTGGAGCAATGGAAGAGATGCAAC CACTGCATGAAGATAATTTCTCACGAGAGAAGACAGCAGAACTTAATGTGCAGGTTCC TGAAGAACCCACCCACTTGGACCAACGTGTGATTGGGGACACTCATGCCTCAGAAGTG TCACAGAAGCCAAATACTGAGAAAGACCTGGACCCAGGGCCAGTTACAACAGAAGACA CTCCTATGGATGCTATTGATGCAAACAAGCAACCAGAGACAGCCGCCGAAGAGCCGGC AAGTGTCACACCTTTGGAAAACGCAATCCTTCTAATATATTCATTCATGTTTTATTTA ACTAAGTCGCTAGTTGCTACATTGCCTGATGATGTTCAGCCTGGGCCTGATTTTTATG GACTGCCATGGAAACCTGTATTTATCACTGCCTTCTTGGGAATTGCTTCGTTTGCCAT TTTCTTATGGAGAACTGTCCTTGTTGTGAAGGATAGAGTATATCAAGTCACGGAACAG CAAATTTCTGAGAAGTTGAAGACTATCATGAAAGAAAATACAGAACTTGTACAAAAAT TGTCAAATTATGAACAGAAGATCAAGGAATCAAAGAAACATGTTCAGGAAACCAGGAA ACAAAATATGATTCTCTCTGATGAAGCAATTAAATATAAGGATAAAATCAAGACACTT GAAAAAAATCAGGAAATTCTGGATGACACAGCTAAAAATCTTCGTGTTATGCTAGAAT CTGAGAGAGAACAGAATGTCAAGAATCAGGACTTGATATCAGAAAACAAGAAATCTAT AGAGAAGTTAAAGGATGTTATTTCAATGAATGCCTCAGAGTTTTCAGAGGTTCAGATT GCACTTAATGAAGCTAAGCTTAGTGAAGAGAAGGTGAAGTCTGAATGCCATCGGGTTC AAGAAGAAAATGCTAGGCTTAAGAAGAAAAAAGAGCAGTTGCAGCAGGAAATCGAAGA CTGGAGTAAATTACATGCTGAGCTCAGTGAGCAAATCAAATCATTTGAGAAGTCTCAG AAAGATTTGGAAGTAGCTCTTACTCACAAGGATGATAATATTAATGCTTTGACTAACT GCATTACACAGTTGAATCTGTTAGAGTGTGAATCTGAATCTGAGGGTCAAAATAAAGG TGGAAATGATTCAGATGAATTAGCAAATGGAGAAGTGGGAGGTGACCGGAATGAGAAG ATGAAAAATCAAATTAAGCAGATGATGGATGTCTCTCGGACACAGACTGCAATATCGG TAGTTGAAGAGGATCTAAAGCTTTTACAGCTTAAGCTAAGAGCCTCCGTGTCCACTCC TCCACCCTTTGGCCCTGGTATGCGTCCACCACTAGGCTTAAGAGAATTTGCACCAGGC GTTCCACCAGGAAGACGGGACCTGCCTCTCCACCCTCGGGGATTTTTACCTGGACACG CACCATTTAGACCTTTAGGTTCACTTGGCCCAAGAGAGTACTTTATTCCTGGTACCCG: ATTACCACCCCCAACCCATGGTCCCCAGGAATACCCACCACCACCTGCTGTAAGAGAC TTACTGCCGTCAGGCTCTAGAGATGAGCCTCCACCTGCCTCTCAGAGCACTAGCCAGG ACTGTTCACAGGCTTTAAAACAGAGCCCATAAAACTATGACCTCTGAGGTTTCATTGG¹

AAAGAAAGTGTACTGTGCATTATCCATTACAGTAAAGGATTTCATTGGCTTCAAAATC

CAAAAGTTTATTTTAAAAGGTTTGTTGTTAGAACTAAGCTGCCTTGGCAGTGTGCATT

TTTGAGCCAAACAATTCAAAAATGTCATTTCTTCCCTAAATAAAAATCACCTTTT

ORF Start: ATG at 13 ORF Stop: TAA at 4786

SEQ ID NO: 66 1591 aa MW at .8kD

!NOV24b, MAAAPGLLVWLLVLRLPWRVPGQLDPSTGRRFSEHKLCADDECSVLMYRGEALEDFTG PDCRFVNFKKGDPVYVYYKLARGWPEVWAGSVGRTFGYFPKDLIQWHEYTKEELQVP CGI 33569-02 TDETDFVCFDGGRDDFHNYNVEELLGFLELYNSAATDSEKAVEKTLQDMEKNPELSKE REPEPEPVEANSEESDSVFSENTEDLQEQFTTQKHHSHANSQANHAQGEQASFESFEE Protein Sequence MLQDKLKVPESENNKTSNSSQVSNEQDKIDAYKLLKKEMTLDLKTKFGSTADALVSDD ETTRLVTSLEDDFDEELDTEYYAVGKEDEENQEDFDELPLLTFTDGEDMKTPAKSGVE KYPTDKEQNSNEEDKVQLTVPPGIKNDDKNILTTWGDTIFSIVTGGEETRDTMDLESS SSEEEKEDDDDALVPDSKQGKPQSATDYSDPDNVDDGLFIVDIPKTNNDKEVNAEHHI KGKGRGVQESKRGLVQDKTELEDENQEGMTVHSSVHSNNLNSMPAAEKGKDTLKSAYD DTENDLKGAAIHISKGMLHEEKPGEQILEGGSESESAQKAAGNQMNDRKIQQESLGSA PLMGDDHPNASRDSVEGDALVNGAKLHTLSVEHQREELKEELVLKTQNQPRFSSPDEI DLPRELEDEVPILGRNLPWQQERDVAATASKQMSEKIRLSEGEAKEDSLDEEFFHHKA MQGTEVGQTDQTDSTGGPAFLSKVEEDDYPSEELLEDENAINAKRSKEKNPGNQGRQF DVNLQVPDRAVLGTIHPDPEIEESKQETSMILDSEKTSETAAKGVNTGGREPNTMVEK ERPLADKKAQRPFERSDFSDSIKIQTPELGEVFQNKDSDYLKNDNPEEHLKTSGLAGE PEGELSKEDHENTEKYMGTESQGSAAAEPEDDSFHWTPHTSVEPGHSDKREDLLIISS FFKEQQSLQRFQKYFNVHELEALLQEMSSKLKSAQQESLPYNMEKVLDKVFRASESQI LSIAEKMLDTRVAENRDLGMNENNIFEEAAVLDDIQDLIYFVRYKHSTAEETATLVMA PPLEEGLGGAMEEMQPLHEDNFSREKTAELNVQVPEEPTHLDQRVIGDTHASEVSQKP NTEKDLDPGPVTTEDTPMDAIDANKQPETAAEEPASVTPLENAILLIYSFMFYLTKSL VATLPDDVQPGPDFYGLPWKPVFITAFLGIASFAIFLWRTVLWKDRVYQVTEQQISE Kekuda et al.

KLKTIMKENTELVQKLSNYEQKIKESKKHVQETRKQNMILSDEAIKYKDKIKTLEKNQ EILDDTAKNLRVMLESEREQNVKNQDLISENKKSIEKLKDVISMNASEFSEVQIALNE AKLSEEKVKSECHRVQEENARLKKKKEQLQQEIEDWSKLHAELSEQIKSFEKSQKDLE VALTHKDDNINALTNCITQLNLLECESESEGQNKGGNDSDELANGEVGGDRNEKMKNQ IKQMMDVSRTQTAISWEEDLKLLQLKLRASVSTPPPFGPGMRPPLGLREFAPGVPPG RRDLPLHPRGFLPGHAPFRPLGSLGPREYFIPGTRLPPPTHGPQEYPPPPAVRDLLPS GSRDEPPPASQSTSQDCSQALKQSP

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 24B.

Further analysis of the NOV24a protein yielded the following properties shown in Table 24C.

Table 24C. Protein Sequence Properties NOV24a

PSort j 0.4600 probability located in plasma membrane; 0.1080 probability located in analysis: ,: nucleus; 0.1000 probability located in endoplasmic reticulum (membrane); } 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 23 and 24 analysis:

A search of the NOV24a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 24D.

Table 24D. Geneseq Results for NOV24a

NOV24a Identities/

Geneseq Protein/Organism/Length Residues/ Similarities for Expect

Identifier [Patent #, Date] Match the Matched Value Residues Region Kekuda et al.

In a BLAST search of public sequence datbases, the NOV24a protein was found to have homology to the proteins shown in the BLASTP data in Table 24E.

Table 24E. Public BLASTP Results for NOV24a

NOV24a Identities/

Protein Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number Residues Portion

Q92580 j KIAA0268 protein - Homo sapiens i 715..1907 1 192/1 193 (99%) 0.0 Kekuda et al.

PFam analysis indicates that the NOV24a protein contains the domains shown in the Table 24F.

Table 24F. Domain Analysis of NOV24a

Identities/

Pfam Domain NOV24a Match Region j Similarities Expect Value for the Matched Region

SH3 48-105 16/61 (26%) 0.026 34/61 (56%) Kekuda et al.

Example 25.

The NOV25 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 25A.

Further analysis of the NOV25a protein yielded the following properties shown in Table 25B.

Table 25B. Protein Sequence Properties NOV25a

PSort 0.5500 probability located in lysosome (lumen); 0.3700 probability located in analysis: outside; 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) Kekuda et al.

A search ofthe NOV25a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 25C.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV25a protein was found to have homology to the proteins shown in the BLASTP data in Table 25D.

PFam analysis indicates that the NOV25a protein contains the domains shown in the Table 25E.

Table 25E. Domain Analysis of NOV25a

Identities/

Pfa Domain NOV25a Match Region Similarities Expect Value for the Matched Region Kekuda et al.

Example 26.

The NOV26 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 26 A.

Kekuda et al.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 26B.

Table 26B. Comparison of NOV26a against NOV26b.

NOV26a Residues/ Identities/ j Protein Sequence Match Residues Similarities for the Matched Region

NOV26b 46-368 299/323 (92%) 19..341 299/323 (92%)

Further analysis ofthe NOV26a protein yielded the following properties shown in Table 26C.

Table 26C. Protein Sequence Properties NOV26a

I PSort ; 0.4500 probability located in cytoplasm; 0.3239 probability located in analysis: j microbody (peroxisome); 0.2643 probability located in lysosome (lumen); 0.1000 probability located in mitochondrial matrix space

SignalP No Known Signal Sequence Indicated analysis:

A search ofthe NOV26a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 26D. Kekuda et al.

In a BLAST search of public sequence datbases, the NOV26a protein was found to have homology to the proteins shown in the BLASTP data in Table 26E.

Table 26E. Public BLASTP Results for NOV26a

Protein Protein/Organism/Length NOV26a Identities/ Expect Kekuda et ul.

PFam analysis indicates that the NOV26a protein contains the domains shown in the Table 26F.

Table 26F. Domain Analysis of NOV26a

Identities/

Pfam Domain NOV26a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 27. The NOV27 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 27A. Kekuda et ul.

Table 27A. NOV27 Sequence Analysis

SEQ ID NO: 73 2195 bp

NOV27a, TTTGTTCCTAACAGATTTCTTGCGACAAGGAAACCGGCAGTCTTCCGCTTCCGGTTGC

TCTGTTGCCATAGTAACCGCACCCATAACAGCCGTGGTGGTTATGGCTGGCCTGAGCG CGI 34403-01 GCGCGCAGATCCCCGACGGGGAGTTCACCGCGGTCGTGTACCGCCTCATCCGCAATGC ACGCTACGCCGAGGCGGTGCAGCTGCTGGGCGGAGAACTGCAGCGGAGCCCTAGGAGC DNA Sequence CGCGCCGGCCTGTCGCTGCTAGGCTACTGCTACTACCGCCTGCAGGAGTTCGCGCTGG CGGCCGAGTGCTATGAGCAGCTGGGCCAGCTGCACCCGGAACTGGAGCAGTACCGCCT GTACCAGGCCCAGGCCCTGTACAAGGCCTGCCTTTATGCGGAGGCCACCCGGGTCGCC TTCCTTCTCCTGGATAACCCCGCCTACCACAGCCGGGTCCTCCGCCTGCAAGCTGCTA TCAAGTACAGCGAGGGCGATCTGCCAGGGTCCAGGAGCCTGGTAGAGCAGCTGCCGAG TAGGGAAGGGGGAGAGGAAAGTGGGGGCGAGAATGAGACCGATGGCCAGATCAACCTG GGTTGTTTGCTCTACAAGGAGGGACAGTATGAAGCTGCATGCTCCAAGTTTTTTGCCG CCCTGCAGGCCTCGGGCTACCAGCCTGACCTTTCCTACAACCTGGCTTTGGCCTATTA CAGCAGCCGACAGTATGCTTCAGCACTGAAGCATATCGCTGAGATTATTGAGCGTGGC ATCCGCCAGCACCCTGAGCTAGGTGTGGGCATGACCACTGAGGGCATTGATGTTCGCA GTGTTGGCAACACCTTAGTCCTCCATCAGACTGCTCTGGTGGAAGCCTTCAACCTTAA GGCAGCTATAGAATACCAACTGAGAAACTATGAGGCAGCTCAAGAAGCCCTCACTGAC ATGCCACCCAGGGCAGAGGAAGAGTTGGACCCTGTGACCCTACACAACCAGGCACTAA TGAACATGGATGCCAGGCCTACAGAAGGGTTTGAAAAGCTACAGTTTTTGCTCCAACA GAATCCCTTTCCTCCAGAGACTTTTGGCAACCTGTTGCTGCTCTACTGTAAATATGAG TATTTTGACCTGGCAGCAGATGTCCTGGCAGAAAATGCCCATTTGATTTATAAGTTCC TCACACCCTATCTCTATGACTTCTTGGACGCTGTGATCACTTGCCAGACAGCTCCTGA AGAGGCTTTCATTAAGCTTGATGGGCTAGCAGGGATGCTGACTGAGGTCCTCCGGAAA CTTACCATACAAGTACAGGAAGCAAGACACAATAGAGATGATGAAGCTATCAAAAAGG CAGTGAATGAATATGATGAAACCATGGAGAAATACATTCCTGTGTTGATGGCTCAGGC AAAAATCTACTGGAATCTTGAAAATTATCCAATGGTGGAAAAGATCTTCCGCAAATCT GTGGAATTCTGTAACGACCATGATGTGTGGAAGTTGAATGTGGCTCATGTTCTGTTCA TGCAGGAAAACAAATACAAAGAAGCCATTGGTTTCTATGAACCCATAGTCAAGAAGCA TTATGATAACATCCTGAATGTCAGTGCTATTGTACTGGCTAATCTCTGTGTTTCCTAT ATTATGACAAGTCAAAATGAAGAAGCAGAGGAGTTGATGAGGAAGATTGAAAAGGAGG AAGAGCAGCTCTCTTATGATGACCCAGATAAGAAAATGTACCATCTCTGCATTGTGAA TTTGGTGATAGGAACTCTTTATTGTGCCAAAGGAAATTATGACTTTGGTATTTCTCGA GTTATCAAAAGCTTGGAACCTTACAACAAAAAGCTGGGAACAGACACCTGGTATTATG CCAAAAGATGCTTCCTGTCCTTGTTAGAAAACATGTCAAAACACACAATCATGCTTCG TGATAGTGTTATTCAAGAATGTGTCCAGTTTCTAGAACACTGTGAACTTCATGGCAGA AACATACCTGCTGTTATTGAACAACCCCTGGAAGAAGAAAGAATGCATGTTGGAAAGA ATACAGTCACATATGAGTCTAGGCAGTTAAAAGCTTTGATTTATGAGATTATAGGATG GAATATATAGTAATAGCTGATAGTGGCATTTATCAAATGGCTTTGTTATGTAAATTTG

CATCGCTTTATTTACCCTTTGGCATCTTTATATTTGTTACATGTTGAAC

ORF Start: ATG at 101 ORF Stop: TAG at 2096

SEQ ID NO: 74 665 aa MW at 76098.0kD

NOV27a, MAGLSGAQIPDGEFTAWYRLIRNARYAEAVQLLGGELQRSPRSRAGLSLLGYCYYRL QEFALAAECYEQLGQLHPELEQYRLYQAQALYKACLYAEATRVAFLLLDNPAYHSRVL CGI 34403-01 RLQAAIKYSEGDLPGSRSLVEQLPSREGGEESGGENETDGQINLGCLLYKEGQYEAAC SKFFAALQASGYQPDLSYNLALAYYSSRQYASALKHIAEIIERGIRQHPELGVGMTTE Protein Sequence GIDVRSVGNTLVLHQTALVEAFNLKAAIEYQLRNYEAAQEALTDMPPRAEEELDPVTL HNQALMNNDARPTEGFEKLQFLLQQNPFPPETFGNLLLLYCKYEYFDLAADVLAENAH LIYKFLTPYLYDFLDAVITCQTAPEEAFIKLDGLAGMLTEVLRKLTIQVQEARHNRDD EAIKKAVNEYDETMEKYIPVLMAQAKIYWNLENYPMVEKIFRKSVEFCNDHDVWKLNV AHVLFMQENKYKEAIGFYEPIVKKHYDNILNVSAIVLANLCVSYIMTSQNEEAEELMR KIEKEEEQLSYDDPDKKMYHLCIVNLVIGTLYCAKGNYDFGISRVIKSLEPYNKKLGT DTWYYAKRCFLSLLENMSKHTIMLRDSVIQECVQFLEHCELHGRNIPAVIEQPLEEER MHVGKNTVTYESRQLKALIYEIIGWNI Kekuda et al.

Further analysis ofthe NOV27a protein yielded the following properties shown in Table 27B.

Table 27B. Protein Sequence Properties NOV27a

PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.6640 analysis: probability located in plasma membrane; 0.3000 probability located in microbody (peroxisome); 0.1000 probability located in mitochondrial inner membrane

I SignalP No Known Signal Sequence Indicated

! analysis:

A search ofthe NOV27a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 27C.

Table 27C. Geneseq Results for NOV27a

NOV27a Identities/

Geneseq Protein/Organism/Length [Patent Residues/ Similarities for Expect Identifier #, Date] Match the Matched Value Residues Region

AAM39821 Human polypeptide SEQ ID NO 1..640 1 636/640 (99%) 0.0 2966 - Homo sapiens, 843 aa. 60-699 637/640 (99%) [WO200153312-A1, 26-JUL-2001]

AAM41607 Human polypeptide SEQ ID NO 356-664 293/309 (94%) e-172 6538 - Homo sapiens, 310 aa. 1..309 302/309 (96%) [WO200153312-A1, 26-JUL-2001]

I ABB61288 Drosophila melanogaster 22..660 301/648 (46%) e-157 polypeptide SEQ ID NO 10656 - i l 8-646 424/648 (64%) Drosophila melanogaster, 652 aa. [WO200171042-A2, 27-SEP-2001]

AAB94836 Human protein sequence SEQ ID 385-664 266/280 (95%) e-156 NO:16004 - Homo sapiens, 281 aa. j 1..280 273/280 (97%) [EP 1074617-A2, 07-FEB-2001 ] j Kekuda et al.

In a BLAST search of public sequence datbases, the NOV27a protein was found to have homology to the proteins shown in the BLASTP data in Table 27D.

PFam analysis indicates that the NOV27a protein contains the domains shown in the Table 27E. Kekuda et al.

Example 28.

The NOV28 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 28A.

Kekuda et al.

SEQ ID NO: 77 1303 bp

NOV28b, GTAACAAAACCGCTCAAGTCTGCCTTAAAGAGCCTTACAAGCCAGCCAGTCCCTGCAG

CTCCACAAACTGACCCATCCTGGGCCTTGTTCTCCACAGAATGGGTCTGCTCCTTCCC CGI 35049-02 CTGGCACTCTGCATCCTAGTCCTGTGCTGCGGAGCAATGTCTCCACCCCAGCTGGCCC TCAACCCCTCGGCTCTGCTCTCCCGGGGCTGCAATGACTCAGATGTGCTGGCAGTTGC DNA Sequence AGGCTTTGCCCTGCGGGATATTAACAAAGACAGAAAGGATGGCTATGTGCTGAGACTC AACCGAGTGAACGACGCCCAGGAATACAGACGGGGTGGCCTGGGATCTCTGTTCTATC TTACACTGGATGTGCTAGACTGTGGAATGAGGATATTTTTTGAATCAGTTTATGGTCA ATGCAAAGCAATATTTTATATGAACAACCCAAGTAGAGTTCTCTATTTAGCTGCTTAT AACTGTACTCTTCGCCCAGTTTCAAAAAAAAAGATTTACATGACGTGCCCTGACTGCC CAAGCTCCATACCCACTGACTCTTCCAATCACCAAGTGCTGGAGGCTGCCACCGAGTC TCTTGCGAAATACAACAATGAGAACACATCCAAGCAGTATTCTCTCTTCAAAGTCACC AGGGCTTCTAGCCAGTGGGTGGTCGGCCCTTCTTACTTTGTGGAATACTTAATTAAAG AATCACCATGTACTAAATCCCAGGCCAGCAGCTGTTCACTTCAGTCCTCCGACTCTGT GCCTGTTGGTCTTTGCAAAGGTTCTCTGACTCGAACACACTGGGAAAAGTTTGTCTCT GTGACTTGTGACTTCTTTGAATCACAGGCTCCAGCCACTGGAAGTGAAAACTCTGCTG TTAACCAGAAACCTACAAACCTTCCCAAGGTGGAAGAATCCCAGCAGAAAAACACCCC CCCAACAGACTCCCCCTCCAAAGCTGGGCCAAGAGGATCTGTCCAATATCTTCCTGAC TTGGATGATAAAAATTCCCAGGAAAAGGGCCCTCAGGAGGCCTTTCCTGTGCATCTGG ACCTAACCACGAATCCCCAGGGAGAAACCCTGGATATTTCCTTCCTCTTCCTGGAGCC TATGGAGGAGAAGCTGGTGGTCCTGCCTTTCCCCAAAGAAAAAGCACGCACTGCTGAG TGCCCAGGGCCAGCCCAGAATGCCAGCCCTCTTGTCCTTCCGCCATGAGAATCACACA

GAGTCTTCTGTAGGGGTATGGTGCGCCGCATGACATGGGAGGCGATGGGGACGATGGA

CAGAGACAGAGCGTGCACACGTAGAGT

ORF Start: ATG at 99 ORF Stop: TGA at 1206

SEQ ID NO: 78 369 aa !MW at 40458.6kD

NOV28b, MGLLLPLALCILVLCCGAMSPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKD GYVLRLNRVNDAQEYRRGGLGSLFYLTLDVLDCGMRIFFESVYGQCKAIFYMNNPSRV CGI 35049-02 LYLAAYNCTLRPVSKKKIYMTCPDCPSSIPTDSSNHQVLEAATESLAKYNNENTSKQY SLFKVTRASSQWWGPSYFVEYLIKESPCTKSQASSCSLQSSDSVPVGLCKGSLTRTH Protein Sequence WEKFVSVTCDFFESQAPATGSENSAVNQKPTNLPKVEESQQKNTPPTDSPSKAGPRGS VQYLPDLDDKNSQEKGPQEAFPVHLDLTTNPQGETLDISFLFLEPMEEKLWLPFPKE KARTAECPGPAQNASPLVLPP

SEQ ID NO: 79 1970 bp

|NOV28c, GTAACAAAACCGCTCAAGTCTGCCTTAAAGAGCCTTACAAGCCAGCCAGTCCCTGCAG

CTCCACAAACTGACCCATCCTGGGCCTTGTTCTCCACAGAATGGGTCTGCTCCTTCCC j CGI 35049-03 CTGGCACTCTGCATCCTAGTCCTGTGCTGCGGAGCAATGTCTCCACCCCAGCTGGCCC j TCAACCCCTCGGCTCTGCTCTCCCGGGGCTGCAATGACTCAGATGTGCTGGCAGTTGC

IDNA Sequence AGGCTTTGCCCTGCGGGATATTAACAAAGACAGAAAGGATGGCTATGTGCTGAGACTC AACCGAGTGAACGACGCCCAGGAATACAGACGGGGTGGCCTGGGATCTCTGTTCTATC TTACACTGGATGTGCTAGACTGTGGAATGAGGATATTTTTTGAATCAGTTTATGGTCA ATGCAAAGCAATATTTTATATGAACAACCCAAGTAGAGTTCTCTATTTAGCTGCTTAT AACTGTACTCTTCGCCCAGTTTCAAAAAAAAAGATTTACATGACGTGCCCTGACTGCC CAAGCTCCATACCCACTGACTCTTCCAATCACCAAGTGCTGGAGGCTGCCACCGAGTC TCTTGCGAAATACAACAATGAGAACACATCCAAGCAGTATTCTCTCTTCAAAGTCACC AGGGCTTCTAGCCAGTGGGTGGTCGGCCCTTCTTACTTTGTGGAATACTTAATTAAAG AATCACCATGTACTAAATCCCAGGCCAGCAGCTGTTCACTTCAGTCCTCCGACTCTGT GCCTGTTGGTCTTTGCAAAGGTTCTCTGACTCGAACACACTGGGAAAAGTTTGTCTCT GTGACTTGTGACTTCTTTGAATCACAGGCTCCAGCCACTGGAAGTGAAAACTCTGCTG TTAACCAGAAACCTACAAACCTTCCCAAGGTGGAAGAATCCCAGCAGAAAAATACCCC CCCAACAGACTCCCCCTCCAAAGCTGGGCCAAGAGGATCTGTCCAATATCTTCCTGAC TTGGATGATAAAAATTCCCAGGAAAAGGGCCCTCAGGAGGCCTTTCCTGTGCATCTGG ACCTAACCACGAATCCCCAGGGAGAAACCCTGGATATTTCCTTCCTCTTCCTGGAGCC TATGGAGGAGAAGCTGGTGGTCCTGCCTTTCCCCAAAGAAAAAGCACGCACTGCTGAG TGCCCAGGGCCAGCCCAGAATGCCAGCCCTCTTGTCCTTCCGCCATGAGAATCACACA Kekuda et al.

GAGTCTTCTGTAGGGGTATGGTGCGCCGCATGACATGGGAGGCGATGGGGACGATGGA CAGAGACAGAGCGTGCACACGTAGAGTACCAGGGGAAGGAGCAGACCCATCCTGGGCC TTGTTCTCCACAGAATGGGTCTGCTCCTTCCCCTGGCACTCTGCATCCTAGTCCTGTG

CTGCGGAGCAATGTCTCCACCCCAGCTGGCCCTCAACCCCTCGGCTCTGCTCTCCCGG

GGCTGCAATGACTCAGATGTGCTGGCAGTTGCAGGCTTTGCCCTGGCGGGATATTAAC AAAGACAGAAAGGATGGCTATGTGCTGAGACTCAACCGAGTGAACGACGCCCAGGAAT

ACAGACGGGGTGGCCTGGGATCTCTGTTCTATCTTACACTGGATGTGCTAGAGACTGA

CTGCCATGTGCTCAGAAAGAAGGCATGGCAAGACTGTGGAATGAGGATATTTTTTGAA

TCAGTTTATGGTCAATGCAAAGCAATATTTTATATGAACAACCCAAGTAGAGTTCTCT

ATTTAGCTGCTTATAACTGTACTCTTCGCCCAGTTTCAAAAAAAAAGATTTACATGAC

GTGCCCTGACTGCCCAAGCTCCATACCCACTGACTCTTCCAATCACCAAGTGCTGGAG

GCTGCCACCGAGTCTCTTGCGAAATACAACAATGAGAACACATCCAAGCAGTATTCTC

TCTTCAAAGTCACCAGGGCTTCTAGCCAGTGGGTGGTCGGCCCTTCTTACTTGTGG

ORF Start: ATG at 99 ORF Stop: TGA at 1206

SEQ ID NO: 80 >69 aa MW at 40458.6kD

!NOV28c, MGLLLPLALCILVLCCGAMSPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKD GYVLRLNRVNDAQEYRRGGLGSLFYLTLDVLDCGMRIFFESVYGQCKAIFYMNNPSRV J CGI 35049-03 LYLAAYNCTLRPVSKKKIYMTCPDCPSSIPTDSSNHQVLEAATESLAKYNNENTSKQY SLFKVTRASSQWWGPSYFVEYLIKESPCTKSQASSCSLQSSDSVPVGLCKGSLTRTH j Protein Sequence WEKFVSVTCDFFESQAPATGSENSAVNQKPTNLPKVEESQQKNTPPTDSPSKAGPRGS VQYLPDLDDKNSQEKGPQEAFPVHLDLTTNPQGETLDISFLFLEPMEEKLWLPFPKE KARTAECPGPAQNASPLVLPP

SEQ ID NO: 81 1427 bp

^<NOV28d, AAAGTCTGCCTTAAAGAGCCTTACAAGCCAGCCAGTCCCTGCAGCTCCACAAACTGAC

CCATCCTGGGCCTTGTTCTCCACAGAATGGGTCTGCTCCTTCCCCTGGCACTCTGCAT ^■ CGI 35049-04 CCTAGTCCTGTGCTGCGGAGCAATGTCTCCACCCCAGCTGGCCCTCAACCCCTCGGCT CTGCTCTCCCGGGGCTGCAATGACTCAGATGTGCTGGCAGTTGCAGGCTTTGCCCTGC ,DNA Sequence GGGATATTAACAAAGACAGAAAGGATGGCTATGTGCTGAGACTCAACCGAGTGAACGA CGCCCAGGAATACAGACGGGCAATTTCAAAAAAAAAGATTTACATGACGTGCCCTGAC TGCCCAAGCTCCATACCCACTGACTCTTCCAATCACCAAGTGCTGGAGGCTGCCACCG AGTCTCTTGCGAAATACAACAATGAGAACACATCCAAGCAGTATTCTCTCTTCAAAGT CACCAGGGCTTCTAGCCAGTGGGTGGTCGGCCCTTCTTACTTTGTGGAATACTTAATT AAAGAATCACCATGTACTAAATCCCAGGCCAGCAGCTGTTCACTTCAGTCCTCCGACT CTGTGCCTGTTGGTCTTTGCAAAGGTTCTCTGACTCGAACACACTGGGAAAAGTTTGT CTCTGTGACTTGTGACTTCTTTGAATCACAGGCTCCAGCCACTGGAAGTGAAAACTCT GCTGTTAACCAGAAACCTACAAACCTTCCCAAGGTGGAAGAATCCCAGCAGAAAAACA CCCCCCCAACAGACTCCCCCTCCAAAGCTGGGCCAAGAGGATCTGTCCAATATCTTCC TGACTTGGATGATAAAAATTCCCAGGAAAAGGGCCCTCAGGAGGCCTTTCCTGTGCAT CTGGACCTAACCACGAATCCCCAGGGAGAAACCCTGGATATTTCCTTCCTCTTCCTGG AGCCTATGGAGGAGAAGCTGGTGGTCCTGCCTTTCCCCAAAGAAAAAGCACGCACTGC TGAGTGCCCAGGGCCAGCCCAGAATGCCAGCCCTCTTGTCCTTCCGCCATGAGAATCA

CACAGAGTCTTCTGTAGGGGTATGGTGCGCCGCATGACATGGGAGGCGATGGGGACGA

TGGACAGAGACAGAGCGTGCACACGTAGAGTGGCTAGTGAAGGACGCCTTTTTGACTC

TTCTTGGTCTCAGCATGTTGACTGGGATTGGAAATAATGAGACTGAGCCCTCGGCTTG

GGCTGCACTCTACCCTGTACACTGCCTTGTACCCTGAGCTGCATCACCTCCTAAACTG

AGCAGTCTCATACCATGGAGAGATGCCTCTCTTATGTCTTCAGCCACTCACTTATAAA

GATACTTATCTTTTCAGCAGTATATATGTGCTGAAATCTCAGCATGAAAGCATTGCAT

GAGTAAAGATACTTTCCCTAAAAAAAAAAAAAAAA

ORF Start: ATG at 85 ORF Stop: TGA at 1036

SEQ ID NO: 82 317 aa MW at 34555.7kD

NOV28d, MGLLLPLALCILVLCCGANSPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKD GYVLRLNRVNDAQEYRRAISKKKIYMTCPDCPSS IPTDSSNHQVLEAATESLAKYNNE CGI 35049-04 NTSKQYSLFKVTRASSQWWGPSYFVEYLIKESPCTKSQASSCSLQSSDSVPVGLCKG Kekuda et al.

Protein Sequence SLTRTHWEKFVSVTCDFFESQAPATGSENSAVNQKPTNLPKVEESQQKNTPPTDSPSK AGPRGSVQYLPDLDDKNSQEKGPQEAFPVHLDLTTNPQGETLDISFLFLEPMEEKLW LPFPKEKARTAECPGPAQNASPLVLPP

SEQ ID NO: 83 1544 bp

NOV28e, AAAGTCTGCCTTAAAGAGCCTTACAAGCCAGCCAGTCCCTGCAGCTCCACAAACTGAC

CCATCCTGGGCCTTGTTCTCCACAGAATGGGTCTGCTCCTTCCCCTGGCACTCTGCAT CGI 35049-05 CCTAGTCCTGTGCTGCGGAGCAATGTCTCCACCCCAGCTGGCCCTCAACCCCTCGGCT CTGCTCTCCCGGGGCTGCAATGACTCAGATGTGCTGGCAGTTGCAGGCTTTGCCCTGC DNA Sequence GGGATATTAACAAAGACAGAAAGGATGGCTATGTGCTGAGACTCAACCGAGTGAACGA CGCCCAGGAATACAGACGGGGTGGCCTGGGATCTCTGTTCTATCTTACACTGGATGTG CTAGAGACTGACTGCCATGTGCTCAGAAAGAAGGCATGGCAAGACTGTGGAATGAGGA TATTTTTTGAATCAGCATCAACAGTTTCAAAAAAAAAGATTTACATGACGTGCCCTGA CTGCCCAAGCTCCATACCCACTGACTCTTCCAATCACCAAGTGCTGGAGGCTGCCACC GAGTCTCTTGCGAAATACAACAATGAGAACACATCCAAGCAGTATTCTCTCTTCAAAG TCACCAGGGCTTCTAGCCAGTGGGTGGTCGGCCCTTCTTACTTTGTGGAATACTTAAT TAAAGAATCACCATGTACTAAATCCCAGGCCAGCAGCTGTTCACTTCAGTCCTCCGAC TCTGTGCCTGTTGGTCTTTGCAAAGGTTCTCTGACTCGAACACACTGGGAAAAGTTTG TCTCTGTGACTTGTGACTTCTTTGAATCACAGGCTCCAGCCACTGGAAGTGAAAACTC TGCTGTTAACCAGAAACCTACAAACCTTCCCAAGGTGGAAGAATCCCAGCAGAAAAAC ACCCCCCCAACAGACTCCCCCTCCAAAGCTGGGCCAAGAGGATCTGTCCAATATCTTC CTGACTTGGATGATAAAAATTCCCAGGAAAAGGGCCCTCAGGAGGCCTTTCCTGTGCA TCTGGACCTAACCACGAATCCCCAGGGAGAAACCCTGGATATTTCCTTCCTCTTCCTG GAGCCTATGGAGGAGAAGCTGGTGGTCCTGCCTTTCCCCAAAGAAAAAGCACGCACTG CTGAGTGCCCAGGGCCAGCCCAGAATGCCAGCCCTCTTGTCCTTCCGCCATGAGAATC

ACACAGAGTCTTCTGTAGGGGTATGGTGCGCCGCATGACATGGGAGGCGATGGGGACG

ATGGACAGAGACAGAGCGTGCACACGTAGAGTGGCTAGTGAAGGACGCCTTTTTGACT

CTTCTTGGTCTCAGCATGTTGACTGGGATTGGAAATAATGAGACTGAGCCCTCGGCTT

GGGCTGCACTCTACCCTGTACACTGCCTTGTACCCTGAGCTGCATCACCTCCTAAACT

GAGCAGTCTCATACCATGGAGAGATGCCTCTCTTATGTCTTCAGCCACTCACTTATAA

AGATACTTATCTTTTCAGCAGTATATATGTGCTGAAATCTCAGCATGAAAGCATTGCA

TGAGTAAAGATACTTTCCCTAAAAAAAAAAAAAAAA

ORF Start: ATG at 85 ORF Stop: TGA at 1 153

SEQ ID NO: 84 356 aa MW at 38961.8kD

I

NOV28e, MGLLLPLALCILVLCCGAMΞPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKD GYVLRLNRVNDAQEYRRGGLGSLFYLTLDVLETDCHVLRKKAWQDCGMRIFFESASTV CGI 35049-05 SKKKIYMTCPDCPSSIPTDSSNHQVLEAATESLAKYNNENTSKQYSLFKVTRASSQWV VGPSYFVEYLIKESPCTKSQASSCSLQSSDSVPVGLCKGSLTRTHWEKFVSVTCDFFE Protein Sequence SQAPATGSENSAVNQKPTNLPKVEESQQKNTPPTDSPSKAGPRGSVQYLPDLDDKNSQ EKGPQEAFPVHLDLTTNPQGETLDISFLFLEP EEKLVVLPFPKEKARTAECPGPAQN ASPLVLPP

SEQ ID NO: 85 1 151 1 bp

NOV28f, AAAGTCTGCCTTAAAGAGCCTTACAAGCCAGCCAGTCCCTGCAGCTCCACAAACTGAC

CCATCCTGGGCCTTGTTCTCCACAGAATGGGTCTGCTCCTTCCCCTGGCACTCTGCAT CGI 35049-06 CCTAGTCCTGTGCTGCGGAGCAATGTCTCCACCCCAGCTGGCCCTCAACCCCTCGGCT CTGCTCTCCCGGGGCTGCAATGACTCAGATGTGCTGGCAGTTGCAGGCTTTGCCCTGC DNA Sequence GGGATATTAACAAAGACAGAAAGGATGGCTATGTGCTGAGACTCAACCGAGTGAACGA CGCCCAGGAATACAGACGGGTTTATGGTCAATGCAAAGCAATATTTTATATGAACAAC CCAAGTAGAGTTCTCTATTTAGCTGCTTATAACTGTACTCTTCGCCCAGTTTCAAAAA AAAAGATTTACATGACGTGCCCTGACTGCCCAAGCTCCATACCCACTGACTCTTCCAA TCACCAAGTGCTGGAGGCTGCCACCGAGTCTCTTGCGAAATACAACAATGAGAACACA TCCAAGCAGTATTCTCTCTTCAAAGTCACCAGGGCTTCTAGCCAGTGGGTGGTCGGCC CTTCTTACTTTGTGGAATACTTAATTAAAGAATCACCATGTACTAAATCCCAGGCCAG CAGCTGTTCACTTCAGTCCTCCGACTCTGTGCCTGTTGGTCTTTGCAAAGGTTCTCTG ACTCGAACACACTGGGAAAAGTTTGTCTCTGTGACTTGTGACTTCTTTGAATCACAGG

2i: Kekuda et al.

CTCCAGCCACTGGAAGTGAAAACTCTGCTGTTAACCAGAAACCTACAAACCTTCCCAA GGTGGAAGAATCCCAGCAGAAAAACACCCCCCCAACAGACTCCCCCTCCAAAGCTGGG CCAAGAGGATCTGTCCAATATCTTCCTGACTTGGATGATAAAAATTCCCAGGAAAAGG GCCCTCAGGAGGCCTTTCCTGTGCATCTGGACCTAACCACGAATCCCCAGGGAGAAAC CCTGGATATTTCCTTCCTCTTCCTGGAGCCTATGGAGGAGAAGCTGGTGGTCCTGCCT TTCCCCAAAGAAAAAGCACGCACTGCTGAGTGCCCAGGGCCAGCCCAGAATGCCAGCC CTCTTGTCCTTCCGCCATGAGAATCACACAGAGTCTTCTGTAGGGGTATGGTGCGCCG

CATGACATGGGAGGCGATGGGGACGATGGACAGAGACAGAGCGTGCACACGTAGAGTG

GCTAGTGAAGGACGCCTTTTTGACTCTTCTTGGTCTCAGCATGTTGACTGGGATTGGA

AATAATGAGACTGAGCCCTCGGCTTGGGCTGCACTCTACCCTGTACACTGCCTTGTAC

CCTGAGCTGCATCACCTCCTAAACTGAGCAGTCTCATACCATGGAGAGATGCCTCTCT

TATGTCTTCAGCCACTCACTTATAAAGATACTTATCTTTTCAGCAGTATATATGTGCT

GAAATCTCAGCATGAAAGCATTGCATGAGTAAAGATACTTTCCCTAAAAAAAAAAAAA

AAA

ORF Start: ATG at 85 ORF Stop: TGA at 1120

SEQ ID NO: 86 345 aa IMW at 37822.5kD

NOV28f, MGLLLPLALCILVLCCGAMSPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKD GWLRLNRVNDAQEYRR\ΛTGQCKAIFYMNNPSRVLYLAAYNCTLRPVSKKKIYMTCPD CGI 35049-06 CPSSIPTDSSNHQVLEAATESLAKYNNENTSKQYSLFKVTRASSQWWGPSYFVEYLI KESPCTKSQASSCSLQSSDSVPVGLCKGSLTRTHWEKFVSVTCDFFESQAPATGSENS Protein Sequence AVNQKPTNLPKVEESQQKNTPPTDSPSKAGPRGSVQYLPDLDDKNSQEKGPQEAFPVH LDLTTNPQGETLDISFLFLEPMEEKLWLPFPKEKARTAECPGPAQNASPLVLPP

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 28B.

I Table 28B. Comparison of NOV28a against NOV28b through NOV28f. j

Further analysis ofthe NOV28a protein yielded the following properties shown in Table 28C. Kekuda et al.

Table 28C. Protein Sequence Properties NOV28a

PSort 0.8200 probability located in outside; 0.1900 probability located in lysosome analysis: (lumen); 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 19 and 20 analysis:

A search ofthe NOV28a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 28D.

Kekuda et ul.

In a BLAST search of public sequence datbases, the NOV28a protein was found to have homology to the proteins shown in the BLASTP data in Table 28E. Kekuda et ul.

PFam analysis indicates that the NOV28a protein contains the domains shown in the Table 28F.

Kekuda et al.

Example 29.

The NOV29 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 29A.

Table 29A. NOV29 Sequence Analysis

SEQ ID NO: 87 2973 bp

NOV29a, CGCCCCGGGCTGGCGATGCTGCGCCGCCCCGCTCCCGCGCTGGCCCCGGCCGCCCGGC

TGCTGCTGGCCGGGCTGCTGTGCGGCGGCGGGGTCTGGGCCGCGCGAGTTAACAAGCA CG54912-02 CAAGCCCTGGCTGGAGCCCACCTACCACGGCATAGTCACAGAGAACGACAACACCGTG

CTCCTCGACCCCCCACTGATCGCGCTGGATAAAGATGCGCCTCTGCGATTTGCAGAGA DNA Sequence GTTTTGAGGTGACAGTCACCAAAGAAGGTGAGATTTGTGGATTTAAAATTCACGGGCA

GAATGTCCCCTTTGATGCAGTGGTAGTGGATAAATCCACTGGTGAGGGAGTCATTCGC

TCCAAAGAGAAACTGGACTGTGAGCTGCAGAAAGACTATTCATTCACCATCCAGGCCT

ATGATTGTGGGAAGGGACCTGATGGCACCAACGTGAAAAAGTCTCATAAAGCAACTGT

TCATATTCAGGTGAACGACGTGAATGAGTACGCGCCCGTGTTCAAGGAGAAGTCCTAC

AAAGCCACGGTCATCGAGGGGAAGCAGTACGACAGCATTTTGAGGGTGGAGGCCGTGG

ATGCCGACTGCTCCCCTCAGTTCAGCCAGATTTGCAGCTACGAAATCATCACTCCAGA:

CGTGCCCTTTACTGTTGACAAAGATGGTTATATAAAAAACACAGAGAAATTAAACTAC

GGGAAAGAACATCAATATAAGCTGACCGTCACTGCCTATGACTGTGGGAAGAAAAGAG

CCACAGAAGATGTTTTGGTGAAGATCAGCATTAAGCCCACCTGCACCCCTGGGTGGCA

AGGATGGAACAACAGGATTGAGTATGAGCCGGGCACCGGCGCGTTGGCCGTCTTTCCA

AATATCCACCTGGAGACATGTGACGAGCCAGTCGCCTCAGTACAGGCCACAGTGGAGC

TAGAAACCAGCCACATAGGGAAAGGCTGCGACCGAGACACCTACTCAGAGAAGTCCCT

CCACCGGCTCTGTGGTGCGGCCGCGGGCACTGCCGAGCTGCTGCCATCCCCGAGTGGA

TCCCTCAACTGGACCATGGGCCTGCCCACCGACAATGGCCACGACAGCGACCAGGTGT

TTGAGTTCAACGGCACCCAGGCAGTGAGGATCCCGGATGGCGTCGTGTCGGTCAGCCC

CAAAGAGCCGTTCACCATCTCGGTGTGGATGAGACATGGGCCATTCGGCAGGAAGAAG

GAGACAATTCTTTGCAGTTCTGATAAAACAGATATGAATCGGCACCACTACTCCCTCT

ATGTCCACGGGTGCCGGCTGATCTTCCTCTTCCGTCAGGATCCTTCTGAGGAGAAGAA

ATACAGACCTGCAGAGTTCCACTGGAAGTTGAATCAGGTCTGTGATGAGGAATGGCAC

CACTACGTCCTCAATGTAGAATTCCCGAGTGTGACTCTCTATGTGGATGGCACGTCCC

ACGAGCCCTTCTCTGTGACTGAGGATTACCCGCTCCATCCATCCAAGATAGAAACTCA

GCTCGTGGTGGGGGCTTGCTGGCAAGAGTTTTCAGGAGTTGAAAATGACAATGAAACT

GAGCCTGTGACTGTGGCCTCTGCAGGTGGCGACCTGCACATGACCCAGTTTTTCCGAG

GCAATCTGGCTGGCTTAACTCTCCGTTCCGGGAAACTCGCGGATAAGAAGGTGATCGA

CTGTCTGTATACCTGCAAGGAGGGGCTGGACCTGCAGGTCCTCGAAGACAGTGGCAGA

GGCGTGCAGATCCAAGCACACCCCAGCCAGTTGGTATTGACCTTGGAGGGAGAAGACC

TCGGGGAATTGGATAAGGCCATGCAGCACATCTCGTACCTGAACTCCCGGCAGTTCCC

CACGCCCGGAATTCGCAGACTCAAAATCACCAGCACAATCAAGTGTTTTAACGAGGCC

ACCTGCATTTCGGTCCCCCCGGTAGATGGCTACGTGATGGTTTTACAGCCCGAGGAGC

CCAAGATCAGCCTGAGTGGCGTCCACCATTTTGCCCGAGCAGCTTCTGAATTTGAAAG

CTCAGAAGGGGTGTTCCTTTTCCCTGAGCTTCGCATCATCAGCACCATCACGAGAGAA

GTGGAGCCTGAAGGGGACGGGGCTGAGGACCCCACAGTTCAAGAATCACTGGTGTCCG

AGGAGATCGTGCACGACCTGGATACCTGTGAGGTCACGGTGGAGGGAGAGGAGCTGAA

CCACGAGCAGGAGAGCCTGGAGGTGGACATGGCCCGCCTGCAGCAGAAGGGCATTGAA

GTGAGCAGCTCTGAACTGGGCATGACCTTCACAGGCGTGGACACCATGGCCAGCTACG

AGGAGGTTTTGCACCTGCTGCGCTATCGGAACTGGCATGCCAGGTCCTTGCTTGACCG

GAAGTTTAAGCTCATCTGCTCAGAGCTGAATGGCCGCTACATCAGCAACGAATTTAAG

GTGGAGGTGAATGTAATCCACACGGCCAACCCCATGGAACACGCCAACCACATGGCTG

CCCAGCCACAGTTCGTGCACCCGGAACACCGCTCCTTTGTTGACCTGTCAGGCCACAA

CCTGGCCAACCCCCACCCGTTCGCAGTCGTCCCCAGCACTGCGACAGTTGTGATCGTG

GTGTGCGTCAGCTTCCTGGTGTTCATGATTATCCTGGGGGTATTTCGGATCCGGGCCG

CACATCGGCGGACCATGCGGGATCAGGACACCGGGAAGGAGAACGAGATGGACTGGGA

CGACTCTGCCCTGACCATCACCGTCAACCCCATGGAGACCTATGAGGACCAGCACAGC

AGTGAGGAGGAGGAGGAAGAGGAAGAGGAAGAGGAAAGCGAGGACGGCGAAGAAGAGG

ATGACATCACCAGCGCCGAGTCGGAGAGCAGCGAGGAGGAGGAGGGGGAGCAGGGCGA Kekuda et al.

Kekuda et al.

Kekuda et ul.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 29B.

Kekuda et al.

2-222 220/231 (94%)

Further analysis ofthe NOV29a protein yielded the following properties shown in Table 29C.

A searc o t e OV29a proten aganst t e eneseq ata ase, a propretary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 29D.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV29a protein was found to have homology to the proteins shown in the BLASTP data in Table 29E.

PFam analysis indicates that the NOV29a protein contains the domains shown in the Table 29F.

222 Kekuda et al.

Example 30.

The NOV30 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 30A.

Kekuda et al.

Kekuda et al.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 30B.

Further analysis ofthe NOV30a protein yielded the following properties shown in Table 30C.

Table 30C. Protein Sequence Properties NOV30a

PSort analysis: j SignalP analysis: ! No Known Signal Sequence Indicated

A search ofthe NOV30a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 30D.

In a BLA T searc o pu c sequence datbases, the NOV30a protein was found have homology to the proteins shown in the BLASTP data in Table 30E. Kekuda et al.

Table 30E. Public BLASTP Results for NOV30a

NOV30a Identities/

Protein

Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number

Residues Portion

No Significant Matches Found

PFam analysis indicates that the NOV30a protein contains the domains shown in the Table 30F.

Table 30F. Domain Analysis of NOV30a

Identities/

Pfam Domain NOV30a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 31.

The NOV31 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 31 A.

Table 31A. NOV31 Sequence Analysis

SEQ ID NO: 1 15 2628 bp

|NOV31a, ACCGTGCCTCTGCGGCCTGCGTGCCCGGAGTCCCCGCCTGTGTCGTCTCTGTCGCCGT

CCCCGTCTCCTGCCAGGCGCGGAGCCCTGCGAGCCGCGGGTGGGCCCCAGGCGCGCAG

|CG56326-01 ACATGGGCTGCTCCGCCAAAGCGCGCTGGGCTGCCGGGGCGCTGGGCGTCGNGGGGCT i ACTGTGCGCTGTGCTGGGCGCTGTCATGATCGTGATGGTGCNGTCGCTCATCAAGCAG

I DNA Sequence CAGGTCCTTAAGAACGTGCGCATCGACCCCAGTAGCCTGTCCTTCAACATGTGGAAGG AGATCCCTATCCCCTTCTATCTCTCCGTCTACTTCTTTGACGTCATGAACCCCAGCGA GATCCTGAAGGGCGAGAAGCCGCAGGTGCGGGAGCCCGGGCCCTACGTGTACAGGGAG TTCAGGCACAAAAGCAACATCACCTTCAACAACAACGACACCGTGTCCTTCCTCGAGT ACCGCACCTTCCAGTTCCAGCCCTCCAAGTCCCACGGCTCGGAGAGCGACTACATCGT CATGCCCAACATCCTGGTCTTGGGTGCGGCGGTGATGATGGAGAATAAGCCCATGACC CTGAAGCTCATCATGACCTTGGCATTCACCACCCTCGGCGAACGTGCCTTCATGAACC GCACTGTGGGTGAGATCATGTGGGGCTACAAGGACCCCCTTGTGAATCTCATCAACAA GTACTTTCCAGGCATGTTCCCCTTCAAGGACAAGTTCGGATTATTTGCTGAGCTCAAC AACTCCGACTCTGGGCTCTTCACGGTGTTCACGGGGGTCCAGAACATCAGCAGGATCC ACCTCGTGGACAAGTGGAACGGGCTGAGCAAGGTTGACTTCTGGCATTCCGATCAGTG CAACATGATCAATGGAACTTCTGGGCAAATGTGGCCGCCCTTCATGACTCCTGAGTCC TCGCTGGAGTTCTACAGCCCGGAGGCCTGCCGATCCATGAAGCTAATGTACAAGGAGT CAGGGGTGTTTGAAGGCATCCCCACCTATCGCTTCGTGGCTCCCAAAACCCTGTTTGN Kekuda et al.

CAACGGGTCCATCTACCCACCCAACGAAGGCTTCTGCCCGTGCCTGGAGTCTGGAATT CAGAACGTCAGCACCTGCAGGTTCAGTGCCCCCTTGTTTCTCTCCCATCCTCACTTCC TCAACGCCGACCCGGTTCTGGCAGAAGNGGTGACTNNCCTGCACNCTAACCAGGAGGC ACACTCCTTGTTCCTGGACATCCACCCGGTCACGGGAATCCCCATGAACTGCTCTGTG AAACTGCAGCTGAGCCTCTACATGAAATCTGTCGCAGGCATTGGACAAACTGGGAAGA TTGAGCCTGTGGTCCTGCCGCTGCTCTGGTTTGCAGAGAGCGGGGCCATGGAGGGGGA GACTCTTCACACATTCTACACTCAGCTGGTGTTGATGCCCAAGGTGATGCACTATGCC CAGTACGTCCTCCTGGCGCTGGGCTGCGTCCTGCTGCTGGTCCCTGTCATCTGCCAAA TCCGGAGCCAAGAGAAATGCTATTTATTTTGGAGTAGTAGTAAAAAGGGCTCAAAGGA TAAGGAGGCCATTCAGGCCTATTCTGAATCCCTGATGACATCAGCTCCCAAGGGCTCT GTGCTGCAGGAAGCAAAACTGTAGGCTCCTGAGGACACCGTGAGCCAGCCAGGCCTGG CCGCTGGGCCTGACCGGCCCCCCAGCCCCTACACNCCGCTTCTCCCGGACTCTCCCAG CAGACAGCCCCCCAGCCCCACAGCCTGAGCCTCCCAGCTGCCATGTCCCTGTTGCACA

CCTGCACACACGCCCTGGCACACATACACACATGCGTGCAGGCTTGTGCAGACACTCA

GGGATGGAGCTGCTGCTGAAGGGACTTGTAGGGAGAGGCTCGTCAACAACCACTGTTC

TGGAACCTTCTCTCCACGTGGCCCACAGGCCTGACCACAGGGGCTGTGGGTCCTGCGT

CCCCTTCCTCGGGTGAGCCTGGCCTGTCCCGTTCAGCCGTTGGGCCCAGGCTTCCTCC

CCTCCAACGTGAAACACTGCAGTCCCGGTGTGGTGGCTCCCCATGCAGGACGGGCCAG

GCTGGGAGTGCCGCCTTCCTGTGCCAAATTCAGTGGGGACTCAGTGCCCAGGCCCTGG

CCACGAGCTTTGGCCTTGGTCTACCTGCCAGGCCAGGCAAAGCGCCTTTACACAGGCC

TCGGAAAACAATGGAGTGAGCACAAGATGCCCTGTGCAGCTGCCCGAGGGTCTCCGCC

CACCCCGGCCGGACTTTGATCCCCCCGAAGTCTTCACAGGCACTCCATCGGGTTGTCT

GGCGCCCTTTTCCTCCAGCCTAAACTGACATCATCCTATGGACTGAGCCGGCCACTTT

GGCCGAAGTGGCCGCAGGCTGTGCCCCCGAGCTGCCCCCACCCCCTCACAGGGTCCCT

CAGATTATAGGTGCCCAGGCTGAGGTGAAGAGGCCTGGGGGCCCTGCCTTCCGGCCGC

TCCTGGACCCTGGGGCAAACCTGTGACCCTTTTCTACTGGAATAGAAATGAGTTTTAT

CATCTTTGAAAAATAATTCACTCTTGAAGTAATAAACGTTTAAAAAAATGGGAAAAAA

AAAAAAAAAAAAAAAAAA

ORF Start: at 218 |ORF Stop: at 1745

SEQ ID NO: 1 16 1509 aa |MW at 56449.3kD

|NOV31a, MGCSAKARWAAGALGVXGLLCAVLGAVMIVMVXSLIKQQVLKNVRIDPSSLSFNMWKE IPIPFYLSVYFFDVMNPSEILKGEKPQVREPGPYVYREFRHKSNITFNNNDTVSFLEY ΪCG56326-01 RTFQFQPSKSHGSESDYIVMPNILVLGAAVMMENKPMTLKLIMTLAFTTLGERAFMNR TVGEIMWGYKDPLVNLINKYFPGMFPFKDKFGLFAELNNSDSGLFTVFTGVQNISRIH I Protein Sequence LVDKWNGLSKVDFWHSDQCNMINGTSGQMWPPFMTPESSLEFYSPEACRSMKLMYKES GVFEGIPTYRFVAPKTLFXNGSIYPPNEGFCPCLESGIQNVSTCRFSAPLFLSHPHFL NADPVLAEXVTXLHXNQEAHSLFLDIHPVTGIPMNCSVKLQLSLYMKSVAGIGQTGKI EPWLPLLWFAESGAMEGETLHTFYTQLVLMPKVMHYAQYVLLALGCVLLLVPVICQI RSQEKCYLFWSSSKKGSKDKEAIQAYSESLMTSAPKGSVLQEAKL

SEQ ID NO: 1 17 1248 bp

NOV31b, AGATCTCTCATCAAGCAGCAGGTCCTTAAGAACGTGCGCATCGACCCCAGTAGCCTGT CCTTCAACATGTGGAAGGAGATCCCTATCCCCTTCTATCTCTCCGTCTACTTCTTTGA 175070268 DNA CGTCATGAACCCCAGCGAGATCCTGAAGGGCGAGAAGCCGCAGGTGCGGGAGCGCGGG CCCTACGTGTACAGGGAGTTCAGGCACAAAAGCAACATCACCTTCAACAACAACGACA Sequence CCGTGTCCTTCCTCGAGTACCGCACCTTCCAGTTCCAGCCCTCCAAGTCCCACGGCTC GGAGAGCGACTACATCGTCATGCCCAACATCCTGGTCTTGGGTGCGGCGGTGATGATG GAGAATAAGCCCATGACCCTGAAGCTCATCATGACCTTGGCATTCACCACCCTCGGCG AACGTGCCTTCATGAACCGCACTGTGGGTGAGATCATGTGGGGCTACAAGGACCCCCT TGTGAATCTCATCAACAAGTACTTTCCAGGCATGTTCCCCTTCAAGGACAAGTTCGGA TTATTTGCTGAGCTCAACAACTCCGACTCTGGGCTCTTCACGGTGTTCACGGGGGTCC AGAACATCAGCAGGATCCACCTCGTGGACAAGTGGAACGGGCTGAGCAAGGTTGACTT CTGGCATTCCGATCAGTGCAACATGATCAATGGAACTTCTGGGCAAATGTGGCCGCCC TTCATGACTCCTGAGTCCTCGCTGGAGTTCTACAGCCCGGAGGCCTGCCGATCCATGA AGCTAATGTACAAGGAGTCAGGGGTGTTTGAAGGCATCCCCACCTATCGCTTCGTGGC TCCCAAAACCCTGTTTGCCAACGGGTCCATCTACCCACCCAACGAAGGCTTCTGCCCG TGCCTGGAGTCTGGAATTCAGAACGTCAGCACCTGCAGGTTCAGTGCCCCCTTGTTTC Kekuda et al.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 3 IB.

Kekuda et al.

Table 31B. Comparison of NOV31a against NOV31b.

NOV31a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV31b 34-447 409/414 (98%) 2..415 409/414 (98%)

Further analysis ofthe NOV31 a protein yielded the following properties shown in Table 3 IC.

Table 31C. Protein Sequence Properties NOV31a

PSort j 0.5644 probability located in microbody (peroxisome); 0.4600 probability analysis: j located in plasma membrane; 0.1000 probability located in endoplasmic

J reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 35 and 36 analysis:

A search ofthe NOV31 a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3 ID.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV31a protein was found to have homology to the proteins shown in the BLASTP data in Table 3 IE.

Table 31E. Public BLASTP Results for NOV31a

NOV31a Identities/ j

Protein ϊ Residues/ Similarities for Ϊ Expect

Accession Protein/Organism/Length

Match the Matched j Value

Number Residues Portion

Q14016 CLA- 1 - Homo sapiens (Human), I ..509 501/509 (98%) 0.0 ! 509 aa. ..509 501/509 (98%)

Q8WTV0 Similar to CD36 antigen (collagen 1..467 460/467 (98%) 0.0 type I receptor, thrombospondin 1..467 460/467 (98%) receptor)-like 1 - Homo sapiens (Human), 552 aa.

Q8SQC1 High density lipoprotein receptor 1..509 437/509 (85%) 0.0 SR-BI - Sus scrofa (Pig), 509 aa. 1..509 474/509 (92%) Kekuda et al.

PFam analysis indicates that the NOV3 la protein contains the domains shown in the Table 3 IF.

Table 31F. Domain Analysis of NOV31a

Identities/

Pfam Domain NOV31a Match Region Similarities Expect Value i for the Matched Region

CD36 5-445 213/567 (38%) 3.6e-227 410/567 (72%)

Example 32.

The NOV32 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 32A.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 32B.

Kekuda et al.

Further analysis ofthe NOV32a protein yielded the following properties shown in Table 32C.

A search ofthe NOV32a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 32D.

Kekuda et ul.

In a BLAST search of public sequence datbases, the NOV32a protein was found to have homology to the proteins shown in the BLASTP data in Table 32E. Kekuda et ul.

PFam analysis indicates that the NOV32a protein contains the domains shown in the Table 32F.

Table 32F. Domain Analysis of NOV32a

Identities/

Pfam Domain NOV32a Match Region 1 Similarities Expect Value

' for the Matched Region serpin 48..424 193/397 (49%) 1.6e-171 317/397 (80%) Kekuda et ul.

Example 33.

The NOV33 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 33A.

Kekuda et al.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 33B.

Kekuda et al.

NOV33d No Significant Alignment Found.

NOV33e No Significant Alignment Found.

NOV33f No Significant Alignment Found.

Further analysis ofthe NOV33a protein yielded the following properties shown in Table 33C.

A search ofthe NOV33a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 33D.

Table 33D. Geneseq Results for NOV33a

NOV33a Identities/

No Significant Matches Found

In a BLAST search of public sequence datbases, the NOV33a protein was found to have homology to the proteins shown in the BLASTP data in Table 33E.

Table 33E. Public BLASTP Results for NOV33a

NOV33a Identities/

Protein Residues/ Similarities for Expect

Accession Protein/Organism/Length

Match the Matched Value

Number Residues Portion

242 Kekuda et al.

PFam analysis indicates that the NOV33a protein contains the domains shown in the Table 33F.

Table 33F. Domain Analysis of NOV33a

Identities/

Pfam Domain NOV33a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 34.

The NOV34 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 34A.

Further analysis ofthe NOV34a protein yielded the following properties shown in

Table 34B.

Table 34B. Protein Sequence Properties NOV34a Kekuda et al.

PSort 0.8500 probability located in lysosome (lumen); 0.5392 probability located in analysis: nucleus; 0.1000 probability located in mitochondrial matrix space; 0.0000 probability located in endoplasmic reticulum (membrane)

SignalP No Known Signal Sequence Indicated analysis:

A search ofthe NOV34a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 34C.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV34a protein was found to have homology to the proteins shown in the BLASTP data in Table 34D.

PFam analysis indicates that the NOV34a protein contains the domains shown in the Table 34E.

Table 34E. Domain Analysis of NOV34a

Identities/

Pfam Domain i NOV34a Match Region Similarities Expect Value for the Matched Region

MHC I 1..24 16/24 (67%) 6.1e-07 Kekuda et ul.

Example 35.

The NOV35 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 35A.

Further analysis ofthe NOV35a protein yielded the following properties shown in Table 35B.

Table 35B. Protein Sequence Properties NOV35a

( PSort 0.8191 probability located in mitochondrial intermembrane space; 0.5581 j

! analysis: probability located in mitochondrial matrix space; 0.5500 probability located in nucleus; 0.3285 probability located in lysosome (lumen)

SignalP No Known Signal Sequence Indicated analysis:

A search ofthe NOV35a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 35C.

Table 35C. Geneseq Results for NOV35a Kekuda et al.

In a BLAST search of public sequence datbases, the NOV35a protein was found to have homology to the proteins shown in the BLASTP data in Table 35D.

Table 35D. Public BLASTP Results for NOV35a

Protein NOV35a Identities/ Expect

Protein/Organism/Length Accession Residues/ Similarities for Value Kekuda et al.

PFam analysis indicates that the NOV35a protein contains the domains shown in the Table 35E.

Table 35E. Domain Analysis of NOV35a

Identities/

Pfa Domain NOV35a Match Region Similarities ] Expect Value

] for the Matched Region j

MHC I .24 13/24 (54%) 0.00021 23/24 (96%)

Example 36.

The NOV36 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 36A.

Table 36A. NOV36 Sequence Analysis Kekuda et ul.

Table 36B.

Table 36B. Protein Sequence Properties NOV36a

PSort 0.8169 probability located in lysosome (lumen); 0.6500 probability located in analysis: cytoplasm; 0.1000 probability located in mitochondrial matrix space; 0.0000 probability located in endoplasmic reticulum (membrane)

SignalP No Known Signal Sequence Indicated j analysis:

A search of the NOV36a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 36C.

Table 36C. Geneseq Results for NOV36a

Identities/

NOV36a

Similarities

Geneseq Protein/Organism/Length Residues/ Expect for the

Identifier i [Patent #, Date] Match Value

Matched Residues

Region

AAU79455 , HLA-G recombinant protein 2 - 1..24 1 24/24 (100%) j 2e-07 •; Homo sapiens, 234 aa. 93..1 16 I 24/24 (100%) j Kekuda et ul.

In a BLAST search of public sequence datbases. the NOV36a protein was found to have homology to the proteins shown in the BLASTP data in Table 36D.

Kekuda et al.

PFam analysis indicates that the NOV36a protein contains the domains shown in the Table 36E.

Kekuda et al.

Example 37.

The NOV37 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 37A.

Kekuda et al.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 37B.

Further analysis ofthe NOV37a protein yielded the following properties shown in

Table 37C. Kekuda et al.

Table 37C. Protein Sequence Properties NOV37a

PSort 0.7480 probability located in microbody (peroxisome); 0.2213 probability analysis: located in lysosome (lumen); 0.1000 probability located in mitochondrial matrix space; 0.0000 probability located in endoplasmic reticulum (membrane)

SignalP No Known Signal Sequence Indicated analysis:

A search ofthe NOV37a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 37D.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV37a protein was found to have homology to the proteins shown in the BLASTP data in Table 37E.

Kekuda et al.

PFam analysis indicates that the NOV37a protein contains the domains shown in the Table 37F.

Table 37F. Domain Analysis of NOV37a

Identities/

Pfam Domain NOV37a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 38.

The NOV38 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 38 A.

Kekuda et al.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 38B.

Table 38B. Comparison of NOV38a against NOV38b.

NOV38a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV38b 18..38 20/21 (95%) 1..21 21/21 (99%)

Further analysis ofthe NOV38a protein yielded the following properties shown in Table 38C.

A search ofthe NOV38a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 38D. Kekuda et al.

In a BLAST search of public sequence datbases, the NOV38a protein was found to have homology to the proteins shown in the BLASTP data in Table 38E. Kekuda et al.

PFam analysis indicates that the NOV38a protein contains the domains shown in the Table 38F. Kekuda et al.

Table 38F. Domain Analysis of NOV38a

Identities/

Pfam Domain NOV38a Match Region Similarities Expect Value for the Matched Region

No Significant Matches Found

Example 39.

The NOV39 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 39A.

Further analysis of the NOV39a protein yielded the following properties shown in Table 39B.

Table 39B. Protein Sequence Properties NOV39a

PSort 0.8500 probability located in lysosome (lumen); 0.7847 probability located in analysis: mitochondrial intermembrane space; 0.4500 probability located in cytoplasm; 0.4488 probability located in mitochondrial matrix space Kekuda et al.

A search ofthe NOV39a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 39C.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV39a protein was found to have homology to the proteins shown in the BLASTP data in Table 39D.

PFam analysis indicates that the NOV39a protein contains the domains shown in the Table 39E.

Table 39E. Domain Analysis of NOV39a

Identities/

Pfam Domain NOV39a Match Region j Similarities Expect Value j for the Matched Region

MHC I 2-24 15/23 (65%) 7.1e-05 21/23 (91%) Kekuda et al.

Example 40.

The NOV40 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 40A.

Further analysis of the NOV40a protein yielded the following properties shown in

Table 40B.

Table 40B. Protein Sequence Properties NOV40a Kekuda et al.

PSort 0.3700 probability located in outside; 0.1440 probability located in microbody analysis: (peroxisome); 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 18 and 19 analysis:

A search of the NOV40a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 40C.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV40a protein was found to have homology to the proteins shown in the BLASTP data in Table 40D.

PFam analysis indicates that the NOV40a protein contains the domains shown in the Table 40E. Kekuda et al.

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 Kekuda et al. 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 of the gene fragment.

2. SeqCalling™ Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen 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.

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 of the 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 Kekuda et al. 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). 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 Kekuda et al. 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 Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component 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 Kekuda et al. 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 Kekuda et al. 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 1 X 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 Kekuda et al. primers were set up as described previously, using 1 X 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 and vl.6 Kekuda et al.

The plates for Panels 1.4, 1.5, and 1.6 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panels 1.4, 1.5, and 1.6 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, and 1.6 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, and 1.6 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 Kekuda et al. 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 surrounding (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 Kekuda et al. 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 ofthe 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 1- 5ng/ml, TNF alpha at approximately 5-1 Ong/ml, IFN gamma at approximately 20-50ng/ml, Kekuda et al.

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 5.5x10^"^ (Gibco), and 1 OmM 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 5.5x10^""M (Gibco), and lOmM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/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 (5.5x10^" "^{^}M) (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°M (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), 1 OOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^*"^ (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 Kekuda et al. 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^"~M (Gibco), and 1 OmM 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 5.5x10°M (Gibco), and 1 OmM 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°M (Gibco), and I OmM 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 5.5x10^"^ (Gibco), and 1 OmM Hepes (Gibco). To activate the cells, we used PWM at 5μg/ml or anti-CD40 (Pharmingen) at approximately lOμg/ml and IL-4 at 5-10ng/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 CD4 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 5.5xlO^"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- Kekuda et al.

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), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10°M (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 0.1 M dbcAMP at SxlO^cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to SxlO^cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^""M (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^"'^ (Gibco), and lOmM Hepes (Gibco). CCDl 106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and lng/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 Corporation) 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 Kekuda et al. 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 Al 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 of the 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 Kekuda et al. 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 Al 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

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: Kekuda et al.

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/Bio Whittaker) 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 Kekuda et al.

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_Vl .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 Kekuda et al. 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 of the 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_V 1.0 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; pateint not demented but showing sever AD-like pathology

SupTemporal Ctx = Superior Temporal Cortex Inf Temporal Ctx = Inferior Temporal Cortex

A. NOVla and NOVlb (CG113254-01 and CG113254-02): Fibulin

Expression of gene CGI 13254-01 and CGI 13254-02 was assessed using the primer-probe sets Agl294b, Ag746, Ag905, Ag4470 and Ag4726, described in Tables AA, AB, AC, AD and AE. Results of the RTQ-PCR runs are shown in Tables AF, AG, AH, Al, Kekuda et al.

AJ, AK, AL and AM. Please note that CGI 13254-02 represents a full-length physical clone and is recognized only by two probes and primer sets: Ag4470 and Ag4726.

Table AB. Probe Name Aε746

'" ⁿ " """"' ""i '^,*'"" " '""'^* ""^™ """"^* ""f i 1 Start | SEQ ID

Primers! Sequences _'Length

: i Position No

Forwardjβ ' -gcattggcagctacaagtgt- 3 ^■ ] 20 690 ] 208

;TET- 5 ' -ctgtcgaactggcttccaccttcat- 3 ' - ^'■ I ^{" " " " "} Probe .; ^ 25 712 209

^•TAMRA ϊ

Reverse '5 ' -cctccgacactcgtttacatc-3 ' • 21 ^" 758^" 210

Table AC. Probe Name Ag905

Start SEQ ID

Primers' Sequences iLength Position No

Forwardi5 ^■ -cattggcagctacaagtgttc- 3 ' ■ 21 _,21 1 lTET- 5 ' -ctgtcgaactggcttccaccttcat- 3 ' - Probe : 212

JTAMRA i ²⁵ 712 Ϊ

Reverse -5 ' - cctccgacactcgtttacatc- 3 ' j 21 758 ^"'213

Table AD. Probe Name Ag4470

Kekuda et al.

Table AE. Probe Name Ag4726

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AG. CNS neurodegeneration vl.O

Kekuda et al.

Kekuda et al.

Table AH. General_screening_panel_vl .4

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table Al. Panel 1.2

Kekuda etal.

Kekuda et al.

Kekuda et al.

Table AJ. Panel 2D

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AK. Panel 4. ID

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AL. Panel 4D Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AM. general oncology screening panel_v_2.4

Kekuda et al.

AI_comprehensive panel_vl.O Summary: Agl294b/Ag4470 Two experiments with two different probe and primer sets Expression of this gene in this panel confirms expression of this gene in cells involved in the immune response. Highest expression of this gene is seen in normal lung (CT=30.5). Please see Panel 4D for discussion of utility of this gene in inflammation.

CNS_neurodegeneration_vl.O Summary: Agl294b/Ag4470/Ag4726 Three experiments with different probe and primer sets produce results that are in reasonable agreement. 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 but significant levels in the brain. Therefore, therapeutic modulation of the 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.

General_screening_panel_vl.4 Summary: Ag4470/Ag4726 Two experiments with different probe and primer sets produce results that are in excellent agreement.

Highest expression of this gene is seen in a liver cancer cell line (CTs=30), with moderate levels of expression seen in fetal and adult liver, and cell lines derived from colon, renal and lung cancers. Thus, expression of this gene could be used to differentiate liver derived tissue from other samples on this panel. Panel 1.2 Summary: Ag746 Two experiments with the same probe and primer set produce results that are in excellent agreement, with highest expression of this gene in a liver cancer cell line (CTs=27). High levels of expression are also seen in fetal and adult liver tissue, a colon cancer cell line and a lung cancer cell line. Thus, expression of this gene could be used to differentiate liver derived samples, the colon cancer cell line and the Kekuda et al. lung cancer cell line from other samples on this panel. Expression of this gene could also be used as a diagnostic marker to detect the presence of colon and lung cancers.

Moderate expression is also seen in the fetal brain, placenta, and endothelial cells. Panel 2D Summary: Ag746 Two experiments with the same probe and primer set produce results that are in excellent agreement, with highest expression of this gene in liver cancer (CTs=31). The prominent expression in liver derived tissue is consistent with the results in Panel 1.2. Moderate levels of expression are also evident in samples from ovarian cancer and kidney cancer. Furthermore, expression of this gene is higher in these cancers than in the normal adjacent tissue. Thus, expression of this gene could be used to differentiate between liver derived samples and other samples on this panel and as a marker to detect the presence of liver, kidney, and ovarian cancer. Furthermore, therapeutic modulation ofthe expression or function of this gene may be effective in the treatment of liver, kidney, and ovarian cancers.

Panel 4. ID Summary: Agl294b/Ag4470/Ag4726 Results from three experiments with three different probe and primer sets are in agreement with the expression profile in Panel 4D, with highest expression of this gene in this experiment in IL-4 treated dermal fibroblasts (CTs=30). In addition, this experiment shows low but significant levels of expresion in resting neutrophils (CT=33.2), a sample absent in Panel 4D. Please see Panel 4D for discussion of utility of this gene in inflammation. Panel 4D Summary: Agl294b Two experiments with the same probe and primer set produce results that are in excellent agreement, with highest expression of this gene in IL-4 treated dermal fibroblasts (CTs=30). In addition, this gene is expressed at moderate levels in IFN gamma stimulated dermal fibroblasts, activated lung fibroblasts, HPAECs, lung and dermal microvasculature, activated small airway and bronchial epithelium, activated NCI-H292 cells, acutely activated T cells, and activated B cells.

Based on these levels of expression in T cells, activated B cells and cells in lung and skin, therapeutics that block the function of this gene product may be useful as therapeutics that reduce or eliminate the symptoms in patients with autoimmune and inflammatory diseases in which activated B cells present antigens in the generation ofthe aberrant immune response and in treating T-cell mediated diseases, including Crohn's disease, ulcerative colitis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, allergy, emphysema, rheumatoid arthritis, or psoriasis. general oncology screening panel_v_2.4 Summary: Ag4470 Highest expression of this gene is seen in kidney cancer (CT=30). In addition, this gene is more highly Kekuda et al. 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. Furthemore, therapeutic modulation ofthe expression or function of this gene product may be useful in the treatment of lung and kidney cancer.

B. NOV2a (CG122729-01): Novel SPTM protein.

Expression of gene CGI 22729-01 was assessed using the primer-probe sets Agl441 , Agl447 and Ag4533, described in Tables BA, BB and BC. Results ofthe RTQ- PCR runs are shown in Tables BD, BE and BF.

Table BA. Probe Name Agl441

Table BB. Probe Name Aεl447

Table BC. Probe Name Aε4 533

Kekuda et al.

Table BE. General_screening_panel_vl .4

116 Kekuda et al.

117 Kekuda et al.

Lung ca. A549 0.0 :Brain (Amygdala) Pool 6.7

Lung ca. NCI-H526 0.0 jBrain (cerebellum) 4.8

Lung ca. NCI-H23 0.0 JBrain (fetal) 2.6 jBrain (Hippocampus)

Lung ca. NCI-H460 0.0 8.2 iPool

Lung ca. HOP-62 0.0 'Cerebral Cortex Pool 6.1 Brain (Substantia nigra)

Lung ca. NCI-H522 0.0 6.1 iPool

Liver 1.3 iBrain (Thalamus) Pool 1 1.5

Fetal Liver 1 1.9 Brain (whole) 12.0

Liver ca. HepG2 0.0 Spinal Cord Pool 15.5

Kidney Pool 8.2 Adrenal Gland 9.2

Fetal Kidney 3.4 ;Pituitary gland Pool 1.8

Renal ca. 786-0 0.0 Salivary Gland 6.9

Renal ca. A498 0.0 Thyroid (female) 1.7

Renal ca. ACHN 0.0 Pancreatic ca. CAPAN2 0.0 - - - — Renal ca. UO-31 Pancreas Pool 7.7 ^{~ ~~}

Table BF. Panel 4. ID

Kekuda et ul.

CNS_neurodegeneration_vl.O Summary: Ag4533 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms Kekuda et nl. the expression of this gene at low levels in the brain. Please see Panel 1.4 for discussion of utility of this gene in the central nervous system.

General_screening_panel_vl.4 Summary: Ag4533 Highest expression of this gene is seen in the spleen (CT=28.4). In addition, low to moderate levels of expression are seen in all regions ofthe CNS examined, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex.

Among tissues with metabolic function, this gene is expressed at moderate to low 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.

Panel 4.1D Summary: Ag4553 Highest expression of this gene is seen in CD40/IL-40 treated B lymphocytes (CT=27.3). In addition, prominent levels of expression are seen in dendritic cells, eosinophils, macrophages, monocytes, and PBMCs. 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 of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

C. NOV3a (CG122777-01): P-type trefoil domain containing protein

Expression of gene CGI 22777-01 was assessed using the primer-probe set Ag4528. described in Table CA. Results ofthe RTQ-PCR runs are shown in Tables CB and CC.

Table CA. Probe Name Ag4528

Table CB. General_screening_panel_vl .4 Kekuda et al.

Kekuda et al.

523 Kekuda et ul.

Table CC. Panel 4. ID

Kekuda et ul.

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag4528 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

General_screening_panel_vl.4 Summary: Ag4528 Highest expression of this gene is seen in the trachea (CT=30.5). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker of this tissue. Low but significant levels of expression are also seen in testis, fetal lung and cell lines derived from gastric, renal, breast, liver and lung cancers.

Panel 4.1D Summary: Ag4528 This gene is only expressed at detectable levels in the kidney (CT=34). Thus, expression of this gene could be used to differentiate the kidney derived sample from other samples on this panel and as a marker of kidney tissue. In addition, therapeutic targeting ofthe expression or function of this gene may modulate kidney function and be important in the treatment of inflammatory or autoimmune diseases that affect the kidney, including lupus and glomerulonephritis.

D. NOV4a (CG124229-01): Insulin like growth factor binding protein 3 Kekuda et al.

Expression of gene CGI 24229-01 was assessed using the primer-probe set Ag6776, described in Table DA. Results of the RTQ-PCR runs are shown in Tables DB, DC, DD and DE.

Table DA. Probe Name Ag6776

Table DB. AI_comprehensive panel vl .0

Kekuda et al.

528 Kekuda et al.

Table DC. CNS_neurodegeneration_vl .O

Kekuda et al.

Kekuda et al.

Table DP. General_screening_panel_vl .6

Kekuda et al.

Kekuda et al.

IBrain (Substantia nigra)

Lung ca. NCI-H522 0.3 0.0 IPool

Liver 1.3 IBrain (Thalamus) Pool 0.1

Fetal Liver 8.7 jBrain (whole) 0.3

!

Liver ca. HepG2 0.0 ΪSpinal Cord Pool 0.1

Kidney Pool 6.8 ^Adrenal Gland 0.2

Fetal Kidney 0.6 iPituitary gland Pool 0.6

Renal ca. 786-0 29.9 ^Salivary Gland 0.1

Renal ca. A498 51.8 Thyroid (female) αi

Renal ca. ACHN 0.3 Tancreatic ca. CAPAN2 5.4

Renal ca. UO-31 0.2 .Pancreas Pool 0.3

Table DE. Panel 4. ID

Kekuda et al.

Kekuda et al.

AI_comprehensive panel_vl.O Summary: Ag6776 Highest expression of this gene is seen in normal tissue adjacent to psoriasis (CT=19.7). Overall, this gene is highly expressed in many samples on this panel, including clusters of samples derived from psoriasis derived tissue. Please see Panel 4. ID for discussion of utility of this gene in autoimmune disease.

CNS_neurodegeneration_vl.O Summary: Ag6776 This panel does not show differential expression of this gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene at moderate levels in the brain. The insulin and insulin- Kekuda et al. like growth factors belong to a family of polypeptides essential for proper regulation of physiologic processes such as energy metabolism, cell proliferation, development, and differentiation. The insulin-like growth factors bind to IGF with high affinity and compete with the IGF receptor for IGF binding. Transgenic mice overexpressing insulin-like growth factor binding proteins (IGFBPs) tend to show brain developmental abnormalities, suggesting a role for these proteins in neurodevelopment. Furthermore, treatment with glycosaminoglycans (which increases muscle re-innervation after motor neuron death) upregulates serum levels of both IGF and IGFBP. Thus, on the basis of its homology to other established IGFBPs, the novel IGFBP encoded by this gene may be useful in the treatment of diseases such as ALS, multiple sclerosis, and peripheral nerve injury on the basis of its homology to other established IGFBPs. [Dave Stone]

General_screening_panel_vl.6 Summary: Ag6776 Highest expression of this gene is seen in a brain cancer cell line (01=20.5). In addition, high levels of expression are seen in a cluster of brain cancer cell lines, melanoma cell lines, renal cancer cell lines, and ovarian cancer cell lines. This gene encodes a putative insulin like growht factor binding protein 3 (IGFBP3). IGFBP-3 enhances the p53-dependent apoptotic response of colorectal cells to DNA damage and is inversely associated with risk for colorectal cancer. Expression of IGFBP-3 induces growth inhibition and differentiation ofthe human colon carcinoma cell line, Caco-2. Thus, therapeutic targeting modulation of this gene product may be useful in the treatment of cancer, especially in those cancer types, like brain and renal tumors where the gene is overexpressed in the tumor cell line compared to the normal tissue sample.

This gene is also expressed at moderate levels in all regions ofthe CNS examined. Please see Panel CNS_neurodegeneration_vl .0 for discussion of utility of this gene in the CNS.

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. Cortizo et. al has suggested that alterations in IGFBP3 levels may result in diabetic complications (Acta Diabetol 1998 Jul;35(2):85-90). This 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.

Panel 4.1D Summary: Ag6776 Highest expression of this gene is seen in TNFalpha stimulated dermal fibroblasts (CT=25.3). In addition, high levels of expression are Kekuda et al. seen in a cluster of treated and untreated samples derived from dermal fibroblasts. Miura has suggested that dermal fibroblasts promote IGFBP mediated keratinocyte proliferation and may contribute to the epidermal hyperplasia manifest in psoriasis (Arch Dermatol Res 2000 Dec;292(12):590-7). Thus, based on the homology of this gene to IGFBP3 and the expression in dermal fibroblasts and psoriasis related tissue on Al comprehensive panel vl .0, modulation ofthe expression or function of this gene may be useful in the clinical management of this disease.

E. NOV5a (CG124445-02): transmembrane kuzbanian

Expression of gene CGI 24445-02 was assessed using the primer-probe set Ag7026, described in Table EA.

Table EA. Probe Name Ag7026

CNS neurodegeneration vl.O Summary: Ag7026 Expression ofthe CGI 24445- 02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel. General_screeningjpanel_vl.6 Summary: Ag7026 Expression of the

CGI 24445-02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

Panel 4.1D Summary: Ag7026 Expression ofthe CG124445-02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

F. NOV6a (CGI 24590-02): INTEGRIN BETA-4 PRECURSOR

Expression of gene CGI 24590-02 was assessed using the primer-probe set Ag6832, described in Table FA. Results ofthe RTQ-PCR runs are shown in Tables FB and FC. Please note that CGI 24590-02 represents a full-length physical clone.

Table FA. Probe Name Ag6832 Kekuda et al.

Table FB. CNS neurodegeneration vl.O

Kekuda et al.

Table FC. Panel 4. ID

Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag6832 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals. Kekuda et al.

However, no differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment.

Expression of this gene in the brain suggests that the protein encoded by this gene may play a role in central nervous system disorders such as Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

General_screening_panel_vl.6 Summary: Ag6832 Results from one experiment with the CGI 24590-02 gene are not included. The amp plot indicates that there were experimental difficulties with this run.

Panel 4.1D Summary: Ag6832 Highest expression ofthe CGI 24590-02 gene is detected in keratinocytes (CT=25). High levels of expression of this gene is also detected in small airway epithelium, cytokine treated bronchial epithelium, and NCI-H292 cells.

Therefore, expression of this gene may be used to distinguish these samples from other samples in this panel. In addition, moderate levels of expression of this gene is also seen in

HPAEC, HUVEC, lung microvascular EC, microvascular dermal EC and neutrophils. Therefore, therapeutic modulation of this gene may be useful in the treatment of autoimmune and inflammatory diseases that involve endothelial cells, such as lupus erythematosus, asthma, emphysema, Crohn's disease, ulcerative colitis, rheumatoid arthritis, osteoarthritis, and psoriasis.

Low to moderate levels of expression of this gene is also seen in normal tissues represented by colon, lung, thymus and kidney. Therefore, therapeutic modulation ofthe protein encoded this gene may be useful in the treatment of autoimmune and inflammatory diseases that affect these tissues.

G. NOV7a (CG124916-01): Selenoprotein P

Expression of gene CGI 24916-01 was assessed using the primer-probe set Ag7029. described in Table GA.

Table GA. Probe Name Ag7029

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag7029 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

General_screening_panel_vl.6 Summary: Ag7029 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

Panel 4.1D Summary: Ag7029 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

H. NOV8a (CG126224-01): Novel Type II Membrane Protein with 3 C2 domains

Expression of gene CG 126224-01 was assessed using the primer-probe set Ag4713, described in Table HA. Results ofthe RTQ-PCR runs are shown in Tables HB, HC and HD.

Table HA. Probe Name Ag4713

Table HB. CNS_neurodegeneration_vl .O

Kekuda et al.

Kekuda et al.

Table HC. General_screeningjpanel_vl .4

Kekuda et al.

Kekuda et al.

Table HD. Panel 4. ID

Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag4713 This panel confirms the expression ofthe CGI 26224-01 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 1.4 for a discussion of the potential utility of this gene in treatment of central nervous system disorders.

General_screening_panel_vl.4 Summary: Ag4713 Highest expression of the CG126224-01 gene is detected in CNS cancer U87-MG cell line (CT=28.8). 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 of the 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, pituitary gland, skeletal muscle. Kekuda et al. heart, fetal 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=32-33) when compared to adult lung and liver (CT=35-37). This observation suggests that expression of this gene can be used to distinguish fetal from adult lung and liver, respectively. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver and lung 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 and lung related diseases.

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.

Panel 4.1D Summary: Ag4713 Highest expression ofthe CGI 26224-01 gene is detected in IFN gamma treated HUVEC cells (CT=28). High to moderate levels of expression in LAK cells, two way MLR, PBMC, B lymphocytes, eosinophils, dendritic cells, monocytes, macrophages, endothelial cells, small airway epithelium, basophils, NCI- H292, lung fibroblast and activated neutrophils. In addition, moderate to low levels of expression of this gene is also seen in liver cirrhosis and normal tissues represented by colon, lung, thymus and kidney. Therefore, therapeutic modulation of this gene may be useful in the treatment of inflammatory and autoimmune diseases such as asthma, allergies. inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, osteoarthritis and liver cirrhosis.

I. NOV9a (CG126233-01): ctl2

Expression of gene CGI 26233-01 was assessed using the primer-probe set Ag4722, described in Table I A. Results of the RTQ-PCR runs are shown in Tables IB, IC and ID. Table IA. Probe Name Ag4722 j ] Start j SEQ ID

Primers Sequences Length] j i Position \ No Kekuda et al.

Table IB. CNS_neurodegeneration_vl .O

Kekuda et al.

Table IC. General_screening_panel_vl .4

Kekuda et al.

Kekuda et al.

Table ID. Panel 4. ID

Kekuda et al.

Kekuda et al.

Kekuda et al.

HUVEC none 3.8 Kidney 0.7

HUVEC starved 7.7

CNS_neurodegeneration_vl.O Summary: Ag4722 This panel does not show differential expression of this gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.4 for discussion of utility of this gene in the central nervous system.

General_screening_panel_vl.4 Summary: Ag4722 This gene is expressed at moderate levels throughout many ofthe samples in this panel. Highest expression is detected in an gastric cancer cell line (CT=29). In addition, this gene is also expressed in a cluster of samples derived from lung cancer cell lines and at low but significant levels in cell lines derived from ovarian, colon and brain cancers. Therefore, therapeutic modulation of this gene or its protein product, through the use of antibodies, might be useful in the treatment of these cancers.

Among tissues involved in metabolic function, this gene is expressed in the pancreas, pituitary, fetal liver, fetal heart and skeletal muscle. Therefore, this gene or its protein product may be important in the pathogenesis and/or treatment of disease of obesity and diabetes.

There is widespread moderate expression of this gene across many ofthe samples derived from the CNS, including the amygdala, hippocampus, thalamus, cerebral cortex, and spinal cord. 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 4.1D Summary: Ag4722 This transcript is most highly expressed in NCI- H292 cells stimulated by IL-9 (CT=32.5). The gene is also expressed in a cluster of treated and untreated samples derived from the NCI-H292 cell line, a human airway epithelial cell line that produces mucins. Mucus overproduction is an important feature of bronchial asthma and chronic obstructive pulmonary disease samples. The transcript is also expressed at lower but still significant levels in small airway epithelium treated with IL-1 beta and TNF-alpha. The expression ofthe transcript in this mucoepidermoid cell line that is often used as a model for airway epithelium (NCI-H292 cells) suggests that this transcript may be important in the proliferation or activation of airway epithelium. Therefore, therapeutics designed with the protein encoded by the transcript may reduce or eliminate symptoms Kekuda et al. caused by inflammation in lung epithelia in chronic obstructive pulmonary disease, asthma, allergy, and emphysema.

J. NOVlOa (CG126600-01): Fibronectin type III Domain-Membrane Protein

Expression of gene CGI 26600-01 was assessed using the primer-probe set Ag7030, described in Table JA. Results ofthe RTQ-PCR runs are shown in Tables JB, JC and JD.

Table JA. Probe Name Ag7030

Kekuda et al.

Table JC. General screening_panel_vl .6

Rel. Exp.(%) Ag7030, I Rel. Exp.(%) Ag7030,

Tissue Name Tissue Name Run 281813484 j Run 281813484

Adipose 10.0 Renal ca. TK-10 | 27.7 Kekuda et al.

Kekuda et nl.

Kekuda et al.

Table JD. Panel 4. ID

562 Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.0 Summary: Ag7030 This panel confirms the expression of the CGI 26600-01 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 1.6 for a discussion ofthe potential utility of this gene in treatment of central nervous system disorders.

General_screening_panel_vl.6 Summary: Ag7030 Highest expression of the CG126600-01 gene is detected in melanoma SK-MEL-5 cell line (CT=25.7). High levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, 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 of the expression or function of this gene may be effective in the treatment of pancreatic, gastric, colon, lung, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers.

Among tissues with metabolic or endocrine function, this gene is expressed at high levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation ofthe activity of this

564 Kekuda et al. gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, this gene is expressed at high 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 4.1D Summary: Ag7030 Highest expression ofthe CG126600-01 gene is detected in activated CD45RA CD4 lymphocyte (CT=26.6). 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. 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 .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.

K. NOVlla (CG127888-01): Novel Secretory Protein

Expression of gene CGI 27888-01 was assessed using the primer-probe set Ag4756, described in Table KA. Results ofthe RTQ-PCR runs are shown in Table KB.

Table KA. Probe Name Ag4756

Kekuda et al.

Table KB. CNS_neurodegeneration_vl.0

566 Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag4756 Low expression of this gene is seen in control temporal cortex (CT=34.6). Therefore, expression of this gene may be used to distinguish this sample from other samples used in this panel. In addition, therapeutic modulation of this gene may be useful for the treatment of neurological disorders.

General_screening_panel_vl.4 Summary: Ag4756 Expression of the CGI 27888-01 gene is low/undetectable (CTs > 35) across all of the samples on this panel.

Panel 4.1D Summary: Ag4756 Expression of the CG127888-01 gene is low/undetectable (CTs > 35) across all of the samples on this panel.

L. NOV12a (CG128249-02): EPHRIN-A4 PRECURSOR

Expression of gene CGI 28249-02 was assessed using the primer-probe set Ag6833, described in Table LA. Results of the RTQ-PCR runs are shown in Table LB. Please note that CGI 28249-02 represents a full-length physical clone.

Table LA. Probe Name Ag6833

Kekuda et al.

568 Kekuda et al.

569 Kekuda et ul.

CNS_neurodegeneration_vl.O Summary: Ag6833 Expression ofthe CGI 28249- 02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

General_screening_panel_vl.6 Summary: Ag6833 Highest expression ofthe CGI 28249-02 gene is detected in ovarian OVCAR-5 cell line (CT=32.8). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Interestingly, this gene is expressed at low/undectactable levels in normal tissues (CTs>35). Thus, expression of this gene could be used to distinguish cancer cell lines from the normal tissue samples in this panel and also as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of pancreatic, gastric, colon, lung, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers.

Panel 4.1D Summary: Ag6833 Expression ofthe CG128249-02 gene is low/undetectable (CTs > 35) across all of the samples on this panel.

M. NOV13a (CG128785-01): alt spliced SPUF

Expression of gene CG128785-01 was assessed using the primer-probe set Ag5883, described in Table MA.

Table MA. Probe Name Ag5883

Kekuda et al.

TAMRA

Reverse 5 ^■ -ttcactgccaagtagatggg-3 20 206 261

General_screeningjpanel_vl.5 Summary: Ag5883 Expression ofthe CGI 28785-01 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

Panel 4.1D Summary: Ag5883 Expression of the CG128785-01 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

N. NOV14a (CG129005-01): 54TM splice variant.

Expression of gene CGI 29005-01 was assessed using the primer-probe set Ag4799, described in Table NA. Results ofthe RTQ-PCR runs are shown in Tables NB and NC.

Table NA. Probe Name Ag4799

Table NB. General_screeningjpanel_vl .4

Kekuda et a I.

Kekuda et al.

Table NC. Panel 4. ID

Kekuda et al.

Kekuda et al.

Kekuda et al.

General_screening_panel_vl.4 Summary: Ag4799 Highest expression ofthe CG129005-01 gene is detected in breast cancer T47D cell line (CT=23.9). High levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, 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 gastric, colon, lung, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Among tissues with metabolic or endocrine function, this gene is expressed at high to 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 high 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.

Panel 4.1D Summary: Ag4799 Highest expression of the CGI 29005-01 gene is detected in lung microvascular EC cells (CT=27.3). 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. 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.4 and also suggests a role for the gene product in cell survival and proliferation. Therefore,

576 Kekuda et al. 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.

O. NOV15a (CG132086-01): Novel Membrane Protein

Expression of gene CGI 32086-01 was assessed using the primer-probe set Ag4809, described in Table OA. Results ofthe RTQ-PCR runs are shown in Table OB.

Table OA. Probe Name Ag4809

Kekuda et al.

Kekuda et al.

General_screening_panel_vl.4 Summary: Ag4809 Results from one experiment with the CGI 32086-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.

Panel 4.1D Summary: Ag4809 Highest expression ofthe CGI 32086-01 gene is detected in LPS treated monocytes and PMA/ionomycin treated basophils (CTs=29.5). This Kekuda et al. 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. 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 expression pattern 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.

P. NOVlόa and NOV16b (CG132297-01 and CG132297-02): ELASTIN

Expression of gene CGI 32297-01 and CGI 32297-02 was assessed using the primer-probe set Ag7016, described in Table PA. Results ofthe RTQ-PCR runs are shown in Tables PB, PC and PD. Please note that CGI 32297-01 represents a full-length physical clone.

Table PA. Probe Name Ag7016

Table PB. CNS_neurodegeneration_vl .O

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table PD. Panel 4. ID

Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag7016 This panel confirms the expression ofthe CG132297-01 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 1.6 for a discussion ofthe potential utility of this gene in treatment of central nervous system disorders.

General_screeningjpanel_vl.6 Summary: Ag7016 Highest expression ofthe CG132297-01 gene of this gene is detected in fetal lung (CT=26.3). Interestingly, this gene is expressed at much higher levels in fetal (CTs=26-31) when compared to adult lung and liver (CT=32-35). This observation suggests that expression of this gene can be used to Kekuda et al. distinguish fetal from adult lung and liver, respectively. In addition, the relative overexpression of this gene in fetal tissues suggests that the elastin encoded by this gene may enhance growth or development of lung and liver in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe elastin encoded by this gene could be useful in treatment of lung and liver related diseases. 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.

Moderate levels of expression of this gene is also seen in colon cancer and in number of cancer cell lines derived from melanoma, brain, and lung cancer cell lines. Therefore, therapeutic modulation ofthe elastin encoded by this gene may be useful in the treatment of melanoma, colon, brain and lung cancer.

Panel 4.1D Summary: Ag7016 Highest expression ofthe CG132297-01 gene of this gene is detected in IL-1 beta treated dermal fibroblasts CCD1070 (CT=28.1). In addition, moderate to low levels of expression of this gene is also seen in dermal and lung fibroblasts, activated CD45RA CD4 lymphocyte and lung. CD45RA CD4 lymphocytes represent activated naive T cells. In activated memory T cells (CD45RO CD4 lymphocyte) or CD4 Thl or Th2 cells, resting CD4 cells (CTs=40), the expression of this gene is strongly down regulated suggesting a role for this putative protein in differentiation or activation of naive T cells. Therefore, modulation ofthe expression and/or activity of this putative protein encoded by this gene might be beneficial for the control of autoimmune diseases and T cell mediated diseases such as COPD, emphysema, atopic asthma, asthma, arthritis, psoriasis, IBD and allergy. Kekuda et al.

Q. NOV17a (CG132343-01): novel transmembrane protein.

Expression of gene CGI 32343-01 was assessed using the primer-probe set Ag4819, described in Table QA. Results ofthe RTQ-PCR runs are shown in Tables QB and QC.

Table OA. Probe Name Ag4819

Start SEQ ID

Primers Sequences {Length Position No

Forward J5 ' -gagttacccatacaccggctat- 3 ' 22 88 271 jTET- 5 ' -atttcacggccaggagagtcctcttt-3 '

Probe 26 1 10 272 JTAMRA

Reverse 15 ' -taaggatgatgcccatacaaag- 3 ' 22 163 272

588 Kekuda et al.

589 Kekuda et al.

Table QC. Panel 4. ID

590 Kekuda et al.

591 Kekuda et al.

General_screening_panel_vl.5 Summary: Ag4819 Expression of this gene is restricted to a few samples in this panel, with highest expression in a breast cancer cell line (CT=29). Low, but significant levels of expression are seen in cell lines derived from brain, Kekuda et al. renal and gastric cancers, as well as in normal testis. Thus, the expression of this gene could be used to distinguish the breast cancer cell line sample from other samples on this panel, and as a marker of breast cancer. In addition, therapeutic modulation of this gene or its protein product may be useful in the treatment of breast, gastric, renal and brain cancers. Panel 4.1D Summary: Ag4819 This gene is only expressed at detectable levels in the kidney (CT=34.5). Thus, expression of this gene could be used to differentiate the kidney derived sample from other samples on this panel and as a marker of kidney tissue. In addition, therapeutic targeting ofthe expression or function of this gene may modulate kidney function and be important in the treatment of inflammatory or autoimmune diseases that affect the kidney, including lupus and glomerulonephritis.

R. NOV18a (CG132423-01): Pregnancy-specific beta-1 -glycoprotein 2 precursor.

Expression of gene CG132423-01 was assessed using the primer-probe set Ag7021, described in Table RA.

Table RA. Probe Name Ag7021

Start I SEQ ID

Primers! Sequences Length

Position j No

-- -- -- ^. -- -- -

FθrwardΪ5 ^■ -aggtccctgatttggacaag-3 ' '274

!

TET-5 ' -aagaacatccttcccctcggacactt-3 ' -

Probe _: : 26 871 J275

; TAMRA

Reverse ]5 ' - ctgcccaagtcatgattgaa-3 ' : ²⁰ 910 |276

CNS_neurodegeneration_vl.O Summary: Ag7021 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

General_screening_panel_vl.6 Summary: Ag7021 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

Panel 4.1D Summary: Ag7021 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

593 Kekuda et al.

S. Novl9a and NOV19b (CG132541-01 and CG132541-02): Protocadherin 16 precursor.

Expression of gene CG132541-01 and CG132541-02 was assessed using the primer-probe sets Agl076, Agl31 1, Ag482, and Ag6709 described in Tables SA, SB, SC, and SD. Results ofthe RTQ-PCR runs are shown in Tables SE, SF, SG, SH, SI, SJ, SK and SL. Please note that probe and primer set Ag6709 is specific for CGI 32541-01 and probe Ag482 is specific for CGI 32541 -02.

Table SA. Probe Name Agl076

Start SEQ ID

Primers! Sequences jLength Position No

Forward 5 ' -tgacagacactgtggtgcttag-3 ' 22 6228 277

TET-5 ' -accatccactgcactcacagaaaagg-3 '

Probe 26 6187 278 TAMRA

Reverse 5 ' -agagaacagtgtcccagctaca-3 ' 22 6165 1279

Table SB. Probe Name Agl 31 1

Kekuda et al.

Table SD. Probe Name Ag6709

Table SE. CNS_neurodegeneration_vl .O

Kekuda et al.

Kekuda et ul.

Kekuda et ul.

Table SG. HASS Panel vl.O

Tissue ReI.Exp.(%)Agl311, Rel. Exp.(%)Agl311,

Tissue Name Name j Run 268362648 Run 268362648

MCF-7CΪ1 0. U87-MGF1 (B) 0.2 Kekuda et ul.

Kekuda et al.

Kekuda et al.

Table SH. Oncology_cell_line_screening_panel_v3.2

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table SI. Panel 1

Kekuda et al.

Kekuda et al.

Table SJ. Panel 1.2

Kekuda et al.

Kekuda et al.

Table SK. Panel 4D

Kekuda et al.

Kekuda et al.

Table SL. general oncology screening panel_v_2.4

Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Agl31 1 This panel confirms the expression of this gene at moderate 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. Ag6709 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

General_screening_panel_vl.4 Summary: Agl31 1 Highest expression of this gene is seen in the fetal brain (CT=25). Thus, expression of this gene could be used to differentiate between fetal and adult brain tissue. Moderate levels of expression are seen in all regions ofthe CNS examined. This gene has homology to cadherin, transmembrane glycoproteins that are involved in many biological processes such as cell adhesion, cytoskeletal organization and morphogenesis. Cadherins can act as axon guidance and cell adhesion proteins, specifically during development and in the response to injury (Ranscht B. Int. J. Dev. Neurosci. 18: 643-651). Therefore, manipulation of levels of this protein may be of use in inducing a compensatory synaptogenic response to neuronal death in Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, progressive supranuclear palsy, ALS, head trauma, stroke, or any other disease/condition associated with neuronal loss.

As in Panel 1.2, this gene is expressed at high to moderate levels in metabolic tissues, including pancreas, pituitary, adipose, adrenal gland, pancreas, thyroid, liver and adult and fetal skeletal muscle, and heart. Please see Panel 1.2 for discussion of utility of this gene in metabolic disease. Moderate levels of expression are also seen in cancer cell lines derived from melanoma, ovarian, lung, colon and brain cancers.

General_screening_panel_vl.6 Summary: Ag6709 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

HASS Panel vl.O Summary: Agl31 1 Highest expression of this gene is detected in glioma cells (CT=27.3). This gene is expressed at a low to moderate level in samples of Kekuda et ul. brain cancer as well as primary astrocytes in culture. Expression is also slightly increased in LnCAP and U87 cells that are subjected to cell stresses such as reduced oxygen, low serum or an acidotic environment which are some ofthe conditions seen in tumors. Oncology_cell_line_screening_panel_v3.2 Summary: Agl31 1 Highest expression of this gene is seen in a lung cancer cell line (CT=27.5). Moderate levels of expression of this gene are also seen in a cluster of samples derived from lung cancer cell lines, bone cancer cell lines and brain cancer cell lines. Please see Panels 1.2 and 2.4 for discussion of utility of this gene in cancer.

Panel 1 Summary: Ag482 Highest expression is seen in ovary (CT=24.3), with high levels of expression in many samples on this panel including melanoma, ovarian, and brain cancer cell lines and normal lung, liver, heart, muscle, brain, pancreas, adrenal, and endothelial cells. This expression is in agreement with results of panels run with Agl31 1. Please see those experiments for discussion of utility of this gene in metabolic and autoimmune disorders and cancer. Panel 1.2 Summary: Agl 31 1 The protein encoded by this gene is homologous to cadherin, a cell-adhesion protein and is highly expressed in a number of samples on panel 1.2. Specifically, the highest expression is detected in fetal heart (CT value = 22.6), although it is also highly expressed in adult heart. This may suggest a potential role for this gene in cardiovascular diseases such as cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (asd), atrioventricular (a-v) canal defect, ductus arteriosus. pulmonary stenosis, subaortic stenosis, ventricular septal defect (vsd), and valve diseases. Overall, gene expression in this panel is associated with normal tissues rather than cancer cell lines. Loss of function ofthe related E-cadherin protein has been described in many tumors, along with an increased invasiveness and a decreased prognosis of many carcinomas, including tumors of endocrine glands and their target systems (ref 1 ). Thus, this gene product might similarly be useful as a protein therapeutic to treat a variety of tumors, since it is found in normal cells but missing from cancer cells.

In addition, this gene is highly expressed in pituitary gland, adrenal gland, thyroid, pancreas, skeletal muscle, and liver, reflecting the widespread role of cadherins in cell-cell adhesion. This observation may suggest that the gene plays a role in normal metabolic and neuroendocrine function and that disregulated expression of this gene may contribute to metabolic diseases (such as obesity and diabetes) or neuroendocrine disorders. Kekuda et nl.

Expression of this gene is also high in many regions ofthe brain, including the amygdala, thalamus, cerebellum, and cerebral cortex, with highest expression in the hippocampus. Expression is also detected in the spinal cord. Cadherins can act as axon guidance and cell adhesion proteins, specifically during development and in the response to injury (ref 2). Manipulation of levels of this protein may be of use in inducing a compensatory synaptogenic response to neuronal death in Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, progressive supranuclear palsy, ALS, head trauma, stroke, or any other disease/condition associated with neuronal loss.

Reference: 1. Potter E., Bergwitz C, Brabant G. (1999) The cadherin-catenin system: implications for growth and differentiation of endocrine tissues. Endocr. Rev. 20: 207-239.

2. Ranscht B. (2000) Cadherins: molecular codes for axon guidance and synapse formation. Int. J. Dev. Neurosci. 18: 643-651.

Panel 4D Summary: Agl 311 Expression of this gene is primarily in endothelial cells and in fibroblasts. However, this gene is also expressed in the kidney, thymus, lung and colon. The expression of this gene is high in normal tissue and untreated cells and is not affected by most treatments with the exception of IL-1 alpha and TNFbeta, which reduce expression of this gene by half in treated HUVECs and reduce expression 10-fold in gamma interferon treated HUVECs. Therefore, the protein encoded for by this gene may be important in normal function of endothelium and fibroblasts. Protein therapeutics designed with the protein encoded for by this transcript could reduce or block inflammation in diseases such as asthma, emphysema, allergy, arthritis, IBD and psoriasis.

Panel 4.1D Summary: Ag6709 Expression of this gene is low/undetectable in all samples on this panel (CTs>35). general oncology screening panel_v_2.4 Summary: Agl 31 1 Highest expression of this gene is seen in a sample from metastatic melanoma (CT=27). Moderate to high levels of expression are also seen samples from colon, kidney, bladder, and prostate cancers. In addition, higher levels of expression are seen in prostate, lung, and kidney cancers when compared to expression in normal adjacent tissue. This gene encodes a putative cadherin, similar to VE cadherin that shows specific expression in mesenchymal cells, fibroblasts and endothelial cells. On Panel 4 this gene shows expression in fibroblasts and endothelial cells and is induced by starvation in Huvec. Activated fibroblasts have shown to be involved in supporting tumor cells (Okada, Lab Invest 2000 Nov;80(l 1): 1617- 28). Corada et al (Blood 2001 Mar 15;97(6): 1679-84) has shown that there are epitopes in Kekuda et nl.

VE Cadherin that are only exposed upon activation of the endothelial cells, probably due to changes in cell-cell adhesions. mAbs against those epitopes have antitumor activities without inducing bleeding. Therefore, based on the expression of this gene in fibroblasts and tumors, and the homology of the protein product to cadherin, targeting of this gene product with a human monoclonal antibody that results in an inhibition of the activity of this protein, preferably as it relates to endothelial and fibroblast activation by tumor cells, may have therapeutic effect on all solid tumors that depend on angiogenesis, and specifically on colon, lung, kidney, melanoma, prostate and bladder. Results from a second experiment with the same probe and primer set, run 263102793, are not included because the amp plot indicates there were experimental difficulties with this run.

T. NOV20a (CG132888-02): M130 ANTIGEN.

Expression of gene CGI 32888-02 was assessed using the primer-probe set Ag4955, described in Table TA. Results of the RTQ-PCR runs are shown in Tables TB, TC and TD.

Table TA. Probe Name Ag4955

Table TB. General_screening_panel_vl .5

Kekuda et ul.

Kekuda et al.

Kekuda et al.

Table TC. Panel 4. ID

Kekuda et al.

Kekuda et al.

Table TD. Panel 5 Islet

Kekuda et al.

Kekuda et al.

General_screening_panel_vl.5 Summary: Ag4955 Highest expression of this gene is detected in bladder (CT=26.8). Therefore, expression of this gene may be useful in distinguishing bladder from other samples used in this panel. In addition, therapeutic modulation of this gene may be useful in the treatment of bladder related diseases. Among tissues with metabolic or endocrine function, this gene is expressed at high to 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 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 4.1D Summary: Ag4955 Highest expression of this gene is detected in LPS treated monocytes (CT=28.3). In addition, moderate to low levels of expression of this gene is also seen in LAK cells, two way MLRs, PBMC, dendritic cells, activated eosinophils and normal tissues represented by colon, lung, thymus and kidney. This gene encodes splice variant of Ml 30 antigen (CD163) precursor. CDl 63 is a macrophage-associated antigen belonging to the scavenger receptor cysteine rich (SRCR) domain family and it scavenges haemoglobin by mediating endocytosis of haptoglobin-haemoglobin complexes (Kristiansen, 2001, Nature 409(6817): 198-201 , PMID: 1 1 196644). CD163 is expressed exclusively on human monocytes and macrophages and it is significantly upregulated by glucocorticoids and IL-10. The highly purified CDl 63 protein is shown to inhibit phorbol ester-induced human T-lymphocyte activation, thus attenuating the immune response to the inflammatory mediator (Hogger P, Sorg C, 2001 , Biochem Biophys Res Commun 2001 Nov 9;288(4):841-3, PMID: 11688984). Furthermore, macrophages expressing the scavenger receptor CD 163 are shown to be increased in synovium and in colonic mucosa in patients with spondyloarthropathy (SpA). Therefore, therapeutic modulation ofthe CD 163 encoded by this gene may be useful in the treatment of asthma, emphysema, inflammatory bowel disease, arthritis, psoriasis and SpA. Kekuda et al.

Moderate levels of expression of this gene is also seen in liver cirrhosis sample. Therefore, therapeutic modulation of this gene may be beneficial in the treatment of liver cirrhosis.

Panel 5 Islet Summary: Ag4955 Highest expression of this gene is detected in placenta (CT=30.2). In addition, moderate to low levels of expression of this gene is also seen in uterus, skeletal muscle, adipose and small intestine. Please see panel 1.5 for the discussion on utility of this gene.

U. NOV22a (CG133508-01): SYNAPTOTAGMIN VI.

Expression of gene CG133508-01 was assessed using the primer-probe set Ag4837, described in Table UA. Results ofthe RTQ-PCR runs are shown in Tables UB, UC and UD.

Table UA. Probe Name Ag4837

Table UB. CNS_neurodegeneration_vl .0

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table UD. Panel 4. ID

Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.0 Summary: Ag4837 Expression of this gene is ubiquitous throughout the samples in this panel, with highest expression in the hippocampus of a patient with Alzheimer's disease (CT=28). While no association between the expression of this gene and the presence of Alzheimer's disease is detected in this panel, these results confirm the expression of this gene in areas that degenerate in Alzheimer's disease, including the cortex, hippocampus, amygdala and thalamus. Synaptotagmin expression is altered in the brain of Alzheimer's patients, possibly explaining impaired synaptogenesis and/or synaptosomal loss secondary to neuronal loss observed in the neurodegenerative disorder. It may also represent, reflect or account for the impaired neuronal transmission in Alzheimer's disease (AD), caused by deterioration ofthe exocytic machinery. Since this gene is a homolog of synaptotagmin, agents that potentiate the expression or function ofthe protein encoded by this gene may be useful in the treatment of Alzheimer's disease.

References:

Sze CI, Bi H, Kleinschmidt-DeMasters BK, Filley CM, Martin LJ. (2000) J Neurol Sci. 175:81-90. Kekuda et al.

Masliah E, Mallory M, Alford M, DeTeresa R, Hansen LA, McKeel DW Jr, Morris JC. (2001)Neurology 56:127-9.

Yoo BC, Cairns N, Fountoulakis M, Lubec G. (2001) Dement Geriatr Cogn Disord. 12:219-25. General_screening_panel_vl.5 Summary: Ag4837 This gene encodes a homolog of synaptotagmin which appears to be almoste exclusively expressed in the brain. This experiment shows moderate to high expression across all brain regions with highest expression in the fetal brain (CT = 28.3). Synaptotagmin is a presynaptic protein involved in synaptic vesicle release, making this an ideal drug target for diseases such as epilepsy, in which reduction of neurotransmission is beneficial. Selective inhibition of this gene or its protein product may therefore be useful in the treatment of seizure disorders. Furthermore, selective inhibition of neural transmission through antagonism ofthe protein encoded by this gene may show therapeutic benefit in psychiatric diseases where it is believed that inappropriate neural connections have been established, such as schizophrenia and bipolar disorder. In addition, antibodies against synaptotagmin may cause Lambert-Eaton myasthenic syndrome. Therefore, peptide fragments ofthe protein encoded by this gene may serve to block the action of these antibodies and treat Lambert-Eaton myasthenic syndrome.

References: Takamori M, Komai K_. Iwasa K. (2000) Am J Med Sci. 319:204-8.

Sokolov BP, Tcherepanov AA, Haroutunian V, Davis KL. (2000) Biol Psychiatry. 48: 184-96.

Panel 4.1 D Summary: Ag4837 This gene is expressed at detectable levels in the kidney (CT=29.8). Thus, expression of this gene could be used to differentiate the kidney derived sample from other samples on this panel and as a marker of kidney tissue. In addition, therapeutic targeting ofthe expression or function of this gene may modulate kidney function and be important in the treatment of inflammatory or autoimmune diseases that affect the kidney, including lupus and glomerulonephritis. Kekuda et al.

V. NOV23a and NOV23b (CG133548-01 and CG133548-02): 1300003P13RIK

Protein Homolog (TmMP)

Expression of gene CGI 33548-01 and CGI 33548-02 was assessed using the primer-probe set Ag4839, described in Table VA. Results ofthe RTQ-PCR runs are shown in Tables VB and VC.

Table VA. Probe Name Ag4839

Table VB. General_screening_panel_vl .5

Kekuda et al.

Kekuda et al.

Table VC. Panel 4. ID

Kekuda et al.

Kekuda et al.

Kekuda et al.

General_screening_panel_vl.5 Summary: Ag4839 Highest expression ofthe CGI 33548-01 gene is detected in ovarian cancer OVCAR-3 cell line (CT=24.8). High to moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, 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 gastric, colon, lung, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers.

Among tissues with metabolic or endocrine function, this gene is expressed at high to 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 high to 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=27.8) when compared to adult kidney (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult kidney. 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 of the protein encoded by this gene could be useful in treatment of kidney related diseases.

Panel 4.1D Summary: Ag4839 Highest expression ofthe CGI 33548-01 gene is detected in lung microvascular EC (CT=27.4). 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 of the 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 Kekuda et al. 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.

W. NOV24a and NOV24b (CG133569-01 and CG133569-02): Type I membrane protein with SH3 domain

Expression of gene CG133569-01 and CG133569-02 was assessed using the primer-probe set Ag4843, described in Table WA. Results ofthe RTQ-PCR runs are shown in Tables WB and WC.

Table WA. Probe Name Ag4843

Start SEQ ID

Primers j Sequences Length Position No

Forward 5 ' -gagcaatggaagagatgcaa-3 ' 20 3170 298

■TET- 5 ' -ccactgcatgaagataatttctcacga- 3 ' r^~~r~ X^'

Probe ; 3190 299

1TAMRA Reverse ,5 ' -cttcaggaacctgcacattaag-3 ' 22 i 3232 300

. J

Table WB. General screening_panel vl .5

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table WC. Panel 4. ID

Kekuda et al.

Kekuda et al.

General_screeningjpanel_vl.5 Summary: Ag4843 Highest expression ofthe CGI 33569-01 gene is detected in CNS cancer SNB-75 cell line (CT=26). High levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, 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, 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 high levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, 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.

Panel 4.1D Summary: Ag4843 Highest expression ofthe CGI 33569-01 gene is detected in PMA/ionomycin treated basophils (CT=29). This gene is expressed at high to moderate levels in a wide range of cell types of significance in the immune response in

442 Kekuda et al. 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.

X. NOV26a and NOV26b (CG134100-01 and CG134100-02): Amidase_2 Domain Protein

Expression of gene CGI 34100-01 and CGI 34100-02 was assessed using the primer-probe sets Ag4387, Ag4893 and Ag4894, described in Tables XA, XB and XC. Results of the RTQ-PCR runs are shown in Tables XD, XE, XF and XG.

Table XA. Probe Name Ag4387

Table XB. Probe Name Ag4893

Kekuda et al.

Table XC. Probe Name Ag4894

Table XD. General_screening_panel_vl .4

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table XF. Oncology_cell_line_screening_panel_v3.1

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table XG. Panel 4. ID

45: Kekuda et al.

Kekuda et al.

CNS_neurodegeneration_vl.O Summary: Ag4387 Expression ofthe CG134100- 01 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

General_screening_panel_vl.4 Summary: Ag4387 Highest expression ofthe CGI 34100-01 gene is detected in bone marrow (CT=30.6). Therefore, expression of this gene may be used to distinguish this sample from other samples used in this panel. In addition, therapeutic modulation of this gene product may be useful in the bone marrow related diseases such as leukemia.

Low levels of expression of this gene is also seen in uterus, trachea and bladder. Therefore, therapeutic modulation of this gene may be useful in the treatment of diseases that affect these tissues.

General_screening_panel_vl.5 Summary: Ag4893/Ag4894 Two experiments with same probe and primer sets are in excellent agreement. Highest expression ofthe CGI 34100-01 gene is detected in bone marrow (CT=30-34). Therefore, expression of this gene may be used to distinguish this sample from other samples used in this panel. In addition, therapeutic modulation of this gene product may be useful in the bone marrow related diseases such as leukemia.

Oncology_cell_line_screening_panel_v3.1 Summary: Ag4893 Highest expression ofthe CGI 34100-01 gene is detected in T cell lymphoma (CT=29.6). In addition, high to moderate levels of expression of this gene is also seen number of cancer samples derived from tongue squamous cell carcinoma, epidermoid carcinoma, bladder carcinoma, pancreatic ductal adenocarcinoma, myelogenous leukemia, uterine and gastric Kekuda et al. carcinoma. Therefore, expression of this gene may be useful as marker to detect the presence of these cancers.

Ag4894 Results from one experiment with this gene are not included. The amp plot indicates that there were experimental difficulties with this run.

Panel 4.1D Summary: Ag4387 Highest expression ofthe CG134100-01 gene is detected in kidney (CT=30.9). Therefore, expression of this gene may be used to distinguish kidney from other samples used in this panel. In addition, therapeutic modulation of this gene may be beneficial in the treatment of autoimmune of inflammatory disease that affect kidney including lupus and glomerulonephritis.

Moderate to low levels of expression of this gene is also seen in thymus, basophils, and small airway epithelium. Therefore, therapeutic modulation of this gene product may be beneficial in the treatment of asthma, allergies, COPD, and emphysema, inflammatory bowel disease, and autoimmune diseases.

Y. NOV27a (CG134403-01): 2510042P03RIK Homolog (TmSP)

Expression of gene CGI 34403-01 was assessed using the primer-probe set Ag4871 , described in Table YA. Results ofthe RTQ-PCR runs are shown in Tables YB and YC. Table YA. Probe Name Ag4871

1 Start SEQ ID

Primers] Sequences Length

Position ^: No

Forwardj5 ' -cctaacagatttcttgcgacaa-3 ' j ^{22 "} '310

JTET- 5 ' -agtcttccgcttccggttgctctgtt - 3 ' -

Probe j \ 26 39 31 1

TAMRA

Reverse 5 ' -tgttatgggtgcggttactatg-3 ' 1 22 67

Table YB. General_screening_panel_vl .5

Kekuda et al.

Kekuda et ul.

Kekuda et ul.

Table YC. Panel 4. ID

Kekuda et ul.

Kekuda et al.

General_screening_panel_vl.5 Summary: Ag4871 Highest expression of this gene is detected in CNS cancer SK-N-AS cell line (CT=28.5). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from gastric, colon, lung, 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 gastric, colon, lung, 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 of the 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 (CTs=32.2-32.9) when compared to adult liver and lung, respectively(CTs=36). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver and lung, respectively. In addition, the relative overexpression of this gene in fetal tissues suggests that the protein product may enhance growth or development of liver and lung 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 and lung related diseases. Kekuda et al.

Panel 4.1D Summary: Ag4871 Highest expression of this gene is detected in IFN gamma treated HUVEC cells (CT=31.9). This gene is expressed at low to moderate levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members of the 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_screeningjpanel_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.

Z. NOV32a (CG56711-01): KALLISTATIN PRECURSOR.

Expression of gene CG56711-01 was assessed using the primer-probe set Agl 689, described in Table ZA. Results of the RTQ-PCR runs are shown in Tables ZB, ZC and ZD. Please note that CG5671 1-01 represents a full-length physical clone

Table ZA. Probe Name Agl 689

Kekuda et al.

Table ZB. Panel 1.3D

Kekuda et al.

Kekuda et al.

Table ZC. Panel 2D

Kekuda et al.

Kekuda et al.

Table ZD. Panel 4D

Kekuda et al.

Kekuda et al.

Kekuda et nl.

CNS_neurodegeneration_vl.O Summary: Agl 689 Expression of this gene is low/undetectable (CTs > 35) across all ofthe samples on this panel. Kekuda et nl.

Panel 1.3D Summary: Agl 689 Two experiment with same probe and primer sets are in excellent agreement with highest expression ofthe CG5671 1-01 gene in adult and fetal liver (CTs=27-29). Therefore, expression of this gene may be used to distinguish these samples from other samples in this panel. Moderate to low expression of this gene is also seen in liver cancer and colon cancer cell line. Therefore, therapeutic modulation of this gene may be useful in the treatment of liver related diseases, liver and colon cancers.

Moderate levels of expression of this gene is also seen in pancrease and stomach. This gene codes for a kallistatin precursor, a serine proteinase inhibitor (serpin) with Phe- Phe residues at the P2 and PI positions. Kallistatin inhibits the proliferation, migration and adhesion of endothelial cells in vitro and angiogenesis in the rat model of hindlimb ischemia. It induces vasorelaxation of isolated aortic rings and reduces renal perfusion pressure in isolated rat kidneys. It also inhibits the proliferation, migration and adhesion of endothelial cells in vitro and angiogenesis in the rat model of hindlimb ischemia (Chao et al., 2001 , Biol Chem 382(1):15-21, PMID: 1 1258665). Furthermore, kallistatin expression is lower in the eye of patients suffering from diabetes and thus may be involved in diabetic retinopathy (Ma et al., 1996, Curr Eye Res 1996 Nov; 15( 1 1):1 1 17-23, PMID: 8950506). Thus, therapeutic modulation ofthe activity ofthe kallistatin precursor encoded by this gene, through the use of protein therapeutics or antibodies, may be useful in the treatment of diabetes, diabetic retinopathy, blood pressure regulation and vascular remodeling. Panel 2D Summary: Agl 689 Highest expression ofthe CG5671 1-01 gene is detected in liver (ODO4309)(CT=25.8). Interestingly, expression of this gene is much lower in the samples derived hepatectomy (ODO4309) metastasis and occular cancer metastasis to liver (ODO4310) (CT=29-40) as compared to corresponding adjacent control samples (CTs=25-26). High levels of expression of this gene is also seen in normal and liver cancer samples. Therefore, therapeutic modulation of expression of this gene or use of the protein encoded by this gene in the form of protein therapeutics may be useful in the treatment of these cancers and their metastasis.

Moderate to low levels of expression of this gene is also seen in gastric and kidney normal tissue samples compared with the adjacent tumor sample. It is also expressed in a sample of uterine and breast cancer . It may thus be used as a marker for these cancers and modulation ofthe activity of this gene or its protein product, through the use of protein therapeutics or antibodies, might be beneficial in the treatment of these cancers.

Panel 4D Summary: Agl 689 Two experiment with same probe and primer sets are in excellent agreement with highest expression of the CG5671 1-01 gene in liver Kekuda et ul. cirrhosis (CTs=27-31). Therefore, expression of this gene may be useful distinguishing this sample from other samples in this panel and also as a marker for the diagnosis of liver cirrhosis. Furthermore, therapeutic modulation of this gene or its product may be beneficial in the treatment of liver cirrhosis.

In addition, moderate levels of expression of this gene is also seen in thymus. Thus, drugs that inhibit the function of this protein may regulate T cell development in the thymus and reduce or eliminate the symptoms of T cell mediated autoimmune or inflammatory diseases, including asthma, allergies, inflammatory bowel disease, lupus erythematosus, or rheumatoid arthritis. Additionally, small molecule or antibody therapeutics designed against this putative protein may disrupt T cell development in the thymus and function as an immunosuppresant for tissue transplant.

AA. NOV40a and NOV21a (CG95205-02 and CG133159-01): TEM-1 splice variant.

Expression of gene CG95205-02 and CG133159-01 was assessed using the primer- probe sets Ag389, Ag4808 and Ag4834, described in Tables AAA, AAB and AAC. Results ofthe RTQ-PCR runs are shown in Tables AAD, AAE, AAF, AAG, AAH, AAI and AAJ. Please note that the probes and primer sets Ag4808 and Ag4834 are specific for CG95205- 02.

Table AAA. Probe Name Ag389

Table AAB. Probe Name Ag4808

Kekuda et ul.

Table AAP. General_screening_panel_vl .4

Kekuda et al.

Kekuda et al.

Table AAE. HASS Panel vl .0

Kekuda et al.

Kekuda et al.

Table AAF. Panel 1.1

Rel. Exp.(%) ] Rel. Exp.(%) ] Rel. Exp.(%) Rel. Exp.(%)

Tissue Name Ag389, Run j Ag389, Run Tissue Name . Ag389, Run Ag389, Run

109668399 ^■ 129785554 109668399 129785554

Renal ca. UO-

Adrenal gland 8.7 8.0 0.1 0.0 31 Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AAG. Panel 1.2

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AAH. Panel 2D

Kekuda et al.

Kekuda et al.

Table AAI. Panel 4D

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table AAJ. Panel 5 Islet

Kekuda et al.

97486_Patient- |94743_Donor 3 U -

3.3 82.4 09sk skeletal muscle jB_Mesenchymal Stem Cells

97487_Patient-

1 1.9 _.94730_Donor 3 AM - A_adipose 100.0 09ut uterus

97488_Patient-

8.3 '94731 _Donor 3 AM - B adipose 67.8 09pl_placenta

97492_Patient-

23.2 _.94732_Donor 3 AM - C_adipose 80.1 l Out uterus

97493_Patient-

15.0 i94733_Donor 3 AD - A adipose 85.9 10pl_placenta

97495_Patient-

6.9 _94734_Donor 3 AD - B adipose 69.7 1 lgo_adipose

97496JPatient-

5.0 _94735_Donor 3 AD - C_adipose 62.4

1 l sk skeletal muscle

97497_Patient-

27.4 77138_Liver_HepG2untreated 4.8 1 1 ut uterus

97498_Patient- 73556_Heart_Cardiac stromal cells

12.8 0.0 1 1 pl_placenta (primary)

]97500_Patient-

72.7 ,81735_Small Intestine 9.4 2go_adipose

*97501_Patient- J2409_Kidney_Proximal

22.2 0.0 12sk skeletal muscle Convoluted Tubule

97502_Patient-

54.7 82685 Small intestine Duodenum 0.9 12ut uterus

97503_Patient- 90650_Adrenal_Adrenocortical

3.5 3.9 12pl_placenta .adenoma

94721_Donor 2 U - A Mesenchymal Stem 49.0 ,72410_Kidney_HRCE 0.0 Cells

94722_Donor 2 U - B Mesenchymal Stem 46.7 ^'7241 1_Kidney_HRE 0.1 Cells

94723_Donor 2 U - i73139_Uterus_Uterine smooth

57.0 28.1 C Mesenchymal Stem ³muscle cells Kekuda et al.

AI_comprehensive panel_vl.O Summary: Ag4834 Expression ofthe CG95205- 02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

General_screening_panel_vl.4 Summary: Ag4808 Highest expression of this gene is detected in melanoma Hs688(B).T cell line (CT=26.7). In addition, high to moderate expression of this is also seen in colon cancer, melanoma melanoma Hs688(A).T cell line, and cell lines derived from brain, liver, lung and breast cancers. This gene codes for endosialin (TEMl) protein, a cell surface glycoprotein identified with monoclonal antibody FB5. It is a highly expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues (Carson- Walter et al., 2001, Cancer Res 61(18):6649-55, PMID: 1 1559528; Christian et al., 2001, J Biol Chem 276(10):7408-14, PMID: 11084048). Therefore, therapeutic modulation ofthe protein encoded by this gene through the use of antibody or small molecule drug, may be beneficial in the treatment of these 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. Interestingly, this gene is expressed at much higher levels in fetal (CT=31.2) when compared to adult liver (CT=37.9). 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 skeletal muscle 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 TEMl encoded by this gene could be useful in treatment of liver related diseases.

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. Kekuda et nl.

General_screeningjpanel_vl.5 Summary: Ag4834 Expression ofthe CG95205- 02 gene (Runs 228726951 and 228783170) is low/undetectable (CTs > 35) across all ofthe samples on this panel.

HASS Panel vl.O Summary: Ag389 Highest expression of this gene is detected in primary melanocytes A5 (CT=29.5). Moderate levels of expression of this gene is detected in a sample of brain cancer, as well as, in cultured primary melanocytes and astrocytes.

Oncology_cell_line_screeningjpanel_v3.1 Summary: Ag4834 Expression ofthe CG95205-02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

Panel 1.1 Summary: Ag4808 Two experiment with same probe and primer sets are in excellent agreement. Highest expression of this gene is detected in melanoma

Hs688(B).T and neuronal metastatic SK-N-AS cell lines (CTs=22-24). In addition, high to moderate expression of this is also seen in colon cancer, melanoma melanoma Hs688(A).T cell line, and cell lines derived from brain, liver, lung and breast cancers. Among tissues with metabolic or endocrine function, this gene is expressed at moderate levels in pancreas, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, fetal liver and the gastrointestinal tract. In addition, this gene is expressed at moderate to low levels in all regions of the central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Please see panel 1.4 for discussion on utility of this gene. Panel 1.2 Summary: Ag389 Two experiment with same probe and primer sets are in excellent agreement. Highest expression of this gene is detected in melanoma Hs688(B).T (CTs=25). In addition, high to moderate expression of this is also seen in colon cancer, melanoma melanoma Hs688(A).T cell line, and cell lines derived from brain, liver, lung and breast cancers. Among tissues with metabolic or endocrine function, this gene is expressed at moderate levels in pancreas, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, fetal liver and the gastrointestinal tract. 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. Please see panel 1.4 for discussion on utility of this gene. Results from two experiments (Runs 138522289 and 138564094) with this gene are not included. The amp plot indicates that there were experimental difficulties with this run.

Panel 2D Summary: Ag389 Highest expression of this gene is detected in normal bladder (CT=30). Moderate to low expression of this gene is seen in both normal and cancer samples derived from colon, stomach, ovary, bladder, liver, thyroid, uterus, kidney, Kekuda et al. lung, and prostate. Therefore, therapeutic modulation ofthe protein encoded by this gene through the use of antibody or small molecule drug, may be beneficial in the treatment of these cancers. Please see panel 1.4 for more discussion.

Panel 4.1D Summary: Ag4834 Expression ofthe CG95205-02 gene is low/undetectable (CTs > 35) across all of the samples on this panel.

Panel 4D Summary: Ag389 Two experiment with same probe and primer sets are in excellent agreement. Highest expression of this gene is detected in IL-4 treated dermal fibroblast (CTs=27.4). In addition, high to moderate expression of this gene is seen in lung and dermal fibroblasts, coronery artery SMC, PHA-L activated PBMC cells, and normal tissues represented by colon, lung, thymus and kidney. Moderate expression of this gene is also detected in CD45RA CD4 lymphocytes, which represents activated naive T cells. Interestingly, the expression of this gene is strongly down regulated in activated memory T cells (CD45RO CD4 lymphocyte) or CD4 Thl or Th2 cells, resting CD4 cells (CTs>35), suggesting a role for this putative protein in differentiation or activation of naive T cells. Therefore, modulation ofthe expression and/or activity of this putative protein encoded by this gene might be beneficial for the control of autoimmune diseases and T cell mediated diseases such as arthritis, IBD, asthma, COPD and skin disorders such as psoriasis and emphysema.

Panel 5 Islet Summary: Ag4808 Highest expression of this gene is detected in midway differentiated adipose (CT=28.3). Moderate to low expression of this gene is also seen in differentiated adipocytes and undifferentiated mesenchymal cells, skeletal muscle, islet cells, small intestine, placenta and uterus. Please see panel 1.4 for further discussion on the utility of this gene.

General oncology screening panel_v_2.4 Summary: Ag4834 Expression of the CG95205-02 gene is low/undetectable (CTs > 35) across all ofthe samples on this panel.

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 Kekuda et al. 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 ofthe 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 Kekuda et nl. 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.

NOVla SNP data:

Four ploymoφhic variants of NOVla have been identified and are shown in Table DI .

NOV4a SNP DATA:

Two polymoφhic variants of NOV4a have been identified and are shown in Table

D2.

Kekuda et al.

NOV5a SNP data:

Two polymoφhic variants of NOV5a have been identified and are shown in Table D3.

NOV6a SNP data:

One polymoφhic variant of NOVόa has been identified and is shown in Table D4.

NOV7a SNP data:

Two polymoφhic variants of NOV7a were identified and are shown in Table D5.

Kekuda et al.

NOV9a SNP data:

One polymoφhic variant of NOV9a has been identified and is shown in Table D6.

NOVlOa SNP data:

Four polymoφhic variants of NOvlOa have been identified and are shown in Table D7.

NOVlla SNP data:

One polymoφhic variant of NOVl la has been identified and is shown in Table D8.

Kekuda et al.

NOV12a SNP data:

Two polymoφhic variants of NOV 12a have been identified and are shown in Table D9.

NOV13a SNP data:

One polymoφhic variant of NOVl 3a has been identified and is shown in Table

D10.

NOV14a SNP data:

Four polymoφhic variants of NOV 14a have been identified and are shown in Table Dl l .

Kekuda et al.

NOVlSa SNP data:

Ten polymoφhic variants of NOV 15a have been identified and are shown in Table D12.

NOVlόa SNP data:

One polymoφhic variant of NOVl 6a has been identified and is shown in Table D13.

Kekuda et al.

NOV22a SNP data:

One polymoφhic variant of NOV22a has been identified and is shown in Table D14.

NOV25a SNP data:

One polymoφhic variant of NOV25a has been identified and is shown in Table D15.

NOV27a SNP data:

Five polymoφhic variants of NOV27a have been identified and are shown in Table D16.

Kekuda et al.

NOV28a SNP data:

Four polymoφhic variants of NOV28a have been identified and are shown in Table D17.

NOV32a SNP data:

Eleven polymorphic variants of NOV32a have been identified and are shown in Table D 18.

Kekuda et al.

NOV40a SNP data:

Two polymoφhic variants of NOV40a have been identified and are shown in Table D19.

Example E:

Each ofthe clones listed below is related to a clone or family of clones listed in Example A. The relationship is identifiable as the clone listed below will have the same NOVX number as the clones to which it is related. For example, NOV30g below is related to the NOV30 family of Example A.

The NOV30g and NOV30h clones were analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table El . Kekuda et al.

The NOV33g clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table E2. Kekuda et al.

The NOV34b clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table E3.

Kekuda et al.

The NOV35b clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table E4.

Kekuda et al.

NOV35b, MV MAPRT LLLS GALTQT ARSHSMRYFYTTMS RPGRGE PRF I S VGYVDYTQFVRFDS DDASPREEPRAP MEREGPEYWDRNTQICKAQARTERENLRIALRYYNQSEGGGSHTMQV CG57668-01 MYGCDVGPDGRF RGYEQHAYDGKDYIA1.NEDLRSWTAADMAAQITKRKWEAARVAEQLR AYLEGEFVEW RRYLENGKETLQRASDPPKTHMTHYPISDHEAT RC ALGFYPAEIT T Protein Sequence WQRDGEDQTTELVETRPAGDGTFQK AAVWPSGEEQRYTCHVQHEGLPEPLTLRWQGQG PSPSPLFPEPSSQPTIPIVGI IAGLVLLVAWTGAWTAVM RKKSSGKEGDGYSTPGGN SAQGSDVSLTA

The NOV36b clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table E5.

Table E5.

SEQ ID NO: 159 1210 bp

NOV36b, jTCGCTCACCCACCCGGACTCATTCTCCCCAGACGCCAAGGATGGTGGTCATGGCACCCCG AACCCTCTTCCTGCTACTCTCGGGGGCCCTGACCCTGACCGAGACCTGGGCGGGCTCCCA

CG59256-01 ] CTCCATGAGGTATTTCAGCGCCGCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCAT

CGCCATGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACTCGGCGTGTCC

DNA Sequence J GAGGATGGAGCCGCGGGCGCCGTGGGTGGAGCAGGAGGGGCCAGAGTATTGGGAAGAGGA

GACACGGAACACCAAGGCCCACGCACAGACTGACAGAATGAACCTGCAGACCCTGCGCGG

CTACTACAACCAGAGCGAGGGGGTGGGGCCAGGTTCTCATACCCTCCAGTGGATGATTGG

JCTGCGACCTGGGGTCCGACGGACGCCTCCTCCGCGGGTATGAACAGTATGCCTACGATGG i CAAGGATTACCTCGCCCTGAACGAGGACCTGCGCTCCTGGACCGCAGCGGACACTGCGGC jTCAGATCTCCAAGCGCAAGTGTGAGGCGGCCAATGTGGCTGAACAAAGGAGAGCCTACCT

GGAGGGCACGTGCGTGGAGTGGCTCCACAGATACCTGGAGAACGGGAAGGAGATGCTGCA

GCGCGCGGACCCCCCCAAGACACACGTGACCCACCACCCTGTCTTTGACTATGAGGCCAC

CCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCATACTGACCTGGCAGCGGGA

TGGGGAGGACCAGACCCAGGACGTGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAAC

CTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCA

5TGTGCAGCATGAGGGGCTGCCGGAGCCCCTCATGCTGAGATGGGAGCAGTCTTCCCTGCC

JCACCATCCCCATCATGGGTATCGTTGCTGGTCTGGTTGTCCTTGCAGCTGTAGTCACTGG lAGCTGCGGTCGCTGCTGTGCTGTGGAGGAAGAAGAGCTCAGGTAAGAAAGGAGGGAGCTA

JcTCTCAGGCTGCAAGTAGTGACAGTGCCCAGGGCTCTAATGTGTCTCTCACGGCTTGTAA

ATGTGACACCCCGGGGGGCCTGATGTGTGTGGGTTGTTGAGGGAAACAGTGGACATAGCT

GTGCTATGAG

SEQ ID NO: 160 379 aa

;NOV36b, MWMAPRTLFL LSGALT TETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRF DSDSACPRMEPRAP VEQEGPEYWEEETRNTKAHAQTDRMNLQTtRGYYNQSEGVGPGSH CG59256-01 TLQWMIGCDLGSDGR LRGYEQYAYDGKDYLALNEDLRS TAADTAAQISKRKCEAANVA EQRRAY EGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEI Protein Sequence ILT QRDGEDQTQDVELVETRPAGDGTFQK AAVWPSGEEQRYTCHVQHEG PEPLMLR WEQSSLPTIPIMGIVAGLWLAAWTGAAVAAVLWRKKSSGKKGGSYSQAASSDSAQGSN VS TACKCDTPGGLMCVGC

The NOV39b clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table E6. Kekuda et al.

Example F: Polynucleotide and Polypeptide Sequences, and Homology Data

Example 1.

The NOV 41 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table FI A.

Kekuda et al.

Kekuda et al.

Kekuda et al.

NOV41d, MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLA VADFLLMICLPFRTDYYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTWAADRYFK CG55676-04 WHPHHAVNTISTRVAAGIVCTL ALVILGTVYLLLENHLCVQETAVSCESFIMESAN G HDIMFQLEFFMPLGI ILFCSFKIV SLRRRQQLARQARMKKATRFIMWAIVFITC Protein Sequence YLPS VSARLYFLWTVPS S ACDPSVHGALHI TLS FTYMNSMLDPLVYYFSS PS FPKFYN KLKI CS LKPKQPGHSKTQRPEEMP I SNLGRRSC I S VANS FQSQSDGQWDPHI VEWH

SEQ ID NO: 185 961 bp

NOV41e, CACCAGATCTAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAG

CCCAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCC CG55676-05 TGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCC CTGCCGAGTGGGGCTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTT DNA Sequence ACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACA CTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCT GGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCC TGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGG AGTTCTTTATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCT GAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATC ATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCT ATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCA CATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTT TCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCA AGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCGAACCTCGG TCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGG GATCCCCACATTGTTGAGTGGCACAAGCTTGGC

ORF Start: at 1 1 ORF Stop: at 953

SEQ ID NO: 186 314 aa MW at 35943.9kD

|NOV41e, NGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRRH AFGDIPCRV GLFTLAMNRAGSIVFLTWAADRYFKWHPHHAVNTISTRVAAGIVCTLWALVILGTV

JCG55676-05 YLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRR

Ϊ QQLARQARMKKATRFIMWAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITL j Protein Sequence SFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEEMPISNLGRRS CISVANSFQSQSDGQWDPHIVEWH

SEQ ID NO: 187 1060 bp

NOV41f, CACCTCGCGAACCATGTACAACGGGTCGTGCTGCCGCATCGAGGGGGACACCATCTCC

CAGGTGATGCCGCCGCTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGGCAATGGGG CG55676-06 TCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCCCAGCACTGTTTACCT TTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGAC DNA Sequence TATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCT TCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGA CAGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTG GCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTT TGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCAT GGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTC GGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGC TGGCCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGT GTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTG CCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCA CCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCC CAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCA AAAACACAAAGGCCGGAAGAGATGCCAATTTCGAACCTCGGTCGCAGGAGTTGCATCA GTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGGGATCCCCACATTGTTGA GTGGCACGTCGACGGC

ORF Start: ATG at 14 ORF Stop: at 1052 Kekuda et al.

Kekuda et al.

Kekuda et al.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table FIB.

Table FIB. Comparison of NOV41a against NOV41b through NOV41k.

NOV41a Residues/ Identities/

Protein Sequence j Match Residues Similarities for the Matched Region

NOV41b 1..346 333/346 (96%) 1..346 333/346 (96%)

NOV41c 1..346 332/346 (95%) 1..346 332/346 (95%)

NOV41d 1..346 334/346 (96%) 1..346 334/346 (96%) Kekuda et al.

Further analysis of the NOV4 la protein yielded the following properties shown in Table FIC.

Table FIC. Protein Sequence Properties NOV41a j PSort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400 « analysis: probability located in plasma membrane; 0.4600 probability located in Golgi body; 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 33 and 34 analysis:

A search of the NOV41a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table FID.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV4 la protein was found to have homology to the proteins shown in the BLASTP data in Table FIE.

Table FIE. Public BLASTP Results for NOV41a

Protein NOV41a Identities/ Expect

PFam analysis predicts that the NOV4 la protein contains the domains shown in the Table FI F.

Example 2.

The NOV42 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table F2A. Kekuda et al.

Table F2A. NOV42 Sequence Analysis

SEQ ID NO: 199 1012 bp

NOV42a, GCATTCACAAGCAGGATGTTCCTTCCCAATGACACCCAGTTTCACCCCTCCTCCTTCC

TGTTGCTGGGGATCCCAGGACTAGAAACACTTCACATCTGGATCGGCTTTCCCTTCTG CG53677-01 TGCTGTGTACATGATCGCACTCATAGGGAACTTCACTATTCTACTTGTGATCAAGACT GACAGCAGCCTACACCAGCCCATGTTCTACTTCCTGGCCATGTTGGCCACCACTGATG DNA Sequence TGGGTCTCTCAACAGCTACCATCCCTAAGATGCTTGGAATCTTCTGGATCAACCTCAG AGGGATCATCTTTGAAGCCTGCCTCACCCAGATGTTTTTTATCCACAACTTCACACTT ATGGAGTCAGCAGTCCTTGTGGCAATGGCTTATGACAGCTATGTGGCCATCTGCAATC CACTCCAATATAGCGCCATCCTCACCAACAAGGTTGTTTCTGTGATTGGTCTTGGTGT GTTTGTGAGGGCTTTAATTTTCGTCATTCCCTCTATACTTCTTATATTGCGGTTGCCC TTCTGTGGGAATCATGTAATTCCCCACACCTACTGTGAGCACATGGGTCTTGCTCATC TATCTTGTGCCAGCATCAAAATCAATATTATTTATGGTTTATGTGCCATTTGTAATCT GGTGTTTGACATCACAGTCATTGCCCTCTCTTATGTGCATATTCTTTGTGCTGTTTTC CGTCTTCCTACTCATGAGCCCCGACTCAAGTCCCTCAGCACATGTGGTTCACATGTGT GTGTAATCCTTGCCTTCTATACACCAGCCCTCTTTTCCTTTATGACTCATTGCTTTGG CCGAAATGTGCCCCGCTATATCCATATACTCCTAGCCAATCTCTATGTTGTGGTGCCA CCAATGCTCAATCCTGTCATATATGGAGTCAGAACCAAGCAGATCTATAAATGTGTAA AGAAAATATTATTGCAGGAACAAGGAATGGAAAAGGAAGAGTACCTAATACATACGAG GTTCTGAATGCAATTTTATGAAATTT

ORF Start: ATG at 16 ORF Stop: TGA at 991

SEQ ID NO: 200 325 aa MW at 36602.5kD

|NOV42a, JMFLPNDTQFHPSSFLLLGIPGLETLHIWIGFPFCAVYMIALIGNFTILLVIKTDSSLH JQPMFYFLAMLATTDVGLSTATIPKMLGIFWINLRGIIFEACLTQMFFIHNFTLMESAV

JCG53677-01 JLVAMAYDSYVAICNPLQYSAILTNKWSVIGLGVFVRALIFVIPSILLILRLPFCGNH i JVIPHTYCEHMGLAHLSCASIKINIIYGLCAICNLVFDITVIALSYVHILCAVFRLPTH

[Protein Sequence [EPRLKSLSTCGSHVCVILAFYTPALFSFMTHCFGRNVPRYIHILLANLYVWPPMLNP IVIYGVRTKQIYKCVKKILLQEQGMEKEEYLIHTRF

SEQ ID NO: 201 988 bp

NOV42b, TAGGATGTTCCTTCCCAATGACACCCAGTTTCACCCCTCCTCCTTCCTGTTGCTGGGG

ATCCCAGGACTAGAAACACTTCACATCTGGATCGGCTTTCCCTTCTGTGCTGTGTACA CG53677-02 TGATCGCACTCATAGGGAACTTCACTATTCTACTTGTGATCAAGACTGACAGCAGCCT ACACCAGCCCATGTTCTACTTCCTGGCCATGTTGGCCACCACTGATGTGGGTCTCTCA DNA Sequence ACAGCTACCATCCCTAAGATGCTTGGAATCTTCTGGATCAACCTCAGAGGGATCATCT TTGAAGCCTGCCTCACCCAGATGTTTTTTATCCACAACTTCACACTTATGGAGTCAGC AGTCCTTGTGGCAATGGCTTATGACAGCTATGTGGCCATCTGCAATCCACTCCAATAT AGCGCCATCCTCACCAACAAGGTTGTTTCTGTGATTGGTCTTGGTGTGTTTGTGAGGG CTTTAATTTTCGTCATTCCCTCTATACTTCTTATATTGCGGTTGCCCTTCTGTGGGAA TCATGTAATTCCCCACACCTACTGTGAGCACATGGGTCTTGCTCATCTATCTTGTGCC AGCATCAAAATCAATATTATTTATGGTTTATGTGCCATTTGTAATCTAGTGTTTGACA TCACAGTCATTGCCCTTTCTTATGTGCATATTCTTTGTGCTGTTTTCCGTCTTCCTAC TCATGAAGCCCGACTCAAGTCCCTCAGCACATGTGGTTCACATGTGTGTGTAATCCTT GCCTTCTATACACCAGCCCTCTTTTCCTTTATGACTCATCGCTTTGGCCGAAATGTGC CCCGCTATATCCATATACTCCTAGCCAATCTCTATGTTGTGGTGCCACCAATGCTCAA TCCTGTCATATATGGAGTCAGAACCAAGCAGATCTATAAATGTGTGAAGAAAATATTA TTGCAGGAACAAGGAATGGAAAAGGAAGAGTACCTAATACATACGAGGTTCTGAATGC AA

ORF Start: ATG at 5 ORF Stop: TGA at 980

SEQ ID NO: 202 325 aa MW at 36629.6kD

NOV42b, MFLPNDTQFHPSSFLLLGIPGLETLHIWIGFPFCAVYMIALIGNFTILLVIKTDSSLH QPMFYFLAMLATTDVGLSTATIPKMLGIFWINLRGIIFEACLTQMFFIHNFTLMESAV Kekuda et al.

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table F2B.

Kekuda et al.

Further analysis ofthe NOV42a protein yielded the following properties shown in Table F2C.

Table F2C. Protein Sequence Properties NOV42a j PSort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400 analysis: probability located in plasma membrane; 0.4600 probability located in Golgi body; 0.1000 probability located in endoplasmic reticulum (lumen)

SignalP Cleavage site between residues 56 and 57 j analysis: A search ofthe NOV42a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table F2D.

Kekuda et al.

In a BLAST search of public sequence datbases, the NOV42a protein was found to have homology to the proteins shown in the BLASTP data in Table F2E.

Kekuda et al.

PFam analysis predicts that the NOV42a protein contains the domains shown in the Table F2F.

Example G: 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 autoimmune diseases), Panel CNSD.01 (containing central nervous system Kekuda et ul. 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.

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. Kekuda et ul.

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 Kekuda et al. 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

The plates for Panel 1.4 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panel 1.4 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 of the 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 Panel 1.4 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 Panel 1.4 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 and 2.2 Kekuda et al.

The plates for Panels 2D and 2.2 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). 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 or CHTN). 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. Panel 3D The plates of Panel 3D 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 and 1.3D are ofthe 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 Kekuda et al. 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 1 - 5ng/ml, TNF alpha at approximately 5-lOng/ml, IFN gamma at approximately 20-50ng/ml, IL-4 at approximately 5-lOng/ml, IL-9 at approximately 5-lOng/ml, IL-13 at approximately 5-lOng/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 Corporation, 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 5.5x10°M (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-lOng/ml, IFN gamma at 20-50ng/ml and IL-18 at 5-lOng/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 5.5x10°M (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). l OOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol (5.5x10^" ⁵M) (Gibco), and 1 OmM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation. Kekuda et al.

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 5.5xlO°M (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, CD 14 and CD 19 cells using CD8, CD56, CD 14 and CD 19 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), 1 OOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^""^ (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 5.5x10°M (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), 1 OOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^"5M (Gibco), and 1 OmM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared. Kekuda et al.

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°M (Gibco), and 1 OmM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately lOμg/ml and IL-4 at 5-lOng/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 CD4 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 5.5x10°M (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), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^" M (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 SxlO^cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to SxlO^cells/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 Kekuda et al. 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). CCDl 106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and lng/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.

Al comprehensive panel_vl.O

The plates for Al comprehensive panel vl .O 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.

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 Kekuda et al. 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-1 anti- 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 Al comprehensive panel vl .O 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

Panels 5D and 51 Kekuda et al.

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 of the infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) of the 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 1 1 Nondiabetic African American and overweight

Patient 12 Diabetic Hispanic on insulin

Adipocyte 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 Kekuda et ul.

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 Kekuda et ul. 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.

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_Vl .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 Kekuda et al. 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_V1.0 panel, the following abbreviations are used:

Control = Control brains; patient not demented, showing no neuropathology

Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology

SupTemporal Ctx = Superior Temporal Cortex

Inf Temporal Ctx = Inferior Temporal Cortex

GA. NOV41b and NOV41c (CG55676-02 and CG55676-03): GPCR-like

Expression of genes CG55676-02 and CG55676-03 were assessed using the primer- probe set Ag2378, described in Table GA. Results ofthe RTQ-PCR runs are shown in Tables GB-GF.

Table GA. Probe Name Ag2378

Table GB. Panel A/I

Kekuda et al.

Kekuda et al.

Kekuda et al.

Table GC. Panel 1.3D

Kekuda et al.

Kekuda et ul.

Kekuda et nl.

Kekuda et nl.

Kekuda et nl.

54: Kekuda et al.

Kekuda et al.

Table GE. Panel 3D

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Kekuda et al.

Expression in panel 4D: CG55676-02 is expressed highly during initial activation and polarization of T cells regardless of whether polarization is to Thl , Th2 or Tri pathway. It is not expressed in untreated CD4 T cells and the level of expression is much less in chronically activated T cells.

Role in inflammation: CG55676-02 is a putative GPCR and may play an important role in the regulation of T cell polarization, differentiation, and T cell trafficking. Potential therapeutic value: Antagonistic antibodies, preferably fully human monoclonal antibodies directed against the protein encoded for by CG55676-02 could reduce or block inflammation by blocking ligand interaction with this putative GPCR and preventing T cell function in diseases such as asthma, emphysema, allergy, arthritis, diabetes, and psoriasis. Alternatively, if this putative GPCR down regulates T cell activation then agonistic antibodies (Ligand-like) could also block inflammation in these diseases (Bromley et al., J. Immunol. 165(1 ) 15-9).

Expression in panel of relevance to Oncology 1.3D and 2D: In Panel 1 .3D, CG55676-02 is expressed in tumor derived cell lines especially from colon, lung, ovarian and breast cancers. In panel 2D it is overexpressed in breast, lung and bladder tumor tissues compared to normal adjacent tissues. Role in inflammation: CG55676-02 is a putative GPCR and may play a role tumor cell growth

Potential therapeutic value: Antagonistic antibodies, preferably fully human monoclonal antibodies directed against the protein encoded for by CG55676-02 could reduce or block tumor growth by blocking ligand interaction with this putative GPCR resulting in therapeutic treatment for tumor like lung, breast, bladder, kidney and colon. Kekuda et al.

A I panel: The transcript of CG55676-03 is found in bone of 4 out of 4 patients with osteoarthritis and in synovium from 1 out of 4 patients.

Role in inflammation: CG55676-03 encodes a transcript for a putative GPCR that is expressed on cells within the bone and in the synovium of patients with osteoarthritis. Potential therapeutic value: Antagonistic antibodies, preferably fully human monoclonal antibodies or small molecule therapeutics directed against the protein encoded for by CG55676-03 could reduce or block inflammation by preventing ligand interaction with this putative GPCR and as asthma, emphysema, allergy, arthritis, diabetes, and psoriasis.

OTHER EMBODIMENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope 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 of the inventions disclosed herein. Other, unclaimed inventions are also contemplated. Applicants reserve the right to pursue such inventions in later claims.

Claims

Kekuda et al.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 102.

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 102.

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

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

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

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

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

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

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

(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.

1 1 . 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 1 1 wherein the agent is a cellular receptor or a downstream effector.

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

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

(b) contacting the cell with a composition comprising a candidate substance; and Kekuda et al.

(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 the polypeptide of claim 1 ;

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

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

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

16. A method for modulating the activity ofthe 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 Kekuda et al. a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject.

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

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

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

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

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

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

24. 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 102.

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 102, or a complement of said nucleotide sequence. Kekuda et al.

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 ofthe 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 of the nucleic acid molecule is used as a marker for cell or tissue type.

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

35. A method for determining the presence of or predisposition to a disease associated with altered levels of expression ofthe nucleic acid molecule of claim 20 in a first mammalian subject, the method comprising: a) measuring the level of expression ofthe nucleic acid in a sample from the first mammalian subject; and Kekuda et al. 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 of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

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

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

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

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

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

41. A method of producing the polypeptide of claim 2, the method comprising culturing a cell under conditions that lead to expression 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 102.

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

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

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

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