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

Signaling proteins may be classified as endocrine effectors, paracrine effectors or autocrine effectors. Endocrine effectors are signaling molecules secreted by a given organ into the circulatory system, which are then transported to a distant target organ or tissue.

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

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

Many pathological conditions involve dysregulation of expression of important effector proteins. In certain classes of pathologies the dysregulation is manifested as diminished or suppressed level of synthesis and secretion of protein effectors. In other classes of pathologies the dysregulation is manifested as increased or up-regulated level of synthesis and secretion of protein effectors. In a clinical setting a subject may be suspected

of suffering from a condition brought on by altered or mis-regulated levels of a protein effector of interest. Therefore there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject, and to compare the level with that characteristic of a nonpathological condition. There also is a need to provide the protein effector as a product of manufacture. Administration of the effector to a subject in need thereof is useful in treatment of the pathological condition. Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished or suppressed levels of the protein effector of interest. In addition, there is a need for a method of treatment of a pathological condition brought on by a increased or up-regulated levels of the protein effector of interest.

Antibodies are 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 of the immunoglobulin molecules resides in the variable domains; the variable domains of one light chain and one heavy chain associate with each other to generate the antigen-binding moiety. Antibodies that bind immunospecifically to a cognate or target antigen bind with high affinities. Accordingly, they are useful in assaying specifically for the presence of the antigen in a sample. In addition, they have the potential of inactivating the activity of the antigen.

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

SUMMARY OF THE INVENTION The invention is based in part upon the discovery of isolated polypeptides including amino acid sequences selected from mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. The novel nucleic acids and polypeptides are referred to herein as NOVX, where X is an identifier for each sequence as shown in Table A below. 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 of the amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed. In another embodiment, the invention includes the amino acid sequences selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. 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 52, 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 52, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% of the residues are changed.

In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic variants of the sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. 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-1, wherein n is an integer between 1 and 52. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution.

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

SEQ ID NO : 2n, wherein n is an integer between 1 and 52 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 52 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 52 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 52 in a first mammalian subject, the method involving measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and comparing the amount of the polypeptide in this sample to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, the disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

In another embodiment, the invention involves a method of identifying an agent that binds to a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, the method including introducing the polypeptide to the agent; and determining whether the agent binds to the polypeptide. The agent could be a cellular receptor or a downstream effector.

In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer

between 1 and 52, the method including providing a cell expressing the polypeptide of the invention and having a property or function ascribable to the polypeptide ; contacting the cell with a composition comprising a candidate substance; and determining whether the substance alters the property or function ascribable to the polypeptide ; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition devoid of the substance, the substance is identified as a potential therapeutic agent.

In another embodiment, the invention involves a method for screening for a modulator of activity or of latency or predisposition to a pathology associated with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, the method including administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of the invention, wherein the test animal recombinantly expresses the polypeptide of the invention; measuring the activity of the polypeptide in the test animal after administering the test compound; and comparing the activity of the protein in the test animal with the activity of the polypeptide in a control animal not administered the polypeptide, wherein a change in the activity of the polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of, or predisposition to, a pathology associated with the polypeptide of the invention. The recombinant test animal could express a test protein transgene or express the transgene under the control of a promoter at an increased level relative to a wild-type test animal The promoter may or may not b the native gene promoter of the transgene.

In another embodiment, the invention involves a method for modulating the activity of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, 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 52, the method including administering the polypeptide to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject. The subject could be human.

In another embodiment, the invention involves a method of treating a pathological state in a mammal, the method including administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52 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 52; 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 52 wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; the amino acid sequence selected from the group consisting of SEQ ID NO : 2n, wherein n is an integer between 1 and 52; 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 52, 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 52 or any variant of the polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and the complement of any of the nucleic acid molecules.

In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.

In another embodiment, the invention involves an isolated nucleic acid molecule including a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52 that encodes a variant polypeptide,

wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.

In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52, 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-1, wherein n is an integer between 1 and 52.

In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52; a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID N0 : 2n-1, wherein n is an integer between 1 and 52. is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52; and a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52, wherein the nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, or a complement of the nucleotide sequence.

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

ID NO : 2n, wherein n is an integer between 1 and 52, wherein the nucleic acid molecule has a nucleotide sequence in which any nucleotide specified in the coding sequence of the chosen nucleotide sequence is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides in the chosen coding sequence are so changed, an isolated second polynucleotide that is a complement of the first polynucleotide, or a fragment of any of them.

In another embodiment, the invention includes a vector involving the nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52. This vector can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.

In another embodiment, the invention involves a method for determining the presence or amount of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52 in a sample, the method including providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount of the probe bound to the nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in the sample. The presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type. The cell type can be cancerous.

In another embodiment, the invention involves a method for determining the presence of or predisposition for a disease associated with altered levels of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO : 2n, wherein n is an integer between 1 and 52 in a first mammalian subject, the method including measuring the amount of the nucleic acid in a sample from the first mammalian subject; and comparing the amount of the nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

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 of the NOVX polypeptide to the antibody less than 1 x 10-9 M. More preferably, the NOVX antibody neutralizes the activity of the NOVX polypeptide.

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

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

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

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

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compounds. The sequences are collectively referred to herein as"NOVX nucleic acids"or"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 of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.

TABLE A. SEQUENCES AND CORRESPONDING SEQ ID NUMBERS SEQ ID NO SEQ ID NO NOVX Internal (nucleic (amino Homology Assignment Identification acid acid NOVla CG108030-01 1 2 Human Sequence NOVlb CG108030-02 3 4 Human Sequence NOV2a CG115907-01 5 6 Trypsin inhibitor precursor NOV2b CG115907-04 7 8 Trypsin inhibitor precursor NOV2c CG115907-03 9 10 Trypsin inhibitor precursor NOV2d CG115907-02 11 12 Trypsin inhibitor precursor NOV3a CG139008-01 13 14 Binding protein NOV3b 233028732 15 16 Binding protein NOV3c CG139008-02 17 18 Binding protein NOV4a CG145877-01 19 20 Hypothetical protein NOV5a CG151161-02 21 22 Myelin and lymphocyte protein NOV5b CG151161-01 23 24 Myelin and lymphocyte protein NOV6a CG155653-01 25 26 Similar to PDZ domain NOV7a CG160093-01 27 28 Leukocyte elastase inhibitor NOV7b CG160093-02 29 30 Leukocyte elastase inhibitor NOV8a CG163133-02 31 32 JM4 protein NOV8b CG163133-01 33 34 JM4 protein NOV9a CG165528-01 35 36 Neurexin 1-alpha precursor NOV9b CG165528-02 37 38 Neurexin 1-alpha precursor NOVlOa CG165666-01 39 40 Similar to TPR-containing protein NOVlla CG165676-01 41 42 Integrin, alpha2 NOV12a CG165719-04 43 44 Neuronal membrane protein M6-B NOV12b CG165719-02 45 46 Neuronal membrane protein M6-B NOV12c CG165719-03 47 48 Neuronal membrane protein M6-B NOV12d CG165719-01 49 50 Neuronal membrane protein M6-B NOV12e CG165719-05 51 52 Neuronal membrane protein M6-B NOV13a CG167488-02 53 54 Human protein NOV13b CG167488-01 55 56 Human protein NOV14a CG173318-01 57 58 Human protein NOV15a CG50970-06 59 60 cerebroglycan NOV15b CG50970-01 61 62 cerebroglycan NOV15d 274054257 63 64 cerebroglycan NOV15e CG50970-03 65 66 cerebroglycan NOV15f 237922026 67 68 cerebroglycan NOV15g 237922511 69 70 cerebroglycan NOV15h 315490136 71 72 cerebroglycan NOVlSi CG50970-02 73 74 cerebroglycan NOV15j CG50970-04 75 76 cerebroglycan NOV15k CG50970-05 77 78 cerebroglycan NOV151 CG50970-07 79 80 cerebroglycan NOV16a CG54443-03 81 82 Hypothetical Protein NOV16b CG54443-07 83 84 Hypothetical Protein NOV16c CG54443-01 85 86 Hypothetical Protein NOV16d CG54443-02 87 88 Hypothetical Protein NOV16e CG54443-04 89 90 Hypothetical Protein NOV16f CG54443-05 91 92 Hypothetical Protein NOV16g CG54443-06 93 94 Hypothetical Protein NOV17a CG58495-01 95 96 pulmonary surfactant protein NOVl7b CG58495-03 97 98 pulmonary surfactant protein NOV17c CG58495-02 99 100 pulmonary surfactant protein NOV18a CG97482-01 101 102 S-100 protein, beta chain NOV18b CG97482-02 103 104 S-100 protein, beta chain

Table A indicates the homology of NOVX polypeptides to known protein families.

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

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

well as conditions such as transplantation, neuroprotection, fertility, or regeneration (in vitro and in vivo).

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

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

Details of the sequence relatedness and domain analysis for each NOVX are presented in Example A.

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

The NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of the 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 of the family to which the NOVX polypeptides belong.

The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e. g. , by protein or gene therapy.

Pathological conditions can be diagnosed by determining the amount of the new protein in a sample or by determining the presence of mutations in the new genes. Specific uses are described for each of the NOVX genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.

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

In one specific embodiment, the invention includes an isolated polypeptide comprising an amino acid sequence selected from the group consisting of : (a) a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 52; (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 52, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 52 ; (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 52, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; and (e) a fragment of any of (a) through (d).

In another specific embodiment, the invention includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of : (a) a mature form of the amino acid sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 52; (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 52, wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 52; (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 52, 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 52, or any variant of said polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and (f) the complement of any of said nucleic acid molecules.

In yet another specific embodiment, the invention includes an isolated nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of : (a) the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 52; (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 52, is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; (c) a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 52; 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-1, wherein n is an integer between 1 and 52, is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

NOVX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e. g., NOVX mRNAs) and fragments for use as PCR

primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term"nucleic acid molecule"is intended to include DNA molecules (e. g., cDNA or genomic DNA), RNA molecules (e. g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.

A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature"form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.

The product"mature"form arises, by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell (e. g. , host cell) in which the gene product arises. Examples of such processing steps leading to a "mature"form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 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 of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 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.

A nucleic acid molecule of the invention, e. g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, 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 : 2n-1, wherein n is an integer between 1 and 52, 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"d 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 of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template with appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e. g., using an automated DNA synthesizer.

As used herein, the term"oligonucleotide"refers to a series of linked nucleotide residues.

A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt

to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, or a portion of this nucleotide sequence (e. g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NO : 2n-1, wherein n is ah integer between 1 and 52, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, 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 of the disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 3' direction of the disclosed sequence.

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

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

A"homologous nucleic acid sequence"or"homologous amino acid sequence,"or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e. g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.

Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous

nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.

A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG"start"codon and terminates with one of the three"stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bona fide 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 : 2n-1, wherein n is an integer between 1 and 52; or an anti-sense strand nucleotide sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52; or of a naturally occurring mutant of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52.

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, eg. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e. g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.

"A polypeptide having a biologically-active portion of a NOVX polypeptide"refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX"can be prepared by isolating a portion of SEQ ID NO : 2n-l, wherein n is an integer between 1 and 52, that encodes a polypeptide having a NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e. g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.

NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52.

In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO : 2n, wherein n is an integer between 1 and 52.

In addition to the human NOVX nucleotide sequences of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e. g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms"gene"and"recombinant gene"refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, are intended to be within the scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants and homologues of the

NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52. In another embodiment, the nucleic acid is at least 10,25, 50,100, 250,500, 750,1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term"hybridizes under stringent conditions"is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 65% homologous to each other typically remain hybridized to each other.

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

As used herein, the phrase"stringent hybridization conditions"refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0. 01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C for short probes, primers or oligonucleotides (e. g., 10 nt to 50 nt) and at least about 60 °C for longer probes, primers and oligonucleotides.

Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds. ), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N. Y. (1989), 6.3. 1-6.3. 6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52 ; corresponds to a naturally-occurring nucleic acid molecule. As used herein, a"naturally-occurring"nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e. g, encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, 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 1X SSC, 0.1% SDS at 37 °C. Other conditions of moderate stringency that may be used are well-known within the art. See, e. g, Ausubel, et al. (eds. ), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990 ; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, 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-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e. g., as employed for cross-species hybridizations). See, e. g., Ausubel, et al. (eds. ), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND

EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.

Proc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, thereby leading to changes in the amino acid sequences of the encoded NOVX protein, without altering the functional ability of that NOVX protein. For example, nucleotide substitutions leading to amino acid substitutions at"non-essential" amino acid residues can be made in the sequence of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. A"non-essential"amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an"essential"amino acid residue is required for such biological activity.

For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.

Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 40% homologous to the amino acid sequences of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO : 2n, wherein n is an integer between 1 and 52; more preferably at least about 70% homologous to SEQ ID NO : 2n, wherein n is an integer between 1 and 52; still more preferably at least about 80% homologous to SEQ ID NO : 2n, wherein n is an integer between 1 and 52; even more preferably at least about 90% homologous to SEQ ID NO : 2n, wherein n is an integer between 1 and 52; and most preferably at least about 95% homologous to SEQ ID N0 : 2n, wherein n is an integer between 1 and 52.

An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID N0 : 2n, wherein n is an integer between 1 and 52, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide

sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced any one of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A"conservative amino acid substitution"is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e. g., lysine, arginin, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e. g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e. g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e. g., threonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan, histidine).

Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved"strong"residues or fully conserved"weak"residues. The"strong"group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, 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 (i) the ability to form protein: protein interactions with other NOVX proteins, other cell-surface proteins, or

biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and a NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof ; (e. g. avidin proteins).

In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e. g., regulation of insulin release).

Interfering RNA In one aspect of the invention, NOVX gene expression can be attenuated by RNA interference. One approach well-known in the art is short interfering RNA (siRNA) mediated gene silencing where expression products of a NOVX gene are targeted by specific double stranded NOVX derived siRNA nucleotide sequences that are complementary to at least a 19-25 nt long segment of the NOVX gene transcript, including the 5'untranslated (UT) region, the ORF, or the 3'UT region. See, e. g. , PCT applications WO00/44895, W099/32619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, W002/16620, and W002/29858, each incorporated by reference herein in their entirety.

Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX gene include, e. g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX polypeptide, and polypeptides involved in a NOVX regulatory pathway.

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

The most efficient silencing is generally observed with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3'overhang. The sequence of the 2-nt 3'overhang makes an additional small contribution

to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases. In one embodiment, the nucleotides in the 3'overhang are ribonucleotides. In an alternative embodiment, the nucleotides in the 3'overhang are deoxyribonucleotides. Using 2'-deoxyribonucleotides in the 3'overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.

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

In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the NOVX sequence may be identical or may be different, such that their expression may be modulated independently, or in a temporal or spatial manner.

In a specific embodiment, siRNAs are transcribed intracellularly by cloning the NOVX gene templates into a vector containing, e. g., a RNA pol III transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA Hl. One example of a vector system is the GeneSuppressor RNA Interference kit (commercially available from Imgenex). The U6 and HI promoters are members of the type in class of Pol III promoters.

The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for HI promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine.

Cleavage at this position generates a 3'IJCJ overhang in the expressed siRNA, which is similar to the 3'overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the

expression of around 21-nucleotide siRNAs in, e. g., an approximately 50-nucleotide RNA stem-loop transcript.

A siRNA vector appears to have an advantage over synthetic siRNAs where long term knock-down of expression is desired. Cells transfected with a siRNA expression vector would experience steady, long-term mRNA inhibition. In contrast, cells transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division. The long-term gene silencing ability of siRNA expression vectors may provide for applications in gene therapy.

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

A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 to 100 nt downstream of the start codon.

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

In one embodiment, a complete NOVX siRNA experiment includes the proper negative control. A negative control siRNA generally has the same nucleotide composition as the NOVX siRNA but lack significant sequence homology to the genome. Typically,

one would scramble the nucleotide sequence of the NOVX siRNA and do a homology search to make sure it lacks homology to any other gene.

Two independent NOVX siRNA duplexes can be used to knock-down a target NOVX gene. This helps to control for specificity of the silencing effect. In addition, expression of two independent genes can be simultaneously knocked down by using equal concentrations of different NOVX siRNA duplexes, e. g., a NOVX siRNA and an siRNA for a regulator of a NOVX gene or polypeptide. Availability of siRNA-associating proteins is believed to be more limiting than target mRNA accessibility.

A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (Nl9) residues (e. g., AA (N19) TT).

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

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

Cell Science 114: 4557-4565, incorporated by reference in its entirety.

Transfection of NOVX siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially available from Invitrogen). An assay for NOVX gene silencing is generally performed approximately 2 days after transfection. No NOVX gene silencing has been observed in

the absence of transfection reagent, allowing for a comparative analysis of the wild-type and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-well plate, approximately 0.84 ug of the siRNA duplex is generally sufficient. Cells are typically seeded the previous day, and are transfected at about 50% confluence. The choice of cell culture media and conditions are routine to those of skill in the art, and will vary with the choice of cell type. The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e. g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful NOVX silencing. The efficiency of transfection needs to be carefully examined for each new cell line to be used.

Preferred cell are derived from a mammal, more preferably from a rodent such as a rat or mouse, and most preferably from a human. Where used for therapeutic treatment, the cells are preferentially autologous, although non-autologous cell sources are also contemplated as within the scope of the present invention.

For a control experiment, transfection of 0.84 jig single-stranded sense NOVX siRNA will have no effect on NOVX silencing, and 0. 84 tig antisense siRNA has a weak silencing effect when compared to 0.84 pg of duplex siRNAs. Control experiments again allow for a comparative analysis of the wild-type and silenced NOVX phenotypes. To control for transfection efficiency, targeting of common proteins is typically performed, for example targeting of lamin A/C or transfection of a CMV-driven EGFP-expression plasmid (e. g. commercially available from Clontech). In the above example, a determination of the fraction of lamin A/C knockdown in cells is determined the next day by such techniques as immunofluorescence, Western blot, Northern blot or other similar assays for protein expression or gene expression. Lamin A/C monoclonal antibodies may be obtained from Santa Cruz Biotechnology.

Depending on the abundance and the half life (or turnover) of the targeted NOVX polynucleotide in a cell, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no NOVX knock-down phenotype is observed, depletion of the NOVX polynucleotide may be observed by immunofluorescence or Western blotting.

If the NOVX polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the targeted protein is observed, it may be desirable to analyze whether the target mRNA (NOVX or a NOVX upstream or downstream gene) was effectively destroyed by the transfected siRNA duplex. Two days after transfection, total RNA is prepared, reverse transcribed using a

target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable NOVX protein may exist in the cell. Multiple transfection in sufficiently long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfection steps are required, cells are split 2 to 3 days after transfection. The cells may be transfected immediately after splitting.

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

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

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

method for determination of a NOVX minus (NOVX) phenotype in the treated subject sample. The NOVX-phenotype observed in the treated subject sample thus serves as a marker for monitoring the course of a disease state during treatment.

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

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

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

The NOVX mRNA is radiolabeled (using known techniques) and its stability is monitored by gel electrophoresis.

In a parallel experiment made with the same conditions, the double stranded RNA is internally radiolabeled with a 32P-ATP. Reactions are stopped by the addition of 2 X proteinase K buffer and deproteinized as described previously (Tuschl et al., Genes Dev., 13: 3191-3197 (1999) ). Products are analyzed by electrophoresis in 15% or 18% polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gels for radioactivity, the natural production of 10 to 25 nt RNAs from the double stranded RNA can be determined.

The band of double stranded RNA, about 21-23 bps, is eluded. The efficacy of these 21-23 mers for suppressing NOVX transcription is assayed in vitro using the same rabbit reticulocyte assay described above using 50 nanomolar of double stranded 21-23 mer for each assay. The sequence of these 21-23 mers is then determined using standard nucleic acid sequencing techniques.

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

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

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

The above method provides a way both for the deduction of NOVX siRNA sequence and the use of such siRNA for in vitro suppression. In vivo suppression may be

performed using the same siRNA using well known in vivo transfection or gene therapy transfection techniques.

Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, or fragments, analogs or derivatives thereof. An"antisense"nucleic acid comprises a nucleotide sequence that is complementary to a"sense"nucleic acid encoding a protein (e. g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10,25, 50, 100,250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0 : 2n-1, wherein n is an integer between 1 and 52,-are additionally provided.

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

Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10,15, 20,25, 30,35, 40,45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using

chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e. g, an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e. g., phosphorothioate derivatives and acridine substituted nucleotides can be used).

Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-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 of the invention includes direct injection at a tissue site.

Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens

expressed on a selected cell surface (e. g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol lI or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-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. FEBSLett. 215: 327-330.

Ribozymes and PNA Moities Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.

These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is a ribozyme.

Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e. g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i. e. , SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA.

See, e. g, U. S. Patent 4,987, 071 to Cech, et al. and U. S. Patent 5,116, 742 to Cech, et al.

NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease

activity from a pool of RNA molecules. See, e. g., Bartel et al., (1993) Science 261: 1411-1418.

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

Bioassays 14: 807-15.

In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e. g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e. g, Hyrup, et al., 1996. BioorgMed 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 artifical restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996. supra) ; or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra ; Perry-O'Keefe, et aL, 1996. supra).

In another embodiment, PNAs of NOVX can be modified, e. g, to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e. g., RNase H and DNA polymerases) to interact with the DNA

portion while the PNA portion would provide high binding affinity and specificity.

PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleotide bases, and orientation (see, Hyrup, et al. , 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e. g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5'end of DNA. See, e. g, Mag, et al., 1989. Nucl Acid Res 17: 5973-5988.

PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5'PNA segment and a 3'DNA segment. See, e. g., Finn, et al., 1996. supra.

Alternatively, chimeric molecules can be synthesized with a 5'DNA segment and a 3'PNA segment. See, e. g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5 : 1119-11124.

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e. g., for targeting host cell receptors ion 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.

W088/09810) 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. Phare. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e. g, a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

NOVX Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in any one of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. 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 : 2n, wherein n is an integer between 1 and 52, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.

In general, a NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other

amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.

One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof.

Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

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

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

chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.

Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e. g., the amino acid sequence of SEQ ID NO : 2n, wherein n is an integer between 1 and 52) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10,25, 50,100 or more amino acid residues in length.

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

In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID NO : 2n, wherein n is an integer between 1 and 52. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO : 2n, wherein n is an integer between 1 and 52, and retains the functional activity of the protein of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, and retains the functional activity of the NOVX proteins of SEQ ID N0 : 2n, wherein n is an integer between 1 and 52.

Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e. g, gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the

molecules are homologous at that position (i. e., as used herein amino acid or nucleic acid "homology"is equivalent to amino acid or nucleic acid"identity").

The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52.

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 52, whereas a"non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e. g. , a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within a NOVX

fusion protein the NOVX polypeptide can correspond to all or a portion of a NOVX protein. In one embodiment, a NOVX fusion protein comprises at least one biologically-active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically-active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically-active portions of a NOVX protein. Within the fusion protein, the term"operatively-linked"is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

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

A NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e. g., by employing blunt-ended or stagger-ended termini for ligation, restriction

enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e. g. , Ausubel, et al. (eds. ) CURRENT PROTOCOLSIN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e. g., a GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.

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

Variants of the NOVX proteins that function as either NOVX agonists (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 performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences.

Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e. g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11 : 477.

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

Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the

screening assays to identify NOVX variants. See, e. g., Arkin and Yourvan, 1992. Proc.

Natl. Acad. Sci. USA 89 : 7811-7815 ; Delgrave, et al., 1993. Protein Engineering 6: 327-331.

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, Fab, Fab'and F (ab') 2 fragments, and an Fab 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 IgGI, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID NO : 2n, wherein n is an integer between 1 and 52, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located. on the surface of the protein, e. g, a hydrophilic region. A hydrophobicity analysis of the human NOVX protein sequence will

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

The term"epitope"includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. A NOVX polypeptide or a fragment thereof comprises at least one antigenic epitope. An anti-NOVX antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (KD) is 1 ; jM, preferably : 5 100 nM, more preferably < 10 nM, and most preferably 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

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

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

Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e. g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with

the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine 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 of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D.

Wilkinson (The Scientist, published by The Scientist, Inc. , Philadelphia PA, Vol. 14, No. 8 (April 17,2000), pp. 25-28).

Monoclonal Antibodies The term"monoclonal antibody" (MAb) or"monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.

59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.

Immunol. , 133: 3001 (1984) ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel 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 of the monoclonal antibody can, for example, be determined by the Scatcbard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

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

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

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

Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 328 : 522-525 (1986) ; Riechmann et al. , Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988) ), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.

(See also U. S. Patent No. 5,225, 539. ) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. , 1986; Riechmann et al. , 1988; and Presta, Curr. Op.

Struct. Biol. , 2: 593-596 (1992)).

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

produced by using human hybridomas (see Cote, et al. , 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al. , 1985 In : MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. , pp.

77-96).

In addition, human antibodies can also be produced using additional techniques, includingphage display libraries (Hoogenboom and Winter, J. Mol. Biol. , 227: 381 (1991) ; Marks et al. , J. Mol. Biol. , 222: 581 (1991) ). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e. g, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.

This approach is described, for example, in U. S. Patent Nos. 5,545, 807; 5,545, 806; 5,569, 825; 5,625, 126; 5,633, 425; 5,661, 016, and in Marks et al. (Bio/Technology 10, 779-783 (1992) ) ; Lonberg et al. (Nature 368 856-859 (1994) ); Morrison (Nature 368, 812-13 (1994) ) ; Fishwild et al, ( Nature Biotechnology 14, 845-51 (1996) ); Neuberger (Nature Biotechnology 14,826 (1996)) ; and Lonberg and Huszar (Intern. Rev. Immunol.

13 65-93 (1995)).

Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.

Additionally, the genes encoding the immunoglobulins with human variable regions can be

recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of, an endogenous immunoglobulin heavy chain is disclosed in U. S.

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

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

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

Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e. g, U. S.

Patent No. 4,946, 778). In addition, methods can be adapted for the construction of Fab expression libraries (see e. g., Huse, et al. , 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab 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 Fab fragment generated by reducing the disulfide bridges of an F (ab) fragment ; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.

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

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

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

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

threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e. g. F (ab') 2 bispecific antibodies). Techniques for generating bispecific / antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describe a procedure wherein intact antibodies are 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 of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

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

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al. , J. Immunol. 148 (5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.

The"diabody"technology described by Hollinger et al. , Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains

of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al. , J. Immunol. 152: 5368 (1994).

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

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic . arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e. g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.

Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies, for example, 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, cystine residue (s) can be introduced into the Fc region, thereby allowing

interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).

See Caron et al. , J. Exp Med. , 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.

See Stevenson et al. , Anti-Cancer Drug Design, 3: 219-230 (1989).

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

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 13lI, l3lIn, 90Y, and 186Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science. 238: 1098 (1987). Carbon-14-labeled

l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

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

Immunoliposomes The antibodies disclosed herein can also be formulated as immunoliposomes.

Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980) ; and U. S. Pat. Nos. 4,485, 045 and 4,544, 545.

Liposomes with enhanced circulation time are disclosed in U. S. Patent No. 5,013, 556.

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

See Gabizon et al., J. National Cancer Inst. , 81 (19): 1484 (1989).

Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention 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 of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to a NOVX protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as "Therapeutics").

An antibody specific for a NOVX protein of the 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 of the 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 (ive., 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, p-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 125I, 131I, 35S or 3H.

Antibody Therapeutics Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed

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

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

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

Pharmaceutical Compositions of Antibodies Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al. , editors) Mack Pub. Co. , Easton, Pa.: 1995 ;

Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa. , 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e. g, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

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

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

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

lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

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

For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

NOVX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term"vector"refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a"plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e. g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e. g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

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

The term"regulatory sequence"is intended to includes promoters, enhancers and other expression control elements (e. g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY : METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory

sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e. g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e. g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using 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 of the recombinant protein. Such fusion vectors typically serve three purposes : (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech 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 (Amrannet al., (1988) Gene 69: 301-315) and pET l ld (Studier et al., GENE ExPRssioION

TECHNOLOGY : METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

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

In another embodiment, the NOVX expression vector is a yeast expression vector.

Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec 1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54 : 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif. ), and picZ (InVitrogen Corp, San Diego, Calif.).

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

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

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e. g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1:

268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33 : 729-740; Queen and Baltimore, 1983. Cell 33 : 741-748), neuron-specific promoters (e. g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e. g. , milk whey promoter ; U. S. Pat. No. 4,873, 316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e. g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation.

That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e. g., Weintraub, et al.,"Antisense RNA as a molecular tool for genetic analysis,"Reviews-Trends in Genetics, Vol. 1 (1) 1986.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms"host cell"and "recombinant host cell"are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

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

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

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

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

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

A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e. g. , by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences, i. e., any one of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence (s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and

microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U. S. Patent Nos. 4,736, 866; 4, 870, 009 ; and 4,873, 191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e. g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e. g., the cDNA of any one of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52), 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 52, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i. e. , no longer encodes a functional protein; also referred to as a"knock out"vector).

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

homologously-recombined with the endogenous NOVX gene are selected. See, e. g., Li, et al., 1992. Cell 69: 915.

The selected cells are then injected into a blastocyst of an animal (e. g., a mouse) to form aggregation chimeras. See, e. g, Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.

113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr.

Opin. Biotechnol. 2: 823-829 ; PCT International Publication Nos. : WO 90/11354 ; WO 91/01140 ; WO 92/0968 ; and WO 93/04169.

In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, See, e. g, Lakso, et al., 1992. Proc. Natl. Acad. Sci.

USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251: 1351-1355.

If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of"double"transgenic animals, e. g, by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997. Nature 385: 810-813. In brief, a cell (e. g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e. g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e. g., the somatic cell) is isolated.

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

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

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous

preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, 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, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

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

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The

tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide ; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

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

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

The compounds can also be prepared in the form of suppositories (e. g, with conventional 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 Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U. S. Patent No. 4,522, 811.

It is especially advantageous to formulate oral or parenteral compositions in dosage r unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to

produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

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

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

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

to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.

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

Screening Assays The invention provides a method (also referred to herein as a"screening assay") for identifying modulators, 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 of the membrane-bound form of a NOVX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including : biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the"one-bead one-compound"library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e. g, Lam, 1997.

Anticancer Drug Design 12: 145.

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

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

Engl. 33: 2059 ; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e. g., Houghten, 1992.

Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U. S. Patent No. 5,223, 409), spores (Ladner, U. S. Patent 5,233, 409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89 : 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990.

Science 249 : 404406 ; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U. S. A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310 ; Ladner, U. S. Patent No. 5,233, 409. ).

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with l25I, 35S, I4C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e. g., stimulate or inhibit) the activity of the NOVX protein or

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

Determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e. g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.

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

interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.

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

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

The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton6 X-1 00, Tritone X-1 14, Thesie, Isotridecypoly (ethylene glycol ether) n, N-dodecyl--N, N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) d*methylamminiol-l-propane sulfonate (CHAPS), or 3- (3-cholamidopropyl) dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).

In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate

separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-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 of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e. g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.

In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX

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

In yet another aspect of the invention, the NOVX proteins can be used as"bait proteins"in a two-hybrid assay or three hybrid assay (see, e. g. , U. S. Patent No. 5,283, 317; Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.

Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins"or"NOVX-bp") and modulate NOVX activity. Such NOVX-binding proteins are also involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX pathway.

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

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

Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing) ; and (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 of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

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

Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e. g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes.

By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or

a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e. g, D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

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

Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.

Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN CHROMOSOMES : A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).

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

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e. g, in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through

linkage analysis (co-inheritance of physically adjacent genes), described in, e. g, Egeland, et al., 1987. Nature, 325: 783-787.

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

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

Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

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

5,272, 057).

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

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

Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.

Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ID NO : 2n-1, wherein n is an integer between 1 and 52, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.

Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e. g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity.

For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.

Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as"pharmacogenomics").

Pharmacogenomics allows for the selection of agents (e. g. , drugs) for therapeutic or

prophylactic treatment of an individual based on the genotype of the individual (e. g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e. g., drugs, compounds) on the expression or activity of NOVX in clinical trials.

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

Diagnostic Assays An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e. g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NO : 2n-l, wherein n is an integer between 1 and 52, or a portion thereof, such as an oligonucleotide of at least 15,30, 50,100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

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

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

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

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

Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression

or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e. g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a"test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e. g, serum), cell sample, or tissue.

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

The methods of the invention can also be used to detect genetic lesions in a NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX gene.

For example, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an addition of one or more nucleotides to a NOVX gene ; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene ; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (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.

In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e. g. , U. S. Patent Nos. 4,683, 195 and 4,683, 202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e. g, Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.

Proc. Natl. Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the 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 of the 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 of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

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

In an alternative embodiment, mutations in a 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 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 of the 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 /DNAheteroduplexes. See, e. g., Myers, et al., 1985. Science 230 : 1242. In general, the art technique of"mismatch cleavage"starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.

For instance, RNAIDNA duplexes can be treated with RNase and DNA/DNA hybrids

treated with Sl nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e. g, Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called"DNA mismatch repair"enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e. g, Hsu, et al., 1994. 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 of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e. g, Keen, et al., 1991. Trends Genet. 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 high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e. g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e. g, Saiki, et al., 1986.

Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

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

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which maybe conveniently used, e. g, in clinical settings to diagnose

patients exhibiting symptoms or family history of a disease or illness involving a NOVX gene.

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

However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX activity (e. g. , NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders. The disorders include but are not limited to, e. g. , those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

In conjunction with such treatment, the pharmacogenomics (i. e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e. g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.

See e. g, Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985 ; Linder, 1997.

Clin. Chem., 43: 254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example,

glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e. g., N-acetyltransferase 2 (NAT 2) and cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious, toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.

Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e. g., drugs, compounds) on the expression or activity of NOVX (e. g. , the ability to modulate aberrant cell proliferation and/or

differentiation) can be applied not only in basic drug screening, but also in clinical trials.

For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a"read out"or markers of the immune responsiveness of a particular cell.

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

In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e. g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration 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 of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample

with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i. e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i. e., to decrease the effectiveness of the agent.

Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity. The disorders include but are not limited to, e. g. , those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

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

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

Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with

Therapeutics that increase (i. e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.

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

Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity.

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

Therapeutic Methods Another aspect of the invention pertains to methods of modulating NOVX expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX

peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell.

In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e. g. , by culturing the cell with the agent) or, alternatively, in vivo (e. g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e. g. , an agent identified by a screening assay described herein), or combination of agents that modulates (e. g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.

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

Determination of the Biological Effect of the Therapeutic In various embodiment-s-of-the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.

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

Prophylactic and Therapeutic Uses of the Compositions of the Invention The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders. The disorders include but are not limited to, e. g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from 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 of the nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i. e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.

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

EXAMPLES Example A: Polynucleotide and Polypeptide Sequences, and Homology Data Example 1.

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

Table 1A. NOV1 Sequence Analysis ISEQ ID NO : 1 2566 bp NOVla, GGNCACGAGCGGCCCTCCACTCCCTGACTGTCGTGTTTGTCTCGCTCTGTGCTGAGGGCT GATG OC 108030 01 DNA CTGAGGACCTCCTTGACTCCTTCCTTAGCAACATTCTACAGGACTGCAGGCACCACCTGT GTGA ACCGGACATGAAACTGGTGTGGCCTAGTGCCAAGCTGTTGCAGGCAGCTGCAGGTGCATC TGCC Sequence CGGGCCTGTGACTCTGTCACCAGCAAGTACTGCCTTTACTGCTGGAACAGTTCCACAAGC ACAG TCAGAGCAGCCAGCGGCGGACAATCCTTGAAATGCTCCTGGGTTTCTTGAAGCTGCAGCA GAAA TGGAGCTATGAAGACAAAGATCAAAGGCCTCTGAATGGCTTCAAGGACCAGCTGTGCTCA CTGG TATTCATGGCTCTAACAGACCCCAGCACCCAGCTTCAGCTTGTTGGCATCCGTACACTGA CAGT SEQ ID NO : 2 389 aa MWat426 NOV1 MLLGFLKLQQKWSYEDKDQRPLNGFKDQLCSLVFMALTDPSTQLQLVGIRTLTVLGAQPD LLSY OC108030 01 EDLEIAVGHLYEI. SFIjKEDSQSCRVARLEASGTLARLYPVAFSSHLVPKLAEELRVGESNI. TNG DEPTQCSRHLCCLQALSAVSTHPSIVKETLPLLLQHLWQVNRGNMVAQSSDVIAVCQSLR QMAE Protein Sequence KCQQDPESCWYFHQTAIPCLLALAVQASMPEKEPSVLRKVLLEDEVLAAMVSVIGTATTH LSPE LAAQSVTHIVPLFLDGNVSFLPENSFPSRFQPFQDGSSGQRRLIALLMAFVCSLPRNVEI PQLN QLMRELLELSCCHSCPFSSTAAAKCFAGLLNKHPAGQQLDEFLQLAVDKVEAGLALGPVV VRPS LFFSG

SEQ m NO : 3 3319 bp SEQ IDNO : 3 03319bp NOVlb, TCGCGTTATGGCCGCTGCCGCGGCTGTGGAGGCGGCGGCGCCTATGGGTGCCCTATGGGG CCTC CG108O30-02 DNA GTGCACGACTTCGTCGTGGGTCAGCAAGAGGGCCCCGCTGACCAGGTGGCTGCAGATGTG AAAT CTGGCAACTATACAGTGTTACAAGTTGTGGAAGCCCTTGGGTCCTCTCTAGAGAATCCAG AACC Sequence CCGAACTCGGGCACGAGGAATCCAGCTTTTGTCACAGGTGCTACTCCACTGTCACACCTT GCTC CTGGAGAAGGAAGTGGTACACCTGATACTGTTCTATGAGAACCGGCTGAAGGACCATCAT CTTG TGATCCCATCTGTCCTGCAGGGTTTGAAGGCACTTAGCCTGTGTGTGGCCCTGCCCCCAG GGCT GGCTGTTTCTGTGCTTAAAGCCATCTTCCAGGAAGTGCATGTACAGTCCCTGCCACAGGT GGAC CGACACACAGTCTACAATATCATCACCAATTTTATGCGAACCCGGGAAGAAGAGCTAAAG AGCC TAGGAGCTGACTTCACCTTTGGCTTCATCCAGGTGATGGATGGGGAAAAGGATCCCCGTA ATCT TCTGGTGGCCTTCCGCATCGTCCATGACCTCATCTCCAGGGACTATAGCCTGGGACCCTT TGTG GAGGAGTTGTTTGAAGTGACATCCTGTTATTTCCCTATCGATTTTACCCCTCCACCTAAT GATC CCCATGGTATCCAGAGAGAAGACCTCATCCTGAGTCTTCGCGCTGTGCTGGCTTCTACAC CACG ATTTGCTGAGTTTCTGCTGCCCCTGTTGATTGAGAAAGTGGATTCTGAGGTTCTGAGTGC CAAG TTGGATTCTCTACAGACTCTGAATGCTTGCTGTGCTGTGTATGGACAGAAGGAACTGAAG GACT TCCTCCCCAGCCTTTGGGCTTCTATCCGCAGAGAGGTGTTCCAGACGGCAAGTGAGCGGG TGGA GGCAGAGGGCCTGGCGGCCCTCCACTCCCTGACTGCGTGTTTGTCTCGCTCTGTGCTGAG GGCT GATGCTGAGGACCTCCTTGACTCCTTCCTTAGCAACATTCTACAGGACTGCAGGCACCAC CTGT GTGAACCGGACATGAAACTGGTGTGGCCTAGTGCAAGCTGTTGCAGGCAGCTGCAGGTGC ATCT GCCCGGGCCTGTGACTCTGTCACCAGCAATGTACTGCCTTTACTGCTGGAACAGTTCCAC AAGC ACAGTCAGAGCAGCCAGCGGCGGGACAATCCTTGAAATGCTCCTGGGTTTCTTGAAGCTG CAGC AGAAATGGAGCTATGAAGACAAAGATCAAAGGCCTCTGAATGGCTTCAAGGACCAGCTGT GCTC ACTGGTATTCATGGCTCTAACAGACCCCAGCACCCAGCTTCAGCTTGTTGGCATCCGTAC ACTG ACAGTCTTGGGTGCCCAGCCAGATCTCCTATCTTATGAGGACTTGGAGCTGGCAGTGGGT CACC TGTACAGACTGAGCTTCCTGAAGGAGGATTCCCAGAGTTGCAGGGTGGCAGCACTGGAAG CATC AGGAACCCTGGCTGCTCTCTACCCTGTGGCCTTCAGCAGCCACCTCGTACCCAAGCTCGC TGAG GAGCTGCGTGTAGGGGAGTCAAATTTGACTAACGGAGATGAGCCCACCCAATGCTCCCGG CATC TGTGCTGTCTGCAAGCCTTGTCAGCTGTATCAACACATCCCAGCATCGTCAAGGAGACAC TGCC TCTGCTGCTGCAGCATCTCTGGCAAGTGAACAGAGGGAATATGGTTGCACAATCCAGTGA CGTT ATTGCTGTCTGTCAGAGCCTCAGACAGATGGCAGAAAAATGTCAGCAGGACCCTGAGAGT TGCT GGTATTTCCACCAGACAGCTATACCTTGCCTGCTTGCCTTGGCTGTGCAGGCCTCTATGC CAGA GAAGGAGCCCTCAGTTCTGAGAAAAGTACTATTGGAGGATGAGGTGTTGGCTGCCATGGT GTCT GTCATTGGCACTGCTACAACCCACCTGAGCCCTGAGTTAGCTGCCCAGAGTGTGACACAC ATTG TGCCCCTCTTCTTGGATGGCAACGTGTCCTTTCTGCCTGAAAACAGCTTCCCGAGCAGAT TCCA GCCATTCCAGGATGGCTCCTCAGGGCAGAGGCGGCTGATTGCACTGCTTATGGCCTTTGT CTGC TCCCTGCCTCGAAATGGCAGCAGCTGGATGAATTCCTACAGCTAGCTGTGGACAAAGTGG AGGC TGGCCTGGACTCTGGGCCCTGTCGTAGTCAGGCCTTCACTCTTCTTCTCTGGGTAACAAA GGCC CTAGTGCTCAGATACCATCCTCTCAGCTCCTGCCTTACAGCCCGGCTCATGGGCCTCCTG AGTG ACCCAGAATTAGGTCCAGCAGCAGCTGATGGCTTCTCTCTGCTCATGTCTGACTGCACTG ATGT GCTGACTCGTGCTGGCCATGCCGAAGTGCGGATCATGTTCCGCCAGCGGTTCTTCACAGA TAAT GTGCCTGCTTTGGTCCAAGACTTCCATGCTGCTCCCCAAGATGTGAAGCCAAACTACTTG AAAG GTCTTTCTCATGTACTTAACAGGCTGCCCAAGCCTGTACTCTTGCCAGAGCTGCCCACGC TTCT TTCCTTGCTGCTGGAGGCCCTGTCCTGCCCTGACTGTGTGGTGCAGCTCTCCACCCTCAG CTGC CTTCAGCCTCTTCTACTGGAAGCACCCCAAGTCATGAGTCTTCACGTGGACACCCTCGTC ACCA AGTTTCTGAACCTCAGCTCTAGCCCTTCCATGGCTGTCCGGATCGCCGCACTGCAGTGCA TGCA TGCTCTCACTCGCCTGCCCACCCCTGTGCTGCTGCCGTACAAACCACAGGTGATTCGGGC CTTA GCCAAACCCCTGGATGACAAGAAGAGACTGGTGCGCAAGGAAGCAGTGTCAGCCAGAGGG GAGT GGTTTCTGTTGGGGAGCCCTGGCAGCTGAGCCCTCAGTCCTGGCCTAGACTGTTCTGACA ATCT AACCTGGGATTACTAACTGTTGAGCCATCTTCCCCAAAGCAGGGAAACCACTGGTCTCTG ACTG CCTTTCCCACAGACACAGCACAAATGCTAGGCCTCTGTTGCATGGCTGTACAAAGAACAT AAGA GTCCATATTTCTAGTGGATTTGTARAATAAGTGTGTGTGAGACACTTGCGTTTGAAGAAA GATC TAGGGTCCTGGGTCTCTTGCATTTATATGTCAGAAAAGGGGCGATATGCTGCTGAGGGGT GAGT GCATATGAGTGTGGCCCTGAGGACCAGGGCTGGCAGATGTTGTCTACCTGCTGAG ORF Start : ATG at 8 ORF Stop : TAG at 2219 _ t} US SEQIDNO : 4 737 aa MWat81317. 6kD NOVlb, MAAAAAVEAAAPMGALWGLVHDFWGQQEGPADQVAADVKSGNYTVIQWEALGSSLENPEP RT CG108030-02 RARGIQLLSQVLLHCHTLLLEKEVVHLILFYENRLKDHHLVIPSVLQGLKALSLCVALPP GLAV SVLKAIFQEVHVQSLPQVDRHTVYNIITNFMRTREEELKSLGADFTFGFIQVMDGEKDPR NLLV ProtemSequence AFRIVHDLISRDYSLGPFVEELFEVTSCYFPIDFTPPPNDPHGIQREDLILSLRAVLAST PRFA EFLLPLLIEKVDSEVLSAKLDSLQTLNACCAVYGQKELKDFLPSLWASIRREVFQTASER VEAE GLAALHSLTACLSRSVLRADAEDLLDSFLSNILQDCRHHLCEPDMKLVWPSASCCRQLQV HLPG PVTLSPAMYCLYCWNSSTSTVRAASGGTILEMLLGFLKLQQKWSYEDKDQRPLNGFKDQL CSLV FMALTDPSTQLQLVGIRTLTVLGAQPDLLSYEDLELAVGHLYRLSFLKEDSQSCRVAALE ASGT LAALYPVAFSSHLVPKLAEELRVGESNLTNGDEPTQCSRHLCCLQALSAVSTHPSIVKET LPLL LQHLWQVNRGNMVAQSSDVIAVCQSLRQMAEKCQQDPESCWYFHQTAIPCLLALAVQASM PEKE PSVLRKVLLEDEVLAAMVSVIGTATTHLSPELAAQSVTHIVPLFLDGNVSFLPENSFPSR FQPF QDGSSGQRRLIALLMAFVCSLPRNGSSWMNSYS

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 1B.

Table 1B. Comparison of NOVla against NOVlb. NOV1a Residues/ Identities/ Protein Sequence Match Residues Similarities for the Matched Region NOVlb 1.. 313 313/313 (100%) 416.. 728 313/313 (100%) Further analysis of the NOVla protein yielded the following properties shown in Table 1C.

Table 1C. Protein Sequence Properties NOVla SignalP analysis: No Known Signal Sequence Indicated PSORT Il PSG: a new signal peptide prediction method analysis : N-region: length 11; pos. chg 2; neg. chg 0 H-region : length 3; peak value-19.72 PSG score:-24. 12 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1) : -3.97 possible cleavage site: between 40 and 41 >>> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation : 1 Tentative number of TMS (s) for the threshold 0.5 : 0 number of TMS (s).. fixed PERIPHERAL Likelihood = 0.58 (at 232) ALOM score: 0.58 (number of TMSs : 0) MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75): 4.03 Hyd Moment (95): 7.41 G content: 1 D/E content: 2 S/T content: 1 Score:-7. 36 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite : none content of basic residues: 7.2% NLS Score : -0.47 KDEL: ER retention motif in the C-terminus : none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif : type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 76.7 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 47.8 % : nuclear 26. 1 % : cytoplasmic 17. 4 % : mitochondrial 4. 3 % : vacuolar 4. 3 % : vesicles.. of secretory system » indication for CG108030-01 is nuc (k=23) A search of the NOVla protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 1D. TablelD. GeneseqResultsforNOV1a NOV1a Identities/ Geneseq Protein/Organism/Length [Patent Expect Residues/ Similarities for Identifier #, Date] Value Residues Region AAB61304 Human transcriptional regulator 1.. 378 376/378 (99%) 0. 0 protein #4-Homo sapiens, 615 aa. 1.. 378 376/378 (99%) [WO200078954-A2, 28-DEC-2000] AAU28025 Novel human secretory protein, Seq 1.. 378 376/378 (99%) 0.0 ID No 194-Homo sapiens, 666 aa. 52.. 429 376/378 (99%) [W0200166689-A2, 13-SEP-20013 AAB93270 Human protein sequence SEQ ID 1.. 378 375/378 (99%) 0.0 NO : 12306-Homo sapiens, 774 aa. 160.. 537 375/378 (99%) [EP1074617-A2,07-FEB-2001] AAM41729 Human polypeptide SEQ ID NO 6660 1.. 314 314/314 (100%) e-180 - Homo sapiens, 398 aa. 67.. 380 314/314 (100%) [W0200153312-A1, 26-JUL-2001] AAM39943 Human polypeptide SEQ ID NO 3088 1.. 314 314/314 (100%) e-180 - Homo sapiens, 383 aa. 52.. 365 314/314 (100%) [W0200153312-Al, 26-JUL-2001] In a BLAST search of public sequence databases, the NOV 1 a protein was found to have homology to the proteins shown in the BLASTP data in Table 1E.

Table lE. Public BLASTP Results for NOVA NOVA Protein Identities/ Accession Protein/Organism/Length Residues/Similarities for the Expect Number Match Matched Portion Value Residues Q96T76 MMS19-Homo sapiens (Human), 1.. 378 376/378 (99%) 0. 0 1030 aa. 416.. 793 376/378 (99%) Q9BUE2 Hypothetical protein-Homo sapiens 1.. 378 376/378 (99%) 0. 0 (Human), 692 aa (fragment). 78 455 376/378 (99%) Q9BYS9 MMS19 protein-Homo sapiens 1.. 378 376/378 (99%) 0. 0 (Human), 1030 aa. 416.. 793 376/378 (99%) Q96DF1 MUS19 (MET18 S. cerevisiae)-like 1 378 376/378 (99%) 0. 0 - Homo sapiens (Human), 666 aa. 52.. 429 376/378 (99%) Q96RK5 Transcriptionalcoactivator MMS19 1.. 378 375/378 (99%) 0. 0 ;-Homo sapiens (Human), 1030 aa. 416.. 793 375/378 (99%) Example 2.

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

Table 2A. NOV2 Sequence Analysis SEQID : 5 3058 bu NOV2a, GCCCCACAGTGAGAGGAAGGAAGGCAACAGTCGCCAGCAGCCGATGTGAAGACCGGACTC CGTG CG115907-01 DNA CGCCCCTCGCCGCCTCTGCCTGGCCACATCGATGTTGTGTCCGCCGCCTGCTCGCCCGGA TCAC GATGAAGCCCCCAAGGCCTGTCCGTACCTGCAGCAAAGTTCTCGTCCTGCTTTCACTGCT GGCC Sequence ATCCACCAGACTACTACTGCCGAAAAGAATGGCATCGACATCTACAGCCTCACCGTGGAC TCCA GGGTCTCATCCCGATTTGCCCACACGGTCGTCACCAGCCGAGTGGTCAATAGGGCCAATA CTGT GCAGGAGGCCACCTTCCAGATGGAGCTGCCCAAGAAAGCCTTCATCACCAACTTCTCCAT GATC ATCGATGGCATGACCTACCCAGGGATCATCAAGGAGAAGGCTGAAGCCCAGGCACAGTAC AGCG CAGCAGTGGCCAAGGGAAAGAGCGCTGGCCTCGTCAAGGCCACCGGGAGAAACATGGAGC AGTT CCAGGTGTCGGTCAGTGTGGCTCCCAATGCCAAGATCACCTTTGAGCTGGTCTATGAGGA GCTG CTCAAGCGGCGTTTGGGGGTGTACGAGCTGCTGCTGAAAGTGCGGCCCCAGCAGCTGGTC AAGC CCTGCAGATGGACATTCACATCTTCGAGCCCCAGGGCATCAGCTTTCTGGAGACAGAGAG CAC CTTCATGACCAACCAGCTGGTAGACGCCCTCACCACCTGGCAGAATAAGACCAAGGCTCA CATC CGGTTCAAGCCAACACTTTCCCAGCAGCAAAAGTCCCCAGAGCAGCAAGAAACAGTCCTG GACG GCAACCTCATTATCCGCTATGATGTGGACCGGGCCATCTCCGGGGGCTCCATTCAGATCG AGAA CGGCTACTTTGTACACTACTTTGCCCCCGAGGGCCTAACCACAATGCCCAAGAATGTGGT CTTT GTCATTGACAAGAGCGGCTCCATGAGTGGCAGGAAAATCCAGCAGACCCGGGAAGCCCTA ATCA AGATCCTGGATGACCTCAGCCCCAGAGACCAGTTCAACCTCATCGTCTTCAGTACAGAAG CAAC TCAGTGGAGGCCATCACTGGTGCCAGCCTCAGCCGAGAACGTGAACAAGGCCAGGAGCTT TGCT GCGGGCATCCAGGCCCTGGGAGGGACCAACATCAATGATGCAATGCTGATGGCTGTGCAG TTGC TGGACAGCAGCAACCAGGAGGAGCGGCTGCCCGAAGGGAGTGTCTCACTCATCATCCTGC TCAC CGATGGCGACCCCACTGTGGGGGAGACTAACCCCAGGAGCATCCAGAATAACGTGCGGGA AGCT GTAAGTGGCCGGTACAGCCTCTTCTGCCTGGGCTTCGGTTTCGACGTCAGCTATGCCTTC CTGG AGAAGCTGGCACTGGACAATGGCGGCCTGGCCCGGCGCATCCATGAGGACTCAGACTCTG CCCT GCAGCTCCAGGACTTCTACCAGGAAGTGGCCAACCCACTGCTGACAGCAGTGACCTTCGA GTAC CCAAGCAATGCCGTGGAGGAGGTCACTCAGAACAACTTCCGGCTCCTCTTCAAGGGCTCA GAGA TGGTGGTGGCTGGGAAGCTCCAGGACCGGGGGCCTGATGTGCTCACAGCCACAGTCAGTG GGAA GCTGCCTACACAGAACATCACTTTCCAAACGGAGTCCAGTGTGGCAGAGCAGGAGGCGGA GTTC CAGAGCCCCAAGTATATCTTCCACAACTTCATGGAGAGGCTCTGGGCATACCTGACTATC CAGC AGCTGCTGGAGCAAACTGTCTCCGCATCCGATGCTGATCAGCAGGCCCTCCGGAACCAAG CGCT GAATTTATCACTTGCCTACAGCTTTGTCACGCCTCTCACATCTATGGTAGTCACCAAACC CGAT GACCAAGAGCAGTCTCAAGTTGCTGAGAAGCCCATGGAAGGCGAAAGTAGAAACAGGAAT GTCC ACTCAGGTTCCACTTTCTTCAAATATTATCTCCAGGGAGCAAAAATACCAAAACCAGAGG CTTC CTTTTCTCCAAGAAGAGGATGGAATAGACAAGCTGGAGCTGCTGGCTCCCGGATGAATTT CAGA CCTGGGGTTCTCAGCTCCAGGCAACTTGGACTCCCAGGACCTCCTGATGTTCCTGACCAT GCTG CTTACCACCCCTTCCGCCGTCTGGCCATCTTGCCTGCTTCAGCACCACCAGCCACCTCAA ATCC TGATCCAGCTGTGTCTCGTGTCATGAATATGAAAATCGAAGAAACAACCATGACAACCCA AACC CCAGCCCCCATACAGGCTCCCTCTGCCATCCTGCCACTGCCTGGGCAGAGTGTGGAGCGG CTCT GTGTGGACCCCAGACACCGCCAGGGGCCAGTGAACCTGCTCTCAGACCCTGAGCAAGGGG TTGA GGTGACTGGCCAGTATGAGAGGGAGAAGGCTGGGTTCTCATGGATCGAAGTGACCTTCAA GAAC CCCCTGGTATGGGTTCACGCATCCCCTGAACACGTGGTGGTGACTCGGAACCGAAGAAGC TCTG CGTACAAGTGGAAGGAGACGCTATTCTCAGTGATGCCCGGCCTGAAGATGACCATGGACA AGAC GGGTCTCCTGCTGCTCAGTGACCCAGACAAAGTGACCATCGGCCTGTTGTTCTGGGATGG CCGT GGGGAGGGGCTCCGGCTCCTTCTGCGTGACACTGACCGCTTCTCCAGCCACGTTGGAGGG ACCC TTGGCCAGTTTTACCAGGAGGTGCTCTGGGGATCTCCAGCAGCATCAGATGACGGCAGAC GCAC GCTGAGGGTTCAGGGCAATGACCACTCTGCCACCAGAGAGCGCAGGCTGGATTACCAGGA GGGG CCCCCGGGAGTGGAGATTTCCTGCTGGTCTGTGGAGCTGTAGTTCTGATGGAAGGAGCTG TGCC CACCCTGTACACTTGGCTTCCCCCTGCAACTGCAGGGCCGCTTCTGGGGCCTGGACCACC ATGG GGAGGAAGAGTCCCACTCATTACAAATAAAGAAAGGTGGTGTGAGCCTGA IU top ; AG t 272 a : ATG at 130 920

gQ-"" -- NOV2a, MKPPRPVRTCSKVLVLLSLLAIHQTTTAEKNGIDIYSLTVDSRVSSRFAHTVVTSRVVNR ANTV CG115907-01 QEATFQMELPKKAFITNFSMIIDGMTYPGIIKEKAEAQAQYSAAVAKGKSAGLyKATGRN MEQF . QVSVSVAPNAKITFELVYEELLKRRLGVYELLLKVRPQQLVKHLQMDIHIFEPQGISFLE TEST q FMTNQLVDALTTWQNKTKAHIRFKPTLSQQQKSPEQQETVLDGNLIIRYDVDRAISGGSI QIEN GYFVHYFAPEGLTTMPKNWFVIDKSGSMSGRKIQQTREALIKILDDLSPRDQFNLIVFST EAT QWRPSLVPASAENVNKARSFAAGIQALGGTNINDAMLMAVQLLDSSNQEERLPEGSVSLI ILLT DGDPTVGETNPRSIQNNVREAVSGRYSLFCLGFGFDVSYAFLEKLALDNGGLARRIHEDS DSAL QLQDFYQEVANPLLTAVTFEYPSNAVEEVTQNNFRLLFKGSEMVVAGKLQDRGPDVLTAT VSGK LPTQNITFQTESSVAEQEAEFQSPKYIFHNFMERLWAYLTIQQLLEQTVSASDADQQALR NQAL NLSLAYSFVTPLTSMVVTKPDDQEQSQVAEKPMEGESRNRNVHSGSTFFKYYLQGAKIPK PEAS FSPRRGWNRQAGAAGSRMNFRPGVLSSRQLGLPGPPDVPDHAAYHPFRRLAILPASAPPA TSNP DPAVSRVt4NMKIEETTMTTQTPAPIQAPSAILPLPGQSVERLCVDPRHRQGPVNLLSDP EQGVE VTGQYEREKAGFSWIEVTFKNPLVWVHASPEHVVVTRNRRSSAYKWKETLFSVMPGLKMT MDKT GLLLLSDPDKVTIGLLFWDGRGEGLRLLLRDTDRFSSHVGGTLGQFYQEVLWGSPAASDD GRRT LRVQGNDHSATRERRLDYQEGPPGVEISCWSVBL s SEQ ID NO : 7 2797 bp NOV2b, GCCCCACAGTGAGAGGAAGGAAGGCAACAGTCGCCAGCAGCCGATGTGAAGACCGGACTC CGTG CG115907-04 DNA CGCCCCTCGCCGCCTCTGCCTGGCCACATCGATGTTGTGTCCGCCGCCTGCTCGCCCGGA TCAC GATGAAGCCCCCAAGGCCTGTCCGTACCTGCAGCAAAGTTCTCGTCCTGCTTTCACTGCT GGCC Sequence ATCCACCAGACTACTACTGCCGAAAAGAATGGCATCGACATCTACAGCCTCACCGTGGAC TCCA GGGTCTCATCCCGATTTGCCCACACGGTCGTCACCAGCCGAGTGGTCAATAGGGCCAATA CTGT GCAGGAGGCCACCTTCCAGATGGAGCTGCCCAAGAAAGCCTTCATCACCAACTTCTCCAT GATC ATCGATGGCATGACCTACCCAGGGATCATCAAGGAGAAGGCTGAAGCCCAGGCACAGTAC AGCG CAGCAGTGGCCAAGGGAAAGAGCGCTGGCCTCGTCAAGGCCACCGGGAGAAACATGGAGC AGTT CCAGGTGTCGGTCAGTGTGGCTCCCAATGCCAAGATCACCTTTGAGCTGGTCTATGAGGA GCTG CTCAAGCGGCGTTTGGGGGTGTACGAGCTGCTGCTGAAAGTGCGGCCCCAGCAGCTGGTC AAGC ACCTGCAGATGGACATTCACATCTTCGAGCCCCAGGGCATCAGCTTTCTGGAGACAGAGA GCAC CTTCATGACCAACCAGCTGGTAGACGCCCTCACCACCTGGCAGAATAAGACCAAGGCTCA CATC CGGTTCAAGCCAACACTTTCCCAGCAGCAAAAGTCCCCAGAGCAGCAAGAAACAGTCCTG GACG GCAACCTCATTATCCGCTATGATGTGGACCGGGCCATCTCCGGGGGCTCCATTCAGATCG AGAA CGGCTACTTTGTACACTACTTTGCCCCCGAGGGCCTAACCACAATGCCCAAGAATGTGGT CTTT GTCATTGACAAGAGCGGCTCCATGAGTGGCAGGAAAATCCAGCAGACCCGGGAAGCCCTA ATCA AGATCCTGGATGACCTCAGCCCCAGAGACCAGTTCAACCTCATCGTCTTCAGTACAGAAG CAAC TCAGTGGAGGCCATCACTGGTGCCAGCCTCAGCCGAGAACGTGAACAAGGCCAGGAGCTT TGCT GCGGGCATCCAGGCCCTGGGAGGGACCAACATCAATGATGCAATGCTGATGGCTGTGCAG TTGC TGGACAGCAGCAACCAGGAGGAGCGGCTGCCCGAAGGGAGTGTCTCACTCATCATCCTGC TCAC CGATGGCGACCCCACTGTGGGGGAGACTAACCCCAGGAGCATCCAGAATAACGTGCGGGA AGCT GTAAGTGGCCGGTACAGCCTCTTCTGCCTGGGCTTCGGTTTCGACGTCAGCTATGCCTTC CTGG AGAAGCTGGCACTGGACAATGGCGGCCTGGCCCGGCGCATCCATGAGGACTCAGACTCTG CCCT GCAGCTCCAGGACTTCTACCAGGAAGTGGCCAACCCACTGCTGACAGCAGTGACCTTCGA GTAC CCAAGCAATGCCGTGGAGGAGGTCACTCAGAACAACTTCCGGCTCCTCTTCAAGGGCTCA GAGA TGGTGGTGGCTGGGAAGCTCCAGGACCGGGGGCCTGATGTGCTCACAGCCACAGTCAGTG GGAA GCTGCCTACACAGAACATCACTTTCCAAACGGAGTCCAGTGTGGCAGAGCAGGAGGCGGA GTTC CAGAGCCCCAAGTATATCTTCCACAACTTCATGGAGAGGCTCTGGGCATACCTGACTATC CAGC AGCTGCTGGAGCAAACTGTCTCCGCATCCGATGCTGATCAGCAGGCCCTCCGGAACCAAG CGCT GAATTTATCACTTGCCTACAGCTTTGTCACGCCTCTCACATCTATGGTAGTCACCAAACC CGAT GACCAAGAGCAGTCTCAAGTTGCTGAGAAGCCCATGGAAGGCGAAAGTAGAAACAGGAAT GTCC ACTCAGCTGGAGCTGCTGGCTCCCGGATGAATTTCAGACCTGGGGTTCTCAGCTCCAGGC AACT TGGACTCCCAGGACCTCCTGATGTTCCTGACCATGCTGCTTACCACCCCTTCCGCCGTCT GGCC ATCTTGCCTGCTTCAGCACCACCAGCCACCTCAAATCCTGATCCAGCTGTGTCTCGTGTC ATGA ATATGCAGTATGAGAGGGAGAAGGCTGGGTTCTCATGGATCGAAGTGACCTTCAAGAACC CCCT GGTATGGGTTCACGCATCCCCTGAACACGTGGTGGTGACTCGGAACCGAAGAAGCTCTGC GTAC AAGTGGAAGGAGACGCTATTCTCAGTGATGCCCGGCCTGAAGATGACCATGGACAAGACG GGTC TCCTGCTGCTCAGTGACCCAGACAAAGTGACCATCGGCCTGTTGTTCTGGGATGGCCGTG GGGA GGGGCTCCGGCTCCTTCTGCGTGACACTGACCGCTTCTCCAGCCACGTTGGAGGGACCCT TGGC CAGTTTTACCAGGAGGTGCTCTGGGGATCTCCAGCAGCATCAGATGACGGCAGACGCACG CTGA GGGTTCAGGGCAATGACCACTCTGCCACCAGAGAGCGCAGGCTGGATTACCAGGAGGGGC CCCC GGGAGTGGAGATTTCCTGCTGGTCTGTGGAGCTGTAGTTCTGATGGAAGGAGCTGTGCCC ACCC TGTACACTTGGCTTCCCCCTGCAACTGCAGGGCCGCTTCTGGGGCCTGGACCACCATGGG GAGG GAGTCCCACTCATTACAAATAAAGAAAGGTGGTGTGAGCCTGA ORF Start: ATG at 130 ORF Stop: TAG at 2659 SEQIDNO : 8843 aa Wat93770. 6kD o NOV2b, MKPPRPVRTCSKVLVLLSLLAIHQTTTAEKNGIDIYSLTVDSRVSSRFAHTVVTSRVVNR ANTV CG115907-04 QEATFQMELPKKAFITNFSMIIDGMTYPGIIKEKAEAQAQYSAAVAKGKSAGLVKATGRN MEQF . QVSVSVAPNAKITFELVYEELLKRRLGVYELLLKVRPQQLVKEILQMDIHIFSPQGISFL ETEST Protein Sequence FMTNQLVDALTTWQNKTKAHIRFKPTLSQQQKSPEQQETVLDGNLIIRYDVDRAISGGSI QIEN GYFVHYFAPEGLTTMPKNWFVIDKSGSMSGRKIQQTREALIKILDDLSPRDQFNLIVFST EAT.. QWRPSLVPASAENVNKARSFAAGIQALGGTNINDAMLMAVQLLDSSNQEERLPEGSVSLI ILLT DGDPTVGETNPRSIQNNVREAVSGRYSLFCLGFGFDVSYAFLEKLALDNGGLARRIHEDS DSAL QLQDFYQEVANPLLTAVTFEYPSNAVEEVTQNNFRLLFKGSEMVVAGKLQDRGPDVLTAT VSGK LPTQNITFQTESSVAEQEAEFQSPKYIFHNFMERLWAYLTIQQLLEQTVSASDADQQALR NQAL NLSLAYSFVTPLTSMWTKPDDQEQSQVAEKPMEGESRNRNVHSAGAAGSRMNFRPGVLSS RQL GLPGPPDVPDHAAYHPFRRLAILPASAPPATSNPDPAVSRVMNMQYEREKAGFSWIEVTF KNPL VWVHASPEHW VTRNRRSSAYKWKETLFSVMPGLKMTMDKTGLT. LLSDPDKVTIGLLFWDGRGE GLRLLLRDTDRFSSHVGGTLGQFYQEVLWGSPAASDDGRRTLRVQGNDHSATRERRLDYQ EGPP GVEISCNSVEL

SEQ ID NO : 9 J2914 bp j NOV2C, GCCCCACAGTGAGAGGAAGGAAGGCAACAGTCGCCAGCAGCCGATGTGAAGACCGGACTC CGTG CG115907-03 DNA CGCCCCTCGCCGCCTCTGCCTGGCCACATCGATGTTGTGTCCGCCGCCTGCTCGCCCGGA TCAC GATGAAGCCCCCAAGGCCTGTCCGTACCTGCAGCAAAGTTCTCGTCCTGCTTTCACTGCT GGCC Sequence ATCCACCAGACTACTACTGCCGAAAAGAATGGCATCGACATCTACAGCCTCACCGTGGAC TCCA GGGTCTCATCCCGATTTGCCCACACGGTCGTCACCAGCCGAGTGGTCAATAGGGCCAATA CTGT GCAGGAGGCCACCTTCCAGATGGAGCTGCCCAAGAAAGCCTTCATCACCAACTTCTCCAT GATC ATCGATGGCATGACCTACCCAGGGATCATCAAGGAGAAGGCTGAAGCCCAGGCACAGTAC AGCG CAGCAGTGGCCAAGGGAAAGAGCGCTGGCCTCGTCAAGGCCACCGGGAGAAACATGGAGC AGTT CCAGGTGTCGGTCAGTGTGGCTCCCAATGCCAAGATCACCTTTGAGCTGGTCTATGAGGA GCTG CTCAAGCGGCGTTTGGGGGTGTACGAGCTGCTGCTGAAAGTGCGGCCCCAGCAGCTGGTC AAGC ACCTGCAGATGGACATTCACATCTTCGAGCCCCAGGGCATCAGCTTTCTGGAGACAGAGA GCAC CTTCATGACCAACCAGCTGGTAGACGCCCTCACCACCTGGCAGAATAAGACCAAGGCTCA CATC CGGTTCAAGCCAACACTTTCCCAGCAGCAAAAGTCCCCAGAGCAGCAAGAAACAGTCCTG GACG GCAACCTCATTATCCGCTATGATGTGGACCGGGCCATCTCCGGGGGCTCCATTCAGATCG AGAA CGGCTACTTTGTACACTACTTTGCCCCCGAGGGCCTAACCACAATGCCCAAGAATGTGGT CTTT GTCATTGACAAGAGCGGCTCCATGAGTGGCAGGAAAATCCAGCAGACCCGGGAAGCCCTA ATCA AGATCCTGGATGACCTCAGCCCCAGAGACCAGTTCAACCTCATCGTCTTCAGTACAGAAG CAAC TCAGTGGAGGCCATCACTGGTGCCAGCCTCAGCCGAGAACGTGAACAAGGCCAGGAGCTT TGCT GCGGGCATCCAGGCCCTGGGAGGGACCAACATCAATGATGCAATGCTGATGGCTGTGCAG TTGC TGGACAGCAGCAACCAGGAGGAGCGGCTGCCCGAAGGGAGTGTCTCACTCATCATCCTGC TCAC CGATGGCGACCCCACTGTGGGGGAGACTAACCCCAGGAGCATCCAGAATAACGTGCGGGA AGCT GTAAGTGGCCGGTACAGCCTCTTCTGCCTGGGCTTCGGTTTCGACGTCAGCTATGCCTTC CTGG AGAAGCTGGCACTGGACAATGGCGGCCTGGCCCGGCGCATCCATGAGGACTCAGACTCTG CCCT GCAGCTCCAGGACTTCTACCAGGAAGTGGCCAACCCACTGCTGACAGCAGTGACCTTCGA GTAC CCAAGCAATGCCGTGGAGGAGGTCACTCAGAACAACTTCCGGCTCCTCTTCAAGGGCTCA GAGA TGGTGGTGGCTGGGAAGCTCCAGGACCGGGGGCCTGATGTGCTCACAGCCACAGTCAGTG GGAA GCTGCCTACACAGAACATCACTTTCCAAACGGAGTCCAGTGTGGCAGAGCAGGAGGCGGA GTTC CAGAGCCCCAAGTATATCTTCCACAACTTCATGGAGAGGCTCTGGGCATACCTGACTATC CAGC AGCTGCTGGAGCAAACTGTCTCCGCATCCGATGCTGATCAGCAGGCCCTCCGGAACCAAG CGCT GAATTTATCACTTGCCTACAGCTTTGTCACGCCTCTCACATCTATGGTAGTCACCAAACC CGAT GACCAAGAGCAGTCTCAAGTTGCTGAGAAGCCCATGGAAGGCGAAAGTAGAAACAGGAAT GTCC ACTCAGCTGGAGCTGCTGGCTCCCGGATGAATTTCAGACCTGGGGTTCTCAGCTCCAGGC AACT TGGACTCCCAGGACCTCCTGATGTTCCTGACCATGCTGCTTACCACCCCTTCCGCCGTCT GGCC ATCTTGCCTGCTTCAGCAACACCAGCCACCTCAAATCCTGATCCAGCTGTGTCTCGTGTC ATGA ATATGTCTGCCATCCTGCCACTGCCTGGGCAGAGTGTGGAGCGGCTCTGTGTGGACCCCA GACA CCGCCAGGGGCCAGTGAACCTGCTCTCAGACCCTGAGCAAGGGGTTGAGGTGACTGGCCA GTAT GAGAGGGAGAAGGCTGGGTTCTCATGGATCGAAGTGACCTTCAAGAACCCCCTGGTATGG GTTC ACGCATCCCCTGAACACGTGGTGGTGACTCGGAACCGAAGAAGCTCTGCGTACAAGTGGA AGGA GACGCTATTCTCAGTGATGCCCGGCCTGAAGATGACCATGGACAAGACGGGTCTCCTGCT GCTC AGTGACCCAGACAAAGTGACCATCGGCCTGTTGTTCTGGGATGGCCGTGGGGAGGGGCTC CGGC TCCTTCTGCGTGACACTGACCGCTTCTCCAGCCACGTTGGAGGGACCCTTGGCCAGTTTT ACCA GGAGGTGCTCTGGGGATCTCCAGCAGCATCAGATGACGGCAGACGCACGCTGAGGGTTCA GGGC AATGACCACTCTGCCACCAGAGAGCGCAGGCTGGATTACCAGGAGGGGCCCCCGGGAGTG GAGA TTTCCTGCTGGTCTGTGGAGCTGTAGTTCTGATGGAAGGAGCTGTGCCCACCCTGTACAC TTGG CTTCCCCCTGCAACTGCAGGGCCGCTTCTGGGGCCTGGACCACCATGGGGAGGAAGAGTC CCAC TCATTACAAATAAAGAAAGGTGGTGTGAGCCTGA ORF Start : ATG at 130 ORF Stop : TAG at 2776 SEQ ID NO : 10 882 aa MW at 97921. 2kD NOV2c, MKPPRPVRTCSKVLVLLSLLAIHQTTTAEKNGIDIYSLTVDSRVSSRFAHTWTSRWVNRA NTV CG115907-03 QEATFQMELPKKAFITNFSMIIDGMTYPGIIKEKAEAQAQYSAAVAKGKSAGLVKATGRN MEQF . QVSVSVAPNAKITFELVYEELLKRRLGVYELLLKVRPQQLVKHLQMDIHIFEPQGISFLE TEST Protem Sequence FMTNQLVDALTTWQNKTKAHIRFKPTLSQQQKSPEQQETVLDGNLIIRYDVDRAISGGSI QIEN GYFVHYFAPEGLTTMPKNVVFVIDKSGSMSGRKIQQTREALIKILDDLSPRDQFNLIVFS TEAT QWRPSLVPASAENVNKARSFAAGIQALGGTNINDAMLMAVQLLDSSNQEERLPEGSVSLI ILLT DGDPTVGETNPRSIQNNVREAVSGRYSLFCLGFGFDVSYAFLEKLALDNGGLARRIHEDS DSAL QLQDFYQEVANPLLTAVTFEYPSNAVEEVTQNNFRLLFKGSEMVVAGKLQDRGPDVLTAT VSGK LPTQNITFQTESSVAEQEAEFQSPKYIFHNFMERLWAYLTIQQLLEQTVSASDADQQALR NQAL NLSLAYSFVTPLTSMVVTKPDDQEQSQVAEKPMEGESRNRNVHSAGAAGSRMNFRPGVLS SRQL GLPGPPDVPDHAAYHPFRRLAILPASATPATSNPDPAVSRVMNMSAIIXPLPGQSVERLC VDPRH RQGPVNLLSDPEQGVEVTGQYEREKAGFSWIEVTFKNPLVWVHASPEHVVVTRNRRSSAY KWKE TLFSVMPGLKMTMDKTGLLLLSDPDKVTIGLLFWDGRGEGLRLLLRDTDRFSSHVGGTLG QFYQ EVLWGSPAASDDGRRTLRVQGNDHSATRERRLDYQEGPPGVEISCWSVEL

SEQ ID NO : 11 12968 bp NOV2d, GCCCCACAGTGAGAGGAAGGAAGGCAACAGTCGCCAGCAGCCGATGTGAAGACCGGACTC CGTG CG115907-02 DNA CGCCCCTCGCCGCCTCTGCCTGGCCACATCGATGTTGTGTCCGCCGCCTGCTCGCCCGGA TCAC GATGAAGCCCCCAAGGCCTGTCCGTACCTGCAGCAAAGTTCTCGTCCTGCTTTCACTGCT GGCC Sequence ATCCACCAGACTACTACTGCCGAAAAGAATGGCATCGACATCTACAGCCTCACCGTGGAC TCCA GGGTCTCATCCCGATTTGCCCACACGGTCGTCACCAGCCGAGTGGTCAATAGGGCCAATA CTGT GCAGGAGGCCACCTTCCAGATGGAGCTGCCCAAGAAAGCCTTCATCACCAACTTCTCCAT GATC ATCGATGGCATGACCTACCCAGGGATCATCAAGGAGAAGGCTGAAGCCCAGGCACAGTAC AGCG CAGCAGTGGCCAAGGGAAAGAGCGCTGGCCTCGTCAAGGCCACCGGGAGAAACATGGAGC AGTT CCAGGTGTCGGTCAGTGTGGCTCCCAATGCCAAGATCACCTTTGAGCTGGTCTATGAGGA GCTG CTCAAGCGGCGTTTGGGGGTGTACGAGCTGCTGCTGAAAGTGCGGCCCCAGCAGCTGGTC AAGC ACCTGCAGATGGACATTCACATCTTCGAGCCCCAGGGCATCAGCTTTCTGGAGACAGAGA GCAC CTTCATGACCAACCAGCTGGTAGACGCCCTCACCACCTGGCAGAATAAGACCAAGGCTCA CATC CGGTTCAAGCCAACACTTTCCCAGCAGCAAAAGTCCCCAGAGCAGCAAGAAACAGTCCTG GACG GCAACCTCATTATCCGCTATGATGTGGACCGGGCCATCTCCGGGGGCTCCATTCAGATCG AGAA CGGCTACTTTGTACACTACTTTGCCCCCGAGGGCCTAACCACAATGCCCAAGAATGTGGT CTTT GTCATTGACAAGAGCGGCTCCATGAGTGGCAGGAAAATCCAGCAGACCCGGGAAGCCCTA ATCA AGATCCTGGATGACCTCAGCCCCAGAGACCAGTTCAACCTCATCGTCTTCAGTACAGAAG CAAC TCAGTGGAGGCCATCACTGGTGCCAGCCTCAGCCGAGAACGTGAACAAGGCCAGGAGCTT TGCT GCGGGCATCCAGGCCCTGGGAGGGACCAACATCAATGATGCAATGCTGATGGCTGTGCAG TTGC TGGACAGCAGCAACCAGGAGGAGCGGCTGCCCGAAGGGAGTGTCTCACTCATCATCCTGC TCAC CGATGGCGACCCCACTGTGGGGGAGACTAACCCCAGGAGCATCCAGAATAACGTGCGGGA AGCT GTAAGTGGCCGGTACAGCCTCTTCTGCCTGGGCTTCGGTTTCGACGTCAGCTATGCCTTC CTGG AGAAGCTGGCACTGGACAATGGCGGCCTGGCCCGGCGCATCCA. TGAGGACTCAGACTCTGCCCT GCAGCTCCAGGACTTCTACCAGGAAGTGGCCAACCCACTGCTGACAGCAGTGACCTTCGA GTAC CCAAGCAATGCCGTGGAGGAGGTCACTCAGAACAACTTCCGGCTCCTCTTCAAGGGCTCA GAGA TGGTGGTGGCTGGGAAGCTCCAGGACCGGGGGCCTGATGTGCTCACAGCCACAGTCAGTG GGAA GCTGCCTACACAGAACATCACTTTCCAAACGGAGTCCAGTGTGGCAGAGCAGGAGGCGGA GTTC CAGAGCCCCAAGTATATCTTCCACAACTTCATGGAGAGGCTCTGGGCATACCTGACTATC CAGC AGCTGCTGGAGCAAACTGTCTCCGCATCCGATGCTGATCAGCAGGCCCTCCGGAACCAAG CGCT GAATTTATCACTTGCCTACAGCTTTGTCACGCCTCTCACATCTATGGTAGTCACCAAACC CGAT GACCAAGAGCAGTCTCAAGTTGCTGAGAAGCCCATGGAAGGCGAAAGTAGAAACAGGAAT GTCC ACTCAGCTGGAGCTGCTGGCTCCCGGATGAATTTCAGACCTGGGGTTCTCAGCTCCAGGC AACT TGGACTCCCAGGACCTCCTGATGTTCCTGACCATGCTGCTTACCACCCCTTCCGCCGTCT GGCC ATCTTGCCTGCTTCAGCACCACCAGCCACCTCAAATCCTGATCCAGCTGTGTCTCGTGTC ATGA ATATGAAAATCGAAGAAACAACCATGACAACCCAAACCCCAGCCCCCATACAGGCTCCCT CTGC CATCCTGCCACTGCCTGGGCAGAGTGTGGAGCGGCTCTGTGTGGACCCCAGACACCGCCA GGGG CCAGTGAACCTGCTCTCAGACCCTGAGCAAGGGGTTGAGGTGACTGGCCAGTATGAGAGG GAGA AGGCTGGGTTCTCATGGATCGAAGTGACCTTCAAGAACCCCCTGGTATGGGTTCACGCAT CCCC TGAACACGTGGTGGTGACTCGGAACCGAAGAAGCTCTGCGTACAAGTGGAAGGAGACGCT ATTC TCAGTGATGCCCGGCCTGAAGATGACCATGGACAAGACGGGTCTCCTGCTGCTCAGTGAC CCAG ACAAAGTGACCATCGGCCTGTTGTTCTGGGATGGCCGTGGGGAGGGGCTCCGGCTCCTTC TGCG TGACACTGACCGCTTCTCCAGCCACGTTGGAGGGACCCTTGGCCAGTTTTACCAGGAGGT GCTC TGGGGATCTCCAGCAGCATCAGATGACGGCAGACGCACGCTGAGGGTTCAGGGCAATGAC CACT CTGCCACCAGAGAGCGCAGGCTGGATTACCAGGAGGGGCCCCCGGGAGTGGAGATTTCCT GCTG GTCTGTGGAGCTGTAGTTCTGATGGAAGGAGCTGTGCCCACCCTGTACACTTGGCTTCCC CCTG CAACTGCAGGGCCGCTTCTGGGGCCTGGACCACCATGGGGAGGAAGAGTCCCACTCATTA CAAA GTGGTGTGAGCCTGA ORF Start : ATG at 130 ORF Stop : TAG at 2830 SEQ m NO : 12 900 aa MW at 99856. 4kD NOV2d, MKPPRPVRTCSKVLVLLSLLAIHQTTTAEKNGIDIYSLTVDSRVSSRFAHTVVTSRVVNR ANTV CG115907-02 QEATFQMELPKKAFITNFSMIIDGMTYPGIIKEKAEAQAQYSAAVAKGKSAGLVKATGRN MEQF . QVSVSVAPNAKITFELVYEELLKRRLGVYELLLKVRPQQLVKHLQMDIHIFEPQGISFLE TEST q FMTNQLVDALTTWQNKTKAHIRFKPTLSQQQKSPEQQETVLDGNLIIRYDVDRAISGGSI QIEN GYFVHYFAPEGLTTMPKNVVFVIDKSGSMSGRKIQQTREALIKILDDLSPRDQFNLIVFS TEAT QWRPSLVPASAENVNKARSFAAGIQALGGTNINDAMLMAVQLLDSSNQEERLPEGSVSLI ILLT DGDPTVGETNPRSIQNNVREAVSGRYSLFCLGFGFDVSYAFLEKLALDNGGLARRIHEDS DSAL QLQDFYQEVANPLITAVTFEYPSNAVEEVTQNNFRLIFKGSEMWAGKLQDRGPDVLTATV SGK LPTQNITFQTESSVAEQEAEFQSPKYIFHNFMERLWAYLTIQQLLEQTVSASDADQQALR NQAL NLSLAYSFVTPLTSMVVTKPDDQEQSQVAEKPMEGESRNRNVHSAGAAGSRMNFRPGVLS SRQL GLPGPPDVPDHAAYHPFRRLAILPASAPPATSNPDPAVSRVMNMKIEETTMTTQTPAPIQ APSA ILPLPGQSVERLCVDPRHRQGPVNLLSDPEQGVEVTGQYEREKAGFSWIEVTFKNPLVWV HASP EHVVVTRNRRSSAYKWKETLFSVMPGLKMTMDKTGLLLLSDPDKVTIGLLFWDGRGEGLR LLLR DTDRFSSHVGGTLGQFYQEVLWGSPAASDDGRRTLRVQGNDHSATRERRLDYQEGPPGVE ISCW SVEL ISIML

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

Table 2B. Comparison of NOV2a against NOV2b through NOV2d. Protein Sequence NOV2a Residues/Identities/ Match Residues Similarities for the Matched Region NOV2b 1.. 930 804/930 (86%) 1.. 843 813/930 (86%) NOV2c 1.. 930 881/930 (94%) 1.. 882 881/930 (94%) NOV2d 1.. 930 900/930 (96%) 1.. 900 900/930 (96%)

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

Table 2C. Protein Sequence Properties NOV2a SingalP analysis: Cleavage site between residues 29 and 30 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 8; pos. chg 3 ; neg. chg 0 H-region: length 3; peak value 3.04 PSG score:-1. 36 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1): 0.51 possible cleavage site: between 27 and 28 >>> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0.. 5: 1 Number of TMS (s) for threshold 0.5 : 0 PERIPHERAL Likelihood = 2.01 (at 578) ALOM score:-0. 64 (number of TMSs : 0) MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75): 3.74 Hyd Moment (95): 8.90 G content: 0 D/E content: 1 S/T content: 6 Score:-1. 36 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 18 VRTJCS NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues: 10. 4% NLS Score:-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus : KPPR none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN-'Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 70.6 COIL : Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 60. 9 %: mitochondrial 8. 7 % : cytoplasmic 8. 7 % : extracellular, including cell wall 8. 7 % : peroxisomal 4. 3 % : vacuolar 4.3 Golgi 4. 3 % : nuclear » indication for CG115907-01 is mit (k=23)

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

Table 2D. Geneseq Results for NOV2a NOV2a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABB09709 Amino acid sequence of a human 1.. 930 930/930 (100%) 0. 0 PK-120 polypeptide-Homo sapiens, 1.. 930 930/930 (100%) 930 aa. [W0200212495-A1, 14-FEB-2002] ABB09708 Sequence of H4P heavy chain of inter 1.. 930 928/930 (99%) 0. 0 alpha trypsin inhibitor-Homo sapiens, 1.. 930 929/930 (99%) 930 aa. [W0200212495-A1, 14-FEB-2002] ABB09711 Sequence of H4P heavy chain of 13.. 930 663/924 (71%) 0. 0 inter-alpha-inhibitor protein-Sos sp, 12.. 921 758/924 (81%) 921 aa. [W0200212495-Al, 14-FEB-2002] ABB09707 Sequence of H4P heavy chain of 1.. 930 615/941 (65%) 0. 0 inter-alpha-inhibitor protein-Rattus 1.. 933 728/941 (77%) sp, 933 aa. [WO200212495-Al, 14-FEB-2002] ABB09706 Sequence of H4P heavy chain of 1.. 930 600/941 (63%) 0. 0 inter-alpha-inhibitorprotein-Rattus 1 932 715/941 (75%) sp, 932 aa. [W0200212495-A1, 14-FEB-20021 In a BLAST search of public sequence databases, the NOV2a protein was found to have homology to the proteins shown in the BLASTP data in Table 2E.

Table 2E. Public BLASTP Results for NOV2a Protein NOV2a Identities/ Accession Protein/OrganismLength Residues/Similarities for Expect Number Match the Matched Value Number Residues Portion Q14624 Inter-alpha-trypsin inhibitor heavy chain-1.. 930 929/930 (99%) 0. 0 H4 precursor (ITI heavy chain H4) 1.. 930 929/930 (99%) (Inter-alpha-inhibitor heavy chain 4) (Inter-alpha-trypsin inhibitor family heavy chain-related protein) (IHRP) (Plasma kallikrein sensitive glycoprotein 120) (PK-120) (GP120) (PRO1851) [Contains : GP57]-Homo sapiens (Human), 930 aa. JX0368 inter-alpha-trypsin inhibitor heavy 1.. 930 928/930 (99%) 0.0 chain-related protein precursor-human, 1.. 930 929/930 (99%) 930 aa. P79263 Inter-alpha-trypsin inhibitor heavy chain 13.. 930 663/924 (71%) 0. 0 H4 precursor (ITI heavy chain H4) 12.. 921 758/924 (81%) (Inter-alpha-inhibitor heavy chain 4) (Inter-alpha-trypsin inhibitor family heavy chain-related protein) (IHRP) (Major acute phase protein) (MAP) -Sus scrofa (Pig), 921 aa. Q9lW60 Inter alpha-trypsin inhibitor, heavy chain 4 1.. 930 625/958 (65%) 0.0 - Mus musculus (Mouse), 941 aa. 1.. 941 743/958 (77%) 054882 PK-120-Mus musculus (Mouse), 942 aa. 1.. 930 621/957 (64%) 0.0 1.. 942 740/957 (76%) PFam analysis indicates that the NOV2a protein contains the domains shown in the Table 2F.

Table2F. DomainAnalysisofNOV2a

Identities/ Pfam Domain NOV2a Match Region Similarities Expect Value for the Matched Region vwa 274.. 457 34/209 (16%) l. le-08 125/209 (60%) Example 3.

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

Table 3A. NOV3 Sequence Analysis SEQIDNO : 13 1365 bp NOV3a, ATGCTGCGGATCCTGTGCCTGGCACTCTGCAGCCTGCTGACTGGCACGCGAGCTGACCCT GGGG CG139008-01 DNA CACTGCTGCGGTTGGGCATGGACATCATGAACCGTGAGGTCCAGAGCGCCATGGATGAGA GTCA TATCCTGGAGAAGATGGCAGCCGAGGCAGGCAAGAAACAGCCAGGGATGAAACCTATCAA GGGC Sequence ATCACCAATTTGAAGGTGAAGGATGTCCAGCTGCCCGTCATCACACTGAACTTTGTACCT GGAG TGGGCATCTTCCAATGTGTGTCCACAGGCATGACCGTCACTGGCAAGAGCTTCATGGGAG GGAA CATGGAGATCATCGTGGCCCTGAACATCACAGCCACCAACCGGCTTCTGCGGGATGAGGA GACA GGCCTCCCCGTGTTCAAGAGTGAGGGCTGTGAGGTCATCCTGGTCAATGTGAAGACTAAC CTGC CTAGCAACATGCTCCCCAAGATGGTCAACAAGTTCCTGGACAGCACCCTGCACAAAGTCC TCCC TGGGCTGATGTGTCCCGCCATCGATGCAGTCCTGGTGTATGTGAACAGGAAGTGGACCAA CCTC AGTGACCCCATGCCTGTGGGCCAGATGGGCACCGTCAAATATGTTCTGATGTCCGCACCA GCCA CCACAGCCAGCTACATCCAACTGGACTTCAGTCCTGTGGTGCAGCAGCAAAAGGGCAAAA CCAT CAAGCTTGCTGATGCCGGGGAGGCCCTCACGTTCCCTGAGGGTTATGCCAAAGGCTCGTC GCAG CTGCTGCTCCCAGCCACCTTCCTCTCTGCAGAGCTTGCCCTTCTGCAGAAGTCCTTTCAT GTGA ATATCCAGGATACAATGATTGGTGAGCTGCCCCCACAAACCACCAAGACCCTGGCTCGCT TCAT TCCTGAAGTGGCTGTAGCTTATCCCAAGTCAAAGCCCTTGACGACCCAGATCAAGATAAA GAAG CCTCCCAAGGTCACTATGAAGACAGGCAAGAGCCTGCTGCACCTCCACAGCACCCTGGAG ATGT TCGCAGCTCGGTGGCGGAGCAAGGCTCCAATGTCCCTCTTTCTCCTAGAAGTGCACTTCA ATCT GAAGGTCCAGTACTCAGTGCATGAGAACCAGCTGCAGATGGCCACTTCTTTGGACAGATT ACTG AGCTTGTCCCGGAAGTCCTCATCGATTGGCAACTTCAATGAGAGGGAATTAACTGGCTTC ATCA CCAGCTATCTCGAAGAAGCCTACATCCCAGTTGTCAATGATGTGCTTCAAGTGGGGCTCC CACT CCCGGACTTTCTGGCCATGAATTACAACCTGGCTGAGCTGGACATAGTAGAGCTTGGGGG CATC ATGGAACCTGCCGACATATGA P. ORF Start : ATG at 1 ORF Stop : TGA at 1363 SNO : 14 r54aa 7-at49801. 1kD NOV3a, MLRILCLALCSLLTGTRADPGALLRLGMDIMNREVQSAMDESHILEKMAAEAGKKQPGMK PIKG CG139008-01 ITNLKVKDVQLPVITLNFVPGVGIFQCVSTGMTVTGKSFMGGNMEIIVALNITATNRLLR DEET GLPVFKSEGCEVILVNVKTNLPSNMLPIOTVNKFLDSTLHKVLPGLMCPAIDAVLVYVNR KWTNL SDPMPVGQMGTVKYVLMSAPATTASYIQLDFSPWQQQKGKTIKLADAGEALTFPEGYAKG SSQ LLLPATFLSAELALLQKSFHVNIQDTMIGELPPQTTKTLARFIPEVAVAYPKSKPLTTQI KIKK PPKVTMKTGKSLLHLHSTLEMFAARWRSKAPMSLFLLEVHFNLKVQYSVHENQLQMATSL DRLL SLSRKSSSIGNFNERELTGFITSYLEEAYIPWNDVLQVGLPLPDFLAMNYNLAELDIVEL GGI MEPADI

SEQ m NO : 15 1374 bp NOV3b, AGATCTATGCTGCGGATCCTGTGCCTGGCACTCTGCAGCCTGCTGACTGGCACGCGAGCT GACC 233028732 DNA CTGGGGCACTGCTGCGGTTGGGCATGGACATCATGAACCGTGAGGTCCAGAGCGCCATGG ATGA GAGTCATATCCTGGAGAAGATGGCAGCCGAGGCAGGCAAGAAACAGCCAGGGATGAAACC TATC Sequence AAGGGCATCACCAATTTGAAGGTGAAGGATGTCCAGCTGCCCGTCATCACACTGAACTTT GTAC CTGGAGTGGGCATCTTCCAATGTGTGTCCACAGGCATGACCGTCACTGGCAAGAGCTTCA TGGG AGGGAACATGGAGATCATCGTGGCCCTGAACATCACAGCCACCAACCGGCTTCTGCGGGA TGAG GAGACAGGCCTCCCCGTGTTCAAGAGTGAGGGCTGTGAGGTCATCCTGGTCAATGTGAAG ACTA ACCTGCCTAGCAACATGCTCCCCAAGATGGTCAACAAGTTCCTGGACAGCACCCTGCACA AAGT CCTCCCTGGGCTGATGTGTCCCGCCATCGATGCAGTCCTGGTGTATGTGAACAGGAAGTG GACC AACCTCAGTGACCCCATGCCTGTGGGCCAGATGGGCACCGTCAAATATGTTCTGATGTCC GCAC CAGCCACCACAGCCAGCTACATCCAACTGGACTTCAGTCCTGTGGTGCAGCAGCAAAAGG GCAA AACCATCAAGCTTGCTGATGCCGGGGAGGCCCTCACGTTCCCTGAGGGTTATGCCAAAGG CTCG TCGCAGCTGCTGCTCCCAGCCACCTTCCTCTCTGCAGAGCTTGCCCTTCTGCAGAAGTCC TTTC ATGTGAATATCCAGGATACAATGATTGGTGAGCTGCCCCCACAAACCACCAAGACCCTGG CTCG CTTCATTCCTGAAGTGGCTGTAGCTTATCCCAAGTCAAAGCCCTTGACGACCCAGATCAA GATA AAGAAGCCTCCCAAGGTCACTATGAAGACAGGCAAGAGCCTGCTGCACCTCCACAGCACC CTGG AGATGTTCGCAGCTCGGTGGCGGAGCAAGGCTCCAATGTCCCTCTTTCTCCTAGAAGTGC ACTT CAATCTGAAGGTCCAGTACTCAGTGCATGAGAACCAGCTGCAGATGGCCACTTCTTTGGA CAGA TTACTGAGCTTGTCCCGGAAGTCCTCATCGATTGGCAACTTCAATGAGAGGGAATTAACT GGCT TCATCACCAGCTATCTCGAAGAAGCCTACATCCCAGTTGTCAATGATGTGCTTCAAGTGG GGCT CCCACTCCCGGACTTTCTGGCCATGAATTACAACCTGGCTGAGCTGGACATAGTAGAGCT TGGG GGCATCATGGAACCTGCCGACATACTCGAG ORF Start : at 1 ORF Stop : end of sequence

S EQ ID NO : 16) 458 aa MW at 50286. 7kD NOV3b, RSMLRILCLALCSLLTGTRADPGALLRLGMDIMNREVQSAMDESHILEKMAAEAGKKQPG MKPI 233028732 Protein KGITNhKVKDVQLPVITLNFVPGVGIFQCVSTGMTVTGKSFMGGNMEIIVALNITATNRL LRDE ETGLPVFKSEGCEVILVNVKTNLPSNMLPKMVNKFLDSTLHKVLPGLMCPAIDAVLVYVN RKWT NLSDPMPVGQMGTVKYVLMSAPATTASYIQLDFSPWQQQKGKTIKLADAGEALTFPEGYA KGS SQLLLPATFLSAELALLQKSFHVNIQDTMIGELPPQTTKTLARFIPEVAVAYPKSKPLTT QIKI KKPPKVTMKTGKSLHSTLEMFAARWRSKAPMSLFLLEVHFNLKVQYSVHENQLQMATSLD R LLSLSRKSSSIGNFNERELTGFITSYLEEAYIPWNDVLQVGIPLPDFLAM7YNLAELDIV ELG GIMEPADILE s SEQ ID NO : 17 11226 bp F- NOV3c, ATGCTGCGGATCCTGTGCCTGGCACTCTGCAGCCTGCTGACTGGCACGCGAGCTGACCCT GGGG CG139008-02 DNA 'CTGCTGCGGTTGGGCATGGACATCATGAACCGTGAGGTCCAGAGCGCCATGGATGAGAG TCA TATCCTGGAGAAGATGGCAGCCGAGGCAGGCAAGAAACAGCCAGGGATGAAACCTATCAA GGGC Sequence ATCACCAATTTGAAGGTGAAGGATGTCCAGCTGCCCGTCATCACACTGAACTTTGTACCT GGAG TGGGCATCTTCCAATGTGTGTCCACAGGCATGACCGTCACTGGCAAGAGCTTCATGGGAG GGAA CATGGAGATCATCGTGGCCCTGAACATCACAGCCACCAACCGGCTTCTGCGGGATGAGGA GACA GGCCTCCCCGTGTTCAAGAGTGAGGGCTGTGAGGTCATCCTGGTCAATGTGAAGACTAAC CTGC CTAGCAACATGCTCCCCAAGATGGTCAACAAGTTCCTGGACAGCACCCTGCACAAAGTCC TCCC TGGGCTGATGTGTCCCGCCATCGATGCAGTCCTGGTGTATGTGAACAGGAAGTGGACCAA CCTC AGTGACCCCATGCCTGTGGGCCAGATGGGCACCGTCAAATATGTTCTGATGTCCGCACCA GCCA CCACAGCCAGCTACATCCAACTGGACTTCAGTCCTGTGGTGCAGCAGCAAAAGGGCAAAA CCAT CAAGCTTGCTGATGCCGGGGAGGCCCTCACGTTCCCTGAGGGTTATGCCAAAGGCTCGTC GCAG CTGCTGCTCCCAGCCACCTTCCTCTCTGCAGAGCTTGCCCTTCTGCAGAAGTCCTTTCAT GTGA ATATCCAGGATACAATGATTGGTGAGCTGCCCCCACAAACCACCAAGACCCTGGCTCGCT TCAT TCCTGAAGTGGCTGTAGCTTATCCCAAGTCAAAGCCCTTGACGACCCAGATCAAGATAAA GAAG CCTCCCAAGGTCACTATGAAGACAGGCAAGAGCCTGCTGCACCTCCACAGCACCCTGGAG ATGT TCGCAGCTCGGTGGCGGAGCAAGGCTCCAATGTCCCTCTTTCTCCTAGAAGTGCACTTCA ATCT GAAGGTCCAGTACTCAGTGCATGAGAACCAGCTGCAGATGGCCACTTCTTTGGACAGGAG AGGG AATTAACTGGCTTCATCACCAGCTATCTCGAAGAGCCTACATCCCAGTTGTCAATGATGT GCTT TCAGTGGGCT ORF Start : ATG at 1 ORF Stop : TAA at 1156 SEQ ID NO : 18 IMW at 42216. 5kD NOV3C, MRIICLALCSLLTGTRADPGALLRLGMDIMNREVQSAMDESHILEKMAAEAGKKQPGMKP IKG . CG139008-02 IT'KVQDVQLPVITLNFVPGVGIFQCVSTGMTVTGKSFMGGNMEIIVAI. NITATNRLLRDEET GLPVFKSEGCEVILVNVKMLPSNMLPKMVNKFLDSTLHKVLPGLMCPAIDAVLVYVNRKW TNL SDPMPVGQMGTVKYVLMSAPATTASYIQLDFSPWQQQKGKTIKLADAGEALTFPEGYAKG SSQ LLLPATFLSAELALLQKSFHVNIQDTMIGELPPQTTKTLARFIPEVAVAYPKSKPLTTQI KIKK PPKVTMKTGKSLLHLHSTLEMFAARWRSKAPMSLFLLEVHFNLKVQYSVHENQLQMATSL DRRG N Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 3B.

Table 3B. Comparison of NOV3a against NOV3b and NOV3c.

Protein Sequence NOV3a Residues/Identities/ Match Residues Similarities for the Matched Region NOV3b 1.. 454 454/454 (100%) 3.. 456 454/454 (100%) NOV3c 1.. 382 382/382 (100%) 1..382 382/382 (100%) Further analysis of the NOV3a protein yielded the following properties shown in Table 3C.

Table 3C. Protein Sequence Properties NOV3a SignalP analysis : Cleavage site between residues 19 and 20 PSORT II PSG: a new signal peptide prediction method' analysis : N-region: length 3 ; pos. chg 1 ; neg. chg 0 H-region: length 13 ; peak value 9.26 PSG score: 4.86 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1): 4.69 possible cleavage site: between 18 and 19 >>> Seems to have a cleavable signal peptide (1 to 18) ALOM : Klein et al's method for TM region allocation Init position for calculation: 19 Tentative number of TMS (s) for the threshold 0.5 : 2 Number of TMS (s) for threshold 0.5 : 1 INTEGRAL Likelihood =-2.87 Transmembrane 169-185 PERIPHERAL Likelihood = 1.59 (at 255) ALOM score:-2. 87 (number of TMSs : 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 9 Charge difference:-2. 0 C (0.0)-N (2.0) N >= C: N-terminal side will be inside >>> membrane topology: type la (cytoplasmic tail 186 to 454) MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 7.85 Hyd Moment (95): 8.62 G content: 1 D/E content: 1 S/T content: 3 Score:-2. 21 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 27 TRA|DP NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: none bipartite: none content of basic residues: 9. 9% NLS Score:-0. 47 KDEL : ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus: LRIL none SKL: peroxisomal targeting signal in the C-terminus: none 114 PTS2: 2nd peroxisomal targeting signal: none VAC : possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: too long tail Dileucine motif in the tail: found LL at 257 LL at 258 LL at 270 LL at 332 LL at 356 checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 89 COIL: Lupas's algorithm to detect coiled-coil regions 27 M 0.73 28 D 0.73 29 I 0.73 30 M 0.73 31 N 0.73 32 R 0.73 33 E 0.73 34 V 0.73 35 Q 0.73 36 S 0.73 37 A 0.73 38 M 0.73 39 D 0.73 40 E 0.73 41 S 0.73 42 H 0.73 43 1 0.73 44 L 0.73 45 E 0.73 46 K 0.73 47 M-0. 73 48 A 0.73 49 A 0.73 50 E 0.73 51 A 0.73 52 G 0. 73 53 K 0.73 54 K 0.73 total: 28 residues -------------------------- Final Results (k = 9/23): 44. 4 % : endoplasmic reticulum 22. 2 %: Golgi 11. 1 % : plasma membrane 11. 1 % : vesicles of secretory system 11. 1 % : extracellular, including cell wall » indication for CG139008-01 is end (k=9) A search of the NOV3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3D.

Table 3D. Geneseq Results for NOV3a NOV3a Identities/ Geneseq Protem/Organism/Length [Patent Residnes/Expect Identifier #, Date] Match Matched Region Value Residue AAM51697 Human new lipid binding protein 2-1.. 454 454/454 (100%) 0. 0 Homo sapiens, 454 aa. 1.. 454 454/454 (100%) [W0200179493-A1, 25-OCT-2001] AAB47337 FCTR14-Homo sapiens, 454 aa. 1.. 454 454/454 (100%) 0. 0 [W0200146231-A2, 28-JUN-2001] 1.. 454 454/454 (100%) ABB08898 Human BPIL 325-3 SEQ ID NO 35-1.. 454 454/454 (100%) 0. 0 Homo sapiens, 454 aa. 1.. 454 454/454 (100%) [W0200136478-A2, 25-MAY-2001] ABB08899 Human BPIL 325-4 SEQ ID NO 45-1.. 444 442/444 (99%) 0. 0 Homo sapiens, 453 aa. 1.. 443 443/444 (99%) [WO200136478-A2, 25-MAY-2001] ABG10878 Novel human diagnostic protein 1.. 454 441/455 (96%) 0. 0 #10869-Homo sapiens, 455 aa. 1.. 455 444/455 (96%) [W0200175067-A2, 11-OCT-2001] In a BLAST search of public sequence databases, the NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3E.

Table 3E. Public BLASTP Results for NOV3a Protein NOV3a Identities/ Accession Protein/Organisn/Length Residues/Similarities for Expect Number Match the Matched Value Residues Portion CAC50178 Sequence 27 from Patent W00146231-1.. 454 454/454 (100%) 0. 0 Homo sapiens (Human), 454 aa. 1.. 454 454/454 (100%) Q8NFQ5 Bactericidal/permeability-increasing 1.. 444 442/444 (99%) 0. 0 protein-like 3-Homo sapiens (Human), 1.. 443 443/444 (99%) 453 aa. Q05704 Potential ligand-binding protein-Rattus 59.. 444 130/395 (32%) 3e-57 rattus (Black rat), 470 aa (fragment). 73.. 463 229/395 (57%) CAD12150 Sequence 3 from Patent W00179269-59.. 444 125/394 (31%) 2e-52 Homo sapiens (Human), 637 aa. 241.. 630 222/394 (55%) CAC18887 DJ726C3. 5 (ortholog of potential 59.. 444 125/394 (31%) 2e-52 ligand binding protein RY2G5 (Rat))-73.. 462 222/394 (55%) Homo sapiens (Human), 469 aa (fragment).

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

Table 3F. Domain Analysis of NOV3a Identities/ Pfam Domain NOV3a Match Region Similarities Expect Value for the Matched Region LBPBPICETPC 291.. 429 41/140 (29%) 1. 3e-11 95/140 (68%) Example 4.

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

Table 4A. NOV4 Sequence Analysis u SEQ ID NO : 19 765 bp NOV4a, TCGCCCTTCATGGTGATGTCCCAGGCCACCTACACGTTCCTCACGTGCTTCGCCGGCTTC TGGC 001477 01 DNA TCATCTGGGGTCTCATCGTCCTGCTCTGCTGCTTCTGCAGCTTCCTGCGCCGCCGCCTCA AACG GCGCCAGGAGGAGCGACTGCGCGAGCAGAACCTGCGCGCCCTAGAGCTGGAGCCCCTCGA ACTC Sequence GAGGGCAGTCTGGCCGGSAGCCCCCCGGGCCTGGCGCCGCCGCAGCCACCACCACACCGT AGCC GCCTGGAGGCGCCGGCTCACGCGCACTCGCATCCGCACGTGCACGTGCACCCGCCGCCTA CGCA CCTGTCGGTGCCGCCACGGCCCTGGAGCTACCCGCGCCAAGCGGAATCGGACATGTCCAA ACCA CCGTGTTACGAAGAGGCGGTGCTGATGGCAGAGCCGCCGCCGCCCTATAGCGAGGTGCTC ACGG ACACGCGCGGCCTCTACCGCAAGATCGTCACGCCCTTCCTGAGTCGCCGCGACAGCGCGG AGAA GCAGGAGCAGCCGCCTCCCAGCTACAAGCCGCTCTTCCTGGACCGGGGCTACACCTCGGC GCTG CACCTGCCCAGCGCCCCTCGGCCCGCGCCGCCCTGCCCAGCCCTCTGCCTGCAGGCCGAC CGTG GCCGCCGGGTCTTCCCCAGCTGGACCGACTCAGAGCTCAGCAGCCGCGAGCCCCTGGAGC ACGG AGCTTGGCGTCTGCCGGTCTCCATCCCCTTGTTCGGGAGGACTACAGCCGTATAGAGGGG C ORF Start : ATG at 10 ORF Stop : TAG at 757 SEQ m NO : 20 lT4 ; ; O. lkD NOV4a, MVMSQATYTFLTCFAGFWLIWGLIVLLCCFCSFLRRRLKRRQEERLREQNLRALELEPLE LEGS CG14S877-O LAGSPPGLAPPQPPPHRSRLEAPAHAHSHPHVHVHPPPTHLSVPPRPWSYPRQAESDMSK PPCY Rtn 1 X C 0r7 1 LAGsppGLAppQpppHRsRLEApAmsHpEpppTHLsvppRpwsypRQAEsDMsKppcy EEAVILMAEPPPPYSEVLTDTRGLYRKIVTPFLSRRDSAEKQEQPPPSYKPLFLDRGYTS ALHLP hotmSequmce H S Further analysis of the NOV4a protein yielded the following properties shown in Table 4B.

Table 4B. Protein Sequence Properties NOV4a Signal ? analysis : Cleavage site between residues 35 and 36 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 0; pos. chg 0; neg. chg 0 H-region: length 34; peak value 11.41 PSG score: 7.01 GvH : von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1): 0.65 possible cleavage site: between 31 and 32 >>> Seems to have a cleavable signal peptide (1 to 31) ALOM: Klein et al's method for TM region allocation Init position for calculation: 32 Tentative number of TMS (s) for the threshold 0.5 : 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 8.33 (at 233) ALOM score: 8.33 (number of TMSs : 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 15 Charge difference: 4.0 C (5.0)-N (1.0) C > N: C-terminal side will be inside >>>Caution : Inconsistent mbop result with signal peptide MITDISC: discrimination of mitochondrial targeting seq R content : 5 Hyd Moment (75): 4.61 Hyd Moment (95): 3.22 G content: 2 D/E content : 1 S/T content: 5 Score:-0. 42 Gavel : indication of cleavage sites for mitochondrial preseq R-2 motif at 51 RRQ|EE NUCDISC: discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 11. 6% NLS Score : -0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL : peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: nuclear Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 43.5 % : mitochondrial 43. 5 % : nuclear 13. 0 % : extracellular, including cell wall » indication for CG145877-01 is mit (k=23) A search of the NOV4a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4C.

Table 4C. Geneseq Results for NOV4a NOV4a Identities/ Geneseq Protein/Organisrn/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABG08144 Novel human diagnostic protein #8135 12.. 77 35/66 (53%) 3e-10 - Homo sapiens, 436 aa. 312.. 376 38/66 (57%) [W0200175067-A2, 11-OCT-2001] ABG27250 Novel human diagnostic protein 65.. 202 43/140 (30%) 4e-06 #27241-Homo sapiens, 406 aa. 23.. 150 54/140 (37%) [WO200175067-A2, 11-OCT-2001] AAG67355 Amino acid sequence of a rat N-WASP 66.. 140 32/80 (40%) le-05 protein-Rattus rattus, 501 aa. 294.. 373 36/80 (45%) [WO200144292-A2, 21-JUN-2001] AAM52319 Rat N-WASP protein-Rattus rattus, 66.. 140 32/80 (40O/O) le-05 501 aa. [W0200171356-A2, 294.. 373 36/80 (45%) 27-SEP-2001] AAW46890 Rat Neural-Wiskott-Aldrich syndrome 66.. 140 32/80 (40%) 1 e-05 protein-Rattus sp, 501 aa. 294.. 373 36/80 (45%) [JP10072494-A, 17-MAR-1998] In a BLAST search of public sequence databases, the NOV4a protein was found to have homology to the proteins shown in the BLASTP data in Table 4D.

Table 4D. Public BLASTP Results for NOV4a Protein NOV4a Identities/ Protein Residues/Similarities for Expect Number Match the Matched Value Residues Portion Q9BTA7 Hypothetical protein-Homo sapiens 1.. 249 245/253 (96%) e-147 (Human), 253 aa. 1.. 253 246/253 (96%) Q8TB68 Hypothetical protein MGC10772-1.. 249 248/274 (90%) e-146 Homo sapiens (Human), 274 aa. 1.. 274 248/274 (90%) Q8WU53 Similar to hypothetical protein 1.. 249 247/274 (90%) e-145 MGC10772-Homo sapiens (Human), 1.. 274 247/274 (90%) 274 aa. P13983 Extensin precursor (Cell wall 69.. 202 38/134 (28%) 2e-06 hydroxyproline-rich glycoprotein)-302.. 414 49/134 (36%) Nicotiana tabacum (Common tobacco), 620 aa. 620 aa. Q94ES6 Nodule extensin-Pisum sativum 64 203 40/144 (27%) 7e-06 (Garden pea), 181 aa. 31.. 169 53/144 (36%)

Example 5.

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

Table 5A. NOV5 Sequence Analysis SEQI NO : 21 11126 bp NOV5a, GGCACGAGGCCCGCGCGCGGGGGCGCCCAGGCCACTGGGCTCCGCGGAGCCAGCGAGAGG TCTG CG151161-02 DNA CGCGGAGTCTGAGCGGCGCTCGTCCCGTCCCAAGGCCGACGCCAGCACGCCGTCAT6GCC CCCG CAGCGGCGACGGGGGGCAGCACCCTGCCCAGTGGCTTCTCGGTCTTCACCACCTTGCCCG ACTT Sequence GCTCTTCATCTTTGAGTTTATCTTCGGGGGCCTGGTGTGGATCCTGGTGGCCTCCTCCCT GGTG CCCTGGCCCCTGGTCCAGGGCTGGGTGATGTTCGTGTCTGTGTTCTGCTTCGTGGCCACC ACCA CCTTGATCATCCTGTACATAATTGGAGCCCACGGTGGAGAGACTTCCTGGGTCACCTTGG ACGC AGCCTACCACTGCACCGCTGCCCTCTTTTACCTCAGCGCCTCAGTCCTGGAGGCCCTGGC CACC ATCACGATGCAAGACGGCTTCACCTACAGGCACTACCATGAAAACATTGCTGCCGTGGTG TTCT CCTACATAGCCACTCTGCTCTACGTGGTCCATGCGGTGTTCTCTTTAATCAGATGGAAGT CTTC ATAAAGCCGCAGTAGAACTTGAGCTGAAAACCCAGATGGTGTTAACTGGCCGCCCCACTT TCCG GCATAACTTTTTAGAAAACAGAAATGCCCTTGATGGTGGAAAAAAGAAAACAACCACCCC CCCA CTGCCCAAAAAAAAAAGCCCTGCCCTGTTGCTCGTGGGTGCTGTGTTTACTCTCCCGTGT GCCT TCGCGTCCGGGTTGGGAGCTTGCTGTGTCTAACCTCCAACTGCTGTGCTGTCTGCTAGGG TCAC CTCCTGTTTGTGAAAGGGGACCTTCTTGTTCGGGGGTGGGAAGTGGCGACCGTGACCTGA GAAG GAAAGAAAGATCCTCTGCTGACCCCTGGAGCAGCTCTCGAGAACTACCTGTTGGTATTGT CCAC AAGCTCTCCCCAGCGCCCCATCTTGTGCCATGTTTTAAGTCTTCATGGATGTTCTGCATG TCAT GGGGACTAAAACTCACCCAACAGATCTTTCCAGAGGTCCATGGTGGAAGACGATAACCCT GTGA AATACTTTATAAAATGTCTTAATGTTCAAAAAAAAAAA ORF Start : ATG at 119 ORF Stop : TAA at 578 B= n SEQBDNO : 22 J3 JMWa " NOV5a MATGGSTLPSGFSVFTTLPDLLFIFEFIFGGLVWILVASSLVPWPLVQGWVMFVSVFCFV 51i61-02 ATTTLIILYIIGAlIGGETSWVTLDAAYHCTAALFYLSASVL13ALATITMQDGFTYRHY HENIAA Protein WFSYIATLLYWK&VFSLIRWKSS ProteinSequence

SEQ m NO : 23 464 bp NOV5b GGCACGAGGCCCGCGCGCGGGGGCGCCCAGGCCACTGGGCTCCGCGGAGCCAGCGAGAGG TCTG CG151161-O1 DNA CGCGGAGTCTGAGCGGCGCTCGTCCCGTCCCAAGGCCGACGCCAGCACGCCGTCATGGCC CCCG CAGCGGCGACGGGGGGCAGCACCCTGCCCAGTGGCTTCTCGGTCTTCACCACCTTGCCCG ACTT Sequence GCTCTTCATCTTTGAGTTTGACGCAACCTACCACTGCACCGCTGCCCTCTTTTACCTCAG CGCC TCAGTCCTGGAGGCCCTGGCCACCATCACGATGCAAGACGGCTTCACCTACAGGCACTAC CATG AAAACATTGCTGCCGTGGTGTTCTCCTACATAGCCACTCTGCTCTACGTGGTCCATGCGG TGTT CTCTTTAATCAGATGGAAGTCTTCATAAAGCCGCAGTAGAACTTGAGCTGAAAACCCAGA TGGT GTTAACTGGCCGCCCC ORF Start : ATG at 119 ORF Stop : TAA at 410

SEQ ID NO : 24 10651. IkD NOV5b, MTSTLPSGFSVFTTLPDLLFIFEFDATYHCTAALFYLSASVLEALATITMQDGFTYR CG151161-01 HYHENIAAWFSYIATLLYWHAVFSLIRWKSS Protein Sequence Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 5B.

Table 5B. Comparison of NOV5a against NOV5b. Protein Sequence NOVSa Residues/Identities/ Match Residues Similarities for the Matched Region NOV5b 1 153 94/153 (61%) 1.. 97 94/153 (61%)

Further analysis of the NOV5a protein yielded the following properties shown in Table 5C.

Table 5C. Protein Sequence Properties NOVSa Signal ? analysis : Cleavage site between residues 67 and 68 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 0; pos. chg 0; neg. chg 0 H-region: length 23; peak value 8.79 PSG score: 4.39 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-1. 89 possible cleavage site: between 53 and 54 >>> Seems to have a cleavable signal peptide (1 to 53) ALOM: Klein et al's method for TM region allocation Init position for calculation: 54 Tentative number of TMS (s) for the threshold 0.5 : 3 INTEGRAL Likelihood =-7.64 Transmembrane 55-71 INTEGRAL Likelihood =-0.90 Transmembrane 95-111 INTEGRAL Likelihood =-4.30 Transmembrane 129-145 PERIPHERAL Likelihood = 10.08 (at 74) ALOM score:-7. 64 (number of TMSs : 3) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 26 Charge difference: 0.0 C (-1. 0)-N (-1. 0) N >= C: N-terminal side will be inside >>i membrane topology: type 3a MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75): 2.25 Hyd Moment (95): 2.24 G content: 3 D/E content: 1 S/T content: 7 Score:-5. 30 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: none bipartite: none content of basic residues: 2.0% NLS Score:-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: KKXX-like motif in the C-terminus: RWKS SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif : none Actinin-type actin-binding motif: type 1: none type 2: none NMYR : N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23): 66. 7 % : endoplasmic reticulum 33. 3 % : mitochondrial # indication for CG151161-02 is end (k=9)

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

Table 5D. Geneseq Results for NOV5a Geneseq Protein/Organism/Length [Patent #, NOVSa Identities/Expect Identifier Value Match the Matched Residues Region ABB50292 T cell differentiation protein Mal 1.. 153 153/153 (100%) 5e-85 ovarian tumour marker protein, #74- 1.. 153 153/153 (100%) Homo sapiens, 153 aa. [WO200175177-A2, 11-OCT-2001] AAP80929 Sequence of human T-cell protein 1.. 153 150/153 (98%) 3e-82 designated MAL-Homo sapiens, 153 1.. 153 151/153 (98%) aa. [WO8807549-A, 06-OCT-1988] AAP81879 Sequence of full-length human T-cell 1.. 153 150/153 (98%) 3e-82 protein derived from mature T cells-1 153 151/153 (98%) Homo sapiens, 153 aa. [W08807549-A, 06-OCT-1988] AAU85517 Clone #18966 of lung tumour protein- 3.. 143 60/141 (42%) 8e-28 Homo sapiens, 148 aa. 2.. 142 91/141 (63%) [WO200204514-A2, 17-JAN-2002] AAB76862 Human lung tumour protein related 3 143 60/141 (42%) 8e-28 protein sequence SEQ ID NO : 338-2.. 142 91/141 (63%) Homo sapiens, 148 aa. [W0200100828-A2, 04-JAN-2001] In a BLAST search of public sequence databases, the NOV5a protein was found to have homology to the proteins shown in the BLASTP data in Table 5E.

Table 5E. Public BLASTP Results for NOV5a Protein NOV5a Identities/ .. . <. rr Residues/Similarities for Expect Accession Residues/Similarities for Expect Number Match the Matched Value Number .. .. Residues Portion P21145 Myelin and lymphocyte protein 1 153 153/153 (100%) le-84 (T-lymphocyte maturation-associated 1.. 153 153/153 (100%) protein)-Homo sapiens (Human), 153 aa. Q64349 Myelin and lymphocyte protein 1.. 153 136/153 (88%) 2e-77 (T-lymphocyte maturation-associated 1.. 153 147/153 (95%) protein) (17 kDa myelin vesicular protein) (MVP17) (NS 3)-Rattus norvegicus (Rat), 153 aa. 009198 Myelin and lymphocyte protein 1.. 153 133/153 (86%) 2e-75 (T-lymphocyte maturation-associated 1.. 153 145/153 (93%) protein)-Mus musculus (Mouse), 153 aa. Q28296 Myelin and lymphobyte protein 1.. 153 135/153 (88%) 2e-75 (T-lymphocyte maturation-associated 1.. 153 146/153 (95%) protein) (VIP17 proteolipid) -Canis familiars (Dog), 153 aa. Q9D2R2 Myelin and lymphocyte protein; T-cell 1.. 153 84/153 (54%) 4e-34 differentiation protein-Mus musculus 1.. 97 91/153 (58%) (Mouse,) 97 aa.

Example 6.

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

TaMe 6A. NOV6 Sequence Analysis SEQ ID NO : 25 14901 bp I NOV6a, CCGCTGCGGGCTCGGGCGCCGCAGCGCGCCGGCCCGAGCCCCTGGACGAGGCCCACGGAG CCGC A TCGCCCCGACCCAGCCGCCCGATGTCCTCAAAATGGAGGCAGCGGGGGCGGCGGCGTGAA GAAA GCGGCGCTGTGGGCGCGGGAGTAGGGGCCCGGGCGGAGGCGGTGGCGGGATGGGGCTGCT GCTC Sequence ATGATCCTGGCGTCGGCCGTGCTGGGTTCCTTCCTCACGCTCCTCGCCCAGTTCTTCCTG CTGT ACCGCAGACAGCCCGAGCCGCCGGCGGACGAGGCCGCCCGCGCGGGCGAGGGCTTCCGCT ACAT CAAGCCAGTGCCGGGCCTGCTCCTAAGGGAGTACCTTTATGGCGGCGGCCGGGATGAGGA GCCC TCCGGAGCGGCCCCTGAGGGCGGCGCGACCCCCACCGCGGCCCCCGAGACCCCCGCCCCG CCGA CGCGGGAGACTTGCTACTTCCTCAACGCCACCATCCTATTCCTGTTCCGGGAGTTGCGGG ACAC CGCGCTGACCCGCCGCTGGGTCACCAAGAAGATCAAGGTGGAGTTCGAGGAGCTGCTGCA GACC AAGACGGCCGGGCGCCTGCTGGAGGGGCTGAGCCTGCGGGACGTGTTCCTGGGCGAGACG GTGC CCTTCATCAAGACCATCCGGCTCGTGCGGCCAGTCGTGCCCTCGGCCACCGGGGAGCCCG ATGG CCCTGAAGGGGAGGCGCTGCCCGCCGCCTGCCCCGAGGAGCTGGCCTTCGAGGCGGAGGT GGAG TACAACGGGGGCTTCCACCTGGCCATCGACGTGGACCTGGTCTTCGGCAAGTCCGCCTAC TTGT TTGTCAAGCTGTCCCGCGTGGTGGGAAGGCTGCGCTTGGTCTTTACGCGCGTGCCCTTCA CCCA CTGGTTCTTCTCCTTCGTGGAAGACCCGCTGATCGACTTCGAGGTGCGCTCCCAGTTTGA AGGG CGGCCCATGCCCCAGCTCACCTCCATCATCGTCAACCAGCTCAAGAAGATCATCAAGCGC AAGC ACACCCTACCGAATTACAAGATCAGGTTTAAGCCGTTTTTTCCATACCAGACCTTGCAAG GATT TGAAGAAGATGAAGAGCATATCCATATACAACAATGGGCACTTACTGAAGGCCGTCTTAA AGTT ACGTTGTTAGAATGTAGCAGGTTACTCATTTTTGGATCCTATGACAGAGAGGCAAATGTT CATT GCACACTTGAGTTAAGCAGTAGTGTTTGGGAAGAAAAACAGAGGAGTTCTATTAAGACGG TTGA P. TTAATAAAAGGAAATTTACAAAGTGTTGGACTTACACTTCGTCTTGTCCAGTCAACTGAT GGG TATGCTGGGCACGTCATCATTGAAACTGTGGCTCCAAACTCGCCTGCTGCAATTGCAGAT CTTC AGCGGGGAGATCGACTTATCGCCATTGGAGGTGTGAAAATCACATCAACACTGCAAGTGT TGAA GCTTATCAAGCAGGCTGGTGACCGAGTCCTGGTGTACTATGAAAGGCCTGTTGGCCAGAG TAAT CAAGGTGCAGTGCTGCAAGATAACTTTGGCCAGTTGGAAGAAAACTTTTTGTCAAGCTCA TGCC AATCGGGTTATGAAGAGGAAGCTGCCGGGTTGACAGTAGATACTGAAAGTAGAGAGCTGG ATTC TGAATTTGAAGACTTGGCAAGTGATGTCAGAGCACAAAATGAGTTCAAAGATGAGGCACA ATCA TTAAGTCATAGTCCCAAACGTGTTCCAACAACACTTTCTATTAAACCCCTTGGAGCTATA TCAC CAGTTTTAAACCGTAAATTAGCTGTAGGAAGTCACCCACTACCACCGAAAATTCAGTCCA AAGA TGGAAATAAACCTCCACCCCTAAAAACTTCTGAGATAACAGACCCAGCACAAGTGTCAAA ACCA ACCCAAGGATCTGCTTTCAAACCACCTGTGCCACCACGACCACAAGCGAAAGTTCCTTTG CCTT CCGCCGATGCTCCAAATCAGGCAGAACCAGATGTTCTCGTTGAAAAGCCAGAGAAGGTGG TGCC ACCTCCTCTTGTAGATAAATCTGCTGAAAAGCAAGCAAAAAATGTGGATGCCATAGACGA TGCA GCTGCACCTAAGCAATTTTTAGCAAAGCAAGAAGTGGCCAAAGATGTCACTTCAGAAACT TCCT GCCCTACTAAGGACAGTTCGGACGACCGTCAAACATGGGAATCATCAGAAATTCTTTATC GTAA TAAGCTAGGAAAATGGACAAGAACCAGAGCATCCTGTTTGTTTGACATAGAAGCCTGTCA CAGG TACTTAAACATTGCATTGTGGTGCAGGGATCCTTTCAAGTTGGGAGGTCTCATCTGTTTG GGGC ATGTTAGTTTAAAACTTGAAGATGTGGCTTTAGGATGCCTAGCTACATCAAACACGGAAT ACCT TTCCAAATTGAGACTGGAAGCCCCCTCACCTAAGGCTATAGTCACTAGAACCGCACTACG CAAT CTGAGTATGCAAAAGGGATTCAATGACAAATTTTGCTATGGTGACATTACTATTCACTTC AAAT ATTTGAAAGAAGGAGAATCAGACCACCATGTAGTTACTAACGTAGAAAAAGAAAAAGAAC CCCA TTTGGTTGAAGAAGTTTCTGTTCTCCCTAAAGAGGAGCAATTTGTTGGACAGATGGGTTT AACA GAAAACAAACACAGTTTTCAGGATACTCAGTTCCAGAACCCAACATGGTGTGACTACTGT AAGA AAAAAGTTTGGACTAAAGCAGCTTCCCAGTGTATGTTTTGTGCTTATGTTTGCCATAAAA AATG TCAAGAAAAGTGTCTAGCTGAGACTTCTGTTTGTGGAGCAACTGATAGGCGAATAGACAG GACA CTGAAAAACCTTAGGCTGGAAGGACAGGAAACCCTCTTAGGCCTGCCTCCTCGTGTTGAT GCTG AAGCTAGCAAGTCAGTCAATAAAACAACAGGTTTGACAAGGCATATTATCAATACTAGTT CTCG TTTATTAAATTTGCGTCAAGTCTCTAAAACTCGCCTTTCTGAACCAGGAACCGATCTCGT AGAA CCTTCACCAAAACACACACCCAACACGTCAGACAACGAAGGCAGTGACACGGAGGTCTGT GGTC CAAACAGTCCTTCTAAACGGGGAAACAGCACAGGAATAAAGTTAGTGAGAAAAGAGGGTG GTCT GGATGACAGTGTTTTCATTGCAGTTAAAGAAATTGGTCGTGATCTGTACAGGGGCTTGCC TACA GAGGAAAGGATCCAGAAACTAGAGTTCATGTTGGATAAGCTACAGAATGAAATTGATCAG GAGT TGGAACACAATAATTCCCTTGTTAGAGAAGAAAAAGAGACAACTGATACAAGGAAAAAAT CACT TCTTTCTGCTGCCTTAGCTAAATCAGGTGAAAGGCTACAAGCTCTAACACTTCTTATGAT TCAC TACAGAGCAGGCATTGAAGATATAGAAACTTTAGAAAGTCTGTCTTTAGACCAGCACTCC AAAA AAATAAGCAAGTACACAGATGATACAGAAGAAGACCTTGATAATGAAATAAGCCAACTAA TAGA CTCTCAGCCATTCAGCAGCATATCAGATGACTTATTTGGCCCATCCGAGTCTGTGTAGCA GACA GGTCTATTTAAACTTTCAAATGAACAGGGTAAAGTTGCATCTAAAGTACCACAGATACAA CCAT GTTTAAATCCTCGTATGCACTCTGGCCTGCTTCTCCAGTTACTTGCTTGTGTAAGAACAA AAAT GAGAAAGGTTGTTTTCCAGTAAAAACATGACCAGCTTACTAATTGGTTGTTTTGGATTGC ATTT ATAGCTATGCTTTTTTGGGTTTATACTGGGAATTTATTTTTACTAAATTATTTAACTTTT CTAA TTATGTAATTATGTAAGCTAGCTTTTCATGTTTATGTATGTATGGTGTCCCCTTGTGTTA TTTT TCTTCCTCTTGGTTTTTGAATTAGTGTTAAATAGAATACTGTCTGGATTCTTAAAATATT TTCA TTTCCATCATGGTTATAACAAATTTGCTGCATGCCCAAACTGACAACAGCAATCACTGAG GGAA CAGGTTTTGAATCTTTCTTTTGTGTTATGAAGTTTATCGTCTCTACTTGCTTGAGATTTT TGTT ATTTTGGGGGTTTGGGGGTGCTTTTTGTTTTGTTTTTGCCAAATGTAACATGAAAGCAGA TGCT GCAGCTTTAGTCTGTTATGCTGATTTAGTAAAAAAAAATTTTTTACATATATTGCTTGCT TTCG ATGCTTCTGTGAAATTTTTTTCTAAAGCTTTTGTGCAGCTGTATGGTAAAAATATGGTGA TTAA TTTGAAGAGCTTACATTGAAAGACAATGTAATAGGAAATAAATGTAGATTGCAGTTGGTC AAGA ATTTTGTAGAGAGGATAACAAGACTTAATTACTGAAAAACAGTAACATAGCATTTTGAAA TATG ATCTTTTAAAATATTGATGCTTTCCTTTTAAATGGAAATTTAAATTTTATAATTAAAAGT TTAA ACATTTATGATAATTTTCCTCATCAGTTCTCCCATAGGAAATAAAGCATGTGAAAGGGTA TTTA AAGTTTTGGAGGACTCTTTTTAAAATGACTGTGTTGATAACTAGTTTGGGCTGGTTTTGT TTTA GAAAAAACATTTTCATGTAGGAGTATTCTGTGAAGGAAAGGAATCATGCAAAATATACTT TTTG CTTTGGCGTCTTACAGTTGTAAAGGAATGGTGATCATTCTGAATACTTCTGTAGTGAGTA TTCA T ORF Start : ATG at 178 LUK/snp lAli fl. 619 SEQ ID NO : 26 11154 aa IMW at 128561. 7kD NOV6a, MGLLI, MILASAVLGSFLTLLAQFFLLYRRQPEPPADEAARAGEGFRYIKPVPGLLLREYLYGGG CG155653-01 DEEPSGAAPEGGATPTAAPETPAPPTRETCYFLNATILFLFRELRDTALTRRWVTKKIKV EFE ELLQTKTAGRLLEGLSLRDVFLGETVPFIKTIRLVRPVVPSATGEPDGPEGEA, LPAACPEELAF E EAEVEYNGGFHLAIDVDLVFGKSAYLFVKLSRWGRLRLVFTRVPFTHWFFSFVEDPLIDF EVR SQFEGRPMPQLTSIIVNQLKKIIKRKHTLPNYKIRFKPFFPYQTLQGFEEDEEHIHIQQW ALTE GRLKVTLLECSRLLIFGSYDREANVHCTLELSSSVWEEKQRSSIKTVELIKGNLQSVGLT LRLV QSTDGYAGHVIIETVAPNSPAAIADLQRGDRLIAIGGVKITSTLQVLKLIKQAGDRVLVY YERP VGQSNQGAVLQDNFGQLEENFLSSSCQSGYEEEAAGLTVDTESRELDSEFEDIASDVRAQ NEFK DEAQSLSHSPKRVPTTLSIKPLGAISPVLNRKLAVGSHPLPPKIQSKDGNKPPPLKTSEI TDPA QVSKPTQGSAFKPPVPPRPQAKVPLPSADAPNQAEPDVLVEKPEKVVPPPLVDKSAEKQA KNVD AIDDAAAPKQFLAKQEVAKDVTSETSCPTKDSSDDRQTWESSEILYRNKLGKWTRTRASC LFDI EACHRYLNIALNCRDPFKLGGLICLGHVSLKLEDVALGCLATSNTEYLSKLRLEAPSPKM VTR TALRNLSMQKGFNDKFCYGDITIHFKYLKEGESDHHVVTNVEKEKEPHLVEEVSVLPKEE QFVG QMGLTENKHSFQDTQFQNPTWCDYCKKKVWTKAASQCMFCAYVCHKKCQEKCLAETSVCG ATDR RIDRTLKNLRLEGQETLLGLPPRVDAEASKSVNKTTGLTRHIINTSSRLLNLRQVSKTRL SEPG TDLVEPSPKHTPNTSDNEGSDTEVCGPNSPSKRGNSTGIKI. VRKEGGLDDSVFIAVKEIGRDLY RGLPTEERIQKLEFMLDKLQNEIDQELEHNNSLVREEKETTDTRKKSLLSAALAKSGERL QALT LLMIHYRAGIEDIETLESLSLDQHSKKISKYTDDTEEDLDNEISQLIDSQPFSSISDDLF GPSE SV Further analysis of the NOV6a protein yielded the following properties shown in Table 6B.

Table 6B. Protein Sequence Properties NOV6a SingalP analysis: Cleavage site between residues 22 and 23 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 0; pos. chg 0; neg. chg 0 H-region: length 27 ; peak value 11.55 PSG score: 7.15 GvH : von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1): 0.74 possible cleavage site: between 14 and 15 >>> Seems to have a cleavable signal peptide (1 to 14) ALOM : Klein et al's method for TM region allocation Init position for calculation: 15 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 1.38 (at 204) ALOM score : 1. 38 (number of TMSs : 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 7 Charge difference:-1. 0 C (0.0)-N (1.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 1.54 Hyd Moment (95): 1.27 G content: 2 D/E content: 1 S/T content: 3 Score:-4. 42 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 39 RRQJPE NUCDISC: discrimination of nuclear localization signals pat4: KRKH (3) at 280 pat7: none bipartite: none content of basic residues : 12. 5% NLS Score:-0. 29 KDEL : ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL : peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC : possible vacuolar targeting motif : found TLPN at 284 RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: nuclear Reliability: 55.5 COIL : Lupas's algorithm to detect coiled-coil regions 1028 T 1.00 1029 E 1.00 1030 E 1.00 1031 R 1. 00 1032 I 1. 00 1033 Q 1.00 1034 K 1.00 1035 L 1.00 1036 E 1.00 1037 F 1.00 1038 M 1.00 1039 L 1.00 1040 D 1.00 1041 K 1.00 1042 L 1.00 1043 Q 1.00 1044 N 1.00 1045 E 1.00 1046 1 1.00 1047 D 1.00 1048 Q 1.00 1049 E 1.00 1050 L 1.00 1051 E 1.00 1052 H 1.00 1053 N 1.00 1054 N 1.00 1055 S 1.00 1056 L 1.00 1057 V 0.99 1058 R 0.99 1059 E 0.99 1060 E 0.94 1061 K 0.94 1062 E 0.94 1063 T 0.83 1064 T 0.83 1065 D 0.83 1066 T 0.83 1067 R 0.67 1068 K 0.67 1069 K 0.67 1070 S 0.67 1071 L 0.67 1072 L 0.67 1073 S 0.67 1074 A 0.67 1075 A 0.67 total: 48 residues -------------------------- Final Results (k = 9/23): 33.3 % : extracellular, including cell wall 33.3 % : nuclear 22.2 % : mitochondrial 11. 1 % : cytoplasmic » indication for CG155653-01 is exc (k=9) A search of the NOV6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6C.

Table 6C. Geneseq Results for NOV6a NOV6a Identities/ Geneseq Protein/Organism/Length [Patent Residues/silarities for the Expect Identifier Date] Match Matched Region Value Residues AAM78475 Humanprotein SEQ IDNO 1137-1 1154 1154/1204 (95%) 0. 0 Homo sapiens, 1204 aa. 1.. 1204 1154/1204 (95%) [WO200157190-A2, 09-AUG-2001] AAU99614 Human glioma antigen KU-GB-5-264.. 1154 890/891 (99%) 0. 0 Homo sapiens, 891 aa. 1.. 891 890/891 (99%) [W0200255695-A1, 18-JUL-2002] ABG39902 Human peptide encoded by 436.. 1147 711/712 (99%) 0. 0 1.. 712 712/712 (99%) SEQ ID 29567-Homo sapiens, 712 aa. [WO200186003-A2, 15-NOV-2001] AAM18088 Peptide #4522 encoded by probe for 436.. 1147 711/712 (99%) 0.0 measuring cervical gene expression-1.. 712 712/712 (99al0) Homo sapiens, 712 aa. [WO200157278-A2, 09-AUG-2001] AAM70260 Human bone marrow expressed probe 436.. 1147 711/712 (99%) 0.0 encoded protein SEQ m NO : 30566-1.. 712 712/712 (99°/O) Homo sapiens, 712 aa. [W0200157276-A2, 09-AUG-2001] In a BLAST search of public sequence databases, the NOV6a protein was found to have homology to the proteins shown in the BLASTP data in Table 6D.

'table 6D. Public BLASTP Results for NOV6a

NOVA Protein Identities/ Accession Protein/Organism/Length Residues/Similarities for the Expect Number Residues Matched Portion Residues Q8NEN9 Similar to PDZ domain proteins-1 1154 1154/1154 (100%) 0. 0 Homo sapiens (Human), 1154 aa. 1.. 1154 1154/1154 (100%) Q9UFF1 Hypothetical protein-Homo 642.. 1154 512/513 (99%) 0. 0 sapiens (Human), 513 aa 1.. 513 512/513 (99%) (fragment). Q9VYR9 CG10362 protein (LD34222p)-3.. 494 148/506 (29%) 2e-46 Drosophila melanogaster (Fruit 8.. 473 242/506 (47%) fly), 1037 aa. T20180 hypothetical protein C53B4. 4a-93 447 112/387 (28%) 4e-42 Caenorhabditis elegans, 1584 aa. 203.. 585 194/387 (49%) Q9U3L2 C53B4. 4c protein-Caenorhabditis 93.. 447 112/387 (28%) 4e-42 elegans, 1449 aa. 68.. 450 194/387 (49%) PFam analysis indicates that the NOV6a protein contains the domains shown in the Table 6E.

Table 6E. Domain Analysis of NOV6a v m Identities/ Pfam Domain NOV6a Match Region Similarities Expect Value for the Matched Region PDZ 366..448 19/88 (22%) 7. 7e-10 62/88 (70%) DAG_PE-bind 841..888 18/51 (35) 2.6e-09 36/51 (71%) 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 : 27 | 1157 bp NOV7a, CTTCGGCCTGTCGGTTTTCACCATGGAGCAGCTGAGCTCAGCAAACACCCGCTTCGCCTT GGAC CG160093 01 DNA CTGTTCCTGGCGTTGAGTGAGAACAATCCGGCTGGAAACATCTTCATCTCTCCCTTCAGC ATTT CATCTGCTATGGCCATGGTTTTTCTGGGGACCAGAGGTAACACGGCAGCACAGCTGTCCA AGAC Sequence TTTCCATTTCAACACGGTTGAAGAGGTTCATTCAAGATTCCAGAGTCTGAATGCTGATAT CAAC AAACGTGGAGCGTCTTATATTCTGAAACTTGCTAATAGATTATATGGAGAGAAAACTTAC AATT TCCTTCCTGAGTTCTTGGTTTCGACTCAGAAAACATATGGTGCTGACCTGGCCAGTGTGG ATTT TCAGCATGCCTCTGAAGATGCAAGGAAGACCATAAACCAGTGGGTTGATAACATGACCAA ACTT GTGCTAGTAAATGCCATCTATTTCAAGGGAAACTGGAAGGATAAATTCATGAAAGAAGCC ACGA CGAATGCACCATTCAGATTGAATAAGAAAGACAGAAAAACTGTGAAAATGATGTATCAGA AGAA AAAATTTGCATATGGCTACATCGAGGACCTTAAGTGCCGTGTGCTGGAACTGCCTTACCA AGGC GAGGAGCTCAGCATGGTCATCCTGCTGCCGGATGACATTGAGGACGAGTCCACGGGCCTG AAGA AGATTGAGGRACAGTTGACTTTGGAAAAGTTGCATGAGTGGACTAAACCTGAGAATCTCG ATTT CATTGAAGTTAATGTCAGCTTGCCCAGGTTCAAACTGGAAGAGAGTTACACTCTCAACTC CGAC CTCGCCCGCCTAGGTGTGCAGGATCTCTTTAACAGTAGCAAGGCTGATCTGTCTGGCATG TCAG GAGCCAGAGATATTTTTATATCAAAAATTGTCCACAAGTCATTTGTGGAAGTGAATGAAG AGGG AACAGAGGCGGCAGCTGCCACAGCAGGCATCGCAACTTTCTGCATGTTGATGCCCGAAGA AAAT TTCACTGCCGACCATCCATTCCTTTTCTTTATTCGGCATAATTCCTCAGGTAGCATCCTA TTCT TGGGGAGATTTTCTTCCCCTTAGAAGAAAGAGACTGTAGCAATACAAAAATCAAGCTTAG TGCT AAGGG ORF Start : ATG at 23 ORF Stop : TAG at 1109 SEQIDNO : 28 2 aa 1001. 3kD NOV7a, MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTAAQLSKTFHF NTVE CG160093-01 EVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYGADLASVDFQHA SEDA RKTINQWVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLNKKDMKTVKMMYQKKKFA YGYI Protein Sequence EDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQLTLEKLHEWTKPENLDFIEV NVSL PRFKLEESYTLNSDLARLGVQDLFNSSKADLSGMSGARDIFISKIVHKSFVEVNEEGTEA AAAT AGIATFCMLMPEENFTADHPFLFFIRHNSSGSILFLGRFSSP

- NOV7b, CGGCGGCCTGTCGGAGCTGTTTGTGACGGTTTCCAGGCAGCCCAGGGCCAGGCCGCGGCT CCTA CG160093-02 DNA TCTGCAGCTGCAGGGAGAGAGAGGAGGAACCCCGTGCGATTCTAGAGACGATTTCACAAC AAGG AGAAATCAGCTTTGTGCTTACATGCCGAGCAGCCAGCACGGTTCTTCTTTGCCTGTCCTC GGGG Sequence GTCAGGGC-TCTGAGAGTGGAGATCGAGATGGGCTAGTGGGTGGCGGATGGGACGCTGCA CG GCCAGACCCTGGACTGTGTTTTCACCATGGAGCAGCTGAGCTCAGCAAACACCCGCTTCG CCTT GGACCTGTTCCTGGCGTTGAGTGAGAACAATCCGGCTGGAAACATCTTCATCTCTCCCTT CAGC ATTTCATCTGCTATGGCCATGGTTTTTCTGGGGACCAGAGGTAACACGGCAGCACAGCTG TCCA AGACTTTCCATTTCAACACGGTTGAAGAGGTTCATTCAAGATTCCAGAGTCTGAATGCTG ATAT CAACAAACGTGGAGCGTCTTATATTCTGAAACTTGCTAATAGATTATATGGAGAGAAAAC TTAC AATTTCCTTCCTGAGTTCTTGGTTTCGACTCAGAAAACATATGGTGCTGACCTGGCCAGT GTGG ATTTTCAGCATGCCTCTGAAGATGCAAGGAAGACCATAAACCAGTGGGTCAAAGGACAGA CAGA AGGAAAAATTCCGGAACTGTTGGCTTCGGGCATGGTTGATAACATGACCAAACTTGTGCT AGTA AATGCCATCTATTTCAAGGGAAACTGGAAGGATAAATTCATGAAAGAAGCCACGACGAAT GCAC CATTCAGATTGAATAAGAAAGACAGAAAAACTGTGAAAATGATGTATCAGAAGAAAAAAT TTGC ATATGGCTACATCGAGGACCTTAAGTGCCGTGTGCTGGAACTGCCTTACCAAGGCGAGGA GCTC AGCATGGTCATCCTGCTGCCGGATGACATTGAGGACGAGTCCACGGGCCTGAAGAAGATT GAGG AACAGTTGACTTTGGAAAAGTTGCATGAGTGGACTAAACCTGAGAATCTCGATTTCATTG AAGT TAATGTCAGCTTGCCCAGGTTCAAACTGGAAGAGAGTTACACTCTCAACTCCGACCTCGC CCGC CTAGGTGTGCAGGATCTCTTTAACAGTAGCAAGGCTGATCTGTCTGGCATGTCAGGAGCC AGAG ATATTTTTATATCAAAAATTGTCCACAAGTCATTTGT, GGAAGTGAATGAAGAGGGAAQGAGGC GGCAGCTGCCACAGCAGGCATCGCAACTTTCTGCATGTTGATGCCCGAAGAAAATTTCAC TGCC GACCATCCATTCCTTTTCTTTATTCGGCATAATTCCTCAGGTAGCATCCTATTCTTGGGG AGAT TTTCTTCCCCTTAGAAGAAAGAGACTGTAGCAATACAAAAATCAAGCTTAGTGCTTTATT ACCT GAGTTTTTAATAGAGCCAATATGTCTTATATCTTTACCAATAAAACCACTGTCCAGAAAC AAAA AAAAAAAAAAAAAA ORF Start : ATG at 283 ORF Stop : TAG at 1420

SEQ ID NO : 30 379 aa MW at 42741. 3kD NOV7b, MEQLSSANTRFALDLFLALSENNPAGNIFISPFSISSAMAMVFLGTRGNTAAQLSKTFHF NTVE CG160093-02 EVHSRFQSLNADINKRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYGADLASVDFQHA SEDA u-v RKTINQWVKGQTEGKIPELLASGMVDNMTKLVLVNAIYFKGNWKDKFMKEATTNAPFRLN KKDR Protein Sequence KTVKMMYQKKKFAYGYIEDLKCRVLELPYQGEELSMVILLPDDIEDESTGLKKIEEQLTL EKLH EWTKPENLDFIEVNVSLPRFKLEESYTLNSDLARLGVQDLFNSSKADLSGMSGARDIFIS KIVH KSFVEVNEEGTEAAAATAGIATFCMLMPEENFTADHPFLFFIRHNSSGSILFLGRFSSP Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 7B.

Table 7B. Comparison of NOV7a against NOV7b. NOV7a Residues/Identities/ Protein Sequence Match Residues Similarities for the Matched Region NOV7b 1.. 362 362/379 (95%) 1.. 379 362/379 (95%)

Further analysis of the NOV7a protein yielded the following properties shown in Table 7C.

Table 7C. Protein Sequence Properties NOV7a SignalP analysis: No Known Signal Sequence Indicated PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 10; pos. chg 1; neg. chg 1 H-region: length 3; peak value 5. 12 PSG score: 0.72 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-4. 07 possible cleavage site: between 48 and 49 >>> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation : 1 Tentative number of TMS (s) for the threshold 0.5 :, 1 Number of TMS (s) for threshold 0.5 : 1 INTEGRAL Likelihood =-2.97 Transmembrane 28-44 PERIPHERAL Likelihood = 2.49 (at 314) ALOM score:-2. 97 (number of TMSs : 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 35 Charge difference: 3.5 C (2. 5)-N (-1. 0) C > N: C-terminal side will be inside >>>Caution : Inconsistent mtop result with signal peptide >>> membrane topology: type lb (cytoplasmic tail 28 to 362) MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75): 7.63 Hyd Moment (95): 4.21 G content: 0 D/E content: 2 S/T content: 3 Score:-5. 24 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: KKDRKTVKMMYQKKKFA at 172 content of basic residues: 11. 3% NLS Score: 0.02 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2 : 2nd peroxisomal targeting signal: none 134 VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2 : none NMYR: N-myristoylation pattern : none Prenylation motif: none. memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: too long tail Dileucine motif in the tail: found LL at 214 checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 89 COIL : Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 34. 8 % : nuclear 21. 7 % : mitochondrial 21. 7 cytoplasmic 8. 7 % : vesicles of secretory system 4. 3 %: vacuolar 4. 3 peroxisomal 4. 3 % : endoplasmic reticulum >> indication for CG160093-01 is nuc (k=23)

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

Table 7D. Geneseq Results for NOV7a NOV7a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region AAB43755 Human cancer associated protein 1.. 362 362/379 (95%) 0. 0 sequence SEQ ID NO : 1200-Homo 59.. 437 362/379 (95%) sapiens, 437 aa. [W0200055350-Al, 21-SEP-2000] AAR94367 Human elastase inhibitor-Homo 1.. 362 362/379 (95%) 0. 0 sapiens, 379 aa. [W09610418-Al, 1.. 379 362/379 (95%) ll-APR-1996] AAR64159 Human elastase inhibitor-Homo 1.. 362 362/379 (95%) 0. 0 sapiens, 379 aa. [US5370991-A, 1.. 379 362/379 (95%) 06-DEC-1994] AAY55841 Human cytoplasmic antiproteinase-3 1 362 186/380 (48%) 5e-98 protein (CAP-3)-Homo sapiens, 376 1 376 250/380 (64%) aa. jazz AAR99254 Cytoplasmic antiproteinase-3 protein-1 362 186/380 (48%) 5e-98 Homo sapiens, 376 aa. 1.. 376 250/380 (64%) [W09624650-A2, 15-AUG-1996] In a BLAST search of public sequence databases, the NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7E.

Table 7E. Public BLASTP Results for NOV7a Protein NOV7a Identities/ Accession Protein/Organism/Length Residues/Similarities for Expect Number Match the Matched Value Residues Portion P30740 Leukocyte elastase inhibitor (LEI) 1.. 362 362/379 (95%) 0. 0 (Monocyte/neutrophil elastase 1 379 362/379 (95%) inhibitor) (M/NEI) (EI)-Homo sapiens (Human), 379 aa. P05619 Leukocyte elastase inhibitor (LEI)-1 362 297/379 (78%) e-169 Equus caballus (Horse), 379 aa. 1.. 379 326/379 (85%) Q9D154 1190005M04Rikprotein (RIKEN 1.. 362 291/379 (76%) e-167 cDNA 1190005M04 gene) (Serine 1.. 379 330/379 (86%) protease inhibitor EIA)-Mus musculus (Mouse), 379 aa. P80229 Leukocyte elastase inhibitor (LEI) 1.. 362 291/379 (76%) e-166 (Leucocyte neutral proteinase inhibitor) 1.. 378 332/379 (86%) (LNPI)-Sus scrofa (Pig), 378 aa. S38962 serpin-pig, 378 aa. 1.. 362 291/379 (76%) e-165 1.. 378 330/379 (86%) PFam analysis indicates that the NOV7a protein contains the domains shown in the Table 7F.

Table 7F. Domain Analysis of NOV7a

Identities/ Pfam Domain NOV7a Match Region Similarities Expect Value for the Matched Region serpin 1.. 136 58/142 (41%) 1. 3e-54 120/142 (85%) serpin 137.. 362 117/233 (50%) 5. 2e-115 208/233 (89%) 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 : 31 NOV8a, CAGTGTGCTGGAATTCGCCCTTAACCGGCAGGATGTCGGAGGTGCGGCTGCCACCGCTAC GCGC CG163133 02 DNA CCTGGACGACTTTGTTCTGGGGTCGGCGCGTCTGGCGGCTCCGGATCCATGCGACCCGCA GCGA TGGTGCCACCGCGTCATCAACAACCTCCTCTACTACCAAACCAACTACCTTCTCTGCTTC GGCA Sequence TCGGCCTCGCTCTCGCCGGGTACGTGCGGCCACTTCATACGCTCCTGAGCGCGCTGGTAG TGGC GGTGGCCCTCGGCGTGCTGGTGTGGGCAGCTGAGACCCGCGCAGCTGTGCGCCGCTGCCG CCGC AGCCACCCTGCAGCCTGCCTGGCCGCAGTGCTTGCCGTCGGCCTCCTGGTGCTCTGGGTC GCGG GCGGCGCTTGCACCTTCCTGTTCAGCATCGCCGGGCCGGTGCTTCTGATCCTGGTGCACG CCTC GTTGCGCCTGCGCAACCTTAAGAACAAGATTGAGAACAAGATCGAGAGCATTGGTCTCAA GCGG ACGCCAATGGGCCTGCTACTAGAGGCACTGGGACAAGAGCAGGAGGCTGGATCCTAGGCC CCTG GGATCTGTACCCAGGACCTGGAGAATACCACCCCACCCCCAGCCCATAATTGGGACCCAG AGCC CTTTCCCAGCACTTAAAACAGGAGCCTAGAGCCGCCTGCCCAAACAAAAAAGGGCGA ORF Start : ATG at 33 ORF Stop : TAG at 567 w SEQ ED NO : 32 ! S aa MW at 19257. 6kD NOV8a, MSEVRLPPLRALDDFVLGSARLAAPDPCDPQRWCHRVINNLLYYQTNYLLCFGIGLALAG YVRP CG163133-02 LHTLLSALVVAVALGVLVWAAETRAAVRRCRRSHPAACLAAVLAVGLLVLWVAGGACTFL FSIA GPVLLILVHASLRLRNLKNKIENKIESIGLKRTPMGLLLEALGQEQEAGS ProtemSequence SEQ ID NO : 33 581 bp NOV8b, GCAGTGTGTGGAATCGCCCTTAACCGGCAGGATGTCGGAGGTGCGGCTGCCACCGCTACG CGCC CG163133-01 DNA CTGGACGACTTTGTTCTGGGGTCGGCGCGTCTGGCGGCTCCGGATCCATGCGACCCGCAG CGAT GGTGCCACCGCGTCATCAACAACCTCCTCTACTACCAAACCAACTACCTTCTCTGCTTCG GCAT Sequence CGGCCTCGCTCTCGCCGGGCACGTGCGGCCACTTCATACGCTCCTAAGCGCGCTGGTAGT GGCG GTGGCCCTCGGCGTGCTGGTGTGGGCAGCTGAGACCCGCGCAGCTGTGCGCCGCTGCCGC CGCA GCCACCCTGCAGCCTGCCTGGCCGCAGTGCTTGCCGTCGGCCTCCTGGTGCACGCCTCGT TGCG CCTGCGCAACCTTAAGAACAAGATTGAGAACAAGATCGAGAGCATTGGTCTCAAGCGGAC GCCA ATGGGCCTGCTACTAGAGGCACTGGGACAAGAGCAGGAGGCTGGATCCTAGGCCCCTGGG ATCT GTACCCAGGACCTGGAGAATACCACCCCACCCCCAGCCCATAATTGGGACCCAGAGCCCT TTCC CAGCA ORF Start : ATG at 32 ORF Stop : TAG at 497 SEQ IDNO : 34 8. 7kD _ _ NOV8b MSEVRLPPLRALDDFVLGSARLAAPDPCDPQRWCHRVINNLLYYQTNYLLCFGIGLA7AG HVRP CG163133-01 LHTLLSALVVAVALGVLVWAAETRAAVRRCRRSHPAACLAAVLAVGLLVHASLRLRNLKN KIEN ''U-'-L'l KIESIGLKRTPMGLLI. EMjGQEQEAGS P » tein Sequence Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 8B.

Table 8B. Comparison of NOV8a against NOV8b.

Protein Sequence NOV8a Residues/Identities/ Match Residues Similarities for the Matched Region NOV8b 1 178 154/178 (86%) 1.. 155 155/178 (86%) Further analysis of the NOV8a protein yielded the following properties shown in Table 8C.

Table 8C. Protein Sequence Properties NOV8a SignalP analysis: No Known Signal Sequence Indicated PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 10; pos. chg 2; neg. chg 1 H-region: length 2; peak value-2.04 PSG score:-6. 44 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-3. 41 possible cleavage site: between 59 and 60 >>> Seems to have no N-terminal signal peptide ALOM : Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0.5 : 4 INTEGRAL Likelihood =-0.59 Transmembrane 49-65. INTEGRAL Likelihood =-9.66 Transmembrane 68-84 INTEGRAL Likelihood =-10.14 Transmembrane 100-116 INTEGRAL Likelihood =-7.38 Transmembrane 120-136 PERIPHERAL Likelihood = 8.43 (at 155) ALOM score:-10. 14 (number of TMSs : 4) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 56 Charge difference: 1.0 C (1.5)-N (0.5) C > N: C-terminal side will be inside >>> membrane topology: type 3b MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75): 4.19 Hyd Moment (95): 1.05 G content: 0 D/E content: 2 S/T content: 1 Score:-5. 78 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: none bipartite: none content of basic residues: 10.1% NLS Score:-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus: SEVR none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR : N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 55. 6 % : endoplasmic reticulum 11.1 % : mitochondrial 11.1 % : Golgi 11. 1 % : vacuolar 11. 1 % : cytoplasmic >> indication for CG163133-02 is end (k=9) A search of the 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 8D.

Table 8D. Geneseq Results for NOV8a NOV8a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region AAE14754 Human CCR5 chemoldne 1 178 178/178 (100%) 2e-98 receptor-interacting protein P2-Homo 1.. 178 178/178 (100%) sapiens, 178 aa. [EP1207202-A1, 22-MAY-2002] ABB97608 2e-98 -Homo sapiens, 178 aa. 1..178 178/178 (100%) [W0200222660-A2, 21-MAR-2002] AAB94612 Human protein sequence SEQ ID 1 178 177/178 (99%) 4e-98 NO : 15456-Homo sapiens, 178 aa. 1.. 178 178/178 (99%) [EP1074617-A2, 07-FEB-2001] AAE14761 Human CCR5 chemokine 1.. 178 177/178 (99%) le-97 receptor-interacting protein P2 mutant 1.. 178 177/178 (99%) (G53A) -Homo sapiens, 178 aa. [EP1207202-Al, 22-MAY-2002] AAE14760 Human CCR5 chemokine 1.. 178 177/178 (99%) 2e-97 receptor-interacting protein P2 mutant 1.. 178 177/178 (99%) (G157R)-Homo sapiens, 178 aa. [EP1207202-A1, 22-MAY-2002] In a BLAST search of public sequence databases, the NOV8a protein was found to have homology to the proteins shown in the BLASTP data in Table 8E.

Table 8E. Public BLASTP Results for NOV8a

NOVX Protein NOV8a Identities/ Accession Protein/Organism/Length Residues/Similarities for the Expect T Match T., f n Value Number Residues Matched Portion Value Residues 060831 JM4 protein-Homo sapiens (Human), 1.. 178 178/178 (100%) Se-98 178 aa. 1.. 178 178/178 (100%) Q9JIG8 DXImx39e protein (DNA segment, 1.. 178 162/178 (91%) 6e-89 Chr X, Imnunex 39, expressed)-Mus 1.. 178 166/178 (93%) musculus (Mouse), 178 aa. Q9ES40 Glutamate transporter EAAC1 3 176 78/174 (44%) 4e-41 interacting protein-Rattus norvegicus 2.. 175 119/174 (67%) (Rat), 188 aa. 075915 JWA protein (HSPC127) (Vitamin A 3.. 175 79/173 (45%) 4e-41 responsive, cytoskeleton related)-2.. 174 117/173 (66%) Homo sapiens (Human), 188 aa. Q9DB37 5930404D22Rik protein (RIKEN 3 175 78/173 (45%) le-40 cDNA 5930404D22 gene) (JWA 2.. 174 118/173 (68%) protein)-Mus musculus (Mouse), 188 aa.

Example 9.

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

Table 9A. NOV9 Sequence Analysis SEQIDNO : 35 16240bP 1 NOV9a, CTTTCTGTCTCTCGGGACCCTTATTTCTTCGTCACGGTGTCCAGGACCATTTTGACCCTG TCGG CCCCGGCACCCCCCCGCCGCACCCCAGCCCCGAGCATGGSGACGGCGCTGCTCCAGCGCG GGGG CTGTTTTCTTCTGTGCCTCTCGCTGCTGCTCCTGGGCTGCTGGGCGGAGCTGGGCAGCGG GCTG Sequence GAGTTTCCGGGCGCCGAGGGCCAATGGACGCGCTTCCCCAAGTGGAACGCCTGCTGCGAG AGCG AGATGAGCTTCCAGCTCAAGACTCGCAGCGCCCGCGGCCTCGTGCTCTACTTCGACGACG AGGG CTTCTGCGACTTCCTGGAGCTGATTCTGACGCGCGGCGGCCGCCTGCAGCTCAGCTTCTC CATC TTCTGCGCTGAGCCTGCGACGCTCCTGGCCGACACGCCGGTTAACGACGGCGCCTGGCAC AGCG TGCGCATCCGCCGCCAGTTCCGCAACACCACGCTCTTCATCGACCAGGTGGAGGCCAAGT GGGT GGAGGTCAAGTCCAAGCGCAGGGACATGACGGTGTTCAGCGGCCTTTTCGTCGGGGGGCT GCCC CCGGAACTGCGCGCCGCGGCGCTCAAGCTCACCCTGGCCTCGGTGAGGGAGCGGGAGCCC TTCA AGGGGTGGATTCGTGACGTGAGGGTCAACTCCTCGCAGGTCCTGCCCGTGGACAGCGGCG AGGT GAAGCTGGACGATGAGCCGCCCAACAGCGGCGGGGGAAGCCCGTGCGAGGCGGGCGAGGA GGGC GAGGGCGGGGTGTGCCTCAACGGAGGTGTGTGCTCCGTGGTGGACGACCAGGCCGTGTGC GACT GCTCGCGAACCGGCTTCCGCGGCAAGGACTGCAGCCAAGAAGACAACAATGTGGAAGGTC TGGC GCACCTGATGATGGGCGACCAAGGTAAAAGTAAAGGAAAAGAAGAATATATTGCCACGTT CAAA GGATCTGAATACTTCTGCTACGACTTGTCTCAAAACCCCATTCAAAGCAGCAGTGATGAA ATAA CTCTGTCATTTAAAACCCTTCAGAGGAATGGACTGATGCTTCACACTGGGAAATCGGCTG ATTA TGTCAATCTTGCCCTGAAAAATGGAGCTGTCTCTCTGGTCATTAATTTGGGATCAGGGGC CTTT GAAGCACTAGTGGAGCCTGTGAATGGAAAGTTTAATGATAATGCCTGGCATGATGTGAAA GTCA CCAGGAATCTGCGTCAGCACTCAGGCATTGGACACGCTATGGTAAACAAACTACATTGTT CGGT GACAATATCAGTGGATGGGATTCTTACCACAACGGGCTACACGCAAGAAGATTATACCAT GCTG GGGTCTGATGACTTTTTCTATGTTGGAGGCAGTCCCAGCACAGCCGACCTTCCAGGGTCA CCAG TCAGTAACAACTTTATGGGCTGTCTCAAAGAGGTTGTATATAAAAATAATGATGTGAGGC TGGA ATTATCTCGACTTGCCAAGCAAGGAGATCCTAAGATGAAGATCCATGGAGTGGTGGCATT TAAA TGTGAGAATGTTGCAACTTTAGACCCAATCACCTTTGAAACCCCAGAGTCTTTCATCTCT TTGC CTAAATGGAATGCAAAGAAAACTGGCTCCATATCATTTGATTTCCGTACAACAGAGCCAA ATGG CCTCATCTTATTTAGCCATGGCAAGCCAAGACATCAGAAAGATGCCAAGCACCCACAGAT GATA AAGGTGGACTTCTTTGCTATTGAGATGCTAGATGGCCACCTCTACCTCCTCCTGGACATG GGGT CAGGTACTATAAAAATAAAAGCCCTGTTGAAGAAAGTGAATGATGGAGAATGGTATCATG TGGA CTTCCAGAGAGACGGACGGTCAGGTACCATTTCTGTCAACACGTTGCGTACTCCCTACAC TGCT CCTGGTGAGAGTGAGATTCTGGACCTGGATGATGAGTTGTACCTGGGGGGGCTGCCAGAA AATA AAGCTGGCCTTGTCTTCCCCACCGAGGTGTGGACTGCTCTGCTCAACTATGGCTACGTGG GCTG CATCAGGGATTTGTTCATCGATGGCCAAAGCAAAGATATCCGGCAAATGGCTGAAGTTCA AAGT ACTGCTGGAGTGAAGCCTTCCTGCTCAAAGGAAACAGCAAAACCGTGCCTTAGCAACCCT TGCA AAAACAATGGCATGTGCAGGGATGGGTGGAACAGATATGTCTGTGATTGTTCCGGAACAG GCTA TCTTGGCAGGTCCTGTGAGAGAGAGGCAACGGTTTTGAGCTATGATGGGAGCATGTTTAT GAAA ATTCAGCTCCCCGTAGTCATGCATACGGAGGCTGAGGATGTTTCCTTACGGTTCCGATCC CAGC GTGCATATGGCATTCTGATGGCAACCACTTCTAGAGACTCTGCTGACACCCTCCGCCTGG AGCT AGACGCAGGACGTGTGAAACTGACGGTCAATCTAGATTGTATCAGGATTAACTGTAATTC CAGC AAAGGTCCCGAGACTCTTTTTGCTGGCTATAACCTCAATGATAACGAGTGGCACACAGTG CGTG TAGTTCGGCGTGGAAAAAGTTTAAAGTTAACAGTGGATGACCAACAGGCCATGACAGGTC AAAT GGCAGGTGATCATACTAGGCTGGAGTTCCATAACATAGAGACTGGCATCATCACAGAACG ACGG TATCTTTCTTCTGTCCCCTCCAACTTCATTGGACACCTGCAGAGCTTGACATTTAATGGA ATGG CATACATTGACCTGTGTAAAAATGGCGACATAGATTACTGTGAGCTTAATGCCAGATTTG GCTT CAGGAACATCATAGCAGATCCTGTCACCTTCAAGACCAAATCGAGCTATGTTGCCTTAGC TACC TTGCAAGCCTACACTTCTATGCATCTTTTTTTCCAGTTCAAGACAACATCCCTAGATGGA TTAA TTCTATATAACAGTGGGGATGGAAATGACTTTATTGTGGTTGAATTAGTTAAAGGGTACT TACA TTACGTGTTTGATTTGGGAAATGGTGCTAACCTCATCAAAGGAAGCTCAAATAAACCTCT CAAT GACAATCAGTGGCACAACGTGATGATATCAAGGGACACCAGCAACCTCCACACTGTAAAG ATTG ACACAAAAATCACAACGCAAATCACCGCCGGAGCCAGGAACTTAGACCTCAAGAGTGACT TATA TATAGGAGGAGTAGCTAAAGAAACATACAAATCCTTACCAAAACTTGTACATGCCAAAGA AGGC TTTCAAGGCTGCCTGGCATCAGTTGATTTAAATGGACGGCTTCCGGACCTCATCTCCGAT GCTC TTTTCTGCAACGGACAGATCGAGAGAGGATGTGAAGGGCCCAGCACAACCTGCCAAGAGG ACTC ATGTTCCAATCAAGGTGTGTGCTTGCAACAATGGGATGGCTTCAGCTGTGACTGTAGTAT GACT TCCTTCAGTGGACCACTCTGCAATGACCCTGGGACGACATATATCTTTAGCAAAGGTGGT GGAC AAATCACGTATAAGTGGCCTCCTAATGACCGACCCAGTACACGAGCAGACAGACTGGCCA TAGG TTTTAGCACTGTTCAGAAAGAAGCCGTATTGGTGCGAGTGGACAGTTCTTCAGGCTTGGG TGAC TACCTAGAACTGCATATACACCAGGGAAAAATTGGAGTTAAGTTTAATGTTGGGACAGAT GACA -. jTACCTAGAACTGC. ATATACACCAGGGA TCGCCATTGAAGAATCCAATGCAATCATTAATGATGGGAAATACCATGTAGTTCGTTTCA CGAG GAGTGGTGGCAATGCCACGTTGCAGGTGGACAGCTGGCCAGTGATCGAGCGCTACCCTGC AGGA AACAATGATAACGAGCGCCTGGCGATTGCTAGACAGCGAATTCCATATCGACTTGGTCGA GTAG TTGATGAATGGCTACTCGACAAAGGGCGTCAGCTCACAATCTTCAATAGCCAAGCAACCA TAAT AATTGGCGGGAAAGAGCAGGGCCAGCCCTTCCAGGGCCAGCTCTCTGGGCTGTACTACAA TGGC TTGAAAGTTCTGAATATGGCAGCCGAAAACGATGCCAACATCGCCATAGTGGGAAATGTG AGAC TGGTTGGTGAAGTGCCTTCCTCTATGACAACTGAGTCAACAGCCACTGCCATGCAATCAG AGAT GTCCACATCAATTATGGAGACTACCACGACCCTGGCTACTAGCACAGCCAGAAGAGGAAA GCCC CCGACAAAAGAACCCATTAGCCAGACCACAGATGACATCCTTGTGGCCTCAGCAGAGTGT CCCA GCGATGATGAGGACATTGACCCCTGTGAGCCGAGCTCAGGTGGGTTAGCCAACCCAACCC GAGC AGGCGGCAGAGAGCCGTATCCAGGCTCAGCAGAAGTGATCCGGGAGTCCAGCAGCACCAC GGGT ATGGTCGTTGGGATAGTAGCCGCTGCCGCCCTGTGCATCCTTATCCTCCTCTATGCCATG TACA AGTACAGAAACCGGGATGAAGGCTCATACCATGTGGACGAGAGTCGAAACTACATCAGTA ACTC AGCACAGTCCAATGGGGCTGTTGTAAAGGAGAAACAACCCAGCAGTGCGAAAAGCTCCAA CAAA AATAAGAAAAACAAGGATAAAGAGTATTATGTCTGATCCCAAGATCTTAAATGGACACTT GTAT AGAAATAGTCTTCATTTTATCTGAGACATAATATAAACTTATTTACTTTCCTTTTTATGA AGCA CATACAAAAGAAGACAGAGAATGCAATCAGGAAGGAAAGACTTTTTAAAAAATAAAAACA AGTA TCTCATGCTCTTGTTTCTCAAAAAAGAAAAACAAAAAACAAAAAACAGGGGCCAATAAAT TCCC TAACATCCACAGTGTTTTCATTTACTCTGCTTGTCTTTATGTTGCTGGAACATTTCTAAA AGAC AGTGATGACCGCACGCATTCATAAAGCAAAGGAGTACTACAGCATCAAGGCACAACACAA AAAC CAACACAAAACATAACACAAAAAAGAAGCTACCTATGATCCTGGATTTAGCCAAAGTGCT AGCG CTTTCCTGAGAAGTCAGTCCAATTGCCAGAGAAGACTGTCCTTTTGAGTGACTCAACCTG CAAA CCTTTAAGAGTTTGCCGCCTGGTGCAACTGGAGCAGTGGTTGGAACTTGCATTTGAAACA AAGT GCTGGCTTTTTTGAAGACTTGTGTAGGAACACATTCAAAAAGCCCCTTTCTGGTTGTGAG AGAG GAAAAAAAAAGTATGGAGGCCTTATTTTCAAAAATGTGAAATATAAGGCACGTTTTCACA CAAA ATTTCAAAACAAAAACAAGAGGGCATAGATGCAATCATTGGGAAATTTTCATGCACGCTT ATTA TGTTATTACATATGTTTATATAAAATCCATCTCTGTGTGCTTTCTGGACTGTGATAAGTG ACGT TTTATAGCCTGTTGTATAGAAAATGCAAAATATATCTCTGCTCTTCAGCCATTTTTGGTA AATT CAATGTTATAAGTGTTGCTAAGTATAGGGAGTTTTATGACATCAGAGCAACAATTATTTC AGTT GGGTTTTTCTTTTTTTTGCCACCATTATAAATTGCCACAATTACTTACTTTTATTTTTTA AAGA AATTACAGTGTAGTGTTTATTCTAAGGAAGATATGTATGAATGTATATACAAAGACTCAG CTAC TTCTTTTCTTATATGTACAGCCTTCATTCTGTTGCAATTAAGTTTTAGTACTTGTATGAA AGGT GTGAATTAGAAAGTCACATATATACATATGTATCTTATAATCTTTTCTCCCTGAAATACT CACA TTCCCACATACATTCACTATTTTCACACACACACACACACACACACACACACACACACAC ACAC ACGAATCCACAGCAATCCATCAGATATGCTGGAAGATCCAAACGTGCATACAGTAGCAAA TATT TATTGACAAATTGAAAAGCAGGAAGGAAGAGGGTTGTGCCAAGGTATTGATGACAAATGG GGTG ATTTGCTTCATTGAGATCTTGCTCCCAGGTAACCTTAAGAAGATTTTAGTCCCTAAAGAA ATGA ACCTTTCCTTATCAAATAGAATATCACTGATATACTGCTGCATGAATAAGAACCATTATG TGGG CAGGTTATGGAAGCAAAATTGGTTAATCTACACCTTAACTCTGGCTGCTGCAATTGAAAA CTTT CTTTCTAATAAAATAATATATATATCTCTGAA ORF Start : ATG at 100 ORF Stop : TGA at 4642 m NOV9a, MGTALLQRGGCFLLCLSLLLLGCWAELGSGLEFPGAEGQWTRFPKWNACCESEMSFQLKT RSAR CG165528-01 GLVLYFDDEGFCDFLELILTRGGRLQLSFSIFCAEPATLLADTPVNDGAWHSVRIRRQFR NTTI. . FIDQVEAKWVEVKSKRRDMTVFSGLFVGGLPPELRAAALKLTLASVREREPFKGWIRDVR VNSS Protem Sequence QVLPVDSGEVKLDDEPPNSGGGSPCEAGEEGEGGVCLNGGVCSWDDQAVCDCSRTGFRGK DCS QEDNNVEGLAHLMMGDQGKSKGKEEYIATFKGSEYFCYDLSQNPIQSSSDEITLSFKTLQ RNGL MLHTGKSADYVNLALKNGAVSLVINLGSGAFEALVEPVNGKFNDNAWHDVKVTRNLRQHS GIGH AMVNKLHCSVTISVDGILTTTGYTQEDYTMLGSDDFFYVGGSPSTADLPGSPVSNNFMGC LKEV VYKNNDVRLELSRLAKQGDPKMKIHGVVAFKCENVATLDPITFETPESFISLPKWNAKKT GSIS FDFRTTEPNGLILFSHGKPRHQKDAKHPQMIKVDFFAIEMLDGHLYLLLDMGSGTIKIKA LLKK VNDGEWYHVDFQRDGRSGTISVNTLRTPYTAPGESEILDLDDELYLGGLPENKAGLVFPT EVWT ALLNYGYVGCIRDLFIDGQSKDIRQMAEVQSTAGVKPSCSKETAKPCLSNPCKNNGMCRD GWNR YVCDCSGTGYLGRSCEREATVLSYDGSMFMKIQLPWMHTEAEDVSLRFRSQRAYGILMAT TSR DSADTLRLELDAGRVKLTVNLDCIRINCNSSKGPETLFAGYNLNDNEWHTVRWRRGKSLK LTV DDQQAMTGQMAGDHTRLEFHNIETGIITERRYLSSVPSNFIGHLQSLTFNGMAYIDLCKN GDID YCELNARFGFRNIIADPVTFKTKSSYVALATLQAYTSMHLFFQFKTTSLDGLILYNSGDG NDFI WELVKGYLHYVFDLGNGANLIKGSSNKPLNDNQWHNVMISRDTSNLHTVKIDTKITTQIT AGA RNLDLKSDLYIGGVAKETYKSLPKLVHAKEGFQGCLASVDLNGRLPDLISDALFCNGQIE RGCE GPSTTCQEDSCSNQGVCLQQWDGFSCDCSMTSFSGPLCNDPGTTYIFSKGGGQITYKWPP NDRP STRADRLAIGFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGVKFNVGTDDIAIEESNA IIND GKYHVVRFTRSGGNATLQVDSWPVIERYPAGNNDNERLAIARQRIPYRLGRVVDEWLLDK GRQL TIFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNVRLVGEVPSS MTTE STATAMQSEMSTSIMETTTTIATSTARRGKPPTKEPISQTTDDILVASAECPSDDEDIDP CEPS SGGLANPTRAGGREPYPGSAEVIRESSSTTGMWGIVAAAALCILILLYAMYKYRNRDEGS YHV DESRNYISNSAQSNGAWKEKQPSSAKSSNKNKKNKDKEYYV SEQ ID NO : 37--11611 bp I NOV9b, AAACTTTGCCTCCCGCGGCGGCTGCCCCTCGGCGGGCGCCCCGCCATGTACCAGAGGATG CTCC CG165528-02 DNA GGTGCGGCGCCGAGCTGGGCTCGCCCGGGGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCG CAGG GGGGCGCCTGGCCCTGCTTTGGATAGTCCCGCTCACCCTCAGCGGCCTCCTAGGAGTGGC GTGG Sequence GGGGCATCCAGTTTGGGAGCGCACCACATCCACCATTTCCATGGCAGCAGCAAGCATCAT TCAG TGCCTATTGCAATCTACAGGTCACCGGCATCCTTGCGAGGCGGACACGCTGGGACGACAT ATAT CTTTAGCAAAGGTGGTGGACAAATCACGTATAAGTGGCCTCCTAATGACCGACCCAGTAC ACGA GCAGACAGACTGGCCATAGGTTTTAGCACTGTTCAGAAAGAAGCCGTATTGGTGCGAGTG GACA GTTCTTCAGGCTTGGGTGACTACCTAGAACTGCATATACACCAGGGAAAAATTGGAGTTA AGTT TAATGTTGGGACAGATGACATCGCCATTGAAGAATCCAATGCAATCATTAATGATGGGAA ATAC CATGTAGTTCGTTTCACGAGGAGTGGTGGCAATGCCACGTTGCAGGTGGACAGCTGGCCA GTGA TCGAGCGCTACCCTGCAGGAAACAATGATAACGAGCGCCTGGCGATTGCTAGACAGCGAA TTCC ATATCGACTTGGTCGAGTAGTTGATGAATGGCTACTCGACAAAGGGCGTCAGCTCACAAT CTTC AATAGCCAAGCAACCATAATAATTGGCGGGAAAGAGCAGGGCCAGCCCTTCCAGGGCCAG CTCT CTGGGCTGTACTACAATGGCTTGAAAGTTCTGAATATGGCAGCCGAAAACGATGCCAACA TCGC CATAGTGGGAAATGTGAGACTGGTTGGTGAAGTGCCTTCCTCTATGACAACTGAGTCAAC AGCC ACTGCCATGCAATCAGAGATGTCCACATCAATTATGGAGACTACCACGACCCTGGCTACT AGCA CAGCCAGAAGAGGAAAGCCCCCGACAAAAGAACCCATTAGCCAGACCACAGATGACATCC TTGT GGCCTCAGCAGAGTGTCCCAGCGATGATGAGGACATTGACCCCTGTGAGCCGAGCTCAGG TGGG TTAGCCAACCCAACCCGAGCAGGCGGCAGAGAGCCGTATCCAGGCTCAGCAGAAGTGATC CGGG AGTCCAGCAGCACCACGGGTATGGTCGTTGGGATAGTAGCCGCTGCCGCCCTGTGCATCC TTAT CCTCCTCTATGCCATGTACAAGTACAGAAACCGGGATGAAGGCTCATACCATGTGGACGA GAGT CGAAACTACATCAGTAACTCAGCACAGTCCAATGGGGCTGTTGTAAAGGAGAAACAACCC AGCA GTGCGAAAAGCTCCAACAAAAATAAGAAAAACAAGGATAAAGAGTATTATGTCTGATCCC AAGA TCTTAAATGGACACTTGTATAGAAATAGTCTTCATTTTATCTGAGACATAATATAAACTT ATTT ACTTTCCTTTTTATGAAGCACATACAAAAGAAGACAGGGAATGCAATCAGGAAGGAAAGA CTTT TTAAAAAATAA ORF Start : ATG at 46 ORF Stop : TGA at 1462

SEQ m NO : 38 472 aa MW at 50423. 1kD 3 NOV9b, MYQRMLRCGAELGSPGGGGGGGGGGGRGGRLALLNIVPLTLSGLLGVAWGASSLGAHHIH HFHG CG165528-02 SSKHHSVPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAIGFSTV QKEA VLVRVDSSSGLGDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGN ATLQ Protein Sequence VDSWPVIERYPAGNNDNERLAIARQRIPYRLGRWDENLLDKGRQLTIFNSCATIIIGGKE QGQ P, FQGQLSGLYYNGLKVLNMAAENDANIAIVGNVRLVGEVPSSMTTESTATAMQSEMSTSIM ETT TTLATSTARRGKPPTKEPISQTTDDILVASAECPSDDEDIDPCEPSSGGLANPTRAGGRE PYPG SAEVIRESSSTTGMWGIVAAAALCILILLYAMYKYRNRDEGSYHVDESRNYISNSAQSNG AW KEKQPSSAKSSNKNKKNKDKEYYV

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 9B.

Table 9B. Comparison of NOV9a against NOV9b. Protein Sequence NOV9a Residues/Identities/ Match Residues Similarities for the Matched Region NOV9b 1130.. 1514 385/385 (100%) 88.. 472 385/385 (100-/.) Further analysis of the NOV9a protein yielded the following properties shown in Table 9C.

Table 9C. Protein Sequence Properties NOV9a SignalP analysis: Cleavage site between residues 26 and 27 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 8; pos. chg 1; neg. chg 0 H-region: length 17; peak value 10. 61 PSG score: 6.21 GvH : von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1): 7.83 possible cleavage site: between 25 and 26 >>> Seems to have a cleavable signal peptide (1 to 25) ALOM : Klein et al's method for TM region allocation Init position for calculation: 26 Tentative number of TMS (s) for the threshold 0.5 : 2 Number of TMS (s) for threshold 0.5 : 1 INTEGRAL Likelihood =-13. 59 Transmembrane 1440-1456 PERIPHERAL Likelihood = 2.44 (at 89) ALOM score : -13.59 (number of TMSs : 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 12 Charge difference : -5. 0 C (-3. 0)-N (2. 0) N >= C: N-terminal side will be inside >>> membrane topology: type la (cytoplasmic tail 1457 to 1514) MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75): 10.18 Hyd Moment (95): 7.78 G content: 4 D/E content: 1 S/T content: 2 Score:-4. 63 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 18 QRG GC NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: none bipartite: none content of basic residues: 10.4% NLS Score:-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: KKXX-like motif in the C-terminus: KEYY SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL : transport motif from cell surface to Golgi: none Tyrosines in the tail: too long tail Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: Leucine zipper pattern (PS00029): *** found *** LQRGGCFLLCLSLLLLGCWAEL at 6 none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 70.6 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 44.4 % : endoplasmic reticulum 22. 2 % : Golgi 11.1 %: plasma membrane 11.1 %: vesicles of secretory system 11. 1 %: extracellular, including cell wall # indication for CG165528-01 is end (k=9) 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 9D.

Table 9D. Geneseq Results for NOV9a NOV9a Identities/ Geneseq Protein/Organism/Length [Patent Residues/Similarities for the Expect Identifier #, Date] Match Matched Region Value Residues AAM79855 Human protein SEQ ID NO 3501-1.. 1514 1465/1517 (96%) 0. 0 Homo sapiens, 1522 aa. 47.. 1522 1466/1517 (96%) [W0200157190-A2, 09-AUG-2001] AAM78871 Human protein SEQ ID NO 1533-147.. 1514 1326/1368 (96%) 0. 0 Homo sapiens, 1327 aa. 1.. 1327 1326/1368 (96%) [W0200157190-A2, 09-AUG-20011 AAE17600 Human extracellular messenger 19.. 1514 1041/1496 (69%) 0. 0 , (S)-2 protein-Homo sapiens, 16.. 1438 1210/1496 (80%) 1438 aa. [W0200194587-A2, 13-DEC-2001] AAU28190 Novel human secretory protein, Seq 16.. 1411 975/1426 (68%) 0. 0 ID No 359-Homo sapiens, 1712 aa. 14.. 1419 1162/1426 (81%) [W0200166689-A2, 13-SEP-2001] AAU14241 Human novel protein #112-Homo 414.. 1514 834/1101 (75%) 0. 0 sapiens, 1091 aa. 1.. 1091 960/1101 (86%) [W0200155437-A2, 02-AUG-2001]

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

Table 9E. Public BLASTP Results for NOV9a NOV9a Protein Identities/ Accession Protein/Organism/Length Match Similarities for the Value Number Residues Matched Portion Residues Q63372 Neurexin 1-alpha precursor 1.. 1514 1496/1514 (98%) 0. 0 (Neurexin I-alpha)-Rattus 1.. 1514 1506/1514 (98%) norvegicus (Rat), 1514 aa. Q28146. Neurexin 1-alpha precursor 1.. 1514 1503/1530 (98%) 0. 0 (Neurexin I-alpha)-Bos taurus 1.. 1530 1508/1530 (98%) (Bovine), 1530 aa. A40228 neurexin I-alpha precursor-rat, 1.. 1514 1489/1514 (98%) 0. 0 1507 aa. 1.. 1507 1499/1514 (98%) BAA25504 KIAA0578 protein-Homo sapiens 1.. 1514 1496/1514 (98%) 0. 0 (Human), 1542 aa (fragment). 47.. 1542 1496/1514 (98%) BAC41433 MKIAA0578 protein-Mus 1.. 1514 1468/1514 (96%) 0. 0 musculus (Mouse), 1525 aa 47.. 1525 1473/1514 (96%) (fragment).

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

Table 9F. Domain Analysis of NOV9a Identities/ Pfam Domain NOV9a Match Region Similarities Expect Value for the Matched Region laniinin_G 58 195 46/167 (28%) 4e-12 101/167 (60%) laminin G 312.. 378 23/81 (28%) 1. 4e-08 47/81 (58%) laminin_G 393 456 17/81 (21%) 0. 013 43/81 (53%) lamillin G 515 662 t 56/169 (33%) l. le-28 114/169 (67%) EGF 687.. 719 13/47 (28%) 0. 00049 24/47 (51%) lamininG 753.. 834 26/97 (27%) 0. 00016 59/97 (61%) s laniinin G 940 1071 43/163 (26%) 6. 4e-16 99/163 (61%) EGF 1094..1126 12/47 (26%) 0.00019 26/47 (55%) laminin G 1163 1236 22/87 (25%) 3.2e-07 48/87 (55%) Example 10.

The NOV10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.

Table 10A. NOV10 Sequence Analysis SEQ m NO : 39 1365 bp NOV1Oa, ACGCGTGGAGTCCTGCGGGCCGTGGCCACCCAGCAGCGCGGCGCCGTGTTCGTGGACAAG GAGA CG165666-01 DNA ACCTCACCATGCCGGGCCTCAGGTTCGACAACATCCAGGGAGATGCAGTTAAAGACTTGA TGCT TCGCTTTCTGGGTGAAAAAGCTGCAGCAAAGAGACAAGTCCTAAATGCCGACTCAGTGGA ACAA Sequence TCTTTTGTTGGATTGAAACAGCTAATCCTCTGGTTTGTCAGGCTGGCACTACTAGTGAAG TTGG GCCTTTTCCAGAATGCTGAGATGGAATTTGAACCCTTCGGAAATCTTGATCAGCCAGATC TTTA TTACGAGTACTACCCGCACGTGTACCCTGGGCGCAGGGGCTCCATGGTCCCCTTCTCGAT GCGC ATCTTGCACGCGGAGCTTCAGCAGTACCTGGGGAACCCACAGGAGTCGCTGGATAGACTG CACA AGGTGAAGACTGTCTGCAGCAAGATCCTGGCCAATTTGGAGCAAGGCTTAGCAGAAGACG GCGG CATGAGCAGCGTGACTCAGGAGGGCAGACAAGCCTCTATCCGGCTGTGGAGGTCACGTCT GGGC CGGGTGATGTACTCCATGGCAAACTGTCTGCTCCTGATGAAGGATTATGTGCTGGCCGTG GAGG CGTATCATTCGGTTATCAAGTATTACCCAGAGCAAGAGCCCCAGCTGCTCAGCGGCATCG GCCG GATTTCCCTGCAGATTGGAGACATAAAAACAGCTGAAAAGTATTTTCAAGACGTTGAGAA AGTA ACACAGAAATTAGATGGACTACAGGGTAAAATCATGGTTTTGATGAACAGCGCGTTCCTT CACC TCGGGCAGAATAACTTTGCAGAAGCCCACAGGTTCTTCACAGAGATCTTAAGGATGGATC CAAG AAACGCAGTGGCCAACAACAACGCTGCCGTGTGTCTGCTCTACCTGGGCAAGCTCAAGGA CTCC CTGCGGCAGCTGGAGGCCATGGTCCAGCAGGACCCCAGGCACTACCTGCACGAGAGCGTG CTCT TCAACCTGACCACCATGTACGAGCTGGAGTCCTCACGGAGCATGCAGAAGAAACAGGCCC TGCT GGAGGCTGTCGCCGGCAAGGAGGGGGACAGCTTCAACACACAGTGCCTCAAGCTGGCCTA GCTG CCTCCAACACACTACGTCAGAAGGACCCGGGTCTTTGAAACTGTGTCTTGAAGCTAATGT ATTA ATGTGACATGGAGGAACTCAATAAAACTCCTGCTTCACTGGTGTCTGCTGCGTGTCTTCT TGGT CCCAAGCCACGGCCCAGCCCAGGACTTCCCCGCAGTTGGTCGGCGTTCAGCCACGCAGTC CCTG CAGCTGGGTCACTGTTCATGA ORF Start : ATG at 73 ORF Stop : TAG at 1147 SEQ m NO :. 40 358 aaMW at 40766. 8kD NOV1 Oa, MPGLRFDNIQGDAVKDLMLRFLGEKAAAKRQVLNADSVEQSFVGLKQLILWFVRLALLVK LGLF CG165666-O1 QNAEMEFEPFGNLDQPDLYYEYYPHVYPGRRGSMVPFSMRILHAELQQYLGNPQESLDRL HKVK TVCSKILARLEQGLAEDGGMSSVTQEGRQASIRLWRSRLGRVKYSMANCLLLMKDYVLAV EAYH Protem Sequence SVIKYYPEQEPQLLSGIGRISLQIGDIKTAEKYFQDVEKVTQKLDGLQGKIMVLMNSAFL HLGQ NNFAEAHRFFTEILRMDPRNAVANNNAAVCLLYLGKLKDSLRQLEAMVQQDPRHYLHESV LFNL TTMYELESSRSMQKKQALLEAVAGKEGDSFNTQCLKLA

Further analysis of the NOVlOa protein yielded the following properties shown in Table lOB.

Table 10B. Protein Sequence Properties NOV10a Signal ? analysis : Cleavage site between residues 65 and 66 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 7; pos. chg 1; neg. chg 1 H-region: length 4; peak value-9.72 PSG score:-14. 12 GvH : von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1) : -6.97 possible cleavage site: between 58 and 59 >>> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0.5 : 1 Number of TMS (s) for threshold 0.5 : 1 INTEGRAL Likelihood =-5.47 Transmembrane 48-64 PERIPHERAL Likelihood = 3.87 (at 174) ALOM score:-5. 47 (number of TMSs : 1) MTOP : Prediction of membrane topology (Hartmann et al.) Center position for calculation: 55 Charge difference:-4. 0 C (-2. 0)-N (2. 0) N >= C: N-terminal side will be inside >>> membrane topology: type 2 (cytoplasmic tail 1 to 48) MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 1.36 Hyd Moment (95): 3.47 G content: 2 D/E content: 2 S/T content: 0 Score:-8. 05 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 15 LRFJDN NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues: 11. 5% NLS Score :-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus: PGLR KKXX-like motif in the C-terminus: CLKL SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: found KLKDSLRQL at 292 VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1 : none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 89 COIL : Lupas's algorithm to detect coiled-coil regions 218 I 0. 66 219 K 0. 67 220 T 0. 71 221 A 0.71 222 E 0.71 223 K 0. 71 224 Y 0.71 225 F 0. 71 226 Q 0. 71 227 D 0. 71 228 V 0.71 229 E 0.71 230 K 0. 71 231 V 0.71 232 T 0. 71 233 Q 0.71 234 K 0. 71 235 L 0.71 236 D 0. 71 237 G 0. 71 238 L 0.71 239 Q 0.71 240 G 0. 71 241 K 0. 71 242 1 0. 71 243 M 0. 71 244 V 0.71 245 L 0.71 246 M 0. 71 ; 247 N 0. 71 248 S 0.71 249 A 0.71 total: 32 residues Final Results (k = 9/23): 39. 1 %: mitochondrial 30. 4 %: cytoplasmic 8. 7 %: Golgi 8. 7 % : nuclear 8. 7 %: endoplasmic reticulum 4. 3 %: vacuolar >> indication for CG165666-01 is mit (k=23) A search of the NOV10a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table l OC.

Table 10C. Geneseq Resutts for NOV10a NOVlOa Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region AAB42120 Human ORFX ORF1884 polypeptide 18.. 358 341/379 (89%) 0. 0 sequence SEQ ID NO : 3768-Homo 1.. 379 341/379 (89%) sapiens, 379 aa. [W0200058473-A2, 05-OCT-2000] ABB90440 Human polypeptide SEQ ID NO 2816 1.. 347 329/385 (85%) 0. 0 - Homo sapiens, 449 aa. 41.. 425 335/385 (86%) [W0200190304-A2, 29-NOV-2001] ABP61860 Human polypeptide SEQ ID NO 214-96 358 263/263 (100%) e-148 Homo sapiens, 271 aa. 9.. 271 263/263 (100%) [US2002065394-A1, 30-MAY-2002] AAW73629 Human secreted protein clone 96.. 358 263/263 (100%) e-148 cd265 11-Homo sapiens, 271 aa. 9.. 271 263/263 (100%) [W09855614-A2, 10-DEC-1998] ABB65708 Drosophila melanogaster polypeptide 1 342 126/386 (32%) 4e-48 SEQ ID NO 23916-Drosophila 95.. 463 185/386 (47%) melanogaster, 484 aa. [W0200171042-A2, 27-SEP-2001] In a BLAST search of public sequence databases, the NOV10a protein was found to have homology to the proteins shown in the BLASTP data in Table 10D.

Table 10D. Public BLASTP Results for NOV10a NOVlb Protein Identities/ Accession Protein/Organism/Length Residues/similarities for the Expect Number Match Matched Portion Value Residues Q8WVT3 Similar to TPR-containing protein-1 358 357/396 (90%) 0. 0 Homo sapiens (Human), 735 aa. 340.. 735 358/396 (90°/a) Q8K2L8 Hypothetical protein-Mus 1.. 358 340/396 (85%) 0. 0 musculus (Mouse), 797 aa. 402.. 797 351/396 (87%) Q8WVW1 CGI-87 protein-Homo sapiens 18.. 358 341/379 (89%) 0. 0 (Human), 379 aa. 1.. 379 341/379 (89%) Q9Y395 CGI-87 protein-Homo sapiens 18.. 358 339/379 (89%) 0. 0 (Human), 379 aa. 1.. 379 340/379 (89%) Q8N9NO Hypothetical protein FLJ36862-1.. 278 276/322 (85%) e-149 Homo sapiens (Human), 696 aa. 323.. 644 277/322 (85oxo)

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

Table 10E. Domain Analysis of NOVlOa Identities/ Pfam Domain NOVlOa Match Region Similarities Expect Value for the Matched Region TPR 168.. 201 8/34 (24%) 0. 0053 23/34 (68-/o)

Example 11.

The NOV11 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11A.

Table llA. NOV11 Sequence Analysis Sequence CTTCAAGTGAACAGTTTGGCTATGCAGTGCAGCAGTTTATAAATCCAAAAGGCAACTGGT TACT GGTTGGTTCACCCTGGAGTGGCTTTCCTGAGAACCGAATGGGAGATGTGTATAAATGTCC TGTT GACCTATCCACTGCCACATGTGAAAAACTAAATTTGCAAACTTCAACAAGCATTCCAAAT GTTA CTGAGATGAAAACCAACATGAGCCTCGGCTTGATCCTCACCAGGAACATGGGAACTGGAG GTTT TCTCACATGTGGTCCTCTGTGGGCACAGCAATGTGGGAATCAGTATTACACAACGGGTGT GTGT TCTGACATCAGTCCTGATTTTCAGCTCTCAGCCAGCTTCTCACCTGCAACTCAGCCCTGC CCTT CCCTCATAGATGTTGTGGTTGTGTGTGATGAATCAAATAGTATTTATCCTTGGGATGCAG TAAA GAATTTTTTGGAAAAATTTGTACAAGGCCTGGATATAGGCCCCACAAAGACACAGGTGGG GTTA ATTCAGTATGCCAATAATCCAAGAGTTGTGTTTAACTTGAACACATATAAAACCAAAGAA GAAA TGATTGTAGCAACATCCCAGACATCCCAATATGGTGGGGACCTCACAAACACATTCGGAG CAAT TCAATATGCAAGAAAATATGCTTATTCAGCAGCTTCTGGTGGGCGACGAAGTGCTACGAA AGTA ATGGTAGTTGTAACTGACGGTGAATCACATGATGGTTCAATGTTGAAAGCTGTGATTGAT CAAT GCAACCATGACAATATACTGAGGTTTGGCATAGCAGTTCTTGGGTACTTAAACAGAAACG CCCT TGATACTAAAAATTTAATAAAAGAAATAAAAGCAATCGCTAGTATTCCAACAGAAAGATA CTTT TTCAATGTGTCTGATGAAGCAGCTCTACTAGAAAAGGCTGGGACATTAGGAGAACAAATT TTCA GCATTGAAGGTACTGTTCAAGGAGGAGACAACTTTCAGATGGAAATGTCACAAGTGGGAT TCAG TGCAGATTACTCTTCTCAAAATGATATTCTGATGCTGGGTGCAGTGGGAGCTTTTGGCTG GAGT GGGACCATTGTCCAGAAGACATCTCATGGCCATTTGATCTTTCCTAAACAAGCCTTTGAC CAAA TTCTGCAGGACAGAAATCACAGTTCATATTTAGGTTACTCTGTGGCTGCAATTTCTACTG GAGA AAGCACTCACTTTGTTGCTGGTGCTCCTCGGGCAAATTATACCGGCCAGATAGTGCTATA TAGT GTGAATGAGAATGGCAATATCACGGTTATTCAGGCTCACCGAGGTGACCAGATTGGCTCC TATT TTGGTAGTGTGCTGTGTTCAGTTGATGTGGATAAAGACACCATTACAGACGTGCTCTTGG TAGG TGCACCAATGTACATGAGTGACCTAAAGAAAGAGGAAGGAAGAGTCTACCTGTTTACTAT CAAA GAGGGCATTTTGGGTCAGCACCAATTTCTTGAAGGCCCCGAGGGCATTGAAAACACTCGA TTTG GTTCAGCAATTGCAGCTCTTTCAGACATCAACATGGATGGCTTTAATGATGTGATTGTTG GTTC ACCACTAGAAAATCAGAATTCTGGAGCTGTATACATTTACAATGGTCATCAGGGCACTAT CCGC ACAAAGTATTCCCAGAAAATCTTGGGATCCGATGGAGCCTTTAGGAGCCATCTCCAGTAC TTTG GGAGGTCCTTGGATGGCTATGGAGATTTAAATGGGGATTCGATCACCGATGTGTGTATTG GTGC CTTTGGACAAGTGGTTCAACTCTGGTCACAAAGTATTGCTGATGTAGCTATAGAAGCTTC ATTC ACACCAGAAAAAATCACTTTGGTCAACAAGAATGCTCAGATAATTCTCAAACTCTGCTTC AGTG CAAAGTTCAGACCTACTAAGCAAAACAATCAAGTGGCCATTGTATATAACATCACACTTG ATGC AGATGGATTTTCATCCAGAGTAACCTCCAGGGGGTTATTTAAAGAAAACAATGAAAGGTG CCTG CAGAAGAATATGGTAGTAAATCAAGCACAGAGTTGCCCCGAGCACATCATTTATATACAG GAGC CCTCTGATGTTGTCAACTCTTTGGATTTGCGTGTGGACATCAGTCTGGAAAACCCTGGCA CTAG CCCTGCCCTTGAAGCCTATTCTGAGACTGCCAAGGTCTTCAGTATTCCTTTCCACAAAGA CTGT GGTGAGGACGGACTTTGCATTTCTGATCTAGTCCTAGATGTCCGACRAATACCRGCTGCT CAAG AACAACCCTTTATTGTCAGCAACCAAAACAAAAGGTTAACATTTTCAGTAACGCTGAAAA ATAA AAGGGAAAGTGCATACAACACTGGAATTGTTGTTGATTTTTCAGAAAACTTGTTTTTTGC ATCA TTCTCCCTGCCGGTTGATGGGACAGAAGTAACATGCCAGGTGGCTGCATCTCAGAAGTCT GTTG CCTGCGATGTAGGCTACCCTGCTTTAAAGAGAGAACAACAGGTGACTTTTACTATTAACT TTGA CTTCAATCTTCAAAACCTTCAGAATCAGGCGTCTCTCAGTTTCCAGGCCTTAAGTGAAAG CCAA GAAGAAAACAAGGCTGATAATTTGGTCAACCTCAAAATTCCTCTCCTGTATGATGCTGAA ATTC ACTTAACAAAGGTAACAACAGGAAGTGTTCCAGTAAGCATGGCAACTGTAATCATCCACA TCCC TCAGTATACCAAAGAAAAGAACCCACTGATGTACCTAACTGGGGTGCAAACAGACAAGGC TGGT GACATCAGTTGTAATGCAGATATCAATCCACTGAAAATAGGACAAACATCTTCTTCTGTA TCTT TCAAAAGTGAAAATTTCAGGCACACCAAAGAATTGAACTGCAGAACTGCTTCCTGTAGTA ATGT TACCTGCTGGTTGAAAGACGTTCACATGAAAGGAGAATACTTTGTTAATGTGACTACCAG AATT TGGAACGGGACTTTCGCATCATCAACGTTCCAGACAGTACAGCTAACGGCAGCTGCAGAA ATCA ACACCTATAACCCTGAGATATATGTGATTGAAGATAACACTGTTACGATTCCCCTGATGA TAAT GAAACCTGATGAGAAAGCCGAAGTACCAACAGGAGTTATAATAGGAAGTATAATTGCTGG AATC CTTTTGCTGTTAGCTCTGGTTGCAATTTTATGGAAGCTCGGCTTCTTCAAAAGAAAATAT GAAA AGATGACCAAAAATCCAGATGAGATTGATGAGACCACAGAGCTCAGTAGCTGAACCAGCA GACC TACCTG ORF Start : ATG at 2 ORF Stop : TGA at 3443 SE... at 125495. 9kD s w NOV1la, MGPERTGAAPLPLLLVLALSQGILNCCLAYNVGLPEAKIFSGPSSEQFGYAVQQFINPKG NWLL CG165676-0 1 VGSPWSGFPENRMGDVYKCPVDLSTATCEKLNLQTSTS IPNVTEMKTNMSLGLIhTRNMGTGGF LTCGPLWAQQCGNQYYTTGVCSDISPDFQLSASFSPATQPCPSLIDVWVCDESNSIYPWD AVK Protein Sequence NFLEKFVQGLDIGPTKTQVGLIQYANNPRVVFNLNTYKTKEEMIVATSQTSQYGGDLTNT FGAI QYARKYAYSAASGGRRSATKVMWVTDGESHDGSMLKAVIDQCNNTDNILRFGIAVLGYLN RNAL DTKNLIKEIKAIASIPTERYFFNVSDEAALLEKAGTLGEQIFSIEGTVQGGDNFQMEMSQ VGFS ADYSSQNDILMLGAVGAFGWSGTIVQKTSHGHLIFPKQAFDQILQDRNHSSYLGYSVAAI STGE STHFVAGAPRANYTGQIVLYSVNENGNITVIQAHRGDQIGSYFGSVLCSVDVDKDTITDV LLVG Further analysis of the NOV1 la protein yielded the following properties shown in Table 11B.

Table 11B. Protein Sequence Properties NOVlla SignalP analysis: Cleavage site between residues 30 and 31 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length S ; pos. chg 1 ; neg. chg 1 H-region : length 30; peak value 9.82 PSG score: 5.42 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1): 1.12 possible cleavage site: between 22 and 23 >>> Seems to have a cleavable signal peptide (1 to 22) ALOM: Klein et al's method for TM region allocation Init position for calculation: 23 Tentative number of TMS (S) for the threshold 0.5 : 2 Number of TMS (S) for threshold 0.5 : 1 INTEGRAL Likelihood =-13.27 Transmembrane 1100-1116 PERIPHERAL Likelihood = 0.95 (at 943) ALOM score:-13. 27 (number of TMSs : 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 11 Charge difference:-2. 0 C (-l. O)-N (1.0) N >= C: N-terminal side will be inside > » membrane topology: type la (cytoplasmic tail 1117 to 1147) MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75): 10.10 Hyd Moment (95): 5.95 G content: 4 D/E content: 2 S/T content: 2 Score:-6. 90 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 15 ERTJGA NUCDISC : discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 7. 7% NLS Score:-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus: GPER none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR : N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail : 1128 Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication : cytoplasmic Reliability: 89 COIL : Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 55.6 %: endoplasmic reticulum 22.2 %: Golgi 11.1 %: plasma membrane 11.1 % : extracellular, including cell wall » indication for CG165676-01 is end (k=9) A search of the NOV1 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 11C.

Table 11C. Geneseq Results for NOVlla NOVlb Geneseq Protein/Organism/Length [Patent Residues/..,..... Expect Identifier #, Date] Match Value Residues Matched Region AAY07729 Armenian hamster alpha-2 integrin 1.. 1147 1139/1184 (96%) 0. 0 subunit protein-Cricetulus 1 1183 1141/1184 (96%) migratorius, 1183 aa. [W09916465-A1, 08-APR-1999] AAW70542 Integrin alpha-2 chain-Homo 1.. 1098 1095/1132 (96%) 0. 0 sapiens, 1367 aa. [W09832771-Al, 1.. 1132 1097/1132 (96%) 30-JULr1998] ABG29239 Novel human diagnostic protein 206.. 1147 937/976 (96%) 0. 0 #29230-Homo sapiens, 979 aa. 4.. 979'940/976 (96%) [W0200175067-A2, 11-OCT-2001] ABB90759 Human Tumour Endothelial Marker 23.. 1131 466/1182 (39%) 0. 0 polypeptide SEQ ID NO 250-Homo 22.. 1175 680/1182 (57%) sapiens, 1179 aa. [W0200210217-A2, 07-FEB-2002] ABB90788 Rat Tumour Endothelial Marker 1.. 1131 471/1202 (39%) 0. 0 polypeptide SEQ ID NO 307-Rattus 1 1176 678/1202 (56%) sp., 1180 aa. [W0200210217-A2, 07-FEB-2002]

In a BLAST search of public sequence databases, the NOV1 la protein was found to have homology to the proteins shown in the BLASTP data in Table 11D.

Table 11D. Public BLASTP Results for NOV11a NOVlb Protein Identities/ Accession Protein/Organism/Length Residues/Similarities for the Expect Number Residues Matched Portion Restftues AAM34795 Integrin, alpha2 (CD49B, alpha2 1 1147 1147/1181 (97%) 0. 0 subunit of VLA-2 receptor)-Homo 1.. 1181 1147/1181 (97%) sapiens (Human), 1181 aa. P17301 Integrin alpha-2 precursor (Platelet 1.. 1147 1146/1181 (97%) 0. 0 membrane glycoprotein la) (GPIa) 1.. 1181 1147/1181 (97%) (Collagen receptor) (VLA-2 alpha chain) (CD49b)-Homo sapiens (Human), 1181 aa. P53710 Integrin alpha-2 precursor (Platelet 12.. 1147 986/1170 (84%) 0. 0 membrane glycoprotein la) (GPIa) 1.. 1170 1069/1170 (91%) (Collagen receptor) (VLA-2 alpha chain) (CD49b)-Bos taurus (Bovine), 1170 aa (fragment). Q62469 Integrin alpha-2 precursor (Platelet 1.. 1147 945/1181 (80%) 0. 0 membrane glycoprotein la) (GPIa) 1.. 1178 1040/1181 (88%) (Collagen receptor) (VLA-2 alpha chain) (CD49b)-Mus musculus (Mouse), 1178 aa. 042094 ALPHA1 integrin-Gallus gallus 29 1131 456/1179 (38%) 0. 0 (Chicken), 1171 aa. 17.. 1167 671/1179 (56%) PFam analysis indicates that the NOV1 la protein contains the domains shown in the Table 11E.

Table 11E. Domain Analysis of NOV11a Mentities/ Identities/ Pfam Domain NOVIla Match Region Similarities Expect Value for the Matched Region FG-GAP 45.. 103 16/65 (25%) 6. 1e-05 38/65 (58%) vwa 174.. 357 71/208 (34%) 1. 8e-63 155/208 (75%) FG-GAP 434.. 486 16/64 (25%) 2. 2e-06 38/64 (59%) FG-GAP 488 549 21/66 (32%) 4. 7e-13 47/66 (71%) RG-GAP 551..610 24/67 (36%) 2. 2e-17 53/67 (79%) FG-GAP 615.. 667 16/66 (24%) 5e-08 42/66 (64%) integrin_A 1121 1135 7/15 (47%) 0. 0055 14/15 (93%) Example 12.

The NOV12 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 12A.

Table 12A. NOV12 Sequence Analysis SEQ ID NO : 43 1105 bp NOV1 2a, kGGAGGAAMACAAGTGTGTGTTGGGGGGAACAGGGGGAMAGCATTTTTGGTGGATGGTAT GA CG165719-04 DNA AGCCAGCCPTGGAAACTGCAGCCGAGGAAAATACTGAACAAAGCCAAGAGAGAAAAGTGA ACAG CAGAGCTGAAATGGAAATTGGCAGGTACCACTGGATGTACCCAGGCTCAAAGAACCACCA GTAC Sequence CATCCCGTGCCAACCCTGGGGGACAGGGCTAGCCCCTTGAGCAGTCCAGGCTGCTTTGAA TGCT GCATCAAGTGTCTGGGAGGAGTCCCCTACGCCTCCCTGGTGGCCACCATCCTCTGCTTCT CCGG GGTGGCCTTATTCTGCGGCTGTGGGCATGTGGCTCTCGCAGGCACCGTGGCGATTCTTGA GCAA CACTTCTCCACCAACGCCAGTGACCATGCCTTGCTGAGCGAGGTGATACAACTGATGCAG TATG TCATCTATGGAATTGCGTCCTTTTTCTTCTTGTATGGGATCATTCTGTTGGCAGAAGGCT TTTA CACCACAAGTGCAGTGAAAGAACTGCACGGTGAGTTTAMACAACCGCTTGTGGCCGATGC ATC AGTGGAATGTTCGTTTTCCTCACCTATGTGCTTGGAGTGGCCTGGCTGGGTGTGTTTGGT TTCT CAGCGGTGCCCGTGTTTATGTTCTACAACATATGGTCAACTTGTGAAGTCATCAAGTCAC CGCA GACCAACGGGACCACGGGTGTGGAGCAGATCTGTGTGGATATCCGACAATACGGTATCAT TCCT TGGAATGCTTTCCCCGGAAAAATATGTGGCTCTGCCCTGGAGAACATCTGCAACACAAAC GAGT TCTACATGTCCTATCACCTGTTCATTGTGGCCTGTGCAGGAGCTGGTGCCACCGTCATTG CCCT GATCCACTTCCTCATGATACTGTCTTCTAACTGGGCTTACTTAAAGGATGCGAGCAAAAT GCAG GCTTACCAGGATATCAAAGCAAAGGAAGAACAGGAACTGCAAGATATCCAGTCTCGGTCA AAAG CAACTCAATTCTTACACATAAATGTTTGCCAGAGTGTTTCGGCCGACGTATTTACAGCTC TG ACAAATCATCAGACAGC ORF Start : ATG at 61 ORF Stop : TAA at 1045 SEQ ID NO : 44 328 aa IMW at 36219. 3kD NOV12a, MKPAMETAAEENTEQSQERKVNSRAEMEIGRYHWMYPGSKNHQYHPVPTLGDRASPLSSP GCFE CG165 ? 19-04 CCIKCLGGVPYASLVATILCFSGVALFCGCGHVALAGTVAILEQHFSTNASDHALLSEVI QLMQ YVIYGIASFFFLYGIILLAEGFYTTSAVKELHGEFKTTACGRCISGMFVFLTYVLGVAWL GVPG ProteinSequence FSAVPVFMFYNIWSTCEVIKSPQTNGTTGVEQICVDIRQYGIIPWNAFPGKICGSALENI CNTN EFYMSYHLFIVACAGAGATVIALIHFLMILSSNWAYLKDASKMQAYQDIKAKEEQELQDI QSRS KEQLNSYT L

SEQ ID NO : 45 0 1 133 bP ... e... NOV12b, AGTGTGTGTTGGGGGGAACAGGGGGAAAAGCATTTTTGGTGGATGGTATGA CG165719-02 DNA AGCCAGCCATGGAAACTGCAGCCGAGGAAAATACTGAACAAAGCCAAGAGAGAAAAGTGA ACAG CAGAGCTGAAATGGAAATTGGCAGGTACCACTGGATGTACCCAGGCTCAAAGAACCACCA GTAC Sequence CATCCCGTGCCAACCCTGGGGGACAGGGCTAGCCCCTTGAGCAGTCCAGGCTGCTTTGAA TGCT GCATCAAGTGTCTGGGAGGAGTCCCCTACGCCTCCCTGGTGGCCACCATCCTCTGCTTCT CCGG GGTGGCCTTATTCTGCGGCTGTGGGCATGTGGCTCTCGCAGGCACCGTGGCGATTCTTGA GCAA CACTTCTCCACCAACGCCAGTGACCATGCCTTGCTGAGCGAGGTGATACAACTGATGCAG TATG TCATCTATGGAATTGCGTCCTTTTTCTTCTTGTATGGGATCATTCTGTTGGCAGAAGGCT TTTA CACCACAAGTGCAGTGAAAGAACTGCACGGTGAGTTTAAAACAACCGCTTGTGGCCGATG CATC AGTGGAATGTTCGTTTTCCTCACCTATGTGCTTGGAGTGGCCTGGCTGGGTGTGTTTGGT TTCT CAGCGGTGCCCGTGTTTATGTTCTACAACATATGGTCAACTTGTGAAGTCATCAAGTCAC CGCA GACCAACGGGACCACGGGTGTGGAGCA. GATCTGTGTGGATATCCGACAATACGGTATCATTCCT TGGAATGCTTTCCCCGGAAAAATATGTGGCTCTGCCCTGGAGAACATCTGCAACACAAAC GAGT TCTACATGTCCTATCACCTGTTCATTGTGGCCTGTGCAGGAGCTGGTGCCACCGTCATTG CCCT GCTGATCTACATGATGGCTACTACATATAACTATGCGGTTTTGAAGTTTAAGAGTCGGGA AGAT TGCTGCACTAAATTCTAAATTGCATAAGGAGTTTTAGAGAGCTATGCTCTGTAGCATGAA ATAT CACTGACACTCCAGACTAAAGCAGAGTCTAGGTTTCTGCAATTTGTTACAGTAATTTGTA ATAG CTTTGTAACTCACCTGCATGTAGATAATAAGATGACTACTGTACA ORF or A ME 01 _ L SEQIDNO : 46 305aa MWat33537. 5kD s NOV12b, MKPAMETAAEENTEQSQERKVNSRAEMEIGRYHNMYPGSKNHQYHPVPTLGDRASPLSSP GCFE CG165719-02 CCIKCLGGVPYASLVATILCFSGVALFCGCGHVALAGTVAILEQHFSTNASDHALLSEVI QLMQ YVIYGIASFFFLYGIILLAEGFYTTSAVKELHGEFKTTACGRCISGMFVFLTYVLGVAWL GVFG Protein Sequence FSAVPVFMFYNIWSTCEVIKSPQTNGTTGVEQICVDIRQYGIIPWNAFPGKICGSALENI CNTN EEYMSYHLFISTACAGAGA1VIAILIYMMATTYNYAVIKFKSREDCCTKF

SEQ ID NO : 47 11182 w w NOV12c, AAGTGTGTGTTGGGGGGAACAGGGGGAAAAGCATTTTTGGTGGATGGTATGA CG165719-03 DNA AGCCAGCCATGGAAACTGCAGCCGAGGAAAATACTGAACAAAGCCAAGAGAGAAAAGGCT GCTT TGAATGCTGCATCAAGTGTCTGGGAGGAGTCCCCTACGCCTCCCTGGTGGCCACCATCCT CTGC Sequence TTCTCCGGGGTGGCCTTATTCTGCGGCTGTGGGCATGTGGCTCTCGCAGGCACCGTGGCG ATTC TTGAGCAACACTTCTCCACCAACGCCAGTGACCATGCCTTGCTGAGCGAGGTGATACAAC TGAT GCAGTATGTCATCTATGGAATTGCGTCCTTTTTCTTCTTGTATGGGATCATTCTGTTGGC AGAA GGCTTTTACACCACAAGTGCAGTGAAAGAACTGCACGGTGAGTTTAAAACAACCGCTTGT GGCC GATGCATCAGTGGAATGTTCGTTTTCCTCACCTATGTGCTTGGAGTGGCCTGGCTGGGTG TGTT TGGTTTCTCAGCGGTGCCCGTGTTTATGTTCTACAACATATGGTCAACTTGTGAAGTCAT CAAG TCACCGCAGACCAACGGGACCACGGGTGTGGAGCAGATCTGTGTGGATATCCGACAATAC GGTA TCATTCCTTGGAATGCTTTCCCCGGAAAAATATGTGGCTCTGCCCTGGAGAACATCTGCA ACAC AAACGAGTTCTACATGTCCTATCACCTGTTCATTGTGGCCTGTGCAGGAGCTGGTGCCAC CGTC ATTGCCCTGATCCACTTCCTCATGATACTGTCTTCTAACTGGGCTTACTTAAAGGATGCG AGCA AAATGCAGGCTTACCAGGATATCAAAGCAAAGGAAGAACAGGAACTGCAAGATATCCAGT CTCG GTCAAAAGAACAACTCAATTCTTACACATAAATGTTTGCCAGAGTGTTTCGGCCGACGTA TTTA CAGCTCTGACAAATCATCAGACAGCTGCTCTGCAGTACAGATGTGTATCCCACCAAACTA ATGT AGATGTACAAACACTTCACTGTCTGTCTCAAGCTGCTGGGATGTATCTCTAGGAAAACCT TCCA GTGGGTAAATCTTTTTCTTTAGAACAAATATTGGAGGTTCATGTTGCCCCATTTAAAGGG CACA CTTTTAQAATGATCGTCATACTTTGGGAT ORF Start : ATG at 61 ORF Stop : TAA at 925

SEQ ID NO : 48 288 aa MW at 31670. 3kD --- NOV12c, MKPAMETAAEENTEQSQERKGCFECCIKCLGGVPYASLVATILCFSGVALFCGCGHVALA GTVA CG165719-03 ILEQHFSTNASDHAILSEVIQLMQYVIYGIASFFFLYGIILLAEGFYTTSAVKELHGEFK TTAC GRCISGMFVFLTYVLGVAWLGVFGFSAVPVFMFYNIWSTCEVIKSPQTNGTTGVEQICVD IRQY Protein Sequence GIIPWNAFPGKICGSALENICNTNEFYMSYHLFIVACAGAGATVIALIHFLMILSSNWAY LKDA SKMQAYQDIKAKEEQELQDIQSRSKEQLNSYT s SEQ ID NO : 49 1302 bp NOV12d, AGGAGGAAAAACAAGTGTGTGTTGGGGGGAACAGGGGGAAAAGCATTTTTGGTGGATGGT ATGA , AGCQGCQTGGAAACTGCAGCCGAGGAAAATACTGAAQAGCQAGAGAGAAAAGTGAACAG CAGAGCTGAAATGGAAATTGGCAGGTACCACTGGATGTACCCAGGCTCAAAGAACCACCA GTAC Sequence CATCCCGTGCCAACCCTGGGGGACAGGGCTAGCCCCTTGAGCAGTCCAGGCTGCTTTGAA TGCT GCATCAAGTGTCTGGGAGGAGTCCCCTACGCCTCCCTGGTGGCCACCATCCTCTGCTTCT CCGG GGTGGCCTTATTCTGCGGCTGTGGGCATGTGGCTCTCGCAGGCACCGTGGCGATTCTTGA GCAA CACTTCTCCACCAACGCCAGTGACCATGCCTTGCTGAGCGAGGTGATACAACTGATGCAG TATG TCATCTATGGAATTGCGTCCTTTTTCTTCTTGTATGGGATCATTCTGTTGGCAGAAGGCT TTTA CACCACAAGTGCAGTGAAAGAACTGCACGGTGAGTTTAAAACAACCGCTTGTGGCCGATG CATC GTGGAATGTTCGTTTTCCTCACCTATGTGCTTGGAGTGGCCTGGCTGGGTGTGTTTGGTT TCT CAGCGGTGCCCGTGTTTATGTTCTACAACATATGGTCAACTTGTGAAGTCATCAAGTCAC CGCA GACCAACGGGACCACGGGTGTGGAGCAGATCTGTGTGGATATCCGACAATACGGTATCAT TCCT TGGAATGCTTTCCCCGGAAAAATATGTGGCTCTGCCCTGGAGAACATCTGCAACACAAAC GAGT TCTACATGTCCTATCACCTGTTCATTGTGGCCTGTGCAGGAGCTGGTGCCACCGTCATTG CCCT GATCCACTTCCTCATGATACTGTCTTCTAACTGGGCTTACTTAAAGGATGCGAGCAAAAT GCAG GCTTACCAGGATATCAAAGCAAAGGAAGAACAGGAACTGCAAGATATCCAGTCTCGGTCA AAAG AACAACTCAATTCTTACACATAAATGTTTGCCAGAGTGTTTCGGCCGACGTATTTACAGC TCTG ACAAATCATCAGACAGCTGCTCTGCAGTACAGATGTGTATCCCACCAAACTAATGTAGAT GTAC AAACACTTCACTGTCTGTCTCAAGCTGCTGGGATGTATCTCTAGGAAAACCTTCCAGTGG GTAA ATCTTTTTCTTTAGAACRAATATTGGAGGTTCATGTTGCCCCATTTAAAGGGCACACTTT TACA AATGATCGTCATAGTTTGGGAT 5 ORF Start : ATG at 61 ORF Stop : TAA at 1045

SEQ IID NO : 50 1328 aa IMW at 36219. 3kD - NOV12d, MK'AEENTEQSQERKVNSRAEMEIGRYHWMYPGSKNHQYHPVPTLGDRASPLSSPGCFE CG165719-01 CCIKCLGGVPYASLVATILCFSGVALFCGCGHVALAGTVAILEQHFSTNASDHALLSEVI QLMQ YVIYGIASFFFLYGIILLAEGFYTTSAVKELHGEFKTTACGRCISGMFVPLTYVLGVAWL GVFG ProteinSequence FSAVPVFMFYNIWSTCEVIKSPQTNGTTGVEQICVDIRQYGIIPWNAFPGKICGSALENI CNTN EFYMSYHLFIVACAGAGATVIALIHFLMILSSNNAYLKDASKMQAYQDIKAKEEQELQDI QSRS KEQLNSYT' KEQLNSYT SEQ ID NO : 51 NOV1 2e, AGGAGGAAAAl&CAAGTGTGTGTTGGGGGGAACAGGGGGAAhAGCATTTTTGGTGGATGG TATGA CG165719 05 DNA AGCCAGCCATGGAAACTGCAGCCGAGGAAAATACTGAACAAAGCCAAGAGAGAAAAGGCT GCTT TGAATGCTGCATCAAGTGTCTGGGAGGAGTCCCCTACGCCTCCCTGGTGGCCACCATCCT CTGC Sequence TTCTCCGGGGTGGCCTTATTCTGCGGCTGTGGGCATGTGGCTCTCGCAGGCACCGTGGCG ATTC TTGAGCAACACTTCTCCACCAACGCCAGTGACCATGCCTTGCTGAGCGAGGTGATACAAC TGAT GCAGTATGTCATCTATGGAATTGCGTCCTTTTTCTTCTTGTATGGGATCATTCTGTTGGC AGAA GGCTTTTACACCACAAGTGCAGTGAAAGAACTGCACGGTGAGTTTAAAACAACCGCTTGT GGCC GATGCATCAGTGGAATGTTCGTTTTCCTCACCTATGTGCTTGGAGTGGCCTGGCTGGGTG TGTT TGGTTTCTCAGCGGTGCCCGTGTTTATGTTCTACAACATATGGTCAACTTGTGAAGTCAT CAAG TCACCGCAGACCAACGGGACCACGGGTGTGGAGCAGATCTGTGTGGATATCCGACAATAC GGTA TCATTCCTTGGAATGCTTTCCCCGGAAAAATATGTGGCTCTGCCCTGGAGMLCATCTGCR ACAG AAACGAGTTCTACATGTCCTATCACCTGTTCATTGTGGCCTGTGCAGGAGCTGGTGCCAC CGTC ATTGCCCTGCTGATCTACATGATGGCTACTACATATAACTATGCGGTTTTGAAGTTTAAG AGTC GGGAAGATTGCTGCACTAAATTCTAAATTGCATAAGGAGTTTTAGAGAGCTATGCTCTGT AGCA TGAAATATCACTGACACTCCAGAAAGGGCGATT ORF Start : ATG at 61 ORF Stop : TAA at 856 SEQ ID NO : 52 9. 5kD I-RL NOV12e, EEENTEQSQERKGCFECCIKCLGGVPYASLVATILCFSGVA FCGCGHFAiAGTVA CG165719-05 ILEQHFSTNASDHALLSEVIQLMQYVIYGIASFFFLYGIILLAEGFYTTSAVKELHGEFK TTAC GRCISGMFVFLTYVLGVAWLGVFGFSAVPVFMFYNIWSTCEVIKSPQTNGTTGVEQICVD IRQY GIIPWNAFPGKICGSALENICNTNEFYMSYHLFIVACAGAGATVIALLIYMh7ATTYNYA VLKFK SREDCCTKF Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 12B.

Table 12B. Comparison of NOV12a against NOV12b through NOV12e.

Protein Sequence NOV12a Residues/Identities/ Match Residues Similarities for the Matched Region NOV12b 1.. 298 285/298 (95%) 1.. 298 291/298 (97%) NOV12c 1.. 328 288/328 (87%) 1.. 288 288/328 (87%) NOV12d 1.. 328 328/328 (100%) 1.. 328 328/328 (100%) NOV12e 1.. 298 245/298 (82%) 1.. 258 251/298 (84%) Further analysis of the NOV12a protein yielded the following properties shown in Table 12C.

Table 12C. Protein Sequence Properties NOV12a Signal ? analysis: No Known Signal Sequence Indicated PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 11 ; pos. chg 1; neg. chg 3 H-region: length 2 ; peak value 0. 00 PSG score:-4. 40 GvH : von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-10. 13 possible cleavage site: between 60 and 61 >>> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0. 5: 4 INTEGRAL Likelihood =-6.26 Transmembrane 78-94 INTEGRAL Likelihood =-6.74 Transmembrane 130-146 INTEGRAL Likelihood =-5.15 Transmembrane 175-191 INTEGRAL Likelihood =-8.17 Transmembrane 264-280 PERIPHERAL Likelihood = 3.07 (at 195) ALOM score :-8. 17 (number of TMSs : 4) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 85 Charge difference: 0.0 C (0. 0)-N (0. 0) N >= C: N-terminal side will be inside >>> membrane topology: type 3a MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75): 7.96 Hyd Moment (95): 11.32 G content: 0 D/E content: 2 S/T content: 1 Score:-5. 88 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite : none content of basic residues: 6.1% NLS Score :-0. 47 KDEL : ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL : peroxisomal targeting signal in the C-terminus : none PTS2 : 2nd peroxisomal targeting signal : none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif : type 1: none type 2: none NMYR : N-myristoylation pattern: none Prenylation motif: none memYQRL : transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs : none checking 71 PROSITE ribosomal protein motifs: none none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication : cytoplasmic Reliability: 94.1 COIL : Lupas's algorithm to detect coiled-coil regions 290 A 0. 60 291 Y 0.75 292 L 0. 75 293 K 0. 88 294 D 0. 93 295 A 0. 98 296 S 0.99 297 K 1.00 298 M 1.00 299 Q 1.00 300 A 1.00 301 Y 1.00 302 Q 1.00 303 D 1.00 304 1 1.00 305 K 1.00 306 A 1.00 307 K 1.00 308 E 1.00 309 E 1. 00 310 Q 1.00 311 E 1.00 312 L 1.00 313 Q 1.00 314 D 1.00 315 1 1.00 316 Q 1.00 317 S 1.00 318 R 1.00 319 S 1.00 320 K 1.00 321 E 1. 00 322 Q 1.00 323 L 1.00 324 N 1.00 325 S 0.97 326 Y 0. 96 327 T 0. 87 total: 38 residues -------------------------- Final results (k = 9/23) : 55.6 % : endoplasmic reticulum 44. 4 % : mitochondrial # indication for CG165719-04 is end (k=9) A search of the NOV12a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 12D.

Table12D. Genese

NOV12a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABG70364 Novel human thrombopoietin variant 27.. 298 259/272 (95%) e-155 protein, NV-23-Homo sapiens, 279 1.. 272 265/272 (97%) aa. [US2002068342-A1, 06-JUN-2002] AAY09510 Human M6bl protein-Homo sapiens, 1.. 298 245/298 (82%) e-138 265 aa. [W09921982-A1, 1.. 258 251/298 (84%) 06-MAY-1999] AAW39215'Human M6 protein-Homo sapiens, 61.. 328 155/276 (56%) 2e-86 278 aa. [JP10014577-A, 20-JAN-1998] 13.. 278 209/276 (75%) ABG02005 Novel human diagnostic protein #1996 49.. 294 134/246 (54%) le-76 - Homo sapiens, 541 aa. 289.. 533 173/246 (69%) [W0200175067-A2, 11-OCT-2001] AAR95171 Murine CNS myelin membrane 61.. 294 130/234 (55%) 8e-76 proteolipid protein isoform DM20-2.. 234 169/234 (71%) Mus musculus, 242 aa. [EP685558-Al, 06-DEC-1995] In a BLAST search of public sequence databases, the NOV12a protein was found to have homology to the proteins shown in the BLASTP data in Table 12E.

Table 12E. Public BLASTP Results for NOV12a NOVl2a Protein Identities/ Accession Protein/Organism/Length Residues/Similarities for the Expect T ivjL<ncn n 11 'value Number Residues Matched Portion Value Residues Q8N956 Hypothetical protein FLJ38338-1 328 328/328 (100%) 0. 0 Homo sapiens (Human), 328 aa. 1.. 328 328/328 (100%) Q9JI65 Neuronal membrane glycoprotein 1.. 328 321/328 (97%) 0. 0 M6-B-Mus musculus (Mouse), 328 1.. 328 324/328 (97%) aa. P35803 Neuronat membrane glycoprotein 1.. 328 284/328 (86%) e-162 M6-b (M6b)-Mus musculus 1 288 287/328 (86%) (Mouse), 288 aa. Q98ST3 Myelin PLP-related membrane 61.. 328 237/268 (88%) e-141 protein DM gammal-Xenopus 2.. 269 250/268 (92%) laevis (African clawed frog), 269 aa. Q8UUS8 DMgamma2-Brachydanio rerio 61 328 218/268 (81%) e-132 (Zebrafish) (Danio rerio), 268 aa. 2..268 244/268 (90%) PFam analysis indicates that the NOV12a protein contains the domains shown in the Table 12F.

I Table 12F. Domain Analysis of NOV12a

Identities/ Pfam Domain NOV12a Match Region Similarities Expect Value Pfam Domain NOVl2a Match Region Sinailarities Expect Value for the Matched Region Myelin PLP 61 305 175/288 (61%) 2. 3e-196 243/288 (84%) Example 13.

The NOV13 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 13A.

Table 13A. NOV13 Sequence Analysis SEQ ID NO : 53 NOV13a, TTTGATCTGAAGACTAGGGGACAATGGATATCATAGAGACAGCAAAACTTGAAGAACATT TGGA CG167488-02 DNA AAATCkACCCAGTI-ATCCTACGAACACTTATGCAAGACCCGCTGAACCTGTTGAAGAAG AAAAC iu/o-u j ( (rF (ccQ (GCTTATC (GTGGGCTGCGRRARGGC&CC&&AA>ACC AAAAATGGCAATGGTAAACCCAAGAGCTTATCCAGTGGGCTGCGAAAAGGCACCAAAAAG TACC SeCuence CGGACTATATCCAAATTGCTATGCCCACTGAATCAAGGAACAAATTTCCACTAGAGTGGT GGAA AACGGGCATTGCCTTCATATATGCAGTTTTCAACCTCGTCTTGACAACCGTCATGATCAC AGTT GTACATGAGAGGGTCCCTCCCAAGGAGCTTAGCCCTCCACTCCCAGACAAGTTTTTTGAT TACA TTGATAGGGTGAAATGGGCATTTTCTGTATCAGAAATAAATGGGATTATATTAGTTGGAT TATG GATCACCCAGTGGCTGTTTCTGAGATACAAGTCAATAGTGGGACGCAGATTCTGTTTTAT TATT GGAACTTTATACCTGTATCGCTGCATTACAATGTATGTTACTACTCTACCTGTGCCTGGA ATGC ATTTCCAGTGTGCTCCAAAGCTCAATGGAGACTCTCAGGCAAAAGTTCAACGGATTCTAC GATT GATTTCTGGTGGTGGATTGTCCATAACTGGATCACATATCTTATGTGGAGACTTCCTCTT CAGC GGTCACACGGTTACGCTGACACTGACTTATTTGTTCATCAAAGAATATTCGCCTCGTCAC TTCT GGTGGTATCATTTAATCTGCTGGCTGCTGAGTGCTGCCGGGATCATCTGCATTCTTGTAG CACA CGAACACTACACTATCGATGTGATCATTGCTTATTATATCACAACACGACTGTTTTGGTG GTAC CATTCAATGGCCAATGAAAAGAACTTGAAGGTCTCTTCACAGACTAATTTCTTATCTCGA GCAT GGTGGTTCCCCATCTTTTATTTTTTTGAGAAAAATGTACAAGGCTCAATTCCTTGCTGCT TCTC CTGGCCGCTGTCTTGGCCTCCTGGCTGCTTCAAATCATCATGCAAAAAGTATTCACGGGT TCAG AAGATTGGTGAAGACAATGAGAAATCGACCTGAGGAGCAAAACAAAGGCATCAGCTCTTA CACC GAGTTAACGCTGTAACCAAAGAAGGGCGATTCCAGCACACTGCGC ORF Start : ATG at 24 ORF Stop : TGA at 1119 SEQ ID NO : 54 365 aa MW at 42279. 71D NOV13a, MDIIETAKLEEHLENQPSDPTNTYARPAEPVEEENKNGNGKPKSLSSGLRKGTKKYPDYI QIAM CG167488 02 PTESRNKFPLEWWKTGIAFIYAVFNLVLTTVMITVVHERVPPKELSPPLPDKFFDYIDRV KWAF SVSEINGIILVGLWITQWLFLRYKSIVGRRFCFIIGTLYLYRCITMYVTTLPVPGMHFQC APKL Protein NGDSQAKVQRILRLISGGGLSITGSHILCGDFLFSGHTVTLTLTYLFIKEYSPRHFWNYH LICN LLSAAGIICILVAHEHYTIDVIIAYYITTRLFWWYHSMANEKNLKVSSQTNFLSRAWWFP IFYF FEKNVQGSIPCCFSWPLSWPPGCFKSSCKKYSRVQKIGEDNEKST, SEQ ID NO : 55 11893 bp I NOV13b, CGGAGCTACCTTATAAAGACCATCTGTACATCCACTGTGAAATGGAGTTTCAAAATCACA AGCT CG167488-01 DNA TCTTTCCCACATGAACATAAGACTAGGAGCACATATGGAAGAGTAAAGTTGAAGGGAATT TGGA TGATGATTTGGCAAGATGCTGTGGGATAGTAACATCTTTTTGAGGGAAGAATTGGCTTCC TTTC Sequence TTGAAAGTGGTGAAGGTACAGCATATAGCTGCATGGAAGAAACAGTAATCGGATGGCTAC CTTC TACATTTTGTATTAGGAAACAAAGTCCATTGTAAGAGTCCATGTTGATCTTGGAAATAGA AGGA TTGAAAAAAGCTAAATTTCCACAAAGAACAAGAACTTGACCATCTCCTTTTTGATCTGAA GACT AGGGGACAATGGATATCATAGAGACAGCAAAACTTGAAGAACATTTGGAAAATCAACCCA GTGA TCCTACGAACACTTATGCAAGACCCGCTGAACCTGTTGAAGAAGAAAACAAAAATGGCAA TTGG TAAACCCAAGAGCTTATCCAGTGGGCTGCGAAAAGGCACCAAAAAGTACCCGGACTATAT CCAA ATTGCTATGCCCACTGAATCAAGGAACAAATTTCCACTAGAGTGGTGGAAAACGGGCATT GCCT TCATATATGCAGTTTTCAACCTCGTCTTGACAACCGTCATGATCACAGTTGTACATGAGA GGGT CCCTCCCAAGGAGCTTAGCCCTCCACTCCCAGACAAGTTTTTTGATTACATTGATAGGGT GAAA TGGGCATTTTCTGTATCAGAAATAAATGGGATTATATTAGTTGGATTATGGATCACCCAG TGGC TGTTTCTGAGATACAAGTCAATAGTGGGACGCAGATTCTGTTTTATTATTGGAACTTTAT ACCT GTATCGCTGCATTACAATGTATGTTACTACTCTACCTGTGCCTGGAATGCATTTCCAGTG TGCT CCAAAGCTCAATGGAGACTCTCAGGCAAAAGTTCAACGGATTCTACGATTGATTTCTGGT GGTG GATTGTCCATAACTGGATCACATATCTTATGTGGAGACTTCCTCTTCAGCGGTCACACGG TTAC GCTGACACTGACTTATTTGTTCATCAAAGAAGATTCGCCTCGTCACTTCTGGTGGTATCA TTTA ATCTGCTGGCTGCTGAGTGCTGCCGGGATCATCTGCATTCTTGTAGCACACGAACACTAC ACTA TCGATGTGATCATTGCTTATTATATCACAACACGACTGTTTTGGTGGTACCATTCAATGG CCAA TGAAAAGAACTTGAAGGTCTCTTCACAGACTAATTTCTTATCTCGAGCATGGTGGTTCCC CATC TTTTATTTTTTTGAGAAAAATGTACAAGGCTCAATTCCTTGCTGCTTCTCCTGGCCGCTG TCTT GGCCTCCTGGCTGCTTCAAATCATCATGCAAAAAGTATTCACGGGTTCAGAAGATTGGTG AAGA CAATGAGAAATCGACCTGAGGAGCAAAACAAAGGCATCAGCTCTTACACCAAAAGAGTTA ACGC TGTAACCAAAGGTATAGTTTTGTTTTTTATTTTAGGAGAACTGACTGGTAAATGAAGAAA TGGA CCAAATTTTGTGTAAACGATTAGAAAGATGAACAAAGTATTGCCCTTTGACTGGTTTTCT TCTT CATCCTGAGAAAGATACATTCTCTTGCAGCTCTTCATTCATTGGTGACAAGCCCCCACCC CGGG ACTTTACTAATGAGCTTGTTAAAGAGGTGCCAAAGAACATATTCCTCCTTTCTTTATTCT TTCT CCACCAAAACCCTCTACTTCAGAATTTTTTCAGGATATTTTTCAGCCCAAGGTCAGAAGA ATGT GTTAATATTTTAAATAAAATATCTGGACATCTACAAA ORF Start : ATG at 463 ORF Stop : TGA at 1489

SEQ m NO : 56 342, MW at 39679. 3kD NOV13b MQDPLNLLKKKTKMAIGKPKSLSSGLRKGTKKYPDYIQIAMPTESRNKFPLEWWKTGIAF IYAV , FNLVLTTVMITVVHERVPPKELSPPLPDKFFDYIDRVKWAFSVSEINGIILVGLWITQWL FLRY KSIVGRRFCFIIGTLYLYRCITMYVTTLPVPGMHFQCAPKLNGDSQAKVQRILRLISGGG LSIT ProteinSequence GSHILCGDFLFSGHTVTLTLTYLFIKEDSPRHFWWYHLICWLLSAAGIICILVAHEHYTI DVII AYYITTRLFWWYHSMANEKNLKVSSQTNFLSRAWWFPIFYFFEKNVQGSIPCCFSWPLSW PPGC 'FKSSCKKYSRVQKIGEDNEKST Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 13B. m Protein Sequence NOV13a Residues/Identities/ Match Residues Similarities for the Matched Region NOV13b 27.. 365 327/339 (96%) 4.. 342 331/339 (97%) Further analysis of the NOV13a protein yielded the following properties shown in Table 13C.

Table 13C. Protein Sequence Properties NOV13a SignalP analysis: No Known Signal Sequence Indicated PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 11; pos. chg 1; neg. chg 4 H-region: length 2; peak value 0.00 PSG score : -4. 40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-13. 01 possible cleavage site: between 25 and 26 >>> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0.5 : 3 INTEGRAL Likelihood =-6. 21 Transmembrane 81-97 INTEGRAL Likelihood =-1.59 Transmembrane 133-149 INTEGRAL Likelihood =-9.98 Transmembrane 253-269 PERIPHERAL Likelihood = 1.11 (at 160) ALOM score:-9. 98 (number of TMSs : 3) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 88 Charge difference:-1. 5 C (-0. 5) -N (1. 0) N >= C: N-terminal side will be inside > » membrane topology: type 3a MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75): 4.36 Hyd Moment (95): 8.65 G content: 0 D/E content: 2 S/T content: 0 Score:-6. 99 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues: 10. 7% NLS Score :-0. 47 KDEL : ER retention motif in the C-terminus: none ER Membrane Retention Signals: KKXX-like motif in the C-terminus: NEKS SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif : none memYQRL : transport motif from cell surface to Golgi: none Tyrosines in the tail : none Pileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN : Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability : 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues ------------------------- Final Results (k = 9/23) : 55.6 % : endoplasmic reticulum 33.3 %: mitochondrial 11.1 %: vesicles of secretory system » indication for CG167488-02 is end (k=9) A search of the NOVI 3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 13D.

Table 13D. Geneseq Results for NOV13a

NOV13a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABB09578 Human cytochrome constitutive protein 1.. 354 215/354 (60%) e-130 45-Homo sapiens, 413 aa. 61.. 409 272/354 (76%) [CN1333280-A, 30-JAN-2002] AAM41726 Human polypeptide SEQ ID NO 6657-1.. 354 215/354 (60%) e-130 Homo sapiens, 430 aa. 78.. 426 272/354 (76%) [W0200153312-Al, 26-JUL-2001] AAM39940 Human polypeptide SEQ ID NO 3085-1.. 354 215/354 (60%) e-130 Homo sapiens, 413 aa. 61.. 409 272/354 (76%) [W0200153312-A1, 26-JUL-2001]. AAG81320 Human AFP protein sequence SEQ ID 136.. 354 150/219 (68%) 2e-93 NO : 158-Homo sapiens, 222 aa. 1.. 218 181/219 (82%) [W0200129221-A2, 26-APR-2001] ABB60637 Drosophila melanogaster polypeptide 97.. 330 107/244 (43%) 5e-57 SEQ ID NO 8703-Drosophila 1.. 240 155/244 (62%) melanogaster, 384 aa. [W0200171042-A2, 27-SEP-2001] In a BLAST search of public sequence databases, the NOV1 3a protein was found to<BR> <BR> have homology to the proteins shown in the BLASTP data in Table 13E.

Table 13E. Public BLASTP Results for NOV13a NOVX Protein Identities/ Accession Protein/Organism/Length Match Similarities for the Value T Match ni. . Vatue Number Residues Matched Portion Restdues Q8NHU3 Similar to putative-Homo sapiens 1.. 365 365/365 (100%) 0. 0 (Human), 365 aa. 1.. 365 365/365 (100%) Q9D4B1 4933405A16Rik protein-Mus 96 365 260/270 (96%) e-162 musculus (Mouse), 270 aa. 1.. 270 267/270 (980/.) Q8VCQ6 Hypothetical 48. 7 kDa protein-1.. 354 216/354 (61%) e-131 Mus musculus (Mouse), 413 aa. 61.. 409 274/354 (77%) CAC38570 Sequence 157 from Patent 136.. 354 150/219 (68%) 6e-93 W00129221-Homo sapiens 1.. 218 181/219 (82%) (Human), 222 aa. Q9DA37 1700010P07Rik protein-Mus 68.. 340 123/273 (45%) 2e-69 musculus (Mouse), 478 aa. 204.. 470 178/273 (65%)

Example 14.

The NOV14 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 14A.

Table 14A. NOV14 Sequence Analysis SEQ ID NO : 57 11785 bp .......... NOV14a, GTCGCCAGCTGAGGCGGTTTGTAAGTTTTGGGTCGCAGTATGCTAGAATTTTGAGGCTCC CTTC CG173318-01 DNA TGATGAAAATTGAGCTGTCCATGCAGCCATGGAACCCGGGTTACAGCAGTGAGGGGGCCA CGGC TCAAGAAACTTACACATGTCCAAAAATGATTGAGATGGAGCAGGCGGAGGCCCAGCTTGC TGAG Sequence TTAGACCTGCTAGCCAGTATGTTCCCTGGTGAGAATGAGCTCATAGTGAATGACCAGCTG GCTG TAGCAGAACTGAAAGATTGTATTGAAAAGAAGACAATGGAGGGGCGATCTTCAAAAGTCT ACTT TACTATCAATATGAACCTGGATGTATCTGACGAAAAAATGGTAATTCAGTTTTGCTTTTA GAGG GATTGAAACATGTTGAGACTTAAAACATTGGTTAGTGCACTTTTTCTTCTTCTCTTTAAT CAGG CGATGTTTTCTCTGGCCTGTATTCTTCCCTTTAAATACCCGGCAGTTCTGCCTGAAATTA CTGT CAGATCAGTATTATTGAGTAGATCCCAGCAGACTCAGCTGAACACAGATCTGACTGCATT CCTG CAAAAACATTGTCATGGAGATGTTTGTATACTGAATGCCACAGAGTGGGTTAGAGAACAC GCCT CTGGCTATGTCAGCAGAGATACTTCATCTTCACCCACCACAGGAAGCACAGTCCAGTCAG TTGA CCTCATCTTCACGAGACTCTGGATCTACAGCCATCATATCTATAACAAATGCAAAAGAAA GAAT ATTCTAGAGTGGGCAAAGGAGCTTTCCCTGTCTGGGTTTAGCATGCCTGGAAAACCTGGT GTTG TTTGTGTGGAAGGCCCACAAAGTGCCTGTGAAGAATTCTGGTCAAGACTCAGAAAATTAA ACTG GAAGAGAATTTTAATTCGCCATCGAGAAGACATTCCTTTTGATGGTACAAATGATGAAAC GGAA GACAAAGGAAATTTTCCATTTTTGAAGAAAAAGTGTTCAGTGTTAATGGAGCCAGGGGAA ACC ACATGGACTTTGGTCAGCTCTATCAGTTCTTAAACACCAAAGGATGTGGGGATGTTTTCC AGAT GTTCTTTGGTGTAGAAGGACAATGACATCAAGAGTAGTTGAAAGTATCTTGCCACTGTTG GCCT TTTGATTTTTTTTTCCCACTTTTTCTTGAAAGATTAAGTAATTTTATTTTAGTTCCATTC TAGA ATGTTGGGGAGTGGGGCACAAGAAAAAATAGTATAGCTGAAATGCATCTGTTAAAAATGT CATG ATTGAAAGCAGAACTGAGTTTCAAATTACAACCTTAAAATTGTTGTTAGATATTTCTTCA CATA TCAGCTGCCCATTTTGAAAAAGAAATTATCCATAAAGGTAATGTTGGTGCTCCAATTTGC CAGC CATTCCCAACCCCCTTCTCCCTTACCTGCCTTCACTAAAGAACCCAGAAAAGCTAATTGC TCCC CTTTCAGCCTCTGTTGCAACTAACAACTCTCAGTGGCCTCAGGACACAGCTTTGGCCTTG GGRA TTCTGGGRAAACTTTTACTTCCTGATTAAAGATACATATGCAGCTAGGCCACCTCCTCCC CCCC TTACTGCCATAAACACCAAAGTGATGACTGGAGCTGGAGGAGTTATTTGAACCACGACGG AAGG GCCAAGAGAACCACGAAGATGCCAGTTGCCACATTGTTGAGCTGCTGACCCAACACCAGC CATT CTAAACATCTTATGAAATAAAACCAATTTTGTT ........, _, IGCCTGTCT ORF Start : ATG at 394 ORF Stop : TGA at 1111

SEQ ID NO : 58) 9. 3kD NOV14a, MLRLKTLVSALFLLLFNQAMFSLACILPPKYPAVLPEITVRSVLLSRSQQTQLNTDLTAF LQKH CG17331R 01 CHGDVCILNATEWVREHa. SGYVSRDTSSSPTTGSTVQSVDI. IFTELWIYSHHIYNKCKRKNII. E WAKELSLSGFSMPGKPGWCVEGPQSACEEFWSRLRKLNWKRILIRHREDIPFDGTNDETE RQR Protein Sequence KFSIFEEKVFSVNGARGNHMDFGQLYQFLNTKGCGDVFQMFFGVEGQ Further analysis of the NOV14a protein yielded the following properties shown in Table 14B.

Table 14B. Protein Sequence Properties NOV14a SignalP analysis : Cleavage site between residues 23 and 24 PSORT II PSG: a new signal peptide prediction method analysis: N-region : length 5 ; pos. chg 2; neg. chg 0 H-region: length 24 ; peak value 10.88 PSG score: 6.47 GvH : von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-2. 51 possible cleavage site: between 24 and 25 >>> Seems to have no N-terminal signal peptide ALOM : Klein et al's method for TM region allocation Init position for calculation : 1 Tentative number of TMS (s) for the threshold 0. 5 : 1 Number of TMS (s) for, threshold 0. 5: 1 INTEGRAL Likelihood =-4.78 Transmembrane 11-27 PERIPHERAL Likelihood = 5.41 (at 133) ALOM score:-4. 78 (number of TMSs : 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 18 Charge difference:-2. 0 C (1.0)-N (3.0) N >= C: N-terminal side will be inside >>> membrane topology: type 2 (cytoplasmic tail 1 to 11) MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75): 4.83 Hyd Moment (95): 3.70 G content: 0 D/E content: 1 S/T content: 3 Score:-3. 64 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 57 SRS|QQ NUCDISC : discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues: 11. 7% NLS Score :-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus: LRLK none SKL : peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination indication: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23): 34. 8 % : mitochondrial 30. 4 % : cytoplasmic 13. 0 % : Golgi 8. 7 %: endoplasmic reticulum 4. 3 % : vacuolar 4. 3 %: extracellular, including cell wall 4.3 % : vesicles of secretory system » indication for CG173318-01 is mit (lk=23) A search of the NOV14a 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.

Table 14C. Geneseq Results for NOV14a

NOV14a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region AAE15253 Human RNA metabolism protein-16 19.. 239 221/221 (100%) e-131 (RMEP-16)-Homo sapiens, 319 aa. 99.. 319 221/221 (100%) [W0200183524-A2, 08-NOV-2001] AAM78405 Human protein SEQ ID NO 1067-19.. 239 221/221 (100%) e-131 Homo sapiens, 319 aa. 99.. 319 221/221 (100%) [W0200157190-A2, 09-AUG-2001] F AAM79389 Human protein SEQ ID NO 3035-19.. 236 215/218 (98%) e-127 Homo sapiens, 354 aa. 137.. 354 216/218 (98%) [W0200157190-A2, 09-AUG-2001] ABB11888 Human novel protein, SEQ ID 19.. 236 215/218 (98%) e-127 NO : 2258-Homo sapiens, 354 aa. 137.. 354 216/218 (98%) [W0200157188-A2, 09-AUG-2001] AAB58229 Lung cancer associated polypeptide 19.. 167 147/149 (98%) 9e-84 sequence SEQ ID 567-Homo 103.. 251 147/149 (98%) sapiens, 305 aa. [W0200055180-A2, 21-SEP-2000] In a BLAST search of public sequence databases, the NOV14a protein was found to have homology to the proteins shown in the BLASTP data in Table 14D.

Table 14D. Public BLASTP Results for NOVMa NOVX Protein Identities/ Accession Protein/Organism/Length Residues/Similarities for the Expect Number Match Matched Portion Value Residues P57060 Protein C21orf6 (GLO1 1)-Homo 19.. 239 221/221 (100%) e-130 sapiens (Human), 319 aa. 99.. 319 221/221 (100%) Q9DCJ3 Open reading frame 5-Mus 21 239 182/219 (83%) e-105 musculus (Mouse), 244 aa. 26.. 244 192/219 (87%) Q99M03 Similar to open reading frame 5-21.. 239 182/219 (83%) e-105 Mus musculus (Mouse), 290 aa. 72.. 290 192/219 (87%) Q9JLH4 Orf5 protein-Mus musculus 21.. 239 181/219 (82%) e-105 (Mouse), 291 aa. 73.. 291 192/219 (87%) Q9D9S3 1700030G20Rik protein-Mus 23.. 239 85/222 (38%) 4e-38 musculus (Mouse), 292 aa. 72.. 288 127/222 (56%)

Example 15.

The NOV15 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 15A.

Table 15A. NOV15 Sequence Analysis SEQ m NO : 59 1776 bp .. _.. ____-. NOV15a, CACCGGATCCACCATGTCCGCGCTGCGACCTCTCCTGCTTCTGCTGCTGCCTCTGTGTCC CGGT Cl50970-06 DNA CCTGGTCCCGGACCCGGGAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAG GTGC TGGGGGCCCGGGGATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACC TCCG Sequence GGTCTGTCCCCAGGAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGA GACT GAGGCCACCTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCT GCCA GGCACAGAAAATTTGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGA CCCA GCTCTTCTCCCACTCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCT GTTC TCTCGGCTGCGAGACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGAT TTCT GGGCACAGCTCCTGGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTG ACTA CCTGCTCTGCCTCTCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGA CTCA CCCCGCCGCCTCCGCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAG GGCC TGGAGACTGGAAGAAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCA GCCA GGCTCTGATGCGTCTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTG CCAG GGCTTCTGCCTCAACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGG GGCA ACTATCTGGATGGTCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGC TGAC GGCCGAGTCCATTGGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGC GAAG GTGTCCGCCCAGGTGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGT CGAG CCCCGCCGCCCCGGGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGC CCAC GACGGCCGCAGGCACCAACCTGCACCGGCTGGTGTGGGAGCTCCGCGAGCGTCTGGCCCG GATG CGGGGCTTCTGGGCCCGGCTGTCCCTGACGGTGTGCGGAGACTCTCGCATGGCAGCGGAC GCCT CGCTGGAGGCGGCGCCCTGCTGGACCGGAGCCGGGCGGGGCCGGTACTTGCCGCCAGTGG TCGG GGGCTCCCCGGCCGAGCAGGTCAACAACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGA TGTC CCGACACGGCGGCGTCGACTACAGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCA CTGG GACACGACCTGGACGGGCAGGACGCGGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGC AGTA TGCAGATGACTGGATGGCTGGGGCTGTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATA CCCT CCTAGAAGGGATGGTTCTGGGGGCAAAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGC CGGA GCAGGAGTGGGGGGGCATCTATTGGTTTTCACACCCAAACCATCCTCATTCTCTCCCTCT CAGC CCTGGCCCTGCTTGGACCTCGACTCGAGGGCAAGGGCGAATTCCAGCA .......... ORF Start : ATG at 14 ORF Stop : at 1751 SEQ ID NO : 60 8. 7kD X NOV15a, MSALRPLLLLLLPLCPGPGPGPGSEAKVTRSCAETRQVLGARGYSLNLIPPALISGEHLR VCPQ CG5 0970-06 EYTCCS SETEQRLIRETEATFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQLFSH . SYGRLYAQHAI, IFNGLFSRLRDFYGESGEGLDDTLADFWAQLLERVFPBLHPQYSFPPDYLLCL q SR SRLASSTDGSLQPFGDSPRRLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQA LMR LIGCPLCRGVPSLMPCQGFCLNVVRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELT AESI GVKISEGLMYLQENSAKVSAQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPT TAAG TNLHRLVWELRERLARMRGFWARLSLTVCGDSRMAADASLEAAPCWTGAGRGRYLPPWGG SPA EQVNNPELKVDASGPDVPTRRRRLQLRAATARMKTAALGHDLDGQDADEDASGSGGGQQY ADDW MAGAVAPPARPPRPPYPPRRDGSGGKGGGGSARYNQGRSRSGGASIGFHTQTILILSLSA LALL

wu wu SEQID : 61 1775 bp NOV15b, ATGTCCGCGCTGCGACCTCTCCTGCTTCTGCTGCTGCCTCTGTGTCCCGGTCCTGGTCCC GGAC CG50970 01 DNA CCGGGAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAGGTGCTGGGGGCCC GGGG ATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACCTCCGGGTCTGTCC CCAG Sequence GAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTGAGGCCACC TTCC GAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAGGCACAGAA AATT TGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGACCCAGCTCTTCTC CCAC TCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCTCTCGGCTG CGAG ACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTGGGCACAGC TCCT GGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTACCTGCTCTG CCTC TCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCACCCCGCCGC CTCC GCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCTGGAGACTG GAAG AAATGTGGTCAGCGAAGCGCTTAAGGTTCCGGTGTCTGAAGGCTGCAGCCAGGCTCTGAT GCGT CTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGGGCTTCTGC CTCA ACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAACTATCTGG ATGG TCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACGGCCGAGTC CATT GGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGGTGTCCGCC CAGG TATTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGCCCCGCCGC CCCG GGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCAACGACCGCCGC AGGC ACCAACCTGCACCGGCTGGTGTGGGAGCTCCGCGAGCGTCTGGCCCGGATGCGGGGCTTC TGGG CCCGGCTGTCCCTGACGGTGTGCGGAGACTCTCGCATGGCAGCGGACGCCTCGCTGGAGG CGGC GCCCTGCTGGACCGGAGCCGGGCGGGGCCGGTACTTGCCGCCAGTGGTCGGGGGCTCCCC GGCC GAGCAGGTCAACAACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGATGTCCCGACACGG CGGC GTCGGCTACAGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCACTGGGACACGACC TGGA CGGGCAGGACGCAGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGCAGTATGCAGATGA CTGG ATGGCTGGGGCTGTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATACCCTCCTAGAAGG GATG GTTCTGGGGGCAAAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGCCGGAGCAGGAGTG GGGG GGCATCTATTGGTTTTCACACCCAAACCATCCTCATTCTCTCCCTCTCAGCCCTGGCCCT GCTT GGACCTCGATAACGGGGGAGGGGTGCCCTAGCATCAGAAGGGTTCATGGCCCTTTCC ORF Start : ATG at 1 ORF Stop : TAA at 1738 SEQ ID NO : 62 1579 aa MW at 62828. 7kD s NOV15b, MSALRPLLLLLLPLCPGPGPGPGSEAKVTRSCAETRQVLGARGYSLNLIPPALISGEHLR VCPQ CG50970-01 EYTCCSSETEQRLIRETEATFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQ LFSH . SYGRLYAQHINI, IFNGLFSRLRDFYGESGEGLDDTLADFWAQLLERVFPLLHPQYSFPPDYLLCL SRLASSTDGSLQPFGDSPRRLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQA LMR LIGCPLCRGVPSLMPCQGFCLNVVRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELT AESI GVKISEGLMYLQENSAKVSAQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPT TAAG TNLHRLVWELRERLARMRGFWARLSLTVCGDSRMAADASLEAAPCWTGAGRGRYLPPWGG SPA EQVNNPELKVDASGPDVPTRRRRLQLRAATARMKTAALGHDLDGQDADEDASGSGGGQQY ADDW MAGAVAPPARPPRPPYPPRRDGSGGKGGGGSARYNQGRSRSGGASIGFHTQTILILSLSA LALL GPR -11648 bp . NOV15d, CACCGGATCCAGCGAGGCAAAGGTCRCCGGGAGTTGTGCRGAGACCCGGCAGGTGCTGGG GGCC 274054257 DNA CGGGGATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACCTCCGGGTC TGTC CCCAGGAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTGAGG CCAC Sequence CTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAGGCA CAGA AAATTTGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGACCCAGCTC TTCT CCCACTCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCTCTC GGCT GCGAGACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTGGGC ACAG CTCCTGGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTACCTG CTCT GCCTCTCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCACCCC GCCG CCTCCGCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCTGGA GACT GGAAGAAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCAGCCAGGCT CTGA TGCGTCTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGGGCT TCTG CCTCAACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAACTA TCTG GATGGTCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACGGCC GAGT CCATTGGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGGTGT CCGC CCAGGTGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGCCCC GCCG CCCCGGGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCCACGACG GCCG CAGGCACCAACCTGCACCGGCTGGTGTGGGAGCTCCGCGAGCGTCTGGCCCGGATGCGGG GCTT CTGGGCCCGGCTGTCCCTGACGGTGTGCGGAGACTCTCGCATGGCAGCGGACGCCTCGCT GGAG GCGGCGCCCTGCTGGACCGGAGCCGGGCGGGGCCGGTACTTGCCGCCAGTGGTCGGGGGC TCCC CGGCCGAGCAGGTCAACAACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGATGTCCCGA CACG GCGGCGTCGGCTACAGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCACTGGGACA CGAC CTGGACGGGCAGGACGCGGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGCAGTATGCA GATG ACTGGATGGCTGGGGCTGTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATACCCTCCTA GAAG GGATGGTTCTGGGGGCAAAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGCCGGAGCAG GAGT GGGGGGGCATCTATTGGTTTTCACACCCAAACCATCCTCCTCGAGGGC ORF Start : at 2 ORF Stop : end of sequence

S # co OVLS5d, TGSSEAICVTRSCAETRQVLGARGYSLNLIPPALISGEHLRVCPQEYTCCSSETEQRLIR ETEAT 274054257 Protein FRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQLFSHSYGRLYAQHALIFNGL FSRL RDFYGESGEGLDDTLADFWAQLLERVFPLLHPQYSFPPDYLLCLSRLASSTDGSLQPFGD SPRR Sequence LRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQALMRLIGCPLCRGVPSLMPCQ GFC LNWRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELTAESIGVKISEGLMYLQENSAK VSA QVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPTTAAGTNLHRLVWELRERLAR MRGF WARLSLTVCGDSRMAADASLEAAPCWTGAGRGRYLPPWGGSPAEQVNNPELKVDASGPDV PTR RRRLQLRAATARMKTAALGHDLDGQDADEDASGSGGGQQYADDWMAGAVAPPARPPRPPY PPRR DGSGGKGGGGSARYNQGRSRSGGASIGFHTQTILLEG SEQ m NO : 65 1613 bp NOV15e, ATGTCCGCGCTGCGACCTCTCCTGCTTCTGCTGCTGCCTCTGTGTCCCGGTCCTGGTCCC GGAC CG50970 03 DNA CCGGGAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAGGTGCTGGGGGCCC GGGG ATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACCTCCGGGTCTGTCC CCAG Sequence GAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTGAGGCCACC TTCC GAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAGGCACAGAA AATT TGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGACCCAGCTCTTCTC CCAC TCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCTCTCGGCTG CGAG ACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTGGGCACAGC TCCT GGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTACCTGCTCTG CCTC TCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCACCCCGCCGC CTCC GCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCTGGAGACTG GAAG AAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCAGCCAGGCTCTGAT GCGT CTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGGGCTTCTGC CTCA ACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAACTATCTGG ATGG TCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACGGCCGAGTC CATT GGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGGTGTCCGCC CAGG TGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGCCCCGCCGC CCCG GGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCCACGACGGCCGC AGGC ACCAACCTGCACCGGCTGGTACTTGCCGCCAGTGGTCGGGGGCTCCCCGGCCGAGCAGGT CAAC AACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGATGTCCCGACACGGCGGCGTCGGCTA CAGC TCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCACTGGGACACGACCTGGACGGGCAGG ACGC GGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGCAGTATGCAGATGACTGGATGGCTGG GGCT GTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATACCCTCCTAGAAGGGATGGTTCTGGG GGCA AAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGCCGGAGCAGGAGTGGGGGGGCATCTA TTGG TTTTCACACCCAAACCATCCTCATTCTCTCCCTCTCAGACCTGGCCCTGCTTGGACCTCG ATAA CGGGGGAGGGGTG , ORF Start : ATG at 1 ORF Stop : TGA at 1348 CG50970-03 EYTCCSSETEQRLIRETEATFRGLVEDSGSFIVHTLAA12FIRKFDEFFLEMLSVAQHSI TQLFSH Prot Se uence SYGRLYAQHALIFNGLFSRLRDFYGESGEGLDDTLADFWAQLLERVFPLLHPQYSFPPDY LLCL SRIASSTDGSLQPFGDSPRRLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQA LMR LIGCPLCRGVPSLMPCQGFCLNWRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELTA ESI GVKISEGLMYLQENSAKVSAQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPT TAAG TNLHRLVLAASGRGLPGRAGQQPRAQGGRLGPRCPDTAASATAPGGHGQNENGRTGTRPG RAGR G G v SEQ ID NO : 67 1297 bp NOV15f, CACCGGATCCACCAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAGGTGCT GGGG 237922026 DNA GCCCGGGGATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTQGGTGAGCACCTCCGGG TCT GTCCCCAGGAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTG AGGC Sequence CACCTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAG GCAC AGAAAATTTGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGACCCAG CTCT TCTCCCACTCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCT CTCG GCTGCGAGACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTG GGCA CAGCTCCTGGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTAC CTGC TCTGCCTCTCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCAC CCCG CCGCCTCCGCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCT GGAG ACTGGAAGAAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCAGCCAG GCTC TGATGCGTCTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGG GCTT CTGCCTCAACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAA CTAT CTGGATGGTCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACG GCCG AGTCCATTGGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGG TGTC CGCCCAGGTGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGC CCCG CCGCCCCGGGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCCACG ACGG CCGCAGGCACCAACCTGCACCGGCTGGTACTTGCCGCCAGTGGTCGGGGGCTCCCCGGCC GAGC AGGTCAACAACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGATGTCCCGACACGGCGGC GTCG GCTACAGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCACTGGGACACGACCTGGA CGGG CAGGACGCGGACTCGAG w ORF Start : at 2 ORF Stop : end of sequence

SEQ 111) NO : 68 lo. gkd ^ NOV15f, TGSTSEAKVTRSCAETRQVLGARGYSLNLIPPALISGEHLRVCPQEYTCCSSETEQRLIR ETEA 237922026 Protein TFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQLFSHSYGRLYAQHALIFNG LFSR LRDFYGESGEGLDDTLADFWAQLLERVFPLLHPQYSFPPDYLLCLSRLASSTDGSLQPFG DSPR Sequence RLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQALMRLIGCPLCRGVPSLMPC QGF CLNVVRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELTAESIGVKISEGLMYLQENS AKVS AQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPTTAAGTNLHRLVLAASGRGL PGRA GQQPRAQGGRLGPRCPDTAASATAPGGHGQNENGRTGTRPGRAGRGLE ffi SEQ ID NO : 69 11126 bp x NOViSg, CACCGGATCCACCAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAGGTGCT GGGG 237922511 DNA GCCCGGGGATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACCTCCGG GTCT GTCCCCAGGAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTG AGGC Sequence CACCTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAG GCAC AGAAAATTTGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGACCCAG CTCT TCTCCCACTCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCT CTCG GCTGCGAGACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTG GGCA CAGCTCCTGGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTAC CTGC TCTGCCTCTCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCAC CCCG CCGCCTCCGCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCT GGAG ACTGGAAGAAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCAGCCAG GCTC TGATGCGTCTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGG GCTT CTGCCTCAACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAA CTAT CTGGATGGTCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACG GCCG AGTCCATTGGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGG TGTC CGCCCAGGTGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGC CCCG CCGCCCCGGGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCCACG ACGG CCGCAGGCACCAACCTGCACCGGCTGGTACTTCTCGAG ORF Start : at 2 ORF Stop : end of sequence

SEQ m NO : 70 375 aa MW at 41526. kD w . NOV15g, TGSTSEAKVTRSCAETRQVLGARGYSLNLIPPALISGEHLRVCPQEYTCCSSETEQRLIR ETEA 2379225i1Protein TFRGhVEDSGSFhVHTLAaRHRKFDEFFhEMLSVAQHShTQhFSHSYGRLYAQHALIFNG hFSR LRDFYGESGEGLDDTLADFWAQLLERVFPLLIiPQYSFPPDYLLCLSRLASSTDGSLQPF GDSPR Sequence RLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQALMRLIGCPLCRGVPSLMPC QGF CLNVVRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELTAESIGVKISEGLMYLQENS AKVS AQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPTTAAGTNLHRLVLLE SEQ ID NO : 71 1776 bp NOV15 CACCGGATCQCCATGTCCGCGCTGCGACCTCTCCTGCTTCTGCTGCTGCCTCTGTGTCCC GGT 315490136 DNA CCTGGTCCCGGACCCGGGAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAG GTGC TGGGGGCCCGGGGATATAGCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACC TCCG Sequence GGTCTGTCCCCAGGAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGA GACT GAGGCCACCTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCT GCCA GGCACAGAAAATTTGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGA CCCA GCTCTTCTCCCACTCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCT GTTC TCTCGGCTGCGAGACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGAT TTCT GGGCACAGCTCCTGGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTG ACTA CCTGCTCTGCCTCTCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGA CTCA CCCCGCCGCCTCCGCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAG GGCC TGGAGACTGGAAGAAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCA GCCA GGCTCTGATGCGTCTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTG CCAG GGCTTCTGCCTCAACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGG GGCA ACTATCTGGATGGTCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGC TGAC GGCCGAGTCCATTGGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGC GAAG GTGTCCGCCCAGGTGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGT CGAG CCCCGCCGCCCCGGGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGC CCAC GACGGCCGCAGGCACCAACCTGCACCGGCTGGTGTGGGAGCTCCGCGAGCGTCTGGCCCG GATG CGGGGCTTCTGGGCCCGGCTGTCCCTGACGGTGTGCGGAGACTCTCGCATGGCAGCGGAC GCCT CGCTGGAGGCGGCGCCCTGCTGGACCGGAGCCGGGCGGGGCCGGTACTTGCCGCCAGTGG TCGG GGGCTCCCCGGCCGAGCAGGTCAACAACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGA TGTC CCGACACGGCGGCGTCGACTACAGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCA CTGG GACACGACCTGGACGGGCAGGACGCGGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGC AGTA TGCAGATGACTGGATGGCTGGGGCTGTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATA CCCT CCTAGAAGGGATGGTTCTGGGGGCAAAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGC CGGA GCAGGAGTGGGGGGGCATCTATTGGTTTTCACACCCAAACCATCCTCATTCTCTCCCTCT CAGC CCTGGCCCTGCTTGGACCTCGACTCGAGGGCAAGGGCGAATTCCAGCA ORF Start : at 2 ORF Stop : end of sequence SEQ ID NO : 72 592 aa IMW at 64064, OkD NOVISIh, TGSTMSALRPLLLLLLPLCPGPGPGPGSEAKVTRSCAETRQVLGARGYSLNLIPPALISG EHLR 315490136 Protein VCPQEYTCCSSETEQRLIRETEATFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQH SLTQ LFSHSYGRLYAQHALIFNGLFSRLRDFYGESGEGLDDTLADFWAQLLERVFPLLHPQYSF PPDY Sequence LLCLSRLASSTDGSLQPFGDSPRRLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEG CSQ ALMRLIGCPLCRGVPSLMPCQGFCLNWRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSF ELT AESIGVKISEGLMYLQENSAKVSAQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEE ERPT TAAGTNLHRLVWELRERLARMRGFWARLSLTVCGDSRMAADASLEAAPCWTGAGRGRYLP PWG GSPAEQVNNPELKVDASGPDVPTRRRRLQLRAATARMKTAALGHDLDGQDADEDASGSGG GQQY ADDWMAGAVAPPARPPRPPYPPRRDGSGGKGGGGSARYNQGRSRSGGASIGFHTQTILIL SLSA LALLGPRLEGKGEFQX

SEQ ID NO : 73 11976 bp I NOVISi, GGCTCTGCTTTCCTCCTTAGGACCCACTTTGCCGTCCTGGGGTGGCTGCAGTTATGTCCG CGCT CG50970-02 DNA GCGACCTCTCCTGCTTCTGCTGCTGCCTCTGTGTCCCGGTCCTGGTCCCGGACCCGGGAG CGAG GCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAGGTGCTGGGGGCCCGGGGATATAGC TTAA Sequence ACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACCTCCGGGTCTGTCCCCAGGAGTACA CCTG CTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTGAGGCCACCTTCCGAGGCCT GGTG GAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAGGCACAGAAAATTTGATGAG TTTT TTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGACCCAGCTCTTCTCCCACTCCTACG GCCG CCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCTCTCGGCTGCGAGACTTCTA TGGG GAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTGGGCACAGCTCCTGGAGAGA GTGT TCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTACCTGCTCTGCCTCTCACGCT TGGC CTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCACCCCGCCGCCTCCGCCTGCA GATA ACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCTGGAGACTGGAAGAAATGTG GTCA GCGAAGCGCTTAAGGTTCCGGTGTCTGAAGGCTGCAGCCAGGCTCTGATGCGTCTCATCG GCTG TCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGGGCTTCTGCCTCAACGTGGT TCGT GGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAACTATCTGGATGGTCTCCTG ATCC TGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACGGCCGAGTCCATTGGGGTGA AGAT CTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGGTGTCCGCCCAGGTATTTCA GGAG TGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGCCCCGCCGCCCCGGGAAGAG GCGG GCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCAAGCGCAGATGAGGATGCCAGCG GCTC TGGAGGGGGACAGCAGTATGCAGATGACTGGATGGCTGGGGCTGTGGCTCCCCCAGCCCG GCCT CCTCGGCCTCCATACCCTCCTAGAAGGGATGGTTCTGGGGGCAAAGGAGGAGGTGGCAGT GCCC GCTACAACCAGGGCCGGAGCAGGAGTGGGGGGGCATCTATTGGTTTTCACACCCAAACCA TCCT CATTCTCTCCCTCTCAGCCCTGGCCCTGCTTGGACCTCGATAACGGGGGAGGGGTGCCCT AGCA TCAGAAGGGTTCATGGCCCTTTCCCCTCCTCCCCCCTCAGCTGGGCCTGGGGAGGAGTCG AAGG GGGCTGCF1GAGAGGGTAGAGAAGGGACTTTGCAGGTGAATGGCTGGGGCCCCAAATCCA GGAGA TTTTCATCAGAGGTGGGTGGGTGTTCACAATATTTATTTTTTCATTTGGTAATGGGAGGG GGGC CTGGGGGTATTTATTTAGGAGGGAGTGTGGTTTCCPTAGAAGGTATAGTCTCTAGCCCTC TAAG GCTGGGGCTGGTGATCAGCCCCAACAGAGAAAATGAGGAGTTTAGAGTTGCAGCTGGGTT CTGT TGAGTTTTTTCAGTATCAATTTCTTAAACCAAATTTTAAAAAAAACAAGGTGGGGGGGTG CTCA TCTCGTGACCTCTGCCACCCACATCCTTCACAAACTCCATGTTTCAGTGTTTGAGTCCAT GTTT ATTCTGCAAATAAATGGTAATGTATTA ORF Start : ATG at 54 ORF Stop :, TAA at 144. 9 tSuS =

SEQ m NO : 74 MW at 50470. 8kD NOV15I MSALRPLLLLLLPLCPGPGPGPGSEAKVTRSCAETRQVLGARGYSLNLIPPALISGEHLR VCPQ NOV1 5i MSALRPhLhhLLPLCPGPGPGPGSEAKVTRSQETRQVLGARGYSLNLI PPALI SGEHLRVCPQ CG50970-02 EYTCCSSETEQRLIRETEATFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQ LFSH . SYGRhYAQHALI FNGhFSRLRDFYGESGEGLDDTLADFWAQLhERVFPhLEPQYSFPPDYLLCh Protem Sequence SRLASSTDGSLQPFGDSPRRLRLQITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQA LMR LIGCPLCRGVPSLMPCQGFCLNVVRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELT AESI GVKISEGLMYLQENSAKVSAQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPS ADED SGSGGGQQYADDWMAGAVAPPARPPRPPYPPRRDGSGGKGGGGSARYNQGRSRSGGASIG FHT QTILILSLSALALLGPR z SEQIDNO : 75725 bpJ' NOV1SJ, CGCCTGGTCCAGCTATCGTGCTCGGTATTCAGTTTTCCGGAGCAGCGCTCTTTCTCTGGC CCGC CGS0970-04 DNA GCGGTCCCGCGGCCGAGTACCGGATTCCCGAGTTTGGGAGGCTCTGCTTTCCTCCTTAGG A CCCACTTTGCCGTCCTGGGGTGGCTGCAGTTATGTCCGCGCTGCGACCTCTCCTGCTTCT GCTG Sequence CTGCCTCTGTGTCCCGGTCCTGGTCCCGGACCCGGGAGCGAGGCAAAGGTCACCCGGAGT TGTG CAGAGACCCGGCAGGTGCTGGGGGCCCGGGGATATAGCTTAAACCTAATCCCTCCCGCCC TGAT CTCAGGTGAGCACCTCCGGGTCTGTCCCCAGGAGTACACCTGCTGTTCCAGTGAGACAGA GCAG AGGCTGATCAGGGAGACTGAGGCCACCTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTT CTGG TTCACACACTGGCTGCCAGGCACAGAAAATTTGATGAGTTTTTTCTGGAGATGCTCTCAG TAGC CCGGCCTCCTCGGCCTCCATACCCTCCTAGAAGGGATGGTTCTGGGGGCAAAGGAGGAGG TGGC AGTGCCCGCTACAACCAGGGCCGGAGCAGGAGTGGGGGGGCATCTATTGGTTTTCACACC CAAA CCATCCTCATTCTCTCCCTCTCAGCCCTGGCCTTGCTTGGACCTCGATAACGGGGGAGGG GTGC CCTAGCATCAGAAGGGTTCAT ORF Start : ATG at 160 ORF Stop : TAA at 688 SEQ ID NO : 76 176 aa MW at 18879. 4kD NOV15j, MSALRPLLLLLLPLCPGPGPGPGSEAKVTRSCAETRQVLQARGYSLNLIPPALISGEHLR VCPQ NOV15'n C'r" EYTCCSSETEQRLIRETEATFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVARPPRPP YPPR RDGSGGKGGGGSARYNQGRSRSGGASIGFHTQTILILSLSALALLGPR Protein Sequence SEQ ID NO. 177 1 s v NOV15k, AGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAGGTGCTGGGGGCCCGGGGA TATA CG50970-05 DNA GCTTAAACCTAATCCCTCCCGCCCTGATCTCAGGTGAGCACCTCCGGGTCTGTCCCCAGG AGTA CACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGAGACTGAGGCCACCTTCCG AGGC Sequence CTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCTGCCAGGCACAGAAAATTT GATG AGTTTTTTCTGGAGATGCTCTCAGTAGCCCAACACTCTCTGACCCAGCTCTTCTCCCACT CCTA CGGCCGCCTGTATGCCCAGCACGCCCTCATATTCAATGGCCTGTTCTCTCGGCTGCGAGA CTTC TATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGATTTCTGGGCACAGCTCCTG GAGA GAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTGACTACCTGCTCTGCCTCT CACG CTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGACTCACCCCGCCGCCTCCG CCTG CAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAGGGCCTGGAGACTGGAAGA AF1TG TGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCAGCCAGGCTCTGATGCGTC TCAT CGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTGCCAGGGCTTCTGCCTCAA CGTG GTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGGGGCAACTATCTGGATGGT CTCC TGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGCTGACGGCCGAGTCCATTG GGGT GAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGCGAAGGTGTCCGCCCAGGT GTTT CAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGTCGAGCCCCGCCGCCCCGG GAAG AGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGCCCACGACGGCCGCAGGCA CCAA CCTGCACCGGCTGGTGTGGGAGCTCCGCGAGCGTCTGGCCCGGATGCGGGGCTTCTGGGC CCGG CTGTCCCTGACGGTGTGCGGAGACTCTCGCATGGCAGCGGACGCCTCGCTGGAGGCAGCG CCCT GCTGGACCGGAGCCGGGCGGGGCCGGTACTTGCCGCCAGTGGTCGGGGGCTCCCCGGCCG AGCA GGTCAACAACCCCGAGCTCAACGTGGACGCCTCGGGCCCCGATGTCCCGACACGGCGGCG TCGG CTACGGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCACTGGGACACGACCTGGAC GGGC AGGACGCGGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGCAGTATGCAGATGACTGGA TGGC TGGGGCTGTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATACCCTCCTAGAAGGGATGG TTCT GGGGGCAAAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGCCGGAGCAGGAGT ORF Start : at 1 ORF Stop : end of sequence

SEQ ID NO : 78 i53 at 57988. 9kD NOVISI5, SEAKVTRSCAETRQVLGARGYSINLIPPALISGEHLRVCPQEYTCCSSETEQRLIRETEA TFRG CG5097 -O LDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQLFSHSYGRLYAQHALIFNGLFSRLR DF V-YGESGEGLDDTLADFWAQLLERVFPLLHPQYSFPPDYLLCLSRLASSTDGSLQPFGDS PRRLRL Protein Sequence QITRTLVAARAFVQGLETGRNWSEALKVPVSEGCSQALMRLIGCPLCRGVPSLMPCQGFC LNV VRGCLSSRGLEPDWGNYLDGLLILADKLQGPFSFELTAESIGVKISEGLMYLQENSAKVS AQVF QECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPTTAAGTNLHRLVWELRERLARMRG FWAR LSLTVCGDSRMAADASLEAAPCWTGAGRGRYLPPWGGSPAEQVNNPELNVDASGPDVPTR RRR SEQIDNO : 791762bp t NOV151, CACCGGATCCACCATGTCCGCGCTGCGACCTCTCCTGCTTCTGCTGCTGCCTCTGTGTCC CGGT CG50970-07 DNA CCTGGTCCCGGACCCGGGAGCGAGGCAAAGGTCACCCGGAGTTGTGCAGAGACCCGGCAG GTGC TGGGGGCCCGGGGATATAGCTTAAA. CCTAATCCCTCCCGCCCTGATCTCAGCTGAGCACCTCCG Sequence GGTCTGTCCCCAGGAGTACACCTGCTGTTCCAGTGAGACAGAGCAGAGGCTGATCAGGGA GACT GAGGCCACCTTCCGAGGCCTGGTGGAGGACAGCGGCTCCTTTCTGGTTCACACACTGGCT GCCA GGCACAGAAAATTTGATGAGTTTTTTCTGGAGATGCTCTCAGTAGCCCAGCACTCTCTGA CCCA GCTCTTCTCCCACTCCTACGGCCGCCTGTATGCCCAGCACGCCCTCATATTCA, ATGGPCTGTTC TCTCGGCTGCGAGACTTCTATGGGGAATCTGGTGAGGGGTTGGATGACACCCTGGCGGAT TTCT GGGCACAGCTCCTGGAGAGAGTGTTCCCGCTGCTGCACCCACAGTACAGCTTCCCCCCTG ACTA CCTGCTCTGCCTCTCACGCTTGGCCTCATCTACCGATGGCTCTCTGCAGCCCTTTGGGGA CTCA CCCCGCCGCCTCCGCCTGCAGATAACCCGGACCCTGGTGGCTGCCCGAGCCTTTGTGCAG GGCC TGGAGACTGGAAGAAATGTGGTCAGCGAAGCGCTTAAGGTGCCGGTGTCTGAAGGCTGCA GCCA GGCTCTGATGCGTCTCATCGGCTGTCCCCTGTGCCGGGGGGTCCCCTCACTTATGCCCTG CCAG GGCTTCTGCCTCAACGTGGTTCGTGGCTGTCTCAGCAGCAGGGGACTGGAGCCTGACTGG GGCA ACTATCTGGATGGTCTCCTGATCCTGGCTGATAAGCTCCAGGGCCCCTTTTCCTTTGAGC TGAC GGCCGAGTCCATTGGGGTGAAGATCTCGGAGGGTTTGATGTACCTGCAGGAAAACAGTGC GAAG GTGTCCGCCCAGGTGTTTCAGGAGTGCGGCCCCCCCGACCCGGTGCCTGCCCGCAACCGT CGAG CCCCGCCGCCCCGGGAAGAGGCGGGCCGGCTGTGGTCGATGGTGACCGAGGAGGAGCGGC CCAC GACGGCCGCAGGCACCAACCTGCACCGGCTGGTGTGGGAGCTCCGCGAGCGTCTGGCCCG GATG CGGGGCTTCTGGGCCCGGCTGTCCCTGACGGTGTGCGGAGACTCTCGCATGGCAGCGGAC GCCT CGCTGGAGGCGGCGCCCTGCTGGACCGGAGCCGGGCGGGGCCGGTACTTGCCGCCAGTGG TCGG GGGCTCCCCGGCCGAGCAGGTCAACAACCCCGAGCTCAAGGTGGACGCCTCGGGCCCCGA TGTC CCGACACGGCGGCGTCGGCTACAGCTCCGGGCGGCCACGGCCAGAATGAAAACGGCCGCA CTGG GACACGACCTGGACGGGCAGGACGCGGATGAGGATGCCAGCGGCTCTGGAGGGGGACAGC AGTA TGCAGATGACTGGATGGCTGGGGCTGTGGCTCCCCCAGCCCGGCCTCCTCGGCCTCCATA CCCT CCTAGAAGGGATGGTTCTGGGGGCAAAGGAGGAGGTGGCAGTGCCCGCTACAACCAGGGC CGGA GCAGGAGTGGGGGGGCATCTATTGGTTTTCACACCCAAACCATCCTCATTCTCTCCCTCT CAGC CCTGGCCCTGCTTGGACCTCGATAGGTCGACGGC ~ u v ORF Start : ATG at 1 ORF Stop : TAG at 1751

SEQ ID NO : 80 1579 aa IMW at 62828. 7kD NOV151, MSALRPLLLLLLPLCPGPGPGPGSEAKVTRSCAETRQVLGARGYSLNLIPPALISGEHLR VCPQ CGS0970-07 EYTCCSSETEQRLIRETEATFRGLVEDSGSFLVHTLAARHRKFDEFFLEMLSVAQHSLTQ LFSH WfiJ v SYGRLYAQHALIFNGLFSRLRDFYGESGBGLDDTLADFWAQLLERVFPLLHPQYSFPPDY LLCL Protein Sequence SRLASSTDGSLQPFGDSPRRLRLQITRTLVAARAFVQGLETSRUWSBALKVPVSEGCSQA LMR LIGCPLCRGVPSLMPCQGFCLNWRGCLSSRGLEPDWGNYLBGLI. IIADKLQGPFSFELTAESI GVKISEGLMYLQENSAKVSAQVFQECGPPDPVPARNRRAPPPREEAGRLWSMVTEEERPT TAAG TNLHRLVWELRERLARMRGFWARLSLTVCGDSRMAADASLEAAPCWTGAGRGRYLPPWGG SPA EQVNNPELKVDASGPDVPTRRRRLQLRAATARMKTAALGHDLDGQDADEDASGSGGGQQY ADDW MAGAVAPPARPPRPPYPPRRDGSGGKGGGGSARYNQGRSRSGGASIGFHTQTILILSLSA IALL GPR Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 15B.

Table 15B. Comparison of NOV15a against NOV15b through NOV151. NOVlSa Residues/Identities/ Match Residues Similarities for the Matched Region NOV15b 1.. 579 579/579 (100%) 1..579 579/579 (100%) NOV15c 1.. 579 579/579 (100%) 1.. 579 579/579 (100%) NOV15d 24.. 567 543/544 (99%) 4.. 547 544/544 (99%) NOV15e 1. 391 391/391 (100%) 1.. 391 391/391 (100%) NOV15f 21.. 391 369/371 (99%) 2.. 372 369/371 (99%) NOV15g 21.. 391 369/371 (99%) 2..372 369/371 (99%) NOV15h 1.. 579 579/579 (100%) 5.. 583 579/579 (100%) NOV15i 1.. 380 379/380 (99%) 1.. 380 380/380 (99%) NOVX 1.. 119 118/119 (99%) 1.. 119 119/119 (99%) NOV15k 24.. 553 528/530 (99%) 1.. 530 529/530 (99%) NOV151 1.. 579 579/579 (100%) 1..579 579/579 (100%) Further analysis of the NOV 15a protein yielded the following properties shown in Table 15C.

Table 15C. Protein Sequence Properties NOV15a Signal ? analysis: Cleavage site between residues 24 and 25 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 5; pos. chg 1; neg. chg 0 H-region: length 19 ; peak value 10. 14 PSG score: 5.74 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-0. 46 possible cleavage site: between 20 and 21 >>> Seems to have a cleavable signal peptide (1 to 20) ALOM : Klein et al's method for TM region allocation Init position for calculation: 21 Tentative number of TMS (s) for the threshold 0. 5: 1 Number of TMS (S) for threshold 0.5 : 0 PERIPHERAL Likelihood = 4.24 (at 254) ALOM score:-0. 85 (number of TMSs : 0) t MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 10 Charge difference : -1.0 C (1.0)-N (2.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75): 8.46 Hyd Moment (95): 7.50 G content: 4 D/E content: 1 S/T content: 2 Score:-4. 90 Gavel: indication of cleavage sites for mitochondrial preseq R-3 motif at 45 ARGYS NUCDISC: discrimination of nuclear localization signals pat4: RHRK (3) at 103 pat4: RRRR (5) at 468 pat7: PRRLRLQ (5) at 210 pat7: PARNRRA (4) at 353 pat7: PTRRRRL (5) at 466 bipartite: none content of basic residues: 10.9% NLS Score: 1.23 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: XXRR-like motif in the N-terminus: SALR none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking. 63 PROSITE DNA binding motifs : none checking 71 PROSITE ribosomal protein motifs : none checking 33 PROSITE prokaryotic DNA binding motifs : none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 55.5 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23) : 33.3 %: extracellular, including cell wall 33. 3 % : mitochondrial 22.2 96 : endoplasmic reticulum 11.1 % : vacuolar # indication for CG50970-06 is ec (k=9) A search of the NOVI 5a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 15D.

Table 15D. Geneseq Results for NOV15a NOV15a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABG70277 Human Glypican-2 Precursor-like 1.. 579 579/579 (100%) 0. 0 protein #1-Homo sapiens, 579 aa. 1.. 579 579/579 (100%) [W0200255702-A2, 18-JUL-2002] ABG70279 Human Glypican-2 Precursor-like 1.. 391 391/391 (100%) 0. 0 protein #3-Homo sapiens, 449 aa. 1.. 391 391/391 (100%) [W0200255702-A2, 18-JUL-2002] ABG70278 Human Glypican-2 Precursor-like 1 380 378/380 (99%) 0. 0 protein #2-Homo sapiens, 465 aa. 1.. 380 379/380 (99%) [W0200255702-A2, 18-JI3L-2002) AAU29071 Human PRO polypeptide sequence 2.. 511 227/512 (44%) e-127 #48-Homo sapiens, 555 aa. 7.. 504 329/512 (63%) [W0200168848-A2, 20-SEP-2001] AAB44256 Human PR0705 (LNQ369) protein 2.. 511 227/512 (44%) e-127 7.. 504 329/512 (63%) sapiens, 555 aa. [WO200053756-A2, 14-SEP-2000] In a BLAST search of public sequence databases, the NOV15a protein was found to have homology to the proteins shown in the BLASTP data in Table 15E.

Table 15E. Public BLASTP Results for NOV15a

NOVlb Protein Identities/ Accession Protein/Organism/Length Residues/ »ilarities for the Expect Number Match Matched Portion Residues Q8N158 Similar to cerebroglycan 1.. 579 579/579 (100%) 0. 0 (Hypothetical protein FLJ38962)-1.. 579 579/579 (100%) Homo sapiens (Human), 579 aa. P51653 Glypican-2 precursor 1.. 579 477/581 (82%) 0. 0 (Cerebroglycan) (HSPG M13)-1.. 579 513/581 (88%) Rattus norvegicus (Rat), 579 aa. Q9R087 Glypican-6 precursor-Mus musculus 2.. 511 227/512 (44%) e-127 (Mouse), 555axa. 7 504 332/512 (64%) Q9Y625 Glypican-6 precursor-Homo sapiens 2.. 511 227/512 (44%) e-127 (Human), 555 aa. 7.. 504 329/512 (63%) Q8R3X6 Similar to glypican 6-Mus musculus 2.. 511 228/522 (43%) e-125 (Mouse), 565 aa. 7.. 514 333/522 (63%) PFam analysis indicates that the NOV15a protein contains the domains shown in the Table 15F.

Table 15F. Domain Analysis of NOV15a Identities/ Pfam Domain NOV15a Match Region Similarities Expect Value for the Matched Region Glypican 3.. 566 271/631 (43%) 6. 7e-291 510/631 (81%)

Example 16.

The NOV16 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 16A.

Table 16A. NOV16 Sequence Analysis SEQ ID NO : 81 11242 bp m u. m NOV16a, ATGGGGTCGACCGACTCCAAGCTGAACTTCCGGAAGGCGGTGATCCAGCTCACCACCAAG ACGCA CG543-O3 DNA GCCCGTGGAAGCCACCGATGATGCCTTTTGGGACCAGTTCTGGGCAGACACAGCCACCTC GGTGC J-tJ-UJ ji A (AJQJGTgggQQ, (JgQ ( ( GGATGTGTTTGCACTGGTGCCGGCAGCAGAGATCCGGGCCGTGCGGGAAGAGTCACCCTC CAAC Sequence TTGGCCACCCTGTGCTACAAGGCCGTTGAGAGGCTGGTGCAGGGAGCTGAGAGTGGCTGC CACTC GGAGAAGGAGAAGCAGATCGTCCTGAACTGCAGCCGGCTGCTCACCCGCGTGCTGCCCTA CATCT TTGAGGACCCCGACTGGAGGGGCTTCTTCTGGTCCACAGTGCCCGGGGCAGGGCGAGGAG GGCAG GGAGAAGAGGATGATGAGCATGCCAGGCCCCTGGCCGAGTCCCTGCTCCTGGCCATTGCT GACCT GCTCTTCTGCCCGGACTTCACGGTTCAGAGCCACCGGAGGAGCACTGTGGACTCGGCAGA GGACG TCCACTCCCTGGACAGCTGTGAATACATCTGGGAGGCTGGTGTGGGCTTCGCTCACTCCC CCCAG CCTAACTACATCCACGATATGAACCGGATGGAGCTGCTGAAACTGCTGCTGACATGCTTC TCCGA GGCCATGTACCTGCCCCCAGCTCCGGAAAGTGGCAGCACCAACCCATGGGTTCAGTTCTT TTGTT CCACGGAGAACAGACATGCCCTGCCCCTCTTCACCTCCCTCCTCAACACCGTGTGTGCCT ATGAC CCTGTGGGCTACGGGATCCCCTACAACCACCTGCTCTTCTCTGACTACCGGGAACCCCTG GTGGA GGAGGCTGCCCAGGTGCTCATTGTCACTTTGGACCACGACAGTGCCAGCAGTGCCAGCCC CACTG TGGACGGCACCACCACTGGCACCGCCATGGATGATGCCGATCCTCCAGGCCCTGAGAACC TGTTT GTGAACTACCTGTCCCGCATCCATCGTGAGGAGGACTTCCAGTTCATCCTCAAGGGTATA GCCCG GCTGCTGTCCAACCCCCTGCTCCAGACCTACCTGCCTAACTCCACCAAGAAGATCCAGTT CCACC AGGAGCTGCTAGTTCTCTTCTGGAAGCTCTGCGACTTCAACAAGAAATTCCTCTTCTTCG TGCTG AAGAGCAGCGACGTCCTAGACATCCTTGTCCCCATCCTCTTCTTCCTCAACGATGCCCGG GCCGA TCAGTCT ORF Start : ATG at 1 ORF Stop : end of sequence

SEQ ID NO : 82 414 aa MW at 46487. 9kD NOV1Sa, MGSTDSKLNFRKAVIQLTTKTQPVEATDDAFWDQFWADTATSVQDVFALVPAAEIRAVRE ESPS CG54443-03 NLITLCYKAVERLVQGAESGCHSEKEKQIVLNCSRLLTRVLPYIFEDPDWRGFFWSTVPG AGRG . GQGEEDDEHARPLA F*SLLbAIADLLFCPDFTVQSHRRS TVDSAEDVHSLDSCEYIWEAGVGFAH SPQPNYIHDMNRMELLKLLLTCFSEAMYLPPAPESGSTNPWVQFFCSTENRHALPLFTSL LNTV CAYDPVGYGIPYNHLLFSDYREPLVEEAAQVLIVTLDHDSASSASPTVDGTTTGTAMDDA DPPG PENLFVNYLSRIHREEDFQFILKGIARLLSNPLLQTYLPNSTKKIQFHQELLVLFWKLCD FNKK FLFFVLKSSDVLDILVPILFFLNDARRDQS SEQ ID NO : 83 1912 bp w i NOV16b, CGAGGGCCGGGGGCGGGGCGCGCCGCTTGTCTCCTGCGAGAGCCGCGGGGGCCGCGGAGC TGGA CG54443 07 DNA GCCGGAGCTGAAGCCGGAGCCGGGTTGGAGTCTTGGGCGGGGGCCGGGCCGGAGCGGGCT CCAG GACATGGGGTCGACCGACTCCAAGCTGAACTTCCGGAAGGCGGTGATCCAGCTCACCACC AAG Sequence ACGCAGCCCGTGGAAGCCACCGATGATGCCTATGACCCTGTGGGCTACGGGATCCCCTAC AACC ACCTGCTCTTCTCTGACACCGGGGAACCCCTGGTGGAGGAGGCTGCCCAGGTGCTCATTG TCAC TTTGGACCACGACAGTGCCAGCAGTGCCAGCCCCACTGTGGACGGCACCACCACTGGCAC CGCC ATGGATGATGCCGATCCTCCAGGCCCTGAGAACCTGTTTGTGAACTACCTGTCCCGCATC CATC GTGAGGAGGACTTCCAGTTCATCCTCAAGGGTATAGCCCGGCTGCTGTCCRACCCCCTGC TCCA GACCTACCTGCCTAACTCCACCAAGAAGATCCAGTTCCACCAGGAGCTGCTAGTTCTCTT CTGG AAGCTCTGCGACTTCAACAAGAAATTCCTCTTCTTCGTGCTGAAGAGCAGCGACGTCCTA GACA TCCTTGTCCCCATCCTCTTCTTCCTCAACGATGCCCGGGCCGATCAGTCTCGGGTGGGCC TGAT GCACATTGGTGTCTTCATCTTGCTSCTTCTGAGCGGGGAGCGGAACTTCGGGGTGCGGCT GAAC AAACCCTACTCAATCCGCGTGCCCATGGACATCCCAGTCTTCACAGGGACCCACGCCGAC CTGC TCATTGTGGTGTTCCACAAGATCATCACCAGCGGGCACCAGCGGTTGCAGCCCCTCTTCG ACTG CCTGCTCACCATCGTGGTCAACGTGTCCCCCTACCTCAAGAGCCTGTCCATGGTGACCGC CAAC AAGTTGCTGCACCTGCTGGAGGCCTTCTCCACCACCTGGTTCCTCTTCTCTGCCGCCCAG AACC ACCACCTGGTCTTCTTCCTCCTGGAGGTCTTCAACAACATCATCCAGTACCAGTTTGATG GCAA CTCCAACCTGGTCTACGCCATCATCCGCAAGCGCAGCATCTTCCACCAGCTGGCCAACCT GCCC ACGGACCCGCCCACCATTCACAAGGCCCTGCAGCGGCGCCGGCGGACACCTGAGCCCTTG TCTC GCACCGGCTCCCAGGAGGGCACCTCCATGGAGGGCTCCCGCCCCGCTGCCCCTGCAGAGC CAGG CACCCTCAAGACCAGTCTGGTGGCTACTCCAGGCATTGACAAGCTGACCGAGAAGTCCCA GGTG TCAGAGGATGGCACCTTGCGGTCCCTGGAACCTGAGCCCCAGCAGAGCTTGGAGGATGGC AGCC CGGCTAAGGGGGAGCCCAGCCAGGCATGGAGGGAGCAGCGGCGACCATCCACCTCATCAG CCAG TGGGCAGTGGAGCCCAACGCCAGAGTGGGTCCTCTCCTGGAAGTCGAAGCTGCCGCTGCA GACC ATCATGAGGCTGCTGCAGGTGCTGGTTCCGCAGGTGGAGAAGATCTGCATCGACAAGGGC CTGA CGGATGAGTCTGAGATCCTGCGGTTCCTGCAGCATGGCACCCTGGTGGGGCTGCTGCCCG TGCC CCACCCCATCCTCATCCGCAAGTACCAGGCCAACTCGGGCACTGCCATGTGGTTCCGCAC CTAC ATGTGGGGCGTCATCTATCTGAGGAATGTGGACCCCCCTGTCTGGTACGACACCGACGTG AAGC TGTTTGAGATACAGCGGGTGTGAGGATGAAGCCGACGAGGGGCTCAGTCTAGGGGAAGGC AGGG CCTTGGTCCCTGAGGCTTCCCCCATCCACCATTCTGAGCTTTAAATTACCACGATC ORF Start : ATG at 133 ORF Stop : TGA at 1813

SEQ ID NO : 84 at 63082. 9kD ." :... n, NOV16b, MGSTDSKLNFRKAVIQLTTKTQPVEATDDAYDPVGYGIPYNHLLFSDTGEPLVEEAAQVL IVTL CG54443-07 DHDSASSASPTVDGTTTGTAMDDADPPGPENLFVNYLSRIHREEDFQFILKGIARLLSNP LLQT _ YLPNSTKKIQFHQELLVLFWKLCDFNKKFLFFVLKSSDVLDILVPILFFLNDARADQSRV GLMH Protein Sequence IGVFILLLLSGERNFGVRLNKPYSIRVPMDIPVFTGTHMH. IWFHKIITSGHQRLQPLFDCI, LTIVVNVSPYLKSLSMVTANKLLHLLEAFSTTWFLFSAAQNHHLVFFLLEVFNNIIQYQF DGNS NLVYAIIRKRSIFHQLANLPTDPPTIHKALQRRRRTPEPLSRTGSQEGTSMEGSRPAAPA EPGT LKTSLVATPGIDKLTEKSQVSEDGTLRSLEPEPQQSLEDGSPAKGEPSQAWREQRRPSTS SASG QWSPTPEWVLSWKSKLPLQTIMRLLQVLVPQVEKICIDKGLTDESEILRFLQHGTLVGLL PVPH PILIRKYQANSGTAMWFRTYMWGVIYLRNVDPPVWYDTDVKLFEIQRV SEQ ID NO : 85 3146 bp NVV16c, ATGGGGTCGACCGACTCCAAGCTGAACTTCCGGAAGGCGGTGATCCAGCTCACCACCAAG ACGC CG54443-01 DNA AGCCCGTGGAAGCCACCGATGATGCCTTTTGGGACCAGTTCTGGGCAGACACAGCCACCT CGGT GCAGGATGTGTTTGCACTGGTGCCGGCAGCAGAGATCCGGGCCGTGCGGGAAGAGTCACC CTCC Sequence AACTTGGCCACCCTGTGCTACAAGGCCGTTGAGAAGCTGGTGCAGGGAGCTGAGAGTGGC TGCC ACTCGGAGAAGGAGAAGCAGATCGTCCTGAACTGCAGCCGGCTGCTCACCCGCGTGCTGC CCTA CATCTTTGAGGACCCCGACTGGAGGGGCTTCTTCTGGTCCACAGTGCCCCAGCAGGGAGA AGAG GATGATGAGCATGCCAGGCCCCTGGCCGAGTCCCTGCTCCTGGCCATTGCTGACCTGCTC TTCT GCCCGGACTTCACGGTTCAGAGCCACCGGAGGAGCACTGTGGACTCGGCAGAGGACGTCC ACTC CCTGSAQGCTGTGAATACATCTGGGAGGCTGGTGTGGGCTTCGCTCACTCCCCCCAGCCT AAC TACATCCACGATATGAACCGGATGGAGCTGCTGAAACTGCTGCTGACATGCTTCTCCGAG GCCA TGTACCTGCCCCCAGCTCCGGAAAGTGGCAGCACCAACCCATGGGTTCAGTTCTTTTGTT CCAC GGAGAACAGACATGCCCTGCCCCTCTTCACCTCCCTCCTCAACACCGTGTGTGCCTATGA CCCT GTGGGCTACGGGATCCCCTACAACCACCTGCTCTTCTCTGACTACCGGGAACCCCTGGTG GAGG AGGCTGCCCAGGTGCTCATTGTCACTTTGGACCACGACAGTGCCAGCAGTGCCAGCCCCA CTGT GGACGGCACCACCACTGGCACCGCCATGGATGATGCCGATGACTTCCAGTTCATCCTCAA GGGT . ATAGCCCGGCTGCTGTCCAACCCCCTGCTCCAGACCTACCTGCCTAACTCCACCAAGAAG ATCC AGTTCCACCAGGAGCTGCTAGTTCTCTTCTGGAAGCTCTGCGACTTCAACAAGAAATTCC TCTT CTTCGTGCTGAAGAGCAGCGACGTCCTAGACATCCTTGTCCCCATCCTCTTCTTCCTCAA CGAT GCCCGGGCCGATCAGTCTCGGGTGGGCCTGATGCACATTGGTGTCTTCATCTTGCTGCTT CTGA GCGGGGAGCGGAACTTCGGGGTGCGGCTGAACAAACCCTACTCAATCCGCGTGCCCATGG ACAT CCCAGTCTTCACAGGGACCCACGCCGACCTGCTCATTGTGGTGTTCCACAAGATCATCAC CAGC GGGCACCAGCGGTTGCAGCCCCTCTTCGACTGCCTGCTCACCATCGTGGTCAACGTGTCC CCCT ACCTCAAGAGCCTGTCCATGGTGACCGCCAACAAGTTGCTGCACCTGCTGGAGGCCTTCT CCAC CACCTGGTTCCTCTTCTCTGCCGCCCAGAACCACCACCTGGTCTTCTTCCTCCTGGAGGT CTTC CAACATCATCCAGTACCAGTTTGATGGCAACTCCAACCTGGTCTACGCCATCATCCGCAA GC GCAGCATCTTCCACCAGCTGGCCAACCTGCCCACGGACCCGCCCACCATTCACAAGGCCC TGCA GCGGCGCCGGCGGACACCTGAGCCCTTGTCTCGCACCGGCTCCCAGGAGGGCACCTCCAT GGAG GGCTCCCGCCCCGCTGCCCCTGCAGAGCCAGGCACCCTCAAGACCAGTCTGGTGGCTACT CCAG GCATTGACAAGCTGACCGAGAAGTCCCAGGTGTCAGAGGATGGCACCTTGCGGTCCCTGG AACC TGAGCCCCAGCAGAGCTTGGAGGATGGCAGCCCGGCTAAGGGGGAGCCCAGCCAGGCATG GAGG GAGCAGCGGCGACCGTCCACCTCATCAGCCAGTGGGCAGTGGAGCCCAACGCCAGAGTGG GTCC TCTCCTGGAAGTCGAAGCTGCCGCTGCAGACCATCATGAGGCTGCTGCAGGTGCTGGTTC CGCA GGTGGAGAAGATCTGCATCGACAAGGGCCTGACGGATGAGTCTGAGATCCTGCGGTTCCT GCAG CATGGCACCCTGGTGGGGCTGCTGCCCGTGCCCCACCCCATCCTCATCCGCAAGTACCAG GCCA ACTCGGGCACTGCCATGTGGTTCCGCACCTACATGTGGGGCGTCATCTATCTGAGGAATG TGGA CCCCCCTGTCTGGTACGACACCGACGTGAAGCTGTTTGAGATACAGCGGGTGTGAGGATG AAGC CGACGAGGGGCTCAGTCTAGGGGAAGGCAGGGCCTTGGTCCCTGAGGCTTCCCCCATCCA CCAT TCTGAGCTTTAAATTACCACGATCAGGGCCTGGAACAGGCAGAGTGGCCCTGAGTGTCAT GCCC TAGAGACCCCTGTGGCCAGGACAATGTGAACTGGCTCAGATCCCCCTCAACCCCTAGGCT GGAC TCACAGGAGCCCCATCTCTGGGGCTATGCCCCCACCAGAGACCACTGCCCCCAACACTCG GACT CCCTCTTTAAGACCTGGCTCAGTGCTGGCCCCTCAGTGCCCACCCACTCCTGTGCTACCC AGCC CCAGAGGCAGAAGCCAAAATGGGTCACTGTGCCCTAAGGGGTTTGACCAGGGAACCACGG GCTG TCCCTTGAGGTGCCTGGACAGGGTAAGGGGGTGCTTCCAGCCTCCTAACCCAAAGCCAGC TGTT CCAGGCTCCAGGGGAAAAAGGTGTGGCCAGGCTGCTCCTCGAGGAGGCTGGGAGCTGGCC GACT GCAAAAGCCAGACTGGGGCACCTCCCGTATCCTTGGGGCATGGTGTGGGGTGGTGAGGGT CTCC TGCTATATTCTCCTGGATCCATGGAAATAGCCTGGCTCCCTCTTACCCAGTAATGAGGGG CAGG GAAGGGAACTGGGAGGCAGCCGTTTAGTCCTCCCTGCCCTGCCCACTGCCTGGATGGGGC GATG CCACCCCTCATCCTTCACCCAGCTCTGGCCTCTGGGTCCCACCACCCAGCCCCCCGTGTC AGAA CAATCTTTGCTCTGTACAATCGGCCTCTTTACAATAAAACCTCCTGCTC w w _~ m em ORF Start : ATG at 1 ORF Stop : TGA at 2293 SEQ ID NO : 86 1764 aa IMW at 86166. 6kD NOV1SC, MGSTDSKLNFRKAVIQLTTKTQPVEATDDAFWDQFWADTATSVQDVFALVPAAEIRAVRE ESPS CG54443-O NLATLCYKAVEKLVQGAESGCHSEKEKQIVLNCSRLLTRVLPYIFEDPDWRGFFWSTVPQ QGEE DDEHARPLAESLLLAIADLLFCPDFTVQSHRRSTVDSAEDVHSLDSCEYIWEAGVGFAHS PQPN Prote Sequence YIHDMNRMELLKLLLTCFSEAMYLPPAPESGSTNPWVQFFCSTENRHALPLFTSLLNTVC AYDP VGYGIPYNHLLFSDYREPLVEEAAQVLIVTLDHDSASSASPTVDGTTTGTAMDDADDFQF ILKG IARLLSNPLLQTYLPNSTKKIQFHQELLVLFWKLCDFNKKFLFFVLKSSDVLDILVPILF FLND ARADQSRVGLMHIGVFILLLLSGERNFGVRLNKPYSIRVPMDIPVFTGTHADLLIVVFHK IITS GHQRLQPLFDCLLTIVVNVSPYLKSLSMVTANKLLHLLEAFSTTWFLFSAAQNHHLVFFL LEVF NNIIQYQFDGNSNLVYAIIRKRSIFHQLANLPTDPPTIHKALQRRRRTPEPLSRTGSQEG TSME GSRPAAPAEPGTLKTSLVATPGIDKLTEKSQVSEDGTLRSLEPEPQQSLEDGSPAKGEPS QAWR EQRRPSTSSASGQWSPTPEWVLSWKSKLPLQTIMRLLQVLVPQVEKICIDKGLTDESEIL RFLQ HGTLVGLLPVPHPILIRKYQANSGTAMWFRTYMWGVIYLRNVDPPVWYDTDVTLFEIQRV H

SEQ ID NO : 87 3314 bp NOV16d, GCGAGAGCCGCGGGGGCCGCGGAGCTGGAGCCGGAGCTGAAGCCGGAGCCGGGTTGGAGT CTGG CG543-02 DNA GCGGGGGCCGGGCCGGAGCGGGCTCCAGAGACATGGGGTCGACCGACTCCAAGCTGAACT TCCG GAAGGCGGTGATCCAGCTCACCACCAAGACGCAGCCCGTGGAAGCCACCGATGATGCCTT TTGG Sequence GACCAGTTCTGGGCAGACACAGCCACCTCGGTGCAGGATGTGTTTGCACTGGTGCCGGCA GCAG AGATCCGGGCCGTGCGGGAAGAGTCACCCTCCAACTTGGCCACCCTGTGCTACAAGGCCG TTGA GAAGCTGGTGCAGGGAGCTGAGAGTGGCTGCCACTCGGAGAAGGAGAAGCAGATCGTCCT GAAC TGCAGCCGGCTGCTCACCCGCGTGCTGCCCTACATCTTTGAGGACCCCGACTGGAGGGGC TTCT TCTGGTCCACAGTGCCCGGGGCAGGGCGAGGAGGGCAGGGAGAAGAGGATGATGAGCATG CCAG GCCCCTGGCCGAGTCCCTGCTCCTGGCCATTGCTGACCTGCTCTTCTGCCCGGACTTCAC GGTT CAGAGCCACCGGAGGAGCACTGTGGACTCGGCAGAGGACGTCCACTCCCTGGACAGCTGT GAAT ACATCTGGGAGGCTGGTGTGGGCTTCGCTCACTCCCCCCAGCCTAACTACATCCACGATA TGAA CCGGATGGAGCTGCTGAAACTGCTGCTGACATGCTTCTCCGAGGCCATGTACCTGCCCCC AGCT CCGGAAAGTGGCAGCACCAACCCATGGGTTCAGTTCTTTTGTTCCACGGAGAACAGACAT GCCC TGCCCCTCTTCACCTCCCTCCTCAACACCGTGTGTGCCTATGACCCTGTGGGCTACGGGA TCCC CTACAACCACCTGCTCTTCTCTGACACCGGGGAACCCCTGGTGGAGGAGGCTGCCCAGGT GCTC ATTGTCACTTTGGACCACGACAGTGCCAGCAGTGCCAGCCCCACTGTGGACGGCACCACC ACTG GCACCGCCATGGATGATGCCGATCCTCCAGGCCCTGAGAACCTGTTTGTGAACTACCTGT CCCG CATCCATCGTGAGGAGGACTTCCAGTTCATCCTCAAGGGTATAGCCCGGCTGCTGTCCAA CCCC CTGCTCCAGACCTACCTGCCTAACTCCACCAAGAAGATCCAGTTCCACCAGGAGCTGCTA GTTC TCTTCTGGAAGCTCTGCGACTTCAACAAGAAATTCCTCTTCTTCGTGCTGAAGAGCAGCG ACGT CCTAGACATCCTTGTCCCCATCCTCTTCTTCCTCAACGATGCCCGGGCCGATCAGTCTCG GGTG GGCCTGATGCACATTGGTGTCTTCATCTTGCTGCTTCTGAGCGGGGAGCGGAACTTCGGG GTGC GGCTGAACAAACCCTACTCAATCCGCGTGCCCATGGACATCCCAGTCTTCACAGGGACCC ACGC CGACCTGCTCATTGTGGTGTTCCACAAGATCATCACCAGCGGGCACCAGCGGTTGCAGCC CCTC TTCGACTGCCTGCTCACCATCGTGGTCAACGTGTCCCCCTACCTCAAGAGCCTGTCCATG GTGA CCGCCAACAAGTTGCTGCACCTGCTGGAGGCCTTCTCCACCACCTGGTTCCTCTTCTCTG CCGC CCAGAACCACCACCTGGTCTTCTTCCTCCTGGAGGTCTTCAACAACATCATCCAGTACCA GTTT GATGGCAACTCCAACCTGGTCTACGCCATCATCCGCAAGCGCAGCATCTTCCACCAGCTG GCCA ACCTGCCCACGGACCCGCCCACCATTCACAAGGCCCTGCAGCGGCGCCGGCGGACACCTG AGCC CTTGTCTCGCACCGGCTCCCAGGAGGGCACCTCCATGGAGGGCTCCCGCCCCGCTGCCCC TGCA GAGCCAGGCACCCTCAAGACCAGTCTGGTGGCTACTCCAGGCATTGACAAGCTGACCGAG AAGT CCCAGGTGTCAGAGGATGGCACCTTGCGGTCCCTGGAACCTGAGCCCCAGCAGAGCTTGG AGGA TGGCAGCCCGGCTAAGGGGGAGCCCAGCCAGGCATGGAGGGAGCAGCGGCGACCATCCAC CTCA TCAGCCAGTGGGCAGTGGAGCCCAACGCCAGAGTGGGTCCTCTCCTGGAAGTCGAAGCTG CCGC TGCAGACCATCATGAGGCTGCTGCAGGTGCTGGTTCCGCAGGTGGAGAAGATCTGCATCG ACAA GGGCCTGACGGATGAGTCTGAGATCCTGCGGTTCCTGCAGCATGGCACCCTGGTGGGGCT GCTG CCCGTGCCCCACCCCATCCTCATCCGCAAGTACCAGGCCAACTCGGGCACTGCCATGTGG TTCC GCACCTACATGTGGGGCGTCATCTATCTGAGGAATGTGGACCCCCCTGTCTGGTACGACA CCGA CGTGAAGCTGTTTGAGATACAGCGGGTGTGAGGATGAAGCCGACGAGGGGCTCAGTCTAG GGGA AGGCAGGGCCTTGGTCCCTGAGGCTTCCCCCATCCACCATTCTGAGCTTTAAATTACCAC GATC AGGGCCTGGAACAGGCAGAGTGGCCCTGAGTGTCATGCCCTAGAGACCCCTGTGGCCAGG ACAA TGTGAACTGGCTCAGATCCCCCTCAACCCCTAGGCTGGACTCACAGGAGCCCCATCTCTG GGGC TATGCCCCCACCAGAGACCACTGCCCCCAACACTCGGACTCCCTCTTTAAGACCTGGCTC AGTG CTGGCCCCTCAGTGCCCACCCACTCCTGTGCTACCCAGCCCCAGAGGCAGAAGCCAAAAT GGGT CACTGTGCCCTAAGGGGTTTGACCAGGGAACCACGGGCTGTCCCTTGAGGTGCCTGGACA GGGT AAGGGGGTGCTTCCAGCCTCCTAACCCAAAGCCAGCTGTTCCAGGCTCCAGGGGAAAAAG GTGT GGCCAGGCTGCTCCTCGAGGAGGCTGGGAGCTGGCCGACTGCAAAAGCCAGACTGGGGCA CCTC CCGTATCCTTGGGGCATGGTGTGGGGTGGTGAGGGTCTCCTGCTATATTCTCCTGGATCC ATGG AAATAGCCTGGCTCCCTCTTACCCAGTAATGAGGGGCAGGGAAGGGAACTGGGAGGCAGC CGTT TAGTCCTCCCTGCCCTGCCCACTGCCTGGATGGGGCGATGCCACCCCTCATCCTTCACCC AGCT CTGGCCTCTGGGTCCCACCACCCAGCCCCCCGTGTCAGAACAATCTTTGCTCTGTACAAT CGGC CTCTTTACAATAAAACCTCCTGCTC S w mcE SEQIDNO : 88 J788aa JMWat88582. 2kD NOV16d, MGSTDSKLNFRKAVIQLTTKTQPVEATDDAFWDQFWADTATSVQDVFALVPAAEIRAVRE ESPS CG54443-02 NLATLCYKAVEKLVQGAESGCHSEKEKQIVLNCSRLLTRVLPYIFEDPDWRGFFWSTVPG AGRG GQGEEDDEHARPLAESLLLAIADLLFCPDFTVQSHRRSTVDSAEDVHSLDSCEYIWEAGV GFAH Protem Sequence SPQPNYIHDMNRMELLKLLLTCFSEAMYLPPAPESGSTNPWVQFFCSTENRHALPLFTSL LNTV CAYDPVGYGIPYNHLLFSDTGEPLVEEAAQVLIVTLDHDSASSASPTVDGTTTGTAMDDA DPPG PENLFVNYLSRIHREEDFQFILKGIARLLSNPLLQTYLPNSTKKIQFHQELLVLFWKLCD FNKK FLFFVLKSSDVLDILVPILFFLNDARADQSRVGLMHIGVFILLLLSGERNFGVRLNKPYS IRVP MDIPVFTGTHADLLIWFHKIITSGHQRLQPLFDCLLTIWNVSPYLKSLSMVTANKLLHLL EA FSTTWFLFSAAQNHHLVFFLLEVFNNIIQYQFDGNSNLVYAIIRKRSIFHQLANLPTDPP TIHK ALQRRRRTPEPLSRTGSQEGTSMEGSRPAAPAEPGTLKTSLVATPGIDKLTEKSQVSEDG TLRS LEPEPQQSLEDGSPAKGEPSQAWREQRRPSTSSASGQWSPTPEWVLSWKSKLPLQTIMRL LQVL VPQVEKICIDKGLTDESEILRFLQHGTLVGLLPVPHPILIRKYQANSGTAMWFRTYMWGV IYLR NVDPPVWYDTDVKLFEIQRV

Sequence TTGGCCACCCTGTGCTACAAGGCCGTTGAGAGGCTGGTGCAGGGAGCTGAGAGTGGCTGC CACTC GGAGAAGGAGAAGCAGATCGTCCTGAACTGCAGCCGGCTGCTCACCCGCGTGCTGCCCTA CATCT TTGAGGACCCCGACTGGAGGGGCTTCTTCTGGTCCACAGTGCCCGGGGCAGGGCGAGGAG GGCAG GGAGAAGAGGATGATGAGCATGCCAGGCCCCTGGCCGAGTCCCTGCTCCTGGCCATTGCT GACCT GCTCTTCTGCCCGGACTTCACGGTTCAGAGCCACCGGAGGAGCACTGTGGACTCGGCAGA GGACG TCCACTCCCTGGACAGCTGTGAATACATCTGGGAGGCTGGTGTGGGCTTCGCTCACTCCC CCCAG CCTAACTACATCCACGATATGAACCGGATGGAGCTGCTGAAACTGCTGCTGACATGCTTC TCCGA GGCCATGTACCTGCCCCCAGCTCCGGAAAGTGGCAGCACCAACCCATGGGTTCAGTTCTT TTGTT CCACGGAGAACAGACATGCCCTGCCCCTCTTCACCTCCCTCCTCAACACCGTGTGTGCCT ATGAC CCTGTGGGCTACGGGATCCCCTACAACCACCTGCTCTTCTCTGACTACCGGGAACCCCTG GTGGA GGAGGCTGCCCAGGTGCTCATTGTCACTTTGGACCACGACAGTGCCAGCAGTGCCAGCCC CACTG TGGACGGCACCACCACTGGCACCGCCATGGATGATGCCGATCCTCCAGGCCCTGAGAACC TGTTT GTGAACTACCTGTCCCGCATCCATCGTGAGGAGGACTTCCAGTTCATCCTCAAGGGTATA GCCCG GCTGCTGTCCAACCCCCTGCTCCAGACCTACCTGCCTAACTCCACCAAGAAGATCCAGTT CCACC AGGAGCTGCTAGTTCTCTTCTGGAAGCTCTGCGACTTCAACAAGAAATTCCTCTTCTTCG TGCTG AAGAGCAGCGACGTCCTAGACATCCTTGTCCCCATCCTCTTCTTCCTCAACGATGCCCGG GCCGA TCAGTCT TCAGTCT ORF Start : ATG at 1 ORF Stop : end of sequence ORF start : ATG at 1 loRF stop : end of sequence S H SEQIDNOO ! 414aa MWat46487. 9 NOV16e, MGSTDSKLNFRKAVIQLTTKTQPVEATDDAFWDQFWADTATSVQDVFALVPAAEIRAVRE ESPS CG54443-04 NLATLCYKAVERLVQGAESGCHSEKEKQIVLNCSRLLTRVLPYIFEDPDWRGFFWSTVPG AGRG . GQGEEDDEHARPLAESLLLAIADLLFCPDFTVQSHRRSTVDSAEDVHSLDSCEYIWEAGV GFAH q SPQPNYIHDMNRMELLKLLLTCFSEAMYLPPAPESGSTNPWVQFFCSTENRHALPLFTSL LNTV CAYDPVGYGIPYNHLLFSDYREPLVEEAAQVLIVTLDHDSASSASPTVDGTTTGTAMDDA DPPG PENLFVNYLSRIHREEDFQFILKGIARLLSNPLLQTYLPNSTKKIQFHQELLVLFWKLCD FNKK FLFFVLKSSDVLDILVPILFFLNDARADQS

SEQID : 91 1242 bp NOV16f, ATGGGGTCGACCGACTCCAAGCTGAACTTCCGGAAGGCGGTGATCCAGCTCACCACCAAG ACGCA CG543-05 DNA GCCCGTGGAAGCCACCGATGATGCCTTTTGGGACCAGTTCTGGGCAGACACAGCCACCTC GGTGC AGGATGTGTTTGCACTGGTGCCGGCAGCAGAGATCCGGGCCGTGCGGGAAGAGTCACCCT CCAAC Sequence TTGGCCACCCTGTGCTACAAGGCCGTTGAGAGGCTGGTGCAGGGAGCTGAGAGTGGCTGC CACTC GGAGAAGGAGAAGCAGATCGTCCTGAACTGCAGCCGGCTGCTCACCCGCGTGCTGCCCTA CATCT TTGAGGACCCCGACTGGAGGGGCTTCTTCTGGTCCACAGTGCCCGGGGCAGGGCGAGGAG GGCAG GGAGAAGAGGATGATGAGCATGCCAGGCCCCTGGCCGAGTCCCTGCTCCTGGCCATTGCT GACCT GCTCTTCTGCCCGGACTTCACGGTTCAGAGCCACCGGAGGAGCACTGTGGACTCGGCAGA GGACG TCCACTCCCTGGACAGCTGTGAATACATCTGGGAGGCTGGTGTGGGCTTCGCTCACTCCC CCCAG CCTAACTACATCCACGATATGAACCGGATGGAGCTGCTGAAACTGCTGCTGACATGCTTC TCCGA GGCCATGTACCTGCCCCCAGCTCCGGAAAGTGGCAGCACCAACCCATGGGTTCAGTTCTT TTGTT CCACGGAGAACAGACATGCCCTGCCCCTCTTCACCTCCCTCCTCAACACCGTGTGTGCCT ATGAC CCTGTGGGCTACGGGATCCCCTACAACCACCTGCTCTTCTCTGACTACCGGGAACCCCTG GTGGA GGAGGCTGCCCAGGTGCTCATTGTCACTTTGGACCACGACAGTGCCAGCAGTGCCAGCCC CACTG TGGACGGCACCACCACTGGCACCGCCATGGATGATGCCGATCCTCCAGGCCCTGAGAACC TGTTT GTGAACTACCTGTCCCGCATCCATCGTGAGGAGGACTTCCAGTTCATCCTCAAGGGTATA GCCCG GCTGCTGTCCAACCCCCTGCTCCAGACCTACCTGCCTAACTCCACCAAGAAGATCCAGTT CCACC AGGAGCTGCTAGTTCTCTTCTGGAAGCTCTGCGACTTCAACAAGAAATTCCTCTTCTTCG TGCTG AAGAGCAGCGACGTCCTAGACATCCTTGTCCCCATCCTCTTCTTCCTCAACGATGCCCGG GCCGA TCAGTCT ORF Start : ATG at 1 ORF Stop : end of sequence SEQ ID NO : 92 at 46487. 9kD NOV16f MGSTDSKLNFRKAVIQLTTKTQPVEATDDAFWDQFWADTATSVQDVFALVPAAEIRAVRE ESPS CG54443-05 NLATLCYKAVERLVQGAESGCHSEKEKQIVLNCSRLLTRVLPYIFEDPDWRGFFWSTVPG AGRG GQGEEDDEHARPLAESLLLAIADLLFCPDFTVQSHRRSTVDSAEDVHSLDSCEYIWEAGV GFAH SPQPNYIHDMNRMELLKLLLTCFSEAMYLPPAPESGSTNPWVQFFCSTENRHALPLFTSL LNTV CAYDPVGYGIPYNHLLFSDYREPLVEEAAQVLIVTLDHDSASSASPTVDGTTTGTAMDDA DPPG PENLFVNYLSRIHREEDFQFILKGIARLLSNPLLQTYLPNSTKKIQFHQELLVLFWKLCD FNKK SEQ ID NO :, 93 11742 bp NOVl6g, ATGGGGTCGACCGACTCCAAGCTGAACTTCCGGAAGGCGGTGATCCAGCTCACCACCAAG ACGCA CG543-06 DNA GCCCGTGGAAGCCACCGATGATGCCTTTTGGGACCAGTTCTGGGCAGACACAGCCACCTC GGTGC AGGATGTGTTTGCACTGGTGCCGGCAGCAGAGATCCGGGCCGTGCGGGAAGAGTCACCCT CCAAC Sequence TTGGCCACCCTGTGCTACAAGGCCGTTGAGAGGCTGGTGCAGGGAGCTGAGAGTGGCTGC CACTC GGAGAAGGAGAAGCAGATCGTCCTGAACTGCAGCCGGCTGCTCACCCGCGTGCTGCCCTA CATCT TTGAGGACCCCGACTGGAGGGGCTTCTTCTGGTCCACAGTGCCCGGGGCAGGGCGAGGAG GGCAG GGAGAAGAGGATGATGAGCATGCCAGGCCCCTGGCCGAGTCCCTGCTCCTGGCCATTGCT GACCT GCTCTTCTGCCCGGACTTCACGGTTCAGAGCCACCGGAGGAGCACTGTGGACTCGGCAGA GGACG TCCACTCCCTGGACAGCTGTGAATACATCTGGGAGGCTGGTGTGGGCTTCGCTCACTCCC CCCAG CCTAACTACATCCACGATATGAACCGGATGGAGCTGCTGAAACTGCTGCTGACATGCTTC TCCGA GGCCATGTACCTGCCCCCAGCTCCGGAAAGTGGCAGCACCAACCCATGGGTTCAGTTCTT TTGTT CCACGGAGAACAGACATGCCCTGCCCCTCTTCACCTCCCTCCTCAACACCGTGTGTGCCT ATGAC CCTGTGGGCTACGGGATCCCCTACAACCACCTGCTCTTCTCTGACTACCGGGAACCCCTG GTGGA GGAGGCTGCCCAGGTGCTCATTGTCACTTTGGACCACGACAGTGCCAGCAGTGCCAGCCC CACTG TGGACGGCACCACCACTGGCACCGCCATGGATGATGCCGATCCTCCAGGCCCTGAGAACC TGTTT GTGAACTACCTGTCCCGCATCCATCGTGAGGAGGACTTCCAGTTCATCCTCAAGGGTATA GCCCG GCTGCTGTCCAACCCCCTGCTCCAGACCTACCTGCCTAACTCCACCAAGAAGATCCAGTT CCACC AGGAGCTGCTAGTTCTCTTCTGGAAGCTCTGCGACTTCAACAAGAAATTCCTCTTCTTCG TGCTG AAGAGAGCGACGTCCTAGACATCCTTGTCCCCATCCTCTTCTTCCTCAACGATGCCCGGG CCGA TCAGTCT ORF Start : ATG at 1 ORF Stop : end of sequence

SEQ ID NO : 94-Tl4 aa 46487. 9ldD NV1 6g, MGSTDSKLNFRKAVIQLTTKTQPVEATDDAFWDQFWADTATSVQDVFALVPAAEIRAVRE ESPS CG54443-06 NLATLCYKAVvQGAESGCHSEKEKQIVLNCSRLLTRVLPYIFEDPDWRGFFWSTVPGAGR G . GQGEEDDEHARPLAESLLLAIADLLFCPDFTVQSSRSTVDSAEDVHSLDSCEYIWEAGVG FAH q SPQPNYIIDMNRMELLKLLLTCFSEAMYLPPAPESGSTNPWVQFFCSTENRHALPLFTSL LNTV CAYDPVGYGIPYNHLLFSDYREPLVEEAAQVLIVTLDHDSASSASPTVDGTTTGTAMDDA DPPG PENLFVNYLSRIHREEDFQFILKGIARLLSNPLLQTYLPNSTKKIQFHQELLVLFWKLCD FNKK S Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 16B.

Table 16B. Comparison of NOV16a against NOV16b through NOV16g. Protein Sequence NOV16a Residues/Identities/ Match Residues Similarities for the Matched Region NOV16b 258.. 414 155/157 (98%) 30.. 186 155/157 (98%) NOV16c 1.. 414 388/414 (93%) 1.. 390 389/414 (93%) NOV16d 1.. 414 411/414 (99%) 1 414 412/414 (99%) NOV16e 1.. 414 414/414 (100%) 1 414 414/414 (100%) NOV16f 1.. 414 414/414 (100%) 1.. 414 414/414 (100%) NOV16g 1.. 414 414/414 (100%) 1.. 414 414/414 (100%) Further analysis of the NOV16a protein yielded the following properties shown in Table 16C.

Table 16C. Protein Sequence Properties NOVX Signal ? analysis: No Known Signal Sequence Indicated PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 11; pos. chg 2; neg. chg 1 H-region: length 0 ; peak value-0.21 PSG score:-4. 61 GvH: von Heijne's method for signal seq. recognition GvH score (threshold :-2. 1) : -5.12 possible cleavage site: between 48 and 49 >>> Seems to have no N-terminal signal peptide ALOM : Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0.5 : 2 Number of TMS (S) for threshold 0. 5: 0 PERIPHERAL Likelihood = 3.13 (at 93) ALOM score :-1. 28 (number of TMSs : 0) MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75): 2.33 Hyd Moment (95): 1.80 G content: 1 D/E content: 2 S/T content: 6 Score : -5.83 Gavel: indication of cleavage sites for mitochondrial preseq R-2 motif at 21 FRKJAV NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues: 7. 7% NLS Score :-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1 : none type 2: none NMYR: N-myristoylation pattern: MGSTDSK Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN : Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23): 43. 5 % : cytoplasmic 30. 4 % : mitochondrial 21.7 %: nuclear 4.3 % : peroxisomal # indication for CG54443-03 is cyt (k=23) A search of the NOV1 6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 16D.

Table 16D. Geneseq Results for NOV16a NOV16a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABG70273 Human CG8841-like protein #2-1.. 414 411/414 (99%) 0. 0 Homo sapiens, 788 aa. 1.. 414 412/414 (99%) [W0200255702-A2, 18-JUL-2002] AAM79253 Human protein SEQ ID NO 1915-1.. 414 412/414 (99%) 0. 0 Homo sapiens, 787 aa. 1.. 413 413/414 (99%) [W0200157190-A2, 09-AUG-2001] ABG70272 Human CG8841-like protein #1-l. Al4 388/414 (93%) 0. 0 Homo sapiens, 764 aa. 1.. 390 389/414 (93%) [W0200255702-A2, 18-JUL-2002] ABB12112 Human secreted protein homologue, 1.. 271 259/272 (95%) e-151 SEQ ID NO : 2482-Homo sapiens, 284 12.. 283 261/272 (95%) aa. [W0200157188-A2, 09-AUG-2001] ABB64025 Drosophila melanogaster polypeptide 1 414 252/414 (60%) e-146 SEQ ID NO 18867-Drosophila 1.. 398 310/414 (74%) melanogaster, 837 aa. [W0200171042-A2, 27-SEP-2001]

In a BLAST search of public sequence databases, the NOV1 6a protein was found to have homology to the proteins shown in the BLASTP data in Table 16E.

Table 16E. Public BLASTP Resuilts for NOV16a NOVX Protein Identitiesl Residuesl Expect Accession Protein/Organism/Length M h SimHarities for the Expect Number Residues Matched Portion Residues AAH35372 Hypothetical protein-Homo 1.. 414 413/414 (99%) 0. 0 sapiens (Human), 788 aa. 1.. 414 414/414 (99%) Q8TE83 Hypothetical protein FLJ23821-1.. 414 413/414 (99%) 0. 0 Homo sapiens (Human), 625 aa. 1.. 414 414/414 (99%) Q8R1F6 Hypothetical 88. 8 kDa protein-1.. 414 400/414 (96%) 0. 0 Mus musculus (Mouse), 788 aa. 1.. 414 410/414 (98%) Q9NT34 Hypothetical protein-Homo 1.. 364 354/364 (97%) 0. 0 sapiens (Human), 380 aa 1.. 363 355/364 (97%) (fragment). Q9V695 CG8841 protein-Drosophila 1.. 414 252/414 (60%) e-146 7 aL 1 398 310/414 (74%)

Example 17.

The NOV17 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 17A.

Table 17A. NOV17 Sequence Analysis SEQ ID NO : 95 1752 bp NOV17a, CTCTGGCCCTCACCCTCATCTTGATGGCAGCCTCTGGTGCTGCGTGCGAAGTGAGGGACG TTTG CG58495-01 DNA TGTTGGAAGCCCTGGTATCCCCGGCACTCCTGGATCCCACGGCCTGCCAGGCAGGGACGG GAGA GATGGTGTCAAAGGAGACCCTGGCCCTCCAGGCCCCATGGGTCCGCCTGGAGAAACACCA TGTC Sequence CTCCTGGGAATAATGGGCTGCCTGGAGCCCCTGGTGTCCCTGGAGAGCGTGGAGAGAAGG GGGA GCCTGGCGAGAGAGGCCCTCCAGGGCTTCCAGCTCATCTAGATGAGGAGCTCCAAGCCAC ACTC CACGACTTCAGACATCAAATCCTGCAGACAAGGGGAGCCCTCAGTCTGCAGGGCTCCATA ATGA CAGTAGGAGAGAAGGTCTTCTCCAGCAATGGGCAGTCCATCACTTTTGATGCCATTCAGG AGGC TGTGCCAGAGCAGGCGGCCGCATTGCTGTCCCAAGGAATCCAGAGGAAAATGAGGCCATT GCA AGCTTCGTGAAGAAGTACAACACATATGCCTATGTAGGCCTGACTGAGGGTCCCAGCCCT GGAG ACTTCCGCTACTCAGATGGGACCCCTGTAAACTACACCAACTGGTACCGAGGGGAGCCTG CAGG TCGGGGAAAAGAGAAGTGTGTGGAGATGTACACAGATGGGCAGTGGAATGACAGGAACTG CCTG TACTCCCGACTGACCATCTGTGAGTTCTGAGAGGCATTTAGGCCATGG ORF Start : at 3 ORF Stop : TGA at 732 SEQ M NU : 96 1243 25592. 3kD NOV17a, LALTLILMAASGAACEVRDVCVGSPGIPGTPGSHGLPGRDGRDGVKGDPGPPGPMGPPGE TPCP CG58495-01 PGNNGLPGPGpGERGEKGEPGERGPPGLPAHLDEELQATLHDFRHQILQTRGALSLQGSI MT Ws-v 1 VGEKVFSSNGQS ITFDAIQEACARAGGRIAVPRNPEENEAIASFVKKYNTYAYVGSTEGPSPGD FRYSDGTPVNYTNWYRGEPAGRGKEKCVEMYTDGQWNDRNCLYSRLTICEF SEQ ID NO : 97 _ 1681 bp _ I NOV17b, CCAAGCACCTGGAGGCTCTGTGTGTGGGTCGCTGATTTCTTGGAGCCTGAAAAGAAGGAG CAGC CG58495-03 DNA GACTGGACCCAGAGCCATGTGGCTGTGCCCTCTGGCCCTCACCCTCATCTTGATGGCAGC CTCT jayj-ujL'A (TQQcQjQQQ ( (Qc (QcCA (CTCCAC (CTT (G&CATC&AATCCTGC GGTGCTGCGTGCGAAGTGAAGGAGCTCCAAGCCACACTCCACGACTTCAGACATCAAATC CTGC' AGACAAGGGGAGCCCTCAGTCTGCAGGGCTCCATAATGACAGTAGGAGACAAGGTCTTCT CTAG CAATGGGCAGTCCATCACTTTTGATGCCATTC, AGGAGGCATGTGCCAGAGCAGGCGGCCGCATT GCTGTCCCAAGGAATCCAGAGGAAAATGAGGCCATTGCAAGCTTCGTGAAGAAGTACAAC ACAT ATGCCTATGTAGGCCTGACTGAGGGTCCCAGCCCTGGAGACTTCCGCTACTCAGATGGGA CCCC TGTAAACTACACCAACTGGTACCGAGGGGAGCCTGCAGGTCGGGGAAAAGAGAAGTGTGT GGAG TGTACACAGATGGGCAGTGGAATGACAGGAACTGCCTGTACTCCCGACTGACCATCTGTG AGT TCTGAGAGGCATTTAGGCCATGGGACAGGGAGGATCCTGTCTGGCCTTCAGTTTCCATCC CCAG GATCCACTTGGTCTGTGAGATGCTAGAACTCCCTTTCAACA ORF Start : ATG at 81 ORF Stop : TGA at 579

SEQ ID NO : 98 166 aa MW at 1838. 6kD w NOV1 7Lv, MWLCPLALTLILMAASGAACEVKELQATLHDFRHQILQTRGALSLQGSIMTVGEKVFSSN GQS I CG58495-03 TFDAIQEACARAGGRIAVPRNPEENEAIASFVKKYNTYAYVGLTEGPSPGDFRYSDGTPV NYTN . WYRGEPAGRGKEKCVEMYTDGQWNDRNCLYSRLTICEF Protem Sequence _ _ __ _

SEQ m NO : 99 1161 bp NOV1 7c, GGCTCTTTCTAGCTATAaAQCTGCTTGCCGCGCTGCACTCCACCACGCCTCCTCCAAGTC CCA CG58495-02 DNA GCGAACCCGCGTGCAACCTGTCCCGACTCTAGCCGCCTCTTCAGCTCACGGATCAATTCC CAAG TCGCTGGAGGCTCTGTGTGTGGGAGCAGCGACTGGACCCAGAGCCATGTGGCTGTGCCCT CTGG Sequence CCCTCAACCTCATCTTGATGGCAGCCTCTGGTGCTGTGTGCGAAGTGAAGGACGTTTGTG TTGG AAGCCCTGGTATCCCCGGCACTCCTGGATCCCACGGCCTGCCAGGCAGGGACGGGAGAGA TGGT GTCAAAGGAGACCCTGGCCCTCCAGGCCCCATGGGTCCACCTGGAGAAATGCCATGTCCT CCTG GAAATGATGGGCTGCCTGGAGCCCCTGGTATCCCTGGAGAGTGTGGAGAGAAGGGGGAGC CTGG CGAGAGGGGCCCTCCAGGGCTTCCAGCTCATCTAGATGAGGAGCTCCAAGCCACACTCCA CGAC TTTAGACATCAAATCCTGCAGACAAGGGGAGCCCTCAGTCTGCAGGGCTCCATAATGACA GTAG GAGAGAAGGTCTTCTCCAGCAATGGGCAGTCCATCACTTTTGATGCCATTCAGGAGGCAT GTGC CAGAGCAGGCGGCCGCATTGCTGTCCCAAGGAATCCAGAGGAAAATGAGGCCATTGCAAG CTTC GTGAAGAAGTACAACACATATGCCTATGTAGGCCTGACTGAGGGTCCCAGCCCTGGAGAC TTCC GCTACTCAGACGGGACCCCTGTAAACTACACCAACTGGTACCGAGGGGAGCCCGCAGGTC GGGG AAAAGAGCAGTGTGTGGAGATGTACACAGATGGGCAGTGGAATGACAGGAACTGCCTGTA CTCC CGACTGACCATCTGTGAGTTCTGAGAGGCATTTAGGCCATGGGACAGGGAGGACGCTCTC TGGC CTCCATCCTGAGGCTCCACTTGGTCTGTGAGATGCTAGAACTCCCTTCAACAGAATTGAT CCCT GCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCTCGAGGAGTACACT AAGA AGCTCAACACCCAGTGAGGCGCCCGCCGCCGCCCCCCTTCCCGGTGCTCAGAATAAACGT TTCC AAAGTGGGA ORF Start : ATG at 174 ORF Stop : TGA at 91 oR SEQ ID NO : 100"1 4iZ"IMW at 26228. 2kD NOVI7C, MWLCPLALNLILMAASGAVCEVKDVCVGSPGIPGTPGSHGLPGRDGRDGVKGDPGPPGPM GPPG CG58495-02 EMPCPPGNDGLPGAPGIPGECGEKGEPGERGPPGLPAHLDEELQATLHDFRHQILQTRGA LSLQ GSIMTVGEKVFSSNGQSITFDAIQEACARAGGRIAVPRNPEEN] EAIASFVKKYNTYAYVGLTEG PSPGDFRYSDGTPVNYTNWYRGEPAGRGKEQCVEMYTDGQWNDRNCLYSRLTICEF

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 17B.

Table 17B. Comparison of NOV17a against NOVl7b and NOV17c. Protein Sequence NOV17a Residues/Identities/ Match Residues Similarities for the Matched Region NOV17b 100.. 243 143/144 (99%) 23.. 166 144/144 (99%) NOV17c 1.. 243 235/243 (96%) 6.. 248 239/243 (97%) Further analysis of the NOV17a protein yielded the following properties shown in Table 17C.

Table 17C. Protein Sequence Properties NOV17a SignalP analysis: Cleavage site between residues 16 and 17 PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 0 ; pos. chg 0 ; neg. chg 0 H-region: length 15; peak value 10.71 PSG score: 6.31 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1) : 2.44 possible cleavage site: between 15 and 16 >>> Seems to have a cleavable signal peptide (1 to 15) ALOM : Klein et al's method for TM region allocation Init position for calculation: 16 Tentative number of TMS (s) for the threshold 0. 5: 0 number of TMS (s).. fixed PERIPHERAL Likelihood = 7.37 (at 113) ALOM score: 7.37 (number of TMSs : 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation :. 7 Charge difference:-2. 0 C (-1. O)-N (1. 0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 2.24 Hyd Moment (95): 0.60 G content: 1 D/E content: 1 S/T content: 2 Score:-6. 05 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC : discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 9. 1% NLS Score :-0. 47 KDEL : ER retention motif in the C-terminus: none ER Membrane Retention Signals : none. SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif : none RNA-binding motif : none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Prenylation motif: none memYQRL : transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Indication: nuclear Reliability: 55.5 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues -------------------------- Final Results (k = 9/23): 66. 7 % : extracellular, including cell wall 22. 2 % : nuclear 11. 1 % : mitochondrial >> indication for CG58495-01 is exc (k=9) A search of the NOV17a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 17D.

Table 17D. Geneseq Results for NOV17a

NOV17a Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region AAU76468 Human lung surfactant protein A-1.. 243 241/243 (99%) e-147 Homo sapiens, 248 aa. 6.. 248 243/243 (99%) [W0200206301-A2, 24-JAN-2002] AAY77989 Human SP-A amino acid sequence-1.. 243 241/243 (99%) e-147 Homo sapiens, 248 aa. 6.. 248 243/243 (99%) [W0200011161-A1, 02-MAR-2000] AAP70662 35kd pulmonary surfactant protein-1.. 243 240/243 (98%) e-146 Homo sapiens, 248 aa. 6.. 248 242/243 (98%) [W08702037-A, 09-APR-1987]'' AAR05091 Vector PSP 35K-lA-10 gene product 1.. 243 239/243 (98%) e-146 encoding pulmonary surfactant protein-6.. 248 242/243 (99%) Homo sapiens, 248 aa. [US4882422-A, 21-NOV-1989] AAB58135 Lung cancer associated polypeptide 1.. 243 238/243 (97%) e-145 sequence SEQ ID 473-Homo sapiens, 17.. 259 239/243 (97%) 259 aa. [W0200055180-A2, 21-SEP-2000] In a BLAST search of public sequence databases, the NOV17a protein was found to have homology to the proteins shown in the BLASTP data in Table 17E.

Table 17E. Public BLASTP Results for NOV17a NOV17a Identities/ Protein NOV17a Identities/ Accession Protein/Organism/Length Residues/Similarities for Expect Number patch the Matched Value Residues Portion LNHUP1 pulmonary surfactant protein A precursor 1.. 243 240/243 (98%) e-146 (clone 1A)-human, 248 aa. 6.. 248 243/243 (99%) I51921 pulmonary surfactant-associated protein 1.. 243 238/243 (97%) e-145 Al-human, 248 aa. 6.. 248 241/243 (98%) P07714 Pulmonary surfactant-associated protein 1.. 243 235/243 (96%) e-143 A precursor (SP-A) (PSP-A) (PSAP) 6.. 248 240/243 (98%) (Alveolar proteinosis protein) (35 kDa pulmonary surfactant-associated protein) - Homo sapiens (Human), 248 aa. LNHUPS pulmonary surfactant protein A precursor 1.. 243 232/243 (95%) e-141 (genomic clone) -human, 248 aa. 6.. 248 237/243 (97%) Q9TT06 Pulmonary surfactant protein A 1.. 243 183/243 (75%) e-114 (Pulmonary surfactant-associated protein 6.. 248 208/243 (85%) A)-Ovis aries (Sheep), 248 aa.

PFam analysis indicates that the NOV17a protein contains the domains shown in the Table 17F.

Table 17F. Domain Analysis of NOV17a Identities/ Pfam Domain NOV17a Match Region Similarities Expect Value for the Matched Region Collagen 32.. 92 34/61 (56%) 0. 00019 49/61 (80%) Xlink 131.. 158 13/32 (41%) 0. 41 19/32 (59%) S lectin c 139.. 243 48/125 (38%) 5e-45 92/125 (74%) Example 18.

The NOV18 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 18A.

Table 18A. NOV18 Sequence Analysis SEQ ID NO : 101 349 bp NOV18a, GGTGAGACAAGGAAGAGGATGTCTGAGCTGGAGAAGGCCATGGTGGCCCTCATCGACGTT TTCC Cl97482 DNA ACCAATATTCTGGAAGGGAGGGAGACAAGCACAAGCTGAAGAAATCCCAACTCAAGGAGC TCAT CAACAATGAGCTTTCCCATTTCTTAGAGGAAATCAAAGAGCAGGAGGTTGTGGACAAAGT CATG Sequence GAAACACTGGACAATGATGGAGACGGCGAATGTGACTTCCAGGAATTCATGGCCTTTGTT GCCA TGGTTACTACTGCCCGCCACGAGTTCTTTGAACATGAGTGAGATTAGAAAGCAGCCAAAC CTTT CCTGTAACAGAGACGGTCATGCAAGAAAG ORF Start : ATG at 19 ORF Stop : TGA at 295 SEQ ID NO : 102 IMW at 10766. OkD SEQ ID NO : 102 92 aa lMW at 10766. 0kD NOVIS, MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMET LDND CG97482-01 GDGECDFQEFMAFVAMVTTARHEFFEHE Protein Sequence s-v SEQ ID NO : 103 1271 bp NOV18b, GGTGAGACAAGGAAGAGGATGTCTGAGCTGGAGAAGGCCATGGTGGCCCTCATCGACGTT TTCC CG97482-02 DNA ACCAATATTCTGGAAGGGAGGGAGACAAGCACAAGCTGAAGAAATCCGAACTCAAGGAGC TCAT CAACAATGAGCTTTCCCATTTCTTAGAGGAAATCAAAGAGCAGGAGGTTGTGGTTACTAC TGCC Sequence TGCCACGAGTTCTTTGAACATGAGTGAGATTAGAAAGCAGCCAAACCTTTCCTGTAACAG AGAC GGTCATGCAAGAAAG S i ORF Start : ATG at 19 ORF Stop : TGA at 217

SEQ m NO : 104 66 aa MW at 7772. 7kD NOV18b, MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVVTTACH EFFE CG97482-02 HE Protein Sequence Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 18B.

Table 18B. Comparison of NOV18a against NOV18b. NOVla Residues/Identities/ Match Residues Similarities for the Matched Region NOV18b 1.. 92 65/92 (70%) 1.. 66 65/92 (70%) Further analysis of the NOV1 8a protein yielded the following properties shown in Table 18C.

Table 18C. Protein Sequence Properties NOV18a Signal ? analysis: No Known Signal Sequence Indicated PSORT II PSG: a new signal peptide prediction method analysis: N-region: length 6; pos. chg 1; neg. chg 2 H-region: length 6; peak value 0.00 PSG score:-4. 40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold:-2. 1):-8. 88 possible cleavage site: between 20 and 21 >>> Seems to have no N-terminal signal peptide ALOM : Klein et ales method for TM region allocation Init position for calculation: 1 Tentative number of TMS (s) for the threshold 0. 5 : 0 number of TMS (s).. fixed PERIPHERAL Likelihood = 7.32 (at 68) ALOM score: 7.32 (number of TMSs : 0) MITDISC : discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 2.53 Hyd Moment (95): 2.95 G content: 0 D/E content: 2 S/T content: 1 Score:-7. 61 Gavel: indication of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC : discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 10. 9% NLS Score :-0. 47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1: none type 2: none NMYR: N-myristoylation pattern: none Brenylation motif: none memYQRL : transport motif from cell surface to Golgi: none Tyrosines in the tail : none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none discrimination Indication : cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total : 0 residues Final Results (k = 9/23): 56. 5 % : cytoplasmic 30. 4 % : nuclear 8. 7 % : mitochondrial 4. 3 % : Golgi >> indication for CG97482-01 is cyt (k=23) A search of the NOV18a 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.

Table 18D. Geneseq Results for NOV18a NOVla Identities/ Geneseq Protein/Organism/Length [Patent #, Residues/Similarities for Expect Identifier Date] Match the Matched Value Residues Region ABB97495 Novel human protein SEQ ID NO : 763 1.. 92 91/92 (98%) 3e-47 - Homo sapiens, 92 aa. 1 92 91/92 (98%) [W0200222660-A2, 21-MAR-2002] ABP51390 Human MDDT SEQ ID NO 412-1.. 92 89/92 (96%) 3e-46 Homo sapiens, 97 aa. 6.. 97 90/92 (97%) [W0200240715-A2, 23-MAY-2002] AAW46607 Human brain protein SlOOb beta 2.. 92 84/91 (92%) 4e-43 subunit-Homo sapiens, 91 aa. 1.. 91 87/91 (95%) [W09801471-A1, 15-JAN-1998] AAM40258 Human polypeptide SEQ ID NO 3403-2.. 89 52/88 (59%) 2e-23 Homo sapiens, 94 aa. 3.. 90 66/88 (74%) [W0200153312-Al, 26-JUL-2001] AAB45531 Human S 100A1 protein-Homo 2.. 89 52/88 (59%) 2e-23 sapiens, 94 aa. [DE19915485-A1, 3.. 90 66/88 (74%) 19-OCT-2000] In a BLAST search of public sequence databases, the NOV18a protein was found to have homology to the proteins shown in the BLASTP data in Table 18E.

Table 18E. Public BLASTP Results for NOV18a Protein NOVla Identities/ Accession el Residues/Similarities for Expect Number Match the Matched Value Residues Portion CAD35011 Sequence 319 from Patent W00222660 1.. 92 91/92 (98%) 7e-47 - Homo sapiens (Human), 92 aa. 1,. 92 91/92 (98%) P04271 S-100 protein, beta chain-Homo 2.. 92 90/91 (98%) 3e-46 sapiens (Human), 91 aa.. 1.. 91 90/91 (98%) A48015 S-100 protein beta chain-mouse, 92 aa. 1 92 90/92 (97%) 4e-46 1.. 92 90/92 (97%) A26557 S-100 protein beta chain-rat, 92 aa. 1.. 92 89/92 (96%) 8e-46 1.. 92 90/92 (97%) AAA72205 SYNTHETIC 1.. 92 88/92 (95%) le-45 CALCIUM-MODULATED PROTEIN 1.. 92 91/92 (98%) 5100-BETA GENE, 5'END-synthetic construct, 92 aa (fragment).

PFam analysis indicates that the NOVla protein contains the domains shown in the Table 18F.

Table 18F. Domain Analysis of NOV18a Identities/ Pfam Domain NOV18a Match Region Similarities Expect Value for the Matched Region S100 4.. 47 28/44 (64%) 3. 6e-23 41/44 (93%) H efhand 53.. 81 9/29 (31%) 0. 0012 25/29 (86%)

Example B: Sequencing Methodology and Identification of NOVX Clones 1. GeneCaflinlem Technology: This is a proprietary method of performing differential gene expression profiling between two or more samples developed at CuraGen and described by Shimkets, et al.,."Gene. expression analysis by transcript profiling coupled to a gene. database query". Nature. Biotechnology 17: 198-803 (1999). cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were

obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then digested with up to as many as 120 pairs of restriction enzymes and pairs of linker-adaptors specific for each pair of restriction enzymes were ligated to the appropriate end. The restriction digestion generates a mixture of unique cDNA gene fragments.

Limited PCR amplification is performed with primers homologous to the linker adapter sequence where one primer is biotinylated and the other is fluorescently labeled. The. doubly labeled material is isolated and the fluorescently labeled single strand is resolved by capillary gel electrophoresis. A computer algorithm compares the electropherograms from an experimental and control group for each of the restriction digestions. This and additional sequence-derived information is used to predict the identity of each differentially expressed gene fragment using a variety of genetic databases. The identity of the gene fragment is confirmed by additional, gene-specific competitive PCR or by isolation and sequencing of the gene fragment.

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

3. PathCallingTM Technology: The NOVX nucleic acid sequences are derived by laboratory screening of cDNA library by the two-hybrid approach. cDNA fragments covering either the full length of the DNA sequence, or part of the sequence, or both, are sequenced. In silico prediction was based on sequences available in CuraGen

Corporation's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.

The laboratory screening was performed using the methods summarized below: cDNA libraries were derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then directionally cloned into the appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). Such cDNA libraries as well as commercially available cDNA libraries from Clontech (Palo Alto, CA) were then transferred from E. coli into a CuraGen Corporation proprietary yeast strain (disclosed in U.

S. Patents 6,057, 101 and 6,083, 693, incorporated herein by reference in their entireties).

Gal4-binding domain (Gal4-BD) fusions of a CuraGen Corportion proprietary library of human sequences was used to screen multiple Gal4-AD fusion cDNA libraries resulting in the selection of yeast. hybrid diploids in each of which the Gal4-AD fusion contains an individual cDNA. Each sample was amplified using the polymerase chain reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such PCR product was sequenced; sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

Physical clone: the cDNA fragment derived by the screening procedure, covering the entire open reading frame is, as a recombinant DNA, cloned into pACT2 plasmid (Clontech) used to make the cDNA library. The recombinant plasmid is inserted into the host and selected by the yeast hybrid diploid generated during the screening procedure by the mating of both CuraGen Corporation proprietary yeast strains N106'and YULH (U. S.

Patents 6,057, 101 and 6,083, 693).

4. RACE: Techniques based on the polymerase chain reaction such as rapid amplification of cDNA ends (RACE), were used to isolate or complete the predicted sequence of the cDNA of the invention. Usually multiple clones were sequenced from one or more human samples to derive the sequences for fragments. Various human tissue samples from different donors were used for the RACE reaction. The sequences derived from these procedures were included in the SeqCalling Assembly process described in preceding paragraphs.

5. Exon Linking: The NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences from other species.

These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain-amygdala, brain-cerebellum, brain- hippocampus, brain-substantia nigra, brain-thalamus, brain-whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma-Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector.

The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs... Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.

6. Physical Clone: Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to. further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.

The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clones used for expression and screening purposes.

Example C. Quantitative expression analysis of clones in various cells and tissues The quantitative expression of various 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), AIcomprehensivepanel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD. O1 (containing samples from normal and diseased brains) and CNSneurodegenerationpanel (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, p-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 Corporation ; Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 g of total RNA were performed in a volume. of 20 111 and incubated for 60 minutes at 42°C.. This. reaction can be scaled up to 50 ug of total RNA in a final volume of 100 ul. sscDNA samples are then normalized to reference nucleic acids as described previously, using 1X 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 of the probe, respectively. Their final concentrations were: forward and reverse primers, 900nM each, and probe, 200nM.

PCR conditions : When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone and another gene-specific set multiplexed with the target probe). PCR reactions were set up using TaqMan@. One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No.

4313803) following manufacturer's instructions. Reverse transcription was performed at

48°C for 30 minutes followed by amplification/PCR cycles as follows: 95°C 10. min,. then 40 cycles of 95°C for 15. seconds, 60°C for 1 minute.. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.

When working with sscDNA samples, normalized sscDNA was used as described previously for RNA samples. PCR reactions containing one or two sets of probe and primers were set up as described previously, using 1X TaqMan (X9. 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 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 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 of the 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, pl. eff = pl effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.

General_screening wanel_vl. 4, vl. 5, v1. 6 and 1.7 The plates for Panels 1.4, 1. 5, 1.6 and 1.7 include 2 control wells (genomic DNA control and chemistry control) and 88 to 94 wells containing cDNA from various samples.

The samples in Panels 1.4, 1.5, 1.6 and 1.7 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers 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 Panels 1. 4, 1.5, 1.6 and 1.7 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panels 1.4, 1.5, 1.6 and 1.7 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the 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 of the patient. These matched margins are taken from the tissue surrounding (i. e. immediately proximal) to the zone of surgery (designated"NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc. ). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.

HASS Panel v 1.0 The HASS panel v 1.0 plates are comprised of 93 cDNA samples and two controls.

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

ARDAIS Panel v 1.0

The plates for ARDAIS panel v 1.0 generally include 2 control wells and 22 test samples composed of RNA isolated from human tissue procured by surgeons working in close cooperation with Ardais Corporation. 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 histopathological assessment of tumor differentiation grade and stage. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical state of the patient.

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 of the 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 bf the most common cell lines used in the scientific literature.

Panels 4D, 4R, and 4. 1D Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4. 1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney (Clontech) was

employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc. , Hayward, CA). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).

Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 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-133 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),. 100, M non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), and lOmM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20ng/ml PMA and 1-211gel 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),. 100µM non essential amino acids. (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5xlO-5M (Gibco), and lOmM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5ug/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),. lOOuM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol

(5. 5x10-5M) (Gibco), and lOmM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1-7 days for RNA preparation.

Monocytes were isolated from mononuclear cells using CD14 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),. lOOuM non essential amino. acids (Gibco),. 1mM 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),. lOOpM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), lOmM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50ng/ml.

Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at lOOng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at lOug/ml for 6. and 12-14 hours.

CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45RO beads were then used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5xlO-5M (Gibco), and lOmM Hepes (Gibco) and plated at 106cells/ml onto Falcon 6 well tissue culture plates that. had been coated overnight with O. Sg/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), lOOuM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), and lOmM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated

6 and 24 hours after the second activation and after 4 days of the second expansion culture.

The isolated NK cells were cultured in DMEM 5% FCS. (Hyclone) lOOuM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), and lOmM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.

To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 106cells/ml in DMEM 5% FCS. (Hyclone), 100liM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), and lOmM Hepes (Gibco). To. activate the. cells, we used PWM at 5ug/ml or anti-CD40 (Pharmingen) at approximately 10µg/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 Trl cells, six-well Falcon plates were coated overnight with lOug/mt anti-CD28 (Pharmingen) and 2llg/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), 100µM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), lOmM Hepes (Gibco) and IL-2 (4ng/ml). IL-12 (5ng/ml) and anti-IL4 (1 ug/ml) were used to direct to Thl, while IL-4 (5ng/ml) and anti-IFN gamma (1 llg/ml). were used to direct to Th2. and IL-10 at 5ng/ml was used to direct to Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS. (Hyclone),. 100µM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), lOmM Hepes (Gibco) and IL-2 (lng/ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD (1 µg/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl 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 Trl 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 Sxl05cells/ml for 8 days, changing the, media every 3 days and adjusting the cell concentration to 5xl05cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by. the ATCC), with the addition of 5% FCS (Hyclone), 1 OOuM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5x10-5M (Gibco), lOmM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 1 Ong/ml and ionomycin at 1 llg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC.. Both were cultured in DMEM 5% FCS (Hyclone), lOOpM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol 5. 5xlO-5M (Gibco), and lOmM Hepes (Gibco). CCD1106 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 rpm 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 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300w1 of RNAse-free water and 35ul buffer (Promega) 5ul DTT, 7ul RNAsin and 8111 DNAse were added., The tube was incubated at 37°C. for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of 100% ethanol.

The RNA was spun down and placed in RNAse free water. RNA was stored at-80°C.

AI comprehensive panel vl. O The plates for AI comprehensive panel vl. 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.

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 of the 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 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 AIcomprehensive panelvl. 0 panel, the following abbreviations are used:

AI = Autoimmunity Syn = Synovial Normal = No apparent disease Rep22/Rep20 = individual patients RA = Rheumatoid arthritis Backus = From Backus Hospital OA = Osteoarthritis (SS) (BA) (MF) = Individual patients Adj = Adjacent tissue Match control = adjacent tissues - M = Male - F = Female COPD = Chronic obstructive pulmonary disease AI. 05 chondrosarcoma The AI. 05 chondrosarcoma plates are comprised of SW1353 cells that had been subjected to serum starvation and treatment with cytokines that are known to induce MMP (1,3 and 13) synthesis (eg. ILlbeta). These treatments include: IL-lbeta (10 ng/ml), IL-lbeta + TNF-alpha (50 ng/ml), IL-lbeta + Oncostatin (50 ng/ml) and PMA (100 ng/ml).

The SW1353 cells were obtained from the ATCC (American Type Culture Collection) and were all cultured under standard recommended conditions. The SW1353 cells were plated at 3 x105 cells/ml (in DMEM medium-10 % FBS) in 6-well plates. The treatment was done in triplicate, for 6 and 18 h. The supernatants were collected for analysis of MMP 1,3 and 13 production and for RNA extraction. RNA was prepared from these samples using the standard procedures.

Panels 5D and SI 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 11: 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 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 SI 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. O1 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 of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear 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 of the 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 CNSNeurodegenerationV1. 0 The plates for Panel CNSNeurodegenerationV1. 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 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 CNSNeurodegenerationV1. 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. CG115907-02 (NOV2d), CG115907-03 (NOV2c), and CG115907-04 (NOV2b): PK-120.

Expression of gene CG115907-02, CG115907-03, and CG115907-04 was assessed using the primer-probe sets Ag6155, Ag6156 and Ag6131, described in Tables AA, AB and AC. Results of the RTQ-PCR runs are shown in Tables AD and AE. Please note that primer-probe set Ag6155 is specific for CG115907-03 and Ag6156 is specific for CG115907-04.

Table AA. Probe Name Ag6155 Primers Sequeces Length stårt SEQ Position ID No Forward 5'-atcttgcctgcttcagcaa-3'19 2113 105 Probe TET-5'-caaatcctgatccagctgtgtctcgt-3'-TAMRA 26 2144 106 Reverse 5'-ggatggcagacatattcatgac-3'222170 107 Table AB. Probe Name Ag6156

Primers Sequnces Length Start SEQ Position ID No Forward 5 F-ggecatcttgcctgctt-3 t 17 2109 108 Probe TET-5'-atcctgatccagctgtgtctcgtgtc-3'-TAMRA 26 2147 109 Reverse 5'-ctccctctcatactgcatattcat-3'24 2173 110 Table AC. Probe Name Ag6131 Primers Sequeces, l"""i' (1 i lLength Start SEQ Position m No Forward 5'-gtccactcagctggagctg-3'19 1981 111 Probe TET-5'-aacttggactcccaggacctcctgat-3'-TAMRA 26 2045 112 Reverse 5'-cagctggatcaggatttgag-3'20 2142 113

Table AD. CNS neurodegeneration vl. O Rel. Rel. Exp.(%) Tissue Name Exp. (%) Tissue Name Ag6131, Ag6131, un 253574594 Run 253574594 AD 1 Hippo 0. 0 Control (Path) 3 Temporal Ctx 3.4 --- AD 2 Hippo 25. 0 Control (Path) 4 Temporal Ctx 51. 8 AD 3 Hippo 0. 0 AD 1 Occipital Ctx 3. 8 AD 4 Hippo 11. 7 AD 2 Occipital Ctx (Missing) 0.0 AD 5 Hippo 99.3 AD 3 Occipital Ctx 0.0 AD 6 Hippo 55. 1 AD 4 Occipital Ctx 10. 6 Control 2 Hippo 37.6 AD 5 Occipital Ctx 22.8 Control 4 Hippo 14. 3 AD 6 Occipital Ctx 29. 7 Control (Path) 3 Hippo 9. 0 Control 1 Occipital Ctx 0.0 AD 1 Temporal Ctx 2.6 Control 2 Occipital Ctx 79.6 AD 2 Temporal Ctx 0.0 Control 3 Occipital Ctx 30.8 AD 3 Temporal Ctx 0.0 Control 4 Occipital Ctx 9. 0 AD 4 Temporal Ctx 22. 1 Control (Path) 1 Occipital Ctx 68.8 AD 5 Inf Temporal Ctx 100.0 Control (Path) 2 Occipital Ctx 17.7 AD 5 Sup Temporal Ctx 28.9 Control (Path) 3 Occipital Ctx 5.0 AD 6 Inf Temporal Ctx 56. 6 Control (Path) 4 Occipital Ctx 20.6 AD 6 Sup Temporal Ctx 39.2 Control 1 Parietal Ctx 0.0 Control 1 Temporal Ctx 0. 0 Control 2 Parietal Ctx 59. 0 Control 2 Temporal Ctx 33.0 Control 3 Parietal Ctx 11. 1 Control 3 Temporal Ctx 19. 9 Control (Path) 1 Parietal Ctx 26.4 Control 3 Temporal Ctx 9.0 Control (Path) 2 Parietal Ctx 13.5 Control (Path) 1 Temporal Ctx 57.0 Control (Path) 3 Parietal Ctx 4.1 Control (Path) 2 Temporal Ctx 55.5 Control (Path) 4 Parietal Ctx 56.3 Table AE. General screening panel v1. 5 Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag6131, issue Name Ag6131, Run Run 253101092 253101092 Adipose 0. 0 Renal ca. TK-10 0. 1 Melanoma* Hs688(A).T 0.0 Bladder 12.3 Melanoma* Hs688(B).T 0.0 Gastric ca. (liver met.) NCI-N87 0.1 Melanoma* M14 0. 1 Gastric ca. KATO in0. 0 Melanoma* LOXIMVI 0.0 Colon ca. SW-948 0. 0 Melanoma* SK-MEL-5 0.0 Colon ca. SW480 0.0 Squamous cell carcinoma SCC-4 0. 0 Colon ca. * (SW480 met) SW620 0.0 Testis Pool 0. 1 Colon ca. HT29 0. 0 Prostate ca. * (bone met) PC-3 0. 0 Colon ca. HCT-116 0.0 Prostate Pool 0.0 Colon ca. CaCo-2 0.2 Placenta 0.0 Colon cancer tissue 0.1 Uterus Pool 0. 0 Colon ca. SW1116 0. 0 OYarianca. OVCAR.-3 0. 0Colon ca. Colo-2050. 0 Ovarian ca. SK-OV-3 0.1 Colon ca. SW-48 0.0 Ovarian ca. vOVCAR-4 00 Colon Pool 0.1 Ovarian ca. OVCAR-5 0. 0 Small Intestine Pool 0. 1 Ovarian ca. IGROV-1 0. 0 Stomach Pool 0. 1 Ovarian ca. OVCAR-8 0. 0 Bone Marrow Pool 0. 1 Ovary 1. 3, Fetal Heart 0. 0 Breast ca. MCF-7 0. 0 Heart Pool 0.2 Breast ca. MDA-MB-231 0. 1 Lymph Node Pool 0. 2 Breast ca. BT 549 0. 1 Fetal Skeletal Muscle 2. 0 Breast ca. T47D 0. 0 Skeletal Muscle Pool 1.7 Breast ca. MDA-N 0. 0 Spleen Pool 0.1 Breast Pool 0. 2 Thymus Pool 0. 4 Trachea 0.2 CNS cancer (glio/astro) U87-MG 0.0 Lung 0.2 CNS cancer (glio/astro) U-118-MG 0.0 Fetal Lung 0.8 CNS cancer (neuro;met) SK-N-AS 0.0 Lung ca. NCI-N417 0. 0 CNS cancer (astro) SF-539 0. 0 Lung ca. LX-1 0. 1 CNS cancer (astro) SNB-75 0.1 Lung ca. NCI-H146 0. 0 CNS cancer (glio) SNB-19 0. 0 Lung ca. SHP-77 0. 0 CNS cancer (glio) SF-295 1. 0 Lung ca. A549 0. 2 Brain (Amygdala) Pool 0. 1 Lung ca. NCI-H526 0. 0 Brain (cerebellum) 0.4 Lung ca. NCI-H23 0.1 Brain (fetal) 0.2 Lung ca. NCI-H460 0.0 Brain (Hippocampus) Pool 0. 1 Lung ca. HOP-62 0.0 Cerebral Cortex Pool 0.0 Lung ca. NCI-H522 0.1 Brain (substantia nigra) Pool 0.3 Liver 100.0 Brain (Thalamus) Pool 0. 2 Fetal Liver 41. 2 Brain (whole) 3.6 Liver ca. HepG2 0.0 Spinal Cord Pool 0.1 Kidney Pool 0.7 Adrenal Gland 0.6 Fetal Kidney 0. 5 Pituitary gland Pool 0.0 Renal ca. 786-0 0. 0 Salivary Gland 0.0 Renal ca. A498 0.0 Thyroid (female) 0.0 Renal ca. ACHN 0.0 Pancreatic ca. CAPAN2 0. 0 Renal ca. UO-31 0.0 Pancreas Pool 2. 3

CNS neurodegeneration v1. 0 Summary: Ag6131 Low levels of expression of this gene is detected in the brains from control and Alzheimer's patients. 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.

Ag61 55/Ag6156 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

Generalscreemngpanelvl. 5 Summary: Ag6131 Highest expression of this gene is detected in liver (CT=25 : 2). High expression of this gene is mainly seen in adult and fetal liver, with moderate to low levels of expression in adult and fetal skeletal muscle, adernal gland and pancrease. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, moderate to low expression of this gene is also seen in whole brain, fetal brain, substantia nigra, thalamus, cerebellum, 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.

Low expression of this gene is also seen in a brain cancer SF-295 cell line.

Therefore, therapeutic modulation of this gene may be useful in the treatment of brain cancer.

Ag6155/Ag6156 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

Ag6155/Ag6156 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

B. CG139008-01 (NOV3a): novel secreted.

Expression of gene CG139008-01 was assessed using the primer-probe sets Ag243 and Ag7477, described in Tables BA and BB. Results of the RTQ-PCR runs are shown in Tables BC and BD.

Table BA. Probe Name Ag243 Start SEQ Primers Sequnces Length Position ID No Forward 5'-caagggcatcaccaatttga-3'20 186 114 Probe TET-5'-aggatgtccagctgcccgtcatca-3'-TAMRA 24 212 115 Reverse 5'-gcccactccaggtacaaagttc-3 221240 116 Table BB. Probe Name Ag7477 Primers Sequnces Len, gth start SEQ Position ID No Forward 5'-ttctttggacagattactgagctt-3'24 1134 " Probe TET-5'-tcctcatcgattggcaacttcaatga-3'-TAMRA 26 1168 118 Reverse = z ~ L 21 1212 119

Table BC. Panel 1.3D Rel. el. Exp.(%) Exp.(%) Tissue Name n'Tissue Name Ag243, Run Run 156536275 Liver adenocarcinoma 0.0 Kidney (fetal) 0.0 pancreas 0.0 Renal ca. 786-0 0.0 Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 0.0 Adrenal gland 0. 0 Renal ca. RXF 393 0. 0 Thyroid 0. 0 Renal ca. ACHN 0. 0 Salivary gland 17.6 Renal ca. UO-31 0.0 Pituitary gland 0.0 Renal ca. TK-10 0.0 Brain (fetal) 0.0 Liver 0.0 Brain (whole) 0.0 Liver (fetal) 0. 0 Brain (amygdala) 0.0 Liver ca. (hepatoblast) HepG2 46.3 Brain (cerebellum) 0.0 Lung 0.0 Brain (hippocampus) 0.0 Lung (fetal) 0.0 Brain (substantia nigra) 0. 0 Lung ca. (small cell) LX-1 0. 0 Brain (thalamus) 0.0 Lung ca. (small cell) NCI-H69 0.0 Cerebral Cortex 0.0 Lung ca. (s. cell var. ) SHP-77 0.0 Spinal cord 0.0 Lung ca. (large cell)NCI-H460 0.0 glio/astro U87-MG 0.0 Lung ca. (non-sm. cell) A549 0.0 glio/astro U-118-MG 0.0 Lung ca. (non-s.cell) NCI-H23 0.0 astrocytoma SW1783 0.0 Lung ca. (non-s.cell) HOP-62 0.0 neuro*; met SK-N-AS 0. 0 Lung ca. (non-s. cl) NCI-H522 0. 0 astrocytoma SF-539 0. 0 Lung ca. (squam. ) SW 900 0. 0 astrocytoma SNB-75 0.0 Lung ca. (squam.) NCI-H596 0.0 glioma SNB-19 0.0 Mammary gland 0.0 glioma U251 0. 0 Breast ca. * (pl. ef) MCF-7 0. 0 glioma SF-295 0.0 Breast ca. * (pl. ei) MDA-MB-231 0.0 Heart (fetal) 0. 0 Breast ca. * (pl. ef) T47D 0. 0 Heart 0.0 Breast ca. BT-549 0. Skeletalmuscle (fetal) 0. 0 Breast ca. MDA-N 0. 0 Skeletal muscle 0. 0 Ovary 0. 0 Bone marrow 0. 0 Ovarian ca. OVCAR-3 0. 0 Thymus 0. 0 Ovarian ca. OVCAR-4 0. 0 Spleen 0. 0 Ovarian ca. OVCAR-5 0. 0 Lymph node 0.0 Ovarian ca. OVCAR-8 0. 0 Colorectal 0. 0 Ovarian ca. IGROV-1 0. 0 Stomach 0. 0 Ovarian ca. * (ascites) SK-OV-3 0. 0 Small intestine 0. 0 Uterus 0. 0 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.* SW620(SW480 met) .0 Prostate 0.0 Colon ca. HT29 0. 0 Prostate ca. * (bone met) PC-3 0.0 Colonca. HCT-1160. 0Testis0. 0 Colon ca. CaCo-2 0.0 Melanoma Hs688(A).T 0.0 Colon ca. tissue (ODO3866) 0.0 Melanoma* (met) Hs688 (B). T 0.0 Colon ca. HCC-2998 0.0 Melanoma UACC-62 0.0 Gastric ca.* (liver met) NCI-N87 0.0 Melanoma M14 0.0 Bladder 0. 0 Melanoma LOX IMVI 0. 0 Trachea 100. 0 Melanoma* (met) SI MEL-S 0. 0 Kidney 0.0 Adipose 0. 0 Table BD. Panel 2D Rel. Rel. Exp. (%) Exp. (%) Ag243, Tissue Name Ag243, Run Run 156536477 156536477 Normal Colon 0. 0 Kidney Margin 8120608 0. 0 CC Well to Mod Diff (OD03866) 0. 0 Kidney Cancer 8120613 0. 0 CC Margin (OD03866) 0.0 Kidney Margin 8120614 0.0 CC Gr. 2 rectosigmoid (OD03868) 0.0 Kidney Cancer 9010320 0. 0 CC Margin (ODO3868) 0.0 Kidney Margin 9010321 0.0 CC Mod Diff (ODO3920) 0.0 Normal Uterus 0.0 CC Margin (ODO3920) 0.0 Uterus Cancer 064011 0.0 CC Gr. 2 ascend colon (OD03921) 0.0 Normal Thyroid 0.0 CC Margin (OD03921) 0. 0Thyroid Cancer 064010 0. 0 CC from Partial Hepatectomy 0. 0 Thyroid Cancer A302152 0.0 (OD04309) Mets Liver Margin (OD04309) 0.0 Thyroid Margin A302153 0.0 Colon mets to lung (OD04451-01) 0.0 Normal Breast 0.0 Lung Margin (OD04451-02) 0.0 Breast Cancer (OD04566) 0.0 Normal Prostate 6546-1 0. 0 Breast Cancer (OD04590-01) 0.0 Prostate Cancer (OD04410) 21.9 Breast Cancer Mets 0.0 (OD04590-03) Breast Cancer Metastasis Prostate Margin (OD04410) 0.0 0.0 (OD04655-05) Prostate Cancer (OD04720-01) 0.0 Breast Cancer 064006 0.0 Prostate Margin (OD04720-02) 0. 0 Breast Cancer 1024 10. 0 Normal Lung 061010 0. 0 Breast Cancer 9100266 100. 0 Lung Met to Muscle (OD04286) 0. 0 Breast Margin 9100265 0. 0 Muscle Margin (ODO4286) 0.0 Breast Cancer A209073 0. 0 Lung Malignant Cancer (OD03126) 15. 8 Breast Margin A209073 0.0 Lung Margin (OD03126) 0. 0 Normal Liver 0. 0 Lung Cancer (OD04404) 0. 0 Liver Cancer 064003 0. 0 Lung Margin (OD04404) 0. 0 Liver Cancer 1025 0. 0 Lung Cancer (OD04565) 0.0 Liver Cancer 102621. 9 Lung Margin (OD04565) 0.0 Liver Cancer 6004-T 0. 0 Lung Cancer (OD04237-01) 0. 0 Liver Tissue 6004-N 0.0 Lung Margin (OD04237-02) 0.0 Liver Cancer 6005-T 0. 0 Ocular Mel Met to Liver (ODO4310) 0. 0Liver Tissue 6005-N0. 0 Liver Margin (OD04310) 0.0 Normal Bladder 0.0 Melanoma Mets to Lung (OD04321) 0.0 Bladder Cancer 1023 0. 0 Lung Margin (OD04321) 0. 0 Bladder Cancer A302173 0. 0 Normal Kidney 0. 0 Bladder Cancer (OD04718-01) 0. 0 Kidney Ca, Nuclear grade 2 Bladder Normal Adjacent (OD04338) (OD04718-03) Kidney Margin (OD04338) 0.0 Normal Ovary 0.0 Kidney Ca Nuclear grade 1/2 0.0 Ovarian Cancer 064008 0.0 (OD04339) Kidney Margin (OD04339) 0. 0 Ovarian Cancer (OD04768-07) 0. 0 Kidney Ca, Clear cell type (OD04340) 0. 0 Ovary Margin (OD04768-08) 0. 0 Kidney Margin (OD04340) 0. 0 Normal Stomach 0. 0 Kidney Ca, Nuclear grade 3 0. 0 Gastric Cancer 9060358 0.0 (ou04348) Kidney Margin (OD04348) 0. 0 Stomach Margin 9060359 0. 0 Kidney Cancer (OD04622-01) 0.0 Gastric Cancer 9060395 0. 0 Kidney Margin (OD04622-03) 0. 0 Stomach Margin 9060394 0. 0 Kidney Cancer (OD04450-01) 0. 0 Gastric Cancer 9060397 0. 0 Kidney Margin (OD04450-03) 0.0 Stomach Margin 9060396 0. 0 Kidney Cancer 81206070. 0Gastric Cancer 0640050. 0

Panel 1 Summary: Ag243 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

Panel 1.3D Summary: Ag243 Expression of this gene is restricted to the trachea and a liver cancer cell line (CTs=33. 5-34.5). Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker to

detect the presence of liver cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of liver cancer.

Panel 2D Summary: Ag243 Expression of this gene is restricted to the a breast cancer cell line (CT=34. 5). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect the presence of breast cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of breast cancer.

Panel 4. 1D Summary: Ag7477 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

Panel 4D Summary: Ag243 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

C. CG165528-01 (NOV9a): Neurexin I alpha precursor.

Expression of gene CG165528-01 was assessed using the primer-probe set Ag5964, described in Table CA. Results of the RTQ-PCR runs are shown in Tables CB and CC.

Table CA. Probe Name Ag5964 Primers Sequeces Length Position ED No Position ID No - Probe TET-5'-ttaccatagcgtgtccaatgcctgag-3'-TAMRA 26 1236 121 Reverse 5'-gatattgtcaccgaacaatgtagttt-3'26 1264 122 Table CB. CNS neurodegeneration vl. O Rel. Rel. Rel. Rel. Exp. (%) o Exp. (%) Exp. (%) Tissue Name Ag5964, Tissue Name Ag5964, Ag5964, Ag5964, Ru 8162714 248162714 268784143 248162714 248162714 268784143 ------- ALD 1 Hippo 11 Control (Path) 2 temporal Ctx Control (Path) 4 Temporal Ctx 62. 4 47. 3 AD 3 Hippo 3.5 7.7 AD 1 Occipital Ctx 11.0 13.1 AD 2 Occipital Ctx AD 4 15.0 12.6 0. 0.0 (Missing) AD 5 hippo 100.0 100.0 AD 3 Occipital Ctx 3.9 1.1 AD 6 Hippo 49. 7 36. 6 AD 4 Occipital Ctx 34. 6 18. 8 Control 2 Hippo 30.6 24.0 AD 5 Occipital Ctx 41.5 29.3 Control 4 Hippo 19.9 17.8 AD 6 Occipital Ctx 41.5 41.8 Control (Path) 3 11. 0 Control 1 Occipital 8. 5 2. 2 Hippo Ctx Control 2 Occipital AD 1 Temporal Ctx 11.4 9.5 66.0 48.6 Ctx Control 3 Occipital 28. 5 15. 4 btx AD 3 Temporal Ctx 4.6 2.9 Controle 4 Occipital 7.7 13.4 btx AD 4 Temporal Ctx 37.9 27.9 Control (Path) 1 92.0 86.5 Occipital Ctx Control (Path) 1 AD 4 Temporal Ctx 37.9 27.9 92.0 86.5 Occipital Ctx AD 5 Inf Temporal Control (Path) 2 97.3 59.9 21.6 12.9 Ctx Occipital Ctx AD 5 SupTemporal Control (Path) 3 45.1 29.3 5.8 4.9 Ctx Occipital Ctx AD 6 Inf Temporal Control (Path) 4 49.3 55.9 19.8 10.9 Occipital Ctx AD 6 Sup Temporal 57. 4 75. 3 Control I Parietal 22. 5 12. 9 Ctx Control 1 Temporal 28. 7 18. 2 Control 2 Parietal 0. 9 24. 1 Ctx Control 2 Temporal 47. 0 21. 5 Control 3 Parietal 20.4 19.1 Ctx Control 3 Temporal Control (Path) 1 5. 9 72. 2 Parietal Ctx Control 4 Temporal 14. 8 17. 2 Control (Path) 2 32. 3 31. 4 Ctx Parietal Ctx Control (Path) 1 80.7 97.9 Control (Path) 3 4.9 12.2 Temporal Ctx Parietal Ctx Control (Path) 2 57.8 49.3 (Path) 4 66. 4 33. Temporal Ctx Parietal Ctx Table CC. General screening panel v1. 5 Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag5964, issue Name Ag5964, Run Run 248163367 248163367 Adipose 3.6 Renal ca. TK-10 0.0 Melanoma* Hs688 (A). T 0. 0 Bladder 8. 1 Melanoma* Hs688(B).T 0. 0 Gastric ca. (liver met.) NCI-N87 0. 0 Melanoma* M14 0.0 Gastric ca. KATO III 0.0 Melanoma* LOXIMVI 0.0 Colon ca. SW-948 0. 0 Melanoma* KS-MEL-5 0.0 Colon ca. SW480 0.0 Squamous cell carcinoma SCC-4 0.0 Colon ca.* (SW480 met) SW620 0.0 Testis Pool 3. 0 Colon ca. HT29 0. 0 Prostate ca. * (bone met) PC-3 0. 0 Colon ca. HCT-116 0. 0 Prostate Pool 2.5 Colon ca. CaCo-2 0.0 Placenta 0. 0 Colon cancer tissue 0. 0 Uterus Pool 5.7 Colon ca. SW1116 0. 0 Ovarian ca. OVCAR-3 0. 0 Colon ca. Colo-205 0.0 Ovarian ca. SK-OV-3 0.0 Colon ca. SW-48 0.0 Ovarian ca. OVCAR-4 0.0 Colon Pool 0.3 Ovarian ca. OVCAR-5 0. 0 Small Intestine Pool 13. 8 Ovarian ca. IGROV-1 0.0 Stomach Pool 4.5 Ovarian ca. OVCAR-8 0. 0 Bone Marrow Pool 4. 3 Ovary 0. 0 Fetal Heart 0. 5 Breast ca. MCF-7 0. 0 Heart Pool 4.2 Breast ca. MDA-MB-231 0.0 Lymph Node Pool 2.8 Breast ca. BT 549 0.0 Fetal Skeletal Muscle 5.4 Breast ca. T47D 0.0 Skeletal Muscle Pool 0.7 Breast ca. MDA-N 0.0 Spleen Pool 1.0 Breast Pool 0. 0 Thymus Pool 1. 3 Trachea 5. 1 CNS cancer (glio/astro) U87-MG 0.0 Lung 0. 0 CNS cancer (glio/astro) U-118-Mg 0.0 Fetal Lung 7. 0 CNS cancer (neuro ; met) SK-N-AS 0.4 Lung ca. NCI-N417 4. 2 CNS cancer (astro) SF-539 0. 0 Lung ca. LX-1 0.0 CNS cancer (astro) SNB-75 0.0 Lung ca. NCI-H146 4.8 CNS cancer (glio) SNB-19 0.0 Lung ca. SHP-77 3. 2 CNS cancer (glio) SF-295 0. 0 Lung ca. A549 0. 0 Brain (Amygdala) Pool 37.4 Lung ca. NCI-H526 0.0 Brain (cerebellum) 43.8 Lung ca. NCI-H23 0.0 Brain (fetal) 100.0 Lung ca. NCI-H460 0. 0 Brain (Hippocampus) Pool 46. 3 Lung ca. HOP-62 0.0 Cerebral Cortex Pool 47.6 Lung ca. NCI-H522 0.0 Brain (Substantia nigra) Pool 35.4 Liver 0. 0 Brain (Thalamus) Pool 61. 1 Fetal Liver 0.7 Brain (whole) 46.3 Liver ca. HepG2 0. 0 Spinal Cord Pool 20. 9 Kidney Pool 2. 0 Adrenal Gland 2. 4 Fetal Kidney 5.6 Pituitary gland Pool 7.6 Renal ca. 786-0 0.0 salivary Gland 2.3 Renal ca. A4980. 0Thyroid (female) 0. 0 :.-- Renal ca. ACHN 0. 0 Pancreatic ca. CAPAN2 0.0 Renalca. UO-310. 0PaiicreasPool13

CNS neurodegeneration v1. 0 Summary: Ag5964 Two experiments with same probe-primer sets are in good agreement. This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene is found to be slightly 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 associated with this disease and neuronal death.

General_screening Sanel_vl. 5 Summary: Ag5964 Expression of this gene is seen exclusively in all the regions of brain region, with highest expression in fetal brain (CT=30.9). Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

D. CG165528-02 (NOV9b): Neurexin I beta.

Expression of gene CG165528-02 was assessed using the primer-probe set Ag7944, described in Table DA. Results of the RTQ-PCR runs are shown in Table DB.

Table DA. Probe Name Ag7944 Primers Sequeces Length Start SEQ Position ID No Forward 5'-gcaccacatccaccatttc-3' 19 213 123 Probe TET-5'-cagcagcaagcatcattcagtgcc-3'-TAMRA 24 237 124 Reverse 5'-gatgccggtgacctgtaga-3'19 269 125 Table DB. CNS neurodegeneration vl. O Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag7944, Tissue Name Ag7944, Run Run 319510463 319510463 AD 1 Hippo 5.9 Control (Path) 3 Temporal Ctx 4.7 AD 2 Hippo 13.2 Control (Path) 4 Temporal Ctx 48.3 AD 3 Hippo 3.5 AD 1 Occipital Ctx 17. 4 i i a S. AD 4 Hippo 4.7 AD 2 Occipital Ctx (Missing) 0.0 AD 5 Hippo 100.0 AD 3 Occipital Ctx 3.3 AD 6 Hippo 31.6 AD 4 Occipital Ctx 23.8 Control 2 Hippo 15.9 AD 5 Occipital Ctx 56.6 Control 2 Hippo 15. 9 AD 5 Occipital Ctx 56. 6 Control 4 Hippo 3.8 AD 6 Occipital Ctx 16.2 Control (Path) 3 Hippo 2.4 Control 1 Occipital Ctx 1.7 AD1 Temporal Ctx 9.2 Control 2 Occipital Ctx 64.6 AD 1 Temporal Ctx 9.2 Control 2 Occipital Ctx 64.6 AD 2 Temporal Ctx 31. 4 Control 3 Occipital Ctx 22. 7 AD 3 Temporal Ctx 6. 4 Control 4 Occipital Ctx 2. 4 AD 4 Temporal Ctx 23.8 Control (Path) 1 Occipital Ctx 72. 2 AD 5 Inf Temporal Ctx 79. 0 Control (Path) 2 Occipital Ctx 12. 9 AD 5 Sup Temporal Ctx 20. 0 Control (Path) 3 Occipital Ctx 0. 9 AD 6 Inf Temporal Ctx 30.8 Control (Path) 4 Occipital Ctx 23.2 AD 6 Sup Temporal Ctx 40.3 Control 1 Parietal Ctx 5.8 Control 1 Temporal Ctx 2.7 Control 2 Parietal Ctx 37.4 Control 1 Temporal Ctx 2. 7 Control 2 Parietal Ctx 37. 4 Control 2 Temporal Ctx 33.4 Control 3 Parietal Ctx 16.6 Control 3 Temporal Ctx 19. 8 Control (Path) 1 Parietal Ctx 81.8 Control 3 Temporal Ctx 7. 7 Control (Path) 2 Parietal Ctx 28. 1 Control (Path) 1 Temporal Ctx 44.8 Control (Path) 3 Parietal Ctx 1.8 Control (Path) 2 Temporal Ctx 47.6 Control (Path) 4 Parietal Ctx 62.4

CNS neurodegeneration v1. 0 Summary: Ag7944 No differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment. However, this panel confirms the expression of this gene at low levels in the brains of an independent group of individuals. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Panel 4. 1D Summary: Ag7944 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

E. CG165666-01 (NOVlOa) : CGI-87 PROTEIN.

Expression of gene CG165666-01 was assessed using the primer-probe set Ag5963, described in Table EA. Results of the RTQ-PCR runs are shown in Tables EB, EC and ED.

Table EA. Probe Name Ag5963 Cf Start SE Pnmer Lengt ... Q Primer Seqences Lengt Positio s h IID No Forward 5'-gagacaagtcctaaatgccgac-3'22 159 126 Probe TET-5'-aacaatcttttgttggattgaaacagctaatcct-3'-34 188 127 TAMRA Reverse 5'-ctagtagtgccagcctgacaaa-3'22 226 128

Table EB. CNS neurodegeneration vl. O Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag5963, Tissue Name Ag5963, Run Run 248162713 248162713 AD 1 Hippo 10.7 Control (Path) 3 Temporal Ctx 3.8 AD 2 Hippo 28. 7 Control (Path) 4 Temporal Ctx 27.5 AD 3 Hippo 7. 5 AD 1 Occipital Ctx 12. 6 AD 4 Hippo 7.4 AD 2 Occipital Ctx (Missing) 0.0 AD 5 Hippo 100.0 AD 3 Occipital Ctx 6.4 AD 6 Hippo 50. 0 AD 4 Occipital Ctx 16. 8 Control 2 Hippo 29.7 AD 5 Occipital Ctx 54.3 Control 4 Hippo 13. 6 AD 6 Occipital Ctx 16. 4 Control (Path) 3 Hippo 4. 8 Control 1 Occipital Ctx 3. 3 AD 1 Temporal Ctx 27. 4 Control 2 Occipital Ctx 57. 4 AD 2 Temporal Ctx 39.0 Control 3 Occipital Ctx 13.7 AD 3 Temporal Ctx 4. 2 Control 4 Occipital Ctx 6. 3 AD 4 Temporal Ctx 23. 2 Control (Path) 1 Occipital Ctx 84.1 AD 5 Inf Temporal Ctx 72.7 Control (Path) 2 Occipital Ctx 11.0 AD 5 Sup Temporal Ctx 40.6 Control (Path) 3 Occipital Ctx 3.0 AD 6 Inf Temporal Ctx 46. 3 Control (Path) 4 Occipital Ctx 21.8 AD 6 Sup Temporal Ctx 42.6 Control 1 Parietal Ctx 8.0 Control 1 Temporal Ctx 5.4 Control 2 Parietal Ctxc 253 Control 2 Temporal Ctx 29.9 Control 3 Parietal Ctx 14. 5 Control 3 Temporal Ctx 9. 3 Control (Path) 1 Parietal Ctx 63.3 Control 3 Temporal Ctx 8. 7 Control (Path) 2 Parietal Ctx 24. 8 Control 3 Temporal Ctx 9.3 Control (Path) 1 Parietal Ctx 63.3 Control 3 Temporal Ctx 8.7 Control (Path) 2 Parietal Ctx 24.8 Control (Path) 1 Temporal Ctx 51.4 Control (Path) 3 Parietal Ctx 1.9 Control (Path) 2 Temporal Ctx 36.6 Control (Path) 4 Parietal Ctx 34.9 Table EC. General screening panel v1. 5 Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag5963, issue Name Ag5963, Run Run 247945158 247945158 Adipose 9.9 Renal ca. TK-10 21.2 Melanoma* Hs688 (A). T 56. 6 Bladder 8. 8 Melanoma* Hs688 (B). T 30. Gastric ca. (liver met. ) NCI-N87 100.0 Melanoma* M14 15. 7 Gastric ca. KATO m 84. 1 Melanoma* LOXIMVI 60. 7 Colon ca. SW-948 9. 1 Melanoma* SK-MEL-5 20.9 Colon ca. SW480 24. 8 Squamous cell carcinoma SCC-4 12. 6 Colon ca. * (SW480 met) SW620 19.5 Testis Pool 15. 3 Colon ca. HT29 13. 6 Prostate ca. * (bone met) PC-3 42. 9 Colon ca. HCT-116 65. 5 Prostate Pool 24. 5 Colon ca. CaCo-2 23. 0 Placenta 5.3 Colon cancer tissue 23.0 uterus Pool 14. 2 Colon ca. SW1116 5. 2 Ovarian ca. OVCAR-3 49. 3 Colon ca. Colo-20516. 7 Ovarian ca. SK-OV-3 7.8 Colon ca. SW-48 6.1 Ovarian ca. OVCAR-4 6.9 Colon Pool 8.5 Ovarian ca. OVCAR-5 26.1 Small Intestine Pool 9. 9. Ovarian ca. IGROV-1 28. 9 Stomach Pool 8.0 Ovarian ca. OVCAR-8 10.1 Bone Marrow Pool 7.8 Ovary 5.2 Fetal Heart 13.9 Breast ca. MCF-7 26. 6 Heart Pool 17. 7 Breast ca. MDA-MB-231 31. 9 Lymph Node Pool 0.0 Breast ca. BT 549 16. 8 Fetal Skeletal Muscle 5. 0 Breast ca. T47D 4. 3 Skeletal Muscle Pool 50. 0 Breast ca. MDA-N 13. 5 Spleen Pool 17.1 Breast Pool 26. 4 Thymus Pool 13. 0 Trachea 16. 2 CNS cancer (glio/astro) U87-MG 27.9 Lung 0. 0 CNS cancer (glio/astro) U-1 18-MG 88. 9 Fetal Lung 47.0 CNS cancer (neuro;met) SK-N-AS 72.7 Lung ca. NCI-N417 2.9 CNS cancer (astro) SF-539 38.7 Lung ca. LX-1 35. 6 CNS cancer (astro) SNB-75 77.4 Lung ca. NCI-H146 8.8 CNS cancer (glio) SNB-19 9.7 Lung ca. SHP-77 61. 1 CNS cancer (glio) SF-295 70. 2 Lung ca. A549 95. 3 Brain (Amygdala) Pool 29. 5 Lung ca. NCI-H526 3. 0 Brain (cerebellum) 47. 3 Lung ca. NCI-H23 34.9 Brain (fetal) 41.8 Lung ca. NCI-H460 21.0 Brain (Hippocampus) Pool 6.2 Lung ca. HOP-62 9. 0 Cerebral Cortex Pool 33. 2 Lung ca. NCI-H52215. 1Brain (Substantia nigra) Pool10. 2 Liver 2.2 Brain (Thalamus) Pool 13.3 Fetal Liver 10.2 Brain (whole) 12.1 Liver ca. HepG2 15.6 Spinal Cord Pool 6. 4 Kidney Pool 14. 1 Adrenal Gland 6. 2 Fetal Kidney 23. 0 Pituitary gland Pool 2. 1 Renal ca. 786-0 12. 1 Salivary Gland 25. 3 Renal ca. A498 6. 6 Thyroid (female) 5. 7 Renal ca. ACHN 42. 3 Pancreatic ca. GAPAN2 14. 3 Renal ca. UO-31 3. 8 Pancreas Pool 30. 6 Table ED. Panel 4.1D Rel. Rel. Exp. (%) Exp. (%) Tissue Name g5963, Tissue Name Ag5963, Run Run 247851482 247851482 Secondary Th1 act 47.0 HUVEC IL-1beta 27.9 Secondary Th2 act 55.5 HUVEC IFN gamma 31.0 Secondary Tr1 act 15.3 HUVEC TNF alpha + IFN gamma 0.0 Secondary Thl rest 0.0 HUVEC TNF alpha + IL4 0.0 Secondary Th2 rest 0.0 HUVEC IL-11 9.5 Secondary Tr1 rest 0.0 Lung Microvascular EC none 84.7 Primary Th1 act 4.8 Lung Microvascular EC TNFalpha+ 17.4 IL-lbeta Primary The act 34.6 Microvascular Dermal EC none 0.0 Primary Tr1 act 29.5 Microsvasular Dermal EC TNFalpha+ 5.5 In-beta Bronchial epithelium TNFalpha + 22.1 IL1beta Primary Th2 rest 11.4 Small airway epithelium none 53.2 Primary Trl rest 0. 0 Small airway epithelium TNFalpha + 54. 0 IL-lbeta CD45RA CD4 lymphocyte act 45.1 Coronery artery SMC rest 30. 1 CD45RO CD4 lymphocyte act 55.5 Coronery artery SMC TNFalpha + 36. 1 IL-1beta CD8 lymphocyte act 2.8 Astrocytes rest 0. 0 Secondary CD8 lymphocyte rest 27.2 Astrocytes TNFalpha+IL-1beta 0. 0 Secondary CD8 lymphocyte act 0.0 KU-812 (Basophil) rest 24.1 CD1 lymphocyte none 2.7 KU-812 (Basophil) PMA/ionomycin 47.3 2ry Th1/Th2/Tr1_anti-CD95 0.0 CCD1106 (Keratinocytes) none 32.5 CH11 LAK cells rest 13.5 CCD1106 (Keratinocytes) TNFalpha+ 35.6 IL-1beta LAK cells IL-2 9. 0 Liver cirrhosis 10. 0 LAK cells IL-2+IL-12 0.0 NCI-H292 none 20. 9 LAK cells IL-2+IFN gamma 12.6 NCI-H292 IL-4 39. 0 LAK cells IL-2+IL-18 12.3 NCI-H292 IL-9 47.0 LAK cells PMA/ionomycin 13.6 NCI-H292 IL-13 69.3 NK Cells IL-2 rest 48. 3 NCI-H292 IFN gamma 17. 4 Two Way MLR 3 day 10. 9 HPAEC none 3. 8 Two Way MLR 5 day 0. 0 HPAEC TNF alpha + IL-1 beta 34. 4 Two Way MLR 7 day 0.0 Lung fibroblast none 31.0 PBMC rest 0.0 Lung fibroblast TNF alpha+IL-1 beta 33.0 PBMC PWM 5. 5 Lung fibroblast IL-4 12A PBMC PHA-L 5.8 Lung fibroblast IL-9 21.6 Ramos (B cell) none 3. 9 Lung fibroblast IL-13 0. 0 Ramos (B cell) ionomycin 21.6 Lung fibroblast IFN gamma 42.9 B lymphocytes PWM 40.3 Dermal fibroblast CCD1070 rest 69.7 B lymphocytes CD40L and IL-4 57. 0 Dermal fibroblast CCD1070 TNF 100. 0 alpha EOL-1 dbcAMP 43.2 Dermal fibroblast CCD1070IL-1 beta 37.1 EOL-1 dbcAMP PMA/ionomycin 6.5 Dermal fibroblast IFN gamma 6. 7 Dendritic cells none 11. 8 Dermal fibroblast IL-4 32. 8 Dendritic cells LPS 0. 0 Dermal Fibroblasts rest 39.0 Dendritic cells anti-CD40 18. 7 Neutrophils TNFa+LPS 0.0 Monocytes rest 6.4 Neutrophils rest 24.8 Monocytes LPS 21. 3 Colon 0. 0 Macrophages rest 12.5 Lung 0.0 Macrophages LPS 0. 0 Thymus 5. 1 HUVEC none 21.3 Kidney 23.3 HUVEC starved 41.2

CNS neurodegenerationvl. O Summary: Ag5963 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals.

This gene is found to be slightly upregulated in the temporal cortex of Alzheimer's disease patients. Blockade of this receptor may be of use in the treatment of this disease and decrease neuronal death.

General_screening_panel_v1. 5 Summary: Ag5963 Higest expression of this gene is detected in a gastric cancer NCI-N87 cell line (CT=31). Moderate to low 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, skeletal muscle, heart, fetal liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, this gene is expressed at moderate to low levels in all regions of the central nervous system examined, including amygdala, 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: Ag5963 Low expression of this gene is detected in TNF alpha activated dermal fibroblast (CT=34.6). Therefore, theratpeutic modulation of this gene may be useful in the treatment of skin disorders, including psoriasis.

F. CG165676-Ol (NOVlla) : INTEGRIN ALPHA-2 PRECURSOR Expression of gene CG165676-01 was assessed using the primer-probe set Ag4510, described in Table FA. Results of the RTQ-PCR runs are shown in Tables FB, FC and FD.

Table FA. Probe Name Ag4510 Primers Sequnces Length Start SEQ Position m No 9 Forward 5'-aaaatttcaggcacaccaaag-3'21 3018 129 Probe TET-5'-aattgaactgcagaactgcttcctgt-3'-TAMRA26 3039 130 Reverse 5'-tctcctttcatgtgaacgtctt-3'22 3087 131 TableFB. AI comprehensivepanel vl. O Rel. ~ Rel. Exp. (%) Exp. (%) Tissue Name Ag4510, Tissue Name Ag4510, Run Run 46953623 246953623 110967 COPD-F 5. 8 112427 Match Control Psoriasis-F 38. 2 110980 COPD-F 8.8 112418 Psoriasis-M 6.4 110968 COPD-M 3.8 112723 Match Control Psoriasis-M 5.3 110977 COPD-M 29. 7 112419 Psoriasis-M 6. 4 110989 Emphysema-F 37. 9 112424 Match Control Psoriasis-M 7.3 110992 Emphysema-F 8. 4 112420 Psoriasis-M 25. 9 110993 Emphysema-F 5.4 112425 Match Control Psoriasis-M 35. 6 110994 Emphysema-F 2.1 104689 (MF) OA Bone-Backus 82. 4 110995 Emphysema-F 22. 4 104690 (MF) Adj "Normal" 16.6 Bone-Backus 110996 Emphysema-F 6.7 104691 (MF) OA Synovium-Backus 19.5 110997 Asthma-M 8.2 104692 (BA) OA Cartilage-Backus 1.7 111001 Ash_ 16. 6 104694 (BA) OA Bone-Backus 100. 0 111002 Asthma-F 26.6 104695 (BA) Adj'Normal"28, 9 Bone-Backus 111003 Atopic Asthma-F 32. 8 104696 (BA) OA Synovium-Backus 12. 8 111004 Atopic Asthma-F 32. 5 104700 (SS) OA Bone-Backus 15. 4 104701 (SS) Adj "Normal" 111005 Atopic Asthma-F 24.0 19.6 Bone-Backus 111006 Atopic Asthma-F 5.1 104702 (SS) OA Synovium-Backus 32.8 111417 Allergy-M 14.8 117093 OA Cartilage Rep7 17.2 112347 Allergy-M 0.0 112672 OA Bone5'16. 8 112349 Normal Lung-F 0.0 112673 OA Synovmm511. 8 112357 Normal Lung-F 16. 3 112674 OA Synovial Fluid cells5 8.0 112354 Normal Lung-M 5. 7 117100 OA Cartilage Repl4 0.8 112374 Crohns-F 5. 3 112756 OA Bone9 30. 8 112389 Match Control Crohns-F 31.6 112757 OA Synovium9 3. 2 112375 Crohns-F 5. 4 112758 OA Synovial Fluid Cells9 8. 1 112732 Match Control Crohns-F 22.7 117125 RA Cartilage Rep2 9.9 112725 Crohns-M 3.3 113492 Bone2 RA 50. 0 112387 Match Control Crohns-M 5.4 113493 Synovium2 RA 16.6 112378 Crohns-M 0.0 113494 Syn Fluid Cells RA 25.5 112390 Match Control Crohns-M 60.3 113499 Cartilage4 RA 28.5 112726 Crohns-M 27. 9 113500 Bone4 RA 42. 6 112731 Match Control Crohns-M 12.0 113501 Synovium4 RA 30.1 112380 Ulcer Col-F 23.3 113502 Syn Fluid Cells4 RA 15.9 112734 Match Control Ulcer 65.5 113495 Cartilage3 RA 25.2 Col-F 112384 Ulcer Col-F 63.3 113496 Bone3 RA 34. 4 241 112737 Match Control Ulcer 13.2 113497 Synovium3 RA 17.4 Col-F 112386 Ulcer Col-F 2.0 113498 Syn Fluid Cells3 RA 45.1 112738 Match Control Ulcer 25.3 117106 Normal Cartilage Rep20 1.5 Col-F 112381 Ulcer Col-M 0.4 113663 Bone3 Normal 0.1 112735 Match Control Ulcer 2.1 113664 Synovium3 Normal 0.0 Col-M 112382 Ulcer Col-M 35.6 113665 Syn Fluid Cells3 Normal 0.0 112394 Match Control Ulcer 1.2 117107 Normal Cartilage Rep22 5.6 Col-M 112383 Ulcer Col-M 39.0 113667 Bone4 Normal 7.5 112736 Match Control Ulcer 16.3 113668 Synovium4 Normal 6.0 Col-M 112423 Psoriasis-F 12.3 113669 Syn Fluid Cells4 Normal 9.1 Table FC. General screening panel v1. 4 Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag4510, issue Name Ag4510, Run Run 222695870 222695870 Adipose 1.7 Renal ca. TK-10 9.2 Melanoma* Hs688 (A). T0. 9 Bladder8. 0 Melanoma* Hs688 (B). T 43 Gastric ca. (liver met.) NCI-N87 35. 1 Melanoma* M14 5. 6 Gastric ca. KATO HI21. 0 Melanoma*LOXIMVI40. 3 Colon ca. SW-948 3. 5 Melanoma* SK-MEL-5 4.3 Colon ca. SW480 12.8 Squamous cell carcinoma SCC-4 22. 1 Colon ca. * (SW480 met) SW620 5.6 Testis Pool 0. 9 Colon ca. HT29 2. 9 Prostate ca. * (bone met) PC-3 36.3 Colon ca. HCT-116 10.4 Prostate Pool 2. 3 Colon ca. CaCo-2 5. 7 Placenta 0. 2 Colon cancer tissue 17. 0 Uterus Pool 0. 4 Colon ca. SW1116 1. 5 Ovarian ca. OVCAR-3 11A Colon ca. Colo-205 2. 7 Ovarian ca. SK-OV-3 2. 2 Colon ca. SW-48 3.5 Ovarian ca. OVCAR-4 0.7 Colon Pool 1. 6 Ovarian ca. OVCAR-5 10. 4 Small Intestine Pool 1.9 Ovarian ca. IGROV-1 7. 4 Stomach Pool 2.3 Ovarian ca. OVCAR-8 1. 2 Bone Marrow Pool 0. 9 Ovary 0. 8 Fetal Heart 1.1 Breast ca. MCF-7 19. 3 Heart Pool 0. 4 Breast ca. MDA-MB-231 64. 6 Lymph Node Pool 1.5 Breast ca. BT 549 0.0 Fetal Skeletal Muscle 1.4 Breast ca. T47D 17.0 Skeletal Muscle Pool 0.1 Breast ca. MDA-N 3.2 Spleen Pool 4.2 Breast ca. MDA-N 3. 2 Spleen Pool 4. 2 Breast Pool 1.8 Thymus Pool 2.0 Trachea 6.6 CNS cancer (glio/astro) U87-MG 23.3 Lung 0. 2 CNS cancer (glio/astro) U-118-MG 18.7 Fetal Lung 7. 2 CNS cancer (neuro; met) SK-N-AS 24.0 Lung ca. NCI-N417 0. 0 CNS cancer (astro) SF-539 0.2 Lung ca. LX-1 6. 0 CNS cancer (astro) SNB-75 1. 9 Lungca. NCI-H1463CNS cancer (glio) SNB-196. 9 Lung ca. SHP-77 0.0 CNS cancer (glio) SF-295 100. 0 Lung ca. A549 10.8 Brain (Amygdala) Pool 1. 9 Lung ca. NCI-H526 0.2 Brain (cerebellum) 0. 1 Lung ca. NCI-H23 9.9 Brain (fetal) 0.8 Lung ca. NCI-H460 1.0 Brain (Hippocampus) Pool 1.4 Lung ca. HOP-62 17. 9 Cerebral Cortex Pool 1. 6 Lung ca. NCI-H522 0. 1 Brain (Substantia nigra) Pool 2. 0 Liver 0. 0 Brain (Thalamus) Pool 2.3 Fetal Liver 0. 6 Brain (whole) 0.4 Liver ca. HepG2 16.7 Spinal Cord Pool 2. 4 Kidney Pool 1.7 Adrenal Gland 3.7 Fetal Kidney 7.1 Pituitary gland Pool 0.2 Renal ca. 786-0 1.1 Salivary Gland 0. 7 Renal ca. A498 2.0 Thyroid (female) 1.0 Renal ca. ACHN 1. 0 Pancreatic ca. CAPAN2 34. 2 Renal ca. UO-31 8. 5 Pancreas Pool 1. 9 Table FD. Panel 4. 1D Rel. Rel. Exp. (%) Exp. (%) Tissue Name g4510, Tissue Name Ag4510, Run Run 246789401 246789401 Secondary Th1 act 9.8 HUVEC IL-1beta 36.1 Secondary Th2 act 10.7 HUVEC IFN gamma 22.8 Secondary Tr1 act 2.4 HUVEC TNF alpha + IFN gamma 3.1 Secondary Thl rest 0. 1 HUVEC TNF alpha + IL4 2. 5 Secondary'Th2 rest 0. 2 HUVEC II.-11 17. 2 Secondary Trl rest 0.0 Lung Microvascular EC none 79.0 Primary Th1 act 0.0 Lung Microvascular EC TNFalpha + 11.2 IL-lbeta Primary Th2 act 0.9 Microvascular Dermal EC none 1. 9 . IMicrosvasular Dermal EC TNFalpha + IL-lbeta Bronchial epithelium TNFalpha + Primary Thl rest 0. 0 26. 4 Primary Th2 rest 0.3 Small airway epithelium none 15.7 Small airway epithelium TNFalpha + Primary Tr1 rest 0.1 42.3 In-beta CD45RA CD4 lymphocyte act 16. 8 Coronery artery SMC rest 27. 0 Coronery artery SMC TNFalpha + CD45RO CD4 lymphocyte act 0.8 45.4 IL-1beta CD8 lymphocyte act 0.0 Astrocytes rest 0.3 Secondary CD8 lymphocyte rest 0. 8 Astrocytes TNFalpha + IL-lbeta 2. 0 Secondary CD8 lymphocyte act 0.4 KU-812 (Basophil) rest 0.0 CD4 lymphocyte none 0.0 KU-812 (Basophil) PMA/ionomycin 1.5 2ry Th1/Th2/Tr1_anti-CD95 0.1 CCD1106 (Keratinocytes) none 48.0 CH11 LAK cells rest 0. 0 CCD1106 (Keratinocytes) TNFalpha + 35. 1 EL-lbeta LAK cells IL-2 0. 4 Liver cirrhosis 2. 4 LAK cells IL-2+IL-12 0.0 NCI-H292 none 22.2 LAK cells IL-2+IFN gamma 0.6 NCI-H292 IL-4 16.0 LAK cells IL-2+IL-18 0.2 NCI-H292 IL-9 30.8 LAK cells PMA/ionomycin 3.7 NCI-H292 IL-13 20.4 NK Cells IL-2 rest 1.9 NCI-H292 IFN gamma 13. 5 Two Way MLR 3 day 0.1 HPAEC none 12. 0 Two Way MLR 5 day 0.1 HPAEC TNF alpha + IL-1 beta 64.2 Two Way MLR 7 day 0.7 Lung fibroblast none 27.2 PBMC rest 0.0 Lung fibroblast TNF alpha + IL-1 beta 55.5 PBMC PWM 0. 2 Lung fibroblast IL4 35.8 PBMC PHA-L 0. 1 Lung fibroblast IL-9 42.6 Ramos (B cell) none 0.0 Lung fibroblast IL-13 2.6 Ramos (B cell) ionomycin 0. 0 Lung fibroblast IFN gamma 100. 0 B lymphocytes PWM 0. 8 Dermal fibroblast CCD1070 rest 31.2 B lymphocytes CD40L and IL4 0. 2 Dermal fibroblast CCD1070 TNF 40. 3 alpha EOL-1 dbcAMP 0.0 Dermal fibroblast CCD1070 IL-1 beta 26.1 EOL-1 dbcAMP PMA/ionomycin 2.8 Dermal fibroblast IFN gamma 3.8 Dendritic cells none 0.0 Dermal fibroblast IL4 1.8 Dendritic cells LPS 0.0 Dermal Fibroblasts rest 2.8 Dendritic cells anti-CD40 0. 0 Neutrophils TNFa+LPS 0. 0 Monocytes rest 0.0 Neutrophils rest 0. 0 Monocytes LPS 0. 8 Colon 0. 5 Macrophages rest 0 Lung 2. Macrophages LPS 0. 1 Thymus 0. 6 HUVEC none 18.0 Kidney 7. 1 HUVEC starved14. 2

AI comprehensive panel vl. O Summary: Ag4510 Highest expression of this gene is detected in orfhoarthritis bone (CT=29.5). This gene shows a widespread expression in this panel. Moderate to low levels of expression of this gene are detected in samples derived from normal and orthoarthitis/rheumatoid arthritis bone, cartilage, synovium and synovial fluid samples, from normal lung, COPD lung, emphysema, atopic asthma, asthma, allergy, Crohn's disease (normal matched control and diseased), ulcerative colitis (normal matched control and diseased), and psoriasis (normal matched control and diseased).

Therefore, therapeutic modulation of this gene product may ameliorate symptoms/conditions associated with autoimmune and inflammatory disorders including psoriasis, allergy, asthma, inflammatory bowel disease, rheumatoid arthritis and osteoarthritis General nel vl. 4 Summary: Ag4510 Highest expression of this gene is detected in a CNS cancer SF-295 cell line (CT=25. 6). Moderate to high 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. Expression of this gene is higher in cancer cell lines compared to the normal tissues. 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 levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, fetal skeletal muscle, heart, fetal liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such. as obesity and diabetes.

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

Interestingly, this gene is expressed at much higher levels in fetal (CT=31-33) when compared to adult liver and skeletal muscle (CTs=35-38). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver and skeletal muscle.

In addition, the relative overexpression of this gene in fetal tissues suggest that the protein product may enhance liver and muscle 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 liver and muscle related diseases.

Panel 4. 1D Summary: Ag4510 Highest expression of this gene is detected in a IFN gamma stimulated lung fibroblasts (CT=28.4). Moderate to low levels of expression of this gene is detected in endothelial cells, keratinocytes, dermal fibroblasts and lung related samples including resting and activated-NCI-H292 mucoepidermoid cells, resting and activated lung fibroblasts, human pulmonary aortic endothelial cells (treated and untreated), small airway epithelium (treated and untreated), treated bronchial epithelium and lung microvascular endothelial cells (treated and untreated). Low expression of this gene is also detected in activated secondary Thl, Th2 and Trl cells, activated eosinophils and activated CD45RA CD4 lymphocyte (CT=30.9), which represent activated naive T cells. In activated memory T cells (CD45RO CD4 lymphocyte) or CD4 Thl or Th2 cells, resting CD4 cells (CTs>35), 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, therapeutic modulation of this gene may be useful in the treatement of autoimune and inflammatory disorders that include arthritis, psoriasis, Crohns disease, ulcerative colitis, asthma, chronic obstructive pulmonary disease, allergy and emphysema.

G. CG165719-01 (NOV12d), CG165719-02 (NOV12b) and CG165719-03 (NOV12c) : NEURONAL MEMBRANE GLYCOPROTEIN M6-B.

Expression of gene CG165719-01, CG165719-02 and CG165719-03 was assessed using the primer-probe sets Ag5977, Ag5978, Ag7810 and Ag7794, described in Tables GA, GB, GC and GD. Results of the RTQ-PCR runs are shown in Tables GE, GF and GG.

Please note that primer-probe set Ag5977 is specific for CG165719-03 and Ag5978 is specific for CG165719-01 and CG165719-02.

Table GA. Probe Name Ag5977 Primers Sequeces Length Start SEQ m Position No Forward 5'-caagagagaaaaggctgctttg-3'22 109 132 Probe TET-5'-ggaggagtcccctacgcctccct-3'-TAMRA 23 151 133 Reverse 19 205 134

Table GB. Probe Name Ag5978 c Start SEQID Primers Sequeces Length start SEQ IID Position No Forward 51-gtgaacagcagagctgaaatg-3 21 121 135 Probe TET-5'-cccgtgccaaccctgggggacag-3'-TAMRA 23 196 136 Reverse 5'-ggggactcctcccagac-3'-17 266 137 Reverse, < s D =L 17 æ 137 Table GC. Probe Name Ag7810 ,, _.... st : lrt SEQ ID Primers Sequeces Length Start SEQ ID Position No Forward 51-gcatcagtggaatgttcgttt-3'21 572 138 Probe TET-5'-cagccaggccactccaagcacat-3'-TAMRA 23 602 139 Reverse 5'-caccgctgagaaaccaaac-3 s 19 630 140

Table GD. Probe Name Ag7794 c T Start SEQID Primers Sequeces Length start SEQ ID Position No Forward 5'-gcgattcttgagcaacactt-3'20 370 141 Probe TET-5'-cacctcgctcagcaaggcatggt-3'-TAMRA 23 407 142 Reverse 5'-ccatagatgacatactgcatcagtt-3'25 434 143 I Table GE. CNS neurodegeneration vl. O Rel. Rel. Rel. Rel. Rel. Rel. T Exp. (%) Exp. (%) Exp. (%) Tissue Exp. (%) Exp. (%) Exp. (%) Tissue Ag5977, A5978, Ag7794, N m Ag5977, Ag5978, Ag7794, Run Run Run Run Run Run 248589057 248589058 312372407 248589057 248589058 312372407 Control AD1 10 oc icn (Path) 3,-.- AD 1 16. 3 8. 5 15. 0 (Path) 3 4. 5 2. 6 4. 5 Hippo Temporal Ctx Control AD 40. 9 28.7 23.3 (Path) 4 26.2 24.5 12.6 PP btx AD 3 AD 1 5.5 4.7 5.0 9.6 3.0 10.7 Hippo Occipital Ctx AD 2 AD 4 10.3 8.4 10.0 Occipital Ctx 0.0 0.0 0.0 (Missing) AD 5 AD 3 28.3 65.1 10.8 4.2 1.5 5.0 uppo Occipital Ctx AD 6 AD 4 100.0 26.6 74.7 19.2 21.6 12.6 Hippo Occipital Ctx Control 2 AD 5 42.0 38.2 22.2 42.0 13.2 12.1 Hippo Occipital Ctx Control 4 AD 6 15.4 17.2 13.4 31.4 46.7 19.1 Hippo Occipital Ctx Control Control 1 (Path) 3 6.2 4.0 4.8 2.2 1.7 2.1 Occipital Ctx Hippo Control 2 Temporal 11. 8 5.4 15.3 Occipital Ctx 56.3 84.1 32.8 Ctx Control 3 Temporal 44. 8 34.2 23.0 Occipital Ctx 15. 0 11. 0 9. 4 ctx Control 4 Temporal 3.6 3. 8 0.0 Occipital Ctx 9.9 9.3 Btx AD 4 Control Temporal 26.2 22.1 15.5 (Path) 1 0.1 100.0 42.6 Occipital Ctx Control Temporal 39.0 82.9 100.0 (Path) 2 7.4 6.4 5. 5 Occipital Ctx AD 5 Control SupTemp 24.8 45.7 56.3 (Path) 3 2.4 1.2 2.7 oral Ctx Occipital Ctx AD 6 Inf Control Temporal 74.7 47.6 45.1 (Path) 4 5.6 4.9 4.7 Ctx Occipital Ctx Cox Temporal 69.3 36.6 38.7 Parietal Ctx 5.5 5.6 7.1 Control 1 Control 2 Temporal 9.3 8.7 6.0 18.3 26.8 31.0 Parietal Ctx Ctx Control 2 Control 3 Temporal 58.2 52.9 23. 8 Parietal Ctx 18.2 17. 1 10. 2 Ctx Control 3 Control Temporal 20.7 14.8 9. 0 (Path) 1 0.1 92.7 36.9 Parietal Ctx Control 4 Control Temporal 17.8 14. 8 9.1 (Path) 2 17.7 18.4 11. 4 Ctx Parietal Ctx Control Control (Path) 1 0. 2 75. 3 24.0 (Path) 3 2.7 1.2 3.2 Temporal Parietal Ctx Ctx Control Control 30. 6 29.9 17.2 (Path) 4 24.1 22. 1 12.4 Temporal Parietal Ctx Ctx Table GF. General screening panel v1. 5 Rel. Rel. Rel. Rel. Exp. (%) Exp. (%) Exp. (%) Exp. (%) Tissue Name Ag5977, g5978, Tissue Name Ag5977, Ag5978, Run Run Run Run 248220118 248445832 248220118 248445832 Adipose 0. 3 0. 1 Renal ca. TK-10 0. 0 0. 0 Melanoma* 0.0 0.0 Bladder 0.6 0.7 Hs688(A).T Melanoma* Gastric ca. (liver 0.0 0.0 0.1 0.0 Hs688 (B).T met.) NCI-N87 Melanoma* Gastric ca. KATO 0.7 3.4 0.0 0.0 M14 III Melanoma* 0.0 0.0 Colon ca. SW-948 0.0 0.0 LOXRAVI Melanoma* 2. 1 11.7 Colon ca. SW480 0.0 0. 0 SK-MEL-5 Squamous cell Colon ca. * (SW480 carcinoma. met) SW620 Suc-4. Testis Pool 0. 5 0. 8 Colon ca. HT29 0. 0 0. 0 Prostate ca. * (bone met) 0.0 0.0 Colon ca. HCT-116 0.0 0.0 PC-3 Prostate Pool 2. 8 2. 0 Colon ca. CaCo-2 0. 0 0. 0 Placenta 0.0 0. 0 Colon cancer tissue 0.0 0.0 Uterus Pool 2. 3 1. 0 Colon ca. SW1116 0. 0 0.0 Ovarian ca. 0.1 0.2 Colon ca. Colo-205 0.0 0.0 OVCAR-3 Ovarian ca. 0.0 0. 1 Colon ca. SW-48 0.0 0.0 SK-OV-3 Ovarian ca. 0.0 0.0 Colon Pool 0.3 0.1 OVCAR-4 Ovarian ca. 0.1 0.0 Small Intestine Pool 1.9 2.6 OVCAR-5 Ovarian ca. 54.0 24.7 Stomach Pool 1.3 1.1 IGROV-1 Ovarian ca. 3 ; 9 5.9 Bone Marrow Pool 0. 8 1.2 OSCAR 8 Ovary 0. 0 0. 0 Fetal Heart 0. 7 0. 5 Breast ca. 0. 0 0.0 Heart Pool 0.7 1.1 MCF-7 Breast ca. MDA-MB-23 0.0 0.0 Lymph Node Pool 0. 4 0.9 1 Breast ca. BT 0.0 0.0 Fetal Skeletal 0.3 0.5 549 Muscle Breast ca. 0.0 0.0 Skeletal Muscle 1.1 1.0 T47D Pool Breast ca. 0. 2 1.8 Spleen Pool 0.1 0. 7 MUA N Breast Pool 0. 1 0.1 Thymus Pool 0.1 0. 5 CNS cancer Trachea 2.2 0. 6 (glio/astro) 0. 0 0.0 U87-MG CNS cancer Lung 0.3 0. 3 (glio/astro) 0. 0 0. 0 U-118-MG CNS cancer Fetal Lung 0.8 1.2 (neuro; met) 0.0 0.3 SK-N-AS Lung ca. 0.0 0.3 CNS cancer (astro) 0. 0 0.0 NCI-N417 SF-539 Lung ca. CNS cancer (astro) 0.0 0.0 62.9 46.3 LX-1 SNB-75 Lung ca. CNS cancer (glio) 0.2 1.7 73.2 27.9 NCI-H146 SNB-19 Lung ca. CNS cancer (glio) 0.0 0.0 0.0 0.0 SHP-77 SF-295 Lung ca. Brain (Amygdala) 0.0 0.0 84.1 43.2 A549 Pool Lung ca. 0.0 Brain (Amygdala) 1 43.2 A549 Pool Lung ca. 0.1 0.0 Brain (cerebellum) 98.6 100.0 NCI-H526 Lung ca. 0.0 0.1 Brain (fetal) 53.6 24.8 NCI-H23 Lung ca. Brain 0.0 0.0 100.0 45.4 NCI-H460 (Hippocampus) Pool Lung ca. Cerebral Cortex 0.0 0.0 89.5 54.0 HOP-62 Pool Lung ca. 0.0 0.0 Brain (Substantia 94.6 41.5 NCI-H522 nigra) Pool Liver 0. 0 0. 0 Brain (Thalamus) 87. 1 70. 2 Pool Fetal Liver 0.0 0.0 Brain (whole) 57.4 48.6 Liver ca. 0.0 0.0 Spinal Cord Pool 57.4 27.0 HepG2 Kidney Pool 0.4 1.1 Adrenal Gland 0.4 0.4 Fetal Kidney 0.1 0.1 Pituitary gland Pool 1.3 1.2 Renal ca. 0.0 0.0 Salivary Gland 0.4 0.8 786-0 Renal ca. 0. 0 0.0 Thyroid (female) 0.3 0.1. A498 Renal 0. 0 0. 0 Pancreatic ca. 0. 0 0. 0 ACHN 0.0 0.0 CAPAN2 0.0 0.0 Renal ca. 0.0 0.0 Pancreas Pool 0.4 0. 6 UO-31 Table GG. Panel 4. 1D Rel. Rel. Rel. Rel. Rel. ReI. Exp. (%) Exp. (%) Exp. (%) Exp. (%) Exp. (%) Exp. (%) Ag578, Ag7794, Ag7810, Ag5978, Ag7794, Ag7810, Tissue Name Run, Run Run Run Run Run 2481226 3123559 3123633 2481226 3123559 3123633 33 78 84 33 78 84 HUVEC Secondary Th1 act 0.0 0.0 0.0 0.0 0.0 0.0 IL-lbeta HUVEC IFN Secondary Th2 act 0.0 0.0 0.0 0.0 0.0 0.0 gamma HUVEC TNF Secondary Tr1 act 0.0 0.0 0.0 alpha + IFN 0.0 0.0 0.0 gamma HUVEC TNF Secondary Th1 rest 0.0 0.0 0.0 0.0 0.0 0.0 alpha + IL4 HUVEC Secondary Th2 rest 0.0 0.0 0.0 0.0 1.4 0.0 Lung Secondary Trl rest 0.0 0.0 0.0 Microvascular 0. 0 0.0 0.0 EC none Lung Primary Thl act 0.0 0.0 0.0 Mcrovascular 0. 0 0.0 0.0 EC TNFalpha + IL-lbeta Microvascular Primary Th2 act 0.0 0.0 0.0 Dermal EC 0.0 0.0 0.0 none Microsvasular Primary Trl act 0.0 0.0 0.0 Dermal EC 0.0 0.4 0.0 TNFalpha + IL-lbeta Bronchial Primary Thl rest 0.0 0. 0 0.0 epithelium 0. 0 0. 0 0. 0 TNFalpha + Il1beta Small airway Primary Th2 rest 0.0 0.0 0. 0 epithelium 0.0 1.7 4.9 none Small airway Primary Trl rest 0.0 0.0 0.0 epithelium 0.0 0.0 0.0 TNFalpha + IL-lbeta CD45RA CD4 Coronery lymphocyte act 0.0 0.0 0.0 artery SMC 0.0 3.0 0.0 zest Coronery CD45RO CD4 artery SMC 0.0 0.0 0.0 0.0 0.0 2.3 lymphocyte act TNFalpha + IL-lbeta CD8 lymphocyte act 0.0 0.0 0.0 Astrocytes 100.0 100.0 100.0 rest Secondary CD8 0.0 0.0 0.0 Astrocytes lymphocyte rest 0.0 0.0 0.0 TNFalpha + 15.3 14.7 34.2 IL-1beta KU-812 Secondary CD8 0.0 0.0 0.0 (Basophil) 0.0 0.0 0.0 lymphocyte act rest KU-812 CD4 lymphocyte 0.0 0.8 0.0 (Basophil) 0.0 0.0 0.0 none PMA/ionomy cm CCD1106 Thl/Th2/Trl anti-C 0.0 0.0 0.0 (Keratinocytes 0. 0 11.9 40.3 D95 CH11 ) none CCD1106 LAK cells rest 0.0 0.0 0. 0 (Keratinocytes 0. 0 1.0 1.1 ) TNFalpha + IL-lbeta LAK cells IL-2 0.0 0.0 0. 0 Liver cirrhosis 0. 0 4. 5 8. 8 LAK cells 0. 0 0.0 0.0 none 0. 0 0.0 0.0 IL-2+IL-12 none LAK cells IL-2+IFN 0.0 0.0 0.0 NCI-H292 0.0 0.0 0.0 gamma LAK cells IL-2+ NCI-H292 0.0 0.0 0.0 0.0 3.0 0.0 IL-18 Il-9 LAK cells 0.0 0.0 0.0 NCI-H292 0.0 0.0 5.5 PMA/ionomycin IL-13 NCI-H292 NK Cells Il-2 rest 0.0 0.0 2.3 0.0 0.0 0.0 IFN gamma Two Way MLR 3 0.0 0.0 0.0 HPAEC none 0.0 0.0 0.0 day HPAEC TNF Two Way MLR 5 0.0 0.0 0.0 alpha + IL-1 0.0 2.3 7.9 day beta Two Way MLR 7 day 0.0 0.8 0.0 fibroblast 0.0 11.6 35.8 none Lung PBMC rest 0.0 0.0 0. 0 fibroblast 0. 0 7.1 7.8 TNF alpha + IL-1 beta Lung PBMC PWM 0.0 0.0 0.0 0.0 5.7 3.0 fibroblast IL-4 Lung PBMC HPA-L 0.0 0.0 0.0 0.0 3.6 11.4 fibroblast IL-9 Lung Ramos (B cell) none 0.0 0.0 0.0 fibroblast 0.0 4. 5 5.4 IL-13 Ramos (B cell) 0.0 0.0 0.0 fibroblast IFN 27. 0 19.1 58.2 ionomycin gemma Dermal B lymphocytes PWM 0. 0 0.0 0.0 fibroblast 0.0 8. 1 1.6 CCD1070 rest Dermal B lymphocytes 0.0 0.0 0.0 fibroblast 29.1 4.0 1.5 CD40L and IL-4 CCD1070 TNF alpha Dermal EOL-1 dbcAMP 0.0 0.0 0.0 fibroblast 0. 02. 9 0.0 CCD1070 IL-1 beta EOL, L dbcANP Dermal 0.0 0.0 0.0 fibroblast IFN 0.0 5.2 19.9 PMA/ionomycin gamma Dermal Dendritic cells none 0.0 0.0 0.0 0.0 8.5 32.3 fibroblast IL-4 Dermal Dendritic cells LPS 0.0 0.0 0.0 Fibroblasts 0.0 0.9 2.6 rest Dendritic cells Neutrophils 0.0 0.0 0.0 0.0 0.0 0.0 anti-CD40 TNFa+LPS Monocytes rest 0.0 0.0 0.0 Neutrophils 0. 0 0. 0 0. 0 rest Monocytes LPS 0. 0 0. 0 0. 0 Colon 16. 0 10. 1 1. 1 Macrophages rest 0.0 0. 0 0. 0 Lung 0.0 6. 0 8. 0 Macrophages LPS 0. 0 0.0 0. 0 Thymus 0. 0 0. 0 0. 0 HUVEC none 0. 0 0. 0 0. 0 Kidney 0. 0 10. 3 64.6 HWEC starved 0.0 0. 0 0.0

CNS_neurodegeneration_v1.0 Summary: Ag5977/Ag5978/Ag7794 Three <BR> <BR> experiments with different probe pimer sets are in good agreement. This panel confirms the expression of this gene at significant 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 1.5 for a discussion of the potential utility of this gene in treatment of central nervous system disorders.

General screening panel v1. 5 Summary: Ag5977/Ag5978 Two experminents with different probe primer sets are in good agreement with highest expression of this gene seen in cerebellum and hippocampus (CTs=27-28. 9). This gene shows preferential expression in all the regions of brain including including amygdala, hippocampus, substantia nigra, thalamus ; cerebellum, cerebral cortex, and spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Moderate expression of this gene is also seen in two of the brain cancer, two ovarian cancer and melanoma cell lines. Therefore, therapeutic modulation of this gene may be useful in the treatment of melanoma, brain, and ovarian cancers.

Low levels of expression of this gene is also seen in pancreas, pituitary gland, skeletal muscle and 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: Ag5978/Ag7794/Ag7810 Multiple experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in resting astrocytes (CTs=31-34. 7). Low expression of this gene is also seen in activated astrocytes and lung fibroblasts. Therefore, therapeutic regulation of this gene or the encoded protein could be important in the treatment of multiple sclerosis or other inflammatory diseases of the CNS and and inflammatory lung disorders including chronic obstructive pulmonary disease, asthma, allergy and emphysema.

Ag5977 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

H. CG167488-01 (NOV13b) : Hypothetical Transmembrane Protein.

Expression of gene CG167488-01 was assessed using the primer-probe set Ag5997, described in Table HA. Results of the RTQ-PCR runs are shown in Tables HB, HC and HD.

Table HA. Probe Name Ag5997 Start SEQ Primers Sequnces Length Position '° No Forward 5'-gagctaccttataaagaccatctgtacat-3'29 3 144 t | h Probe TET-5'-ccactgtgaaatggagtttcaaaatcaca-3'-TAMR 29 32 145 A Reverse 5'-atatgtgctcctagtcttatgttcatgt-3'28 73 146

Table HB. CNS neurodegeneration vl. O Rel. Rel. Exp.(%) Tissue Name Exp.(%) Tissue Name Ag5997, Ag5997 Run 248589037 Run 248589037 Control (Path) 3 Temporal AD 1 Hippo 0.0 2.6 Ctx Control (Path) 4 Temporal AD 2 Hippo 28.9 54.3 Ctx AD 3 Hippo 1.6 AD 1 Occipital Ctx 4.6 AD 4 Hippo 16.4 AD 2 Occipital Ctx (Missing) 0.0 AD 5 hippo 92.7 AD 3 Occipital Ctx 0.0 AD 6 Hippo 31.4 AD 4 Occipital Ctx 36.1 Control 2 Hippo 35.4 AD 5 Occipital Ctx 48.0 Control 4 Hippo 16.8 AD 6 Occipital Ctx 41.5 Control (Path) 3 Hippo 28.9 Control 1 Occipital Ctx 0.0 AD 1 Temporal Ctx 4.6 Control 2 Occipital Ctx 45.4 AD 2 Temporal Ctx 35.4 Control 3 Occipital Ctx 20.0 AD 3 Temporal Ctx 9.1 Control 4 Occipital Ctx 3.3 Control (Path) 1 Occipital AD 4 Temporal Ctx 30.1 99.3 Ctx Control (Path) 2 Occipital AD 5 Inf Temporal Ctx 83.5 12.9 Ctx Control (Path) 3 Occipital AD 5 SupTemporal Ctx 68.8 0.0 Ctx Control (Path) 4 Occipital AD 6 Inf Temporal Ctx 88.3 31.2 Ctx AD 6 Sup Temporal Ctx 59.5 Control 1 Parietal Ctx 1.5 Control 1 Temporal Ctx 1.2 Control 2 Parietal Ctx 47.6 Control 2 Temporal Ctx 34.2 Control 3 Parietal Ctx 20.6 Control 3 Temporal Ctx 36.3 Control (Path) 1 Parietal Ctx 100.0 Control 4 Temporal Ctx 13.8 Control (Path) 2 Parietal Ctx 29.9 Control (Path) 1 Temporal Ctx 90.8 Control (Path) 3 Parietal Ctx 0.0 Control (Path) 2 Temporal Ctx 52.9 Control (Path) 4 Parietal Ctx 75.3 Table HC. General screening panel v1. 5 Rel. Rel. Exp.(%) Exp. (%) Tissue Name Ag5997, issue Name Ag5997, Run Run 248592793 248592793 Adipose 36.3 Renal ca. TK-10 9.2 Melanoma* Hs688(A).T 2.9 Bladder 14.2 Melanoma* Hs688(B).T 7.2 Gastric ca. (liver met.) NCI-N87 22.4 Melanoma* M14 0. 0 Gastric ca. KATO III 12.5 Melanoma* LOXIMVI 3.4 Colon ca. SW-948 6. 2 Melanoma* SK-MEL-5 3. 5 Colon ca. SW480 10. 7 Squamous cell carcinoma SCC-4 5.4 Colon ca. * (SW480 met) SW620 1.5 Testis Pool 8. 2 Colon ca. HT29 7.3 Prostate ca. * (bone met) PC-3 5.0 Colon ca. HCT-116 12. 3 Prostate Pool 13. 2 Colon ca. CaCo-2 4.2 Placenta 8. 8 Colon cancer tissue 11.8 uterus Pool 5. 9 Colon ca. SW1116 3. 0 Ovarian ca. OVCAR-3 12.6 Colon ca. Colo-205 0.0 Ovarian ca. SK-OV-3 22.1 Colon ca. SW-48 OA Ovarian ca. OVCAR-4 3. 1 Colon Pool 12.2 Ovarian ca. OVCAR-5 25.9 Small Intestine Pool 5.2 Ovarian ca. IGROV-1 6. 0 Stomach Pool 13. 4 Ovarian ca. OVCAR-8 7. 2 Bone Marrow Pool 3. 7 Ovary 1.6 Fetal Heart 0. 8 Breast ca. MCF-7 0.6 Heart Pool 7. 0 Breast ca. MDA-MB-231 30.6 Lymph Node Pool 9.7 Breast ca. BT 549 7.9 Fetal Skeletal Muscle 3. 2 Breast ca. T47D 0. 0 Skeletal Muscle Pool 18. 0 Breast ca. MDA-N 0. 1 Spleen Pool 1. 3 Breast Pool 13. 8 Thymus Pool 6. 7 Trachea 12.1 CNS cancer (glio/astro) U87-MG 12.8 Lung 0.5 CNS cancer (glio/astro) U-1 18-MG 14.8 Fetal Lung 100.0 CNS cancer (neuro; met) SK-N-AS 0.0 Lung ca. NCI-N417 0. 0CNS cancer (astro) SF-539 6.8 Lung ca. LX-1 3. 4 CNS cancer (astro) SNB-75. 7 Lung ca. NCI-H146 4.1 CNS cancer (glio) SNB-19 5.1 Lung ca. SHP-77 12.4 CNS cancer (glio) SF-295 14.9 Lung ca. A549 10. 3 Brain (Amygdala) Pool 5. 4 Lung ca. NCI-H526 0.0 Brain (cerebellum) 3.5 Lung ca. NCI-H23 6.0 Brain (fetal) 0.1 Lung ca. NCI-H460 7.6 Brain (Hippocampus) Pool 6. 8 Lung ca. HOP-62 3.9 Cerebral Cortex Pool 7.7 Lung ca. NCI-H522 11.5 Brain (Substantia nigra) Pool 4.0 Liver0. 0Brain (Thalamus) Pool10. 7 Fetal Liver 1. 8BrMn (whole) 6. 3 Liver ca. HepG2 1.3 Spinal Cord Pool 5.9 Kidney Pool 10.8 Adrenal Gland 0.1 Fetal Kidney 5. 6 Pituitary gland Pool 5. 8 Renal ca. 786-0 10. 9 Salivary Gland 2. 3 Renal ca. A498 3.8 Thyroid (female) 6.6 Renal ca. ACHN 4. 3 Pancreatic ca. CAPAN2 21. 8 Renal ca. UO-31 7.9 Pancreas Pool 17. 2 Table HD. Panel 5D Rel. Rel. Exp. (%) Exp. (%) Ag5997, Tissue Name Ag5997, Run Run 263248222 263248222 97457_Patient-02go_adipose 13.4 94709_Donor 2 AM - A_adipose 0.0 97476_Patient-07sk_skeletal 0.0 94710_Donor 2 AM - B_adipose 9. 5 muscle 97477 Patient-07ut uterus 0. 0 94711 Donor 2 AM-C adipose 0.0 97478_Patient-07pl_placenta 22. 8 94712 Donor 2 AD-A adipose 0.0 97481_Patient-08sk_skeletal 1.6 94713_Donor 2 AD - B_adipose 7.2 muscle 97482_Patient-08ut_uterus 0.0 94714_Donor 2 AD - C_adipose 0.0 97483_Patient-08pl_placenta 11.2 94742_Donor 3 U - A_Mesenchymal 0.0 - Stem Cells 97486_Patient-09sk_skeletal 94743_Donor 3 U - B_Mesenchymal 0.0 5.6 muscle Stem Cells 97487_Patient-09ut_uterus 13.8 94730_Donor 3 AM - A_adipose 4.5 97488Patient-09plplacenta 0.0 94731 Donor 3 AM-B adipose 0.0 97492_Patient-10ut_uterus 29.5 94732_Donor 3 AM - C_adipose 3.2 97493_Patient-10pl_placenta 0.0 94733_Donor 3 AD - A_adipose 0.0 97495_Patient-11go_adipose 17.7 94734_Donor 3 AD - B_adipose 0.0 97496 Patient-llskskeletal 94735 Donor3AD-C adipose 9. 2 muscle 97497 Patient-l lut uterus 158. 6 77138 Liver HepG2untreated 8. 2 73556_Heart_Cardiac stromal cells 97498_Patient-11pl_placenta 100.0 0.0 (primary) 97500_Patient-12go_adipose 0.0 81735_Small Intestine 5.4 97501_Patient-12sk_skeletal 72409_Kidney_Proximal Convoluted muscle Tubule 97502 Patient-12ut uterus 5.9 82685 Small intestine Duodenum 0.0 90650_Adrenal_Adrenocortical 97503_Patient-12pl_placenta 6.2 0.0 adenoma 94721Donor2U-72410 Kidney HRCE 21. 2 A Mesenehymal Stem Cells- 94722 Donor 2 U-0. 0 72411 Kidney EDlE 5.8 B Mesenchymal Stem Cells 94723 Donor 2 U - 73139 Uterus Uterine smooth , C_Mesenchymal Stem Cells muscle cells I I

CNS_neurodegeneration_v1.0 Summary: Ag5997 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals.

However, no differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment. Please see Panel 1.5 for a discussion of the potential utility of this gene in treatment of central nervous system disorders.

General_screening_panel_v1. 5 Summary: Ag5997 Highest expression of this gene is detected in fetal lung (CT=29.4). Interestingly, this gene is expressed at much higher levels in fetal compared to adult lung (CT=37). This observation suggests that expression of this gene can be used to distinguish fetal from adult lung. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance lung 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 lung related diseases.

Moderate to low 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 levels in pancreas, adipose, thyroid, pituitary gland, skeletal muscle, heart, and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

In addition, this gene is expressed. at low 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 5D Summary: Ag5997 Low expression of this gene is exclusively seen in placenta of non-diabetic but obese patient (CT=33.9). Therefore, expression of this gene may be used to distinguish placenta from other samples used in this panel.

I. CG50970-01 (NOV15b) and CG50970-02 (NOV15i) : Glypican-2 precursor.

Expression of gene CG50970-01 and CG50970-03 was assessed using the primer-probe sets Agl309 and Ag2251, described in Tables IA and IB. Results of the RTQ-PCR runs are shown in Tables IC, ID, IE, IF, IG, IH, II, IJ and IK. Please note that CG50970-03 represents a full-length physical clone.

Table IA. Probe Name Ag1309 Primers Sequnces Length Start SEQ m Position No Forward 5'-actctctgacccagctcttctc-3'22 359 147 Probe TET-5'-ccactcctacggccgcctgtatg-3'-TAMRA 23 381 148 ffi 22 416 149

Table IB. Probe Name Ag2251 Primers Sequnces Length start SEQ m Position No Forward 5'-actctctgacccagctcttctc-3'22 359 150 Probe TET-5'-ccactcctacggccgcctgtatg-3'-TAMRA 23 381 151 Reverse s 22 416 152 Table IC. AI comprehensive panel vl. O ReL Rel. Exp. (%) Exp. (%) Tissue Name Ag2251, Tissue Name Ag2251, Run Run 44570248 244570248 110967 COPD-F 20.7 112427 Match Control Psoriasis-F 29.3 110980 COPD-F 112418 Psoriasis-M 4. 7 110968 COPD-M 4.4 112723 Match Control Psoriasis-M 27.9 110977 COPD-M 8.8 112419 Psoriasis-M 2.1 110989 Emphysema-F 12.6 112426 Match Control Psoriasis-M 2. 9 110992 Emphysema-F 2. 9 112420 Psoriasis-M 20. 4 110993 16. 4 1 i2425 Match Controi Psoriasis-M 23.8 110994 Emphysema-F 3.8 104689 (ME OA Bone-Backus 8. 7 110995 Emphysema-F 19.3 104690 (MF) Adj "Normal" Bone-Backus 110996 Emphysema-F 2.4 104691 (MF) OA Synovium-Backus 4. 3 110997 Asthma-M 5.6 104692 (BA) OA Cartilage-Backus 2. 4 111001 Asthma-F 14.8 104694 (BA) OA Bone-Backus 4. 6 104695 (BA) Adj "Normal" 111002 Asthma-F 16.4 7.7 Bone-Backus 111003 Atopic Asthma-F 16.2 104696 (BA) OA Synovium-Backus 2.7 111004 Atopic Asthma-F 28.3 104700 (SS) OA Bone-Backus 9.0 111005 Atopic Asthma-F 7. 2 104701 (SS) Adj"Normal" Bone-Backus 111006 Atopic Asthma-F 4.4 104702 (SS) OA Synovium-Backus 7.5 111417 Allergy-M 11.0 117093 OA Cartilage Rep7 14. 7 112347 Allergy-M 7.5 112672 OA Bone5 57. 0 112349 Normal Lung-F 9. 4 112673 OA Synovium5 27.9 112357 Normal Lung-F 34. 2 112674 OA Synovial Fluid cells5 24. 8 112354 Normal Lung-M 9.2 117100 OA Cartilage Repl4 4.0 112374 Crohns-F 10.3 112756 OA Bone9 100.0 112389 Match Control Crohns-F 6.0 112757 OA Synovium9 17.9 112375 Crohns-F 22.2 112758 OA Synovial Fluid Cells9 9.2 112375 Crohns-F 22.2 112758 OA Synovial Fluid Cells9 9. 2 112732 Match Control Crolms-F 7. 5117125 RA Cartilage Rep26. 9 112725 Crobns-M 0. 0 113492 Bone2 RA 3.7 112387 Match Control Crohns-M 3.0 113493 Synovium2 RA 0. 6 112378 Crohns-M 10. 4 113494 Syn Fluid Cells RA 3.0 112390 Match Control Crohns-M 40.6 113499 Cartilage4 RA 1. 3 112726 Crohns-M 6. 7 113500 Bone4 RA 2.2 112731 Match Control Crohns-M 9.0 113501 Synovium4 RA 2.8 112380 Ulcer Col-F 25.5 113502 Syn Fluid Cells4 RA 5.3 112734 Match Control Ulcer 9. 5 113495 Cartilage3 RA 0.0 Col-F 112384 Ulcer Col-F 22.2 113496 Bone3 RA 2.9 112737 Match Control Ulcer 5.3 113497 Synovium3 RA 0.0 Col-F 112386 Ulcer Col-F 0. 0 113498 Syn Fluid Cells3 RA 0. 0 112738 Match Control Ulcer p. 0 117106 Normal Cartilage Rep20 0. 0 Col-F 112381 Ulcer Col-M 2.0 113663 Bone3 Normal 10.3 112735 Match Control Ulcer 6.9 113664 Synovium3 Normal 6.5 Col-M 112382 Ulcer Col-M 15. 8 113665 Syn Fluid Cells3 Normal 3.6 112394 Match Control Ulcer 5. 6 117107 Normal Cartilage Rep22 10.5 Col-M 112383 Ulcer Col-M 13. 3 113667 Bone4 Normal 9.3 112736 Match Control Ulcer Cool-M 112423 Psoriasis-F 4.1 113669 Syn Fluid Cells4 Normal 12.2 Table ID. CNS neurodegeneration vl. O Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag225, Tissue Name Ag2251, Run Run 206265375 206265375 AD 1 Hippo 19. 8 Control (Path) 3 Temporal Ctx 3.5 AD 2 Hippo 35. 8 Control (Path) 4 Temporal Ctx 34.2 AD 3 Hippo 7.2 AD 1 Occipital Ctx 19.5 AD 4 Hippo 9. 7 AD 2 Occipital Ctx (Missing) 0.0 AD 5 hippo 59. 0 AD 3 Occipital Ctx 17. 3 AD 6 Hippo 97. 9 AD 4 Occipital Ctx 19. 2 Control 2 Hippo 37.1 AD 5 Occipital Ctx 52. 9 Control 4 Hippo 34. 9 AD 6 Occipital Ctx 42. 0 Control (Path) 3 Hippo 17. 2 Control 1 Occipital Ctx 6. 3 AD 1 Temporal Ctx 20.6 Control 2 Occipital Ctx 51. 8 AD 2 Temporal Ctx 33. 7 Control 3 Occipital Ctx 23. 0 AD 3 Temporal Ctx 9. 9 Control 4 Occipital Ctx 6.6 AD 4 Temporal Ctx 36. 1 Control (Path) 1 Occipital Ctx 73. 7 AD 5 Inf Temporal Ctx 76. 8 Control (Path) 2 Occipital Ctx 16.8 AD 5 SupTemporal Ctx 97. 9 Control (Path) 3 Occipital Ctx 11. 8 AD 6 Inf Temporal Ctx_ 159. 9 Control (Path) 4 Occipital Ctx 28.1 AD 6 Sup Temporal Ctx100. 0 Control 1 Parietal Ctx 12. 1 Control 1 Temporal Ctx 9. 9 Control 2 Parietal Ctx 62.4 Control 2 Temporal Ctx 29. 9 Control 3 Parietal Ctx 20.4 Control 3 Temporal Ctx 10.5 Control (Path) 1 Parietal Ctx 43.8 Control 4 Temporal Ctx 34.2 Control (Path) 2 Parietal Ctrx 14.9 Control (Path) 1 Temporal Ctx 63.7 Control (Path) 3 Parietal Ctx 7.8 Control (Path) 2 Temporal Ctx 13. 8 Control (Path) 4 Parietal Ctx 29. 9 Table IE. General screening panel v1. 5 Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag2251, issue Name Ag2251, Run Run 246733742 246733742 Adipose 0.2 Renal ca. TK-10 9.2 Melanoma* Hs688 (A). T 0.8 Bladder 0.6 melanoma* Hs688(B).T 0.9 Gastric ca. (liver met.) NCI-N87 0.3 melanoma* M14 4.5 Gastric ca. KATO III 1.4 Melanoma* LOXIMVI 0.6 Colon ca. SW-948 0.1 Melanoma* SK-MEL-5 4.3 Colon ca. SW480 3. 8 Squamous cell carcinoma SCC-4 0. 4 Colon ca. * (SW480 met) SW620 1.6 Testis Pool 8.1 Colon ca. HT29 0.9 Prostate ca. * (bone met) PC-3 3. 6 Colon ca. HCT-116 2. 5 Prostate Pool 0. 0 Colon ca. CaCo-2 4. 3 Placenta 0.7 Colon cancer tissue 0.7 Uterus Pool 0.1 Colon ca. SW1116 0. 7. Ovarian ca. OVCAR-3 2. 9 Colon ca. Colo-205 0. 2 Ovarian ca. SK-OV-3 0. 5 Colon ca. SW-48 0. 8 Ovarian ca. OVCAR-4 0.6 Colon Pool 0.8 Ovarian ca. OVCAR-5 1.2 Small Intestine Pool 0.9 Ovarian ca. IGROV-1 5. 5 Stomach Pool 0. 4 Ovarian ca. OVCAR-8 1.6 Bone Marrow Pool 0.3 Ovary 0. 8 Fetal Heart 2. 0 Breast ca. MCF-7 1. 6 Heart Pool 0.3 Breast ca. MDA-MB-231 0. 2 Lymph Node Pool 0.8 Breast ca. BT 549 22. 5 Fetal Skeletal Muscle 2. 8 Breast ca. T47D 0.2 Skeletal Muscle Pool 0.3 Breast ca. MDA-N 2.6 Spleen Pool 0.3 Breast Pool 1.3 Thymus Pool 9.9 Trachea 0. 3 CNS cancer (glio/astro) U87-MG 3.0 Lung 0. 4 CNS cancer (glio/astro) U-1 18-MG 0.8 Fetal Lung 2.9 CNS cancer (neuro ; met) SK-N-AS 22.4 Lung ca. NCI-N417 5. 0 CNS cancer (astro) SF-539 0. 3 Lung ca. LX-1 5.3 CNS cancer (astro) SNB-75 17.2 Lung ca. NCI-H146 62.9 CNS cancer (glio) SNB-19 7.3 Lung ca. SHP-77 12. 4 CNS cancer (glio) SF-295 7. 6 Lung ca. A549 1.5 Brain (Amygdala) Pool 1.5 Lung ca. NCI-H526 25. 9 Brain (cerebellum) 3. 1 Lung ca. NCI-H23 7.0 Brain (fetal) 100.0 Lung ca. NCI-H460 6.7y Brain (Hippocampus) Pool 1.0 Lung ca. HOP-62 1. 3 Cerebral Cortex Pool 1. 3 Lung ca. NCI-H522 36.3 Brain (Substantia nigra) Pool 0. 8 Liver 0. 0 Brain (Thalamus) Pool 2. 1 Fetal Liver 0. 4 Brain (whole) 2.2 Liver ca. HepG2 0. 2 Spinal Cord Pool 1. 7 Kidney Pool 0.9 Adrenal Gland 0.3 Fetal Kidney 9.3 Pituitary gland Pool 0.1 Renal ca. 786-0 0.8 Salivary Gland 0.2 Renal ca. A498 0.7 Thyroid (female) 0.0 Renal ca. ACHN 1.0 Pancreatic ca. CAPAN2 0.8 Renal ca. UO-31 5. 7 Pancreas Pool 0. 9 Table IF. Oncology cell line screening panel v3.2 Rel. Rel. Exp.(%) Exp. (%) g2251, Ag2251, Tissue Name Tissue Name Run Run 2482021 2482021 32 32 94905_Daoy_Medulloblastoma/Cerebellu 94954 Ca Ski Cervical epidermoid m sscDNA. carcinoma (metastasis) sscDNA 10.7 94906_TE671_Medulloblastom/Cerebellu 14.8 94955_ES-2_Ovarian clear cell m sscDNA carcinoma sscDNA 94907 D7R' ! 94957_Ramos/6h stim_ Stimulated Med-Medulloblastoma/Cerebellum_sscDN 16.6 4.0 with PMA/ionomycin 6h_sscDNA A 94958_Ramos/14h stim_ 94908_PFSK-1_Primitive 1.7 Stimulated with PMA/ionomycin 2.9 Neuroectodermal/Cerebellum_sscDNA 14h_sscDNA 94962 MEG-Ol Chronic 94909_XF-498_CNS_sscDNA 1.2 myelogenous leukemia 0.3 (megokaryoblast) sscDNA 94963_Raji_Burkitt's 94910 SNB-78 CNS/glioma sscDNA 1. 0 1.2 l 94911 SF-268 CNS/glioblastoma sscDN 94964 Daudi Burkitt's A'lymphomasscDNA 94965_U266_B-cell 94912_T98G_Glioblastoma_sscDNA 1.3 0.0 plasmacytoma/myeloma_sscDNA 96776 SK-N-SH Neuroblastoma 94968 CA46 Burkitt's (metastasis) sscDNAlymphomasscDNA 94913_SF-295_CNS/glioblastoma_sscDN 94970_RL_non-Hodgkin's B-cell A lymphoma sscDNA 94972_JM1_pre-B-cell 132565_NT2 pool_sscDNA 16.6 5.5 lymphoma/leukemia_sscDNA 94973_Jurkat_T cell 94914_Cerebellum_sscDNA 1.3 5.1 leukemia_sscDNA 94974 TF-1 Erythroleukemia ssc 96777_Cerebellum_sscDNA 1.7 1.5 DNA 94916_NCI-H292_Mucoepidermoid lung 94975_HUT 78_T-cell 0.3 2.5 carcinoma_sscDNA lymphoma_sscDNA 94917_DMA-114_Small cell lung 94977_U937_Histiocytic cancersscDNA'ymphomasscDNA 94918_DMS-79_Small cell lung 94980_KU-812_Myelogenous 100.0 0.0 cancer/neuroendocrine_sscDNA leukemia_sscDNA 94919 NCI-H146 Small cell lung 94981 769-P Clear cell renal cancer/neuroendGcrinesscDNA'carcinomasscDNA 94920NCI-H526Small cell lung 94983 Caki-2 Clear cell renal cancer/neuroendocrine_sscDNA 79.0 carcinoma_sscDNA 0.1 94921 NCI-N417 Small cell lung 94984 SW 839 Clear cell renal cancer/neuroendocrinesscDNA'carcinomasscDNA 94923 NCI-H82 Small cell lung 94986G401Wihns' cancer/neuroendocnnesscDNA tumorsscDNA 94924 NCI-H157 Squamous cell lung 0. 2 126768 293 cells sscDNA 1. 9 cancer (metastasis) sscDNA 94987_Hs766T_Pancreatic 94925_NCI-H1155_Large cell lung 19.5 carcinoma (LN 0.7 cancer/neuroendocrine_sscDNA metastasis)_sscDNA 94988_CAPAN-1_Pancreatic 94926_NCI-H1299_Large cell lung 9.3 adenocarcinoma (liver 0.1 cancer/neuroendocrine sscDNA - metastasis) sscDNA 94989 SU86. 86 Pancreatic catcinoid sscDNA - metastasis) sscDNA 94928_NCI-UMC-11_Lung 94990_BxPC-3_Pancreatic carcinoid sscDNA adenocarcinoma sscDNA 94929_LX-1_Small cell lung 94991_HPAC_Pancreatic cancer sscDNA adenocarcinoma-sscDNA 94992_MIA PaCa-2_Pancreatic 94930_Colo-205_Colon cancer_sscDNA 0.0 0.0 carcinoma_sscDNA 94993 CFPAC-1 Pancreatic ductal adenocarcinoma sscDNA 94994_PANC-1_Pancreatic 94932_KM20L2_Colon cancer_sscDNA 0.2 epithelioid ductal 0.2 carcinoma_sscDNA 94996 T24 Bladder carcinma 94933 NCI-H716 Colon sscDNA5.6 0.2 - (transitional cell) sscDNA 94935_SW-8_Colon 94997_5637_Bladder 0.2 2.4 adenocarcinoma_sscDNA carcinoma_sscDNA 94936_SW1116_Colon 94998_HT-1197_Bladder 0.3 0.7 adenocarcinoma_sscDNA carcinoma_sscDNA 94999_UM-UC-3_Bladder 94937 LS 174T Colon - 0. 3 careinma 0. 2 cell)_sscDNA 94938_SW-948_Colon 95000_A204_Rhabdomyosarcoma_ adenocarcinoma sscDNA. sscDNA. 94939_SW-480_Colon 95001_HT-1080_Fibrosarcoma_ssc adenocarcinoma sscDNA. DNA. 94940_NCI-SNU-5_Gastric 95002_MG-63_Osteosarcoma 0.9 0.0 carcinoma_sscDNA (bone)_sscDNA 95003_SK-LMS-1_Leiomyosarcom 112197_KATO III_Stomach_sscDNA 0.0 1.3 a (vulva)_sscDNA 94943_NCI-SNU-16_Gastric 95004_SJRH30_Rhabdomyosarco carcinoma sscDNA 0.2 ma (met to bone marrow) sscDNA' 94944 NCI-SNU-1_Gastric 95005_A431_Epidermoid carcinoma sscDNA 1'S sscDNA0. 2 94946_RF-1_Gastric 95007_WM266-4_Melanoma_sscD 0.3 0.9 adenocarcinoma_sscDNA NA 94947_RF-48_Gastric 0.6 112195 DU 145 Prostate sscDNA 0.2 adenocarcinoma sscDNA 96778_MKN-45_Gastric 95012_MDA-MB-468_Breast 0.6 0.5 carcinoma_sscDNA adenocarcinoma_sscDNA 94949_NCI-N87_Gastric 0.6 112196_SSC-4_Tongue_sscDNA 0.9 carcinoma_sscDNA 94951_OVCAR-5_Ovarian 0.0 112194_SSC-9_Tongue_sscDNA 1.3 carcinoma_sscDNA 94952_RL95-2_Uterine 0. 3 112191_SSC-15_Tongue_sscDNA 0. 3 carcinoma-sscDNA carcinoma_sscDNA 94953_HelaS3_cervical 95017_CAL 27_Squamous cell 0.2 0.0 adenocarcinoma_sscDNA carcinoma of tongue_sscDNA Table IG. Panel 1. 3D Rel. Rel. Exp. (%) Exp. (%) Ag2251, Tissue Name Ag2251, Run Run 159074821 159074821 Liver adenocarcinoma 0.9 Kidney (fetal) 1.9 Pancreas 0.4 Renal ca. 786-0 1.0 Pancreatic ca. CAPAN 2 0. 4 Renal ca. A498 4. 5 Adrenal gland 0. 6 Renal ca. RXF 393 0.0 Thyroid 0.4 Renal ca. ACHN 0. 3 Salivary gland 1.2 Renal ca. UO-31 2. 8 Pituitary gland 0. 7 Renal ca. TK-10 3.8 Brain (fetal) 73.7 Liver 0. 0 Brain (whole) 4.6 Liver (fetal) 1.7 Brain (amygdala) 6.4 Liver ca. (hepatoblast) HepG2 1. 8 Brain (cerebellum) 1. 8 Lung 0.0 Brain ippocampus) 22. 2 Lung (fetal) 3. 1 Brain (substantia nigra) 2.1 Lung ca. (small cell) LX-1 4. 5 Brain (thalamus) 4. 5 Lung ca. (small cell) NCI-H69 8.7 Cerebral Cortex 3. 5 Lung ca. (s.cell var.) SHP-77 25.7 Spinal cord 3.2 Lung ca. (large cell)NCI-H460 2.5 glio/astro U87-MG 4.3 Lung ca. (non-sm. cell) A549 2.8 glio/astro U-118-MG 2.2 Lung ca.(non-s.cell) NCI-H23 12.4 astrocytoma SW1783 14. 3 Lung ca : (non-s. cell) HOP-62 1. 7 neuro*; met SK-N-AS 100.0 Lung ca. (non-s.cl) NCI-H522 28.1 astrocytoma SF-539 0.5 Lung ca. (squam.) SW 900 2.1 astrocytoma SNB-75 13. 0 Lung ca. (squam.) NCI-H596 0.7 glioma SNB-19 14.7 Mammary gland 1.0 gjioma U251 3. 6 Breast ca. * (pl. ef) MCF-7 4. 0 glioma SF-295 3.6 Breast ca.* (pl.ef) MDA-MB-231 1.1 Heart (fetal) 3.4 Breast ca.* (pl.ef) T47D 1.1 Heart 0. 0 Breast ca. BT-549 16.3 Skeletal muscle (fetal) 15. 2 Breast ca. MDA-N 6. 4 Skeletal muscle 0. 0 Ovary 3. 2 Bone marrow 1.8 Ovarian ca. OVCAR-3 1. 7 Thymus 21. 2 Ovarian ca. OVCAR-4 0. 8 Spleen 0. 8 Ovarian ca. OVCAR-5 2. 3 Lymph node 1. 1 Ovarian ca. OVCAR-8 7. 3 Colorectal 0.8 Ovarian ca. IGROV-1 2.4 Stomach 0.6 Ovarian ca.* (ascites) SK-OV-3 0.6 Small intestine 2.6 Uterus 0. 8 Colon ca. SW480 2. 5 Placenta 0. 8 Colon ca. * SW620 (SW480 met) 1.5 Prostate 1.1 Colon ca. HT29 1.7 Prostate ca. * (bone met) PC-3 3. 2 Colon ca. HCT-116 2.4 Testis 69. 7 Colon ca. CaCo-2 2. 5 Melanoma Hs688 (A). T 0. 0 Colon ca. tissue (OD03866) 2. 2 Melanoma* (met) Hs688 (B). T 0. 0 Colon ca. HCC-29982. 0Melanoma UACC-620. 4 Gastric ca.* (liver met) NCI-N87 0. 8 Melanoma M14 2.6 Bladder 1.0 Melanoma LOX IMVI 0. 7 Trachea 1.8 Melanoma* (met) SK-MEL-5 5.6 Kidney 0.7 Adipose 0.0 Table IH. Panel 2D Rel. Rel. Exp. (%) Exp. (%) Ag2251, Tissue Name Ag2251, Run Run 159075939 159075939 Normal Colon 5.5 Kidney Margin 8120608 0.0 CC Well to Mod Diff (ODO3866) 4.5 Kidney Cancer 8120613 0.0 CC Margin (ODO3866) 2.6 Kidney Margin 8120614 0.6 CC Gr.2 rectosigmoid (ODO3868) 1.2 Kidney Cancer 9010320 0.0 CC Margin (OD03868) 1. 1 Kidney Margin 9010321 1. 3 CC Mod Diff (OD03920) 5.8 Normal Uterus 1.1 CC Margin (ODO3920) 2.3 Uterus Cancer 064011 3.0 CC Gr.2 ascend colon (ODO3921) 4.1 Normal Thyroid 0.6 CC Margin (ODO3921) 0.0 Thyroid Cancer 064010 0.6 CC from Partial Hepatectomy 1.3 Thyroid Cancer A302152 0.4 (OD04309) Mets Liver Margin (OD04309) 0. 0 Thyroid Margin A302153 2. 3 Colon mets to lung (OD04451-01) 4.3 Normal Breast 4.4 Lung Margin (OD04451-02) 0. 0 Breast Cancer (OD04566) 1.2 Normal Prostate 6546-1 0.0 Breast Cancer (OD04590-01) 10.0 Breast Cancer Mets Prostate Cancer (OD04410) 3.4 1.5 (OD04590-03) Prostate Margin (OD04410) 0. 0 Breast Cancer Metastasis 3. 7 (OD04655-05) Prostate Cancer (OD04720-01) 0.6 Breast Cancer 064006 6.8 Prostate Margin (OD04720-02) 1.8 Breast Cancer 1024 10. 4 Normal Lung 061010 5. 1 Breast Cancer 9100266 6. 6 Lung Met to Muscle (ODO4286) 0.0 Breast Margin 9100265 3.4 Muscle Margin (ODO4286) 0.6 Breast Cancer A209073 7.9 Lung Malignant Cancer (OD03126) 3.9 Breast Margin A209073 2.5 Lung Margin (OD03126) 0.0 Normal Liver 0.0 Lung Cancer (OD04404) 0.0 Liver Cancer 064003 0.6 Lung Margin (OD04404) 0.6 Liver Cancer 1025 0.0 Lung Cancer (OD04565) 0.6 Liver Cancer 1026 0.6 Lung Margin (OD04565) 0.0 Liver Cancer 6004-T 0. 0 Lung Cancer (OD04237-01) 99. 3 Liver Tissue 6004-N 0. 6 Lung Margin (OD04237-02) 2. 4 Liver Cancer 6005-T 1. 1 Ocular Mel Met to Liver (OD04310) 0.7 Liver Tissue 6005-N 0.0 Liver Margin (OD04310) 0. 0 Normal Bladder 1. 8 Melanoma Mets to Lung (OD04321) 18.0 Bladder Cancer 1023 2. 8 Lung Margin (OD04321) 0.6 Bladder Cancer A302173 13. 2 Normal Kidney 1.4 Bladder Cancer (OD04718-01) 0.0 Kidney Ca, Nuclear grade 2 Bladder Normal Adjacent (OD04338) 8.0 (OD04718-03) 1.3 Kidney Margin (OD04338) 0.0 Normal Ovary 2.8 Kidney Ca Nuclear grade 1/2 2.4 Ovarian Cancer 064008 4.3 (OD04339) Kidney Margin (OD04339) 0. 0 Ovarian Cancer (OD04768-07) 4. 0 Kidney Ca, Clear cell type (OD04340) 1.2 Ovary Margin (OD04768-08) 0.0 Kidney Margin (OD04340) 1.0 Normal Stomach 0.8 Kidney Ca, Nuclear grade 3 0.0 Gastric Cancer 9060358 0.3 (OD04348) Kidney Margin (OD04348) 0.8 Stomach Margin 9060359 1. 2 Kidney Cancer (OD04622-0l) 1. 1 Gastric Cancer 9060395 0. 0 Kidney Margin (OD04622-03) 0. 0 Stomach Margin 9060394 1.5 Kidney Cancer (OD04450-01) 4. 6 Gastric Cancer 9060397 6.8 Kidney Margin (OD04450-03) 0. 6 Stomach Margin 9060396 0. 0 Kidney Cancer 8120607 0.6 Gastric Cancer 064005 2.5 Table II. Panel 4. 1D Rel. Rel. Exp. (%) Exp. (%) Ag2251, Tissue Name Ag2251, Run Run 244570228 244570228 Secondary Th1 act 12.4 HUVEC IL-1beta 26.,4 Secondary Th2 act 16.0 HUVEC IFN gamma 16.5 Secondary Tr1 act 0.0 HUVEC TNF alpha + IFN gamma 3.3 Secondary Th1 rest 0.0 HUVEC TNF alpha + IL4 8.9 Secondary Th2 rest 0.0 HnjVECIL-ll 39. 8 Secondary Tri rest 0.0 Lung Microvascular EC none 22.2 Primary Thl act 0. 0 Lung Microvascular EC TNFalpha + 2. 9 IL-lbeta Primary Th2 act 11.2 Microvascular Dermal EC none 0. 0 Primary Tr1 act 6.7 Microsvasular Dermal EC TNFalpha + 0. 0 IL-lbeta Bronchial epithelium TNEalpha + IL1beta Primary Th2 rest 0.0 Small airway epithelium none 0. 0 Primary Trl rest 0. 0 Small airway epithelium TNFalpha+-, Small afrway epineflum TNFalpha + Primary Tr1 rest 0.0 1.6 IL-1beta CD45RA CD4 lymphocyte act 15.3 Coronery artery SMC rest 8.1 Coronery artery SMC TNFalpha + CD45RO CD4 lymphocyte act 27.7 15.1 IL-lbeta CD8 lymphocyte act 0.0 Astrocytes rest 31.6 Secondary CD8 lymphocyte rest 19.9 Astrocytes TNFalpha + IL-1beta 6.4 Secondary CD8 lymphocyte act 4.1 KU-812 (Basophil) rest 0.0 CD4 lymphocyte none 0.0 KU-812 (Basophil) PMA/ionomycin 2. 9 2ry Th1/Th2/Tr1_anti-CD95 5.2 CCD1106 (Keratinocytes) none 21.6 CH11 CCD1106 (Keratinocytes) TNFalpha + LAK cells rest 0.0 16.3 IL-lbeta LAK cells IL-2 0.0 Liver cirrhosis 0. 0 LAK cells IL-2+IL-12 0.0 NCI-H292 none 0.0 LAK cells IL-2+IFN gamma 0.0 NCI-H292IL-4 3.2 LAK cells IL-2+IL-18 2.7 NCI-H292 IL-9 5. 8 LAK cells PMA/ionomycin 6. 5NCI-H292 IL-13 5. 9 NK Cells IL-2 rest 5.6 NCI-H292 IFN gamma 0.0 Two Way MLR 3 day 3.5 HPAEC none 3.3 Two Way MLR 5 day 0.0 HPAEC TNF alpha + IL-1 beta 8.2 Two Way MLR 7 day 2.5 Lung fibroblast none 21. 3 PBMC rest 0. 0 Lung fibroblast TNF alpha + IL-1 beta 7.7 PBMC PWM 5.3 Lung fibroblast IL-4 10. 7 PBMC PHA-L 9. 0 Lung fibroblast IL-9 11. 2 Ramos (B cell) none 0. 0 Lung fibroblast IL-13 0. 0 Ramos (B cell) ionomycin 100.0 Lung fibroblast IFN gamma 3.8 B lymphocytes PWM _ 7. 3 Dermal fibroblast CCD1070 rest 14. 3 Dermal fibroblast CCD1070 TNF B lymphocytes CD40L and IL-4 14.0 3.2 alpha EOL-1 dbcAMP 24.8 Dermal fibroblast CCD1070 IL-1 beta 3.7 EOL-1 dbcAMP PMA/ionomycin 0.0 Dermal fibroblast IFN gamma 3. 7 Dendritic cells none 3. 8 Dermal fibroblast ILA 5.9 Dendritic cells LPS 0.0 Dermal Fibroblasts rest 3.3 Dendritic cells anti-CD40 0.0 Neutrophils TNFa+LPS 2. 7 Monocytes rest 0.0 Neutrophils rest 6. 6 Monocytes LPS 8. 2 Colon 0. 0 Macrophages rest 0.0 Lung 0. 0 Macrophages LPS 0. 0 Thymus 37. 4 HUVEC none 30. 8 Kidney 0.0 HUVEC starved 59.9 Table IJ. Panel 4D Rel. Rel. Rel. Rel. Exp.(%) Exp.(%) Exp.(%) Exp.(%) Tissue Name g1309, Ag2251, Tissue Name Ag1309, Ag2251, Run Run Run Run 138960659 159076647 138960659 159076647 Secondary Th1 act 1.5 1.6 HUVEC IL-1beta 1.3 1.6 HUVEC IFN Secondary Th2 act 1.0 1.2 2.9 2.5 gamma HUVEC TNF alpha Secondary Tr1 act 2.0 1.7 1.5 0.1 + IFN gamma HUVEC TNF alpha Secondary Th1 rest 1.7 0.5 3.5 2.6 + IL4 Secondary Th2 rest 1.4 0.6 HUVEC IL-11 4.4 1.4 Lung Secondary Tr1 rest 1.4 1.2 Microvascular EC 1.3 2.3 none Lung Microvascular EC Primary Th1 act 1.7 2.7 2.1 1.7 TNFalpha + IL-1beta Microvascular Primary Th2 act 3.4 1.9 6.1 2.6 Dermal EC none Microsvasular Primary Tr1 act 5.9 1.2 2.0 1.3 TNFalpha + IL-lbeta Bronchial Primary Thl rest 12.5 17.1 epithelium 2.9 1.6 TNFalpha + ILlbeta Primary Th2 rest 6.5 8. 6 Small airway 0. 8 0.4 epithelium none Small airway Primary Trl rest 3.7 2. 4 epithelium 3.1 1. 5 TNFalpha + IL-lbeta CD45RA CD4 lymphocyte 3.2 1.2 Coronery artery 1.3 1.1 act SMC rest CD45RO CD4 lymphocyte Coronery artery act 4. 3 2.7 SMC TNFalpha + 1.4 2.0 IL-lbeta CD8 lymphocyte act 1.1 1.9 Astrocytes rest 17.8 22.5 Secondary CD8 Astrocytes Secondary CD8 1.7 0.9 TNFalpha + 6.2 4.7 lymphocyte rest IL-1beta Secondary CD8 1.1 1.0 KU-812 (Basophil) 0. 3 0. 2 lymphocyte act. rest CD4 lymphocyte none 1.4 0.5 KU-812 (Basophil) 1.2 0.3 PMA/ionomycin 2ry CCD1106 Th1/Th2/Tr1_anti-CD95 4.5 1.7 (Keratinocytes) 3.9 3.9 CH1 none CCD1106 LAK cells rest 1.2 1.6 (Keratinocytes) 19. 5 3. 1 TNFalpha + IL-lbeta LAK cells IL-2 3.1 1.8 Liver cirrhosis 2.0 2.6 LAK cells IL-2+IL-12 1.8 0.7 Lupus kidney 0.3 0.0 LAK cells IL-2+IFN 1.7 1.3 NCI-H292 none 0.7 0.4 gamma LAK cells IL-2+ IL-18 1.5 1.4 NCI-H292 IL-4 1.7 0.4 LAK cells PMA/ionomycin 0.8 0.0 NCI-H292 IL-9 0.0 1.6 NK Cells IL-2 rest 0.9 1.1 NCI-H292 IL-13 0.6 1.6 NCI-H292 IFN Two Way MLR 3 day 1.6 2.3 0.0 0.3 gamma Two Way MLR 5 day 1.7 0.3 HPAEC none 3.3 2.0 HPAEC TNF alpha Two Way MLR 7 day 0.8 0.4 1.6 0.6 + IL-1 beta Lung fibroblast PBMC rest 0.4 0.0 3.7 3.4 none Lung fibroblast PBMC PWM 6.1 2.1 TNF alpha + IL-1 1.6 1.5 beta Lung fibroblast PBMC PHA-L 9.9 5.7 3.6 2.8 IL-4 Lung fibroblast Ramos (B cell) none 13.6 6.3 2.6 3.2 IL-9 Lung fibroblast Ramos (B cell) ionomycin 34.9 24.3 2.7 2.8 IL-13 Lung fibroblast IFn B lymphocytes PWM 7.4 7.3 0.5 1.9 gamma B lymphocytes CD40L and Dermal fibroblast IL-4.. CCD1070 rest. Dermal fibroblast EOL-1 dbcAMP 2.6 2.3 CCD1070 TNF 2.4 4.2 alpha EOL-1 dbcAMP Dermal fibroblast PMA/ionomycin CCD1070IL-lbeta Dermal fibroblast Dendritic cells none 0.5 0.8 0.7 0.2 IFN gamma Dermal fibroblast Dendritic cells LPS 0.0 0.0 0.7 0.8 IL-4 Dendritic cells anti-CD40 0.3 0.0 IBD Colitis 2 0.2 0.0 Monocytes rest 0. 3 0. 0 IBD Crohn's 0. 0 0.0 Monocytes LPS 1.1 0.3 Colon 3.1 4. 2 Macrophages rest 0.6 1.3 Lung 1.5 1.3 Macrophages LPS 0. 0 0. 0 Thymus 1. 6 0. 3 HUVEC none 5. 3 3.5 Kidney 100. 0 100.0 HUVEC starved12. 7 12. 4 Table IK general oncology screening panel v 2.4 Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag2251, Tissu Name Ag2251, Run Run 259733199 259733199 Colon cancer 1 Bladder NAT 2 0. 0 Colon NAT 1 0. 0 Bladder NAT 3 0. 2 Colon cancer 2 3.1 Bladder NAT 4 0.0 Colon NAT 2 0. 0 Prostate adenocarcinoma 1 4. 8 Colon cancer 3, 8. 8 Prostate adenocarcinoma 2 0. 0 Colon NAT 3 1.6 Prostate adenocarcinoma 3 1.1 Colon malignant cancer 4 2. 4 Prostate adenocarcinoma 4 7. 0 Colon NAT 4 0. 0 Prostate NAT 5 1A Lung cancer 1 20. 4 Prostate adenocarcinoma 6 0. 5 Lung NAT 1 0. 0 Prostate adenocarcinoma 7 0. 6 Lung cancer 2 100.0 Prostate adenocarcinoma 8 0. 0 Lung NAT 2 0.6 Prostate adenocarcinoma 9 1.0 Squamous cell carcinoma 3 3.6 Prostate NAT 10 0.4 Lung NAT 3 0. 0 Kidney cancer 1 4. 8 Metastatic melanoma 1 5.0 Kidney NAT 1 0.3 Melanoma 2 1.8 Kidney cancer 2 5.5 Melanoma 3 2.7 Kidney NAT 2 0.0 Metastatic melanoma 4 22.2 Kidney cancer 3 4.9 Metastatic melanoma 5 17.8 Kidney NAT 3 1.2 Bladder cancer 1 0.7 Kidney cander 4 0.0 Bladder NAT 100Kidney NAT 400 Bladder cancer 2 0. 0

AI_comprehensive panel_vl. O Summary: Ag2251 Highest expression of this gene is detected in orthoarthritis bone (CT=31. 6). Low expression of this gene is also seen in in samples derived from normal and orthoarthitis bone, synovium samples, from normal lung, COPD lung, emphysema, atopic asthma, asthma, allergy, Crohn's disease (normal matched control and diseased), ulcerative colitis (normal matched control and diseased), and psoriasis (normal matched control and diseased). Therefore, therapeutic modulation of this gene. product may ameliorate symptoms/conditions associated with autoimmune and inflammatory disorders including psoriasis, allergy, asthma, inflammatory bowel diseases (Crohn's and ulcerative colitis), and osteoarthritis.

CNS_neurodegeneration_v1.0 Summary: Ag2251 Highest expression of this gene in this panel is detected in the cerebral cortex of an Alzheimer's patient (CT=32.7).

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. Please see Panel 1.3D and 1.5 for a discussion of potential utility of this gene in the central nervous system.

Generaljscreemngjpanelyl. 5 Summary: Ag2251 Highest expression of this gene is detected in fetal brain (CT=27.1). Low expression of this gene is also seen all the regions of adult brain including amygdala, hippocampus, substantia nigra, thalamus,

cerebellum, cerebral cortex, and spinal cord. Interestingly, expression of this gene is higher in fetal compared to the adult whole brain (32.6). This gene represents the human ortholog of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain. Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is apparently expressed in only one tissue in the rat: the nervous system and it is really present only during fetal development in immature neurons. Expression of this gene in human fetal and all the regions of adult brain regions suggest that this gene may play a role in central nervous system disorders such as.

Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

In addition, significant expression of this gene is also seen in number of cancer cell lines derived from pancreatic, gastric, colon, lung, renal, breast, ovarian, prostate, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Expression of this gene is higher in cell lines derived especially from breast and lung cancers and also in fetal tissues including lung, heart, kidney and skeletal muscle (CTs=30-32. 7) compared to respective adult tissues (CTs=33-35.5). Thus, this gene may play role growth or development of the cells, especially during tumorogenesis and may also act in a regenerative capacity in the adult.

Therefore, therapeutic modulation of the expression or function of this gene through the use of antibodies may be effective in the treatment of these cancers, especially breast and lung cancers.

Oncology_cell_line_screeningwanel_v3. 2Summary : Ag2251 Highest expression of this gene is detected in small cell lung cancer DMS-79 cell line (CT=28.7).

High expression of this gene is seen in number of cell lines derived from lung cancer.

Moderate to low expression of this gene is also seen in number of cell lines derived from brain, colon, cervical, bladder and bone cancers, T and B cell lymphomas. Please see panel 1.5 and 1.3D for further discussion on the utility of this gene.

Panel 1.3D Summary: Ag2251 The highest level of expression of this gene is seen in a CNS cancer cell line SK-N-AS (CT=29.6). The gene is also expressed at higher levels

in cell lines derived from lung, prostate, and breast cancers compared to the normal tissues and may play a role in these cancers. Thus, expression of this gene could be used as a marker or as a therapeutic for lung, prostate and breast cancer. In addition, therapeutic modulation of the activity of the product of this gene, through the use of peptides, antibodies, chimeric molecules or small molecule drugs, may be useful in the treatment of these cancers.

This gene is also expressed at higher levels in fetal liver, lung, skeletal muscle, and heart (CTs=32-35) when compared to the expression in adult tissues (CTs=40). These results suggest that expression of this gene could potentially be used to distinguish between the adult and fetal phenotypes of these tissues. Furthermore, the difference in expression in fetal and adult tissue may also indicate an involvement of the gene product in the differentiation processes leading to the formation of the adult organs. Therefore, the protein encoded by this gene could potentially play a role in the regeneration of these tissues in the adult.

This gene, a glypican homolog, is expressed at moderate to low levels across many regions of the brain. These regions include the hippocampus, amygdala, thalamus and cerebral cortex, all of which are key regions subject to Alzheimer's disease neurodegeneration. Furthermore, glypican is expressed in senile plaques and neurofibrillary tangles, also indicating a role in Alzheimer's disease. Therefore, the expression profile of this gene suggests that antibodies against the protein encoded by this gene can be used to. distinguish neurodegenerative disease in the human brain. Furthermore, since glycopican are components of senile plaques which are thought to give rise to the dementia pathology of Alzheimer's disease, agents that target this gene and disrupt its role in senile plaques, (Ref. 1) may have utility in treating the cause and symptoms or Alzheimer's disease as well as other neurodegenerative diseases that involve this glypican.

Reference: 1. Verbeek MM, Otte-Holler L van den Born J, van den Heuvel LP, David G, Wesseling P, de Waal RM. (1999) Agrin is a major heparan sulfate proteoglycan accumulating in Alzheimer's disease brain. Am J Pathol. 155: 2115-25. PMID : 10595940

Panel 2D Summary: Ag2251 The highest expression of this gene is seen in a breast cancer sample (CT = 30.3). The expression of this gene appears to show an association with samples derived from colon, lung, kidney, breast, bladder and gastric cancers when compared to the matched normal tissue. Thus, expression of this gene could be used as a marker for these cancers. In addition, therapeutic activity of the product of this gene, through the use of peptides, antibodies, chimeric molecules or small molecule drugs, may be useful in the treatment of colon, lung, kidney, breast, bladder and gastric cancers.

Panel 4. 1D Summary : Ag2251 Highest expression of this gene is seen in ionomycin activated Ramos B cells (CT=31.4). Expression of this gene is low or undectable in resting Ramos B cells (CT=40). B. cells represent a principle component of immunity and contribute to the immune response in a number of important functional roles, including antibody production. Production of antibodies against self-antigens is a major component in autoimmune disorders. In addition, low expression of this gene is also seen in eosinophils, HUVEC cells, activated secondary Thl and Th2 cells, naive and memory T cells, lung and dermal fibroblast and thymus. Therefore, therapeutic modulation of this gene product may reduce or eliminate the symptoms of patients suffering from asthma, allergies, chronic obstructive pulmonary disease, emphysema, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, osteoarthritis, systemic lupus erythematosus and other autoimmune disorders.

Panel 4D Summary : Ag2251/Agl309 Two experiments using two different probe and primer sets produce results that are in very good agreement, with highest expression seen in the kidney (CTs=28-29). This high level of expression in the kidney suggests that expression of this gene can serve as a marker for kidney tissue. This gene is also expressed at low level in activated Ramos B cell line, in activated primary B cells, Thl T cells, activated HUVEC and keratinocytes. This gene encodes for a protein that is a homolog of a glypican molecule, which belongs to the family of HSPG (heparan sulfate proteoglycans).

Glypicans can regulate the activity of a wide variety of growth and survival factors.

Therefore, therapeutic modulation of the expression or function of this gene or gene product, through the use of antibody drugs could potentially prevent T and B cell activation in the treatment of autoimmune mediated diseases such as insulin-dependent diabetes mellitus, rheumatoid arthritis, Crohn's disease, allergies, delayed type hypersensitivity, asthma, and psoriasis.

general oncology screening panels2. 4 Summary: Ag2251 Highest expression of this gene is detected in lung cancer2 (CT=30.7). Moderate to low expression of this gene is also seen in lung cancer, two metastatic melanoma and prostate cancer samples.

Therefore, expression of this gene may be used as a diagnostic marker to detect the presence of these cancers and also, therapeutic modulation of this gene or its product through the use of antibodies or small molecule drug may be useful in the treatment of metastatic melanoma, lung and prostate cancers.

J. CG54443-07 (NOV16b) : CG8841 PROTEIN-like protein.

Expression of gene CG54443-07 was assessed using the primer-probe sets Ag2000 and Ag6688, described in Tables JA and JB. Results of the RTQ-PCR runs are shown in Tables JC, JD and JE.

Table JA. Probe Name Au2000 c. T Start SEQ Primers Sequnces Length Start SEQ Position ID No Forward 5'-actccaccaagaagatccagtt-3'22 527 153 Probe TET-5'-tctcttctggaagctctgcgacttca-3-TAMRA 26 567 154 Reverse SM 22 595 155

Table JB. Probe Name Ag6688 Start SEQ IID Primers Sequeces Length ... ' Position No Forward 5'-ccaccaagacgcagc-3'15 185 156 Probe TET-5'-aagccaccgatgatgcctatg-3'-TAMRA 21 206 157 f ms w ma w==l Reverse 5'-gagcaggtggttgtaggg-3'18 247158 Table JC. Panel 1. 3D Rel. Rel. Exp. (%) Exp. (%) Tissue Name g2000, Tissue Name Ag2000, Run Run 147805564 147805564 Liver adenocarcinoma 9. 8 Kidney (fetal) 6.0 Pancreas 24. 8 Renal ca. 786-0 0. 0 Pancreatic ca. CAPAN2 1. 3 Renal ca. A498 1. 0 Adrenal gland 3.3 Renal ca. RXF 393 0.0 Thyroid 11.0 Renal ca. ACHN 1.5 Salivary gland 30.6 Renal ca. UO-31 1. 1 Pituitary gland 30. 4 Renal ca. TK-10 2.4 Brain (fetal) 13. 0 Liver 0. 7 Brain (whole) 39.2 Liver (fetal) 2. 5 Brain (amygdala) 23. 7 Liver ca. (hepatoblast) HepG2 8. 8 Brain (cerebellum) 21.0 Lung 12.9 Brain (hippocampus) 46. 7 Lung (fetal) 30.4 Brain (substantia nigra) 10. 4 Lung ca. (small cell) LX-1 8. 7 Brain (thalamus) 33. 2 Lung ca. (small cell) NCI-H69 29. 5 Cerebral Cortex 100. 0. Lung ca. (s. cell var.) SHP-77 33.0 Spinal cord 14. 6 Lung ca. (large cell) NCI-H460 0.9 glio/astro U87-MG 0. 1 Lung ca. (non-sm. cell) A549 15.9 glio/astro U-118-MG 0. 3 Lung ca. (non-s. cell) NCI-H23 2. 3 astrocytoma SW1783 0. 0 Lung ca. (non-s. cell) HOP-62 3. 3 neuro*; met SK-N-AS 4. 3 Lung ca. (non-s. cl) NCI-H522 1.8 astrocytoma SF-539 0. 0 Lung ca. (squam. ) SW 900 20.2 astrocytoma SNB-75 35.6 Lung ca. (squam.) NCI-H596 3.3 glioma SNB-19 5.7 Mammary gland 40.1 glioma U251 2. 1-Breast ca. * (pl. ef) MCF-7 42. 0 glioma SF-295 2.6 Breast ca. * (pl. ef) MDA-MB-231 6.3 Heart (fetal) 44. Breast ca. * (pl. ef) T47D 73. 2 Heart 3.6 Breast ca. BT-549 0.0 Skeletal muscle (fetal) 69.3 Breast ca. MUA N 0.2 Skeletal muscle 0.6 Ovary 17.6 Bone marrow 1.8 Ovarian ca. OVCAR-3 23.5 Thymus 2.9 Ovarian ca. OVCAR-4 9.2 Spleen 14.8 Ovarian ca. OVCAR-5 13.0 Lymph node 8.6 Ovarian ca. OVCAR-8 2.8 Colorectal 18.9 Ovarian ca. IGOV-1 1.9 Stomach 68.3 Ovarian ca.* (ascites) SK-OV-3 2.7 Small intestine 21.9 Uterus 9.9 Colon ca. SW480 10. 0 Placenta 27. 2 Colon ca. * SW620 (SW480 met) 2.9 Prostate 25. 9 Colon ca. HT29 16. 8 Prostate ca. * (bone met) PC-3 18. 7 Colon ca. HCT-116 5. 5 Testis 7.4 Colon ca. CaCo-2 11.6 Melanoma Hs688 (A). T 0. 0 Colon ca. tissue (OD03866) 27.0 Melanoma* (met) Hs688 (B). T 0. 1 Colon ca. HCC-2998 17. 2 Melanoma UACC-62 0. 0 Gastric ca. * (liver met) NCI-N87 48.6 Melanoma M14 0. 0 Bladder 10. 7 Melanoma LOX IMVI 0. 0 Trachea 36. 1 Melanoma* (met) SK-MEL-5 0.7 Kidney 1. 9 Adipose 4.3 Table JD. Panel 2.2 Rel.-Rel. Exp. (%) Exp. (%) Ag2000, Tissue Name Ag2000, Run Run 174232799 174232799 Normal Colon 14.0 Kidney Margin (OD04348) 10.7 Colon cancer (OD06064) 21.3 Kidney malignant cancer 29.7 (OD06204B) Colon Margin (OD06064) 24.5 Kidney normal adjacent tissue (OD06204E) Colon cancer (OD06159) 7. 0 Kidney Cancer (OD04450-01) 4. 1 Colon Margin (OD06159) 11.0 Kidney Margin (OD04450-03) 5. 0 Colon cancer (OD06297-04) 8. 7 Kidney Cancer 8120613 1.3 Colon Margin (OD06297-05) 14.1 Kidney Margin 8120614 7. 6 CC Gr. 2 ascend colon (OD03921) 9A Kidney Cancer 9010320 2. 7 CC Margin (OD03921) 4. 8 Kidney Margin 9010321 2. 9 Colon cancer metastasis (OD06104) 3. 1 Kidney Cancer 8120607 9. 0 Lung Margin (OD06104) 10.2 Kidney Margin 8120608 3. 0 Colon mets to lung (OD04451-01) 10.8 Normal Uterus 9.0 Lung Margin (OD04451-02) 8.3 Uterine Cancer 064011 4.9 Normal Prostate 42. 9 Normal Thyroid 5. 4 Prostate Cancer (OD04410) 17. 2 Thyroid Cancer 064010 2. 8 Prostate Margin (OD04410) 10.4 Thyroid Cancer A302152 6.3 Normal Ovary 7. 6Thyroid Margin A302153 4. 6 Ovarian cancer (OD06283-03) 9.5 Normal Breast 19. 6 Ovarian Margin (OD06283-07) 4. 7 Breast Cancer (OD04566) 15. 8 Ovarian Cancer 064008 7. 3 Breast Cancer 1024 22. 4 Ovarian cancer (OD06145) 0.4 Breast Cancer (OD04590-01) 47.6 Ovarian Margin (OD06145) 7.3 Breast Cancer Mets (OD04590-03) 41.2 Ovarian cancer (OD06455-03) 18.0 Breast Cancer Metastasis 100.0 (OD04655-05) Ovarian Margin (OD06455-07) 2. 4 Breast Cancer 064006 11. 1 Normal Lung 18.6 Breast Cancer 9100266 49.0 Invasive poor diff. lung adeno 10.0 Breast Margin 9100265 20.7 (ODO4945-01 Lung Margin (OD04945-03) 5.7 Breast Cancer A209073 18. 6 Lung Malignant Cancer (OD03126) 17. 6 Breast Margin A2090734 21.5 Lung Margin (OD03126) 3.9 Breast cancer (OD06083) 81.2 Breast cancer node metastasis Lung Cancer (OD05014A) 11.3 66.0 (OD06083) Lung Margin (OD05014B) 0.2 Normal Liver 2.4 Lung cancer (OD06081) 4.2 Liver Cancer 1026 4.4 Lung Margin (OD06081) 6. 3 Liver Cancer 1025 4. 6 Lung Cancer (OD04237-01) 4.6 Liver Cancer 6004-T 3.8 Lung Margin (OD04237-02) 9.1 Liver Tissue 6004-N 1.5 Ocular Melanoma Metastasis 0. 7 Liver Cancer 6005-T 12. 1 Ocular Melanoma Margin (Liver) 2. 8 Liver Tissue 6005-N 9. 6 Melanoma Metastasis 0. 3 Liver Cancer 064003 1. 5 Melanoma Margin (Lung) 9.2 Normal Bladder 19.6 Normal Kidney 2. 5 Bladder Cancer 102363 Kidney Ca, Nuclear grade 2 9.7 Bladder Cancer A302173 8.7 (OD04338) Kidney Margin (OD04338) 1. 7 Normal Stomach 62. 4 Kidney Ca Nuclear grade 1/2 4.2 Gastric Cancer 9060397 5.1 (OD04339) Kidney Margin (OD04339) 4.1 Stomach Margin 9060396 38.4 Kidney Ca. Clear cell type 2. 7 Gastric Cancer 9060395 21.5 (OD04340) Kidney Margin (OD04340) 6.7 Stomach Margin 9060394 43.5 Kidney Ca, Nuclear grade 3 0.6 Gastric Cancer 064005 11. 4 (OD04348) Table JE. Panel 4D Rel. Rel. Exp. (%) Exp. (%) Ag2000, Tissue Name Ag2000, Run Run 165822435 165822435 Secondary Thl act 0.2 HUVEC IL-1beta 4. 8 Secondary Th2 act 0.3 HUVEC IFN gamma14. 7 Secondary Trl act 0.6 HUVEC TNF alpha + IFN gamma 1.9 Secondary Thl rest 0.1 HUVEC TNF alpha + IL4 4. 0 Secondary Th2 rest 0.7 HUVEC IL-11 15. 6 Secondary Trl rest 0.3 Lung Microvascular EC none 14.4 Primary Thl act 0. 1 Lung Microvascular EC TNFalpha + 6. 3 IL-lbeta Primary Th2 act 0.2 Microvascular Dermal EC none 15.4 Microsvasular Dermal EC TNFalpha + Primary Tr1 act 0.1 5.1 IL-1beta Bronchial epithelium TNFalpha + IL1beta Primary Th2 rest 0. 2 Small airway epithelium none 0. 8 Primary Trl rest 0. 1 Small airway epithelium TNFalpha + 3.4 IL-1beta CD45RA CD4 lymphocyte act 0.3 Coronery artery SMC rest 0.1 Coronery artery SMC TNFalpha + CD45RO CD4 lymphocyte act 0.7 0.3 IL-1beta CD8 lymphocyte act 0.8 Astrocytes rest 3.6 Secondary CD8 lymphocyte rest 0.9 Astrocytes TNFalpha + IL-1beta 6.9 Secondary CD8 lymphocyte act 0.0 KU-812 (Basophil) rest 0.0 CD4 lymphocyte none 2.7 KU-812 (Basophil) PMA/ionomycin 0.0 2ry Th1/Th2/Tr1_anti-Cd95 0.0 CCD1106 (Keratinocytes) none 0.3 CH11 CCD1106 (Keratinocytes) TNFalpha + LAK cells rest 1.4 1.5 J-beta LAK cells IL-2 2.6 Liver cirrhosis 11.2 LAK cells IL-2+IL-12 1.4 Lupus kidney 9.2 LAK cells IL-2+IFN gamma 1.2 NCI-H292 none 20.2 LAK cells IL-2+ IL-18 1.6 NCI-H292 IL-4 17.4 LAK cells PMA/ionomycin 0. 3 NCI-H292 IL-9 21. 6 NK Cells IL-2 rest 0.4NCI-H292 IL-13 9.5 Two Way MLR 3 day 1. 2 NCI-H292 IFN gamma 10. 3 Two Way MLR 5 day 0. 4 HPAEC none 13. 7 Two Way MLR 7 day 0. 0 HPAEC TNF alpha + IL-1 beta 9.2 PBMC rest 0. 8 Lung fibroblast none 0. 2 PBMC PWM 0.2 Lune fibroblast TNF alpha + IL-1 beta 0.8 PBMC PHA-L 0. 3 LungfibroblastIL-4 0.1 Ramos (B cell) none 0.5 Lung fibroblast IL-9 0. 2 Ramos (B cell) ionomycin 0.7 Lung fibroblast IL-13 0.2 B lymphocytes PWM 0.8 Lung fibroblast IFN gamma 0.3 B lymphocytes CD40L and IL-4 5. 8 Dermal fibroblast CCD1070 rest 0.1 EOL-1 dbcAMP 0.0 Dermal fibroblast CCD1070 TNF 0.0 alpha EOL-1 dbcAMP 0.0 0.0 alpha EOL-1 dbcAMP PMA/ionomycin 0.0 Dermal fibroblast CCD1070 IL-1 beta 0.1 Dendritic cells none 0.2 Dermal fibroblast IFN gamma 0.1 Dendritic cells LPS 0. 0 Dermal fibroblast IL-4 0.1 Dendritic cells anti-CD40 0.0 IBD Colitis 2 2.9 Monocytes rest 0 IBD Crohn's 9.2 Monocytes LPS 0 Colon 100.0 Macrophagesrest Od Lung 19.3 Macrophages LPS 0.0 Thymus 11.1 HUVEC none 7. 3 Kidney 6. 4 HUVEC starved 17.7

CNS_neurodegeneration_v1.0 Summary: Ag6688 Expression of this gene is limited to a single sample from the parietal cortex (CT=34).

Generalscreeningjpanel vl. 6 Summary: Ag6688 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

Panel 1.3D Summary: Ag2000 Highest expression of this gene, a homolog of a transmembrane multi-pass protein, is seen in the cerebral cortex (CT=26.8), with moderate

expression detectable across all regions of the brain. Because this gene shows a large down-regulation in brain cancers, its absence would be an excellent marker to determine if brain tissue was pre-cancerous in the examining and classifying of postmortem tissue Expression of this gene is also widespread among tissues with metabolic relevance, including adipose, pancreas, adult and fetal heart, adult and fetal liver, adult and fetal skeletal muscle, and the adrenal, pituitary, and thyroid glands. The gene is expressed at . much higher levels in fetal heart and skeletal muscle (CTs=28) than in adult heart and skeletal muscle (CTs=31-34). This differential expression pattern suggests that this gene expression could be used to differentiate between the two tissue sources for heart and skeletal muscle. Furthermore, the significantly higher level of expression of the gene in fetal skeletal muscle suggestes that this gene product may be involved in muscular growth or development in the fetus and could potentially act in a regenerative capacity in an adult.

Therefore, therapeutic modulation of this gene could be useful in the treatment of muscle related diseases and the treatment of week or dystrophic muscle.

This gene is also expressed at significant levels in cell lines derived from ovarian, breast, lung, gastric, prostate and colon cancers compared to the normal tissues. Thus, the expression of this gene could be of use as a marker or as a therapeutic for ovarian, breast, lung, gastric, prostate and colon. In addition, therapeutic modulation of the product of this gene, through the use of peptides, chimeric molecules or small molecule drugs, may be useful in the treatment of these cancers.

Panel 2.2 Summary: Ag2000 Highest expression of this gene is seen in breast cancer (CT=28) as is seen in Panel 1.3D. In addition, there is significant overexpression of this gene in a cluster of breast, lung, and ovarian cancer samples when compared to corresponding normal tissues. Thus, expression of this gene could be used to differentiate breast, ovarian and lung cancers from normal tissue and as a marker for the presence of these cancers. Furthermore, therapeutic modulation of the protein product of this gene could be beneficial in the treatment of breast, ovarian and lung cancers. The expression of this gene also shows a reverse association with some normal stomach samples when compared to the matched gastric cancer tissue. This suggests that the this gene could be used to distinguish between normal and cancerous gastric tissue and that therapeutic modulation of the gene product may be useful in the treatment of gastric cancer.

Panel 4. 1D Summary: Ag6688 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

Panel 4D Summary: Ag2000 The highest expression of this gene is found in the colon (CT=26.2), with modest expression detectable in the muco-epidremoid cell line H292, and the lung. It is also expressed at moderate levels on HUVEC and lung microvasculature regardless of their activation status. The protein encoded by this gene is homologous to an epidermal growth factor related protein (fibropellin like) and could be used as a marker of lung muco-epidermoid cells, colon or vasculature. The putative protein encoded by the transcript may also play an important role in the normal homeostasis of these tissues. Small molecule or antibody therapeutics designed with this gene product could be important for maintaining or restoring normal function to these organs during inflammation associated with asthma and emphysema.

K. CG58495-03 (NOV 17b): Pulmonary surgactant-associated protein A precursor.

Expression of gene CG58495-03 was assessed using the primer-probe set Ag7945, described in Table KA.

Table KA. Probe Name Ag7945 c. T Start SEQ Primers Sequeces Length start SEQ Position ID No Forward S'-gcgtgcgaagtgaagga-3'. 17 135 159 Probe TET-5'-ctccaagccacactccacgacttcag-3'-TAMRA 26 153 160 Reverse 5'-ctgagggctccccttgtc-3'18194 161

CNS neurodegeneration v1. 0 Summary: Ag7945 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

Panel 4. 1D Summary: Ag7945 Expression of this gene is low/undetectable (CTs > 35) across all of the samples on this panel.

L. CG97482-02 (NOV18b) : S100 Calcium-Binding Protein-like.

Expression of gene CG97482-02 was assessed using the primer-probe set Ag6384, described in Table LA. Results of the RTQ-PCR runs are shown in Table LB. Please note that CG97482-02 represents a full length physical clone.

Table LA. Probe Name Ag6384 Start SEQ Primers Sequnces Length position S Forward 5'-tggccctcatcgacgt-3'16 44 162 Probe TET-5'-agctcatcaacaatgagctttcccatt-3'-TAMRA 27 122 163 Reverse 5'-gcagtagtaaccacaacctcct-3'22 170 164 Table LB. CNS neurodegeneration vl. O Rel. Rel. Exp. (%) Exp. (%) Tissue Name Ag6384, Tissue Name Ag6384, Run Run 269253944 269253944 AD 1 Hippo 28.3 Control (Path) 3 Temporal Ctx 5.1 AD 2 Hippo 92. 7 Control (Path) 4 Temporal Ctx 25. 9 AD 3 Hippo 4. 2 AD 1 Occipital Ctx 19.5 AD 4 Hippo 17. 7 AD 2 Occipital Ctx (Missing) 0.0 AD 5. Hippo 36. 1 AD 3 Occipital Ctx 7. 7 AD 6 Hippo 100.0 AD 4 Occipital Ctx 37. 1 Control 2 Hippo 82.9 AD 5 Occipital Ctx 68. 3 Control 4 Hippo 65. 1 AD 6 Occipital Ctx 39. 2 Control (Path) 3 Hippo 13. 7 Control 1 Occipital Ctx 9. 7 AD 1 Temporal Ctx 22. 4 Control 2 Occipital Ctx 61. 6 AD 2 Temporal Ctx 74. 7 Control 3 Occipital Ctx 35. 8 AD 3 Temporal Ctx 4. 7 Control 4 Occipital Ctx 47. 3 AD 4 Temporal Ctx 42.0 Control (Path) 1 Occipital Ctx 97.3 AD 5 Inf Temporal Ctx 68. 3 Control (Path) 2 Occipital Ctx 25.5 AD 5 Sup Temporal Ctx 75. 8 Control (Path) 3 Occipital Ctx 10.4 AD 6 Inf Temporal Ctx 57. 0 Control (Path) 4 Occipital Ctx 12. 1 AD 6 Sup Temporal Ctx 45. 4 Control 1 Parietal Ctx 26.2 Control 1 Temporal Ctx 21.8 Control 2 Parietal Ctx 46.3 Control 2 Temporal Ctx 56. 3 Control 3 Parietal Ctx 51. 1 Control 3 Temporal Ctx 35.4 Control (Path) 1 Parietal Ctx 58.2 Control 3 Temporal Ctx 30.6 Control (Path) 2 Parietal Ctx 59.9 Control (Path) 1 Temporal Ctx 42.0 Control (Path) 3 Parietal Ctx 6. 2

Control (Path) 2 Temporal Ctx 39.8 Control (Path) 4 Parietal Ctx 37.9 CNS neurodegeneration v1. 0 Summary: Ag6384 No differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment. However, this panel confirms the expression of this gene at low levels in the brains of an independent group of individuals. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

Panel 4. 1D Summary: Ag6384 Expression of this gene is low/undetectable (CTs > 35) across all of the 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 polymorphism (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 polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic 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 of the amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result of the redundancy of the 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 of the 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 of the 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, IMMER, 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 of the 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 Corporation's human SeqCalling database. SeqCalling fragments suitable for inclusion were identified by the CuraTools program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.

The regions defined by the procedures described above were then manually integrated and corrected for apparent inconsistencies that may have arisen, for example, from miscalled bases in the original fragments or from discrepancies between predicted exon junctions, EST locations and regions of sequence similarity, to derive the final sequence disclosed herein. When necessary, the process to identify and analyze SeqCalling assemblies and genomic clones was reiterated to derive the full length sequence (Alderborn et al., Determination of Single Nucleotide Polymorphisms 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 of the invention.

NOVlb SNP Data (CG108030-02).

Seven polymorphic variants of NOVlb have been identified and are shown in Table 19A.

Table 19A. Variant of NOVlb. Variant Nucleotides Amino Acids Variant Position-Initial Modified Position Initial Modified 13381876 354'T C 116 Leu Pro 13381877 627 T C 207 Leu Pro 13381845 2249 A G 0 N/A N/A 13381844 2454 C T 0 N/A N/A 13381881 2949 T C 0 N/A N/A 13381882 2959 A G 0 N/A N/A 13381883 3124 A G 0 N/A N/A

NOV2d SNP Data (CG115907-02).

Four polymorphic variants of NOV2d have been identified and are shown in Table 19B.

Table 19B. Variant of NOV2d.

Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13381868 204 T C 25 Thr Thr 13381869 1892 T A 588 Leu His 13381842 2131 C A 668 Pro Thr 13381871 2544 G A 805 Leu Leu

NOV6a SNP Data (CG155653-01).

Three polymorphic variants of NOV6a have been identified and are shown in Table 19C.

Table 19C. Variant of NOV6a. Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13381864 301 G A 42 Gly Ser 13381889 1260 G T 361 Arg Ser 13381867 4013 G A 0 N/A N/A

NOV7a SNP Data (CG160093-01).

Three polymorphic variants of NOV7a have been identified and are shown in Table 19D.

Table 19D. Variant of NOV7a.

. Variant Nucleotides Amino Acids Position Initial Modified Position Initial Modified 13381888 966 A G 315 Glu Gly 13381887 980 A G 320 Thr Ala 13381886 1008 T C 329 Leu Ser

NOV9a SNP Data (CG165528-01).

Four polymorphic variants of NOV9a have been identified and are shown in Table 19E.

Table 19E. Variant of NOV9a. Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13381837 78 C T 0 N/A N/A 13381838 4640 T C 1514 Val Ala 13381839 4754 A G 0 N/A N/A 13381840 4936 A G 0 N/A N/A NOV12d SNP Data (CG165719-01).

Four polymorphic variants of NOV12d have been identified and are shown in Table 19F.

Table 19F. Variant of NOV12d.

Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13381873 291 C A 77 Ser Ser 13381875 475 T C 139 Phe Leu 13381874 559 G A 167 Ala Thr 13381884 631 T C 191 Phe Leu

NOV17b SNP Data (CG58495-03).

Three polymorphic variants of NOV17b have been identified and are shown in Table 19G.

Table 19G. Variant of NOV17b. Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13376633 151 A G 24 Glu Gly 13381911 386 T C 102 Tyr Tyr 13376634 501 A C 141 Lys Gln

NOV18b SNP Data (CG97482-02).

One polymorphic variant of NOV1 8b has been identified and is shown in Table 19H.

Table 19H. Variant of NOV18b. Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13376808 176 T C 53 Val Ala

Example E. CG50970-01, NOV15b.

Role in inflammation: This transcript encodes glypican 2 a glycosylphosphatidylinositol (gpi) achored cell surface heparan sulfate proteoglycan. This type of proteoglycan can bind cytokines and is potentially involved in lymphocytic migration and activation (1). Additionally, this molecule is also found in bone marrow and cartilage (2-3) and may be involved in osteoblast function and hematopoiesis.

Therapeutic function: Antibody therapeutics which antagonized the function of the protein encoded for by this transcript could be used to reduce or inhibit lymphocyte extravasation associated with inflammation due to asthma, emphysema, rheumatoid arthritis, IBD or psoriasis. Antibodies may also block tissue changes associated with osteoarthritis (4).

Example E1 : Gene Expression analysis using CuraChip in human tissues from tumors and from equivalent normal tissues CuraGen has developed a gene microarray (CuraChip 1.2) for target identification.

It provides a high-throughput means of global mRNA expression analyses of CuraGen's collection of cDNA sequences representing the Pharmaceutically Tractable Genome (PTG).

This sequence set includes genes which can be developed into protein therapeutics, or used to develop antibody or small molecule therapeutics. CuraChip 1, 2 contains-11, 000 oligos representing approximately 8,500 gene loci, including (but not restricted to) kinases, ion channels, G-protein coupled receptors (GPCRs), nuclear hormone receptors, proteases, transporters, metabolic enzymes, hormones, growth factors, chemokines, cytokines, complement and coagulation factors, and cell surface receptors.

The CuraChip cDNAs were represented as 30-mer oligodeoxyribonucleotides (oligos) on a glass microchip. Hybridization methods using the longer CuraChip oligos are more specific compared to methods using 25-mer oligos. CuraChip oligos were synthesized with a linker, purified to remove truncated oligos (which can influence hybridization strength and specificity), and spotted on a glass slide. Oligo-dT primers were used to generate cRNA probes for hybridization from samples of interest. A biotin-avidin conjugation system was used to detect hybridized probes with a fluorophore-labeled secondary antibody. Gene expression was analyzed using clustering and correlation bioinformatics tools such as Spotfire@. (Spotfire, Inc.,, 212 Elm Street,. Somerville, MA 02144) and statistical tools such as multivariate analysis (MVA).

Results of PTG Chip 1.2 : One hundred seventy-eight samples of RNA from tissues obtained from surgically dissected tumors, non-diseased tissues from the corresponding organs and tumor xenografts grown in nude nu/nu mices were used to generate probes and run on PTG Chip 1, 2. An oligo (optg20011299) that corresponds to CG50970 on the PTG Chip 1.2 was scrutinized for its expression profile. The statistical analysis identify significant over-expression in a subset of lung tumors, about 30%, compared with corresponding normal lung tissue and strong expression in breast cancers, also about 30%, which do not have matched normal tissue. It is also useful that the expression of this gene is mantained when human tumor cell lines are grown as tumor xenografts in nude mice, especially by the lung tumor cell lines NCI-H82 and NCI-H69.

Therfore these tumor xenografts can be used as animal models.

Thus, based upon its profile, the expression of this gene could be of use as a marker for subsets of lung and breast cancers. In addition, therapeutic inhibition of the activity of the product of this gene, through the use of antibodies or small molecule drugs, may be useful in the therapy of lung and breast cancers that express CG50970.

Table E1a: CG50970 expression in CuraChip Oncology samples

Example E2. Protein expression and purification CG50970-05 is expressed and purified in the CHO stable cell system using the Wave bioreactor.

To separate the glycanated form of the proteoglycan from the unglycanatedcore protein,. the conditioned medium was applied to. a 0. 9 x 8-cm column of DEAE-Sephacel equilibrated with 150 mM NaCl, 50 mM Tris-HCl, pH 8.0. After elution with 50 mM Tris-HCl (pH 8.0) containing 0.6 M NaC, the glycanated glypican-1-Fc was bound to protein A-Sepharose beads and eluted with 0.1 M glycine, pH 3.0.

Procedure 1. Transfected into attached CHO stable cells with Lipofectamine 2000 in Opti-MEM 1.

Overlay with DMEM media with 5% FBS after 4 hours.

2. Harvested after 3,5 and 7 days incubation at 37°C.

Cell Lysis/Protein Recovery Procedure 1. Centrifuged at 3000 rpm for 10 min and filter with 0.2 um pore size.

Procedure 1. Metal Affinity Chromatography-Pharmacia 50ml and 5 ml Metal Chelate-Running buffer 20 mM phosphate, pH 7.4, 0.5 MNaCl. Wash with 20mM, 50mM, and lOOmM Imidazole. Elute with 500mM Imidazole.

2. HS Cation Exchange Chromatography-Poros HS 1.6 ml column-30 mM Tris-CI, pH 8.0, 0.05% CHAPS. Elute with 0-2 M NaCl gradient.

3. Dialysis-@ 4C using 3,500 MWCO against 20mM Tris-HCl, pH7.4 + 150mM NaCl.

PROTEIN QUALITY CONTROL Western Blot Procedure Antibody name, catalog # and supplier: Anti-VS-HRP Antibody, 46-0708, Invitrogen (Carlsbad.

CA), * S-protein HRP coniutate, 69047. Novazen (Madison, WI) Antibody dilution buffer: PBS/5% milk/0. 1% Tween-20 Wash buffer : PBS/0. 1% Tween-20 Detection reagents: ECL (Amersham Biosciences Corp. , Piscataway, NJ) 1. The blot was covered with antibody dilution buffer and incubated on a rocker for one hour at room temperature.

2. The blocking solution was replaced with antibody dilution buffer containing the appropriate amount of conjugate, and the blot was incubated on a rocking platform for one hour at room temperature.

3. The antibody solution was decanted, and the blot was washed quickly with two quick rinses of wash buffer. The blot was then covered with wash buffer and incubated on the rocking platform for five minutes, and the wash buffer was decanted. This process was repeated twice for a total of three five-minute washes.

4. The blot was developed using ECL reagents (Amersham Biosciences Corp., Piscataway, NJ) as per manufacturer instructons and luminescence was then digitized on a Kodak Image Sciences Imaging Station.

Expression of CG50970-05 in stable CHO-K1 cells.

A 1590 bp long BamHI-XhoI fragment containing the CG50970-05 sequence was subcloned into BamHI-XhoI digested pEE14.4Sec2 and pEE14.4SecFc3. The resulting plasmids are transfected into CHO-K1 cells using the LipofectaminePlus reagent following the manufacturer's instructions (Invitrogen/Gibco)... The cell pellet and supernatant are harvested 72h post transfection and examined for CG50970-05 expression by Western blot (reducing conditions) using an anti-V5 antibody.

MSX resistant clones are selected using the GS system (Lonza Biologicals) The culture media in the selection process was: Glutamin-free DMEM (JRH), 10% dialyzed FBS, lx GS supplement (JRH), 25uM MSX (JRH).

A high expressor clone, is selected for scale up in 10 LWave bioreactors. Two reactors were inoculated. 30 L conditioned media was collected from each reactors yielding batches 2 and 3.

The culture media was harvested 120h after inoculating the Wave bioreactor and examined for CG50970-05 expression by Western blot (reducing conditions) using an anti-V5 antibody

Example E3. Growth factor mediated proliferation assays Several growth factors require the presence of heparan sulfate for high affinity binding to their tyrosine kinase receptors and therefore use HSPG's as coreceptors. in their. signaling. We determine whether it is possible to modulate responsiveness to heparin-binding growth factors by altering CG50970 protein levels, either-increasing them or decreasing them. Kleeff et al (J. Clin. Invest. Volume 102, Number 9, November 1998,, 1662-1673) and Matsuda et al (Cancer Research 61,5562-5569, July 15,2001) used this approach to demonstrate the activity of Glypican-1.

Tumor cell lines with low level of CG50970 are transiently transfected with mature forms of CG50970, variants 06 and 07. The increase in expression of CG50970 is then monitored by western blot analysis. Next, the effects of growth factors on cell growth are determined during the 48-96 h interval after transfection, when CG50970 protein levels are maximally increased. Cells are treated with several growth factors like FGF2, HB-EGF.

Cells expressing CG50970 will have a higher rate of proliferation in response to the growth factors than control cells.

. Tumor cell lines with high level of CG50970 are transiently transfected with antisense oligos directed against CG50970. The decrease in expression of CG50970 is then monitored by PCR-based methods. Next, the effects of growth factors on cell growth are determined during the 48-96 h interval after transfection, when CG50970 protein levels are maximally decrease. Cells are treated with Fetal Bovine Serum or individual growth factors like FGF2, HB-EGF. As shown in table E3a below, cells treated with CG50970 antisense 1 and stimulated with with Fetal Bovine Serum have a lower rate of proliferation in response to the growth factors than control cells.

Table E3a: Proliferation assay on NCI-H146 lung cancer cells treated with antisense oligos

Sequences of the antisense oligos, relative position and length that correspond to Table E3a.

Example E4: Preparation of Antibodies that Bind CG50970 As described above, inhibiting CG50970 activity has utility in cancer therapy and specifically in inhibiting lung and breast cancers. It is know in the art that antibodies that bind HSPGs factors like CG50970 can inhibit their activity in a process called neutralization. Specifically, neutralizing monoclonal antibodies that bind syndecan-3 interfered with FGF-2 mitogenic action, but not that of insulin-like growth factor-1 or parathyroid hormone (Kirsch et al. J Biol Chem2002 Nov 1; 277 (44): 42171-7). Therefore production of polyclonal and monoclonal antibodies directed against CG57094 has utility in cancer therapy and specifically in inhibiting kidney, lung, melanomas and breast cancers.

As opposed to VEGF, that is needed only for tumor induced endothelial cell growth and

survival, CG57094 is required for cell growth and survival both by endothelial and tumor cells, therefore inhibition of CG57094 activity could have a more pronounced therapeutic effect.

Method: Techniques for producing the antibodies are known in the art and are described, for example, in"Antibodies, a Laboratory Manual". Eds Harlow and Lane, Cold Spring Harbor publisher. Both rabbits and mice are suitable for the production of polyclonal antibodies, while mice are also suitable for the production of monoclonal antibodies. Mice where the human immunoglubolin genes have replaced the mouse immunoglubolin genes can be used to produce fully human monoclonal antibodies. These antibodies have better pharmaceutical characteristic, no or minimal antibody directed immune reactions that results in loss of therapeutic efficacy and have been shown to eradicate tumor in animal model (Yang XD, Jia XC, Corvalan JR, Wang P, Davis CG, Jakobovits A Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Res 1999 Mar 15 ; 59 (6): 1236-43).

Generation of rabbit polyclonal antibodies Rabbit are immunized with the immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally or intramuscolar in an amount from 50-1000 micrograms. The immunized rabbits are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the rabbits. might also be boosted with additional immunization injections. Serum samples may be periodically obtained from the rabbit by bleeding of the ear for testing ELISA assays to detect the antibodies.

Generation of human monoclonal antibodies Fully human monoclonal antibodies (MAb), direct against CG50970-05 are generated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and kappa light chain loci as previously described in Yang et al. (Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy.

Cancer Res 1999 Mar 15; 59 (6): 1236-43).

Elisa assay is then used to determine the specificity of the antibodies.

Example E5 : ELISA Protocol todetermine binding of the antibodies Solution Preparation # Coating Buffer (O. 1M Carbonate, pH9.5) 8. 4 g. NaHC03,3. 56g. Na2C03, pH to 9.5, and dilute to 1 L. with ddH20 ASSAY DILUENT . Pharmin, gen &num 26411E PROTOCOL # Coat a 96-well high protein binding ELISA plate (Corning Costar #3590) with 50 ul. of protein at a concentration of 5ug/mL. in coating buffer overnight at 4 degrees.

Following day wash the cells 5X 200-300 ul. of 0.5% Tween-20 in PBS.

Block plates with 200ul. of assay diluent for at least 1 hour at room temperature.

Dilute antibodies in assay diluent.

Wash plate as in step 2.

# Add 50ul. of each antibody dilution to the proper wells for at least 2 hours at room temp.

Wash plate as in step 2.

# Add 50ul. of secondary antibody and incubate for 1 hour at room temp.

Wash plate as in step 2.

# Develop assay with l00ul. of TMB substrate solution/well. (1: 1 ratio of solution A+B) (Pharmingen #2642KK) Stop reaction with 50ul. sulfuric acid Read plate at 450nm with a correction of 550nm.

Example 6: Identification of CG50970 neutralizing antibodies As mentioned above, proteoglycans like CG50970 have affinity for glycosaminoglycan (GAG) -binding proteins like laminin-1 and midkine. Specifically, Herndon et al (Glycobiology 1999 Feb; 9 (2): 143-55) have previously shown that rat glypican-2 has an high affinity for laminin-1, while Kurosawa et al. (Glycoconj J 2001 Jun; l 8 (6): 499-507) have shown that rat glypican-2 has an high affinity for midkine.

As previously discussed, the identification of antibodies, preferably fully human monoclonal antibodies that bind to CG50970 and neutralize its activity, limiting or abolishing its ability to bind to glycosaminoglycan (GAG)-binding proteins like laminin-1 and midkine, would be very beneficial because these antibodies will have therapeutic effect against tumors, specifically against lung and breast cancers. To determine whether an antibody can neutralize CG50970 activity, various amounts of such antibody are added to the Receptor-ligand Elisa assay as described in the method below.

Receptor-ligand Elisa assay Protocol--96-well plates (Corning Costar, catalog no. 9018) are coated overnight with the laminin-1 (BT-276 from BTI website at btiinc. com/page/catal. html&num Laminin) or midkine (258-MD from R&D system) at a saturating concentration of in phosphate-buffered saline. After removing the unbound protein by washing with TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), the wells were blocked with 10% fetal bovine serum in TBST for 2 h and then incubated for 18 h at room temperature with varying concentrations of glycanated CG50970-05-Fc in phosphate-buffered saline in the presence or absence of various amounts of monoclonal antibodies that bind to CG50970. The CG50970-05-Fc bound to laminin-1 proteins was detected using a biotinylated anti-human Fc antibody (Jackson ImmunoResearch Laboratories, Inc. ; 1: 250,000 in TBST, for 2 h) followed by incubation for 20 min with horseradish peroxidase-conjugated streptavidin (1: 20,000 in TBST). The colorimetric reaction product from the o-phenylenediamine substrate was measured at 450 nm using a Dynatech MRX ELISA plate reader. Nonspecific binding was calculated as the binding of glypican-2-Fc to wells coated with 100 pg of bovine serum albumin.

Antibodies identified with this assay are then tested at various concentrations in the growth factor mediated proliferation assay described in example 4 to determine whether they can inhibit cellular proliferation.

Antibody that can neutralize the CG50970-05-Fc biochemical activity and have anti-proliferative activity can be useful as therapeutic agents.

Example 7: Quantification of membrane bound CG50970 by Flow Cytometry CG50970 is a type 1 membrane protein, therefore Mabs binding to this protein could be able to stain the membrane of cells expressing CG50970 in a Flow Cytometry

assay (FACS). It is known in the art that not all antibodies that recognize a recombinant protein in Elisa or IHC assays will also work in FACS. At the same time those antibodies that do are preferred because they have a higher chance to recognize the antigen in-vivo in patients and therefore have a potential use as therapeutic or ex-vivo diagnostic agents. We therefore set-up a FACS assay using cell lines that express CG50970, like lung cancer NCI-H146, NCI-H526 or breast cancer BT 549 and one that express it at much lower level, lung ca. ncer HOP-62 and breast cancer T47D.

Flow Cytometry Protocol for Adherent Cells (ver. l) 11-25-02 KT 1. Wash cells with lx PBS (Ca and Mg free) twice.

2. Add Versene and incubate at 37°C until the cells detach.

3. Count cells. Use <1 million cells per assay tube.

4. Wash the cells twice with ice-cold FACS buffer.

5. Resuspend cells in 100 ul of ice-cold FACS buffer. Mix.

6. Add primary mAb. Incubate on ice for 30 min.

7. Wash cells 2-3 times with 1 ml of ice-cold FACS buffer.

8. Resuspend cells in 100 ul ice-cold FACS buffer. Mix.

9. Add secondary (conjugated) mAb. Incubate on ice for 30 min with a cover.

10. Wash cells 2-3 times with 1 ml of ice-cold FACS buffer.

11. Fix cells with 0.5-1 ml of 1 % formaldehyde (in PBS) and analyze by Flow Cytometry.

FACS buffer: 0. 01 M HEPES (pH 7. 4) 0.15 M NaCl --------------------- (may be substituted by PBS) 0.1% NaN3 4% FBS Example 8: Preparing and testing of chemotherapy and radioimmunoconjugated antibodies Cytotoxic chemotherapy or radiotherapy of cancer is limited by serious, sometimes life-threatening, side effects that arise from toxicities to sensitive normal cells because the therapies are not selective for malignant cells. There therefore the need to improve the

selectivity. One strategy is to couple the therapeutics to antibodies that recognize tumour-associated antigens. This increases the exposure of the malignant cells, and reduces the exposure of normal cells, to the ligand-targeted therapeutics (reviewed in Allen Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer 2002 Oct; 2 (10): 750-63) CG56972-03 is one of these tumour-associated antigen, as shown by its specific expression on cellular membranes of tumor cells by FACS and IHC.

Therefore the fully human monoclonal antibodies direct against CG50970-05 disclosed in this application could be coupled to cytotoxic chemotherapic agents or radiotherapic agents to generate anti-tumor therapeutics.

Depending on the intended use of the antibody, i. e. , as a diagnostic or therapeutic reagent, radiolabels are known in the art and have been used for similar purposes. For instance, radionuclides which have been used in clinical diagnosis include. sup. 131 I, . sup. 125 I,. sup. 123 I,. sup. 99 Tc,. sup. 67 Ga, as well as. sup. lll In. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. (1987) The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer. Immunol. Cell Biol65 : 111-125). These radionuclides include. sup. 188 Re and. sup. 186 Re as well as. sup. 90 Y, and to a lesser extent. sup. 199 Au and. sup. 67 Cu.

I- (131) has also been used for therapeutic purposes. U. S. Pat. No. 5,460, 785 provides a listing of such radioisotopes and is herein incorporated by reference.

Patents relating to radiotherapeutic chelators and chelator conjugates are known in the art. For instance, U. S. Pat. No. 4,831, 175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation. U. S. Pat. Nos. 5,099, 069,5, 246,692, 5,286, 850, and 5,124, 471 of Gansow also relate to polysubstituted DTPA chelates These patents are incorporated herein in their entirety.

Cytotoxic chemotherapy are known in the art and have been used for similar purposes. For instance. U. S. Pat. No 6,441, 163 describes the process for the production of cytotoxic conjugates of maytansinoids and antibodies. The anti-tumro activity of a new tubulin polymerization inhibitor, auristatin PE, is know in the art (Mohammad et al. Int J Oncol 1999 Aug ; 15 (2): 367-72).

Once these conjugates of chemotherapy or radiolabels and antibodies is made, it is tested for its cytotoxic activity on CG50970-05 positive cells, using methods know in the art like by MTS, Cell counts and clonogenic assays.

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