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

Abstract

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

Description

FIELD OF THE INVENTION

[0002] The present invention relates to novel polypeptides, and the nucleic acids encoding them, having properties related to stimulation of biochemical or physiological responses in a cell, a tissue, an organ or an organism. More particularly, the novel polypeptides are gene products of novel genes, or are specified biologically active fragments or derivatives thereof. Methods of use encompass diagnostic and prognostic assay procedures as well as methods of treating diverse pathological conditions.

BACKGROUND OF THE INVENTION

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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

[0009] 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 37. The novel nucleic acids and polypeptides are referred to herein as NOV1a, NOV1b, NOV2a, NOV2b, etc. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as “NOVX” nucleic acid or polypeptide sequences.

[0010] 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 37, 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 37. In another embodiment, the invention also comprises variants of the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed. The invention also involves fragments of any of the mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% of the residues are changed.

[0011] 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 37. These allelic variants include amino acid sequences that are the translations of nucleic acid sequences differing by a single nucleotide from nucleic acid sequences selected from the group consisting of SEQ ID NOS: 2n−1, wherein n is an integer between 1 and 37. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution.

[0012] In another embodiment, the invention comprises a pharmaceutical composition involving a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 and a pharmaceutically acceptable carrier. In another embodiment, the invention involves a kit, including, in one or more containers, this pharmaceutical composition.

[0013] In another embodiment, the invention includes the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease being selected from a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 wherein said therapeutic is the polypeptide selected from this group.

[0014] In another embodiment, the invention comprises a method for determining the presence or amount of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 in a sample, the method involving providing the sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, thereby determining the presence or amount of polypeptide in the sample.

[0015] In another embodiment, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 in a first mammalian subject, the method involving measuring the level of expression 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.

[0016] In another embodiment, the invention involves a method of identifying an agent that binds to a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, the method including introducing the polypeptide to the agent; and determining whether the agent binds to the polypeptide. The agent could be a cellular receptor or a downstream effector.

[0017] In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, the method including providing a cell expressing the polypeptide 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.

[0018] In another embodiment, the invention involves a method for screening for a modulator of activity or of latency or predisposition to a pathology associated with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, the method including administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide 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.

[0019] In another embodiment, the invention involves a method for modulating the activity of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, the method including introducing a cell sample expressing the polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide.

[0020] In another embodiment, the invention involves a method of treating or preventing a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37, the method including administering the polypeptide to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject. The subject could be human.

[0021] In another embodiment, the invention involves a method of treating a pathological state in a mammal, the method including administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 or a biologically active fragment thereof.

[0022] 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 37; 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 37 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 37; 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 37, 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 37 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.

[0023] 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 37, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.

[0024] 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 37 that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.

[0025] 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 37, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2n−1, wherein n is an integer between 1 and 37.

[0026] 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 37, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37; a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37 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 37; 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 37 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.

[0027] 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 37, 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 37, or a complement of the nucleotide sequence.

[0028] 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 37, wherein the nucleic acid molecule has a nucleotide sequence in which any nucleotide specified in the coding sequence of the chosen nucleotide sequence is changed from that selected from the group consisting 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.

[0029] 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 37. This vector can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.

[0030] 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 37 in a sample, the method including providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount 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.

[0031] 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 37 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.

[0032] 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 1×10−9 M. More preferably, the NOVX antibody neutralizes the activity of the NOVX polypeptide.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIG. 1. Inhibition of OVCAR-5 cell proliferation by antisense knockdown of CG52414-01

DETAILED DESCRIPTION OF THE INVENTION

[0038] 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
Assignment	Identification	acid)	acid)	Homology
NOV1a	CG126472-01	1	2	TEM7, Tumor endothelial marker 7
				precursor (Tumor endothelial
				marker 3 precursor) - Homo sapiens
NOV1b	CG126472-02	3	4	TEM7, Tumor endothelial marker 7
				precursor (Tumor endothelial
				marker 3 precursor) - Homo sapiens
NOV2a	CG138751-02	5	6	Human transporter protein - Homo
				sapiens
NOV2b	CG138751-01	7	8	Human transporter protein - Homo
				sapiens
NOV3a	CG170490-01	9	10	Ubiquitin ligase E3 alpha-I - Homo
				sapiens
NOV4a	CG170667-01	11	12	Neuronal cell adhesion molecule
				homolog - Homo sapiens
NOV4b	CG170667-02	13	14	Neuronal cell adhesion molecule
				homolog - Homo sapiens
NOV5a	CG170791-01	15	16	Acetyl-coenzyme A transporter
				(Similar to acetyl-coenzyme A
				transporter) - Homo sapiens
NOV6a	CG171174-01	17	18	Plasma Membrane Protein - Homo
				sapiens
NOV7a	CG172318-01	19	20	Secreted protein homolog - Homo
				sapiens
NOV8a	CG172921-01	21	22	Interleukin-5 receptor precursor -
				Homo sapiens
NOV9a	CG173919-01	23	24	Pyrin Domain containing protein -
				Homo sapiens
NOV10a	CG174858-02	25	26	Homolog of secreted protein -
				Homo sapiens
NOV10b	CG174858-01	27	28	Homolog of secreted protein -
				Homo sapiens
NOV11a	CG176203-01	29	30	membrane protein -Homo sapiens
				(similar to hypothetical protein
				FLJ32731 - Mus musculus)
NOV12a	CG176213-01	31	32	Cochlin precursor (COCH-5B2) -
				Homo sapiens
NOV13a	CG50691-05	33	34	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog - Homo sapiens
NOV13b	CG50691-04	35	36	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog- Homo sapiens
NOV13c	CG50691-02	37	38	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog - Homo sapiens
NOV13d	CG50691-03	39	40	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog- Homo sapiens
NOV13e	308482339	41	42	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog- Homo sapiens
NOV13f	CG50691-01	43	44	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog- Homo sapiens
NOV13g	CG50691-06	45	46	Cysteine-rich repeat-containing
				protein S52 precursor (CRIM1
				protein) homolog- Homo sapiens
NOV14a	CG51905-03	47	48	Novel protein
NOV14b	CG51905-01	49	50	Novel protein
NOV14c	278699747	51	52	Novel protein
NOV14d	310912740	53	54	Novel protein
NOV14e	CG51905-02	55	56	Novel protein
NOV15a	CG52414-03	57	58	Rhomboid
NOV15b	CG52414-01	59	60	Rhomboid
NOV15c	CG52414-02	61	62	Rhomboid
NOV16a	CG52552-06	63	64	PP1201 protein - Homo sapiens
NOV16b	CG52552-04	65	66	PP1201 protein - Homo sapiens
NOV16c	CG52552-01	67	68	PP1201 protein - Homo sapiens
NOV16d	CG52552-02	69	70	PP1201 protein - Homo sapiens
NOV16e	CG52552-03	71	72	PP1201 protein - Homo sapiens
NOV16f	CG52552-05	73	74	PP1201 protein - Homo sapiens

[0039] 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.

[0040] Pathologies, diseases, disorders and condition and the like that are associated with NOVX sequences include, but are not limited to: e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), vascular calcification, fibrosis, atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, metabolic disturbances associated with obesity, transplantation, osteoarthritis, rheumatoid arthritis, osteochondrodysplasia, adrenoleukodystrophy, congenital adrenal 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Additional utilities for NOVX nucleic acids and polypeptides according to the invention are disclosed herein.

[0046] NOVX Clones

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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 37; (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 37, 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 37; (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 37 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; and (e) a fragment of any of (a) through (d).

[0051] 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 37; (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 37 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 37; (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 37, 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 37 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.

[0052] 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 37; (b) a nucleotide sequence 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 37 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 37; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n−1, wherein n is an integer between 1 and 37 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.

[0053] NOVX Nucleic Acids and Polypeptides

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 37, or a complement of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)

[0059] 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.

[0060] 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 37, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

[0061] 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 37, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, thereby forming a stable duplex.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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, N.Y., 1993, and below.

[0067] 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 37, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.

[0068] 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.

[0069] 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 37; or an anti-sense strand nucleotide sequence of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37; or of a naturally occurring mutant of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37.

[0070] Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe has a detectable label attached, e.g. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.

[0071] “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−1, wherein n is an integer between 1 and 37, 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.

[0072] NOVX Nucleic Acid and Polypeptide Variants

[0073] 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 37, 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 37. 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 37.

[0074] In addition to the human NOVX nucleotide sequences of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, 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 (eg., 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.

[0075] 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 37, 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.

[0076] 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 37. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule 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.

[0077] 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.

[0078] 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.

[0079] 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 6×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 0.2×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 37, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0080] 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 37, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5× Reinhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×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.

[0081] 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 37, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×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 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.

[0082] Conservative Mutations

[0083] 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 37, 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 37. 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.

[0084] 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 37, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 40% homologous to the amino acid sequences of SEQ ID NO:2n, wherein n is an integer between 1 and 37. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; more preferably at least about 70% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; still more preferably at least about 80% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; even more preferably at least about 90% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37; and most preferably at least about 95% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37.

[0085] An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID NO:2n, wherein n is an integer between 1 and 37, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

[0086] Mutations can be introduced any one of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

[0087] 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.

[0088] 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).

[0089] 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).

[0090] Interfering RNA

[0091] In one aspect of the invention, NOVX gene expression can be attenuated by RNA interference. One approach well-known in the art is short interfering RNA (siRNA) mediated gene silencing where expression products of a NOVX gene are targeted by specific double stranded NOVX derived siRNA nucleotide sequences that are complementary to at least a 19-25 nt long segment of the NOVX gene transcript, including the 5′ untranslated (UT) region, the ORF, or the 3′ UT region. See, e.g., PCT applications WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, WO02/16620, and WO02/29858, each incorporated by reference herein in their entirety. Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX gene include, e.g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX polypeptide, and polypeptides involved in a NOVX regulatory pathway.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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 H1. One example of a vector system is the GeneSuppressor™ RNA Interference kit (commercially available from Imgenex). The U6 and H1 promoters are members of the type III class of Pol III promoters. The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for H1 promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3′ UU overhang in the expressed siRNA, which is similar to the 3′ overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript.

[0096] 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.

[0097] In general, siRNAs are chopped from longer dsRNA by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular proteins into an endonuclease complex. In vitro studies in Drosophila suggest that the siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex, called an RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER. RISC uses the sequence encoded by the antisense siRNA strand to find and destroy mRNAs of complementary sequence. The siRNA thus acts as a guide, restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g., AA(N19)TT). A desirable spacer region has a G/C-content of approximately 30% to 70%, and more preferably of about 50%. If the sequence AA(N19)TT is not present in the target sequence, an alternative target region would be AA(N21). The sequence of the NOVX sense siRNA corresponds to (N19)TT or N21, respectively. In the latter case, conversion of the 3′ end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the NOVX polynucleotide. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3′ overhangs. Symmetric 3′ overhangs may help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200, incorporated by reference herein in its entirely. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition.

[0102] 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.

[0103] 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 μg of the siRNA duplex is generally sufficient. Cells are typically seeded the previous day, and are transfected at about 50% confluence. The choice of cell culture media and conditions are routine to those of skill in the art, and will vary with the choice of cell type. The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful NOVX silencing. The efficiency of transfection needs to be carefully examined for each new cell line to be used. Preferred cell are derived from a mammal, more preferably from a rodent such as a rat or mouse, and most preferably from a human. Where used for therapeutic treatment, the cells are preferentially autologous, although non-autologous cell sources are also contemplated as within the scope of the present invention.

[0104] For a control experiment, transfection of 0.84 μg single-stranded sense NOVX siRNA will have no effect on NOVX silencing, and 0.84 μg antisense siRNA has a weak silencing effect when compared to 0.84 μg of duplex siRNAs. Control experiments again allow for a comparative analysis 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] Production of RNAs

[0111] Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using known methods such as transcription in RNA expression vectors. In the initial experiments, the sense and antisense RNA are about 500 bases in length each. The produced ssRNA and asRNA (0.5 μM) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCl were heated to 95° C. for 1 min then cooled and annealed at room temperature for 12 to 16 h. The RNAs are precipitated and resuspended in lysis buffer (below). To monitor annealing, RNAs are electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide. See, e.g., Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989).

[0112] Lysate Preparation

[0113] 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.

[0114] 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× 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.

[0115] 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.

[0116] RNA Preparation

[0117] 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)).

[0118] These RNAs (20 μM) single strands are incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C.

[0119] Cell Culture

[0120] 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 (1-3×105 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.

[0121] 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.

[0122] Antisense Nucleic Acids

[0123] 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 37, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO:2n, wherein n is an integer between 1 and 37, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, are additionally provided.

[0124] 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).

[0125] 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).

[0126] 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-N-6-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).

[0127] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0128] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.

[0129] Ribozymes and PNA Moieties

[0130] 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.

[0131] 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 37). 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. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No. 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.

[0132] 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.

[0133] In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleotide bases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomer can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.

[0134] PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., 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).

[0135] 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.

[0136] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

[0137] NOVX Polypeptides

[0138] A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in any one of SEQ ID NO:2n, wherein n is an integer between 1 and 37. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID NO:2n, wherein n is an integer between 1 and 37, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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 37) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity 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.

[0144] 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.

[0145] In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 37. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 37, and retains the functional activity of the protein of SEQ ID NO:2n, wherein n is an integer between 1 and 37, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 37, and retains the functional activity of the NOVX proteins of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

[0146] Determining Homology Between Two or More Sequences

[0147] 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”).

[0148] The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region 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 37.

[0149] 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.

[0150] Chimeric and Fusion Proteins

[0151] The invention also provides NOVX chimeric or fusion proteins. As used herein, a NOVX “chimeric protein” or “fusion protein” comprises a NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An “NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a NOVX protein of SEQ ID NO:2n, wherein n is an integer between 1 and 37, whereas a “non-NOVX polypeptide” refers to a polypeptide having an amino acid sequence 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] A NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.

[0156] NOVX Agonists and Antagonists

[0157] The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the NOVX proteins.

[0158] 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.

[0159] Polypeptide Libraries

[0160] 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 S1 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.

[0161] 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.

[0162] Anti-NOVX Antibodies

[0163] 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 IgG1, 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.

[0164] 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 37, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

[0165] 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.

[0166] 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 μM, preferably ≦100 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0167] 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.

[0168] 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, N.Y., incorporated herein by reference). Some of these antibodies are discussed below.

[0169] Polyclonal Antibodies

[0170] 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).

[0171] 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 (Apr. 17, 2000), pp. 25-28).

[0172] Monoclonal Antibodies

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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, Calif. and the American Type Culture Collection, Manassas, Va. 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).

[0177] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[0178] 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.

[0179] 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.

[0180] The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. 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. Pat. 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.

[0181] Humanized Antibodies

[0182] The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. 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)).

[0183] Human Antibodies

[0184] 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).

[0185] In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. 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)).

[0186] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

[0187] 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. Pat. 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.

[0188] A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. 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.

[0189] 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.

[0190] Fab Fragments and Single Chain Antibodies

[0191] 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. Pat. 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)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F, fragments.

[0192] Bispecific Antibodies

[0193] 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.

[0194] 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).

[0195] 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).

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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).

[0200] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0201] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (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).

[0202] Heteroconjugate Antibodies

[0203] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. 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. Pat. No. 4,676,980.

[0204] Effector Function Engineering

[0205] It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

[0206] Immunoconjugates

[0207] 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).

[0208] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.

[0209] 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 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

[0210] 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.

[0211] Immunoliposomes

[0212] 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. Pat. No. 5,013,556.

[0213] 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).

[0214] Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention

[0215] 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.

[0216] 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”).

[0217] 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 (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0218] Antibody Therapeutics

[0219] 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.

[0220] 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.

[0221] 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.

[0222] Pharmaceutical Compositions of Antibodies

[0223] 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.

[0224] 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.

[0225] 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.

[0226] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0227] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0228] ELISA Assay

[0229] 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, N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 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.

[0230] NOVX Recombinant Expression Vectors and Host Cells

[0231] 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.

[0232] 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).

[0233] 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.).

[0234] 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.

[0235] 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.

[0236] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0237] 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.

[0238] In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kudjan 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.).

[0239] 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).

[0240] 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.

[0241] 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 (Banecji, 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 α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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).

[0247] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ie., 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.

[0248] Transgenic NOVX Animals

[0249] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0250] 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 37, 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. Pat. 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.

[0251] 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 37), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector).

[0252] Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5′- and 3′-termini by additional nucleic acid of the NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′- and 3′-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al., 1992. Cell 69: 915.

[0253] 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.

[0254] 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.

[0255] 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 G0 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.

[0256] Pharmaceutical Compositions

[0257] 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.

[0258] 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 (ie., 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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. Pat. No. 4,522,811.

[0266] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0267] 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. Pat. 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.

[0268] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0269] Screening and Detection Methods

[0270] 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.

[0271] The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.

[0272] Screening Assays

[0273] 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.

[0274] 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.

[0275] 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.

[0276] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

[0277] 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. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No. 5,233,409.).

[0278] 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 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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, Tritone X-100, Tritone X-114, Thesite, Isotridecypoly(ethylene glycol ether)n, N-dodecyl—N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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. Pat. 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.

[0289] 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.

[0290] The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.

[0291] Detection Assays

[0292] 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.

[0293] Chromosome Mapping

[0294] 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 37, 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.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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).

[0300] 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.

[0301] 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.

[0302] 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.

[0303] Tissue Typing

[0304] 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. Pat. No. 5,272,057).

[0305] 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.

[0306] 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).

[0307] 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 37, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0308] Predictive Medicine

[0309] 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.

[0310] 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.

[0311] These and other agents are described in further detail in the following sections.

[0312] Diagnostic Assays

[0313] 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−1, wherein n is an integer between 1 and 37, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[0314] An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] Prognostic Assays

[0319] 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.

[0320] 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).

[0321] 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.

[0322] 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. Pat. 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.

[0323] Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0324] 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. Pat. No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0325] 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.

[0326] 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).

[0327] Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230:1242. In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 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.

[0328] 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. Pat. No. 5,459,039.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a NOVX gene.

[0334] 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.

[0335] Pharmacogenomics

[0336] 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.

[0337] 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.

[0338] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0339] 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.

[0340] 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.

[0341] Monitoring of Effects During Clinical Trials

[0342] 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.

[0343] 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.

[0344] 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.

[0345] Methods of Treatment

[0346] 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.

[0347] These methods of treatment will be discussed more fully, below.

[0348] Diseases and Disorders

[0349] 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 (ie., 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.

[0350] 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.

[0351] Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).

[0352] Prophylactic Methods

[0353] 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.

[0354] Therapeutic Methods

[0355] 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.

[0356] 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).

[0357] Determination of the Biological Effect of the Therapeutic

[0358] In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.

[0359] 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.

[0360] Prophylactic and Therapeutic Uses of the Compositions of the Invention

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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 1

[0365] The NOV1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1A.

TABLE 1A
NOV1 Sequence Analysis
	SEQ ID NO: 1	2322 bp
NOV1a,	GGGCGGCCGCAGCCTGAGCCAGGGCCCCCTCCCTCGTCAGGACCGGGGCAGCAAGCAGGCCGGG
CG126472-01	GGCAGGTCCCGGCACCCACCATGCGAGGCGAGCTCTGGCTCCTGGTGCTGGTGCTCAGGGAGGC
DNA Sequence	TGCCCGGGCGCTGAGCCCCCAGCCCGGAGCAGGTCACGATGAGGGCCCAGGCTCTGGATGGGCT
	GCCAAAGGGACCGTGCGGGGCTGGAACCGGAGAGCCCGAGAGAGCCCTGGGCATGTGTCAGAGC
	CGGACAGGACCCAGCTGAGCCAGGACCTGGGTGGGGGCACCCTGGCCATGGACACGCTGCCAGA
	TAACAGGACCAGGGTCGTGGAGGACAACCACAGCTATTATGTGTCCCGTCTCTATGGCCCCAGC
	GAGCCCCACAGCCGGGAACTGTGGGTAGATGTGGCCGAGGCCAACCGGAGCCAAGTGAAGATCC
	ACACAATACTCTCCAACACCCACCGGCAGGCTTCGAGAGTGGTCTTGTCCTTTGATTTCCCTTT
	CTACGGGCATCCTCTGCGGCAGATCACCATAGCAACTGGAGGCTTCATCTTCATGGGGGACGTG
	ATCCATCGGATGCTCACAGCTACTCAGTATGTGGCGCCCCTGATGGCCAACTTCAACCCTGGCT
	ACTCCGACAACTCCACAGTTGTTTACTTTGACAATGGGACAGTCTTTGTGGTTCAGTCGGACCA
	CGTTTATCTCCAAGGCTGGGAAGACAAGGGCAGTTTCACCTTCCAGGCAGCTCTGCACCATGAC
	GGCCGCATTGTCTTTGCCTATAAAGAGATCCCTATGTCTGTCCCGGAAATCAGCTCCTCCCAGC
	ATCCTGTCAAAACCGGCCTATCGGATGCCTTCATGATTCTCAATCCATCCCCGGATGTGCCAGA
	ATCTCGGCGAAGGAGCATCTTTGAATACCACCGCATAGAGCTGGACCCCAGCAAGGTCACCAGC
	ATGTCGGCCGTGGAGTTCACCCCATTGCCGACCTGCCTGCAGCATAGGAGCTGTGACGCCTGCA
	TGTCCTCAGACCTGACCTTCAACTGCAGCTGGTGCCATGTCCTCCAGAGATGCTCCAGTGGCTT
	TGACCGCTATCGCCAGGAGTGGATGGACTATGGCTGTGCACAGGAGGCAGAGGGCAGGATGTGC
	GAGGACTTCCAGGATGAGGACCACGACTCAGCCTCCCCTGACACTTCCTTCAGCCCCTATGATG
	GAGACCTCACCACTACCTCCTCCTCCCTCTTCATCGACAGCCTCACCACAGAAGATGACACCAA
	GTTGAATCCCTATGCAGGAGGAGACGGCCTTCAGAACAACCTGTCCCCCAAGACAAAGGGCACT
	CCTGTGCACCTGGGCACCATCGTGGGCATCGTGCTCGCAGTCCTCCTCGTGGCGGCCATCATCC
	TGGCTGGAATTTACATCAATGGCCACCCCACATCCAATGCTGCGCTCTTCTTCATCGAGCGTAG
	ACCTCACCACTGGCCAGCCATGAAGTTTCGCAGCCACCCTGACCATTCCACCTATGCGGAGGTG
	GAGCCCTCGGGCCATGAGAAGGAGGGCTTCATGGAGGCTGAGCAGTGCTGAGAACACCAAGTCT
	CCCCTTTGAAGACTTTGAGGCCACAGAAAAGACAGTTAAAGCAAAGAAGAGAAGTGACTTTTCC
	TGGCCTCTCCCAGCATGCCCTGGGCTGAGATGAGATGGTGGTTTATGGCTCCAGAGCTGCTGTT
	CGCTTCGTCAGCACACCCCGAATATTGAAGAGGGGGCCAAAAAACAACCACATGGATTTTTTAT
	AGGAACAACAACCTAATCTCATCCTGTTTTGATGCAAGGGTTCTCTTCTGTGTCTTGTAACCAT
	GAAACAGCAGAAGAACTAACATAACTAACTCCATTTTTGTTTAAGGGGCCTTTACCTATTCCTG
	CACCTAGGCTAGGATAACTTTAGAGCACTGACATAAAACGCAAAAACAGGAATCATGCCGTTTG
	CAAAACTAACTCTGGGATTAAAGGGGAAGCATGTAAACAGCTAACTGTTTTTGTTAAAGATTTA
	TAGGAATGAGGAGGTTTGGCTATTGTCACATGACAGACTGTTAGCCAAGGACAAAGAAGTTCTG
	CAAACCTCCCCTGGACCCTTGCTGGTGTCCAGATGTCTGCGGTTGTCAGCCCCTTCCTTTCCCC
	CGACCTAAACATAAAAGACAAGGCAAAGCCCGCATAATTTTAAGACGGTTCTTTAGGACATTAG
	TCCACCATCTTCTTGGTTTGCTGGCTCTCCGAAATAAAGTCCCTTTCCTTGCTCCAAAAAAAAA
	AAAAAAAAAAAAAAAAAA
	ORF Start: ATG at 85		ORF Stop: TGA at 1585
	SEQ ID NO: 2	500 aa	MW at 55759.7 kD
NOV1a,	MRGELWLLVLVLREAARALSPQPGAGHDEGPGSGWAAKGTVRGWNRRARESPGHVSEPDRTQLS
CG126472-01	QDLGGGTLAMDTLPDNRTRVVEDNHSYYVSRLYGPSEPHSRELWVDVAEANRSQVKIHTILSNT
Protein Sequence	HRQASRVVLSFDFPFYGHPLRQITIATGGFIFMGDVIHRMLTATQYVAPLMANFNPGYSDNSTV
	VYFDNGTVFVVQWDHVYLQGWEDKGSFTFQAALHHDGRIVFAYKEIPMSVPEISSSQHPVKTGL
	SDAFMILNPSPDVPESRRRSIFEYHRIELDPSKVTSMSAVEFTPLPTCLQHRSCDACMSSDLTF
	NCSWCHVLQRCSSGFDRYRQEWMDYGCAQEAEGRMCEDFQDEDHDSASPDTSFSPYDGDLTTTS
	SSLFIDSLTTEDDTKLNPYAGGDGLQNNLSPKTKGTPVHLGTIVGIVLAVLLVAAIILAGIYIN
	GHPTSNAALFFIERRPHHWPAMKFRSHPDHSTYAEVEPSGHEKEGFMEAEQC
	SEQ ID NO: 3	2286 bp
NOV1b,	GGGCGGCCGCAGCCTGAGCCAGGGCCCCCTCCCTCGTCAGGACCGGGGCAGCAAGCAGGCCGGG
CG126472-02	GGCAGGTCCGGGCACCCACCATGCGAGGCGAGCTCTGGCTCCTGGTGCTGGTGCTCAGGGAGGC
DNA Sequence	TGCCCGGGCGCTGAGCCCCCAGCCCGGAGCAGGTCACGATGAGGGCCCAGGCTCTCGATGGGCT
	GCCAAAGGGACCGTGCGGGGCTGGAACCGGAGAGCCCGAGAGAGCCCTGGGCATGTGTCAGAGC
	CGGACAGGACCCAGCTGAGCCAGGACCTGGGTGGGGGCACCCTGGCCATGGACACGCTGCCAGA
	TAACAGGACCAGGGTGGTGGAGGACAACCACAGCTATTATGTGTCCCGTCTCTATGGCCCCAGC
	GAGCCCCACAGCCGGGAACTGTGGGTAGATGTGGCCGAGGCCAACCGGAGCCAAGTGAAGATCC
	ACACAATACTCTCCAACACCCACCGGCAGGCTTCGAGAGTGGTCTTGTCCTTTGATTTCCCTTT
	CTACGGGCATCCTCTGCGGCAGATCACCATAGCAACTGGAGGCTTCATCTTCATGGGGGACGTG
	ATCCATCGGATGCTCACAGCTACTCAGTATGTGGCGCCCCTGATGGCCAACTTCAACCCTGGCT
	ACTCCGACAACTCCACAGTTGTTTACTTTGACAATGGGACAGTCTTTGTGGTTCAGTGGGACCA
	CGTTTATCTCCAAGGCTGGGAAGACAAGGGCAGTTTCACCTTCCAGGCAGCTCTGCACCATGAC
	GGCCGCATTGTCTTTGCCTATAAAGAGATCCCTATGTCTGTCCCCGAAATCAGCTCCTCCCAGC
	ATCCTGTCAAAACCGGCCTATCGGATGCCTTCATGATTCTCAATCCATCCCCGGATGTGCCAGA
	ATCTCGGCGAAGGAGCATCTTTGAATACCACCGCATAGAGCTGGACCCCAGCAAGGTCACCAGC
	ATGTCGGCCGTGGAGTTCACCCCATTGCCGACCTGCCTGCAGCATAGGAGCTGTGACGCCTGCA
	TGTCCTCAGACCTGACCTTCAACTGCAGCTGGTGCCATGTCCTCCAGAGATGTTCCAGTGGCTT
	TGACCGCTATCGCCAGGAGTGGATGGACTATGGCTGTGCACAGGAGGCAGAGGGCAGGATGTGC
	GAGGACTTCCAGGATGAGGACCACGACTCAGCCTCCCCTGACACTTCCTTCAGCCCCTATGATG
	GAGACCTCACCACTACCTCCTCCTCCCTCTTCATCGACAGCCTCACCACAGAAGGCCTTCAGAA
	CAACCTGTCCCCCAAGACAAAGGGCACTCCTGTGCACCTGGGCACCATCGTGGGCATCGTGCTG
	GCAGTCCTCCTCGTGGCGGCCATCATCCTGGCTGGAATTTACATCAATGGCCACCCCACATCCA
	ATGCTGCGCTCTTCTTCATCGAGCGTAGACCTCACCACTGGCCAGCCATGAAGTTTCGCAGCCA
	CCCTAACCATTCCACCTATGCGGAGGTGGAGCCCTCGGGCCATGAGAAGGAGGGCTTCATGGAG
	GCTGAGCAGTGCTGAGAACACCAAGTCTCCCCTTTGAAGACTTTGAGGCCACAGAAAAGACACT
	TAAAGCAAAGAAGAGAAGTGACTTTTCCTGGCCTCTCCCAGCATGCCCTGGGCTGAGATGAGAT
	GGTGGTTTATGGCTCCAGAGCTGCTGTTCGCTTCGTCAGCACACCCCGAATATTGAAGAGGGGG
	CCAAAAAACAACCACATGGATTTTTTATAGGAACAACAACCTAATCTCATCCTGTTTTGATGCA
	AGGGTTCTCTTCTGTGTCTTGTAACCATGAAACAGCAGAAGAACTAACATAACTAACTCCATTT
	TTGTTTAAGGGGCCTTTACCTATTCCTGCACCTAGGCTAGGATAACTTTAGAGCACTGACATAA
	AACGCAAAAACAGGAATCATGCCGTTTGCAAAACTAACTCTGGGATTAAAGGGGAAGCATGTAA
	ACAGCTAACTGTTTTTGTTAAAGATTTATAGGAATGAGGAGGTTTGGCTATTGTCACATGACAG
	ACTGTTAGCCAAGGACAAAGAAGTTCTGCAAACCTCCCCTGGACCCTTGCTGGTGTCCAGATGT
	CTGCGGTTGTCAGCCCCTTCCTTTCCCCCGACCTAAACATAAAAGACAAGGCAAAGCCCGCATA
	ATTTTAAGACGGTTCTTTAGGACATTAGTCCACCATCTTCTTGGTTTGCTGGCTCTCCGAAATA
	AAGTCCCTTTCCTTGCTCCAAAAAAAAAAAAAAAAAAAAAAAAAAA
	ORF Start: ATG at 85		ORF Stop: TGA at 1549
	SEQ ID NO: 4	488 aa	MW at 54511.5 kD
NOV1b,	MRGELWLLVLVLREAARALSPQPGAGHDEGPGSGWAAKGTVRGWNRRARESPGHVSEPDRTQLS
CG126472-02	QDLGGGTLAMDTLPDNRTRVVEDNHSYYVSRLYGPSEPHSRELWVDVAEANRSQVKIHTILSNT
Protein Sequence	HRQASRVVLSFDFPFYGHPLRQITIATGGFIFMGDVIHRNLTATQYVAPLMANFNPGYSDNSTV
	VYFDNGTVFVVQWDHVYLQGWEDKGSFTFQAALHHDGRIVFAYKEIPMSVPEISSSQHPVKTGL
	SDAFMILNPSPDVPESRRRSIFEYHRIELDPSKVTSMSAVEFTPLPTCLQHRSCDACMSSDLTF
	NCSWCHVLQRCSSGFDRYRQEWMDYGCAQEAEGRMCEDFQDEDHDSASPDTSFSPYDGDLTTTS
	SSLFIDSLTTEGLQNNLSPKTKGTPVHLGTIVGIVLAVLLVAAIILAGIYINGHPTSNAALFFI
	ERRPHHWPAMKFRSHPNHSTYAEVEPSGHEKEGFMEAEQC

[0366] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 1B.

TABLE 1B
Comparison of NOV1a against NOV1b.
			Identities/
	Protein	NOV1a Residues/	Similarities for the
	Sequence	Match Residues	Matched Region
	NOV1b	1 . . . 500	487/500 (97%)
		1 . . . 488	488/500 (97%)

[0367] Further analysis of the NOV1a protein yielded the following properties shown in Table 1C.

TABLE 1C
Protein Sequence Properties NOV1a
SignalP	Cleavage site between residues 19 and 20
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 4; pos. chg 1; neg. chg 1
	H-region: length 8; peak value 11.03
	PSG score: 6.62
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): 0.95
	possible cleavage site: between 18 and 19
	>>> Seems to have a cleavable signal peptide (1 to 18)
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 19
	Tentative number of TMS(s) for the threshold 0.5: 1
	Number of TMS(s) for threshold 0.5: 1
	INTEGRAL	Likelihood =	−15.87	Transmembrane 427-443
	PERIPHERAL	Likelihood =	3.87 (at 153)
	ALOM score: −15.87 (number of TMSs: 1)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 9
	Charge difference: 0.5 C(1.5)-N(1.0)
	C > N: C-terminal side will be inside
	>>>Caution: Inconsistent mtop result with signal peptide
	>>> membrane topology: type 1a (cytoplasmic tail 444 to 500)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	2	Hyd Moment (75):	3.62
	Hyd Moment(95):	3.15	G content:	1
	D/E content:	2	S/T content:	0
	Score: −6.39
	Gavel: prediction of cleavage sites for mitochondrial
	preseq
	R-2 motif at 12 MRG|EL
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: PESRRRS (4) at 270
	bipartite: none
	content of basic residues: 8.2%
	NLS Score: −0.13
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	XXRR-like motif in the N-terminus: RGEL
	none
	SKL: peroxisomal targeting signal in the C-terminus: none
	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: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 76.7
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	55.6%: endoplasmic reticulum
	22.2%: Golgi
	11.1%: plasma membrane
	11.1%: extracellular, including cell wall
	>> prediction for CG126472-01 is end (k = 9)

[0368] A search of the NOV1a 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.

TABLE 1D
Geneseq Results for NOV1a
		NOV1a	Identities/
Geneseq	Protein/Organism/Length [Patent #,	Residues/	Similarities for	Expect
Identifier	Date]	Match Residues	the Matched Region	Value
ABB90749	Human Tumor Endothelial Marker	1 . . . 500	500/500 (100%)	0.0
	polypeptide SEQ ID NO 230 - Homo	1 . . . 500	500/500 (100%)
	sapiens, 500 aa. [WO200210217-A2,
	07 FEB. 2002]
ABB90723	Human Tumour Endothelial Marker	1 . . . 500	500/500 (100%)	0.0
	polypeptide SEQ ID NO 179 - Homo	503 . . . 1002	500/500 (100%)
	sapiens, 1002 aa. [WO200210217-A2,
	07 FEB. 2002]
ABB90783	Mouse Tumour Endothelial Marker	1 . . . 500	409/501 (81%)	0.0
	polypeptide SEQ ID NO 297 - Mus	1 . . . 500	455/501 (90%)
	musculus, 500 aa. [WO200210217-A2,
	07 FEB. 2002]
ABB90729	Mouse Tumour Endothelial Marker	1 . . . 500	409/501 (81%)	0.0
	polypeptide SEQ ID NO 192 - Mus	1 . . . 500	455/501 (90%)
	musculus, 500 aa. [WO200210217-A2,
	07 FEB. 2002]
AAB85400	Tumour endothelial marker 7 precursor	72 . . . 500	414/431 (96%)	0.0
	protein - Homo sapiens, 431 aa.	1 . . . 431	415/431 (96%)
	[WO200153500-A1, 26 JUL. 2001]

[0369] In a BLAST search of public sequence databases, the NOV1a protein was found to have homology to the proteins shown in the BLASTP data in Table 1E.

TABLE 1E
Public BLASTP Results for NOV1a
Protein		NOV1a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q9HCT9	Tumor endothelial marker 7 precursor	1 . . . 500	500/500 (100%)	0.0
	(Tumor endothelial marker 3 precursor) -	1 . . . 500	500/500 (100%)
	Homo sapiens (Human), 500 aa.
AAH36059	Tumor endothelial marker 7 precursor -	1 . . . 500	499/500 (99%)	0.0
	Homo sapiens (Human), 500 aa.	1 . . . 500	500/500 (99%)
Q91ZV7	Tumor endothelial marker 7 precursor -	1 . . . 500	409/501 (81%)	0.0
	Mus musculus (Mouse), 500 aa.	1 . . . 500	455/501 (90%)
Q9CWV5	2410003I07Rik protein - Mus musculus	1 . . . 500	408/501 (81%)	0.0
	(Mouse), 500 aa.	1 . . . 500	454/501 (90%)
BAC29318	16 days neonate cerebellum cDNA,	1 . . . 500	408/508 (80%)	0.0
	RIKEN full-length enriched library,	1 . . . 507	454/508 (89%)
	clone: 9630040L07 product: TUMOR
	ENDOTHELIAL MARKER 7 PRECURSOR
	homolog - Mus musculus (Mouse), 507 aa.

[0370] PFam analysis predicts that the NOV1a protein contains the domains shown in the Table 1F.

TABLE 1F
Domain Analysis of NOV1a
		Identities/
	NOV1a Match	Similarities for	Expect
Pfam Domain	Region	the Matched Region	Value
Plexin_repeat	303 . . . 348	15/67 (22%)	0.02
		31/67 (46%)

Example 2

[0371] The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.

TABLE 2A
NOV2 Sequence Analysis
	SEQ ID NO: 5	1573 bp
NOV2a,	GACTCAGCCTTAGGTACCGGTCAGGCAAAATGCGGTCCTCCCTGGCTCCGGGAGTCTGGTTCTT
CG138751-02	CCGGGCCTTCTCCAGGGACAGCTGGTTCCGAGGCCTCATCCTGCTGCTGACCTTCCTAATTTAC
DNA Sequence	GCCTGCTATCACATGTCCAGGAAGCCTATCAGTATCGTCAAGAGCCGTCTGCACCAGAACTGCT
	CGGAGCAGATCAAACCCATCAATGATACTCACAGTCTCAATGACACCATGTGGTGCAGCTGGGC
	CCCATTTGACAACGACAACTATAAGGAGTTACTAGGGGGCGTGGACAACGCCTTCCTCATCGCC
	TATGCCATCGGCATGTTCATCAGTGGGGTTTTTGGGGAGCGGCTTCCGCTCCGTTACTACCTCT
	CAGCTGGAATGCTGCTCAGTCGCCTTTTCACCTCGCTCTTTGGCCTGGGATATTTCTGGAACAT
	CCACGAGCTCTGGTACTTTGTGGTCATCCAGGTCTGTAATGGACTCGTCCAGACCACAGGCTGG
	CCCTCTGTGGTGACCTGTGTTGGCAACTGGTTCGGGAAGGGGAAGCGGGGGTTCATCATGGGCA
	TCTGGAATTCCCACACATCTGTGGGCAACATCCTGGGCTCCCTGATCGCCGGCATCTGGGTGAA
	CGGGCAGTGGGGCCTGTCGTTCATCGTGCCTGGCATCATTACTGCCGTCATGGGCGTCATCACC
	TTCCTCTTCCTCATCGAACACCCAGAAGATGTGGACTGCGCCCCTCCTCAGCACCACGGTGAGC
	CAGCTGAGAACCAGGACAACCCTGAGGACCCTGCGAACAGTCCCTGCTCTATCAGGGAGAGCGG
	CCTTGAGACTGTGGCCAAATGCTCCAAGGGGCCATGCGAAGAGCCTGCTGCCATCAGCTTCTTT
	GGGGCGCTCCGGATCCCAGGCGTGGTCGAGTTCTCTCTGTGTCTGCTGTTTGCCAAGCTGGTCA
	GTTACACCTTCCTCTACTGGCTGCCCCTCTACATCGCCAATGTGGCTCACTTTAGTGCCAAGGA
	GGCTGGGGACCTGTCTACACTCTTCGATGTTGGTGGCATCATAGGCGGCATCGTGGCAGGGCTC
	GTCTCTGACTACACCAATGGCAGGGCCACCACTTGCTGTGTCATGCTCATCTTGGCTGCCCCCA
	TGATGTTCCTGTACAACTACATTGGCCAGGACGGGATTGCCAGCTCCATAGTGATGCTGATCAT
	CTGTGGGGGCCTGGTCAATGGCCCATACGCGCTCATCACCACTGCTGTCTCTGCTGATCTGGGG
	ACTCACAAGAGCCTGAAGGGCAACGCCAAAGCCCTGTCCACGGTCACGGCCATCATTGACGGCA
	CCGGCTCCATAGGTGCGGCTCTGGGGCCTCTGCTGGCTGGGCTCATCTCCCCCACGGGCTGGAA
	CAATGTCTTCTACATGCTCATCTCTGCCGACGTCCTAGCCTGCTTGCTCCTTTGCCGGTTAGTA
	TACAAAGAGATCTTGGCCTGGAAGGTGTCCCTGAGCAGAGGCAGCGGGTGAGTCCGGGGAGCTG
	AAGCTGCCCCTCTACCAACCTCATTTCTCGTGGGAAT
	ORF Start: ATG at 30		ORF Stop: TGA at 1521
	SEQ ID NO: 6	497 aa	MW at 53902.2 kD
NOV2a,	MRSSLAPGVWFFRAFSRDSWFRGLILLLTFLIYACYHMSRKPISIVKSRLHQNCSEQIKPINDT
CG138751-02	HSLNDTMWCSWAPFDKDNYKELLGGVDNAFLIAYAIGMFISGVFGERLPLRYYLSAGMLLSGLF
Protein Sequence	TSLFGLGYFWNIHELWYFVVIQVCNGLVQTTGWPSVVTCVGNWFGKGKRGFIMGIWNSHTSVGN
	ILGSLIAGIWVNGQWGLSFIVPGIITAVMGVITFLFLIEHPEDVDCAPPGHHGEPAENQDNPED
	PGNSPCSIRESGLETVAKCSKGPCEEPAAISFFGALRIPGVVEFSLCLLPAKLVSYTFLYWLPL
	YIANVAHFSAKEAGDLSTLFDVGGIIGGIVAGLVSDYTNGRATTCCVMLILAAPMMFLYNYIGQ
	DGIASSIVMLIICGGLVNGPYALITTAVSADLGTHKSLKGNAKALSTVTAIIDGTGSIGAALGP
	LLAGLISPTGWNNVFYMLISADVLACLLLCRLVYKEILAWKVSLSRGSG
	SEQ ID NO: 7	1638 bp
NOV2b,	ACACGCGCCCAGCTCTGTAGCCTCCTCCGTCGACTCAGCCTTACGTACCGGTCAGGCAAAATGC
CG138751-01	GGTCCTCCCTGGCTCCGGGAGTCTGGTTCTTCCGGGCCTTCTCCAGGGACAGCTGGTTCCCAGG
DNA Sequence	CCTCATCCTGCTGCTGACCTTCCTAATTTACGCCTGCTATCACATGTCCAGGAAGCCTATCAGT
	ATCGTCAAGAGCCGTCTGCACCAGAACTGCTCGGAGCAGATCAAACCCATCAATGATACTCACA
	GTCTCAATGACACCATGTGGTGCAGCTGGGCCCCATTTGACAAGGACAACTATAAGGAGTTACT
	AGGGGGCGTGGACAACGCCTTCCTCATCGCCTATGCCATCGGCATGTTCATCAGTGGGGTTTTT
	GGGGAGCGGCTTCCGCTCCGTTACTACCTCTCAGCTGGAATGCTGCTCAGTGGCCTTTTCACCT
	CGCTCTTTGGCCTGGGATATTTCTGGAACATCCACGAGCTCTGGTACTTTGTGGTCATCCAGGT
	CTGTAATGGACTCGTCCAGACCACAGGCTGGCCCTCTGTGGTGACCTGTGTTGGCAACTGGTTC
	GGGAAGGGGAAGCGGGGGTTCATCATGGGCATCTGGAATTCCCACACATCTGTGGGCAACATCC
	TGGGCTCCCTGATCGCCGGCATCTGGGTGAACGGGCAGTGGGGCCTGTCGTTCATCGTGCCTGG
	CATCATTACTGCCGTCATGGGCGTCATCACCTTCCTCTTCCTCATCGAACACCCAGAAGATGTG
	GACTGCGCCCCTCCTCAGCACCACGGTGAGCCAGCTGAGAACCAGGACAACCCTGAGGACCCTG
	GGAACAGTCCCTGCTCTATCAGGGAGAGCGGCCTTGAGACTGTGGCCAAATGCTCCAAGGGGCC
	ATGCGAAGAGCCTGCTGCCATCAGCTTCTTTGGGGCGCTCCGGATCCCAGGCGTGGTCGAGTTC
	TCTCTGTGTCTGCTGTTTGCCAAGCTGGTCAGTTACACCTTCCTCTACTGGCTGCCCCTCTACA
	TCGCCAATGTGGCTCACTTTAGTGCCAAGGAGGCTGGGGACCTGTCTACACTCTTCGATGTTGG
	TGGCATCATAGGCGGCATCGTGGCAGGGCTCGTCTCTGACTACACCAATGGCAGGGCCACCACT
	TGCTGTGTCATGCTCATCTTGGCTGCCCCCATGATGTTCCTGTACAACTACATTGGCCAGGACG
	GGATTGCCAGCTCCATAGGTGAGGTCCCAGTGATGCTGATCATCTGTGGGGGCCTGGTCAATGG
	CCCATACGCGCTCATCACCACTGCTGTCTCTGCTGATCTGGGGACTCACAAGAGCCTGAAGGGC
	ACAGCCAAAGCCCTGTCCACGGTCACGGCCATCATTGACGGCACCGGCTCCATAGGTGCGGCTC
	TGGGGCCTCTGCTGGCTGGGCTCATCTCCCCCACGGGCTGGAACAATGTCTTCTACATGCTCAT
	CTCTGCCGACGTCCTAGCCTGCTTGGTCCTTTGCCGGTTAGTATACAAAGAGATCTTGGCCTGG
	AAGGTGTCCCTGAGCAGAGGCAGCGGGTGAGTCCGGGGAGCTGAAGCTGCCCCTCTACCAACCT
	CATTTCTCGTGGGAATCAGCCCAGCGCTCAGTTTCTCC
	ORF Start: ATG at 61		ORF Stop: TGA at 1564
	SEQ ID NO: 8	501 aa	MW at 54257.6 kd
NOV2b,	MRSSLAPGVWFFRAFSRDSWFRGLILLLTFLIYACYHMSRKPISIVKSRLHQNCSEQIKPINDT
CG138751-01	HSLNDTMWCSWAPFDKDNYKELLGGVDNAFLIAYAIGMFISGVFGERLPLRYYLSAGMLLSGLF
Protein Sequence	TSLFGLGYFWNIHELWYFVVIQVCNGLVQTTGWPSVVTCVGNWFGKGKRGFIMGIWNSHTSVGN
	ILGSLIAGIWVNGQWGLSFIVPGIITAVMGVITFLFLIEHPEDVDCAPPQHHGEPAENQDNPED
	PGNSPCSIRESGLETVMKCSKGPCEEPAAISFFGALRIPGVVEFSLCLLFAKLVSYTFLYWLPL
	YIANVAHFSAKEAGDLSTLFDVGGIIGGIVAGLVSDYTNGRATTCCVMLILAAPMMFLYNYIGQ
	DGIASSIGEVPVMLIICGGLVNGPYALITTAVSADLGTHKSLKGTAKALSTVTAIIDGTGSIGA
	ALGPLLAGLISPTGWNNVFYMLISADVLACLVLCRLVYKEILAWKVSLSRGSG

[0372] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 2B.

TABLE 2B
Comparison of NOV2a against NOV2b.
			Identities/
	Protein	NOV2a Residues/	Similarities for
	Sequence	Match Residues	the Matched Region
	NOV2b	1 . . . 497	495/501 (98%)
		1 . . . 501	496/501 (98%)

[0373] Further analysis of the NOV2a protein yielded the following properties shown in Table 2C.

TABLE 2C
Protein Sequence Properties NOV2a
SignalP	Cleavage site between residues 37 and 38
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 2; pos. chg 1; neg. chg 0
	H-region: length 10; peak value 8.95
	PSG score: 4.55
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −0.87
	possible cleavage site: between 34 and 35
	>>> Seems to have a cleavable signal peptide (1 to 34)
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 35
	Tentative number of TMS(s) for the threshold 0.5: 8
	INTEGRAL	Likelihood =	−3.72	Transmembrane 93-109
	INTEGRAL	Likelihood =	−0.48	Transmembrane 118-134
	INTEGRAL	Likelihood =	−6.90	Transmembrane 211-227
	INTEGRAL	Likelihood =	−2.71	Transmembrane 294-310
	INTEGRAL	Likelihood =	−2.76	Transmembrane 338-354
	INTEGRAL	Likelihood =	−4.83	Transmembrane 362-378
	INTEGRAL	Likelihood =	−3.72	Transmembrane 385-401
	INTEGRAL	Likelihood =	−6.74	Transmembrane 462-478
	PERIPHERAL	Likelihood =	1.27 (at 438)
	ALOM score: −6.90 (number of TMSs: 8)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 17
	Charge difference: −1.0 C(0.0)-N(1.0)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 3a
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	3	Hyd Moment (75):	7.33
	Hyd Moment (95):	6.86	G content:	1
	D/E content:	1	S/T content:	3
	Score: −1.57
	Gavel: prediction of cleavage sites for mitochondrial preseq
	R-10 motif at 23 FRA FS
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: none
	bipartite: none
	content of basic residues: 6.0%
	NLS Score: −0.47
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	XXRR-like motif in the N-terminus: RSSL none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	66.7%: endoplasmic reticulum
	22.2%: mitochondrial
	11.1%: nuclear
	>> prediction for CG138751-02 is end (k = 9)

[0374] 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	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
ABB98201	Human transporter protein SEQ ID	1 . . . 497	497/497 (100%)	0.0
	NO 2 - Homo sapiens, 497 aa.	1 . . . 497	497/497 (100%)
	[WO200242456-A2, 30 MAY 2002]
AAM00776	Human bone marrow protein, SEQ ID	181 . . . 391	205/211 (97%)	e−118
	NO: 139 - Homo sapiens, 211 aa.	1 . . . 211	206/211 (97%)
	[WO200153453-A2, 26 JUL. 2001]
AAM00889	Human bone marrow protein, SEQ ID	170 . . . 368	193/199 (96%)	e−113
	NO: 365 - Homo sapiens, 201 aa.	3 . . . 201	195/199 (97%)
	[WO200153453-A2, 26 JUL. 2001]
AAG31980	Arabidopsis thaliana protein fragment	24 . . . 485	221/466 (47%)	e−111
	SEQ ID NO: 38498 - Arabidopsis	31 . . . 462	297/466 (63%)
	thaliana, 476 aa. [EP1033405-A2,
	06 SEP. 2000]
AAB42327	Human ORFX ORF2091 polypeptide sequence	295 . . . 485	187/191 (97%)	e−103
	SEQ ID NO: 4182 - Homo sapiens, 192 aa.	2 . . . 192	188/191 (97%)
	[WO200058473-A2, 05 OCT. 2000]

[0375] 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		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q8TED4	Hypothetical protein FLJ23627 - Homo	1 . . . 497	496/497 (99%)	0.0
	sapiens (Human), 501 aa.	1 . . . 497	496/497 (99%)
BAC26224	10 days neonate skin cDNA, RIKEN	1 . . . 497	437/497 (87%)	0.0
	full-length enriched library,	1 . . . 497	462/497 (92%)
	clone: 4732478E01 product: solute
	carrier family 37 (glycerol-3-phosphate
	transporter), member 1, full insert
	sequence - Mus musculus (Mouse), 506 aa.
Q9WU81	cAMP inducible 2 protein - Mus	1 . . . 497	437/497 (87%)	0.0
	musculus (Mouse), 501 aa.	1 . . . 497	462/497 (92%)
BAC37639	Adult male bone cDNA, RIKEN	1 . . . 497	436/497 (87%)	0.0
	full-length enriched library,	1 . . . 497	462/497 (92%)
	clone: 9830146N24 product: solute
	carrier family 37 (glycerol-3-phosphate
	transporter), member 1, full insert
	sequence - Mus musculus (Mouse), 501 aa.
Q8TEM2	FLJ00171 protein - Homo sapiens	1 . . . 346	346/346 (100%)	0.0
	(Human), 396 aa (fragment).	12 . . . 357	346/346 (100%)

[0376] PFam analysis predicts that the NOV2a protein contains the domains shown in the Table 2F.

TABLE 2F
Domain Analysis of NOV2a
		Identities/
	NOV2a	Similarities for	Expect
Pfam Domain	Match Region	the Matched Region	Value
sugar_tr	9 . . . 490	66/549 (12%)	0.31
		301/549 (55%)

Example 3

[0377] The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.

TABLE 3A
NOV3 Sequence Analysis
	SEQ ID NO: 5	1573 bp
NOV3a,	CGGCCACAGGTTTCCGCTTGCCTCTGGCCGGGGGTCGGCAACTGCAGGCGTCAGTTTCCCTCAA
CG170490-01	GATGGCGGACGAGGAGGCTGGAGGTACTGAGAGGATGGAAATCAGCGCGGAGTTACCCCAGACC
DNA Sequence	CCTCAGCGTCTGGCATCTTGGTGGGATCAGCAAGTTGATTTTTATACTGCTTTCTTGCATCATT
	TGGCACAATTGGTGCCAGAAATTTACTTTGCTGAAATGGACCCAGACTTGGAAAAGCAGGAGGA
	AAGTGTACAAATGTCAATATTCACTCCACTGGAATGGTACTTATTTGGAGAAGATCCAGATATT
	TGCTTAGAGAAATTGAAGCACAGTGGAGCATTTCAGCTTTGTGGGAGGGTTTTCAAAAGTGGAG
	AGACAACCTATTCTTGCAGGGATTGTGCAATTGATCCAACATGTGTACTCTGTATGGACTGCTT
	CCAGGACAGTGTTCATAAAAATCATCGTTACAAGATGCATACTTCTACTGGAGGAGGGTTCTGT
	GACTGTGGAGACACAGAGGCATGGAAAACTGGCCCTTTTTGTGTAAATCATGAACCTGGAAGAG
	CAGGTACTATAAAAGAGAATTCACGCTGTCCGTTGAATGAAGAGGTAATTGTCCAAGCCAGGAA
	AATATTTCCTTCAGTGATAAAATATGTCGTAGAAATGACTATATGGGAAGAGGAAAAAGAACTG
	CCTCCTGAACTCCAGATAAGGGAGAAAAATGAAAGATACTATTGTGTCCTTTTCAATGATGAAC
	ACCATTCATATGACCACGTCATATACAGCCTACAAAGAGCTCTTGACTGTGAGCTCGCAGAGGC
	CCAGTTGCATACCACTGCCATTGACAAAGAGGGTCGTCGGGCTGTTAAAGCGGGAGCTTATGCT
	GCTTGCCAGGAAGCAAAGGAAGATATAAAGAGTCATTCAGAAAATGTCTCTCAACATCCACTTC
	ATGTAGAAGTATTACACTCAGAGATTATGGCTCATCAGAAATTTGCTTTGCGTCTTGGTTCCTG
	GATGAACAAAATTATGAGCTATTCAAGTGACTTTAGGCAGATCTTTTGCCAAGCATGCCTTAGA
	GAAGAACCTGACTCGGAGAATCCCTGTCTCATAAGCAGGTTAATGCTTTGGGATGCAAAGCTTT
	ATAAAGGTGCCCGTAAGATCCTTCATGAATTGATCTTCAGCAGTTTTTTTATGGAGATGGAATA
	CAAAAAACTCTTTGCTATGGAATTTGTGAAGTATTATAAACAACTGCAGAAAGAATATATCAGT
	GATGATCATGACAGAAGTATCTCTATAACTGCACTTTCAGTTCAGATGTTTACTGTTCCTACTC
	TGGCTCGACATCTTATTGAAGAGCAGAATGTTATCTCTGTCATTACTGAAACTCTGCTAGAAGT
	TTTACCTGAGTACTTGGACAGGAACAATAAATTCAACTTCCAGGGTTATAGCCAGGACAAATTG
	GGAAGAGTATATGCAGTAATATGTGACCTAAAGTATATCCTGATCAGCAAACCCACAATATGGA
	CAGAAAGATTAAGAATGCAGTTCCTTGAAGGTTTTCGATCTTTTTTGAAGATTCTTACCTGTAT
	GCAGGGAATGGAAGAAATCCGAAGACAGGTTGGGCAACACATTGAAGTGGATCCTGATTGGGAG
	GCTGCCATTGCTATACAGATGCAATTGAAGAATATTTTACTCATGTTCCAAGAGTGGTGTGCTT
	GTGATGAAGAACTCTTACTTGTGGCTTATAAAGAATGTCACAAAGCTGTGATGAGGTGCAGTAC
	CAGTTTCATATCTAGTAGCAAGACAGTAGTACAATCGTGTGGACATAGTTTGGAAACAAAGTCC
	TACAGAGTATCTGAGGATCTTGTAAGCATACATCTGCCACTCTCTAGGACCCTTGCTGGTCTTC
	ATGTACGTTTAAGCAGGCTGGGTGCTGTTTCAAGACTGCATGAATTTGTGTCTTTTGAGGACTT
	TCAAGTAGAGGTACTAGTGGAATATCCTTTACGTTGTCTGGTGTTGGTTGCCCAGGTTGTTGCT
	GAGATGTGGCGAAGAAATGGACTGTCTCTTATTAGCCAGGTGTTTTATTACCAAGATGTTAAGT
	GCAGAGAAGAAATGTATGATAAAGATATCATCATGCTTCAGATTGGTGCATCTTTAATGGATCC
	CAATAAGTTCTTGTTACTGGTACTTCAGAGGTATGAACTTGCCGAGGCTTTTAACAAGACCATA
	TCTACAAAAGACCAGGATTTGATTAAACAATATAATACACTAATAGAAGAAATGCTTCAGGTCC
	TCATCTATATTGTGGGTGAGCGTTATGTACCTGGAGTGGGAAATGTGACCAAAGAAGAGGTCAC
	AATGAGAGAAATCATTCACTTGCTTTGCATTGAACCCATGCCACACAGTGCCATTGCCAAAAAT
	TTACCTGAGAATGAAAATAATGAAACTGGCTTAGAGAATGTCATAAACAAAGTGGCCACATTTA
	AGAAACCAGGTGTATCAGGCCATGGAGTTTATGAACTAAAAGATGAATCACTGAAAGACTTCAA
	TATGTACTTTTATCATTACTCCAAAACCCAGCATAGCAAGGCTGAACATATGCAGAAGAAAAGG
	AGAAAACAAGAAAACAAAGATGAAGCATTGCCGCCACCACCACCTCCTGAATTCTGCCCTGCTT
	TCAGCAAAGTGATTAACCTTCTCAACTGTGATATCATGATGTACATTCTCAGGACCGTATTTGA
	GCGGGCAATAGACACAGATTCTAACTTGTGGACCGAAGGGATGCTCCAAATGGCTTTTCATATT
	CTGGCATTGGGTTTACTAGAAGAGAAGCAACAGCTTCAAAAAGCTCCTGAAGAAGAAGTAACAT
	TTGACTTTTATCATAAGGCTTCAAGATTGGGAAGTTCAGCCATGAATATACAAATGCTTTTGGA
	AAAACTCAAAGGAATTCCCCAGTTAGAAGGCCAGAAGGACATGATAACGTGGATACTTCAGATG
	TTTGACACAGTGAAGCGATTAAGAGAAAAATCTTGTTTAATTGTAGCAACCACATCAGGATCGG
	AATCTATTAAGAATGATGAGATTACTCATGATAAAGAAAAAGCAGAACGAAAAAGAAAAGCTGA
	AGCTGCTAGGCTACATCGCCAGAAGATCATGGCTCAGATGTCTGCCTTACAGAAAAACTTCATT
	GAAACTCATAAACTCATGTATGACAATACATCAGAAATGCCTGGGAAAGAAGATTCCATTATGG
	AGGAAGAGAGCACCCCAGCAGTCAGTGACTACTCTAGAATTGCTTTGGGTCCTAAACGGGGTCC
	ATCTGTTACTGAAAAGGAGGTGCTGACGTGCATCCTTTGCCAAGAAGAACAGGAGGTGAAAATA
	GAAAATAATGCCATGGTATTATCGGCCTGTGTCCAGAAATCTACTGCCTTAACCCAGCACAGGG
	GAAAACCCATAGAACTCTCAGGAGAAGCCCTAGACCCACTTTTCATGGATCCAGACTTGGCATA
	TGGAACTTATACAGGAAGCTGTGGTCATGTAATGCACGCAGTGTGCTGGCAGAAGTATTTTGAA
	GCTGTACAGCTGAGCTCTCAGCAGCGCATTCATGTTGACCTTTTTGACTTGGAAAGTGGAGAAT
	ATCTTTGCCCTCTTTGCAAATCTCTGTGCAATACTGTGATCCCCATTATTCCTTTGCAACCTCA
	AAAGATAAACAGTGAGAATGCAGATGCTCTTGCTCAACTTTTGACCCTGGCACGGTGGATACAG
	ACTGTTCTGGCCAGAATATCAGGTTATAATATAAGACATGCTAAAGGAGAAAACCCAATTCCTA
	TTTTCTTTAATCAAGGAATGGGAGATTCTACTTTGGAGTTCCATTCCATCCTGAGTTTTGGCGT
	TGAGTCTTCGATTAAATATTCAAATAGCATCAAGGAAATGGTTATTCTCTTTGCCACAACAATT
	TATAGAATTGGATTGAAAGTGCCACCTGATGAAAGGGATCCTCGAGTCCCCATGCTGACCTGGA
	GCACCTGCGCTTTCACTATCCAGGCAATTGAAAATCTATTGGGAGATGAAGGAAAACCTCTGTT
	TGGAGCACTTCAAAATAGGCAGCATAATGGTCTGAAAGCATTAATGCAGTTTGCAGTTGCACAG
	AGGATTACCTGTCCTCAGGTCCTGATACAGAAACATCTGGTTCGTCTTCTATCAGTTGTTCTTC
	CTAACATAAAATCAGAAGATACACCATGCCTTCTGTCTATAGATCTGTTTCATGTTTTGGTGGG
	TGCTGTGTTAGCATTCCCATCCTTGTATTGGGATGACCCTGTTGATCTGCAGCCTTCTTCAGTT
	AGTTCTTCCTATAACCACCTTTATCTCTTCCATTTGATCACCATGGCACACATGCTTCAGATAC
	TACTTACAGTAGACACAGGCCTACCCCTTGCTCAGGTTCAAGAAGACAGTGAAGAGGCTCATTC
	CGCATCTTCTTTCTTTGCAGAAATTTCTCAATATACAAGTGGCTCCATTGGGTGTGATATTCCT
	GGCTGGTATTTGTGGGTCTCACTGAAGAATGGCATCACCCCTTATCTTCGCTGTGCTGCATTGT
	TTTTCCACTATTTACTTGGGGTAACTCCGCCTGAGGAACTGCATACCAATTCTGCAGAAGGAGA
	GTACAGTGCACTCTGTAGCTATCTATCTTTACCTACAAATTTGTTCCTGCTCTTCCAGGAATAT
	TGGGATACTGTAAGGCCCTTGCTCCAGAGGTGGTGTGCAGATCCTGCCTTACTAAACTGTTTGA
	AGCAAAAAAACACCGTGGTCAGGTACCCTAGAAAAAGAAATAGTTTGATAGAGCTTCCTGATGA
	CTATAGCTGCCTCCTGAATCAAGCTTCTCATTTCAGGTGCCCACGGTCTGCAGATGATGAGCGA
	AAGCATCCTGTCCTCTGCCTTTTCTGTGGGGCTATACTATGTTCTCAGAACATTTGCTGCCAGG
	AAATTGTGAACGGGGAAGAGGTTGGAGCTTGCATTTTTCACGCACTTCACTGTGGAGCCGGAGT
	CTGCATTTTCCTAAAAATCAGAGAATGCCGAGTGGTCCTGGTTGAAGGTAAAGCCAGAGGCTGT
	GCCTATCCAGCTCCTTACTTGGATGAATATGGAGAAACAGACCCTGGCCTGAAGAGGGGCAACC
	CCCTTCATTTATCTCGTGAGCGGTATCGGAAGCTCCATTTGGTCTGGCAACAACACTGCATTAT
	AGAAGAGATTGCTAGGAGCCAAGAGACTAATCAGATGTTATTTGGATTCAACTGGCAGTTACTG
	TGAGCTCCAACTCTGCCTCAAGACAATCACAAATGACGACAGTAGTAAAGGCTGATTCAAAATT
	ATGGAAAACTTTCTGAGGGCTGGGAAAGTATTGGAGGGTCTTTTGCTCCATGTCCAGGTTCACT
	TACATCAATAAAATATTTCTTAATGG
	ORF Start: ATG at 66		ORF Stop: TGA at 5313
	SEQ ID NO: 10	11749 aa	MW at 200208.3 kD
NOV3a,	MADEEAGGTERMEISAELPQTPGRLASWWDGQVDFYTAFLHHLAQLVPEIYFAEMDPDLEKQEE
CG170490-01	SVQMSIFTPLEWYLFGEDPDICLEKLKHSGAFQLCGRVFKSGETTYSCRDCAIDPTCVLCMDCF
Protein Sequence	QDSVHKNHRYKMHTSTGGGFCDCGDTEAWKTGPFCVNHEPGRAGTIKENSRCPLNEEVIVQARK
	IFPSVIKYVVEMTIWEEEKELPPELQIREKNERYYCVLFNDEHHSYDHVIYSLQRALDCELAEA
	QLHTTAIDKEGRRAVKAGAYAACQEAKEDIKSHSENVSQHPLHVEVLHSEIMAHQKFALRLGSW
	MNKIMSYSSDFRQIFCQACLREEPDSENPCLISRLMLWDAKLYKGARKILHELIFSSFFMEMEY
	KKLFAMEFVKYYKQLQKEYISDDHDRSISITALSVQMFTVPTLARHLIEEQNVISVITETLLEV
	LPEYLDRNNKFNFQGYSQDKLGRVYAVICDLKYILISKPTIWTERLRMQFLEGFRSFLKILTCM
	QGMEEIRRQVGQHIEVDPDWEAAIAIQMQLKNILLMFQEWCACDEELLLVAYKECHKAVMRCST
	SFISSSKTVVQSCGHSLETKSYRVSEDLVSIHLPLSRTLAGLHVRLSRLGAVSRLHEFVSFEDF
	QVEVLVEYPLRCLVLVAQVVAEMWRRNGLSLISQVFYYQDVKCREEMYDKDIIMLQIGASLMDP
	NKFLLLVLQRYELAEAFNKTISTKDQDLIKQYNTLIEEMLQVLIYIVGERYVPGVGNVTKEEVT
	MREIIHLLCIEPMPHSAIAKNLPENENNETGLENVINKVATFKKPGVSGHGVYELKDESLKDFN
	MYFYHYSKTQHSKAEHMQKKRRKQENKDEALPPPPPPEFCPAFSKVINLLNCDIMMYILRTVFE
	RAIDTDSNLWTEGMLQMAFHILALGLLEEKQQLQKAPEEEVTFDFYHKASRLGSSAMNIQMLLE
	KLKGIPQLEGQKDMITWILQMFDTVKRLREKSCLIVATTSGSESIKNDEITHDKEKAERKRKAE
	AARLHRQKIMAQMSALQKNFIETHKLMYDNTSEMPGKEDSIMEEESTPAVSDYSRIALGPKRGP
	SVTEKEVLTCILCQEEQEVKIENNAMVLSACVQKSTALTQHRGKPIELSGEALDPLFMDPDLAY
	GTYTGSCGHVMHAVCWQKYFEAVQLSSQQRIHVDLFDLESGEYLCPLCKSLCNTVIPIIPLQPQ
	KINSENADALAQLLTLARWIQTVLARISGYNIRHAKGENPIPIFFNQGMGDSTLEFHSILSFGV
	ESSIKYSNSIKEMVILFATTIYRIGLKVPPDERDPRVPMLTWSTCAFTIQAIENLLGDEGKPLF
	GALQNRQHNGLKALMQFAVAQRITCPQVLIQKHLVRLLSVVLPNIKSEDTPCLLSIDLFHVLVG
	AVLAFPSLYWDDPVDLQPSSVSSSYNHLYLFHLITMAHMLQILLTVDTGLPLAQVQEDSEEAHS
	ASSFFAEISQYTSGSIGCDIPGWYLWVSLKNGITPYLRCAALFFHYLLGVTPPEELHTNSAEGE
	YSALCSYLSLPTNLFLLFQEYWDTVRPLLQRWCADPALLNCLKQKNTVVRYPRKRNSLIELPDD
	YSCLLNQASHFRCPRSADDERKHPVLCLFCGAILCSQNICCQEIVNGEEVGACIFHALHCGAGV
	CIFLKIRECRVVLVEGKARGCAYPAPYLDEYGETDPGLKRGNPLHLSRERYRKLHLVWQQHCII
	EEIARSQETNQMLFGFNWQLL

[0378] Further analysis of the NOV3a protein yielded the following properties shown in Table 3B.

TABLE 3B
Protein Sequence Properties NOV3a
SignalP	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 11; pos. chg 1; neg. chg 4
	H-region: length 1; peak value 0.00
	PSG score: −4.40
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −7.16
	possible cleavage site: between 52 and 53
	>>> Seems to have no N-terminal signal peptide
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 1
	Tentative number of TMS(s) for the threshold 0.5: 5
	INTEGRAL	Likelihood =	−1.81	Transmembrane 644-660
	INTEGRAL	Likelihood =	−5.57	Transmembrane 1397-1413
	INTEGRAL	Likelihood =	−1.28	Transmembrane 1438-1454
	INTEGRAL	Likelihood =	−3.50	Transmembrane 1625-1641
	INTEGRAL	Likelihood =	−2.76	Transmembrane 1652-1668
	PERIPHERAL	Likelihood =	1.27 (at 412)
	ALOM score: −5.57 (number of TMSs: 5)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 651
	Charge difference: 4.0 C(1.0)-N(−3.0)
	C > N: C-terminal side will be inside
	>>> membrane topology: type 3b
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment(75):	9.30
	Hyd Moment (95):	7.89	G content:	0
	D/E content:	2	S/T content:	0
	Score: −6.40
	Gavel: prediction of cleavage sites for mitochondrial preseq
	cleavage site motif not found
	NUCDISC: discrimination of nuclear localization signals
	pat4: KKRR (5) at 851
	pat4: KRRK (5) at 852
	pat4: RKRK (5) at 1019
	pat4: PRKR (4) at 1588
	pat7: PRKRNSL (5) at 1588
	bipartite: none
	content of basic residues: 10.2%
	NLS Score: 1.15
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals: none
	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:
	Leucine zipper pattern (PS00029): *** found ***
	LVSIHLPLSRTLAGLHVRLSRL at 604
	none
	checking 71 PROSITE ribosomal protein motifs:
	Ribosomal protein S16 signature (PS00732): *** found ***
	LHVRLSRLGA at 618
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear
	discrimination
	Prediction: cytoplasmic
	Reliability: 76.7
	COIL: Lupas's algorithm to detect coiled-coil regions
	1004 I 0.56
	1005 K 0.56
	1006 N 0.56
	1007 D 0.56
	1008 E 0.56
	1009 I 0.56
	1010 T 0.56
	1011 H 0.56
	1012 D 0.56
	1013 K 0.56
	1014 E 0.56
	1015 K 0.56
	1016 A 0.56
	1017 E 0.56
	1018 R 0.56
	1019 K 0.56
	1020 R 0.56
	1021 K 0.56
	1022 A 0.56
	1023 E 0.56
	1024 A 0.56
	1025 A 0.56
	1026 R 0.56
	1027 L 0.56
	1028 H 0.56
	1029 R 0.56
	1030 Q 0.56
	1031 K 0.56
	1032 I 0.56
	total: 29 residues
	Final Results (k = {fraction (9/23)}):
	56.5%: endoplasmic reticulum
	13.0%: vacuolar
	8.7%: mitochondrial
	8.7%: nuclear
	4.3%: Golgi
	4.3%: vesicles of secretory system
	4.3%: cytoplasmic
	>> prediction for CG170490-01 is end (k = 23)

[0379] A search of the NOV3a protein against the Gene seq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3C.

TABLE 3C
Geneseq Results for NOV3a
		NOV3a	Identities/
Geneseq	Protein/Organism/Length [Patent #,	Residues/	Similarities for	Expect
Identifier	Date]	Match Residues	the Matched Region	Value
AAB31162	Amino acid sequence of Mouse	1 . . . 1749	1619/1757 (92%)	0.0
	Ubr1 protein - Mus sp, 1757 aa.	1 . . . 1757	1683/1757 (95%)
	[US6159732-A, 12 DEC. 2000]
AAW84351	Murine ubiquitin-protein ligase Ubr1 -	1 . . . 1749	1619/1757 (92%)	0.0
	Mus sp, 1757 aa. [US5861312-A,	1 . . . 1757	1683/1757 (95%)
	19 JAN. 1999]
AAB93464	Human protein sequence SEQ ID	204 . . . 1014	811/811 (100%)	0.0
	NO: 12732 - Homo sapiens, 811 aa.	1 . . . 811	811/811 (100%)
	[EP1074617-A2, 07 FEB. 2001]
AAM78576	Human protein SEQ ID NO 1238 -	356 . . . 1749	672/1417 (47%)	0.0
	Homo sapiens, 1400 aa.	1 . . . 1400	947/1417 (66%)
	[WO200157190-A2, 09 AUG. 2001]
AAM79560	Human protein SEQ ID NO 3206 -	357 . . . 1749	671/1416 (47%)	0.0
	Homo sapiens, 1400 aa.	2 . . . 1400	946/1416 (66%)
	[WO200157190-A2, 09 AUG. 2001]

[0380] In a BLAST search of public sequence databases, the NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3D.

TABLE 3D
Public BLASTP Results for NOV3a
Protein		NOV3a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
AAL32103	Ubiquitin ligase E3 alpha-I -	1 . . . 1749	1749/1749 (100%)	0.0
	Homo sapiens (Human), 1749 aa.	1 . . . 1749	1749/1749 (100%)
AAO14997	UBR1 E3a ligase - Homo sapiens	18 . . . 1727	1697/1710 (99%)	0.0
	(Human), 1709 aa (fragment).	1 . . . 1709	1698/1710 (99%)
O70481	Ubiquitin-protein ligase E3	1 . . . 1749	1619/1757 (92%)	0.0
	COMPONEN N-recognin	1 . . . 1757	1683/1757 (95%)
	(Ubiquitin-protein ligase E3-alpha) -
	Mus musculus (Mouse), 1757 aa.
AAL32101	Ubiquitin ligase E3 alpha-II - Homo	23 . . . 1749	833/1752 (47%)	0.0
	sapiens (Human), 1755 aa.	22 . . . 1755	1168/1752 (66%)
BAC40933	Activated spleen cDNA, RIKEN	1 . . . 849	801/849 (94%)	0.0
	full-length enriched library,	1 . . . 849	821/849 (96%)
	clone: F830005C07 product: ubiquitin
	protein ligase E3 component
	n-recognin 1, full insert sequence -
	Mus musculus (Mouse), 849 aa (fragment).

[0381] PFam analysis predicts that the NOV3a protein contains the domains shown in the Table 3E.

TABLE 3E
Domain Analysis of NOV3a
		Identities/
	NOV3a	Similarities for	Expect
Pfam Domain	Match Region	the Matched Region	Value
zf-UBR1	97 . . . 167	42/80 (52%)	1.2e−37
		68/80 (85%)
DUF174	214 . . . 299	17/87 (20%)	0.18
		39/87 (45%)

Example 4

[0382] The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.

TABLE 4A
NOV4 Sequence Analysis
NOV4a,	CTGTGAGCCGCGAGAGGCCCGGGAGCCGCGCGTCGCCGAGCCGAGCTGACCGAGAGCCCCATGG
CG170667-01	CTGTGCAGCGCGCCGCGTCTCCGCGCCGCCCGCCCGCCCCGCTCTGGCCCCCGCTCCTGCTCCC
DNA Sequence	GCTGCTGTTGCTGCTGCTGCCCGCGCCGAGCGAGGGTCTTGGCCACTCTGCTGAACTGGCATTT
	GCTGTGGAGCCAAGTGATGATGTTGCCGTCCCCGGGCAGCCTATAGTGCTGGACTGCAGGGTGG
	AGGGGACCCCTCCAGTGCGAATCACCTCGAGGAAGAATGGGGTAGAGCTGCCAGAGAGTACCCA
	CTCCACCTTGCTGGCCAATGGGTCCTTGATGATCCGTCACTTCAGGCTGGAGCCGGGAGGCAGC
	CCTTCGGATGAAGGTGACTATGAGTGTGTGGCCCAGAACCGCTTTGGGCTGGTGGTCAGCCGGA
	AGGCTCGCATCCAAGCTGCAACCATGTCCGACTTCCACGTGCATCCCCAGGCCACCGTGGGTGA
	GGAGGGTGGTGTGGCCCGCTTCCAGTGCCAAATCCATGGGCTTCCCAAACCCCTGATCACTTGG
	GAGAAGAACAGAGTCCCAATTGACACGGACAATGAGAGGTACACATTGCTGCCCAAGGGGGTCC
	TGCAGATCACAGGACTTCGAGCTGAGGACGGTGGCATCTTCCACTGTGTGGCCTCAAACATCGC
	CAGTATCCCGATCAGCCACGGGGCCAGGCTCACTGTGTCAGGCTCGGGCTCTGGGGCCTACAAG
	GAGCCAGCCATCCTCGTGGGGCCTGAGAACCTCACCCTGACAGTGCACCAGACCGCGCTGCTTG
	AGTGTGTCGCCACGGGCAACCCGCGCCCCATTGTGTCCTGGAGCCGCCTGGATGGTCGCCCTAT
	CGGGGTGGAGGGCATCCAGGTGCTGCGCACAGGAAACCTCATCATCTCAGACGTGACGGTCCAG
	CACTCTGGCGTCTACGTCTGTGCAGCCAACAGACCTGGCACCCGGGTGAGGACAACGGCACAGG
	GCCGGCTGGTGGTGCAAGCCCCAGCTGAGTTTGTGCAGCATCCCCAGTCCATCTCCAGGCCAGC
	TGGGACCACAGCCATGTTCACCTGCCAAGCCCAGGGTGAGCCACCGCCTCATGTCACGTGGCTG
	AAAAATGGACAGGTGCTGGGGCCAGGAGGCCACGTCAGGCTCAAGAATAACAACAGCACACTGA
	CCATTTCTGGAATCGGTCCTGAGGATGAAGCCATTTATCAGTGTGTGGCCGAGAACAGTGCGGG
	CTCATCACAGGCCAGTGCCAGGCTGACCGTACTGTGGGCTGAGGGGCTCCCCGGGCCTCCCCGC
	AATGTGCGGGCAGTCTCTGTGTCTTCCACTGAGGTGCGTGTGTCCTGGAGTGAGCCGCTGGCCA
	ACACCAAGGAGATCATCGGCTACGTCCTGCACATCAGGAAGGCTGCTGACCCACCGGAGCTGGA
	GTATCAGGAGGCAGTCAGCAAGAGCACCTTTCAGCACCTGGTCAGCGACCTGGAGCCCTCCACA
	GCCTACAGTTTCTACATCAACGCCTACACACCAAGGGGGGCCAGCTCAGCCTCTGTGCCCACCC
	TAGCTAGCACCCTGGGTGAAGCCCCTGCCCCACCCCCACTGTCAGTGCGAGTCCTGGGCAGCTC
	CTCCTTGCAGCTGCTGTGGGAGCCTTGGCCCCGGCTGGCCCAGCACGAGGGCGGCTTCAAGCTG
	TTTTACCGCCCAGCAAGCAAGACCTCCTTCACCGGCCCCATCCTGCTGCCTGGAACCGTCTCCT
	CCTACAACCTCAGCCAGCTCGACCCCACTGCAGTGTATGAGGTGAAGCTGCTCGCCTACAGCCA
	GCATGGAGATGGCAATGCCACAGTCCGCTTTGTGTCTTTGAGGGGAGCATCTGAGAGGACAGGC
	ATCGTCATCGGCATCCACATCGGGGTCACTTGCATCATCTTCTGTGTCCTCTTCCTCCTGTTCG
	GCCAAAGGGGCAGGGTCCTCCTGTGTAAAGATGTGGAAAACCAGCTGTCCCCTCCACAGGGTCC
	CCGGAGCCAGAGGGACCCTGGCATTCTGGCCCTAAATGGGGCGAGACGGGGACAGCGGGGCCAG
	CTGGGCCGAGACGAGAAACGTGTGGATATGAAGGAGCTGGAGCAGCTGTTCCCCCCGGCCAGCG
	CAGCAGGGCAGCCGGACCCCAGACCCACACAGGATCCTGCAGCCCCCGCTCCGTGTGAGGAGAC
	CCAGCTCTCCGTGCTGCCACTTCAGGGGTGCGGCCTGATGGAGGGGAAGACGACGGACGCGAAG
	ACCACAGAGGCCACGGCTCCCTGCGCCGGCCTGGCGGCTGCCCCACCACCCCCAGATGGAGGCC
	CTGGCCTCCTCAGTGAAGGCCAGGCTTCCAGGCCTGCAGCGGCCCGCGTTACCCAGCCAGCTCA
	CTCGGAACAGTAGCCAGTGTCTGGCAGGCTCCAGAGGGTCGACGGAGCGGGGCCCATTCTCAGG
	TCAAAAGCAAGATTTCTACTGTCATGTGGGATTTGGATGGTCCTGGGGGCTCCCCAGCATTTCT
	ATCCTGACTGCCTCTTGGGTTGTCAAAACCCAAGGCAGCCTTGACAGGGACCCCCCGGCCCTAA
	CACCCATCAGGAGTTGGAGCAGTTCCTGCAGGAGCCTGTTCCTTCCCTGGGCTGACGCCCCCTT
	GCCTCTGCCTGGTACCCACATGACTTGGAACTGAACTAACATTTTTCTTTAAAAAGCAAA
	ORF Start: ATG at 61		ORF Stop: TAG at 2443
	SEQ ID NO: 12	794 aa	MW at 84591.3 kD
NOV4a,	MAVQRAASPRRPPAPLWPRLLLPLLLLLLPAPSEGLGHSAELAFAVEPSDDVAVPGQPIVLDCR
CG170667-01	VEGTPPVRITWRKNGVELPESTHSTLLANGSLMIRHFRLEPGGSPSDEGDYECVAQNRFGLVVS
Protein Sequence	RKARIQAATMSDFHVHPQATVGEEGGVARFQCQIHGLPKPLITWEKNRVPIDTDNERYTLLPKG
	VLQITGLRAEDGGIFHCVASNIASIRISHGARLTVSGSGSGAYKEPAILVGPENLTLTVHQTAV
	LECVATGNPRPIVSWSRLDGRPIGVEGIQVLGTGNLIISDVTVQHSGVYVCAANRPGTRVRRTA
	QGRLVVQAPAEFVQHPQSISRPAGTTAMFTCQAQGEPPPHVTWLKNGQVLGPGGHVRLKNNNST
	LTISGIGPEDEAIYQCVAENSAGSSQASARLTVLWAEGLPGPPRNVRAVSVSSTEVRVSWSEPL
	ANTKEIIGYVLHIRKAADPPELEYQEAVSKSTFQHLVSDLEPSTAYSFYIKAYTPRGASSASVP
	TLASTLGEAPAPPPLSVRVLGSSSLQLLWEPWPRLAWHEGGFKLFYRPASKTSFTGPILLPGTV
	SSYNLSQLDPTAVYEVKLLAYSQHGDGNATVRFVSLRGASERTGIVIGIHIGVTCIIFCVLFLL
	FGQRGRVLLCKDVENQLSPPQGPRSQRDPGILALNGARRGQRGQLGRDEKRVDMKELEQLFPPA
	SAAGQPDPRPTQDPAAPAPCEETQLSVLPLQGCGLMEGKTTEAKTTEATAPCAGLAAAPPPPDG
	GPGLLSEGQASRPAAARVTQPAHSEQ
	SEQ ID NO: 13	2805 bp
NOV4b,	CTGTGAGCCGCGAGAGGCCCCGGAGCCGCGCGTCGCCGAGCCGAGCTGACCGAGAGCCCCATGG
CG170667-02	CTGTCCAGCGCGCCGCGTCTCCGCGCCGCCCGCCCGCCCCGCTCTGGCCCCGGCTCCTGCTGCC
DNA Sequence	GCTGCTGTTGCTGCTGCTGCCCGCGCCGAGCGAGGGTCTTGGCCACTCTGCTGAACTGGCATTT
	GCTGTGGAGCCAAGTGATGATGTTGCCGTCCCCGGGCAGCCTATAGTGCTGGACTGCAGGGTGG
	AGGGGACCCCTCCAGTGCGAATCACCTGGAGGAAGAATGGGGTAGAGCTGCCAGAGAGTACCCA
	CTCCACCTTGCTGGCCAATGGGTCCTTGATGATCCGTCACTTCAGGCTGGAGCCGGGAGGCAGC
	CCTTCGGATGAAGGTGACTATGAGTGTGTGGCCCAGAACCGCTTTGGGCTGGTGGTCAGCCGGA
	AGGCTCCCATCCAAGCTGCAACCATGTCGGACTTCCACGTGCATCCCCAGGCCACCGTCGGTGA
	GGAGGGTGGTGTGGCCCGCTTCCAGTGCCAAATCCATGGGCTTCCCAAACCCCTGATCACTTGG
	GAGAAGAACAGAGTCCCAATTGACACGGACAATGAGAGGTACACATTGCTGCCCAACGGGGTCC
	TGCAGATCACAGGACTTCGAGCTGAGGACGGTGGCATCTTCCACTGTGTGGCCTCAAACATCGC
	CAGTATCCGGATCAGCCACGGGGCCAGGCTCACTGTGTCAGGCTCGGGCTCTGGGGCCTACAAG
	GAGCCAGCCATCCTCGTGGGGCCTGAGAACCTCACCCTGACAGTGCACCACACCGCGGTGCTTG
	AGTGTGTCGCCACGGGCAACCCGCGCCCCATTGTGTCCTGGAGCCGCCTGGATGGTCGCCCTAT
	CGGGGTGGAGGGCATCCAGGTGCTGCGCACAGGAAACCTCATCATCTCAGACGTGACCGTCCAG
	CACTCTGGCGTCTACGTCTGTGCAGCCAACAGACCTGGCACCCGGGTGAGGAGAACGGCACAGG
	GCCGGCTGGTGGTGCAAGCCCCAGCTGAGTTTGTGCAGCATCCCCAGTCCATCTCCAGGCCAGC
	TGGGACCACAGCCATGTTCACCTGCCAAGCCCAGCGTGAGCCACCGCCTCATGTCACGTGGCTG
	AAAAATGGACAGGTGCTGGGGCCAGGAGGCCACGTCAGGCTCAAGAATAACAACAGCACACTGA
	CCATTTCTGGAATCGGTCCTGAGGATGAAGCCATTTATCAGTGTGTGGCCGAGAACAGTGCGGG
	CTCATCACAGGCCAGTGCCAGGCTGACCGTACTGTGGGCTGAGGGGCTCCCCGGGCCTCCCCGC
	AATGTGCGGGCAGTCTCTGTGTCTTCCACTGAGGTGCGTGTGTCCTGGAGTGAGCCGCTGGCCA
	ACACCAAGGAGATCATCGGCTACGTCCTGCACATCACGAAGGCTGCTGACCCACCGGAGCTGGA
	GTATCAGGAGGCAGTCAGCAAGAGCACCTTTCAGCACCTCGTCAGCGACCTCGAGCCCTCCACA
	GCCTACAGTTTCTACATCAAGGCCTACACACCAAGGGGGGCCAGCTCAGCCTCTGTGCCCACCC
	TAGCTAGCACCCTGGGTGAAGCCCCTGCCCCACCCCCACTGTCAGTGCGAGTCCTGGGCAGCTC
	CTCCTTGCAGCTGCTGTCGGAGCCTTGGCCCCCGCTGGCCCAGCACGAGGGCGGCTTCAAGCTG
	TTTTACCGCCCAGCAAGCAAGACCTCCTTCACCGGCCCCATCCTGCTGCCTGGAACCGTCTCCT
	CCTACAACCTCAGCCAGCTCGACCCCACTGCAGTGTATGAGGTGAAGCTGCTCGCCTACAGCCA
	GCATGGAGATGGCAATGCCACAGTCCGCTTTGTGTCTTTGAGGGGAGCATCTGAGAGGACAGCC
	TTGAGCCCACCATGTGACTGCCGGAAGGAGGAGGCCGCCAACCAGACGTCCACCACAGGCATCG
	TCATCGGCATCCACATCGGGGTCGCTTCCATCATCTTCTGTGTCCTCTTCCTCCTGTTCGGCCA
	AAGGGGCAGGGTCCTCCTGTGTAAAGATGTGGAAAACCAGCTGTCCCCTCCACAGGGTCCCCGG
	AGCCAGAGGGACCCTGGCATTCTGGCCCTAAATGGGGCGAGACGGGGACAGCGGGGCCAGCTGG
	GCCGAGACGAGAAACGTGTGGATATGAAGGAGCTGGAGCAGCTGTTCCCCCCGGCCAGCGCAGC
	AGGGCGGCCGGACCCCAGACCCACACAGGATCCTGCAGCCCCCGCTCCGTGTGAGGAGACCCAG
	CTCTCCTTGCTGCCACTTCAGGGGTGCGGCCTGATGGACGGGAAGACGACGGAGGCGAAGACCA
	CAGAGGCCACGGCTCCCTGCGCCGGCCTGGCGGCTGCCCCACCACCCCCAGATGGAGGCCCTGG
	CCTCCTCAGTGAAGGCCAGGCTTCCAGGCCTGCAGCGGCCCGGGTTACCCAGCCAGCTCACTCG
	GAACAGTAGCCAGTGTCTGGCAGGCTCCAGAGGGTGGACGGAGCGGGGCCCATTCTCAGGTCAA
	AAGCAAGATTTCTACTGTCATGTGGGATTTGGATGGTCCTGGGGGCTCCCCAGCATTTCTATCC
	TGACTGCCTCTTGGGTTGTCAAAACCCAAGGCAGCCTTGACAGGGACCCCCCCGCCCTAACACC
	CATCAGGAGTTGGAGCAGTTCCTGCAGGAGCCTGTTCCTTCCCTGGGCTGACGCCCCCTTGCCT
	CTGCCTGGTACCCACATGACTTGGAACTGAACTAACATTTTTCTTTAAAAAGC
	ORF Start: ATG at 61		ORF Stop: TAG at 2503
	SEQ ID NO: 14	814 aa	MW at 86707.6kD
NOV4b,	MAVQRAASPRRPPAPLWPRLLLPLLLLLLPAPSEGLGHSAELAFAVEPSDDVAVPGQPIVLDCR
CG170667-02	VEGTPPVRITWRKNGVELPESTHSTLLANGSLMIRHFRLEPGGSPSDEGDYECVAQNRFGLVVS
Protein Sequence	RKARIQAATMSDFHVHPQATVGEEGGVARFQCQIHGLPKPLITWEKNRVPIDTDNERYTLLPKG
	VLQITGLRAEDGGIFHCVASNIASIRISHGARLTVSGSGSGAYKEPAILVCPENLTLTVHQTAV
	LECVATGNPRPIVSWSRLDGRPIGVEGIQVLGTGNLIISDVTVQHSGVYVCAANRPGTRVRRTA
	QGRLVVQAPAEFVQHPQSISRPAGTTAMFTCQAQGEPPPHVTWLKNGQVLGPGGHVRLKNNNST
	LTISGIGPEDEAIYQCVAENSAGSSQASARLTVLWAEGLPGPPRNVRAVSVSSTEVRVSWSEPL
	ANTKEIIGYVLHIRKAADPPELEYQEAVSKSTFQHLVSDLEPSTAYSFYIKAYTPRGASSASVP
	TLASTLGEAPAPPPLSVRVLGSSSLQLLWEPWPRLAQHEGGFKLPYRPASKTSFTGPILLPGTV
	SSYNLSQLDPTAVYEVKLLAYSQHGDGNATVRFVSLRGASERTALSPPCDCRKEEAANQTSTTG
	IVIGIHIGVACIIFCVLFLLFGQRGRVLLCKDVENQLSPPQGPRSQRDPGILALNGARRGQRGQ
	LGRDEKRVDMKELEQLFPPASAAGRPDPRPTQDPAAPAPCEETQLSLLPLQGCGLMEGKTTEAK
	TTEATAPCAGLAAAPPPPDGGPGLLSEGQASRPAAARVTQPAHSEQ

[0383] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 4B.

TABLE 4B
Comparison of NOV4a against NOV4b.
			Identities/
	Protein	NOV4a Residues/	Similarities for the
	Sequence	Match Residues	Matched Region
	NOV4b	1 . . . 794	791/814 (97%)
		1 . . . 814	793/814 (97%)

[0384] Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.

TABLE 4C
Protein Sequence Properties NOV4a
SignalP	Cleavage site between residues 36 and 37
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 11; pos. chg 3; neg. chg 0
	H-region: length 7; peak value −3.55
	PSG score: −7.95
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): 3.23
	possible cleavage site: between 35 and 36
	>>> Seems to have no N-terminal signal peptide
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 1
	Tentative number of TMS(s) for the threshold 0.5: 2
	Number of TMS(s) for threshold 0.5: 1
	INTEGRAL	Likelihood =	−10.61	Transmembrane 625-641
	PERIPHERAL	Likelihood =	3.45 (at 279)
	ALOM score: −10.61 (number of TMSs: 1)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 632
	Charge difference: −0.5 C(1.0)-N(1.5)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 2 (cytoplasmic tail 1 to 625)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	4	Hyd Moment (75):	9.73
	Hyd Moment(95):	7.17	G content:	0
	D/E content:	1	S/T content:	2
	Score: −0.12
	Gavel: prediction of cleavage sites for mitochondrial preseq
	R-2 motif at 29 PRL|LL
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: PGTRVRR (3) at 312
	bipartite: none
	content of basic residues: 9.1%
	NLS Score: −0.22
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	XXRR-like motif in the N-terminus: AVQR none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	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 20
	LL at 21
	LL at 24
	LL at 25
	LL at 26
	LL at 27
	LL at 28
	LL at 90
	LL at 188
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 55.5
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	43.5%: mitochondrial
	26.1%: cytoplasmic
	8.7%: vacuolar
	8.7%: endoplasmic reticulum
	4.3%: Golgi
	4.3%: vesicles of secretory system
	4.3%: nuclear
	>> prediction for CG170667-01 is mit (k = 23)

[0385] A search of the NOV4a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4D.

TABLE 4D
Geneseq Results for NOV4a
		NOV4a	Identities/
Geneseq	Protein/Organism/Length [Patent #,	Residues/	Similarities for	Expect
Identifier	Date]	Match Residues	the Matched Region	Value
AAE14781	Human immunoglobulin superfamily	1 . . . 794	791/794 (99%)	0.0
	protein (IGSFP)-1 - Homo sapiens, 793	1 . . . 793	793/794 (99%)
	aa. [WO200240671-A2, 23 MAY 2002]
ABG12152	Novel human diagnostic protein	138 . . . 785	646/668 (96%)	0.0
	#12143 - Homo sapiens, 898 aa.	69 . . . 736	648/668 (96%)
	[WO200175067-A2, 11 OCT. 2001]
AAG65914	Amino acid sequence of GSK gene Id	19 . . . 603	244/605 (40%)	e−111
	27142 - Homo sapiens, 1250 aa.	10 . . . 610	335/605 (55%)
	[WO200172961-A2, 04 OCT. 2001]
AAE05252	Mouse Nope (neighbour of punc ell)	29 . . . 603	236/594 (39%)	e−108
	extracellular domain - Mus musculus,	3 . . . 588	330/594 (54%)
	932 aa. [WO200149714-A2, 12 JUL. 2001]
AAE05251	Mouse Nope (neighbour of punc ell)	29 . . . 603	236/594 (39%)	e−108
	protein - Mus musculus, 1252 aa.	24 . . . 609	330/594 (54%)
	[WO200149714-A2, 12 JUL. 2001]

[0386] In a BLAST search of public sequence databases, the NOV4a protein was found to have homology to the proteins shown in the BLASTP data in Table 4E.

TABLE 4E
Public BLASTP Results for NOV4a
Protein		NOV4a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
AAH42054	Similar to putative neuronal cell adhesion	1 . . . 794	792/814 (97%)	0.0
	molecule - Homo sapiens (Human), 814 aa.	1 . . . 814	794/814 (97%)
O70246	Putative neuronal cell adhesion molecule	1 . . . 784	688/796 (86%)	0.0
	(PUNC) (Putative neuronal cell adhesion	1 . . . 790	718/796 (89%)
	molecule, short form) - Mus musculus
	(Mouse), 793 aa.
BAC34502	9 days embryo whole body cDNA,	1 . . . 784	688/816 (84%)	0.0
	RIKEN full-length enriched library,	1 . . . 810	718/816 (87%)
	clone: D030056K15 product: putative
	neuronal cell adhesion molecule, full
	insert sequence - Mus musculus (Mouse), 813 aa.
O95215	Putative neuronal cell adhesion molecule -	507 . . . 794	284/308 (92%)	e−161
	Homo sapiens (Human), 308 aa (fragment).	1 . . . 308	286/308 (92%)
Q8TDY8	HDDM36 - Homo sapiens (Human), 1250 aa.	19 . . . 603	244/605 (40%)	e−110
		10 . . . 610	335/605 (55%)

[0387] PFam analysis predicts that the NOV4a protein contains the domains shown in the Table 4F.

TABLE 4F
Domain Analysis of NOV4a
			Identities/
	Pfam	NOV4a	Similarities for	Expect
	Domain	Match Region	the Matched Region	Value
	ig	56 . . . 119	17/67 (25%)	2.5e−07
			46/67 (69%)
	ig	153 . . . 211	15/62 (24%)	1e−05
			41/62 (66%)
	ig	252 . . . 309	16/61 (26%)	1.8e−08
			42/61 (69%)
	ig	344 . . . 402	21/62 (34%)	3e−13
			47/62 (76%)
	fn3	424 . . . 510	24/88 (27%)	6.6e−18
			68/88 (77%)
	fn3	521 . . . 606	28/87 (32%)	6e−07
			61/87 (70%)

Example 5

[0388] The NOV5 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 5A.

TABLE 5A
NOV5 Sequence Analysis
	SEQ ID NO: 15	1439 bP
NOV5a,	ACGCTCAGCCTCGGCCCCCCACAGACGGGGCTCTGCATCGTCTCTGATATGTCACCCACCATCT
CG170791-01	CCCACAAGGACAGCAGCCGGCAACGGCGGCCAGGGAATTTCAGTCACTCTCTGGATATGAAGAG
DNA Sequence	CGGTCCCCTGCCGCCAGGCGGTTGGGATGACAGTCATTTGGACTCAGCGGGCCGGGAAGGGGAC
	AGAGAAGCTCTTCTGGGGGATACCGGCACTGGCGACTTCTTAAAAGCCCCACAGAGCTTCCGGG
	CCGAACTAAGCAGCATTTTGCTACTACTCTTTCTTTACGTGCTTCAGGGTATTCCCCTGGGCTT
	GGCGGGAAGCATCCCACTCATTTTGCAAAGCAAAAATGTTAGCTATACAGACCAAGCTTTCTTC
	AGTTTTGTCTTTTGGCCCTTCAGTCTCAAATTACTCTGGGCCCCGTTGGTTGATGCGGTCTACG
	TTAAGAACTTCGGTCGTCGCAAATCTTGGCTTGTCCCGACACAGTATATACTAGGACTCTTCAT
	GATCTATTTATCCACTCAGGTGGACCGTTTGCTTGGGAATACCGATGACAGAACACCCGACGTG
	ATTGCTCTCACTGTGGCGTTCTTTTTGTTTGAATTCTTGGCCGCCACTCAGGACATTGCCGTCG
	ATGGTTGGGCGTTAACTATGTTATCCAGGGAAAATGTGGGTTATGCTTCTACTTGCAATTCGGT
	GGGCCAAACAGCGGGTTACTTTTTGGGCAATGTTTTGTTTTTGGCCCTTGAATCTGCCGACTTT
	TGTAACAAATATTTGCGGTTTCAGCCTCAACCCAGAGGAATCGTTACTCTTTCAGATTTCCTTT
	TTTTCTGGGGAACTGTATTTTTAATAACAACAACATTGGTTGCCCTTCTGAAAAAAGAAAACGA
	AGTATCAGTAGTAAAAGAAGAAACACAAGGGATCACAGATACTTACAAGCTGCTTTTTGCAATT
	ATAAAAATGCCAGCAGTTCTGACATTTTGCCTTCTGATTCTAACTGCAAAGGTTACAGTGTACA
	GCATGTATGTTTCTATAATGGCTTTCAATGCAAAGGTTAGTGATCCACTTATTGGAGGAACATA
	CATGACCCTTTTAAATACCGTGTCCAATCTGGGAGGAAACTGGCCTTCTACAGTAGCTCTTTGG
	CTTGTAGATCCCCTCACAGTAAAAGAGTGTGTAGGAGCATCAAACCAGAATTGTCGAACACCTG
	ATGCTGTTGAGCTTTGCAAAAAACTGGGTGGCTCATGTGTTACAGCCCTGGATGGTTATTATGT
	GGAGTCCATTATTTGTGTTTTCATTGGATTTGGTTGGTGGTTCTTTCTTGGTCCAAAATTTAAA
	AAGTTACAGGATGAAGGATCATCTTCGTGGAAATGCAAAAGGAACAATTAATATATATGCTACT
	GGACATTCTAGCAAGGTTGAATTTTAGAGTG
	ORF Start: ATG at 49		ORF Stop: TAA at 1393
	SEQ ID NO: 16	448 aa	MW at 49630.9 kD
NOV5a,	MSPTISHKDSSRQRRPGNFSHSLDMKSGPLPPGGWDDSHLDSAGREGDREALLGDTGTGDFLKA
CG170791-01	PQSFRAELSSILLLLFLYVLQGIPLGLAGSIPLILQSKNVSYTDQAFFSFVFWPFSLKLLWAPL
Protein Sequence	VDAVYVKNFGRRKSWLVPTQYILGLFMIYLSTQVDRLLGNTDDRTPDVIALTVAFFLFEFLAAT
	QDIAVDGWALTMLSRENVGYASTCNSVGQTAGYFLGNVLFLALESADFCNKYLRFQPQPRGIVT
	LSDFLFFWGTVFLITTTLVALLKKENEVSVVKEETQGITDTYKLLFAIIKMPAVLTFCLLILTA
	KVTVYSMYVSIMAFNAKVSDPLIGGTYMTLLNTVSNLGGNWPSTVALWLVDPLTVKECVGASNQ
	NCRTPDAVELCKKLGGSCVTALDGYYVESIICVFIGFGWWFFLGPKFKKLQDEGSSSWKCKRNN

[0389] Further analysis of the NOV5a protein yielded the following properties shown in Table 5B.

TABLE 5B
Protein Sequence Properties NOV5a
Signal	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 9; pos. chg 1; neg. chg 1
	H-region: length 2; peak value −20.41
	PSG score: −24.81
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −13.86
	possible cleavage site: between 32 and 33
	>>> Seems to have no N-terminal signal peptide
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 1
	Tentative number of TMS(s) for the threshold 0.5: 5
	INTEGRAL	Likelihood =	−6.53	Transmembrane	75-91
	INTEGRAL	Likelihood =	−5.79	Transmembrane	176-192
	INTEGRAL	Likelihood =	−3.88	Transmembrane	261-277
	INTEGRAL	Likelihood =	−7.11	Transmembrane	301-317
	INTEGRAL	Likelihood =	−2.97	Transmembrane	411-427
	PERIPHERAL	Likelihood =	1.38 (at 110)
	ALOM score: −7.11 (number of TMSs: 5)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 82
	Charge difference: 0.0 C(0.0)-N(0.0)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 3a
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	3	Hyd Moment (75):	8.26
	Hyd Moment(95):	5.54	G content:	1
	D/E content:	2	S/T content:	7
	Score: −2.44
	Gavel: prediction of cleavage sites for mitochondrial
	preseq
	R-2 motif at 25 RRP|GN
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: PKFKKLQ (5) at 429
	bipartite: none
	content of basic residues: 8.5%
	NLS Score: −0.04
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	KKXX-like motif in the C-terminus: CKRN
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	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:
	Leucine zipper pattern (PS00029): *** found ***
	LVPTQYILGLFMIYLSTQVDRL at 144
	none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	39.1%: mitochondrial
	39.1%: endoplasmic reticulum
	13.0%: nuclear
	4.3%: plasma membrane
	4.3%: vesicles of secretory system
	>> prediction for CG170791-01 is mit (k = 23)

[0390] 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 5C.

TABLE 5C
Geneseq Results for NOV5a
		NOV5a	Identities/
Geneseq	Protein/Organism/Length [Patent #,	Residues/	Similarities for	Expect
Identifier	Date]	Match Residues	the Matched Region	Value
AAW70898	Acetyl-coenzyme A transporter (AT)	1 . . . 322	321/322 (99%)	0.0
	protein - Homo sapiens, 549 aa.	1 . . . 322	322/322 (99%)
	[US5851788-A, 22 DEC. 1998]
AAW69948	Human acetyl coenzyme A transporter	1 . . . 322	321/322 (99%)	0.0
	(AT-1) protein - Homo sapiens, 549 aa.	1 . . . 322	322/322 (99%)
	[WO9833816-A1, 06 AUG. 1998]
ABB64604	Drosophila melanogaster polypeptide	44 . . . 323	157/289 (54%)	5e−84
	SEQ ID NO 20604 - Drosophila	10 . . . 297	203/289 (69%)
	melanogaster, 525 aa.
	[WO200171042-A2, 27 SEP. 2001]
AAO00018	Human polypeptide SEQ ID NO 13910 -	321 . . . 432	94/112 (83%)	1e−47
	Homo sapiens, 151 aa.	40 . . . 151	97/112 (85%)
	[WO200164835-A2, 07 SEP. 2001]
ABP00614	Human ORFX protein sequence SEQ	313 . . . 362	26/53 (49%)	5e−08
	ID NO: 1210 - Homo sapiens, 91 aa.	39 . . . 91	37/53 (69%)
	[WO200192523-A2, 06 DEC. 2001]

[0391] In a BLAST search of public sequence databases, the NOV5a protein was found to have homology to the proteins shown in the BLASTP data in Table 5D.

TABLE 5D
Public BLASTP Results for NOV5a
Protein		NOV5a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
O00400	Acetyl-coenzyme A transporter	1 . . . 322	321/322 (99%)	0.0
	(Similar to acetyl-coenzyme A	1 . . . 322	322/322 (99%)
	transporter) - Homo sapiens (Human),
	549 aa.
Q99J27	Similar to acetyl-coenzyme A	1 . . . 322	294/323 (91%)	e−169
	transporter - Mus musculus (Mouse),	1 . . . 323	304/323 (94%)
	550 aa.
Q9JM68	Acetyl-CoA transporter - Rattus	1 . . . 322	293/323 (90%)	e−168
	norvegicus (Rat), 550 aa.	1 . . . 323	303/323 (93%)
Q9WTN1	Acetyl-CoA transporter - Mus	1 . . . 322	288/323 (89%)	e−165
	musculus (Mouse), 550 aa.	1 . . . 323	299/323 (92%)
Q95TG1	SD08430p - Drosophila melanogaster	44 . . . 323	157/289 (54%)	1e−83
	(Fruit fly), 525 aa.	10 . . . 297	204/289 (70%)

[0392] PFam analysis predicts that the NOV5a protein contains the domains shown in the Table 5E.

TABLE 5E
Domain Analysis of NOV5a
Pfam Domain	NOV5a	Identities/	Expect
	Match Region	Similarities for	Value
		the Matched Region

Example 6

[0393] The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.

TABLE 6A
NOV6 Sequence Analysis
	SEQ ID NO: 17	8172 bp
NOV6a,	ATGGCGAGGCCCGGCCGGGGGGTCCTGTCGGGCGGCGCGGGAGAGCGCGGGGGGGGCGTCGCTT
CG171174-01	CCACAGCGCCCGAGAGGTCCTCGCCCGCGTCCCTGTATTTCGTGGTTGGTGTTGGTGCCTCGAT
DNA Sequence	CGTGTGTTCCTTTGAAGTGGAAATGCCCCCGTTCTCAACAGTTGAGTTGAACGCAGGGGCCAGC
	TCTGGGGGCCGGCCCGTGGGGCAGCGTGCGGCCGCAGAGCAAGAGGCCCAGGAAGGATCCTCCG
	AGCGCTGTGGGGAGCGGCAACGCCGGTGGCTCGGGGCCCCGCGGAAAAGGTTTGTTGTTCATGG
	TTCAGAAGCCCTGGACCTTGAATCCAGCCGGCATTCGTCCCCCATGTCCCTGGCCTCCAACCTT
	GCTCTGCCCCTTCACCCTCTTGGAGATGCTTTTCTGTCGGGTGTCCTCACCTGGGGCTCGCCCT
	CCTCCTCCCGGAACTTAGGGTCTTCTGGTGGCGAGAAGGAAGAAGGCAAAAAGGTCCGGCGGCA
	GTGGGAGTCGTGGAGCACAGAGGACAAGAACACCTTCTTCGAGGGGCTGTACGAGCATGGGAAA
	GACTTTGAAGCGATTCAGAACAACATTGCGCTGAAGTACAAGAAGAAAGGCAAGCCAGCAAGCA
	TGGTGAAGAACAAGGAGCAGGTCCGCCACTTCTACTACCGCACCTGGCACAAGATCACCAAGTA
	CATCGACTTTGATCATGTGTTCTCTCGAGGCCTGAAGAAGTCATCCCAGGAACTGTATGGCCTG
	ATCTGCTATGGCGAGCTGCGCAAGAAGATTGGGGGCTGTATGGATGACAAGAATGCAACAAAGC
	TGAATGAACTCATTCAGGTTGGGATCCATACTTGGGGCAAATCTTACTTCACCTTTTATTTCAT
	TTCCTCCATGATTGATCGAATGAAGCCAGAGTTCCAGACTCTTTGCTGTATGCTTGAGGACGGT
	GCACAGAGTGGCCTGTCCGATGAGCGTTCCTTTTGCCAAAACACAGATGTGCTGCCCAGCGGGG
	GCGTGGTGGGCACCTGCAGCGCCATCCGCGGGAGAACTTATGCCTCAGCGTTCCTTCAAAACTC
	TTTTCCCAGGGCCACCACTGTACGTTACAAAGGGCGGAACCTGCGGATCAAAGCGCCCATGTGC
	CGGGCCCTGAAGAAGCTGTGCGATCCAGATGGCTTGAGTGATGAAGACGACCAGAAGCCAGTGC
	GCCTGCCTCTGAAAGTCCCTATAGAGCTACAGCCGCGGAACAACCACGCCTGGGCCCGTGTGCA
	GAGCCTTGCCCAGAACCCACGCCTCAGGAACTTCCACGAGAAGCAGGTCCACCCCTATGCTCTG
	TCATCACACGAGGACGCAGCAGTGTGGAGGCGACTGGAGTCCAGGGAGCACTGGGCTGCAGTCC
	TGTATCTGGGCAGGGATCGCCCAACCTGTGTTCAGGCCGTGGAGGGGATGTCGCGGATGATCGT
	GGAGCTACATCGAAAGGTCTCCAGCCTCATCGAATTCTTGAAGCAGAAGTGGGCGCTCCATGAG
	CATCCCGACCTCAGTGCTAGCCAGTGTGGGCCTTCCTTGACGGGCACTCAGCGGAAGACACTCG
	AGGAGCGGCAGCTGCAGGACTCATGCTCCGCACCGATGCAGGAGAAGGTGACACTGCACTTGTT
	CCCAGGCGAGAACTGTACACTGACACCGCTGCCGGGCGTGCCTCGCGTGGTGCACTCCAAGGCC
	TTCTGCACAGTGCACTGGCAGGAGGGCGGCCGGTGCAAGCAGAGTGCCAAGGACGCCCACGTGC
	TGCCCCCAGCCCAGATCCTGGGCATCCAGAGTGGGCAGGGCACGGCCCGGGGCCAGGTGAAATG
	CCCGCGGAGCGGAGCTGAGGGCAAGGGTGTGGGGCGGCCCCCTCCTGCGGCTGACGCCTTGCAG
	AGCTCCGGAGAGAGTTCCCCCGAAAGCGCCCCCGGGGAGGGGGCTGCCCTAAGCTTGAGCAGCC
	CGGACGCTCCTGACAGGCCTCCTCCCAGGCACCAGGACACTGGGCCATGTCTTGAGAAGACCCC
	TGCAGAAGGCAGGGACAGTCCCACCCGGGAGCCAGGGGCCTTGCCGTGTGCCTGTGGCCAGCTC
	CCAGACCTGGAGGACGAGCTCTCGCTTCTAGACCCCTTGCCCCGCTACCTAAAGTCCTGTCAGG
	ACCTCATTGTCCCCGAGCAGTGCCGCTGTGCGGACACACGGCCTGGGAGCGAGCAGCCCCCTCT
	GGGCGGGGCGGCCTCCCCAGAGGTGCTCGCTCCTGTCAGCAAGGAGGCTGCTGACCTTGCTCCC
	ACTCGCCCATCCCCGAGGCCCGGCCCCGGGCTCCTGCTGGATGTTTGCACTAAAGACTTGGCAG
	ATGCACCTGCGGAGGAGCTCCAGGAGAAGGGGAGCCCCGCGGGGCCTCCGCCGTCTCAGCGACA
	GCCTGCCGCCAGGCCCCCGAAGGAGGTCCCCGCCAGCCGGCTGGCTCAGCAGCTCCGTGAGGAG
	GGCTGGAACCTGCAGACCTCCGAAAGCCTCACGCTGGCCGAAGTCTACCTCATGATGGGCAAGC
	CCAGCAACCTGCAGCTGGAGTACGACTGGCTGGGGCCCGGCCGCCAGGACCCCCGCCCCGGCTC
	CCTACCCACCGCCCTCCACAAGCAGCGCCTCCTCAGCTGCCTCCTGAAGCTCATTTCCACCGAG
	GTCAACCCCAAGCTGGCTCTGGAAGCAAACACCATCTCTACAGCCTCAGTAAGGCCCGCCCAGG
	AGGAGCAGTCGATGACGCCCCCAGGGAAGGTGGTGACCGTCAGCTCTCGCAGCCCCCGCTGCCC
	TCGGAACCAGGCCTCCCTCCGCAGCAGCAAGACCTTCCCGCCCAGCTCTGCACCCTGCTCCTCA
	GGTTTGAGAAACCCTCCAAGACCCCTCTTGGTGCCTGGTCCCTCCAGCACAGGAAGCAATGACT
	CAGATGGAGGCCTTTTTGCTGTCCCGACAACCTTGCCACCCAACAGCCGACACGGGAAGCTCTT
	CTCTCCCAGTAAAGAAGCAGAGCTGACTTTCCGCCAGCATCTGAACTCCATCAGTATGCAGTCG
	GATTTCTTCCTGCCAAAGCCCCGGAAGCTGCCGAACCGGCACCTGCGGAAGCCACTGGTGGTCC
	AGAGAACACTGCTCCCTAGACCATCGGAAAACCAGTCCCACAACGTTTGTTCCTTCTCCATCCT
	GTCTAACTCTTCCGTAACTGGGAGAGGTTCGTTCCGGCCCATCCAGTCTTCTCTGACCAAAGCA
	GCTCTGTCTCGGCCGATCGTGCCCAAGGTCCTTCCACCCCAGGCCACGAGTCACCTGGCCAGTG
	CTATCGACTTAGCAGCTACAAGTGCCGGCATCCTTTCCGGGAACCCCCTCCCTGCCTTGGACAC
	CGAGGGCTTGTCTGGCATCTCTCCACTGTCTTCAGACGAGGTGACGGGTGCCATCTCGGGGCAG
	GACTCTACTGGAACTCACCAGGATGGAGACACCCTCCCCACCGTGGGGGGCTCCGACCCATTTG
	TCAGCATCCCTTCGAGGCCTGAGCAGGAGCCAGTGGCAGACAGTTTCCACGGCTCATCTGTTCT
	CTCCTTATCTGAGCTGCCCAAGGCCCCTCTCCAGAATGGCCTCTCCATACCGCTGTCCTCGTCA
	GAGAGCTCCAGCACCCGGCTGTCTCCACCAGACGTCTCTGCTCTGCTCGACATCTCCCTGCCCG
	GCCCACCTGAGGATGCGCTGTCACAGGGCGAGCCTGCCACACACATTAGCCACTCCATCATTGA
	GATCGCCATCACCTCCGGTCAGTACGGTGAACGAGTCCCTCTTTCCCCAGCAAAACTGAATGGC
	AGTGACAGTTCCAAGAGCCTTCCCTCCCCGTCCAGCAGCCCCCAGCCACACTGGATCGCCTCTC
	CCACCCACGACCCCCAGTGGTACCCCAGTGACTCCACCGACTCCTCGCTCAGCAGCCTGTTTGC
	AAGCTTCATCTCCCCAGACAAGAGCCGGAAGATGTTGCCGACTCCCATTGGGACCAACAGTGGC
	ACTTCCTTGCTTGGCCCCAGCTTGTTGGATCGAAACTCGCGGGACTCATTTGTGTCCAGGTCCC
	TGGCTGACGTTGCAGAGGTTGTGGATTCCCAGCTGGTGTGCATGATGAACGAAAACAGCATTGA
	TTACATTTCTCGGTTCAATGACCTGGCCCAAGAGCTGTCCATCGCTGAGCCTGGCCGCCGAGAA
	GCTCTGTTTGATGGTGGTGGAGGCGGCCCCGCTGTCAGTGACCTGTCCCAGTGACCACACGTCC
	TGGTGGCGGATGAAGCCCTCTTCGAGCTAGAGAAAAATAGATAAGCCCAGCAGCCCCAGAAGAT
	GGTCTGAACAGAGGCATCTCCGCACCCAAGACTGTGCAACGGGCAGGAACGTGGTCACAGAGCT
	GCTTCCCCACGAGCAGCAGGCAACGGCGTCCAAGGAGACTAGGATGAGTTCTTGGCAAGGGCCA
	GCGTTAGAAATCACTGTGGTACTAGAGCCGTTCTTCACCACGCCTGGGCCCATGTTAGGGTCTG
	CATAATGATCCCATTTCAGCCTGTGCTCTGCCTCGATTGTTGTGTTGGACATTCCCGTGGCATT
	TCCTTCTGAGACAAGGGAGTATGTGTGCCTTGGTGTAGTTGCTGTGCACTAGGAGCTGTGATCT
	CCCTCTCTGCAGGGACGCCCCAGCCCCTGCTGCTTGCTTTCTGCCAAACCTGTGCTATGCATCA
	GCTGTGCCCTCTGTGGACTGTAACGGGCAGGACAGTTGGGTGTGGCCTGGGCTCATGCCTGGTG
	GTGTCACATCCCAAGGCAGCAAGAGCATGGATACCGATCACAGGGCTGCTGCGGAGTCGTGGGG
	CCCTGGGCTGGTGCCTCCCCTCCCTAGAGGTTTTGTTCGTACTCTTAACAGGGAGTGGGGGCAG
	GAAGAGTCCTGTACTATGCAGGTTGTGTGGACTTTACATGGGACCCTGCTAAGCTGGTTGAAAA
	TGTTTTTCTTGTGTTTTAAGAATTAGGAGACATGGAAGAGGAAGAACAAAGTCCCCTCTGTAGT
	TGGTTTCCTTCCTGTGTCCCTTTGCAAGCTTCCAGGCGATCTAAGGTGTCATTTCTCCCTCCTG
	GGGTGACCCTTACGCGCTAATATGATTACAGCGAAGACTTTCCTGATAAGTTCTCAAACTCGAT
	GTGTGACTGTTTGGCACTTGAGACAAACCTGCCTTTGCAGGGAAAGTGTCTCTCACGGGCATTG
	GTGTGGGCGTGCCTGACATACGTGTTCAGTCCCTTGCATACCTTTGCCTTGAGACTTCTGTGTC
	TCCTTCCCATTTGGGACACCCAGGTGAGGGCCCAGACATCTGGATGTGGTCAGACCTCACCAAA
	TATATGCCTTCGTGGTGGTCTCCCTCCTTGCGCCCTCTTGGGTGGCCAGCGTTCCTACTGCAGA
	CGGCCCAACATCCAGTCTTTCCCCAGGACAGAGCTAACAAGGGCCCCTTTGCCTTCTCATCCTC
	AGGAGTTCCAGGCACATGAGTCACCGTCCATCCACATCCAGTGTGGCCTGGAGCTGCTACAGAG
	GTGTTGGGCAGGCCATGCCTGTGCCGCCATCTCTCCCTTCCTGCCTCATTTCATCCCCCGCAGC
	AGCCGGGATTGATTGTGCTTTCCTAACCCCCTTGGACCTACTCTCGCTCCTCCCCACCATTCCT
	CTTCCCCCACATGTGTGGCACGCTGCAGCCCTCAAGGCCAGCCCTGGCCCCTCCACTGCTTCTC
	TCCCCATCCACAATGGAGAAGGTGAAAAGAGGAGGGAAAGGCCTTTGGTGTGGACAAGCATGTG
	GACGCCCTCCGTCCTGCAGTCTTGCCAGCCCACCACAGCCACTGTAGACCACAGGCAGGCCGTG
	TACTGCACCACTGGGAGGACGTGGAGAGGACAGTGAACTTCCAGGCAAGAGCTTCCTTCTTTTG
	TCTCACGAGTTTTTCTTAGAGCTCTTGCCTGAGCTGGCTTCCCTCCTTCAGACATTGACATGAG
	ATCTTAAGCAAACAGTCCCAAACCTCTTAGGGGTGAAAAAAGAAACATGCCACTTGATTAGGAG
	AGAGACAGCAGTGTTTGAACTACAGCATCTTTACACTAGCTTGTGTTTTGTGCTACGTATACCA
	GCTTCCAAAATTAGCATCTCATTGAGCCAGAGAAGACAAGGAGATCTCCCTCTGGGCATCTGGC
	TTTGCTGCGTCTCTAGAGGGTTAGGATACCAGGCCGACTTCAGGCCACTGCTAGCTTTCTCATA
	CTCCCACAGGCTAGACCAGAGATGCCAAGTCCCAACAGCACTGAGCTGTGTGCACTGTGCCAGG
	GACAGGAGGGTTTGTGAACTGCCTGTCAGGGTACCTGTTAGCCCCTGACAACTCAGTGGGGTGA
	AGTTTTGGAGGTCAGAGTCTGCTTTCGTAGGCTCTTTAGACAGCACCTACCACTTGGTTCTCCA
	GCGTAGACTCCTGGGAGCAGCCAACTGCAGCCATTGCCATCCAGTGGGGAGATGGGTTAGGGAG
	GAGGACCGGCTGACTCCTCTCCTGTAATAAAGCTGACAAGAGTTCTAGAGGATTCTGCTTCTCT
	AGTAACTAGACAGGTGATACGCATTTGCTTGCCACATTAAGGGAAAATGGTGTCATTTGTTGCA
	GAAAAACAATGGATACATTTTCTTCTGGCCTAAATGAATATTTATGTGCAAACATAGGCAACTG
	TTAAAGGCTGGAATTTTCAAAAGATCCAAACAGAGACTTCCTGCATCTTCTGCCTTTCCAACAG
	AAGCGGTGATCGTCTAAGTATGAGCCTGTGGCTTCCTTTGTGCATTTGAGCATGCTGTAATTAA
	GATGAGATCAGTTTCTTAGAAAAAGCTTTCCTGAATCCCTCTGACGTTGCCTGGGATCTTTCTG
	TTGATTCGTCTTTTCTGGAGATTGGGACAGAGCATCTGTGGTCCAGGGAAGTTAGTCCTCTGGC
	CTCAATTCTGTTGTGGATGTGCAGTGATAAGCGGGCATTGCGTGCCTCGGGGGATGCCTAGTTC
	GTGGCTTCCTGGCTGTTTTGTCCTTCTGTGTCTTGTAGCTGTAGGGTGCCAGCTCAGGGAGTGG
	GGTGTTGGCGGCGTTTCCGCGGTTGGCCTCCTTGCTTTGCCGCACCTCCAGGTTCTGGGCATGA
	GAGGCCGTGGCCTCATTTCTGGTGGATAACCTTTTTAGTTTAATAGCATCTTTAATTAGATCAC
	AGCATTGAATTCAAAATTTCTTCTGCAAAGAAAGTTGTGGGGCATAAGACACCGGGAATGAGGG
	AGGAGGAAGACAGTTGTGTTTTCTCTTTAAACCTTGAGCTCTAGCCGATGCATTTGTCAGGAAA
	TACAGCACTTTGTCTTAAGAAAACAAGGAAGGAGGCCGGGCGCAGTGGCTCACGCCTGTAATCC
	CAGCACTTTGGGAGGCCGAGGCGGGCGGATCACCTGAGGTGGGGAGTATGAGACCACCCTGACT
	AACATGGAGAGACCCTGTCTCTACTAAAAGTACAGAATTAGCCGGGCGTGGTGGCGCATGCCCA
	TAATCCCAGCTACTGAGGAGACTTCAGGTAGGAGAATCACTTGAACCTCAGCCGCGGAGGTTGC
	AGTGAGTCGAGATCGCGCCAGTGCACTCCAGCCTGGGCAAGAAGAGCGAAACTGGGTCTCAAGT
	TAAAAAAAGAAAGCAAGGAAAGAGTAATTTACAACGAAGGAAAAAAACCCACAGCACACCCTTC
	GCGGCTGTCAGCGCTCTCCTGATGTCACAGTGGCGGCGTGTCCTTGGGGTGGGTGAGGTGTGGG
	GAGCCCAGCCCCTGGCCCTGCCTCCCGCGCCCCGCTCCCCTTCTCTCTCTTACTCGGTTAAGCC
	ATAGCGAGGCCTCCGCTCGTTTCAGATATGAATTTGTTTTATAGATTATAAATATGCATATACA
	GTGTATGTATAAAGCAGAATGCCTGCCTTTCCTGGTTATTTTTTGTACCATATTGTAAATTATA
	TTATTTATTCTTTACCAATTTTGGGAATAAAAGGTGTTTTGGTTATTTAATATAATAAGAGCTG
	TTAAACTTCTGTTTAAATTTCCAGTTCAACTTGTAAATGTTTTTATTGTGCATAAATACATACT
	AATGTTGATCTAAAAAAAAAAAAAAAAAAAAAAGGGCGGCCGCT
	ORF Start: ATG at 1		ORF Stop: TGA at 4276
	SEQ ID NO: 18	1425 aa	MW at 153017.4 kD
NOV6a,	MARPGRGVLSGGAGERGGGVASTAPERSSPASLYFVVGVGASIVCSFEVEMPPFSTVELNAGAS
CG171174-01	SGGRRVGQRAAAEQEAQEGSSERCGERQRRWLGAPRKRFVVHGSEALDLESSRHSSPMSLASNL
Protein Sequence	ALPLHPLGDAFLSGVLTWGSRSSSRNLGSSGGEKEEGKKVRRQWESWSTEDKNTFFEGLYEHGK
	DFEAIQNNIALKYKKKGKPASMVKNKEQVRHFYYRTWHKITKYIDFDHVFSRGLKKSSQELYGL
	ICYGELRKKIGGCMDDKNATKLNELIQVGIHTWGKSYFTFYFISSMIDGMKPEFQTLCCMLEDG
	AQSGLSDERSFCQNTDVLPSGGVVGTCSAIRGRTYASAFLQNSFPRATTVRYKGRNLRIKAPMC
	RALKKLCDPDGLSDEEDQKPVRLPLKVPIELQPRNNHAWARVQSLAQNPRLRNFQEKQVHPYAL
	SSHEDAAVWRRLESREHWAAVLYLGRDRPTCVQAVEGMSRMIVELHRKVSSLIEFLKQKWALHE
	HPDLSASQCGPSLTGTQRKTLEERQLQDSCSAPMQEKVTLHLFPGENCTLTPLPGVARVVHSKA
	FCTVHWQEGGRCKQSAKDAHVLPPAQILGIQSGQGTARGQVKCPRSGAEGKGVGRPPPAADALQ
	SSGESSPESAPGEGAALSLSSPDAPDRPPPRHQDTGPCLEKTPAEGRDSPTREPGALPCACGQL
	PDLEDELSLLDPLPRYLKSCQDLIVPEQCRCADTRPGSEQPPLGGAASPEVLAPVSKEAADLAP
	TGPSPRPGPGLLLDVCTKDLADAPAEELQEKGSPAGPPPSQGQPAARPPKEVPASRLAQQLREE
	GWNLQTSESLTLAEVYLMMGKPSKLQLEYDWLGPGRQDPRPGSLPTALHKQRLLSCLLKLISTE
	VNPKLALEANTISTASVRPAQEEQSMTPPGKVVTVSSRSPRCPRNQASLRSSKTFPPSSAPCSS
	GLRNPPRPLLVPGPSSTGSNDSDGGLFAVPTTLPPNSRHGKLFSPSKEAELTFRQHLNSISMQS
	DFFLPKPRKLRNRHLRKPLVVQRTLLPRPSENQSHNVCSFSILSNSSVTGRGSFRPIQSSLTKA
	ALSRPIVPKVLPPQATSHLASAIDLAATSAGILSGNPLPALDTEGLSGISPLSSDEVTGAISGQ
	DSTGTHQDGDTLPTVGGSDPFVSIPSRPEQEPVADSFQGSSVLSLSELPKAPLQNGLSIPLSSS
	ESSSTRLSPPDVSALLDISLPGPPEDALSQGEPATHISDSIIEIAISSGQYGEGVPLSPAKLNG
	SDSSKSLPSPSSSPQPHWIASPTHDPQWYPSDSTDSSLSSLFASFISPEKSRKMLPTPIGTNSG
	TSLLGPSLLDGNSRDSFVSRSLADVAEVVDSQLVCMMNENSIDYISRFNDLAQELSIAEPGRRE
	ALFDGGGGGPAVSDLSQ

[0394] Further analysis of the NOV6a protein yielded the following properties shown in Table 6B.

TABLE 6B
Protein Sequence Properties NOV6a
SignalP	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 6; pos. chg 2; neg. chg 0
	H-region: length 8; peak value 6.08
	PSG score: 1.68
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −7.17
	possible cleavage site: between 41 and 42
	>>> Seems to have no N-terminal signal peptide
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 1
	Tentative number of TMS(s) for the threshold 0.5: 1
	Number of TMS(s) for threshold 0.5: 1
	INTEGRAL	Likelihood =	−2.87	Transmembrane 31-47
	PERIPHERAL	Likelihood =	2.60 (at 1107)
	ALOM score: −2.87 (number of TMSs: 1)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 38
	Charge difference: −3.0 C(−3.0)-N(0.0)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 2 (cytoplasmic tail 1 to 31)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	3	Hyd Moment (75):	3.60
	Hyd Moment (95):	12.59	G content:	8
	D/E content:	2	S/T content:	3
	Score: −6.80
	Gavel: prediction of cleavage sites for mitochondrial preseq
	R-2 motif at 16 GRG|VL
	NUCDISC: discrimination of nuclear localization signals
	pat4: PRKR (4) at 99
	pat4: KPRK (4) at 1030
	pat7: PRKRFVV (5) at 99
	pat7: PKPRKLR (5) at 1029
	pat7: PRKLRNR (5) at 1031
	pat7: PEKSRKM (4) at 1328
	bipartite: none
	content of basic residues: 10.8%
	NLS Score: 1.67
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	XXRR-like motif in the N-terminus: ARPG none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear
	discrimination
	Prediction: nuclear
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	47.8%: nuclear
	26.1%: mitochondrial
	8.7%: cytoplasmic
	4.3%: extracellular, including cell wall
	4.3%: Golgi
	4.3%: plasma membrane
	4.3%: peroxisomal
	>> prediction for CG171174-01 is nuc (k = 23)

[0395] 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/	Similarities for	Expect
Identifier	Date]	Match Residues	the Matched Region	Value
ABB67401	Drosophila melanogaster polypeptide	174 . . . 285	50/113 (44%)	4e−17
	SEQ ID NO 28995 - Drosophila	113 . . . 220	71/113 (62%)
	melanogaster, 982 aa.
	[WO200171042-A2, 27 SEP. 2001]
ABB59353	Drosophila melanogaster polypeptide	174 . . . 285	50/113 (44%)	4e−17
	SEQ ID NO 4851 - Drosophila	113 . . . 220	71/113 (62%)
	melanogaster, 982 aa.
	[WO200171042-A2, 27 SEP. 2001]
ABB71513	Drosophila melanogaster polypeptide	599 . . . 1302	182/750 (24%)	5e−13
	SEQ ID NO 41331 - Drosophila	255 . . . 903	247/750 (32%)
	melanogaster, 950 aa.
	[WO200171042-A2, 27 SEP. 2001]
AAM89174	Human immune/haematopoietic	1005 . . . 1046	29/47 (61%)	5e−07
	antigen SEQ ID NO: 16767 - Homo	23 . . . 69	32/47 (67%)
	sapiens, 170 aa. [WO200157182-A2,
	09 AUG. 2001]
AAB40945	Human ORFX ORF709 polypeptide	899 . . . 1305	103/443 (23%)	5e−05
	sequence SEQ ID NO: 1418 - Homo	648 . . . 1050	156/443 (34%)
	sapiens, 1532 aa. [WO200058473-A2,
	05 OCT. 2000]

[0396] 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
Protein		NOV6a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q96RY5	Hypothetical protein - Homo	56 . . . 1425	1111/1560 (71%)	0.0
	sapiens (Human), 1587 aa.	39 . . . 1587	1164/1560 (74%)
Q9P2C1	Hypothetical protein KIAA1426 -	668 . . . 1425	758/758 (100%)	0.0
	Homo sapiens (Human), 758 aa	1 . . . 758	758/758 (100%)
	(fragment).
Q8NDN1	Hypothetical protein - Homo	786 . . . 1425	636/640 (99%)	0.0
	sapiens (Human), 644 aa.	8 . . . 644	637/640 (99%)
Q8MX88	Cramped protein - Drosophila	174 . . . 285	50/113 (44%)	4e−17
	sechellia (Fruit fly), 975 aa.	113 . . . 220	72/113 (63%)
Q8MM71	Cramped - Drosophila melanogaster	174 . . . 285	50/113 (44%)	1e−16
	(Fruit fly), 982 aa.	113 . . . 220	71/113 (62%)

[0397] PFam analysis predicts that the NOV6a protein contains the domains shown in the Table 6E.

TABLE 6E
Domain Analysis of NOV6a
		Identities/
	NOV6a	Similarities for
Pfam Domain	Match Region	the Matched Region	Expect Value
myb_DNA-binding	172 . . . 229	12/59 (20%)	0.00021
		42/59 (71%)

Example 7

[0398] The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.

TABLE 7A
NOV7 Sequence Analysis
	SEQ ID NO: 19	1047 bp
NOV7a,	GCCTTGAGGTGCAGTGTTGGGGATCCAAAGCCATGTCGGACCTGCTACTACTGGGCCTGATTGG
CG172318-01	GGGCCTGACTCTCTTACTGCTGCTGACGCTGCTGGCCTTTGCCGGGTACTCAGGGCTACTGGCT
DNA Sequence	GGGGTGGAAGTGAGTGCTGGGTCACCCCCCATCCGCAACGTCACTGTGGCCTACAAGTTCCACA
	TGGGGCTCTATGGTGAGACTGGGCGGCTTTTCACTGAGAGCTGCAGCATCTCTCCCAAGCTCCG
	CTCCATCGCTGTCTACTATGACAACCCCCACATGGTGCCCCCTGATAAGTGCCGATGTGCCGTG
	GGCAGCATCCTGAGTGAAGGTGAGGAATCGCCCTCCCCTGAGCTCATCGACCTCTACCAGAAAT
	TTGGCTTCAAGGTGTTCTCCTTCCCGGCACCCAGCCATGTGGTGACAGCCACCTTCCCCTACAC
	CACCATTCTGTCCATCTGGCTGGCTACCCGCCGTGTCCATCCTGCCTTGGACACCTACATCAAG
	GAGCGGAAGCTGTGTGCCTATCCTCGGCTGGAGATCTACCAGGAAGACCAGATCCATTTCATGT
	GCCCACTGGCACGGCAGGGAGACTTCTATGTGCCTGAGATGAAGGAGACAGAGTGGAAATGGCG
	GGGGCTTGTGGAGGCCATTGACACCCAGGTGGATGGCACAGGTACAGAAGGAGCTGACACAATG
	AGTGACACGAGTTCTGTAAGCTTGGAAGTGAGCCCTGGCAGCCGGGAGACTTCAGCTGCCACAC
	TGTCACCTGGGGCGAGCAGCCGTGGCTGGGATGACGGTGACACCCGCAGCGAGCACAGCTACAG
	CGAGTCAGGTGCCAGCGGCTCCTCTTTTGAGGAGCTGGACTTGGAGGGCGAGGGGCCCTTAGGG
	GAGTCACGGCTGGACCCTGGGACTGAGCCCCTGGGGACTACCAAGTGGCTCTGGGAGCCCACTG
	CCCCTGAGAAGGGCAAGGAGTAACCCATGGCCTGCACCCTCCTGCAGTGCAGTTGCTGAGGAAC
	TGAGCAGACTCTCCAGCAGACTC
	ORF Start: ATG at 33		ORF Stop: TAA at 981
	SEQ ID NO: 20	316 aa	MW at 34475.4 kD
NOV7a,	MSDLLLLGLIGGLTLLLLLTLLAFAGYSGLLAGVEVSAGSPPIRNVTVAYKFHMGLYGETGRLF
CG172318-01	TESCSISPKLRSIAVYYDNPHMVPPDKCRCAVGSILSEGEESPSPELIDLYQKFGFKVFSFPAP
Protein Sequence	SHVVTATFPYTTILSIWLATRRVHPALDTYIKERKLCAYPRLEIYQEDQIHFMCPLARQGDFYV
	PEMKETEWKWRGLVEAIDTQVDGTGTEGADTMSDTSSVSLEVSPGSRETSAATLSPGASSRGWD
	DGDTRSEHSYSESGASGSSFEELDLEGEGPLGESRLDPGTEPLGTTKWLWEPTAPEKGKE

[0399] Further analysis of the NOV7a protein yielded the following properties shown in Table 7B.

TABLE 7B
Protein Sequence Properties NOV7a
SignalP	Cleavage site between residues 26 and 27
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 3; pos. chg 0; neg. chg 1
	H-region: length 31; peak value 0.00
	PSG score: −4.40
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): 6.68
	possible cleavage site: between 25 and 26
	>>> Seems to have no N-terminal signal peptide
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 1
	Tentative number of TMS(s) for the threshold 0.5: 1
	Number of TMS(s) for threshold 0.5: 1
	INTEGRAL	Likelihood =	−8.92	Transmembrane 4-20
	PERIPHERAL	Likelihood =	1.43 (at 21)
	ALOM score: −8.92 (number of TMSs: 1)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 11
	Charge difference: 0.0 C(0.0)-N(0.0)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 2 (cytoplasmic tail 1 to 4)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment (75):	7.06
	Hyd Moment (95):	6.67	G content:	6
	D/E content:	2	S/T content:	4
	Score: −8.50
	Gavel: prediction of cleavage sites for mitochondrial
	preseq
	R-2 motif at 54 IRN|VT
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: none
	bipartite: none
	content of basic residues: 8.2%
	NLS Score: −0.47
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	KKXX-like motif in the C-terminus: EKGK
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear
	discrimination
	Prediction: cytoplasmic
	Reliability: 76.7
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	34.8%: mitochondrial
	30.4%: cytoplasmic
	8.7%: Golgi
	8.7%: vacuolar
	8.7%: endoplasmic reticulum
	4.3%: extracellular, including cell wall
	4.3%: vesicles of secretory system
	>> prediction for CG172318-01 is mit (k = 23)

[0400] 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 7C.

TABLE 7C
Geneseq Results for NOV7a
		NOV7a	Identities/
Geneseq	Protein/Organism/Length [Patent #,	Residues/	Similarities for	Expect
Identifier	Date]	Match Residues	the Matched Region	Value
AAU81960	Human PRO536 - Homo sapiens, 313 aa.	1 . . . 316	313/316 (99%)	0.0
	[WO200109327-A2, 08 FEB. 2001]	1 . . . 313	313/316 (99%)
AAB65173	Human PRO536 (UNQ337) protein sequence	1 . . . 316	313/316 (99%)	0.0
	SEQ ID NO: 97 - Homo sapiens, 313 aa.	1 . . . 313	313/316 (99%)
	[WO200073454-A1, 07 DEC. 2000]
AAB94830	Human protein sequence SEQ ID NO: 15991 -	1 . . . 316	313/316 (99%)	0.0
	Homo sapiens, 313 aa. [EP1074617-A2,	1 . . . 313	313/316 (99%)
	07 FEB. 2001]
AAU12370	Human PRO536 polypeptide sequence -	1 . . . 316	313/316 (99%)	0.0
	Homo sapiens, 313 aa. [WO200140466-A2,	1 . . . 313	313/316 (99%)
	07 JUN. 2001]
AAY50944	Human adult heart cDNA clone vf1_1	1 . . . 316	313/316 (99%)	0.0
	derived protein - Homo sapiens, 313 aa.	1 . . . 313	313/316 (99%)
	[WO9955721-A1, 04 NOV. 1999]

[0401] In a BLAST search of public sequence databases, the NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7D.

TABLE 7D
Public BLASTP Results for NOV7a
		NOV7a	Identities/
Protein		Residues/	Similarities for
Accession		Match	the Matched	Expect
Number	Protein/Organism/Length	Residues	Portion	Value
Q9Y6I9	Putative secreted protein ZSIG11	1 . . . 316	313/316 (99%)	0.0
	precursor - Homo sapiens (Human),	1 . . . 313	313/316 (99%)
	313 aa.
CAC25002	Sequence 46 from Patent WO0100806	1 . . . 316	312/316 (98%)	0.0
	precursor - Homo sapiens (Human),	1 . . . 312	312/316 (98%)
	312 aa.
Q99LS5	Similar to putative secreted protein	1 . . . 316	261/316 (82%)	e−147
	(Unknown) (Protein for MGC:7091) -	1 . . . 309	279/316 (87%)
	Mus musculus (Mouse), 309 aa.
Q9D7D9	Adult male tongue cDNA, RIKEN	1 . . . 316	258/316 (81%)	e−145
	full-length enriched library,	1 . . . 309	277/316 (87%)
	clone:2310012P03, full insert sequence -
	Mus musculus (Mouse), 309 aa.
AAN47632	Conserved hypothetical protein -	15 . . . 184	53/181 (29%)	3e−06
	Leptospira interrogans serovar lai str.	9 . . . 180	84/181 (46%)
	56601, 181 aa.

[0402] PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7E.

TABLE 7E
Domain Analysis of NOV7a
Pfam Domain	NOV7a Match Region	Identities/	Expect
		Similarities	Value
		for the Matched
		Region

Example 8

[0403] The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.

TABLE 8A
NOV8 Sequence Analysis
	SEQ ID NO: 21	1372 bp
NOV8a,	TAATGAAAAGGATATGATCATCGTGGCGCATGTATTACTCATCCTTTTGGGGGCCACTGAGATA
CG172921-01	CTGCAAGCTGACTTACTTCCTGATGAAAAGATTTCACTTCTCCCACCTGTCAATTTCACCATTA
DNA Sequence	AAGTTACTGGTTTGGCTCAAGCTCTTTTACAATGGAAACCAAATCCTGATCAAGAGCAAAGGAA
	TGTTAATCTAGAATATCAAGTGAAAATAAACGCTCCAAAAGAAGATGACTATGAAACCAGAATC
	ACTGAAAGCAAATGTGTAACCATCCTCCACAAAGGCTTTTCAGCAAGTGTGCGGACCATCCTGC
	AGAACGACCACTCACTACTGGCCAGCAGCTGGGCTTCTGCTGAACTTCATGCCCCACCAGGGTC
	TCCTCGAACCTCAATTGTGAATTTAACTTGCACCACAAACACTACAGAAGACAATTATTCACGT
	TTAAGGTCATACCAAGTTTCCCTTCACTGCACCTGGCTTGTTGGCACAGATGCCCCTGAGGACA
	CGCAGTATTTTCTCTACTATAGGTATGGCTCTTGGACTGAAGAATGCCAAGAATACAGCAAAGA
	CACACTGGGGAGAAATATCGCATGCTGGTTTCCCAGGACTTTTATCCTCAGCAAAGGGCGTGAC
	TGGCTTGCGGTGCTTGTTAACGGCTCCAGCAAGCACTCTGCTATCAGGCCCTTTGATCAGCTGT
	TTGCCCTTCACGCCATTGATCAAATAAATCCTCCACTGAATGTCACAGCAGAGATTGAAGGAAC
	TCGTCTCTCTATCCAATGGGAGAAACCAGTGTCTGCTTTTCCAATCCATTGCTTTGATTATGAA
	GTAAAAATACACAATACAAGGAATGGATATTTGCAGATAGAAAAATTGATGACCAATGCATTCA
	TCTCAATAATTGATGATCTTTCTAAGTACGATGTTCAAGTGAGAGCAGCAGTGAGCTCCATGTG
	CAGAGAGGCAGGGCTCTGGAGTGAGTGGAGCCAACCTATTTATGTGGGAAATGATGAACACAAG
	CCCTTGAGAGAGTGGTTTGTCATTGTGATTATGGCAACCATCTGCTTCATCTTGTTAATTCTCT
	CGCTTATCTGTAAAATATGTCATTTATGGATCAAGTTGTTTCCACCAATTCCAGCACCAAAAAG
	TAATATCAAAGATCTCTTTGTAACCACTAACTATGAGGTCCTCTGCATTTTCATATACATCTTA
	GATTCGGCTGACAATTTTCTACAAAAAAAGAAAGCTGGGTCCAGTGAGACGGAAATTGAAGTCA
	TCTGTTATATAGAGAAGCCTGGAGTTGAGACCCTGGAGGATTCTGTGTTTTGACTGTCACTTTG
	GCATCCTCTGATGAACTCACACATGCCT
	ORF Start: ATG at 14		ORF Stop: TGA at 1331
	SEQ ID NO: 22	439 aa	MW at 49881.7 kD
NOV8a,	MIIVAHVLLILLGATEILQADLLPDEKISLLPPVNFTIKVTGLAQALLQWKPNPDQEQRNVNLE
G172921-01	YQVKINAPKEDDYETRITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGTS
Protein Sequence	IVNLTCTTNTTEDNYSRLRSYQVSLHCTWLVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTLGR
	NIACWFPRTFILSKGRDWLAVLVNGSSKHSAIRPFDQLFALHAIDQINPPLNVTAEIEGTRLSI
	QWEKPVSAFPIHCFDYEVKIHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAG
	LWSEWSQPIYVGNDEHKPLREWFVIVIMATICFILLILSLICKICHLWIKLFPPIPAPKSNIKD
	LFVTTNYEVLCIFIYILDSADNFLQKKKAGSSETEIEVICYIEKPGVETLEDSVF

[0404] Further analysis of the NOV8a protein yielded the following properties shown in Table 8B.

TABLE 8B
Protein Sequence Properties NOV8a
SignalP	Cleavage site between residues 21 and 22
analysis:
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 10.20
		PSG score: 5.80
	GvH:	von Heijne's method for signal seq. recognition
		GvH score (threshold: −2.1): −1.33
		possible cleavage site: between 19 and 20
	>>> Seems to have a cleavable signal peptide (1 to 19)
	ALOM:	Klein et al's method for TM region allocation
		Init position for calculation: 20
		Tentative number of TMS(s) for the threshold 0.5: 2
		Number of TMS(s) for threshold 0.5: 1
		INTEGRAL Likelihood = −14.33 Transmembrane
		345-361
		PERIPHERAL Likelihood = 3.55 (at 28)
		ALOM score: −14.33 (number of TMSs: 1)
	MTOP:	Prediction of membrane topology (Hartmann et al.)
		Center position for calculation: 9
		Charge difference: −4.5 C(−3.0) − N(1.5)
		N >= C: N-terminal side will be inside
	>>> membrane topology: type 1a (cytoplasmic tail 362 to
	439)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment(75):	3.37
	Hyd Moment(95):	3.35	G content:	1
	D/E content:	1	S/T content:	1
	Score: −5.87
	Gavel: prediction 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: 8.7%
		NLS Score: −0.47
	KDEL:	ER retention motif in the C-terminus: none
	ER Membrane Retention Signals: none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
		type 1: none
		type 2: none
	NMYR:	N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: too long tail
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear
	discrimination
	Prediction: cytoplasmic
	Reliability: 89
	COIL:	Lupas's algorithm to detect coiled-coil regions
		total: 0 residues
	Final Results (k = 9/23):
	44.4%: endoplasmic reticulum
	22.2%: Golgi
	22.2%: extracellular, including cell wall
	11.1%: plasma membrane
	>> prediction for CG172921-01 is end (k = 9)

[0405] 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 8C.

TABLE 8C
Geneseq Results for NOV8a
		NOV8a	Identities/
		Residues/	Similarities for
Geneseq	Protein/Organism/Length [Patent #,	Match	the Matched	Expect
Identifier	Date]	Residues	Region	Value
AAW82842	Human interleukin-5 receptor protein	1 . . . 439	418/439 (95%)	0.0
	sequence - Homo sapiens, 420 aa.	1 . . . 420	419/439 (95%)
	[WO9847923-A1, 29-OCT-1998]
AAR25064	Human IL-5 receptor alpha chain -	1 . . . 439	418/439 (95%)	0.0
	Homo sapiens, 421 aa. [EP492214-A,	1 . . . 420	419/439 (95%)
	01-JUL-1992]
AAR22219	Sequence of secretory interleukin 5	1 . . . 439	417/439 (94%)	0.0
	receptor (HSIL-5R) - Homo sapiens,	1 . . . 420	418/439 (94%)
	420 aa. [EP475746-A, 18-MAR-1992]
AAR22215	Sequence of human interleukin 5 (IL-5)	1 . . . 439	415/439 (94%)	0.0
	receptor with signal peptide (from	1 . . . 420	418/439 (94%)
	healthy volunteers) - Mouse, 420 aa.
	[EP475746-A, 18-MAR-1992]
AAR22216	Sequence of human interleukin 5	1 . . . 392	390/392 (99%)	0.0
	receptor with signal peptide (from a	1 . . . 392	391/392 (99%)
	patient of eosinophilia) - Mouse, 396 aa.
	[EP475746-A, 18-MAR-1992]

[0406] In a BLAST search of public sequence databases, the NOV8a protein was found to have homology to the proteins shown in the BLASTP data in Table 8D.

TABLE 8D
Public BLASTP Results for NOV8a
		NOV8a	Identities/
Protein		Residues/	Similarities for
Accession		Match	the Matched	Expect
Number	Protein/Organism/Length	Residues	Portion	Value
Q14633	Interleukin-5 receptor precursor -	1 . . . 439	418/439 (95%)	0.0
	Homo sapiens (Human), 420 aa.	1 . . . 420	419/439 (95%)
Q01344	Interleukin-5 receptor alpha chain	1 . . . 439	418/439 (95%)	0.0
	precursor (IL-5R-alpha) (CD125	1 . . . 420	419/439 (95%)
	antigen) - Homo sapiens (Human), 420
	aa.
Q14631	Interleukin-5 receptor type 2 precursor -	1 . . . 392	391/392 (99%)	0.0
	Homo sapiens (Human), 396 aa.	1 . . . 392	391/392 (99%)
Q15469	Soluble interleukin-5 receptor	1 . . . 332	331/332 (99%)	0.0
	precursor - Homo sapiens (Human),	1 . . . 332	331/332 (99%)
	333 aa.
E967751	HUMAN SOLUBLE IL	1 . . . 332	330/332 (99%)	0.0
	5-RECEPTOR ALPHA CHAIN -	1 . . . 332	331/332 (99%)
	Homo sapiens (Human), 335 aa.

[0407] PFam analysis predicts that the NOV8a protein contains the domains shown in the Table 8E.

TABLE 8E
Domain Analysis of NOV8a
Pfam Domain	NOV8a	Identities/	Expect
	Match Region	Similarities	Value
		for the Matched Region

Example 9

[0408] The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.

TABLE 9A
NOV9 Sequence Analysis
	SEQ ID NO: 23	2919 bP
NOV9a,	ATGAGTGACGTGAATCCACCCTCTGACACCCCCATTCCCTTTTCATCCTCCTCCACTCACAGTT
CG173919-01	CTCATATTCCGCCCTGGACATTCTCTTGCTACCCCGGCTCCCCATGTGAAAATGGGGTCATGCT
DNA Sequence	GTACATGAGAAACGTGAGCCATGAGGAGCTACAACGGTTCAAGCAGCTCTTACTGACTGAGCTC
	AGTACTGGCACCATGCCCATCACCTGGGACCAGGTCGAGACAGCCAGCTGGGCAGAGGTGGTTC
	ATCTCTTGATAGAGCGTTTCCCTGGACGACGCGCTTGGGATGTGACTTCGAACATCTTTGCCAT
	TATGAACTGTGATAAAATGTGTGTTGTAGTCCGCAGAGAGATAAATGCCATTCTGCCTACCTTG
	GAACCAGAGGACTTGAATGTGGGAGAAACACAGGTGAATCTGGAGGAAGGAGAATCTGGTAAAA
	TACGGCGGTATAAATCGAATGTGATGGAAAAGTTTTTCCCCATATGGGACATTACGACTTGGCC
	TGGAAACCAGAGGGACTTCTTCTACCAAGGTGTACACAGGCACGAGGAGTACTTACCATGTCTG
	CTTCTGCCCAAAAGACCCCAGGGTAGACAGCCCAAGACCGTGGCCATACAGGGAGCTCCTGGGA
	TCGGAAAAACAATCCTGGCCAAAAAGGTGATGTTTGAGTGGGCCAGAAACAAGTTCTACGCCCA
	CAAGCGCTGGTGTGCTTTCTACTTCCATTGCCAACAGGTGAACCAGACGACAGACCAGAGCTTC
	TCCGAGCTGATTGAGCAAAAGTGGCCTGGATCTCAGGACCTCGTGTCAAAGATTATGTCCAAAC
	CCGACCAACTTCTGCTGCTCTTGGATGGCTTTGAGGAGCTCACATCTACCCTCATTGACAGACT
	GGAGGACCTGAGTGAAGACTGGAGGCAGAAATTGCCTCGGTCTGTCCTACTGAGCAGTTTGCTG
	AGCAAAACGATGCTTCCAGAGGCCACGCTACTGATCATGATAAGATTTACCTCTTGGCAGACAT
	GCAAGCCCTTGCTGAAATGTCCCTCTCTCGTAACCCTTCCGGGGTTTAATACGATGGAAAAAAT
	CAAGTATTTCCAGATGTATTTTGGACACACAGAGGAGGGAGACCAAGTCTTGAGTTTCGCCATG
	GAAAACACCATTCTCTTCTCCATGTGCCGGGTCCCTGTGGTTTGCTGGATGGTCTGCTCTGGTC
	TGAAACAGCAAATGGAGAGAGGAAACAATCTCACACAGTCATGTCCAAATGCCACCTCTGTGTT
	CGTCCGGTATATTTCTAGCTTGTTTCCCACCAGAGCTGAGAACTTTTCCAGAAAGATCCACCAA
	GCACAACTGGAAGGTCTGTGTCACTTGGCCGCAGACAGCATGTGGCACAGGAAATGGGTGTTAG
	GTAAAGAAGATCTTGAGGAAGCCAAGCTGGATCAGACGGGAGTCACCGCCTTCCTTGGCATGAG
	TATTCTTCGGAGAATTGCAGGTGAGGAAGACCACTATGTCTTTACCCTCGTGACTTTTCAGGAA
	TTTTTTGCGGCCTTGTTTTATGTTCTCTGTTTCCCACAAAGACTCAAAAATTTTCATGTGTTGA
	GCCACGTGAATATCCAGCGCCTGATAGCGAGTCCCAGAGGAAGCAAAAGCTATCTCTCTCACAT
	GGGACTTTTCTTATTCGGTTTTCTGAACGAGGCCTGCGCTTCGGCCGTGGAACAGTCATTCCAA
	TGCAAGGTGTCTTTCGGTAATAAGAGGAAACTGCTGAAAGTCATACCTCTGTTGCATAAATGTG
	ACCCACCTTCTCCGGGCAGTGGGGTCCCGCAGTTATTCTACTGTCTGCATGAAATCCGGGAGGA
	AGCCTTTGTAACCCAAGCCCTAAATGATTATCATAAAGTTGTCTTGAGAATTGGCAACAACAAA
	GAAGTTCAAGTGTCTGCTTTTTGCCTGAAGCGGTGTCAATATTTGCATGAGGTGGAACTGACCG
	TCACCCTGAACTTCATGAACGTGTGGAAGCTCAGCTCCAGCTCCCATCCTGGCTCTGACCTAAG
	GCGTGTGAATAGCACCATGTTGAACCAGGACTTAATCGGTGTTTTGACGGGGAACCAGCATCTG
	AGATACTTGGAAATACAACATGTGGAAGTGGAGTCCAAAGCTGTGAAGCTTCTATGCAGGGTGC
	TGAGATCCCCCCGGTGCCGTCTGCAGTGTCTCAGGTTGGAAGACTGCTTGGCCACCCCTAGAAT
	TTGGACTGATCTTGGCAATAATCTTCAAGGTAACGGGCATCTAAAGACTCTCATACTAAGAAAA
	AACTCCCTGGAGAACTGTGGGGCGTATTACCTGTCTGTGGCCCAGCTGGAGAGGCTGTCGCAGA
	GTAAGATGCTGACCCACCTGAGCTTGGCAGAAAACGCCTTGAAAGATGAAGGGGCCAAGCATAT
	TTGGAATGCCCTGCCACACCTGAGATGTCCTCTGCAGAGGCTGGTACTGAGAAAGTGTGACTTG
	ACCTTTAATTGCTGTCAGGATATGATCTCTGCGCTCTGTAAAAATAAAACCCTGAAAAGTCTTG
	ACCTAAGTTTTAATAGCCTGAAGGATGATGGGGTGATCCTGCTGTGTGAGGCCCTGAAGAACCC
	TGACTGTACATTACAGATCCTGGAGCTGGAAAACTGCCTGTTCACCTCCATCTGCTGCCAGGCC
	ATGGCTTCCATGCTCCGCAAAAACCAACATCTGAGACATCTGGACTTGAGCAAGAATGCGATTG
	GAGTCTATGGTATTCTGACCTTGTGCGACGCCTTCTCAAGCCAAAAGAAGAGAGAAGAGGTCAT
	TTTCTGTATTCCTGCCTGGACTCGAATAACTAGCTTCTCCCCAACTCCTCACCCACCCGACTTC
	ACGGGAAAAAGTGACTGCCTATCCCAGATTAATCCTTAG
	ORF Start: ATG at 1		ORF Stop: TAG at 2917
	SEQ ID NO: 24	972 aa	MW at 110966.4 kD
NOV9a,	MSDVNPPSDTPIPPSSSSTHSSHIPPWTFSCYPGSPCENGVMLYMRNVSHEELQRFKQLLLTEL
CG173919-01	STGTMPITWDQVETASWAEVVHLLIERFPGRRAWDVTSNIFAIMNCDKMCVVVRREINAILPTL
Protein Sequence	EPEDLNVGETQVNLEEGESGKIRRYKSNVMEKFFPIWDITTWPGNQRDFFYQGVHRHEEYLPCL
	LLPKRPQGRQPKTVAIQGAPGIGKTILAKKVMFEWARNKFYAHKRWCAFYFHCQEVNQTTDQSF
	SELIEQKWPGSQDLVSKIMSKPDQLLLLLDGFEELTSTLIDRLEDLSEDWRQKLPGSVLLSSLL
	SKTMLPEATLLIMIRFTSWQTCKPLLKCPSLVTLPGFNTMEKIKYFQMYFGHTEEGDQVLSFAM
	ENTILFSMCRVPVVCWMVCSGLKQQMERGNNLTQSCPNATSVFVRYISSLFPTRAENFSRKIHQ
	AQLEQLCHLAADSMWHRKWVLGKEDLEEAKLDQTGVTAFLGMSILRRIAGEEDHYVFTLVTFQE
	FFAALFYVLCFPQRLKNFHVLSHVNIQRLIASPRGSKSYLSHMGLFLFGFLNEACASAVEQSFQ
	CKVSFGNKRKLLKVIPLLHKCDPPSPGSGVPQLFYCLHEIREEAFVSQALNDYHKVVLRIGNNK
	EVQVSAFCLKRCQYLHEVELTVTLNFMNVWKLSSSSHPGSDLRRVNSTMLNQDLIGVLTGNQHL
	RYLEIQHVEVESKAVKLLCRVLRSPRCRLQCLRLEDCLATPRIWTDLGNNLQGWQHLKTLILRK
	NSLENCGAYYLSVAQLERLSQSKMLTHLSLAENALKDEGAKHIWNALPHLRCPLQRLVLRKCDL
	TFNCCQDMISALCKNKTLKSLDLSFNSLKDDGVILLCEALKNPDCTLQILELENCLFTSICCQA
	MASMLRKNQHLRHLDLSKNAIGVYGILTLCEAFSSQKKREEVIFCIPAWTRITSFSPTPHPPDF
	TGKSDCLSQINP

[0409] Further analysis of the NOV9a protein yielded the following properties shown in Table 9B.

TABLE 9B
Protein Sequence Properties NOV9a
SignalP	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG:	a new signal peptide prediction method
analysis:		N-region: length 9; pos.chg 0; neg.chg 2
		H-region: length 28; peak value 0.00
		PSG score: −4.40
	GvH:	von Heijne's method for signal seq. recognition
		GvH score (threshold: −2.1): −6.94
		possible cleavage site: between 30 and 31
	>>> Seems to have no N-terminal signal peptide
	ALOM:	Klein et al's method for TM region allocation
		Init position for calculation: 1
		Tentative number of TMS(s) for the threshold 0.5: 2
		INTEGRAL Likelihood = −2.02 Transmembrane
		387-403
		INTEGRAL Likelihood = −2.13 Transmembrane
		507-523
		PERIPHERAL Likelihood = 0.90 (at 555)
		ALOM score: −2.13 (number of TMSs: 2)
	MTOP:	Prediction of membrane topology (Hartmann et al.)
		Center position for calculation: 394
		Charge difference: 1.0 C(1.0) − N(0.0)
		C > N: C-terminal side will be inside
	>>> membrane topology: type 3b
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment(75):	3.53
	Hyd Moment(95):	4.97	G content:	0
	D/E content:	2	S/T content:	2
	Score: −6.93
	Gavel: prediction 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: found
		KIMSKPDQL at 273
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
		type 1: none
		type 2: none
	NMYR:	N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear
	discrimination
		Prediction: cytoplasmic
		Reliability: 70.6
	COIL:	Lupas's algorithm to detect coiled-coil regions
		total: 0 residues
	Final Results (k = 9/23):
	22.2%: vacuolar
	22.2%: nuclear
	22.2%: endoplasmic reticulum
	11.1%: Golgi
	11.1%: cytoplasmic
	11.1%: mitochondrial
	>> prediction for CG173919-01 is vac (k = 9)

[0410] A search of the NOV9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9C.

TABLE 9C
Geneseq Results for NOV9a
		NOV9a	Identities/
		Residues/	Similarities for
Geneseq	Protein/Organism/Length [Patent #,	Match	the Matched	Expect
Identifier	Date]	Residues	Region	Value
AAO17859	Pyrin domain containing protein	1 . . . 942	941/942 (99%)	0.0
	NALP5/Py8-hs - Unidentified, 2312	1 . . . 942	941/942 (99%)
	aa. [WO200240668-A2,
	23-MAY-2002]
AAO17863	Pyrin domain containing protein	1 . . . 681	678/681 (99%)	0.0
	[WO200240668-A2, 23-MAY-2002]	1 . . . 681	680/681 (99%)
AAO15591	Human PYRIN-10 protein - Homo	605 . . . 972	367/368 (99%)	0.0
	sapiens, 481 aa. [WO200261049-A2,	114 . . . 481	367/368 (99%)
	08-AUG-2002]
ABP53254	Human MDDT-13 protein SEQ ID	40 . . . 884	305/874 (34%)	e−142
	NO: 13 - Homo sapiens, 920 aa.	14 . . . 854	458/874 (51%)
	[WO200264792-A2, 22-AUG-2002]
AAE07514	Human PYRIN-1 protein - Homo	44 . . . 932	304/989 (30%)	e−118
	sapiens, 1034 aa. [WO200161005-A2,	11 . . . 994	474/989 (47%)
	23-AUG-2001]

[0411] In a BLAST search of public sequence databases, the NOV9a protein was found to have homology to the proteins shown in the BLASTP data in Table 9D.

TABLE 9D
Public BLASTP Results for NOV9a
		NOV9a	Identities/
Protein		Residues/	Similarities for
Accession		Match	the Matched	Expect
Number	Protein/Organism/Length	Residues	Portion	Value
CAD35284	Sequence 23 from Patent WO0226780 -	44 . . . 927	311/933 (33%)	e−120
	Homo sapiens (Human), 1035 aa.	16 . . . 937	471/933 (50%)
P59046	PYRIN-containing APAF1-like protein 7	44 . . . 927	311/959 (32%)	e−119
	(Monarch-1) - Homo sapiens (Human),	16 . . . 964	475/959 (49%)
	1062 aa.
AAH28069	Hypothetical 120.2 kDa protein - Homo	44 . . . 927	310/958 (32%)	e−118
	sapiens (Human), 1061 aa.	16 . . . 963	473/958 (49%)
Q96P20	Cold autoinflammatory syndrome 1	44 . . . 932	304/989 (30%)	e−117
	protein (Cryopyrin) (NACHT-, LRR- and	11 . . . 994	474/989 (47%)
	PYD-containing protein 3)
	(PYRIN-containing APAF1-like protein
	1) (Angiotensin/vasopressin receptor
	AII/AVP-like) - Homo sapiens (Human),
	1034 aa.
Q8WX94	PYRIN-containing APAF1-like protein 3 -	45 . . . 931	275/931 (29%)	e−105
	Homo sapiens (Human), 980 aa.	14 . . . 928	453/931 (48%)

[0412] PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9E.

TABLE 9E
Domain Analysis of NOV9a
		Identities/
	NOV9a	Similarities	Expect
Pfam Domain	Match Region	for the Matched Region	Value
PAAD_DAPIN	35 . . . 123	18/92 (20%)	0.018
		55/92 (60%)

Example 10

[0413] The NOV10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.

TABLE 10A
NOV10 Sequence Analysis
	SEQ ID NO: 25	487 bp
NOV10a,	GGGCTCCGGGCCCCTGGCCTCGCGGTGCCATGCTGCCCCGGCGGCGGCGCTGAAGGATGGCGAC
CG174858-02	GCCGCTGCCTCCGCCCTCCCCGCGGCACCTGCGGCTGCTGCGGCTGCTGCTCTCCGGCCTCGTC
DNA Sequence	CTCGGCGCCGCCCTGCGTGGAGCCGCCGCCGGCCACCCGGATGTAGCCGCCTGTCCCGGGAGCC
	TGGACTGTGCCCTGAAGAGGCGGGCAAGGTGTCCTCCTGGTGCACATGCCTGTGGGCCCTGCCT
	TCAGCCCTTCCAGGAGGACCAGCAAGGGCTCTGTGTGCCCAGGATGCGCCGGCCTCCAGGCGGG
	GGCCGGCCCCAGCCCAGACTGGAAGATGAGATTGACTTCACGGTGTACGAGTGCCCGGGCCTGG
	CCCCGACCGGGGAAATGGAGGTGCGCAACCCTCTGTTCGACCACGCCGCACTGTCCGCGCCCCT
	GCCGGCCCCCAGCTCACCGCCTGCACTGCCATGACCTGG
	ORF Start: ATG at 57		ORF Stop: TGA at 480
	SEQ ID NO: 26	141 aa	MW at 14812.0 kD
NOV10a,	MATPLPPPSPRHLRLLRLLLSGLVLGAALRGAAAGHPDVAACPGSLDCALKRRARCPPGAHACG
CG174858-02	PCLQPFQEDQQGLCVPRMRRPPGGGRPQPRLEDEIDFTVYECPGLAPTGEMEVRNPLFDHAALS
Protein Sequence	APLPAPSSPPALP
	SEQ ID NO: 27	1441 bp
NOV10b,	GGCACGAGGGCCTCTTCTTCCTCCTGCGTCCTCCCCCGCTGCCTCCGCTGCTCCCGACGCGGAG
CG174858-01	CCCGGAGCCCGCGCCGAGCCCCTGGCCTCGCGGTGCCATGCTGCCCCGGCGGCGGCGCTGAAGG
DNA Sequence	ATGGCGACGCCGCTGCCTCCGCCCTCCCCGCGGCACCTGCGGCTGCTGCGGCTGCTGCTCTCCG
	GCCTCGTCCTCGGCGCCGCCCTGCGTGGAGCCGCCGCCGGCCACCCGGATGTAGCCGCCTGTCC
	CGGGAGCCTGGACTGTGCCCTGAAGAGGCGGGCAAGGTGTCCTCCTGGTGCACATGCCTGTGGG
	CCCTGCCTTCAGCCCTTCCAGGAGGACCAGCAAGGGCTCTGTGTGCCCAGGATGCGCCGGCCTC
	CAGGCGGGGGCCGGCCCCAGCCCAGACTGGAAGATGAGATTGACTTCCTGGCCCAGGAGCTTGC
	CCGGAAGGAGTCTGGACACTCAACTCCGCCCCTACCCAAGGACCGACAGCGGCTCCCGGAGCCT
	GCCACCCTGGGCTTCTCGGCACGGGGGCAGGGGCTGGAGCTGGGCCTCCCCTCCACTCCAGGAA
	CCCCCACGCCCACGCCCCACACCTCCCTGGGCTCCCCTGTGTCATCCGACCCGGTGCACATGTC
	GCCCCTGGAGCCCCGGGGAGGGCAAGGCGACGGCCTCGCCCTTGTGCTGATCCTGGCGTTCTGT
	GTGGCCGGTGCAGCCGCCCTCTCCGTAGCCTCCCTCTGCTGGTGCAGGCTGCAGCGTGAGATCC
	GCCTGACTCAGAAGGCCGACTACGCCACTGCGAAGGCCCCTGGCTCACCTGCAGCTCCCCGGAT
	CTCGCCTGGGGACCAGCGGCTGGCACAGAGCGCGGAGATGTACCACTACCAGCACCAACGGCAA
	CAGATGCTGTGCCTGGAGCGGCATAAAGAGCCACCCAAGGAGCTGGACACGGCCTCCTCGGATG
	AGGAGAATGAGGACGGAGACTTCACGGTGTACGAGTGCCCGGGCCTGGCCCCGACCGGGGAAAT
	GGAGGTGCGCAACCCTCTGTTCGACCACGCCGCACTGTCCGCGCCCCTGCCGGCCCCCAGCTCA
	CCGCCTGCACTGCCATGACCTGGAGGCAGACAGACGCCCACCTGCTCCCCGACCTCGAGGCCCC
	CGGGGAGGGGCAGGGCCTGGAGCTTCCCACTAAAAACATGTTTTGATGCTGTGTGCTTTTGGCT
	GGGCCTCGGGCTCCAGGCCCTGGGACCCCTTGCCAGGGAGACCCCCGAACCTTTGTGCCAGGAC
	ACCTCCTGGTCCCCTGCACCTCTCCTGTTCGGTTTAGACCCCCAAACTGGAGGGGGCATGGAGA
	ACCGTAGAGCGCAGGAACGGGTGGGTAATTCTAGAGACAAAAGCCAATTAAAGTCCATTTCAGA
	CCTGCGGCTTCTGAAAAAAAAAAAAAAAAAAAA
ORF Start: ATG at 129		ORF Stop: TGA at 1104
SEQ ID NO: 28	1325 aa	MW at 34515.9 kD
NOV10b,	MATPLPPPSPRHLRLLRLLLSGLVLGAALRGAAAGHPDVAACPGSLDCALKRRARCPPGAHACG
CG174858-01	PCLQPFQEDQQGLCVPRMRRPPGGGRPQPRLEDEIDFLAQELARKESGHSTPPLPKDRQRLPEP
Protein Sequence	ATLGFSARGQGLELGLPSTPGTPTPTPHTSLGSPVSSDPVHMSPLEPRGGQGDGLALVLILAFC
	VAGAAALSVASLCWCRLQREIRLTQKADYATAKAPGSPAAPRISPGDQRLAQSAEMYHYQHQRQ
	QMLCLERHKEPPKELDTASSDEENEDGDFTVYECPGLAPTGEMEVRNPLFDHAALSAPLPAPSS
	PPALP

[0414] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 10B.

TABLE 10B
Comparison of NOV10a against NOV10b.
		NOV10a	Identities/
	Protein	Residues/	Similarities for
	Sequence	Match Residues	the Matched Region
	NOV10b	1 . . . 105	102/105 (97%)
		1 . . . 105	102/105 (97%)

[0415] Further analysis of the NOV10a protein yielded the following properties shown in Table loc.

TABLE 10C
Protein Sequence Properties NOV10a
SignalP	Cleavage site between residues 35 and 36
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 11; pos. chg 1; neg. chg 0
	H-region: length 2; peak value −7.14
	PSG score: −11.54
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): 0.98
	possible cleavage site: between 26 and 27
	>>> 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 =	−2.39	Transmembrane 13-29
	PERIPHERAL	Likelihood =	9.49 (at 34)
	ALOM score: −2.39 (number of TMSs: 1)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 20
	Charge difference: −3.0 C(0.5)-N(3.5)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 2 (cytoplasmic tail 1 to 13)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	4	Hyd Moment (75):	2.26
	Hyd Moment (95):	2.85	G content:	4
	D/E content:	1	S/T content:	3
	Score: −3.26
	Gavel: prediction of cleavage sites for mitochondrial preseq
	R-2 motif at 40 LRG|AA
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: PRMRRPP (5) at 80
	bipartite: none
	content of basic residues: 9.9%
	NLS Score: −0.04
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals: none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: nuclear
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	43.5%: nuclear
	26.1%: mitochondrial
	8.7%: Golgi
	8.7%: cytoplasmic
	4.3%: extracellular, including cell wall
	4.3%: plasma membrane
	4.3%: peroxisomal
	>> prediction for CG174858-02 is nuc (k = 23)

[0416] 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 10D.

TABLE 10D
Geneseq Results for NOV10a
		NOV10a	Identities/
Geneseq	Protein/Organism/Length	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
AAB75349	Human secreted protein #8 - Homo	1 . . . 105	102/105 (97%)	2e−57
	sapiens, 325 aa. [WO200100806-A2,	1 . . . 105	102/105 (97%)
	04 JAN. 2001]
AAM93794	Human polypeptide, SEQ ID NO: 3823 -	1 . . . 105	102/105 (97%)	2e−57
	Homo sapiens, 325 aa.	1 . . . 105	102/105 (97%)
	[EP1130094-A2, 05 SEP. 2001]
AAB25763	Human secreted protein SEQ ID #75 -	1 . . . 105	102/105 (97%)	2e−57
	Homo sapiens, 325 aa.	1 . . . 105	102/105 (97%)
	[WO200037491-A2, 29 JUN. 2000]
AAB43904	Human cancer associated protein	1 . . . 105	102/105 (97%)	2e−57
	sequence SEQ ID NO: 1349 - Homo	2 . . . 106	102/105 (97%)
	sapiens, 326 aa. [WO200055350-A1,
	21 SEP. 2000]
AAU27661	Human protein AFP485790 - Homo	1 . . . 105	97/105 (92%)	2e−54
	sapiens, 325 aa. [WO200166748-A2,	1 . . . 105	97/105 (92%)
	13 SEP. 2001]

[0417] 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 10E.

TABLE 10E
Public BLASTP Results for NOV10a
Protein		NOV10a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q8NCE1	Hypothetical protein FLJ90312 -	1 . . . 105	102/105 (97%)	6e−57
	Homo sapiens (Human), 325 aa.	1 . . . 105	102/105 (97%)
Q9NQX5	Neural proliferation differentiation and	1 . . . 105	102/105 (97%)	6e−57
	control protein-1 precursor (NPDC-1	1 . . . 105	102/105 (97%)
	protein) - Homo sapiens (Human), 325 aa.
Q8WXX4	NPDC-1 protein - Homo sapiens	1 . . . 105	101/105 (96%)	2e−56
	(Human), 325 aa.	1 . . . 105	101/105 (96%)
CAC88643	Sequence 95 from Patent WO0166748 -	1 . . . 105	97/105 (92%)	7e−54
	Homo sapiens (Human), 325 aa.	1 . . . 105	97/105 (92%)
Q925Q2	Neural proliferation differentiation	1 . . . 105	80/105 (76%)	9e−43
	control-1 protein - Mus musculus (Mouse),	1 . . . 105	85/105 (80%)
	332 aa.

[0418] PFam analysis predicts that the NOV10a protein contains the domains shown in the Table 10F.

TABLE 10F
Domain Analysis of NOV10a
	Pfam	NOV10a	Identities/	Expect
	Domain	Match Region	Similarities for	Value
			the Matched Region

Example 11

[0419] The NOV11 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11A.

TABLE 11A
NOV11 Sequence Analysis
	SEQ ID NO: 29	3751 bp
NOV11a,	ATGGGGGTGGGCAGGGCGCTCGCCGCGCTGCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGC
CG76203-01	TGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGCGCCGCCACGAGACTTAGACAAAAA
DNA Sequence	AAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCCATAATGAACTTCTCTGGACC
	AACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTTTCAGGTTCTGGTAAACG
	TTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAGCACCCAGCACGG
	ATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAATACAGATTT
	GGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATTGCCT
	GTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTAT
	TGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAAC
	AATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGAT
	CTCCCAGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCT
	GCCGCCCCGCCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTC
	AATTATGGAGGAGGAAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTG
	ACCTCGTGTTCCCGTGGTTTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTAT
	ACTGCAACGGGGGTGTTCAAAATTCAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTA
	ATCTGCATAGGAATTATCATTGTGAATCCCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGG
	TGCGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATACTTTGTGGTTAAGACTGTGTTGGA
	GCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAGCTGCCTTTCTCTTCGA
	GACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCTGTGGCTGGGCT
	TGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCATTGGAGA
	TTTCTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGGG
	AGACGATCACCTTTACCAGCACCCATCTTCTAAGCTGTACTTTACCACACCGAGGTGGCCTATG
	ACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGG
	AAAAATACTATTGTTCATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGT
	GTTGTATTCTTGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCC
	AGTAAACAAAAATCTCTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATC
	CTGCTGGTCTGTACCAGTTGTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCA
	GGAATGAATTCCATTCTGGTATACGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGT
	GGAAGCTGAAGGACAACCAGTCCCACAAGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCT
	CTCGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGATTTTTTGGAAAATCTGATGGCTCCCA
	CTGAGATGTGCTGCTGGAAGACTCTAGTAGGCCTGCAGGGAGGACTGAAGCAGCCTTTGTTAAA
	GGGAAGCATTCATTAGGAAATTGACTGGCTGCGTGTTTACAGACTCTGGGGGAAGACACTGATG
	TCCTCAAACTGGTTAACTGTGACACGGCTCGCCAGAACTCTGCCTGTCTATTTGTGACTTACAG
	ATTTGAAATGTAATTGTCTTTTTTCCTCCATCTTCTGTGGAAATGGATGTCTTTGGAACTTCAT
	TCCGAGGAGATAAGCTTTAACTTTCCAAAAGGGAATTGCCATGGGTGTTTTTCTTCTGTGGTGA
	GTGAAACAATCTGAGGTCTGGTTCTTGCTGACCTTGTTGCCCTGCAAACTTCCTTTCCACGTGT
	ACGCGCACACCAACACGAAATGCCATCACTCCTACTGCGGCTGCTATGAAGCTTACTGGTTGTG
	ATGTGTTATAATTTAGTCTGTTTTTTTGATTGAATGCAGTTTAATGTTTCCAGAAAGCCAAAGT
	AATTTTCTTTTCAGATATGCAAGGCTTTGGTGGGTCCAAAAAATGTCTATCACAAGCCATTTTT
	TCCTTTTCCTCTCTCGAAAAGTTAAAATATCTATGTGTTATTCCCAAACCCTCTTACCTATGTA
	TCTGCCTGTCTGTCCATCATCTTCCTTCCTCCCTATCTCTGTGTATCTGGATGGCAGCCGCTGC
	CCAGGGGAGTGGCTGTGGGGAGGGCAGGTACTGTCTTTGCCTGTGGGTCCAGCTGAGCCATCCC
	TGCTGGGTGATGCTGGGCAAGACCCTTGGCCCGTCTGGGCCTTGGCTTCCTCACTTGTGAAATG
	AGCGGGAAGATGACTCTCAGTTCCTTCCACCTCTTAGACATGGTGAGGTAACAGACATCAAAAG
	CTTTTCTGAAATCTTCAGAAGAAATAGTTCCATTACAGAAAACTCTTCAAAATAAATAGTAGTG
	AAAACTTTTAAAAACTCTCATTGGAGTAAGTCTTTTCAAGATGATCCTCCACAATGGAGGCAGC
	GTTCCTACTTGTCATCACACAGCTGAAGACATTGTTTCTTAGGTGTGAAATCGGGGACAAAGGA
	CAAACAGAGACACACGGCATTGTTCATGGGAGGCATCGTCACCCTCCTGGGTGTTCTGTGGGAA
	TTTCCTGTGTGAGGAAAACGTGGCCACAGGGTTGTGCTGTACCCACCCTTCCCCGGCGAGATGG
	CCCTCGGCCTGTGCCGCTGCTTCCACCCTCGCCACTCCATGGCAGCTTTTGGTCTGTTTCCGGC
	TCTGCCCTCTGCCCTGAACTCTCATCCGGCTTGTACCTGCCTGCTGGACCCCTCCACCTGGAGG
	CCAGCCCATGTCTCAGGCCCAGCCCTAGCCTCTTCTCCTCAAATTCTAAGTGTTTTCTCTTTAG
	GTTTCCCTGGCTTTGTGAATGGATCATGTGTCTCTAGGTATAAACCTGACATCATCTTTCCACC
	CGGCTTACCTCCACCAGATCTCCCCAGTTCTGTCTCCATCTTCTACCTGCAGCTGCTCTGTTCT
	CATGGTCACTGCTGCATCACTGAGTCTGGACCCTTGTTATCATTTTCAAACTGGCCTCCTTCCC
	TCGTTCCCCACTTCTTAAAGTCACCTGTCCATTGCCACCAGATTAAGCTTTCTCCAGCCAGATC
	ACCTCTCTCTGAGAAACCTCCATTGACATGGAAACACCATTGTCTGGCACACATACTCACATAC
	TCACCTTCCCGTCTTGATCCCCACACATCTTTCCAGCCTCCCCTCCCACTCCACTCCCTGCTCC
	CTCCTCCACCTCCCCATCCTCTTGTCTCCCCTCCCCTCT
	ORF Start: ATG at 1		ORF Stop: TAA at 1375
	SEQ ID NO: 30	458 aa	MW at 50588.4 kD
NOV11a,	MGVGRALAALLLAASVLSAALLAPGGSSGRDAQAAPPRDLDKKRHAELKMDQALLLIHNELLWT
CG176203-01	NLTVYWKSECCYHCLFQVLVNVPQSPKAGKPSAAAASVSTQHGSILQLNDTLEEKEVCRLEYRF
Protein Sequence	GEFGNYSLLVKNIHNGVSEIACDLAVNEDPVDSNLPVSIAFLIGLAVIIVISFLRLLLSLDDFN
	NWISKAISSRETDRLINSELGSPSRTDPLDGDVQPATWRLSALPPRLRSVDTFRGIALILMVFV
	NYGGGKYWYFKHASWNGLTVADLVFPWFVFIMGSSIFLSMTSILQRGCSKFRLLGKIAWRSFLL
	ICIGIIIVNPNYCLGPLSWDKVRIPGVLQRLGVTYFVVKTVLELLFAKPVPEHCASERSCLSLR
	DITSSWPQWLLILVLEGLWLGLTFLLPVPGCPTGYLGPGGIGDFWQVSKLHWRSCRLHRPPAAG
	RRSPLPAPIF

[0420] Further analysis of the NOV11a protein yielded the following properties shown in Table 11B.

TABLE 11B
Protein Sequence Properties NOV11a
SignalP	Cleavage site between residues 30 and 31
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 5; pos. chg 1; neg. chg 0
	H-region: length 24; peak value 9.20
	PSG score: 4.80
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): 1.28
	possible cleavage site: between 18 and 19
	>>> Seems to have a cleavable signal peptide (1 to 18)
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 19
	Tentative number of TMS(s) for the threshold 0.5: 5
	INTEGRAL	Likelihood =	−12.31	Transmembrane 163-179
	INTEGRAL	Likelihood =	−3.35	Transmembrane 284-300
	INTEGRAL	Likelihood =	−4.73	Transmembrane 313-329
	INTEGRAL	Likelihood =	−1.06	Transmembrane 351-367
	INTEGRAL	Likelihood =	−6.85	Transmembrane 394-410
	PERIPHERAL	Likelihood =	0.95 (at 241)
	ALOM score: −12.31 (number of TMSs: 5)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 9
	Charge difference: 0.0 C(2.0)-N(2.0)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 3a
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	2	Hyd Moment (75):	8.77
	Hyd Moment (95):	7.34	G content:	5
	D/E content:	1	S/T content:	4
	Score: -3.83
	Gavel : prediction of cleavage sites for mitochondrial
	preseq
	R-2 motif at 15 GRA|LA
	NUCDISC: discrimination of nuclear localization signals
	pat4: KKRH (3) at 42
	pat7: none
	bipartite: none
	content of basic residues: 9.4%
	NLS Score: −0.29
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	XXRR-like motif in the N-terminus: GVGR 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: MGVGRAL
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	77.8%: endoplasmic reticulum
	22.2%: mitochondrial
	>> prediction for CG176203-01 is end (k = 9)

[0421] A search of the NOV11a 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 NOV11a
		NOV11a	Identities/
Geneseq	Protein/Organism/Length	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
ABB62779	Drosophila melanogaster polypeptide	42 . . . 428	139/406 (34%)	9e−40
	SEQ ID NO 15129 - Drosophila	11 . . . 369	206/406 (50%)
	melanogaster, 576 aa. [WO200171042-A2,
	27 SEP. 2001]
ABG24189	Novel human diagnostic protein	210 . . . 249	20/45 (44%)	0.98
	#24180 - Homo sapiens, 871 aa.	376 . . . 420	23/45 (50%)
	[WO200175067-A2, 11 OCT. 2001]
ABG63379	Human albumin fusion protein #54 -	372 . . . 421	21/51 (41%)	4.9
	Homo sapiens, 561 aa. [WO200177137-A1,	31 . . . 80	27/51 (52%)
	18 OCT. 2001]
AAE03429	Human gene 3 encoded secreted protein	372 . . . 421	21/51 (41%)	4.9
	HETDB76, SEQ ID NO: 112 - Homo	31 . . . 80	27/51 (52%)
	sapiens, 561 aa. [WO200132675-A1,
	10 MAY 2001]
AAU29269	Human PRO polypeptide sequence	372 . . . 421	21/51 (41%)	4.9
	#246 - Homo sapiens, 300 aa.	46 . . . 95	27/51 (52%)
	[WO200168848-A2, 20 SEP. 2001]

[0422] In a BLAST search of public sequence databases, the NOV11a protein was found to have homology to the proteins shown in the BLASTP data in Table 11D.

TABLE 11D
Public BLASTP Results for NOV11a
Protein		NOV11a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
AAH24084	Similar to hypothetical protein	1 . . . 427	330/427 (77%)	0.0
	FLJ32731 - Mus musculus (Mouse),	1 . . . 416	358/427 (83%)
	624 aa.
BAC29006	Adult male urinary bladder cDNA,	1 . . . 427	329/427 (77%)	0.0
	RIKEN full-length enriched library,	1 . . . 416	357/427 (83%)
	clone: 9530006P14
	product: hypothetical protein, full insert
	sequence - Mus musculus (Mouse), 624 aa.
AAH42037	Hypothetical protein - Homo sapiens	3 . . . 186	184/184 (100%)	e−100
	(Human), 219 aa (fragment).	1 . . . 184	184/184 (100%)
Q96M97	Hypothetical protein FLJ32731 - Homo	288 . . . 428	100/141 (70%)	5e−47
	sapiens (Human), 367 aa.	28 . . . 160	109/141 (76%)
Q9W4F7	CG6903 protein (LD22376p) -	42 . . . 428	139/406 (34%)	3e−39
	Drosophila melanogaster (Fruit fly), 576 aa.	11 . . . 369	206/406 (50%)

[0423] PFam analysis predicts that the NOV11a protein contains the domains shown in the Table 11E.

TABLE 11E
Domain Analysis of NOV11a
	Pfam	NOV11a	Identities/	Expect
	Domain	Match Region	Similarities for	Value
			the Matched Region

Example 12

[0424] 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: 31	1760 bp
NOV12a,	GCGGCCGCGGGGGCCTTGCCTTCCGCACTCGGGCGCAGCCGGGTGGATCTCGAGCAGGTGCGGA
CG176213-01	GCCCCGGGCGGCGGGCGCGGGTGCGAGGGATCCCTGACGCCTCTGTCCCTGTTTCTTTGTCGCT
DNA Sequence	CCCAGCCTGTCTGTCGTCGTTTTGGCGCCCCCGCCTCCCCGCGGTGCGGGGTTGCACACCGATC
	CTGGGCTTCGCTCGATTTGCCGCCGAGGCGCCTCCCAGACCTAGAGGGGCGCTGGCCTGGAGCA
	GCGGGTCGTCTGTGTCCTCTCTCCTCTGCGCCGCGCCCGGGGATCCGAAGGGTGCGGGGCTCTG
	AGGAGGTGACGCGCGGGGCCTCCCGCACCCTGGCCTTGCCCGCATTCTCCCTCTCTCCCAGGTG
	TGAGCAGCCTATCAGTCACCATGTCCGCAGCCTGGATCCCGGCTCTCGGCCTCGGTGTGTGTCT
	GCTGCTGCTGCCGGGGCCCGCGGGCAGCGAGGGAGCCGGTAAACGACTAAAGAAAACACCCGAG
	AAGAAAACTGGCAATAAAGATTGTAAAGCAGACATTGCATTTCTGATTGATGGAAGCTTTAATA
	TTGGGCAGCGCCGATTTAATTTACAGAAGAATTTTGTTGGAAAAGTGGCTCTAATGTTGGGAAT
	TGGAACAGAAGGACCACATGTGGGCCTTGTTCAAGCCAGTGAACATCCCAAAATAGAATTTTAC
	TTGAAAAACTTTACATCAGCCAAAGATGTTTTGTTTGCCATAAAGGAAGTAGGTTTCAGAGGGG
	GTAATTCCAATACAGGAAAAGCCTTGAAGCATACTGCTCAGAAATTCTTCACGGTAGATGCTGG
	AGTAAGAAAAGGGATCCCCAAAGTGGTGGTGGTATTTATTGATGGTTGGCCTTCTGATGACATC
	GAGGAAGCAGGCATTGTGGCCAGAGAGTTTGGTGTCAATGTATTTATAGTTTCTGTGGCCAAGC
	CTATCCCTGAAGAACTGGGGATGGTTCAGGATGTCACATTTGTTGACAAGGCTGTCTGTCGGAA
	TAATGGCTTCTTCTCTTACCACATGCCCAACTGGTTTGGCACCACAAAATACGTAAAGCCTCTG
	GTACAGAAGCTGTGCACTCATGAACAAATGATGTGCAGCAAGACCTGTTATAACTCAGTGAACA
	TTGCCTTTCTAATTGATGGCTCCAGCAGTGTTGGAGATAGCAATTTCCGCCTCATGCTTGAATT
	TGTTTCCAACATAGCCAAGACTTTTGAAATCTCGGACATTGGTGCCAAGATAGCTGCTGTACAG
	TTTACTTATGATCAGCGCACGGAGTTCAGTTTCACTGACTATAGCACCAAAGAGAATGTCCTAG
	CTGTCATCAGAAACATCCGCTATATGAGTGGTGGAACAGCTACTGGTGATGCCATTTCCTTCAC
	TGTTAGAAATGTGTTTGGCCCTATAAGGGAGAGCCCCAACAAGAACTTCCTAGTAATTGTCACA
	GATGGGCAGTCCTATGATGATGTCCAAGGCCCTGCAGCTGCTGCACATGATGCAGGAATCACTA
	TCTTCTCTGTTGGTGTGGCTTGGGCACCTCTGGATGACCTGAAAGATATGGCTTCTAAACCGAA
	GGAGTCTCATGCTTTCTTCACAAGAGAGTTCACAGGATTAGAACCAATTGTTTCTGATGTCATC
	AGAGGCATTTGTAGAGATTTCTTAGAATCCCAGCAATAATGGTAACATTTTGACAACTGAAAGA
	AAAAGTACAAGGGGATCCAGTGTGTAAATTGT
	ORF Start: ATG at 405		ORF Stop: TAA at 1701
	SEQ ID NO: 32	432 aa	MW at 47177.7 kD
NOV12a,	MSAAWIPALGLGVCLLLLPGPAGSEGAGKRLKKTPEKKTGNKDCKADIAFLIDGSFNIGQRRFN
CG176213-01	LQKNFVGKVALMLGIGTEGPHVGLVQASEHPKIEFYLKNFTSAKDVLFAIKEVGFRGGNSNTGK
Protein Sequence	ALKHTAQKFFTVDAGVRKGIPKVVVVFIDGWPSDDIEEAGIVAREFGVNVFIVSVAKPIPEELG
	MVQDVTFVDKAVCRNNGFFSYHMPNWFGTTKYVKPLVQKLCTHEQMMCSKTCYNSVNIAFLIDG
	SSSVGDSNFRLMLEFVSNIAKTFEISDIGAKIAAVQFTYDQRTEFSFTDYSTKENVLAVIRNIR
	YMSGGTATGDAISFTVRNVFGPIRESPNKNFLVIVTDGQSYDDVQGPAAAAHDAGITIFSVGVA
	WAPLDDLKDMASKPKESHAFFTREFTGLEPIVSDVIRGICRDFLESQQ

[0425] Further analysis of the NOV12a protein yielded the following properties shown in Table 12B.

TABLE 12B
Protein Sequence Properties NOV12a
SignalP	Cleavage site between residues 25 and 26
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 0; pos. chg 0; neg. chg 0
	H-region: length 24; peak value 10.25
	PSG score: 5.85
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): 1.68
	possible cleavage site: between 24 and 25
	>>> Seems to have a cleavable signal peptide (1 to 24)
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 25
	Tentative number of TMS(s) for the threshold 0.5: 1
	Number of TMS(s) for threshold 0.5: 0
	PERIPHERAL	Likelihood = 2.60 (at 368)
	ALOM score: 0.42 (number of TMSs: 0)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 12
	Charge difference: 3.0 C(4.0)-N(1.0)
	C > N: C-terminal side will be inside
	>>>Caution: Inconsistent mtop result with signal peptide
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment(75):	1.80
	Hyd Moment (95):	2.02	G content:	4
	D/E content:	1	S/T content:	2
	Score: −7.37
	Gavel: prediction 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
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	43.5%: mitochondrial
	13.0%: extracellular, including cell wall
	13.0%: cytoplasmic
	13.0%: endoplasmic reticulum
	8.7%: vacuolar
	8.7%: nuclear
	>> prediction for CG176213-01 is mit (k = 23)

[0426] 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 12C.

TABLE 12C
Geneseq Results for NOV12a
		NOV12a	Identities/
Geneseq	Protein/Organism/Length	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
AAB50430	Human mutant COCH5B2 protein -	28 . . . 432	405/405 (100%)	0.0
	Homo sapiens, 550 aa. [WO200071081-A2,	146 . . . 550	405/405 (100%)
	30 NOV. 2000]
AAB50429	Human COCH5B2 protein - Homo	28 . . . 432	405/405 (100%)	0.0
	sapiens, 550 aa. [WO200071081-A2,	146 . . . 550	405/405 (100%)
	30 NOV. 2000]
AAB80251	Human PRO294 protein - Homo	28 . . . 432	405/405 (100%)	0.0
	sapiens, 550 aa. [WO200104311-A1,	146 . . . 550	405/405 (100%)
	18 JAN. 2001]
AAU29046	Human PRO polypeptide sequence #23 -	28 . . . 432	405/405 (100%)	0.0
	Homo sapiens, 550 aa. [WO200168848-A2,	146 . . . 550	405/405 (100%)
	20 SEP. 2001]
AAY84405	Amino acid sequence of human COCH5B2	28 . . . 432	405/405 (100%)	0.0
	polypeptide - Homo sapiens, 550 aa.	146 . . . 550	405/405 (100%)
	[WO200018211-A2, 06 APR. 2000]

[0427] 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 12D.

TABLE 12D
Public BLASTP Results for NOV12a
Protein		NOV12a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
O43405	Cochlin precursor (COCH-5B2) -	28 . . . 432	405/405 (100%)	0.0
	Homo sapiens (Human), 550 aa.	146 . . . 550	405/405 (100%)
Q62507	Cochlin precursor (COCH-5B2) - Mus	27 . . . 432	395/406 (97%)	0.0
	musculus (Mouse), 552 aa.	147 . . . 552	402/406 (98%)
O42163	Cochlin precursor (COCH-5B2) -	28 . . . 432	345/405 (85%)	0.0
	Gallus gallus (Chicken), 547 aa.	143 . . . 547	381/405 (93%)
Q96IU6	Coagulation factor C (Limulus	28 . . . 374	347/347 (100%)	0.0
	polyphemus) homology (Cochlin) -	146 . . . 492	347/347 (100%)
	Homo sapiens (Human), 494 aa.
CAD58748	SI: dZ234G15.4 (novel protein similar	29 . . . 429	254/401 (63%)	e−155
	to coagulation factor C homolog	152 . . . 551	325/401 (80%)
	(cochlin, COCH)) - Brachydanio rerio
	(Zebrafish) (Danio rerio), 553 aa.

[0428] PFam analysis predicts that the NOV12a protein contains the domains shown in the Table 12E.

TABLE 12E
Domain Analysis of NOV12a
			Identities/
	Pfam	NOV12a	Similarities for	Expect
	Domain	Match Region	the Matched Region	Value
	vwa	47 . . . 228	57/207 (28%)	4.9e−29
			135/207 (65%)
	vwa	249 . . . 419	60/202 (30%)	1.1e−40
			136/202 (67%)

Example 13

[0429] 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: 33	2689 bp
NOV13a,	CACCAAGCTTCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGCCCG
CG50691-05	GGAGCATCGTGCAGGGCGTCTGCGGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGAGCT
DNA Sequence	GCGGCGGCACCTTCGGGATTTACGGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCCCCC
	GCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACTGAT
	GACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCAATG
	GGAAATGTGAATGTAACACCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGATAT
	GTGCCTTTCAGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGTGAA
	GTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTGGGG
	AGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCA
	GCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTC
	TATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGCAGA
	CCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTACTTT
	GCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCCACT
	CCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTA
	ATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCGAAT
	GGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGT
	GAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATCCAG
	TGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCG
	GTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACC
	GTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCGAAG
	AACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGACAGG
	GAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAAC
	ACCGAGGAACTATGTTCAGAACGTAAACAAGGCTGCACCTTGAACTGTCCCTTCGGTTTCCTTA
	CTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCATAAT
	CTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGT
	AAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCACG
	GCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCAC
	TTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGGGAA
	TGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTGGCA
	ACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCC
	AGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAAACG
	TGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGG
	TGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTAC
	AGATCAACCTTTTCGGCCTTCCTTGTCCCGCAATAACAGCGTACCTAATTACTGCAAAAATGAT
	GAAGGGGATATATTCCTGGCAGCTGAGTCCTGGAAGCCTGACGTTTGTACCAGCTGCATCTGCA
	TTGATAGCGTAATTAGCTGTTTCTCTGAGTCCTGCCCTTCTGTATCCTGTGAAAGACCTGTCTT
	GAGAAAAGGCCAGTGTTGTCCCTACTGCATAGAAGACACAATTCCAAAGAAGGTGGTGTGCCAC
	TTCAGTGGGAAGGCCTATGCCGACGAGGAGCGGTGGGACCTTGACAGCTGCACCCACTGCTACT
	GCCTGCAGGGCCAGACCCTCTGCTCGACCGTCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCAT
	CAACGTGGAAGGAAGTTGCTGCCCAATGTGTCCAGAAATGTATGTCCCAGAACCAACCAATATA
	CCCATTGAGAAGACAAACCATCGAGGAGAGGTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTA
	GTGAAAATGATATCGTCCATCTCCCTAGAGATATGGGTCACCTCCAGGTAGATTACAGACTCGA
	G
	ORF Start: at 11		ORF Stop: at 2684
	SEQ ID NO: 34	891 aa	MW at 97577.0 kD
NOV13a,	LVCLPCDESKCEEPRNCPGSIVQGVCGCCYTCASQRNESCGGTFGIYGTCDRGLRCVIRPPLNG
CG50691-05	DSLTEYEAGVCEDENWTDDQLLGFKPCNENLIAGCNIINGKCECNTIRTCSNPFEFPSQDMCLS
Protein Sequence	ALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVCQPGN
	LNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCTLPTR
	CECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFRMDNC
	RFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGDRWRE
	DDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLTGKDC
	INGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPIICDK
	YCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSHGCLICKCREASASAGPPILSGTCLT
	VDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFVVQKPELS
	TPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQCTDQP
	FRPSLSRNNSVPNYCKNDEGDIFLAAESWKPDVCTSCICIDSVISCFSESCPSVSCERPVLRKG
	QCCPYCIEDTIPKKVVCHFSGKAYADEERWDLDSCTHCYCLQGQTLCSTVSCPPLPCVEPINVE
	GSCCPMCPEMYVPEPTNIPIEKTNHRGEVDLEVPLWPTPSENDIVHLPRDMGHLQVDYR
	SEQ ID NO: 35	5379 bp
NOV13b,	GGCCCGGCTGCGAGGAGGAGGCGGCGGCGGCGCAGGAGGATGTACTTGGTGGCGGGGGACAGGG
CG50691-04	GGTTGGCCGGCTGCGGGCACCTCCTGGTCTCGCTGCTGGGGCTGCTGCTGCTGCTGGCGCGCTC
DNA Sequence	CGGCACCCGGGCGCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGC
	CCGGGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGA
	GCTGCGGCGGCACCTTCGGGATTTACGGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCC
	CCCGCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACT
	GATGACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCA
	ATGGGAAATGTGAATGTAACACCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGA
	TATGTGCCTTTCAGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGT
	GAAGTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTG
	GGGAGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTG
	CCAGCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGAC
	CTCTATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGC
	AGACCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTAC
	TTTGCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCC
	ACTCCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTG
	TTAATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCG
	AATGGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGT
	GGTGAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATC
	CAGTGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGA
	CCGGTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCG
	ACCGTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCG
	AAGAACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGAC
	AGGGAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATA
	AACACCGAGGAACTATGTTCAGAACGTAAACAAGGCTGCACCTTGAACTGTCCCTTCGGTTTCC
	TTACTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCAT
	AATCTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGC
	TGTAAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTC
	ACGGCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGG
	CACTTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGG
	GAATGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTG
	GCAACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAA
	GCCAGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAA
	ACGTGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAG
	AGGTGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTG
	TACAGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTATGCCGACGAG
	GAGCGGTGGGACCTTGACAGCTGCACCCACTGCTACTGCCTGCAGGGCCAGACCCTCTGCTCGA
	CCGTCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTGCTGCCCAAT
	GTGTCCAGAAATGTATGTCCCAGAACCAACCAATATACCCATTGAGAAGACAAACCATCGAGGA
	GAGGTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTAGTGAAAATGATATCGTCCATCTCCCTA
	GAGATATGGGTCACCTCCAGGTAGATTACAGAGATAACAGGCTGCACCCAAGTGAAGATTCTTC
	ACTGGACTCCATTGCCTCAGTTGTGGTTCCCATAATTATATGCCTCTCTATTATAATAGCATTC
	CTATTCATCAATCAGAAGAAACAGTGGATACCACTGCTTTGCTGGTATCGAACACCAACTAAGC
	CTTCTTCCTTAAATAATCAGCTAGTATCTGTGGACTGCAAGAAAGGAACCAGAGTCCAGGTGGA
	CAGTTCCCAGAGAATGCTAAGAATTGCAGAACCAGATGCAAGATTCAGTGGCTTCTACAGCATG
	CAAAAACAGAACCATCTACAGGCAGACAATTTCTACCAAACAGTGTGAAGAAAGGCAACTAGGA
	TGAGGTTTCAAAAGACGGAAGACGACTAAATCTGCTCTAAAAAGTAAACTAGAATTTGTGCACT
	TGCTTAGTGGATTGTATTGGATTGTGACTTGATGTACAGCGCTAAGACCTTACTGGGATGGGCT
	CTGTCTACAGCAATGTGCAGAACAAGCATTCCCACTTTTCCTCAAGATAACTGACCAAGTGTTT
	TCTTAGAACCAAAGTTTTTAAAGTTGCTAAGATATATTTGCCTGTAAGATAGCTGTAGAGATAT
	TTGGGGTGGGGACAGTGAGTTTGGATGGGGAAATGGGTGGGAGGGTGGTGTTGGGAAGAAAAAT
	TGGTCAGCTTGGCTCGGGGAGAAACCTGGTAACATAAAAGCAGTTCAGTGGCCCAGACGTTATT
	TTTTTCCTATTGCTCTGAAGACTGCACTGGTTGCTGCAAAGCTCAGGCCTGAATGAGCAGGAAA
	CAAAAAAGGCCTTGCGACCCAGCTGCcATAACCACCTTAGAACTACCAGACGAGCACATCAGAA
	CCCTTTGACAGCCATCCCAGGTCTAAAGCCACAAGTTTCTTTTCTATACAGTCACAACTGCAGT
	AGGCAGTGAGGAAGCCAGAGAAATGCGATAGCGGCATTTCTCTAAAGCGGGTTATTAAGGATAT
	ATACAGTTACACTTTTTGCTCCTTTTATTTTCTTCCAAGCCAATCAATCAGCCAGTTCCTACCA
	GAGTCAGCACATGAACAAGATCTAAGTCATTTCTTGATGTGAGCACTGGAGCTTTTTTTTTTTT
	ACAACGTGACAGGAAGACGAGGGAGAGGGTGACGAACACCAGGCATTTCCAGGGGCTATATTTC
	ACTGTTTGTTGTTGCTTTGTTCTGTTATATTGTTGGTTGTTCATAGTTTTTGTTGAAGCTCTAG
	CTTAAGAAGAAACTTTTTTTAAAAAGACTGTTTGGGGATTCTTTTTCCTTATTATATACTGATT
	CTACAAAATACAAACTACTTCATTTTAATTGTATATTATTCAAGCACCTTTGTTGAAGCTCAAA
	AAAAATGATGCCTCTTTAAACTTTAGCAATTATAGGAGTATTTATGTAACTATCTTATGCTTCA
	AAAAACAAAAGTATTTGTGTGCATGTGTATATAATATATATATATACATATATATTTATACACA
	TACAATTTATGTTTTCCTGTTGAATGTATTTTTATGAGATTTTAACCAGAACAAAGGCAGATAA
	ACAGGCATTCCATAGCAGTGCTTTTGATCACTTACAAATTTTTTGAATAACACAAAATCTCATT
	CTACCTGCAGTTTAATTGGAAAGATGTGTGTGTGAGAGTATGTATGTGTGTGTGTGTGTGTGTG
	TGTGTGCGCGCGCACGCACGCCTTCAGCAGTCAGCATTGCACCTGCTATCGAGAAGGGTATTCC
	TTTATTAAAATCTTCCTCATTTGGATTTGCTTTCAGTTGGTTTTCAATTTGCTCACTCGCCAGA
	GACATTGATGGCAGTTCTTATCTGCATCACTAATCACCTCCTCGATTTTTTTTTTTTTTTTTTC
	AAACAATGGTTTGAAACAACTACTGGAATATTCTCCACAATAAGCTGGAAGTTTGTTGTAGTAT
	GCCTCAAATATAACTGACTGTATACTATAGTGGTAACTTTTCAAACAGCCCTTAGCACTTTTAT
	ACTAATTAACCCATTTGTGCATTGAGTTTTCTTTTAAAAATGCTTGTTGTGAAAGACACAGATA
	CCCAGTATGCTTAACGTGAAAAGAAAATGTGTTCTGTTTTGTAAAGGAACTTTCAAGTATTGTT
	GTAAATACTTGGACAGAGGTTGCTCAACTTTAAAAAAAATTAATTTATTATTATAATGACCTAA
	TTTATTAATCTGAAGATTAACCATTTTTTTGTCTTAGAATATCAAAAAGAAAAAGAAAAAGGTG
	TTCTAGCTGTTTGCATCAAAGGAAAAAAAGATTTATTATCAAGGGGCAATATTTTTATCTTTTC
	CAAAATAAATTTGTTAATGATACATTACAAAAATAGATTGACATCAGCCTGATTAGTATAAATT
	TTGTTGGTAATTAATCCATTCCTGGCATAAAAAGTCTTTATCAAAAAAAATTGTAGATGCTTGC
	TTTTTGTTTTTTCAATCATGGCCATATTATGAAAATACTAACAGGATATAGGACAAGGTGTAAA
	TTTTTTTATTATTATTTTAAAGATATGATTTATCCTGAGTGCTGTATCTATTACTCTTTTACTT
	TGGTTCCTGTTGTGCTCTTGTAAAAGAAAAATATAATTTCCTGAAGAATAAAATAGATATATGG
	CACTTGGAGTGCATCATAGTTCTACAGTTTGTTTTTGTTTTCTTCAAAAAAGCTGTAAGAGAAT
	TATCTGCAACTTGATTCTTGGCAGGAAATAAACATTTTGAGTTGAAATCAAAAAAAAAAAAAAA
	AAA
	ORF Start: ATG at 40		ORF Stop: TGA at 2926
	SEQ ID NO: 36	962 aa	MW at 105541.4 kD
NOV13b,	MYLVAGDRGLAGCGHLLVSLLGLLLLLARSGTRALVCLPCDESKCEEPRNCPGSIVQGVCGCCY
CG50691-04	TCASQRNESCGGTFGIYGTCDRGLRCVIRPPLNGDSLTEYEAGVCEDENWTDDQLLGFKPCNEN
Protein Sequence	LIAGCNIINGKCECNTIRTCSNPFEFPSQDMCLSALKRIEEEKPDCSKARCEVQFSPRCPEDSV
	LIEGYAPPGECCPLPSRCVCNPAGCLRKVCQPGNLNILVSKASGKPGECCDLYECKPVFGVDCR
	TVECPPVQQTACPPDSYETQVRLTADGCCTLPTRCECLSGLCGFPVCEVGSTPRIVSRGDGTPG
	KCCDVFECVNDTKPACVFNNVEYYDGDMFRMDNCRFCRCQGGVAICFTAQCGEINCERYYVPEG
	ECCPVCEDPVYPFNNPAGCYANGLILAHGDRWREDDCTFCQCVNGERHCVATVCGQTCTNPVKV
	PGECCPVCEEPTIITVDPPACGELSNCTLTGKDCINGFKRDHNGCRTCQCINTEELCSERKQGC
	TLNCPFGFLTDAQNCEICECRPRPKKCRPIICDKYCPLGLLKNKHGCDICRCKKCPELSCSKIC
	PLGFQQDSHGCLICKCREASASAGPPILSGTCLTVDGHHHKNEESWHDGCRECYCLNGREMCAL
	ITCPVPACGNPTIHPGQCCPSCADDFVVQKPELSTPSICHAPGGEYFVEGETWNIDSCTQCTCH
	SGRVLCETEVCPPLLCQNPSRTQDSCCPQCTEDTIPKKVVCHFSGKAYADDERWDLDSCTHCYC
	LQGQTLCSTVSCPPLPCVEPINVEGSCCPMCPEMYVPEPTNIPIEKTNHRGEVDLEVPLWPTPS
	ENDIVHLPRDMGHLQVDYRDNRLHPSEDSSLDSIASVVVPIIICLSIIIAFLFINQKKQWIPLL
	CWYRTPTKPSSLNNQLVSVDCKKGTRVQVDSSQRMLRIAEPDARFSGFYSMQKQNHLQADNFYQ
	TV
	SEQ ID NO: 37	3045 bp
NOV13c,	GGCCCGGCTGCGAGGAGGAGGCGGCGGCGGCGCAGGAGGATGTACTTGGTGGCGGGGGACAGGG
CG50691-02	GGTTGGCCGGCTGCGGGCACCTCCTGGTCTCGCTGCTGGGGCTGCTGCTGCTGCTGGCGCGCTC
DNA Sequence	CGGCACCCGGGCGCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGC
	CCGGGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGA
	CCCGCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACT
	GATGACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCA
	ATGGGAAATGTGAATGTAACACCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGA
	TATGTGCCTTTCAGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGT
	GAAGTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTG
	GGGAGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTG
	CCAGCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGAC
	CTCTATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGC
	AGACCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTAC
	TTTGCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCC
	ACTCCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTG
	TTAATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATCGAGACATGTTTCG
	AATGGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGT
	GGTGAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATC
	CAGTGTATCCTTTTAATAATCCCGCTCGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGA
	CCGGTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCG
	ACCGTCTGCGGACAGACCTGCACAAACCCTGTCAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCG
	AAGAACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGAC
	AGGCAAGGACTGCATTAATGGTTTCAAACQCGATCACAATGGTTGTCGGACCTGTCAGTGCATA
	AACACCGAGGAACTATGTTCAGAACGTAAACAAGGCTGCACCTTGAACTGTCCCTTCGGTTTCC
	TTACTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCAT
	AATCTGTGACAAGTATTGTCCACTTCGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGC
	TGTAAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTC
	ACGGCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGG
	CACTTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGG
	GAATGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTG
	GCAACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTCGTGCAGAA
	GCCAGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAA
	ACGTGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTCAGACAG
	AGGTGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTG
	TACAGATCAACCTTTTCGGCCTTCCTTGTCCCGCAATAACAGCGTACCTAATTACTGCAAAAAT
	GATGAAGGGGATATATTCCTGGCAGCTGAGTCCTCGAAGCCTGACGTTTGTACCAGCTGCATCT
	GCATTGATAGCGTAATTAGCTGTTTCTCTGAGTCCTGCCCTTCTGTATCCTGTGAAAGACCTGT
	CTTGAGAAAAGGCCAGTGTTGTCCCTACTGCATAGAAATGTATGTCCCAGAACCAACCAATATA
	CCCATTGAGAAGACAAACCATCGAGGAGAGGTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTA
	GTGAAAATGATATCGTCCATCTCCCTAGAGATATGGGTCACCTCCAGGTAGATTACAGAGATAA
	CAGGCTGCACCCAAGTGAAGATTCTTCACTGGACTCCATTGCCTCAGTTGTGGTTCCCATAATT
	ATATGCCTCTCTATTATAATAGCATTCCTATTCATCAATCAGAAGAAACAGTGGATACCACTGC
	TTTGCTGGTATCGAACACCAACTAAGCCTTCTTCCTTAAATAATCAGCTAGTATCTGTGGACTG
	CAAGAAAGGAACCAGAGTCCAGGTGGACAGTTCCCAGAGAATGCTAAGAATTGCAGAACCAGAT
	GCAAGATTCAGTGGCTTCTACAGCATGCAAAAACAGAACCATCTACAGGCAGACAATTTCTACC
	AAACAGTGTGAAGAAAGGCAACTAGGATGAGGTTTCAAAAGACCGAAGACGACTAAATCTGCTC
	TAAAAAGTAAACTAGAATTTGTGCACTTGCTTAGTGG
	ORF Start: ATG at 40		ORF Stop: TGA at 2953
	SEQ ID NO: 38	971 aa	MW at 106615.5 kD
NOV13c,	MYLVAGDRGLAGCGHLLVSLLGLLLLLARSGTRALVCLPCDESKCEEPRNCPGSIVQGVCGCCY
CG50691-02	TCASQRNESCGGTFGIYGTCDRGLRCVIRPPLNGDSLTEYEAGVCEDENWTDDQLLGFKPCNEN
Protein Sequence	LIAGCNIINGKCECNTIRTCSNPFEFPSQDMCLSALKRIEEEKPDCSKARCEVQFSPRCPEDSV
	TVECPPVQQTACPPDSYETQVRLTADGCCTLPTRCECLSGLCGFPVCEVGSTPRIVSRGDGTPG
	KCCDVFECVNDTKPACVFNNVEYYDGDMFRMDNCRFCRCQGGVAICFTAQCGEINCERYYVPEG
	ECCPVCEDPVYPFNNPAGCYANGLILAHGDRWREDDCTFCQCVNGERHCVATVCGQTCTNPVKV
	PGECCPVCEEPTIITVDPPACGELSNCTLTGKDCINGFKRDHNGCRTCQCINTEELCSERKQGC
	TLNCPFGFLTDAQNCEICECRPRPKKCRPIICDKYCPLGLLKNKHGCDICRCKKCPELSCSKIC
	PLGFQQDSHGCLICKCREASASAGPPILSGTCLTVDGHHHKNEESWHDGCRECYCLNGREMCAL
	ITCPVPACGNPTIHPGQCCPSCADDFVVQKPELSTPSICHAPGGEYFVEGETWNIDSCTQCTCH
	SGRVLCETEVCPPLLCQNPSRTQDSCCPQCTDQPFRPSLSRNNSVPNYCKNDEGDIFLAAESWK
	PDVCTSCICIDSVISCFSESCPSVSCERPVLRKGQCCPYCIEMYVPEPTNIPIEKTNHRGEVDL
	EVPLWPTPSENDIVHLPRDMGHLQVDYRDNRLHPSEDSSLDSIASVVVPIIICLSIIIAFLFIN
	QKKQWIPLLCWYRTPTKPSSLNNQLVSVDCKKGTRVQVDSSQRMLRIAEPDARFSGFYSMQKQN
	HLQADNFYQTV
	SEQ ID NO: 39	3026 bp
NOV 13d,	GGCCCGGCTGCGAGGAGGAGGCGGCGGCGGCGCAGGAGGATGTACTTGGTGGCGGGGGACAGGG
CG50691-03	GGTTGGCCGGCTGCGGGCACCTCCTGGTCTCGCTGCTGGGGCTGCTGCTGCTGCTGGCGCGCTC
DNA Sequence	CGGCACCCGGGCGCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGC
	CCGGGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGA
	GCTGCGGCGGCACCTTCGGGATTTACGGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCC
	CCCGCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGAAGAGAAGCCAGAT
	TGCTCCAAGGCCCGCTGTGAAGTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCG
	AGGGTTATGCTCCTCCTGCGGAGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGG
	CTGTCTGCGCAAAGTCTGCCAGCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAG
	CCGGGAGAGTGCTGTGACCTCTATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTGG
	AATGCCCTCCTGTTCAGCAGACCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAAC
	TGCAGATGGTTGCTGTACTTTGCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCC
	GTGTGTGAGGTGGGATCCACTCCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCT
	GTGATGTCTTTGAATGTGTTAATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTA
	TGATGGAGACATGTTTCGAATGGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATC
	TGCTTCACTGCCCAGTGTGGTGACATAAACTGCGACAGGTACTACGTGCCCGAAGGAGAGTGCT
	GCCAGTGTGTGAAGATCCAGTGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCT
	GATCCTTGCCCACGGAGACCGGTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGT
	GAACGCCACTGCGTTGCGACCGTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTCGGG
	AGTGTTGCCCTGTGTGCGAAGAACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTT
	ATCAAACTGCACTCTGACAGGGAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGT
	CGGACCTGTCAGTGCATAAACACCGACGAACTATGTTCAGAACGTAAACAAGGCTGCACCTTGA
	ACTGTCCCTTCGGTTTCCTTACTGATGCCCAAAACTGTGAGATCTGTGAGTCCCGCCCAAGGCC
	CAAGAAGTGCAGACCCATAATCTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCAC
	GGCTGTGACATCTGTCGCTGTAAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGG
	GTTTCCAGCAGGACACTCACGGCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGG
	GCCACCCATCCTGTCGGGCACTTGTCTCACCGTCGATGGTCATCATCATAAAAATGAGGAGAGC
	TGGCACGATGGGTGCCGGGAATGCTACTGTCTCAATGGACGGGAAATCTGTGCCCTGATCACCT
	GCCCGGTGCCTGCCTGTGGCAACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGA
	TGACTTTGTGGTGCAGAAGCCAGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAA
	TACTTTGTGGAAGGAGAAACGTGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGAC
	GGGTGCTGTGTGAGACAGAGGTGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGA
	TTCCTGCTGCCCACAGTGTACAGATCAACCTTTTCGGCCTTCCTTGTCCCGCAATAACAGCGTA
	CCTAATTACTGCAAAAATGATGAAGGGGATATATTCCTGGCAGCTGAGTCCTGGAAGCCTGACG
	TTTGTACCAGCTGCATCTGCATTGATAGCGTAATTAGCTGTTTCTCTGAGTCCTGCCCTTCTGT
	ATCCTGTGAAAGACCTGTCTTGAGAAAAGGCCAGTGTTGTCCCTACTGCATAGAAGACACAATT
	CCAAAGAAGGTGGTGTCCCACTTCAGTGGGAAGGCCTATGCCGACGAGGAGCGGTGGGACCTTG
	ACAGCTGCACCCACTGCTACTGCCTGCAGGGCCAGACCCTCTGCTCGACCGTCAGCTGCCCCCC
	TCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTGCTGCCCAATGTGTCCAGAAATGTAT
	GTCCCAGAACCAACCAATATACCCATTGAGAAGACAAACCATCGAGGAGAGGTTGACCTGGAGG
	TTCCCCTGTGGCCCACGCCTAGTGAAAATGATATCGTCCATCTCCCTAGAGATATGGGTCACCT
	CCAGGTAGATTACAGAGATAACAGGCTGCACCCAAGTGAAGATTCTTCACTGGACTCCATTGCC
	TCAGTTGTGGTTCCCATAATTATATGCCTCTCTATTATAATAGCATTCCTATTCATCAATCAGA
	AGAAACAGTGGATACCACTGCTTTGCTGGTATCGAACACCAACTAAGCCTTCTTCCTTAAATAA
	TCAGCTAGTATCTGTGGACTGCAAGAAAGGAACCAGAGTCCACGTGGACAGTTCCCAGAGAATG
	CTAAGAATTGCAGAACCAGATGCAAGATTCAGTGGCTTCTACAGCATGCAAAAACAGAACCATC
	TACAGGCAGACAATTTCTACCAAACAGTGTGAAGAAAGGCAACTAGGATGAGGTTTCAAAAGAC
	GGAAGACGACTAAATCTG
	ORF Start: ATG at 40		ORF Stop: TGA at 2974
	SEQ ID NO: 40	978 aa	MW at 107200.3 kD
NOV13d,	MYLVAGDRGLAGCGHLLVSLLGLLLLLARSGTRALVCLPCDESKCEEPRNCPGSIVQGVCGCCY
CG50691-03	TCASQRNESCGGTFGIYGTCDRGLRCVIRPPLNGDSLTEYEAGVCEEEKPDCSKARCEVQFSPR
Protein Sequence	CPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVCQPGNLNILVSKASGKPGECCDLYECKPV
	FGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCTLPTRCECLSGLCGFPVCEVGSTPRIVSR
	GDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFRMDNCRFCRCQGGVAICFTAQCGEINCER
	YYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGDRWREDDCTFCQCVNGERHCVATVCGQTC
	TNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLTGKDCINGFKRDHNGCRTCQCINTEELCS
	ERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPIICDKYCPLGLLKNKHGCDICRCKKCPEL
	SCSKICPLGFQQDSHGCLICKCREASASAGPPILSGTCLTVDGHHHKNEESWHDGCRECYCLNG
	REMCALITCPVPACGNPTIHPGQCCPSCADDFVVQKPELSTPSICHAPGGEYFVEGETWNIDSC
	TQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQCTDQPFRPSLSRNNSVPNYCKNDEGDIFL
	AAESWKPDVCTSCICIDSVISCFSESCPSVSCERPVLRKGQCCPYCIEDTIPKKVVCHFSGKAY
	ADEERWDLDSCTHCYCLQCQTLCSTVSCPPLPCVEPINVEGSCCPMCPEMYVPEPTNIPIEKTN
	HRGEVDLEVPLWPTPSENDIVHLPRDMGHLQVDYRDNRLHPSEDSSLDSIASVVVPIIICLSII
	IAFLFINQKKQWIPLLCWYRTPTKPSSLNNQLVSVDCKKGTRVQVDSSQRMLRIAEPDARFSGF
	YSMQKQNHLQADNFYQTV
	SEQ ID NO: 41		2470 bP
NOV13e,	CACCAAGCTTCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGCCCG
308482339	GGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGAGCT
DNA Sequence	GCGCCGGCACCTTCGGGATTTACGGAACCTGCGACCCGCGGCTGCGTTGTGTCATCCGCCCCCC
	GCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACTGAT
	GACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCAATG
	GGAAATGTGAATGTAACACCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGATAT
	GTGCCTTTCGGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGTGAA
	GTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTGGGG
	AGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCA
	GCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTC
	TATGAGTGCAAACCAGTTTTCGGCGTGGACTGCAGGACTGTCGAATGCCCTCCTGTTCAGCAGA
	CCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTACTTT
	GCCAACAAGATGCGAGTGTCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCCACT
	CCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTA
	ATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCGAAT
	GGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGT
	GAGATAAACTGCGAGAGGTACTACGTGCCCGAAGGAGAGTGCTGCCCAGTCTGTGAAGATCCAG
	TGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCG
	GTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACC
	GTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTcCGAAG
	AACCAACCATCATCACAGTTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTGACAGG
	GAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAAC
	ACCGAGGAACTATGTTCAGAACGTAAACAACGCTGCACCTTGAACTGTCCCTTCGGTTTCCTTA
	CTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCATAAT
	CTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGT
	AAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCACG
	GCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCAC
	TTGTCTCACCGTGGATGGTCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGGGAA
	TGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTGGCA
	ACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCC
	AGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAAACG
	TGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGG
	TGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTAC
	AGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTATGCCGACGAGGAG
	CGGTCGGACCTTGACAGCTGCACCCACTGCTACTGCCTGCAGGGCCAGACCCTCTGCTCGACCG
	TCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTGCTGCCCAATGTG
	TCCAGAAATGTATGTCCCAGAACCAACCAATATACCCATTGAGAAGACAAACCATCGAGGAGAG
	GTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTAGTGAAAATGATATCGTCCATCTCCCTAGAG
	ATATGGGTCACCTCCAGGTAGATTACAGACTCGAGGGC
	ORF Start: at 2		ORF Stop: end of sequence
	SEQ ID NO: 42	823 aa	MW at 90023.6 kD
NOV13e,	TKLLVCLPCDESKCEEPRNCPGSIVQGVCGCCYTCASQRNESCGGTFGIYGTCDRGLRCVIRPP
308482339	LNGDSLTEYEAGVCEDENWTDDQLLGFKPCNENLIAGCNIINGKCECNTIRTCSNPFEFPSQDM
Protein Sequence	CLSALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVCQ
	PGNLNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCTL
	PTRCECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFRM
	DNCRFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGDR
	WREDDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLTG
	KDCINGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPII
	CDKYCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSHGCLICKCREASASAGPPILSGT
	CLTVDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFVVQKP
	ELSTPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQCT
	EDTIPKKVVCHFSGKAYADEERWDLDSCTHCYCLQGQTLCSTVSCPPLPCVEPINVEGSCCPMC
	PEMYVPEPTNIPIEKTNHRGEVDLEVPLWPTPSENDIVHLPRDMGHLQVDYRLEG
	SEQ ID NO: 43	3361 bp
NOV13f	AAAGAGAGTCTCACCCTGTTTCCCAGACCGGAATGCAGTGGCGTGATCAACCTCGTGGCCTCAA
CG50691-01	GTGATCCTCCCACCTCAAACTCCTGAGTGCTGGGACCACAGGCATGCACAACCATTCCCAGCTA
DNA Sequence	ATTTTTTGTTTTGTTTTTGTAGAGACTGGGTCTCACTGTGTTGCCCACGCTGGTCATGAACTCC
	TGGGCTCAAGTAATCCCCGTGCCTTCGTCTCTGAAAGTGTTGGGATTACACGCATGAGCCACTG
	TGCCTGGCCAAAAAAGAGCTCTTTAAAAAATAATTTTGTAGATTGACAAATGTGACTCTTGTAA
	TTTTATTGAACATGAAAAAACCCAGGAATCTTTATTTGATATTAAACATTTTTAAAGGCATCTC
	AGTTGTTGTTGTAATAACACATTAAGAGAAGTAGTGGTTTTTTATTTCCAACCTTTGTGCATAT
	AGCTATTTAATGCCTACATGGATGGCTATTATTTCACTTTTTTCAGTTATTATGAAGAGATTGG
	GTTTCATTCATTTGTAAAGTTTCAGCCAGACTGCCTTTCACAAATTGATTTGTCAAAATTGAAT
	GTTAATCTTGACATCCCAGTGCGTTTTTGCCCCCGAACAGGCCTTTGAATCAAGCTGCAAACAC
	ACATTATCTGGTTGTTAATTGTTTTACAGATGAGAACTGGACTGATGACCAACTGCTTGGTTTT
	AAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCAATGGGAAATGTGAATGTAACA
	CCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGATATGTGCCTTTCACCTTTAAA
	GAGAATTGAACAAGACAAGCCAGATTGCTCCAACGCCCGCTGTGAAGTCCAGTTCTCTCCACGT
	TGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTGGGGAGTGCTGTCCCTTACCCA
	GCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCACCCGGGAAACCTGAACAT
	ACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTCTATCAGTCCAAACCAGTT
	TTCGGCGTGGACTGCAGGACTGTGGAATGCCCTCCTGTTCAGCAGACCGCGTGTCCCCCGGACA
	GCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTCCTGTACTTTGCCAACAAGATCCGAGTG
	TCTCTCTGGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCCACTCCCCGCATAGTCTCTCGT
	GGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTAATGATACAAAGCCAGCCT
	GGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTAATGATACAAAGCCAGCCT
	TCGATGCCAACGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGTGAGATAAACTGCGAGAGG
	TACTACGTGCCCGAAGGAGAGTGCTGCCCAGTGTGTGAAGATCCAGTGTATCCTTTTAATAATC
	CCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCGGTGGCGGGAAGACGACTG
	CACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACCGTCTGCGGACAGACCTGC
	ACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCGAAGAACCAACCATCATCACAG
	TTGATCCACCTGCATGTGGGGAGTTATCAAACTGCACTCTCACAGGGAAGGACTGCATTAATGG
	TTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAACACCGAGGAACTATGTTCA
	GAACGTAAACAAGGCTGCACCTTGAACTGTCCCTTCGGTTTCCTTACTGATGCCCAAAACTGTG
	AGATCTGTGAGTGCCGCCCAAGGCCCAAGAAGTGCAGACCCATAATCTGTGACAAGTATTGTCC
	ACTTGGATTCCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGTAAGAAATGTCCAGAGCTC
	TCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCGCGGCTGTCTTATCTGCAAGT
	GCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCACTTGTCTCACCGTGGATGG
	TCATCATCATAAAAATGAGGAGAGCTGGCACGATGGGTGCCGGGAATGCTACTGTCTCAATGGA
	CGGGAAATGTGTGCCCTGATCACCTGCCCGCTGCCTGCCTGTGGCAACCCCACCATTCACCCTG
	GACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCCAGAGCTCAGTACTCCCTC
	CATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGACAAACGTGGAACATTGACTCCTGT
	ACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGGTGTGCCCACCGCTGCTCT
	GCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTACAGATCAACCTTTTCGGCC
	TTCCTTGTCCCGCAATAACAGCGTACCTAATTACTGCAAAAATGATGAAGGGGATATATTCCTG
	GCAGCTGAGTCCTGGAAGCCTGACGTTTGTACCAGCTGCATCTGCATTGATAGCGTAATTAGCT
	GTTTCTCTGAGTCCTGCCCTTCTGTATCCTGTGAAAGACCTGTCTTGAGAAAAGGCCAGTGTTG
	TCCCTACTGCATAGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTAT
	GCCGACGAGGAGCGGTGGGACCTTGACAGCTGCACCCACTACTACTGCCTGCAGGGCCAGACCC
	TCTGCTCGACCGTCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTG
	CTGCCCAATGTGTCCAGTTTCACCTTTACCATCTTTGGATATGAGTACAGAACCTATGAGCTGT
	TAGGTGATTAGCACCTGTCTCTTTACAGAAGAAACTGAGGCTCAGGAAAGAGCCCCTGTGGGAA
	GAGGACTCACTGTCATGCCTCAGCTTGGTGGAGTTTCACCGGAAATCTACCCATATGCAGGGTC
	AAGGCAAAAGAATTCCAAAGTTACGTCTCTCCCTCTCACTCAGGAAAAAACCTGAGGTGGAACT
	GAATCAATCCCAGCTCTGGGGCCTCTGCAGAAACTTTTACTACTTAGCCATTGACATTTACAGT
	ATAATACCTATCTGATCAAACTGGATAATGTAAATATATTTACTGAAGATCAGCTTCTAATCTA
	AATGGTTCCAGTGGTAACATAATGGACATCTGA
	ORF Start: ATG at 813		ORF Stop: TAG at 3009
	SEQ ID NO: 44	732 aa	MW at 79910.4 kD
NOV13f,	MCLSALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVC
CG50691-01	QPGNLNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCT
Protein Sequence	LPTRCECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFR
	MDNCRFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGD
	RWREDDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLT
	GKDCINGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPI
	ICDKYCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSRGCLICKCREASASAGPPILSG
	TCLTVDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFVVQK
	PELSTPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQC
	TDQPFRPSLSRNNSVPNYCKNDEGDIFLAAESWKPDVCTSCICIDSVISCFSESCPSVSCERPV
	LRKGQCCPYCIEDTIPKKVVCHFSGKAYADEERWDLDSCTHYYCLQGQTLCSTVSCPPLPCVEP
	INVEGSCCPMCPVSPLPSLDMSTEPMSC
	SEQ ID NO: 45	2470 bp
NOV13g,	CACCAAGCTTCTGGTCTGCCTGCCCTGTGACGAGTCCAAGTGCGAGGAGCCCAGGAACTGCCCG
CG50691-06	GGGAGCATCGTGCAGGGCGTCTGCGGCTGCTGCTACACGTGCGCCAGCCAGAGGAACGAGAGCT
DNA Sequence	GCGGCGGCACCTTCGGGATTTACCGAACCTGCGACCGGGGGCTGCGTTGTGTCATCCGCCCCCC
	GCTCAATGGCGACTCCCTCACCGAGTACGAAGCGGGCGTTTGCGAAGATGAGAACTGGACTGAT
	GACCAACTGCTTGGTTTTAAACCATGCAATGAAAACCTTATTGCTGGCTGCAATATAATCAATG
	GGAAATGTGAATGTAACACCATTCGAACCTGCAGCAATCCCTTTGAGTTTCCAAGTCAGGATAT
	GTGCCTTTCGGCTTTAAAGAGAATTGAAGAAGAGAAGCCAGATTGCTCCAAGGCCCGCTGTGAA
	GTCCAGTTCTCTCCACGTTGTCCTGAAGATTCTGTTCTGATCGAGGGTTATGCTCCTCCTGGGG
	AGTGCTGTCCCTTACCCAGCCGCTGCGTGTGCAACCCCGCAGGCTGTCTGCGCAAAGTCTGCCA
	GCCGGGAAACCTGAACATACTAGTGTCAAAAGCCTCAGGGAAGCCGGGAGAGTGCTGTGACCTC
	TATGAGTGCAAACCAGTTTTCGGCGTGGACTGCACGACTGTGGAATGCCCTCCTGTTCAGCAGA
	CCGCGTGTCCCCCGGACAGCTATGAAACTCAAGTCAGACTAACTGCAGATGGTTGCTGTACTTT
	GCCAACAAGATGCGAGTGTCTCTCTCGCTTATGTGGTTTCCCCGTGTGTGAGGTGGGATCCACT
	CCCCGCATAGTCTCTCGTGGCGATGGGACACCTGGAAAGTGCTGTGATGTCTTTGAATGTGTTA
	ATGATACAAAGCCAGCCTGCGTATTTAACAATGTGGAATATTATGATGGAGACATGTTTCGAAT
	GGACAACTGTCGGTTCTGTCGATGCCAAGGGGGCGTTGCCATCTGCTTCACTGCCCAGTGTGGT
	GAGATAAACTGCGAGAGGTACTACGTGCCCGAACGAGAGTGCTGCCCAGTGTGTGAAGATCCAG
	TGTATCCTTTTAATAATCCCGCTGGCTGCTATGCCAATGGCCTGATCCTTGCCCACGGAGACCG
	GTGGCGGGAAGACGACTGCACATTCTGCCAGTGCGTCAACGGTGAACGCCACTGCGTTGCGACC
	GTCTGCGGACAGACCTGCACAAACCCTGTGAAAGTGCCTGGGGAGTGTTGCCCTGTGTGCGAAG
	AACCAACCATCATCACAGTTGATCCACCTGCATGTGCGGAGTTATCAAACTGCACTCTGACAGG
	GAAGGACTGCATTAATGGTTTCAAACGCGATCACAATGGTTGTCGGACCTGTCAGTGCATAAAC
	ACCGAGGAACTATGTTCAGAACGTAAACAACGCTGCACCTTGAACTGTCCCTTCGGTTTCCTTA
	CTGATGCCCAAAACTGTGAGATCTGTGAGTGCCGCCCAACGCCCAAGAAGTGCAGACCCATAAT
	CTGTGACAAGTATTGTCCACTTGGATTGCTGAAGAATAAGCACGGCTGTGACATCTGTCGCTGT
	AAGAAATGTCCAGAGCTCTCATGCAGTAAGATCTGCCCCTTGGGTTTCCAGCAGGACAGTCACG
	GCTGTCTTATCTGCAAGTGCAGAGAGGCCTCTGCTTCAGCTGGGCCACCCATCCTGTCGGGCAC
	TTGTCTCACCGTGGATGGTCATCATCATAAAAATGACGAGAGCTGGCACGATGGGTGCCGGGAA
	TGCTACTGTCTCAATGGACGGGAAATGTGTGCCCTGATCACCTGCCCGGTGCCTGCCTGTGGCA
	ACCCCACCATTCACCCTGGACAGTGCTGCCCATCATGTGCAGATGACTTTGTGGTGCAGAAGCC
	AGAGCTCAGTACTCCCTCCATTTGCCACGCCCCTGGAGGAGAATACTTTGTGGAAGGAGAAACG
	TGGAACATTGACTCCTGTACTCAGTGCACCTGCCACAGCGGACGGGTGCTGTGTGAGACAGAGG
	TGTGCCCACCGCTGCTCTGCCAGAACCCCTCACGCACCCAGGATTCCTGCTGCCCACAGTGTAC
	AGAAGACACAATTCCAAAGAAGGTGGTGTGCCACTTCAGTGGGAAGGCCTATGCCGACGAGGAG
	CGGTGGCACCTTGACAGCTGCACCCACTGCTACTGCCTGCAGGGCCAGACCCTCTGCTCGACCG
	TCAGCTGCCCCCCTCTGCCCTGTGTTGAGCCCATCAACGTGGAAGGAAGTTGCTGCCCAATGTG
	TCCAGAAATGTATGTCCCAGAACCAACCAATATACCCATTGAGAAGACAAACCATCGAGGAGAG
	GTTGACCTGGAGGTTCCCCTGTGGCCCACGCCTAGTGAAAATGATATCGTCCATCTCCCTAGAG
	ATATGGGTCACCTCCAGGTAGATTACAGACTCGAGGGC
	ORF Start: at 11		ORF Stop: at 2462
	SEQ ID NO: 46	817 aa	MW at 89381.8 kD
NOV13g,	LVCLPCDESKCEEPRNCPGSIVQGVCGCCYTCASQRNESCGGTFGIYGTCDRGLRCVIRPPLNG
CG50691-06	DSLTEYEAGVCEDENWTDDQLLGFKPCNENLIAGCNIINGKCECNTIRTCSNPFEFPSQDMCLS
Protein Sequence	ALKRIEEEKPDCSKARCEVQFSPRCPEDSVLIEGYAPPGECCPLPSRCVCNPAGCLRKVCQPGN
	LNILVSKASGKPGECCDLYECKPVFGVDCRTVECPPVQQTACPPDSYETQVRLTADGCCTLPTR
	CECLSGLCGFPVCEVGSTPRIVSRGDGTPGKCCDVFECVNDTKPACVFNNVEYYDGDMFRMDNC
	RFCRCQGGVAICFTAQCGEINCERYYVPEGECCPVCEDPVYPFNNPAGCYANGLILAHGDRWRE
	DDCTFCQCVNGERHCVATVCGQTCTNPVKVPGECCPVCEEPTIITVDPPACGELSNCTLTGKDC
	INGFKRDHNGCRTCQCINTEELCSERKQGCTLNCPFGFLTDAQNCEICECRPRPKKCRPIICDK
	YCPLGLLKNKHGCDICRCKKCPELSCSKICPLGFQQDSHGCLICKCREASASAGPPILSGTCLT
	VDGHHHKNEESWHDGCRECYCLNGREMCALITCPVPACGNPTIHPGQCCPSCADDFVVQKPELS
	TPSICHAPGGEYFVEGETWNIDSCTQCTCHSGRVLCETEVCPPLLCQNPSRTQDSCCPQCTEDT
	IPKKVVCHFSGKAYADEERWDLDSCTHCYCLQGQTLCSTVSCPPLPCVEPINVEGSCCPMCPEM
	YVPEPTNIPIEKTNHRGEVDLEVPLWPTPSENDIVHLPRDMGHLQVDYR

[0430] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 13B.

TABLE 13B
Comparison of NOV13a against NOV13b through NOV13g.
			Identities/
	Protein	NOV13a Residues/	Similarities for
	Sequence	Match Residues	the Matched Region
	NOV13b	1 . . . 781	723/781 (92%)
		35 . . . 806	734/781 (93%)
	NOV13c	1 . . . 781	777/781 (99%)
		35 . . . 815	779/781 (99%)
	NOV13d	1 . . . 891	827/891 (92%)
		35 . . . 867	830/891 (92%)
	NOV13e	1 . . . 781	723/781 (92%)
		4 . . . 775	734/781 (93%)
	NOV13f	125 . . . 845	715/721 (99%)
		1 . . . 721	716/721 (99%)
	NOV13g	1 . . . 781	723/781 (92%)
		1 . . . 772	734/781 (93%)

[0431] Further analysis of the NOV13a protein yielded the following properties shown in Table 13C.

TABLE 13C
Protein Sequence Properties NOV13a
SignalP	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 10; pos. chg 1; neg. chg 2
	H-region: length 1; peak value 0.00
	PSG score: −4.40
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −6.37
	possible cleavage site: between 33 and 34
	>>> Seems to have no N-terminal signal peptide
	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 = 1.64 (at 736)
	ALOM score: 1.64 (number of TMSs: 0)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment (75):	6.88
	Hyd Moment (95):	6.86	G content:	0
	D/E content:	2	S/T content:	0
	Score: −6.85
	Gavel: prediction of cleavage sites for mitochondrial preseq
	cleavage site motif not found
	NUCDISC: discrimination of nuclear localization signals
	pat4: RPKK (4) at 501
	pat7: PRPKKCR (5) at 500
	pat7: PKKCRPI (5) at 502
	bipartite: none
	content of basic residues: 8.6%
	NLS Score: 0.64
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals: none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: nuclear
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	82.6%: nuclear
	13.0%: cytoplasmic
	4.3%: peroxisomal
	>> prediction for CG50691-05 is nuc (k = 23)

[0432] A search of the NOV13a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 13D.

TABLE 13D
Geneseq Results for NOV13a
		NOV13a	Identities/
Geneseq	Protein/Organism/Length	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
AAU12242	Human PRO4330 polypeptide	1 . . . 891	891/891 (100%)	0.0
	sequence - Homo sapiens, 1036 aa.	35 . . . 925	891/891 (100%)
	[WO200140466-A2, 07 JUN. 2001]
AAY53034	Human secreted protein clone	1 . . . 891	891/891 (100%)	0.0
	dj167_19 protein sequence SEQ ID	35 . . . 925	891/891 (100%)
	NO: 74 - Homo sapiens, 1036 aa.
	[WO9957132-A1, 11 NOV. 1999]
AAY82776	Human chordin related protein (Clone	1 . . . 891	891/891 (100%)	0.0
	dj167_19) - Homo sapiens, 1036 aa.	35 . . . 925	891/891 (100%)
	[WO200009551-A1, 24 FEB. 2000]
AAU07141	Human CRIM1 protein - Homo	1 . . . 891	890/891 (99%)	0.0
	sapiens, 1036 aa. [WO200138519-A1,	35 . . . 925	891/891 (99%)
	31 MAY 2001]
AAE18852	Human pharmaceutical compound	1 . . . 891	885/891 (99%)	0.0
	protein for cancer treatment - Homo	35 . . . 925	887/891 (99%)
	sapiens, 1036 aa. [WO200197850-A2,
	27 DEC. 2001]

[0433] In a BLAST search of public sequence databases, the NOV13a protein was found to have homology to the proteins shown in the BLASTP data in Table 13E.

TABLE 13E
Public BLASTP Results for NOV13a
Protein		NOV13a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q9NZV1	Cysteine-rich repeat-containing	1 . . . 891	891/891 (100%)	0.0
	protein S52 precursor (CRIM1	35 . . . 925	891/891 (100%)
	protein) - Homo sapiens (Human), 1036 aa.
Q9JLL0	Cysteine-rich repeat-containing	1 . . . 891	787/891 (88%)	0.0
	protein CRIM1 precursor - Mus	26 . . . 916	835/891 (93%)
	musculus (Mouse), 1028 aa (fragment).
AAM28339	Cysteine-rich motorneuron 1 - Gallus	1 . . . 891	747/891 (83%)	0.0
	gallus (Chicken), 1048 aa.	47 . . . 937	813/891 (90%)
CAC22521	Sequence 19 from Patent	125 . . . 845	715/721 (99%)	0.0
	WO0075321 - Homo sapiens	1 . . . 721	716/721 (99%)
	(Human), 732 aa.
Q8MM07	B0024.14b protein - Caenorhabditis	2 . . . 841	284/886 (32%)	e−131
	elegans, 960 aa.	26 . . . 793	394/886 (44%)

[0434] PFam analysis predicts that the NOV13a protein contains the domains shown in the Table 13F.

TABLE 13F
Domain Analysis of NOV13a
		Identities/
	NOV13a	Similarities for	Expect
Pfam Domain	Match Region	the Matched Region	Value
toxin_2	102 . . . 108	5/7 (71%)	0.86
		7/7 (100%)
vwc	302 . . . 356	22/84 (26%)	8.8e−08
		38/84 (45%)
vwc	369 . . . 422	25/84 (30%)	8.9e−09
		39/84 (46%)
Antistasin	435 . . . 464	13/34 (38%)	0.98
		21/34 (62%)
Antistasin	471 . . . 498	10/32 (31%)	0.091
		20/32 (62%)
Antistasin	505 . . . 530	9/30 (30%)	0.21
		18/30 (60%)
Antistasin	533 . . . 558	13/30 (43%)	5.9e−05
		18/30 (60%)
vwc	574 . . . 628	24/85 (28%)	1.3e−09
		41/85 (48%)
vwc	645 . . . 700	28/85 (33%)	2.4e−10
		41/85 (48%)
vwc	719 . . . 774	23/85 (27%)	8.5e−09
		39/85 (46%)
vwc	785 . . . 839	25/84 (30%)	1.7e−13
		42/84 (50%)

Example 14

[0435] 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: 47	499 bp
NOV14a,	TATGGAATAAAGAACCATGACGGAGTCCCATGCGCAGCCAGAGAAGAGACCACCACCCGAGAGA
CG51905-03	GGTTTCATCCTACCATGTAACTCTGCTTACAGCCTACTTGCTTCTCACCGGCGTGCTGGGGACA
DNA Sequence	GCAAAGTCTGAGGACTCTGGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAA
	GGCCTCTGCACAGCTCTAGAAGCTTCAATCCCATTTCCACCCATACATCTCTTTGTGCTCTCAC
	ACCCCCACAGCCCTTCTGGAATAAGACCATCACAGCACAGGGTTTGCAAGATGTCTAATGCCAG
	TCATTCACAGGGCAGCTCAGACCCTGGCCTGCGGTGCATACTAGGTGACTCCACATGAGGTGTC
	ATGCTAGATCCTGCAGGGAGAATAAGCACACACAGGCCCGTGACCCATGCTGTGGACTTCATGT
	TCTAGGAGGTAGAGGGAGACAGACAAGAATCAAATGACTGTACTAGGCCGG
	ORF Start: ATG at 30		ORF Stop: TAA at 312
	SEQ ID NO: 48	94 aa	MW at 10354.6 kD
NOV14a,	MRSQRRDHHPREVSSYHVTLLTAYLLLTGVLGTAKSEDSGWCGPVCKESSGHGIRPLHSSRSFN
CG51905-03	PISTHTSLCALTPPQPFWNKTITAQGLQDV
Protein Sequence
	SEQ ID NO: 49	579 bp
NOV 14b,	TATGGAATAAAGAACCATGACGGAGTCCCATGCGCAGCCAGAGAAGAGACCACCACCCGAGAGA
CG51905-01	GGTTTCATCCTACCATGTAACTCTGCTTACAGCCTACTTGCTTCTCACCGGCGTGCTGGGGACA
DNA Sequence	GCAAAGTCTGAGGACTCTGGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAA
	GGCCTCTGCACAGCTCTAGAAGCTTCAATCCCATTTCCACCCATACATCTCTTTGTGCTCTCAC
	ACCCCCACAGCCCTTCTGGAATAAGACCATCACAGCACAGGGTTTGCAAGATGTCTAATGCCAG
	TCATTCACAGGGCAGCTCAGACCCTGGCCTGCGGTGCATACTAGGTGACTCCACATGAGGTGTC
	ATGCTAGATCCTGCAGGGAGAATAAGCACACACAGGCCCGTGACCCATGCTGTGGACTTCATGT
	TCTAGGAGGTAGAGGGAGACAGACAAGAATCAAATGACTGTACTAGGCCGGGCGCACTGGCTCA
	CGCCTGTAATCCCAGCACTTTGGGGAGGCCGAGGCAGGTGGATCACGAGGCCAGGCGTTCGAGA
	CCA
	ORF Start: ATG at 30		ORF Stop: TAA at 312
	SEQ ID NO: 50	94 aa	MW at 10354.6 kD
NOV14b,	MRSQRRDHHPREVSSYHVTLLTAYLLLTGVLGTAKSEDSGWCGPVCKESSGHGIRPLHSSRSFN
CG51905-01	PISTHTSLCALTPPQPFWNKTITAQGLQDV
Protein Sequence
	SEQ ID NO: 51	193 bp
NOV14c,	CACCGGATCCGAGGACTCTGGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAA
278699747	GGCCTCTGCACAGCTCTAGAAGCTTCAATCCCATTTCCACCCATACATCTCTTTGTGCTCTCACA
DNA Sequence	CCCCCACAGCCCTTCTGGAATAAGACCATCACAGCACAGGGTTTGCAAGATGTCCTCGAGGGC
	ORF Start: at 2		ORF Stop: end of sequence
	SEQ ID NO: 52	64 aa	MW at 6822.5 kD
NOV14c,	TGSEDSGWCGPVCKESSGHGIRPLHSSRSFNPISTHTSLCALTPPQPFWNKTITAQGLQDVLEG
278699747
Protein Sequence
	SEQ ID NO: 53	304 bp
NOV14d,	CACCGGATCCACCATGCGCAGCCAGAGAAGAGACCACCACCCGAGAGAGGTTTCATCCTACCATG
310912740	TAACTCTGCTTACAGCCTACTTGCTTCTCACCGGCGTGCTGGGGACAGCAAAGTCTGAGGACTCT
DNA Sequence	GGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAAGGCCTCTGCACAGCTCTAG
	AAGCTTCAATCCCATTTCCACCCATACATCTCTTTGTGCTCTCACACCCCCACAGCCCTTCTGGA
	ATAAGACCATCACAGCACAGGGTTTGCAAGATGTCGTCGACGGC
	ORF Start: at 2		ORF Stop: end of sequence
	SEQ ID NO: 54	101 aa	MW at 10972.2 kD
NOV14d,	TGSTMRSQRRDHHPREVSSYHVTLLTAYLLLTGVLGTAKSEDSGWCGPVCKESSGHGIRPLHSS
310912740	RSFNPISTHTSLCALTPPQPFWNKTITAQGLQDVVDG
Protein Sequence
	SEQ ID NO: 55	174 bp
NOV14e,	GAGGACTCTGGTTGGTGTGGGCCTGTGTGCAAGGAGAGCAGTGGCCATGGGATAAGGCCTCTGCA
CG51905-02	CAGCTCTAGAAGCTTCAATCCCATTTCCACCCATACATCTCTTTGTGCTCTCACACCCCCACAGC
DNA Sequence	CCTTCTGGAATAAGACCATCACAGCACAGGGTTTGCAAGATGTC
	ORF Start: at 1		ORF Stop: end of sequence
	SEQ ID NO: 56	58 aa	MW at 6277.9 kD
NOV14e,	EDSGWCGPVCKESSGHGIRPLHSSRSFNPISTHTSLCALTPPQPFWNKTITAQGLQDV
CG51905-02
Protein Sequence

[0436] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 14B.

TABLE 14B
Comparison of NOV14a against NOV14b through NOV14e.
			Identities/
	Protein	NOV14a Residues/	Similarities for
	Sequence	Match Residues	the Matched Region
	NOV14b	1 . . . 94	94/94 (100%)
		1 . . . 94	94/94 (100%)
	NOV14c	36 . . . 94	59/59 (100%)
		3 . . . 61	59/59 (100%)
	NOV14d	1 . . . 94	94/94 (100%)
		5 . . . 98	94/94 (100%)
	NOV14e	37 . . . 94	58/58 (100%)
		1 . . . 58	58/58 (100%)

[0437] Further analysis of the NOV14a protein yielded the following properties shown in Table 14C.

TABLE 14C
Protein Sequence Properties NOV14a
SignalP	Cleavage site between residues 37 and 38
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 11; pos. chg 4; neg. chg 1
	H-region: length 0; peak value −2.25
	PSG score: −6.65
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −0.05
	possible cleavage site: between 32 and 33
	>>> Seems to have no N-terminal signal peptide
	ALOM: Klein et al's method for TM region allocation
	Init position for calculation: 1
	Tentative number of TMS(s) for the threshold 0.5: 1
	Number of TMS(s) for threshold 0.5: 0
	PERIPHERAL	Likelihood = 12.57 (at 62)
	ALOM score: −1.59 (number of TMSs: 0)
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	4	Hyd Moment (75):	8.79
	Hyd Moment (95):	8.84	G content:	0
	D/E content:	2	S/T content:	1
	Score: −2.40
	Gavel: prediction of cleavage sites for mitochondrial preseq
	R-2 motif at 15 QRR|DH
	NUCDISC: discrimination of nuclear localization signals
	pat4: none
	pat7: none
	bipartite: none
	content of basic residues: 9.6%
	NLS Score: −0.47
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals:
	XXRR-like motif in the N-terminus: RSQR none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: nuclear
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	56.5%: mitochondrial
	30.4%: nuclear
	8.7%: extracellular, including cell wall
	4.3%: cytoplasmic
	>> prediction for CG51905-03 is mit (k = 23)

[0438] 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 14D.

TABLE 14D
Geneseq Results for NOV14a
		NOV14a	Identities/
Geneseq	Protein/Organism/Length	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
AAE08920	Human NOVX1 protein from clone	1 . . . 94	94/94 (100%)	3e−52
	28804279.0.7 - Homo sapiens, 94 aa.	1 . . . 94	94/94 (100%)
	[WO200161008-A2, 23 AUG. 2001]
AAE04787	Vigna unguiculata neoxanthin cleavage	44 . . . 87	18/48 (37%)	2.8
	enzyme, CPRD65 - Vigna unguiculata,	33 . . . 80	23/48 (47%)
	612 aa. [EP1116794-A2, 18 JUL. 2001]
AAU56331	Propionibacterium acnes immunogenic	12 . . . 77	21/66 (31%)	6.4
	protein #17227 - Propionibacterium	9 . . . 67	29/66 (43%)
	acnes, 133 aa. [WO200181581-A2,
	01 NOV. 2001]

[0439] 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 14E.

TABLE 14E
Public BLASTP Results for NOV14a
Protein		NOV14a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q9HT01	Vng0005h - Halobacterium sp.	7 . . . 41	16/35 (45%)	4.7
	(strain NRC-1), 773 aa.	245 . . . 278	21/35 (59%)
Q9FS24	Neoxanthin cleavage enzyme - Vigna	44 . . . 87	18/48 (37%)	8.0
	unguiculata (Cowpea), 612 aa.	33 . . . 80	23/48 (47%)

[0440] PFam analysis predicts that the NOV14a protein contains the domains shown in the Table 14F.

TABLE 14F
Domain Analysis of NOV14a
	Pfam	NOV14a	Identities/
	Domain	Match Region	Similarities for	Expect
			the Matched Region	Value

Example 15

[0441] The NOV15 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 15A.

TABLE 15A
NOV15 Sequence Analysis
	SEQ ID NO: 57	2516 bp
NOV15a,	CACCAGATCTCCCACCATGGCCTCTGCTGACAAGAATGGCGGGAGCGTGTCCTCTGTGTCCAGC
CG52414-03	AGCCGCCTGCAGAGCCGGAAGCCACCCAACCTCTCCATCACCATCCCGCCACCCGAGAAAGAGA
DNA Sequence	CCCAGGCCCCTGGCGAGCAGGACAGCATGCTGCCTGAGAGGAAGAACCCAGCCTACTTGAAGAG
	CGTCAGCCTCCAGGAGCCACGCAGCCGATGGCAGGAGAGTTCAGAGAAGCGCCCTGGCTTCCGC
	CGCCAGGCCTCACTGTCCCAGAGCATCCGCAAGGGCGCAGCCCAGTGGTTTGGAGTCAGCGGCG
	ACTGGGAGGGGCAGCGGCAGCAGTGGCAGCGCCGCAGCCTGCACCACTGCAGCATGCGCTACGG
	CCGCCTGAAGGCCTCGTGCCAGCGTGACCTGGAGCTCCCCAGCCAGGAGGCACCGTCCTTCCAG
	GGCACTGAGTCCCCAAAGCCCTGCAAGATGCCCAAGATTGTGGATCCGCTGGCCCGGGGCCGGG
	CCTTCCGCCACCCGGAGGAGATGGACAGGCCCCACGCCCCGCACCCACCGCTGACCCCCGGAGT
	CCTGTCCCTCACCTCCTTCACCAGTGTCCGTTCTGGCTACTCCCACCTGCCACGCCGCAAGAGA
	ATGTCTGTGGCCCACATGAGCTTGCAAGCTGCCGCTGCCCTCCTCAAGGGGCGCTCGGTGCTGG
	ATGCCACCGGACAGCGGTGCCGGGTGGTCAAGCGCAGCTTTGCCTTCCCGAGCTTCCTGGAGGA
	GGATGTGGTCGATGGGGCAGACACGTTTGACTCCTCCTTTTTTAGTAAGGAAGAAATGAGCTCC
	ATGCCTGATGATGTCTTTGAGTCCCCCCCACTCTCTGCCAGCTACTTCCGAGGGATCCCACACT
	CAGCCTCCCCTGTCTCCCCCGATGGGGTGCAAATCCCTCTGAAGGAGTATGGCCGAGCCCCAGT
	CCCCGGGCCCCGGCGCGGCAAGCGCATCGCCTCCAAGGTGAAGCACTTTGCCTTTGATCGGAAG
	AAGCGGCACTACGGCCTCGGCGTGGTGGGCAACTGGCTGAACCGCAGCTACCGCCGCAGCATCA
	GCAGCACTGTGCAGCGGCAGCTGGAGAGCTTCGACAGCCACCGGCCCTACTTCACCTACTGGCT
	GACCTTCGTCCATGTCATCATCACGCTGCTGGTGATTTGCACGTATGGCATCGCACCCGTGGGC
	TTTGCCCAGCACGTCACCACCCAGCTGGTGCTGCGGAACAAAGGTGTGTACGAGAGCGTGAAGT
	ACATCCAGCAGGAGAACTTCTGGGTTGGCCCCAGCTCGATTGACCTGATCCACCTGGGGGCCAA
	GTTCTCACCCTGCATCCGGAAGGACGGGCAGATCGAGCAGCTGGTGCTGCGCGAGCCAGACCTG
	GAGCGGGACTCAGGCTGCTGTGTCCAGAATGACCACTCCGGATGCATCCAGACCCAGCGGAAGG
	ACTGCTCGGAGACTTTGGCCACTTTTGTCAAGTGGCAGGATGACACTGGGCCCCCCATGGACAA
	GTCTGATCTGGGCCAGAAGCGGACTTCGGGGGCTGTCTGCCACCAGGACCCCAGGACCTGCGAG
	GAGCCAGCCTCCAGCGGTGCCCACATCTGGCCCGATGACATCACTAAGTGGCCGATCTGCACAG
	AGCACGCCAGGAGCAACCACACAGGCTTCCTGCACATGGACTGCGAGATCAAGGGCCGCCCCTG
	CTGCATCGGCACCAAGGGCAGCTGTGAGATCACCACCCGGGAATACTGTGAGTTCATGCACGGC
	TATTTCCATGAGGAAGCAACACTCTGCTCCCAGGTGCACTGCTTGGACAAGGTGTGTGGGCTGC
	TGCCCTTCCTCAACCCTGAGGTCCCAGATCAGTTCTACAGGCTCTGGCTGTCTCTCTTCCTACA
	TGCTGGCGTGGTGCACTGCCTCGTGTCTGTCGTCTTTCAAATGACCATCCTGAGGGACCTGGAC
	AAGCTGGCCGGCTGGCACCGTATCGCCATCATCTTCATCCTCAGTGGCATCACAGGCAACCTCG
	CCAGTGCCATCTTTCTCCCATACCGGGCAGAGGTGGGCCCGGCCGGCTCACAGTTCGGCCTCCT
	CGCCTGCCTCTTCGTGGAGCTCTTCCAGAGCTGGCCGCTGCTGGAGAGGCCCTGGAAGGCCTTC
	CTCAACCTCTCGGCCATCGTGCTCTTCCTGTTCATCTGTCGCCTCCTGCCCTGGATCGACAACA
	TCGCCCACATCTTCGGCTTCCTCAGTGGCCTGCTGCTGGCCTTCGCCTTCCTGCCCTACATCAC
	CTTCGGCACCAGCGACAAGTACCGCAAGCGGGCACTCATCCTGGTGTCACTGCTGGCCTTTGCC
	GGCCTCTTCGCCGCCCTCGTGCTGTGGCTGTACATCTACCCCATTAACTCGCCCTGGATCGAGC
	ACCTCACCTGCTTCCCCTTCACCAGCCGCTTCTGCGAGAAGTATGAGCTGGACCAGGTGCTGCA
	CCTCGAGGGCAAGGGTTTAA
	ORF Start: ATG at 17		ORF Stop: at 2498
	SEQ ID NO: 58	827 aa	MW at 93378.2 kD
NOV15a,	MASADKNGGSVSSVSSSRLQSRKPPNLSITIPPPEKETQAPGEQDSMLPERKNPAYLKSVSLQE
CG52414-03	PRSRWQESSEKRPGFRRQASLSQSIRKGAAQWFGVSGDWEGQRQQWQRRSLHHCSMRYGRLKAS
Protein Sequence	CQRDLELPSQEAPSFQGTESPKPCKMPKIVDPLARGRAFRHPEEMDRPHAPHPPLTPGVLSLTS
	FTSVRSGYSHLPRRKRMSVAHMSLQAAAALLKGRSVLDATGQRCRVVKRSFAFPSFLEEDVVDG
	ADTFDSSFFSKEEMSSMPDDVFESPPLSASYFRGIPHSASPVSPDGVQIPLKEYGRAPVPGPRR
	GKRIASKVRMFAFDRKKRHYGLGVVGNWLNRSYRRSISSTVQRQLESPDSHRPYFTYWLTFVHV
	IITLLVICTYGIAPVGFAQHVTTQLVLRNKGVYESVKYIQQENFWVGPSSIDLIHLGAKFSPCI
	RKDGQIEQLVLRERDLERDSGCCVQNDHSGCIQTQRKDCSETLATFVKWQDDTGPPMDKSDLGQ
	KRTSGAVCHQDPRTCEEPASSGAHIWPDDITKWPICTEQARSNHTGFLHMDCEIKGRPCCIGTK
	GSCEITTREYCEFMHGYFHEEATLCSQVHCLDKVCGLLPFLNPEVPDQFYRLWLSLFLHAGVVH
	CLVSVVFQMTILRDLEKLAGWHRIAIIFILSGITGNLASAIFLPYRAEVGPAGSQFGLLACLFV
	ELFQSWPLLERPWKAFLNLSAIVLFLFICGLLPWIDNIAHIFGFLSGLLLAFAFLPYITFGTSD
	KYRKRALILVSLLAFAGLFAALVLWLYIYPINWPWIEHLTCFPFTSRFCEKYELDQVLH
	SEQ ID NO: 59	2596 bp
NOV15b,	TCAATTGACTTGATATGATTTATTATTTTTACTACTTATAAGAATGGAAATAAGTTCTCCTTAG
CG52414-01	TTTTTTTCTTGGAGAAAGTCTGACATGTGAGGCACAGATGAGTTATTAAAGGCAGATGACTTTC
DNA Sequence	CAGCCTTGTCTTAAATGTTCCATTCTTTACCTTAGAAATTATTTAAATTTGTGTCCTGTCCCAG
	AGCATCCGCAAGGGCGCAGCCCAGTGGTTTGGAGTCAGCGGCGACTGGGAGGGGCAGCGGCAGC
	AGTGGCAGCGCCGCAGCCTGCACCACTGCAGCATGCGCTACGGCCGCCTGAAGGCCTCGTGCCA
	GCGTGACCTGGAGCTCCCCAGCCAGGACGCACCGTCCTTCCAGCGCACTGAGTCCCCAAAGCCC
	TGCAAGATCCCCAAGATTGTGGATCCGCTGGCCCGGGGCCGGGCCTTCCGCCACCCGGAGGAGA
	TGGACAGGCCCCACGCCCTCCACCCACCGCTGACCCCCGGAGTCCTGTCCCTCACCTCCTTCAC
	CAGTGTCCGTTCTGGCTACTCCCACCTGCCACGCCGCAAGAGAATGTCTGTGGCCCACATGAGC
	TTGCAAGCTGCCGCTGCCCTCCTCAAGGGGCGCTCGGTGCTGGATGCCACCGGACAGCGGTGCC
	GGGTGGTCAAGCGCAGCTTTGCCTTCCCGAGCTTCCTGGAGGAGGATGTGGTCGATGGGGCAGA
	CACGTTTGACTCCTCCTTTTTTAGTAAGGAAGAAATGAGCTCCATGCCTGATGATGTCTTTGAG
	TCCCCCCCACTCTCTGCCAGCTACTTCCGAGGGATCCCACACTCAGCCTCCCCTGTCTCCCCCG
	ATGGGGTGCAAATCCCTCTGAAGGAGTATGGCCGAGCCCCAGTCCCCGGGCCCCGGCGCGGCAA
	GCGCATCGCCTCCAACGTGAAGCACTTTGCCTTTGATCGGAAGAAGCGGCACTACGGCCTCGGC
	GTGGTGGGCAACTGGCTGAACCGCAGCTACCGCCGCAGCATCAGCAGCACTGTGCAGCCGCAGC
	TGGAGAGCTTCGACAGCCACCGGCCCTACTTCACCTACTGGCTGACCTTCGTCCATGTCATCAT
	CACGCTGCTGGTGATTTGCACGTATGGCATCGCACCCGTGGGCTTTGCCCAGCACGTCACCACC
	CAGCTGGTGCTGCGGAACAAAGGTGTGTACGAGAGCGTGAAGTACATCCAGCAGGAGAACTTCT
	GGGTTGGCCCCAGCTCGATTGACCTGATCCACCTGGGGGCCAAGTTCTCACCCTGCATCCGGAA
	GGACGGGCAGATCGAGCAGCTGGTGCTGCGCGAGCGAGACCTGGAGCGGGACTCAGGCTGCTGT
	GTCCAGAATGACCACTCCGGCTGCATCCAGACCCAGCGGAACGACTGCTCGGAGACTTTGGCCA
	CTTTTGTCAAGTGGCAGGATGACACTGGGCCCCCCATGGACAAGTCTGATCTGGGCCAGAAGCG
	GACTTCGGGGGCTGTCTGCCACCAGGACCCCAGGACCTGCGAGGAGCCAGCCTCCAGCGGTCCC
	CACATCTGGCCCGATGACATCACTAAGTGGCCGATCTGCACAGAGCACGCCAGGAGCAACCACA
	CAGGCTTCCTGCACATGGACTGCGAGATCAAGGGCCGCCCCTGCTGCATCGGCACCAAGGGCAG
	CTGTGAGATCACCACCCGGGAATACTCTGAGTTCATGCACGGCTATTTCCATGAGGAAGCAACA
	CTCTGCTCCCAGGTGCACTGCTTGGACAAGGTGTGTGGGCTGCTGCCCTTCCTCAACCCTGAGG
	TCCCAGATCAGTTCTACAGGCTCTGGCTGTCTCTCTTCCTACATGCTCGCGTGGTGCACTGCCT
	CGTGTCTGTGGTCTTTCAAATGACCATCCTGAGGGACCTGGAGAAGCTGGCCGGCTGGCACCGT
	ATCGCCATCATCTTCATCCTCAGTGGCATCACAGGCAACCTCGCCAGTACCATCTTTCTCCCAT
	ACCCGGCAGAGGTGGGCCCGGCCGGCTCACAGTTCGGCCTCCTCGCCTGCCTCTTCGTGGAGCT
	CTTCCAGAGCTGGCCGCTGCTGGAGAGGCCCTGGAAGGCCTTCCTCAACCTCTCGACCATCGTG
	CTCTTCCTGTTCATCTGTGGCCTCCTGCCCTGGATCGACAACATCGCCCACATCTTCGGCTTCC
	TCAGTGGCCTGCTGCTGGCCTTCGCCTTCCTGCCCTACATCACCTTCGGCACCAGCGACAAGTA
	CCGCAAGCGGGCACTCATCCTGGTGTCACTGCTGGCCTTTGCCGGCCTCTTCGCCGCCCTCGTG
	CTGTGGCTGTACATCTACCCCATTAACTGGCCCTGGATCGAGCACCTCACCTGCTTCCCCTTCA
	CCAGCCGCTTCTGCGAGAAGTATGAGCTGGACCAGGTGCTGCACTGACCGCTGGGCCACACGGC
	TGCCCCTCAGCCCTGCTGGAACAGGGTCTGCCTGCGAGGGCTGCCCTCTGCAGAGCGCTCTCTG
	TGTGCCAGAGAGCCAGAGACCCAAGACAGGGCCCCGCCTCTGGACCTGGGTGCCCCCCTGCCAG
	GCGACGCTGACTCCGCGTGAGATGGTTGGTTAAGGC
	ORF Start: ATG at 289		ORF Stop: TGA at 2413
	SEQ ID NO: 60	708 aa	MW at 80098.6 kD
NOV15b,	MRYGRLKASCQRDLELPSQEAPSFQGTESPKPCKMPKIVDPLARGRAFRHPEEMDRPHALHPPL
CG52414-01	TPGVLSLTSFTSVRSGYSHLPRRKRMSVAHMSLQAAAALLKGRSVLDATGQRCRVVKRSFAFPS
Protein Sequence	FLEEDVVDGADTFDSSFFSKEEMSSMPDDVFESPPLSASYFRGIPHSASPVSPDGVQIPLKEYG
	RAPVPGPRRGKRIASKVKHFAFDRKKRHYGLGVVGNWLNRSYRRSISSTVQRQLESFDSHRPYF
	TYWLTFVHVIITLLVICTYGIAPVGFAQHVTTQLVLRNKGVYESVKYIQQENFWVGPSSIDLIH
	LGAKFSPCIRKDGQIEQLVLRERDLERDSGCCVQNDHSGCIQTQRKDCSETLATFVKWQDDTGT
	PMDKSDLGQKRTSGAVCHQDPRTCEEPASSGAHIWPDDITKWPICTEQARSNHTGFLHMDCEIK
	GRPCCIGTKGSCEITTREYCEFMHGYFHEEATLCSQVHCLDKVCGLLPFLNPEVPDQFYRLWLS
	LFLHAGVVHCLVSVVFQMTILRDLEKLAGWHRIAIIFILSGITGNLASTIFLPYRAEVGPAGSQ
	FGLLACLFVELFQSWPLLERPWKAFLNLSTIVLFLFICGLLPWIDNIAHIFGFLSGLLLAFAFL
	PYITFGTSDKYRKRALILVSLLAFAGLFAALVLWLYIYPINWPWIEHLTCFPFTSRFCEKYELD
	QVLH
	SEQ ID NO: 61	3040 bp
NOV15c,	TTTGGGGCCGCAGGGAGGTTCCCAGACCAGAGGACTGTTGTTAGGTGATTGGCTGTGAACGCCC
CG52414-02	TGAGGCCAGTGCCCCTCGCTGCTTGGcACTCCGAGATGCCTGATTAGCACCTTTAATCCCTTAC
DNA Sequence	CAATGAGGCAGGTGGAATTGGCCCCATTTTACAGATCGGGAGACTGAGCCACCTGTCTGTCCAG
	CCACCCTTCCACAGACTGAGGCTTGACACCGGAGCATCTGTACAGAGCAAGGAGAAGACAAGAA
	CATGCTCTAAAGCCCTTCAGAGCAAGACCCAGGAAGCCGCGGGCAAACTCAGACTCGAAGCCCT
	CCCACCTCCTGCCCACAATGGCCTCTGCTGACAAGAATGGCGGGAGCGTGTCCTCTGTGTCCAG
	CAGCCGCCTGCAGAGCCGGAAGCCACCCAACCTCTCCATCACCATCCCGCCACCCGAGAAAGAG
	ACCCAGGCCCCTGGCGAGCAGGACAGCATGCTGCCTGAGAGGAAGAACCCAGCCTACTTGAAGA
	GCGTCAGCCTCCAGGAGCCACGCAGCCGATGGCAGGAGAGTTCAGAGAAGCGCCCTGGCTTCCG
	CCGCCAGGCCTCACTGTCCCAGAGCATCCGCAAGGGCGCAGCCCAGTGGTTTGGAGTCAGCGGC
	GACTCGGACGGGCAGCGGCAGCAGTGGCAGCGCCGCAGCCTGCACCACTGCAGCATGCGCTACG
	GCCGCCTGAAGGCCTCGTGCCAGCGTGACCTGGAGCTCCCCAGCCAGGAGGCACCGTCCTTCCA
	GGGCACTGAGTCCCCAAAGCCCTGCAAGATGCCCAAGATTGTGGATCCGCTGGCCCGGGGCCGG
	GCCTTCCGCCACCCGGAGGAGATGGACAGGCCCCACGCCCCGCACCCACCGCTGACCCCCGGAG
	TCCTGTCCCTCACCTCCTTCACCAGTGTCCGTTCTCGCTACTCCCACCTGCCACGCCCCAAGAG
	AATGTCTGTGGCCCACATGAGCTTGCAAGCTGCCGCTGCCCTCCTCAAGGGGCGCTCGGTGCTG
	GATGCCACCGGACAGCGGTGCCGGGTGGTCAAGCGCAGCTTTGCCTTCCCGAGCTTCCTGGAGG
	AGGATGTGGTCGATGGGGCAGACACGTTTGACTCCTCCTTTTTTAGTAAGGAAGAAATGAGCTC
	CATGCCTGATGATGTCTTTGAGTCCCCCCCACTCTCTGCCAGCTACTTCCGAGGGATCCCACAC
	TCAGCCTCCCCTGTCTCCCCCGATGGGGTGCAAATCCCTCTGAAGGAGTATGGCCGAGCCCCAG
	TCCCCGGGCCCCGGCCCGGCAAGCGCATCGCCTCCAAGGTGAAGCACTTTGCCTTTGATCGGAA
	GAAGCGGCACTACGGCCTCGGCGTGGTCGGCAACTGGCTGAACCGCAGCTACCGCCGCAGCATC
	AGCAGCACTGTGCAGCGGCAGCTGGAGAGCTTCGACAGCCACCGGCCCTACTTCACCTACTGGC
	TGACCTTCGTCCATGTCATCATCACGCTGCTGGTGATTTGCACGTATGGCATCGCACCCGTGGG
	CTTTGCCCAGCACGTCACCACCCAGCTGGTGCTGCGGAACAAAGGTGTGTACGAGAGCGTGAAG
	TACATCCAGCAGGAGAACTTCTGGGTTGGCCCCAGCTCGATTGACCTGATCCACCTGGGGGCCA
	AGTTCTCACCCTGCATCCGGAAGGACGGGCAGATCGAGCAGCTGGTGCTGCGCGAGCGAGACCT
	GGAGCGGGACTCAGGCTGCTGTGTCCAGAATGACCACTCCGGATGCATCCAGACCCAGCGGAAG
	GACTGCTCGGAGACTTTGGCCACTTTTGTCAAGTGGCAGGATGACACTGGGCCCCCCATGGACA
	AGTTCTGATCTGCGCCAGAAGCGACTTCCGGGGCTGTCTGCCACCAGGACCCCAGGACCTGCGA
	GGAGCCAGCCTCCAGCGGTGCCCACATCTGGCCCGATGACATCACTAAGTGGCCGATCTGCACA
	GAGCAGGCCAGGAGCAACCACACAGGCTTCCTGCACATGGACTGCGAGATCAAGGGCCGCCCCT
	GCTGCATCGGCACCAAGGGCAGCTGTGAGATCACCACCCGGGAATACTGTGAGTTCATGCACGG
	CTATTTCCATGAGGAAGCAACACTCTGCTCCCAGGTGCACTGCTTGGACAAGGTGTGTGGGCTG
	CTGCCCTTCCTCAACCCTGAGGTCCCAGATCAGTTCTACAGGCTCTGGCTGTCTCTCTTCCTAC
	ATGCTGGGGTGGTGCACTGCCTCGTGTCTGTGGTCTTTCAAATGACCATCCTGAGGGACCTGGA
	GAAGCTGGCCGGCTGGCACCGTATCGCCATCATCTTCATCCTCAGTGGCATCACAGGCAACCTC
	GCCAGTGCCATCTTTCTCCCATACCGGGCAGAGGTAGGCCCGGCCGGCTCACAGTTCGGCCTCC
	TCGCCTGCCTCTTCGTGGAGCTCTTCCAGAGCTGGCCGCTGCTGGAGAGGCCCTGGAAGGCCTT
	CCTCAACCTCTCGGCCATCGTGCTCTTCCTGTTCATCTGTGGCCTCCTGCCCTGGATCGACAAC
	ATCGCCCACATCTTCGGCTTCCTCAGTGGCCTGCTGCTGGCCTTCGCCTTCCTGCCCTACATCA
	CCTTCGGCACCAGCGACAAGTACCGCAAGCGGGCACTCATCCTGGTGTCACTGCTGGCCTTTGC
	CGGCCTCTTCGCCGCCCTCGTGCTGTGGCTGTACATCTACCCCATTAACTGGCCCTGGATCGAG
	CACCTCACCTGCTTCCCCTTCACCAGCCGCTTCTGCGAGAAGTATGAGCTGGACCAGGTGCTGC
	ACTGACCGCTGGGCCACACGGCTGCCCCTCAGCCCTGCTGGAACAGGGTCTGCCTGCGAGGGCT
	GCCCTCTGCAGAGCGCTCTCTGTGTGCCAGAGAGCCAGAGACCCAAGACAGGGCCCGGGCTCTG
	GACCTGGGTGCCCCCCTGCCAGGCGAGGCTGACTCCGCGTGAGATAGATGGTTGGTTAAGGCGG
	GGTTTTTCCGGGCCGCGCCCCCCCCCTCTAAA
	ORF Start: ATG at 338		ORF Stop: TGA at 2819
	SEQ ID NO: 62	827 aa	MW at 93378.2 kD
NOV15c,	MASADKNGGSVSSVSSSRLQSRKPPNLSITIPPPEKETQAPGEQDSMLPERKNPAYLKSVSLQE
CG52414-02	PRSRWQESSEKRPGFRRQASLSQSIRKGAAQWFGVSGDWEGQRQQWQRRSLHHCSMRYGRLKAS
Protein Sequence	CQRDLELPSQEAPSFQGTESPKPCKMPKIVDPLARGRAFRHPEEMDRPHAPHPPLTPGVLSLTS
	FTSVRSGYSHLPRRKRMSVAHMSLQAAAALLKGRSVLDATGQRCRVVKRSFAPPSFLEEDVVDG
	ADTFDSSFFSKEEMSSMPDDVFESPPLSASYFRGIPHSASPVSPDGVQIPLKEYGRAPVPGPRR
	GKRIASKVKHFAFDRKKRHYGLGVVGNWLNRSYRRSISSTVQRQLESFDSHRPYFTYWLTFVHV
	IITLLVICTYGIAPVGFAQHVTTQLVLRNKGVYESVKYIQQENFWVGPSSIDLIHLGAKFSPCI
	RKDGQIEQLVLRERDLERDSGCCVQNDHSGCIQTQRKDCSETLATFVKWQDDTGPPMDKSDLGQ
	KRTSGAVCHQDPRTCEEPASSGAHIWPDDITKWPICTEQARSNHTGFLHMDCEIKGRPCCIGTK
	GSCEITTREYCEFMHGYFHEEATLCSQVHCLDKVCGLLPFLNPEVPDQFYRLWLSLFLHAGVVH
	CLVSVVFQMTILRDLEKLAGWHRIAIIFILSGITGNLASAIFLPYRAEVGPAGSQFGLLACLFV
	ELFQSWPLLERPWKAFLNLSAIVLFLFICGLLPWIDNIAHIFGFLSGLLLAFAFLPYITFGTSD

[0442] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 15B.

TABLE 15B
Comparison of NOV15a against NOV15b and NOV15c.
			Identities/
	Protein	NOV15a Residues/	Similarities for
	Sequence	Match Residues	the Matched Region
	NOV15b	120 . . . 827	705/708 (99%)
		1 . . . 708	705/708 (99%)
	NOV15c	1 . . . 827	827/827 (100%)
		1 . . . 827	827/827 (100%)

[0443] Further analysis of the NOV15a protein yielded the following properties shown in Table 15C.

TABLE 15C
Protein Sequence Properties NOV15a
SignalP	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 6; pos. chg 1; neg. chg 1
	H-region: length 11; peak value 5.03
	PSG score: 0.62
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −7.64
	possible cleavage site: between 21 and 22
	>>> 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: 7
	INTEGRAL	Likelihood =	−6.42	Transmembrane 381-397
	INTEGRAL	Likelihood =	−4.25	Transmembrane 630-646
	INTEGRAL	Likelihood =	−3.08	Transmembrane 666-682
	INTEGRAL	Likelihood =	0.37	Transmembrane 697-713
	INTEGRAL	Likelihood =	−9.08	Transmembrane 720-736
	INTEGRAL	Likelihood =	−4.83	Transmembrane 742-758
	INTEGRAL	Likelihood =	−10.83	Transmembrane 775-791
	PERIPHERAL	Likelihood =	5.25 (at 600)
	ALOM score: −10.83 (number of TMSs: 7)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 388
	Charge difference: 0.5 C(1.5)-N(1.0)
	C > N: C-terminal side will be inside
	>>>Caution: Inconsistent mtop result with signal peptide
	>>> membrane topology: type 3b
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	2	Hyd Moment (75):	6.30
	Hyd Moment (95):	5.24	G content:	2
	D/E content:	2	S/T content:	10
	Score: −3.23
	Gavel: prediction of cleavage sites for mitochondrial preseq
	R-2 motif at 32 SRK|PP
	NUCDISC: discrimination of nuclear localization signals
	pat4: PRRK (4) at 204
	pat4: RRKR (5) at 205
	pat4: RKKR (5) at 335
	pat4: KKRH (3) at 336
	pat7: PRRKRMS (5) at 204
	pat7: PGPRRGK (3) at 316
	pat7: PRRGKRI (5) at 318
	bipartite: KRIASKVKHFAFDRKKR at 322
	content of basic residues: 11.6%
	NLS Score: 2.37
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals: none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 70.6
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	55.6%: endoplasmic reticulum
	11.1%: vacuolar
	11.1%: mitochondrial
	11.1%: vesicles of secretory system
	11.1%: Golgi
	>> prediction for CG52414-03 is end (k = 9)

[0444] A search of the NOV15a 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	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
AAB61148	Human NOV17 protein - Homo	120 . . . 827	705/708 (99%)	0.0
	sapiens, 708 aa. [WO200075321-A2,	1 . . . 708	705/708 (99%)
	14 DEC. 2000]
AAB61147	Human NOV16 protein - Homo	120 . . . 604	484/485 (99%)	0.0
	sapiens, 578 aa. [WO200075321-A2,	1 . . . 485	484/485 (99%)
	14 DEC. 2000]
ABG64458	Human albumin fusion protein #1133 -	498 . . . 827	328/330 (99%)	0.0
	Homo sapiens, 349 aa.	20 . . . 349	330/330 (99%)
	[WO200177137-A1, 18 OCT. 2001]
AAE03323	Human gene 7 encoded secreted protein	498 . . . 827	328/330 (99%)	0.0
	HCRNC80, SEQ ID NO: 97 - Homo	20 . . . 349	330/330 (99%)
	sapiens, 349 aa. [WO200134800-A1,
	17 MAY 2001]
ABB90342	Human polypeptide SEQ ID NO 2718 -	505 . . . 827	322/323 (99%)	0.0
	Homo sapiens, 323 aa.	1 . . . 323	323/323 (99%)
	[W0200190304-A2, 29 NOV. 2001]

[0445] 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
Protein		NOV15a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
BAC26163	10 days neonate skin cDNA, RIKEN	1 . . . 827	759/830 (91%)	0.0
	full-length enriched library,	1 . . . 827	790/830 (94%)
	clone: 4732465I17
	product: EPIDERMAL GROWTH
	FACTOR RECEPTOR-RELATED
	PROTEIN (FRAGMENT) homolog -
	Mus musculus (Mouse), 827 aa.
CAC22528	Sequence 33 from Patent WO0075321 -	120 . . . 827	705/708 (99%)	0.0
	Homo sapiens (Human), 708 aa.	1 . . . 708	705/708 (99%)
Q9H6E9	Hypothetical protein FLJ22341 - Homo	209 . . . 827	619/619 (100%)	0.0
	sapiens (Human), 619 aa.	1 . . . 619	619/619 (100%)
BAB84860	FLJ00080 protein - Homo sapiens	80 . . . 689	603/613 (98%)	0.0
	(Human), 716 aa (fragment).	52 . . . 664	605/613 (98%)
Q8K2I7	Similar to hypothetical protein	222 . . . 827	564/608 (92%)	0.0
	FLJ22341 - Mus musculus (Mouse),	1 . . . 607	585/608 (95%)
	607 aa (fragment).

[0446] PFam analysis predicts that the NOV15a protein contains the domains shown in the Table 15F.

TABLE 15F
Domain Analysis of NOV15a
		Identities/
	NOV15a	Similarities for	Expect
Pfam Domain	Match Region	the Matched Region	Value
Rhomboid	619 . . . 761	51/159 (32%)	1.1e−19
		104/159 (65%)

Example 16

[0447] 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: 63	1778 bp
NOV16a,	GAGGCCAGGAGCGCTCCGTCTGGAACGGCGCAGGTCCCAACAGCTGCGGTTCCCCCTCAGCCCG
CG52552-06	TGAGCAGCCATGTCCAACCCCAACGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAG
DNA Sequence	GCCCTCTGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCC
	TGGCTACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCACCCAC
	CCGATGCCCATGAACTACGGCCCACGCCATGGCTATGATCGCGAGGAGAGAGCGGTGAGTGATA
	GCTTCGCGCCTGGAGAGTGGGATGACCGGAAAGTGCGACACACTTTTATCCGAAAGGTTTACTC
	CATCATCTCCCTGCAGCTGCTCATCACTGTGGCCATCATTGCTATCTTCACCTTTGTGGAACCT
	GTCAGCGCCTTTGTGAGGAGAAATGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCGTTGTCA
	CCTACCTGATCCTTGCCTGCTGCCAGGGACCCAGACGCCGTTTCCCATGGAACATCATTCTGCT
	GACCCTTTTTACTTTTGCCATGGGCTTCATGACCGGCACCATTTCCAGTATGTACCAAACCAAA
	GCCGTCATCATTGCAATGATCATCACTGCGGTGGTATCCATTTCAGTCACCATCTTCTGCTTTC
AGACCAAGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTCCTGGT
GACTGGGATTGTCACTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCTCCACATGCTCTAT
GCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGGCTTACAACACACAGCTGGTCCTGGGGA
	ACCGGAAGCACACCATCAGCCCCGAGGACTACATCACTGGCGCCCTGCAGATTTACACAGACAT
	CATCTACATCTTCACCTTTCTGCTGCAGCTGATGGGGGATCGCAATTAAGGAGCAAGCCCCCAT
	TTTCACCCGATCCTGGGCTCTCCCTTCCAAGCTAGAGGGCTGGGCCCTATGACTGTGGTCTGGG
	CTTTAGGCCCCTTTCCTTCCCCTTGAGTAACATGCCCAGTTTCCTTTCTGTCCTGGAGACAGGT
	GGCCTCTCTGGCTATGGATGTGTGGGTACTTGGTGGGGCACGGAGGAGCTAGGGACTAACTGTT
	GCTCTTGGTGGGCTTGGCAGGGACTAGGCTGAAGATGTGTCTTCTCCCCGCCACCTACTGTATG
	ACACCATTCTTCCTAACAGCTGGGGTTGTGAGGAATATGAAAAGAGCCTATTCGATAGCTAG
	AAGGGAATATGAAAGGTAGAAGTGACTTCAAGGTCACGAGGTTCCCCTCCCACCTCTGTCACAG
	GCTTCTTGACTACGTAGTTGGAGCTATTTCTTCCCCCAGCAAAGCCAGAGAGCTTTGTCCCCGG
	CCTCCTGGACACATAGGCCATTATCCTGTATTCCTTTGGCTTGGCATCTTTTAGCTCAGGAAGG
	TAGAAGAGATCTGTGCCCATGGGTCTCCTTGCTTCAATCCCTTCTTGTTTCAGTGACATATGTA
	TTGTTTATCTGGGTTAGGGATGGGGGACAGATAATAGAACGAGCAAAGTAACCTATACAGGCCA
	GCATGGAACAGCATCTCCCCTGCGCTTGCTCCTGGCTTGTGACGCTATAAGACAGAGCAGGCCA
	CATGTGGCCATCTGCTCCCCATTCTTGAAAGCTGCTGGGGCCTCCTTGCA
	ORF Start: ATG at 74		ORF Stop: TAA at 1007
	SEQ ID NO: 64	311 aa	MW AT 34649.2 kD
NOV 16a,	MSNPNAPPPYEDRNPLYPGPLPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP
CG52552-06	MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSA
Protein Sequence	FVRRNVAVYYVSYAVFVVTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVI
	IAMIITAVVSISVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAAL
	GAICFTLFLAYNTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN
	SEQ IDNO: 65	908 bp
NOV16b,	CGGCCATGTCCAACCCCAGCGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAGGCCC
CG52552-04	TCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCCTGGC
DNA Sequence	TACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCCATGGCTATG
	ATGGGGAGGAGAGAGCAGTGAGTGATAGCTTCGGGCCTGGAGAGTGGGATGACCGGAAAGTGCG
	ACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCGTGCAGCTGCTCATCACTGTGGCCATC
	ATTGCTATCTTCACCTTTGTGGAACCTGTCAGCGCCTTTGTGAGGAGAAATGTGGCTGTCTACT
	ACGTGTCCTATGCTGTCTTCGTTGTCACCTACCTGATCCTTGCCTGCTGCCAGGGACCCAGACG
	CCGTTTCCCATGGAACATCATTCTGCTGACCCTTTTTACTTTTGCCATGGGCTTCATGACGGGC
	ACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCATTGCAATGATCATCACTGCGGTGGTAT
	CCATTTCAGTCACCATCTTCTGCTTTCAGACCAAGGTGGACTTCACCTCGTGCACAGGCCTCTT
	CTGTGTCCTGGGAATTGTGCTCCTGGTGACTGGGATTGTCACTAGCATTGTGCTCTACTTCCAA
	TACGTTTACTGGCTCCACATGCTCTATGCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGG
	CTTACGACACACAGCTGGTCCTGGGGAACCGGAAGCACACCATCAGCCCCGAGGACTACATCAC
	TGGCGCCCTGCAGATTTACACAGACATCATCTACATCTTCACCTTTGTGCTGCAGCTGATGGGG
	GATCGCAATTAA
	ORF Start: ATG at 6		ORF Stop: TAA at 906
	SEQ ID NO: 66	300 aa	MW at 33423.7 kD
NOV16b,	MSNPSAPPPYEDRNPLYPGPPPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPHGYDG
CG52552-04	EERAVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSAFVRRNVAVYYV
Protein Sequence	SYAVFVVTYLILACCQGPRRRFPNNIILLTLFTFAMGFMTGTISSMYQTKAVIIAMIITAVVSI
	SVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSTVLYFQYVYWLHNLYAALGAICFTLFLAY
	DTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN
	SEQ ID NO: 67	1767 bp
NOV16c,	AACGGCGCAGGTCCCAGCAGCTGGGGTTCCCCCTCAGCCCGTGAGCAGCCATGTCCAACCCCAG
CG52552-01	CGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAGGCCCTCTGCCCCCTGGGGGCTAT
DNA Sequence	GGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCCTGGCTACCCGCAGCCTGGCTACG
	GTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCACCCACCCGATGCCCATGAACTACGGCCC
	AGGCCATGGCTATGATGGGGAGGAGAGAGCGGTGAGTGATAGCTTCGGGCCTGGAGAATGGGAT
	GACCGGAAAGTGCGACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCGGGCAGCTGCTCA
	TCACTGGGGCCATCATTGCTATCTTCACCTTTGGGGAACCTGTCAGCGCCTTTGGCAGGAGAAA
	TGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCAGTGTCACCTACCTGATCCTTGCCTGCTGC
	CAGGGACCCAGACGCCGTTTCCCATGGAACATCATTCTGCTGACCCTTTTTACTTTTGCCATGG
	GCTTCATGACGGGCACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCATTGCAATGATCAT
	CACTGCCGTGGTATCCATTTCAGTCACCATCTTCTGCTTTCAGACCAAGGTGGACTTCACCTCG
	TGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTCCTGGTGACTGGGATTGTCACTAGCATTG
	TGCTCTACTTCCAATACGTTTACTGGCTCCACATGCTCTATGCTGCTCTGGGGGCCATTTGTTT
	CACCCTGTTCCTGGCTTACGACACACAGCTGGTCCTCGGGAACCGGAAGCACACCATCAGCCCC
	GAGGACTACATCACTGGCGCCCTGCAGATTTACACAGACATCATCTACATCTTCACCTTTGTGC
	TGCAGCTGATGGGGGATCGCAATTAAGGAGCAAGCCCCCATTTTCACCCGATCCTCGGCTCTCC
	CTTCCAAGCTAGAGGGCTGGGCCCTATGACTGTGGTCTGGGCTTTAGGCCCCTTTCCTTCCCCT
	TCAGTAACATGCCCAGTTTCCTTTCTGTCCTGGAGACAGGTGGCCTCTCTGGCTATGGATGTGT
	GGGTACTTGCTCGGGACGGAGGAGCTAGGGACTAACTGTTGCTCTTGGTGGGCTTGGCAGGGAC
	TAGGCTGAAGATGTGTCTTCTCCCCGCCACCTACTGTATGACACCACATTCTTCCTAACAGCTG
	GGGTTGTGAGGAATATGAAAAGAGCCTATTCGATAGCTAGAAGGGAATATGAAAGGTAGAAGTG
	ACTTCAAGGTCACGAGGTTCCCCTCCCACCTCTGTCACAGGCTTCTTGACTACGTAGTTGGAGC
	TATTTCTTCCCCCAGCAAAGCCAGAGAGCTTTGTCCCCGGCCTCCTGGACACATAGGCCATTAT
	CCTGTATTCCTTTGGCTTGGCATCTTTTAGCTCAGGAAGGTAGAAGAGATCTGTGCCCATCGGT
	CTCCTTGCTTCAATCCCTTCTTGTTTCAGTGACATATGTATTGTTTATCTGGGTTAGGGATGGG
	GGACAGATAATAGAACGAGCAAAGTAACCTATACAGGCCACCATGGAACAGCATCTCCCCTGGG
	CTTGCTCCTGGCTTGTGACGCTATAAGACAGAGCACGCCACATGTGGCCATCTGCTCCCCATTC
	TTGAAAGCTGCTGGGGCCTCCTTGCAGGCTTCTGGATCC
	ORF Start: ATG at 51		ORF Stop: TAA at 984
	SEQ ID NO: 68	311 aa	MW at 34442.7 kD
NOV16c,	MSNPSAPPPYEDRNPLYPGPLPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP
CG52552-01	MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFIRKVYSIISCQLLITGAIIAIFTFGEPVSA
Protein Sequence	FGRRNVAVYYVSYAVFSVTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVI
	IAMIITAVVSISVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAAL
	GAICFTLFLAYDTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN
	SEQ ID NO: 69	2059 bp
NOV16d,	CCCTCCGTCTGGAACGGCGCAGGTCCCAGCAGCTGGGGTTCCCCCTCAGCCCGTGACCAGCCAT
CG52552-02	GTCCAACCCCAGCGCCCCACCACCATATGAAGACCGCAACCCCCTGTACCCAGGCCCTCTGCCC
DNA Sequence	CCTGGGCGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTATCCTGCCTACCCTGGCTACCCGC
	AGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATGCCCCCCACCCACCCGATGCCCAT
	GAACTACGGCCCAGGCCATGGCTATGATGGGGAGGAGAGACCGGTGAGTGATAGCTTCGGGCCT
	GGAGAGTGGGATGACCGGAAAGTGCGACACACTTTTATCCGAAAGGTTTACTCCATCATCTCCG
	TGCACCTGCTCATCACTGTGGCCATCATTGCTATCTTCACCTTTGTGGAACCTGTCAGCGCCTT
	TGTGAGGAGAAATGTGGCTGTCTACTACGTGTCCTATGCTGTCTTCGTTGTCACCTACCTGATC
	CTTGCCTGCTGCCAGGGACCCAGACGCCGTTTCCCATGGAACATCATTCTGCTGACCCTTTTTA
	CTTTTGCCATGGGCTTCATGACGGGCACCATTTCCAGTATGTACCAAACCAAAGCCGTCATCAT
	TGCAATGATCATCACTGCGGTGGTATCCATTTCAGTCACCATCTTCTGCTTTCAGACCAAGGTG
	AGGGCATGGAGGGCCCTTCCCTGGCCCCCCGACTCCCCTTTCTTATCAGGCCCGGACCCCGGTA
	CACTAGGGATGTTCCCTAGAGACCTGATCCCCTTCTCCTCATCCGCACCTACAAAACTGTGTCC
	TGTTTCTGTCCTTAGAATGTTGTGGACATTCCCATACCCCCTAGGAGGCAGCACTGGGACTCCC
	TGGCAGGGCCAGTCTGACTGGGCTGGTTGTCACAGCCATCTGACAGGTGCCTCTTTCTTGCTTC
	CTGGCAGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTCCTGGTG
	ACTGGGATTGTCACTAGCATTGTGCTCTTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCT
	CCACATGCTCTATGCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGGCTTACGACACACAG
	CTGGTCCTGGGGAACCGGAAGCACACCATCAGCCCCGAGGACTACATCACTGGCGCCCTGCAGA
	TTTACACAGACATCATCTACATCTTCACCTTTGTGCTGCAGCTGATGGGGGATCGCAATTAAGG
	AGCAAGCCCCCATTTTCACCCGATCCTGGGCTCTCCCTTCCAAGCTAGAGGACTGGGCCCTATG
	ACTGTGGTCTGGGCTTTAGGCCCCTTTCCTTCCCCTTGAGTAACATGCCCAGTTTCCTTTCTGT
	CCTGGAGACAGGTGGCCTCTCTGGCTATGGATGTGTGGGTACTTGGTGGGGACGGAGGAGCTAG
	GGACTAACTGTTGCTCTTGGTGGGCTTCGCAGGGACTAGGCTGAAGATGTGTCTTCTCCCCGCC
	ACCTACTGTATGACACCACATTCTTCCTAACAGCTGGGGTTGTGAGGAATATGAAAAGAGCCTA
	TTCGATAGCTAGAAGGGAATATGAAAGGTAGAAGTGACTTCAAGGTCACGAGGTTCCCCTCCCA
	CCTCTGTCACAGGCTTCTTGACTACGTAGTTGGAGCTATTTCTTCCCCCAGCAAAGCCAGAGAG
	CTTTGTCCCCGGCCTCCTGGACACATAGGCCATTATCCTGTATTCCTTTGGCTTGGCATCTTTT
	AGCTCAGGAAGGTAGAAGAGATCTGTGCCCATGCGTCTCCTTGCTTCAATCCCTTCTTGTTTCA
	GTGACATATGTATTGTTTATCTGGGTTAGGGATGGGGGACAGATAATAGAACGAGCAAAGTAAC
	CTATACAGGCCAGCATGGAACAGCATCTCCCCTGGGCTTGCTCCTGGCTTGTGACGCTATAAGA
	CAGAGCAGGCCACATGTGGCCATCTGCTCCCCATTCTTGAAAGCTGCTGGGGCCTCCTTGCACG
	CTTCTGGATCC
	ORF Start: ATG at 63		ORF Stop: TGA at 1023
	SEQ ID NO: 70	320 aa	MW at 35204.3 kD
NOV16d,	MSNPSAPPPYEDRNPLYPGPLPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP
CG52552-02	MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSA
Protein Sequence	FVRRNVAVYYVSYAVFVVTYLILACCQGPRRRFPWNIILLTLETFANGFMTGTISSMYQTKAVI
	IAMIITAVVSISVTIFCFQTKVRAWRALPWPPDSPFLSGPDPGTLGMFPRDLIPFSSSAPTKLC
	PVSVLRMLWTFPYPLGGSTGTPWQGQSDWAGCHSHLTGASFLLPGRWTSPRAQASSVSWELCSW
	SEQ ID NO: 71	2437 bp
NOV16e,	ATGCCAGCCCCAAACCTCATCCCTAGTGGAGGCCTTGCTGATGTGGAAGTGGCCAGGGCCCTCA
CG52552-03	TGGTAGGCTGGGCAGAAGCCCAAGAACAGGCTCTAAAGCTGCTAAACCCCGCAGTCCTGGTCCC
DNA Sequence	CGGAGGCTCTTGCCAGTCTGACAGTGTTCTTGGCACTCCTCAAAGGTCCCAGCAGCTGGGGTTC
	CCCGTCAGCCCGTGAGCGGCCATGTCCAACCCCAGCGCCCCACCACCATATGAAGACCGCAACC
	CCCTGTACCCAGGCCCTCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCTGCCAGGAGGGTA
	TCCTGCCTACCCTGGCTACCCGCAGCCTGGCTACGGTCACCCTGCTGGCTACCCACAGCCCATG
	CCCCCCACCCACCCGATGCCCATGAACTACGGCCCAGGCCATGGCTATGATGGGGAGGAGAGAG
	CGGTGAGTCATAGCTTCGGGCCTGGAGAGTGGGATGACCGGAAAGTGCGACACACTTTTATCCG
	AAAGGTTTACTCCATCATCTCCGTGCAGCTGCTCATCACTGTGGCCATCATTGCTATCTTCACC
	TTTGTGGAACCTGTCAGCGCCTTTGTGAGGAGAAATGTGGCTGTCTACTACGTGTCCTATGCTG
	TCTTCGTTGTCACCTACCTGATCCTTGCCTGCTGCCAGGGACCCAGACGCCGTTTCCCATGGAA
	CATCATTCTGCTGACCCTTTTTACTTTTGCCATGGGCTTCATGACGGGCACCATTTCCAGTATG
	TACCAAACCAAAGCCGTCATCATTGCAATGATCATCACTGCGCTGGTATCCATTTCAGTCACCA
	TCTTCTGCTTTCAGACCAAGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAAT
	TGTGCTCCTGGTGACTGGGATTGTCACTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCTC
	CACATGCTCTATGCTGCTCTGGGGGCCATTTGTTTCACCCTGTTCCTGGCTTACGACACACAGC
	TGGTCCTGGGGAACCGGAAGCACACCATCAGCCCCGAGGACTACATCACTGGCGCCCTGCAGAT
	TTACACAGACATCATCTACATCTTCACCTTTGTGCTGCACCTGATGGGGGATCGCAATTAAGGA
	GCAAGCCCCCATTTTCACCCGATCCTGGGCTCTCCCTTCCAAGCTAGAGGGCTGGGCCCTATGA
	CTGTGGTCTGGGCTTTAGGCCCCTTTCCTTCCCCTTGAGTAACATGCCCAGTTTCCTTTCTGTC
	CTGGAGACAGGTGGCCTCTCTGGCTATGGATGTGTGGGTACTTGGTGGGGACGGAGGAGCTAGG
	GACTAACTGTTGCTCTTGGTGGCCTTGGCAGGCACTAGGCTGAAGATGTGTCTTCTCCCCGCCA
	CCTACTGTATGACACCACATTCTTCCTAACAGCTGGGGTTGTGAGGAATATGAAAAGAGCCTAT
	TCGATAGCTAGAAGGGAATATGAAACGTAGAAGTGACTTCAAGGTCACGAGGTTCCCCTCCCAC
	CTCTGTCACAGGCTTCTTGACTACGTAGTTGGAGCTATTTCTTCCCCCAGCAAAGCCAGAGAGC
	TTTGTCCCCGGCCTCCTGGACACATAGGCCATTATCCTGTATTCCTTTGGCTTGGCATCTTTTA
	GCTCAGGAAGGTAGAAGAGATCTGTGCCCATGGQTCTCCTTGCTTCAATCCCTTCTTGTTTCAG
	TGACATATGTATTGTTTATCTGGGTTAGGGATGGGGGACAGATAATAGAACGAGCAAAGTAACC
	TATACAGGCCAGCATGGAACAGCATCTCCCCTGGGCTTGCTCCTGGCTTGTGACGCTATAAGAC
	AGAGCAGGCCACATGTGGCCATCTGCTCCCCATTCTTGAAAGCTGCTGGGGCCTCCTTGCAGGC
	TTCTGGATCTCTGGTCAGAGTGAACTCTTGCTTCCTGTATTCAGGCAGCTCAGAGCAGAAAGTA
	AGGGGCAGAGTCATACGTGTGGCCAGGAAGTAGCCAGGGTGAAGAGAGACTCGGTGCGGGCAGG
	GAGAATGCCTGGGGGTCCCTCACCTGGCTAGGGAGATACCGAAGCCTACTGTGGTACTGAAGAC
	TTCTGGGTTCTTTCCTTCTGCTAACCCAGGGAGGGTCCTAAGAGGAAGGTGACTTCTCTCTGTT
	TGTCTTAAGTTGCACTGGGGGATTTCTGACTTGAGGCCCATCTCTCCAGCCAGCCACTGCCTTC
	TTTGTAATATTAAGTGCCTTGAGCTGGAATGGGGAAGGGGGACAAGGGTCAGTCTGTCGGGTGG
	GGGCAGAAATCAAATCAGCCCAAGGATATAGTTAGGATTAATTACTTAATAGAGAAATCCTAAC
	TATATCACACAAAGGGATACAACTATAAATGTAATAAAATTTATGTCTAGAAGTTAAAAAAAAA
	AAAAA
	ORF Start: ATG at 214		ORF Stop: TAA at 1147
	SEQ ID NO: 72	311 aa	MW at 34607.1 kD
NOV16e,	MSNPSAPPPYEDRNPLYPGPPPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP
CG52552-03	MNYGPGHGYDGEERAVSDSFGPCEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSA
Protein Sequence	FVRRNVAVYYVSYAVFVVTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISSMYQTKAVI
	IAMIITAVVSISVTIFCFQTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQYVYWLHMLYAAL
	GAICFTLFLAYDTQLVLGNRKHTISPEDYITGALQIYTDIIYIFTFVLQLMGDRN
	SEQ ID NO: 73	719 bp
NOV16f,	CAGCTGGGGTTCCCCGTCAGCCCGTGAGCGGCCATGTCCAACCCCAGCGCCCCACCACCATATG
CG52552-05	AAGACCGCAACCCCCTGTACCCAGGCCCTCCGCCCCCTGGGGGCTATGGGCAGCCATCTGTCCT
DNA Sequence	GCCAGGAGGGTATCCTGCCTACCCTGGCTACCCGCAGCCTCGCTACGGTCACCCTGCTGGCTAC
	CCACAGCCCATGCCCCCCACCCACCCGATGCCCATGAACTACGGCCCAGGCCATCGCTATGATG
	GGGAGGAGAGAGCGGTGAGTGATAGCTTCGGGCCTGGAGAGTGGGATGACCGGAAAGTGCGACA
	CACTTTTATCCGAAAGGTTTACTCCATCATCTCCGTGCAGCTGCTCATCACTGTGGCCATCATT
	GCTATCTTCACCTTTGTGGAACCTGTCAGTGCCTTTGTGAGGAGAAATGTGGCTGTCTACTACG
	TGTCCTATGCTGTCTTCGTTGTCACCTACCTGATCCTTGCCTGCTGCCAGGGACCCACACGCCG
	TTTCCCATGGAACATCATTCTGCTGACCCTTTTTACTTTTGCCATGCGCTTCATGACGGGCACC
	ATTTCCAACCAAGGTGGACTTCACCTCGTGCACAGGCCTCTTCTGTGTCCTGGGAATTGTGCTC
	CTGGTGACTGGGATTGTCACTAGCATTGTGCTCTACTTCCAATACGTTTACTGGCTCCACATGC
	TCTATGCTGCTCTGG
	ORF Start: ATG at 34		ORF Stop: TAG at 661
	SEQ ID NO: 74	209 aa	MW at 23010.2 kD
NOV16f,	MSNPSAPPPYEDRNPLYPGPPPPGGYGQPSVLPGGYPAYPGYPQPGYGHPAGYPQPMPPTHPMP
CG52552-05	MNYGPGHGYDGEERAVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLITVAIIAIFTFVEPVSA
Protein Sequence	FVRRNVAVYYVSYAVFVVTYLILACCQGPRRRFPWNIILLTLFTFAMGFMTGTISNQGGLHLVH
	RPLLCPGNCAPGDWDCH

[0448] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 16B.

TABLE 16B
Comparison of NOV16a against NOV16b through NOV16f.
			Identities/
	Protein	NOV16a Residues/	Similarities for
	Sequence	Match Residues	the Matched Region
	NOV16b	1 . . . 311	297/311 (95%)
		1 . . . 300	299/311 (95%)
	NOV16c	1 . . . 311	304/311 (97%)
		1 . . . 311	306/311 (97%)
	NOV16d	1 . . . 214	213/214 (99%)
		1 . . . 214	214/214 (99%)
	NOV16e	1 . . . 311	308/311 (99%)
		1 . . . 311	310/311 (99%)
	NOV16f	1 . . . 184	181/184 (98%)
		1 . . . 184	183/184 (99%)

[0449] Further analysis of the NOV16a protein yielded the following properties shown in Table 16C.

TABLE 16C
Protein Sequence Properties NOV16a
SignalP	No Known Signal Sequence Predicted
analysis:
PSORT II	PSG: a new signal peptide prediction method
analysis:	N-region: length 11; pos. chg 0; neg. chg 1
	H-region: length 0; peak value 0.00
	PSG score: −4.40
	GvH: von Heijne's method for signal seq. recognition
	GvH score (threshold: −2.1): −5.29
	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: 6
	INTEGRAL	Likelihood =	−8.92	Transmembrane 105-121
	INTEGRAL	Likelihood =	−4.25	Transmembrane 138-154
	INTEGRAL	Likelihood =	−2.07	Transmembrane 165-181
	INTEGRAL	Likelihood =	−10.30	Transmembrane 191-207
	INTEGRAL	Likelihood =	−10.51	Transmembrane 225-241
	INTEGRAL	Likelihood =	−3.40	Transmembrane 249-265
	PERIPHERAL	Likelihood =	0.53 (at 292)
	ALOM score: −10.51 (number of TMSs: 6)
	MTOP: Prediction of membrane topology (Hartmann et al.)
	Center position for calculation: 112
	Charge difference: −1.5 C(1.0)-N(2.5)
	N >= C: N-terminal side will be inside
	>>> membrane topology: type 3a
	MITDISC: discrimination of mitochondrial targeting seq
	R content:	0	Hyd Moment (75):	4.00
	Hyd Moment (95):	1.75	G content:	0
	D/E content:	2	S/T content:	1
	Score: −7.55
	Gavel: prediction of cleavage sites for mitochondrial preseq
	cleavage site motif not found
	NUCDISC: discrimination of nuclear localization signals
	pat4: PRRR (4) at 157
	pat7: PRRRFPW (5) at 157
	bipartite: none
	content of basic residues: 5.5%
	NLS Score: 0.21
	KDEL: ER retention motif in the C-terminus: none
	ER Membrane Retention Signals: none
	SKL: peroxisomal targeting signal in the C-terminus: none
	PTS2: 2nd peroxisomal targeting signal: none
	VAC: possible vacuolar targeting motif: none
	RNA-binding motif: none
	Actinin-type actin-binding motif:
	type 1: none
	type 2: none
	NMYR: N-myristoylation pattern: none
	Prenylation motif: none
	memYQRL: transport motif from cell surface to Golgi: none
	Tyrosines in the tail: none
	Dileucine motif in the tail: none
	checking 63 PROSITE DNA binding motifs: none
	checking 71 PROSITE ribosomal protein motifs: none
	checking 33 PROSITE prokaryotic DNA binding motifs: none
	NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
	Prediction: cytoplasmic
	Reliability: 94.1
	COIL: Lupas's algorithm to detect coiled-coil regions
	total: 0 residues
	Final Results (k = {fraction (9/23)}):
	55.6%: endoplasmic reticulum
	22.2%: mitochondrial
	11.1%: nuclear
	11.1%: vesicles of secretory system
	>> prediction for CG52552-06 is end (k = 9)

[0450] A search of the NOV16a 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	Residues/	Similarities for	Expect
Identifier	[Patent #, Date]	Match Residues	the Matched Region	Value
AAY08659	WO9927094 Seq ID 10 - Homo	1 . . . 311	309/311 (99%)	0.0
	sapiens, 311 aa. [WO9927094-A2,	1 . . . 311	311/311 (99%)
	03 JUN. 1999]
AAY08656	Human transmembrane domain	1 . . . 311	309/311 (99%)	0.0
	containing protein from clone	1 . . . 311	311/311 (99%)
	HP01862 - Homo sapiens, 311 aa.
	[WO9927094-A2, 03 JUN. 1999]
AAB58328	Lung cancer associated polypeptide	1 . . . 311	308/311 (99%)	0.0
	sequence SEQ ID 666 - Homo sapiens,	3 . . . 313	310/311 (99%)
	313 aa. [WO200055180-A2,
	21 SEP. 2000]
AAB90754	Human shear stress-response protein	1 . . . 311	308/311 (99%)	0.0
	SEQ ID NO: 8 - Homo sapiens, 311 aa.	1 . . . 311	310/311 (99%)
	[WO200125427-A1, 12 APR. 2001]
AAW69738	Human proline-rich membrane protein -	1 . . . 311	308/311 (99%)	0.0
	Homo sapiens, 311 aa.	1 . . . 311	310/311 (99%)
	[WO9833910-A1, 06 AUG. 1998]

[0451] In a BLAST search of public sequence databases, the NOV16a protein was found to have homology to the proteins shown in the BLASTP data in Table 16E.

TABLE 16E
Public BLASTP Results for NOV16a
Protein		NOV16a	Identities/
Accession		Residues/	Similarities for	Expect
Number	Protein/Organism/Length	Match Residues	the Matched Portion	Value
Q969X1	PP1201 protein (Hypothetical protein) -	1 . . . 311	309/311 (99%)	0.0
	Homo sapiens (Human), 311 aa.	1 . . . 311	311/311 (99%)
Q8TAM3	PP1201 protein - Homo sapiens	1 . . . 311	308/311 (99%)	0.0
	(Human), 311 aa.	1 . . . 311	310/311 (99%)
BAC43762	RECS1 - Mus musculus (Mouse),	1 . . . 311	273/311 (87%)	e−164
	309 aa.	1 . . . 309	292/311 (93%)
BAC36957	8 days embryo whole body cDNA,	1 . . . 311	271/311 (87%)	e−162
	RIKEN full-length enriched library,	1 . . . 309	291/311 (93%)
	clone: 5730523J24 product: PP1201
	PROTEIN homolog - Mus musculus
	(Mouse), 309 aa.
Q8N1R3	Hypothetical protein FLJ37951 - Homo	60 . . . 311	250/252 (99%)	e−143
	sapiens (Human), 303 aa.	52 . . . 303	251/252 (99%)

[0452] PFam analysis predicts that the NOV16a protein contains the domains shown in the Table 16F.

TABLE 16F
Domain Analysis of NOV16a
			Identities/
	Pfam	NOV16a	Similarities for	Expect
	Domain	Match Region	the Matched Region	Value
	UPF0005	120 . . . 311	72/208 (35%)	2.3e−51
			157/208 (75%)

Example B

Sequencing Methodology and Identification of NOVX Clones

[0453] 1. GeneCalling™ Technology: This is a proprietary method of performing differential gene expression profiling between two or more samples developed at CuraGen and described by Shimkets, et al., “Gene expression analysis by transcript profiling coupled to a gene database query” Nature Biotechnology 17:198-803 (1999). cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then digested with up to as many as 120 pairs of restriction enzymes and pairs of linker-adaptors specific for each pair of restriction enzymes were ligated to the appropriate end. The restriction digestion generates a mixture of unique cDNA gene fragments. Limited PCR amplification is performed with primers homologous to the linker adapter sequence where one primer is biotinylated and the other is fluorescently labeled. The doubly labeled material is isolated and the fluorescently labeled single strand is resolved by capillary gel electrophoresis. A computer algorithm compares the electropherograms from an experimental and control group for each of the restriction digestions. This and additional sequence-derived information is used to predict the identity of each differentially expressed gene fragment using a variety of genetic databases. The identity of the gene fragment is confirmed by additional, gene-specific competitive PCR or by isolation and sequencing of the gene fragment.

[0454] 2. SeqCalling™ Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

[0455] 3. PathCalling™ Technology: The NOVX nucleic acid sequences are derived by laboratory screening of cDNA library by the two-hybrid approach. cDNA fragments covering either the full length of the DNA sequence, or part of the sequence, or both, are sequenced. In silico prediction was based on sequences available in CuraGen Corporation's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.

[0456] The laboratory screening was performed using the methods summarized below:

[0457] 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, Calif.) were then transferred from E. coli into a CuraGen Corporation proprietary yeast strain (disclosed in U.S. Pat. Nos. 6,057,101 and 6,083,693, incorporated herein by reference in their entireties).

[0458] Gal4-binding domain (Gal4-BD) fusions of a CuraGen Corporation proprietary library of human sequences was used to screen multiple Gal4-AD fusion cDNA libraries resulting in the selection of yeast hybrid diploids in each of which the Gal4-AD fusion contains an individual cDNA. Each sample was amplified using the polymerase chain reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such PCR product was sequenced; sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

[0459] 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. Pat. Nos. 6,057,101 and 6,083,693).

[0460] 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.

[0461] 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.

[0462] 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.

[0463] 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

[0464] 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 PRISMS 7700 or an ABI PRISM® 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and Alzheimer's diseased brains).

[0465] 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.

[0466] First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions.

[0467] 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 μg of total RNA were performed in a volume of 20 μl and incubated for 60 minutes at 42° C. This reaction can be scaled up to 50 μg of total RNA in a final volume of 100 μl. sscDNA samples are then normalized to reference nucleic acids as described previously, using 1× TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions.

[0468] 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 75 bp to 100 bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, Tex., 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, 900 nM each, and probe, 200 nM.

[0469] 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.

[0470] 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 1× TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions. PCR amplification was performed as follows: 95° C. 10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute. Results were analyzed and processed as described previously.

[0471] 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.

[0472] In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used:

[0473] ca. =carcinoma,

[0474] *=established from metastasis,

[0475] met=metastasis,

[0476] s cell var=small cell variant,

[0477] non-s=non-sm=non-small,

[0478] squam=squamous,

[0479] pl. eff=pl effusion=pleural effusion,

[0480] glio=glioma,

[0481] astro=astrocytoma, and

[0482] neuro=neuroblastoma.

[0483] General_screening_panel_v1.4, v1.5, v1.6 and 1.7

[0484] 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.

[0485] Panels 2D, 2.2, 2.3 and 2.4

[0486] 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, Calif.), Research Genetics, and Invitrogen.

[0487] HASS Panel v 1.0

[0488] 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.

[0489] ARDAIS Panel v 1.0

[0490] 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.

[0491] ARDAIS Prostate v 1.0

[0492] The plates for ARDAIS prostate 1.0 generally include 2 control wells and 68 test samples composed of RNA isolated from human tissue procured by surgeons working in close cooperation with Ardais Corporation. The tissues are derived from human prostate malignancies and in cases where indicated malignant samples have “matched margins” obtained from noncancerous prostate tissue just adjacent to the tumor. These matched margins are taken from the tissue surrounding (i.e., immediately proximal) to the zone of surgery (designated “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). RNA from unmatched malignant and non-malignant prostate samples were also obtained from Ardais. Additional information from Ardais provides a gross histopathological assessment of tumor differentiation grade and stage. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical state of the patient.

[0493] Panel 3D, 3.1 and 3.2

[0494] 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 of the most common cell lines used in the scientific literature.

[0495] Panels 4D, 4R, and 4.1D

[0496] 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, Calif.) 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, Calif.). 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.).

[0497] 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 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.

[0498] 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 μM non essential amino acids (Gibco/Life Technologies, Rockville, Md.), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml PMA and 1-2 μg/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2×106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5×10−5M) (Gibco), and 10 mM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1-7 days for RNA preparation.

[0499] 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, Utah), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours.

[0500] 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), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and plated at 106 cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3 ug/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), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×105M (Gibco), and 10 mM 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), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.

[0501] 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 106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 10 μg/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.

[0502] To prepare the primary and secondary Th1/Th2 and Tr1 cells, six-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, Md.) were cultured at 105-106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×1 0−5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL-4 (1 μg/ml) were used to direct to Th1, while IL-4 (5 ng/ml) and anti-IFN gamma (1 μg/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Tr1. After 4-5 days, the activated Th1, Th2 and Tr1 lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Th1, Th2 and Tr1 lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 μg/ml) to prevent apoptosis. After 4-5 days, the Th1, Th2 and Tr1 lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Th1 and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Th1, Th2 and Tr1 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.

[0503] The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5×10−5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5×10−5 cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 μg/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), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.

[0504] For these cell lines and blood cells, RNA was prepared by lysing approximately 107 cells/ml using Trizol (Gibco BRL). Briefly, {fraction (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 15 ml 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 300 μl of RNAse-free water and 35 μl buffer (Promega) 5 μl DTT, 7 μl RNAsin and 8 μl 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 {fraction (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.

[0505] AI_comprehensive Panel_v1.0

[0506] The plates for AI_comprehensive panel_v1.0 include two control wells and 89 test samples comprised of cDNA isolated from surgical and postmortem human tissues obtained from the Backus Hospital and Clinomics (Frederick, Md.). Total RNA was extracted from tissue samples from the Backus Hospital in the Facility at CuraGen. Total RNA from other tissues was obtained from Clinomics.

[0507] 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.

[0508] 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.

[0509] 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.

[0510] Total RNA from post mortem lung tissue from trauma victims with no disease or with emphysema, asthma or COPD was purchased from Clinomics. Emphysema patients ranged in age from 40-70 and all were smokers, this age range was chosen to focus on patients with cigarette-linked emphysema and to avoid those patients with alpha-1 anti-trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded smokers to prevent those patients that could also have COPD. COPD patients ranged in age from 35-80 and included both smokers and non-smokers. Most patients were taking corticosteroids, and bronchodilators.

[0511] In the labels employed to identify tissues in the Al_comprehensive panel_v1.0 panel, the following abbreviations are used:

[0512] AI=Autoimmunity

[0513] Syn=Synovial

[0514] Normal=No apparent disease

[0515] Rep22/Rep20=individual patients

[0516] RA=Rheumatoid arthritis

[0517] Backus=From Backus Hospital

[0518] OA=Osteoarthritis

[0519] (SS)(BA)(MF)=Individual patients

[0520] Adj=Adjacent tissue

[0521] Match control=adjacent tissues

[0522] -M=Male

[0523] -F=Female

[0524] COPD=Chronic obstructive pulmonary disease

[0525] AI.05 Chondrosarcoma

[0526] 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 (e.g. IL1beta). These treatments include: IL-1beta (10 ng/ml), IL-1beta+TNF-alpha (50 ng/ml), IL-1beta+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 3×105 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.

[0527] Panels 5D and 5I

[0528] The plates for Panel 5D and 5I 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.

[0529] 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:

[0530] Patient 2: Diabetic Hispanic, overweight, not on insulin

[0531] Patient 7-9: Nondiabetic Caucasian and obese (BMI>30)

[0532] Patient 10: Diabetic Hispanic, overweight, on insulin

[0533] Patient 11: Nondiabetic African American and overweight

[0534] Patient 12: Diabetic Hispanic on insulin

[0535] Adiocyte differentiation was induced in donor progenitor cells obtained from Osirus (a division of Clonetics/BioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr. 2 1999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows:

[0536] Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose

[0537] Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated

[0538] Donor 2 and 3 AD: Adipose, Adipose Differentiated

[0539] 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.

[0540] Panel 5I 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 5I.

[0541] In the labels employed to identify tissues in the 5D and 5I panels, the following abbreviations are used:

[0542] GO Adipose=Greater Omentum Adipose

[0543] SK=Skeletal Muscle

[0544] UT=Uterus

[0545] PL=Placenta

[0546] AD Adipose Differentiated

[0547] AM=Adipose Midway Differentiated

[0548] U=Undifferentiated Stem Cells

[0549] Panel CNSD.01

[0550] The plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

[0551] 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.

[0552] In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:

[0553] PSP=Progressive supranuclear palsy

[0554] Sub Nigra=Substantia nigra

[0555] Glob Palladus=Globus palladus

[0556] Temp Pole=Temporal pole

[0557] Cing Gyr=Cingulate gyrus

[0558] BA 4=Brodman Area 4

[0559] Panel CNS_Neurodegeneration_V1.0

[0560] The plates for Panel CNS_Neurodegeneration_V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

[0561] 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.

[0562] In the labels employed to identify tissues in the CNS_Neurodegeneration_V1.0 panel, the following abbreviations are used:

[0563] AD=Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy

[0564] Control=Control brains; patient not demented, showing no neuropathology

[0565] Control (Path)=Control brains; patient not demented but showing sever AD-like pathology

[0566] SupTemporal Ctx=Superior Temporal Cortex

[0567] Inf Temporal Ctx=Inferior Temporal Cortex

[0568] Panel CNS_Neurodegeneration_V2.0

[0569] The plates for Panel CNS_Neurodegeneration_V2.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

[0570] Disease diagnoses are taken from patient records. The panel contains sixteen brains from Alzheimer's disease (AD) patients, and twenty-nine brains from “Normal controls” who showed no evidence of dementia prior to death. The twenty-nine normal control brains are divided into two categories: Fourteen controls with no dementia and no Alzheimer's like pathology (Controls) and fifteen controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0=no evidence of plaques, 3=severe AD senile plaque load). Tissue from the temporal cotex (Broddmann Area 21) was selected for all samples from the Harvard Brain Tissue Resource Center; from the two sample from the Human Brain and Spinal Fluid Resource Center (samples 1 and 2) tissue from the inferior and superior temporal cortex was used; each sample on the panel represents a pool of inferior and superior temporal cortex from an individual patient. The temporal cortex was chosen as it shows a loss of neurons in the intermediate stages of the disease. Selection of a region which is affected in the early stages of Alzheimer's disease (e.g., hippocampus or entorhinal cortex) could potentially result in the examination of gene expression after vulnerable neurons are lost, and missing genes involved in the actual neurodegeneration process.

[0571] In the labels employed to identify tissues in the CNS_Neurodegeneration_V2.0 panel, the following abbreviations are used:

[0572] AD=Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy

[0573] Control=Control brains; patient not demented, showing no neuropathology

[0574] AH3=Control brains; patient not demented but showing sever AD-like pathology

[0575] Inf & Sup Temp Ctx Pool=Pool of inferior and superior temporal cortex for a given individual

[0576] A. CG126472-02: TEM7.

[0577] Expression of gene CG126472-02 was assessed using the primer-probe set Ag4727, described in Table AA. Results of the RTQ-PCR runs are shown in Tables AB, AC, AD, AE and AF.

TABLE AA
Probe Name Ag4727
			Start	SEQ
Primers	Sequences	Length	Position	ID No
Forward	5′-cagtgctgagaacacca	22	1543	75
	agtct-3′
Probe	TET-5′-ccctttgaagact	26	1566	76
	ttgaggccacaga-3′-
	TAMRA
Reverse	5′-gccaggaaaagtcactt	22	1611	77
	ctctt-3′

[0578]

TABLE AB
CNS neurodegeneration v1.0
		Rel. Exp. (%)	Rel. Exp. (%)
		Ag4727, Run	Ag4727, Run
	Tissue Name	218649157	268784120
	AD 1 Hippo	7.8	8.4
	AD 2 Hippo	11.7	9.0
	AD 3 Hippo	6.2	6.5
	AD 4 Hippo	5.9	8.8
	AD 5 Hippo	100.0	100.0
	AD 6 Hippo	22.7	25.3
	Control 2 Hippo	13.2	17.7
	Control 4 Hippo	8.1	7.1
	Control (Path) 3 Hippo	5.3	3.6
	AD 1 Temporal Ctx	14.6	18.9
	AD 2 Temporal Ctx	19.3	19.3
	AD 3 Temporal Ctx	10.3	9.8
	AD 4 Temporal Ctx	24.0	23.0
	AD 5 Inf Temporal Ctx	75.8	50.0
	AD 5 Sup Temporal Ctx	20.7	18.3
	AD 6 Inf Temporal Ctx	28.9	26.4
	AD 6 Sup Temporal Ctx	40.1	36.3
	Control 1 Temporal Ctx	11.3	12.9
	Control 2 Temporal Ctx	29.9	39.2
	Control 3 Temporal Ctx	28.3	22.7
	Control 3 Temporal Ctx	11.9	11.4
	Control (Path) 1	62.4	62.9
	Temporal Ctx
	Control (Path) 2	46.3	47.3
	Temporal Ctx
	Control (Path) 3	6.9	9.1
	Temporal Ctx
	Control (Path) 4	44.1	39.2
	Temporal Ctx
	AD 1 Occipital Ctx	20.2	18.8
	AD 2 Occipital Ctx	0.0	0.4
	(Missing)
	AD 3 Occipital Ctx	7.9	9.5
	AD 4 Occipital Ctx	25.7	24.3
	AD 5 Occipital Ctx	45.4	18.4
	AD 6 Occipital Ctx	14.4	48.0
	Control 1 Occipital Ctx	9.1	7.9
	Control 2 Occipital Ctx	58.2	67.8
	Control 3 Occipital Ctx	34.9	34.6
	Control 4 Occipital Ctx	7.4	7.5
	Control (Path) 1	82.4	79.6
	Occipital Ctx
	Control (Path) 2	22.5	24.8
	Occipital Ctx
	Control (Path) 3	7.3	9.8
	Occipital Ctx
	Control (Path) 4	39.0	42.9
	Occipital Ctx
	Control 1 Parietal Ctx	11.6	13.3
	Control 2 Parietal Ctx	35.4	32.5
	Control 3 Parietal Ctx	22.5	24.7
	Control (Path) 1	72.2	83.5
	Parietal Ctx
	Control (Path) 2	34.6	33.9
	Parietal Ctx
	Control (Path) 3	7.6	6.7
	Parietal Ctx
	Control (Path) 4	57.4	81.8
	Parietal Ctx

[0579]

TABLE AC
General screening panel v1.4
                                 Rel. Exp.(%)
                                 Ag4727,
Tissue Name                      Run 218713021
Adipose                          16.8
Melanoma* Hs688(A).T             1.2
Melanoma* Hs688(B).T             1.6
Melanoma* M14                    0.1
Melanoma* LOXIMVI                0.1
Melanoma* SK-MEL-5               4.1
Squamous cell carcinoma SCC-4    0.2
Testis Pool                      11.7
Prostate ca.* (bone met) PC-3    0.3
Prostate Pool                    6.9
Placenta                         2.4
Uterus Pool                      16.2
Ovarian ca. OVCAR-3              0.5
Ovarian ca. SK-OV-3              0.2
Ovarian ca. OVCAR-4              0.2
Ovarian ca. OVCAR-5              2.3
Ovarian ca. IGROV-1              0.4
Ovarian ca. OVCAR-8              1.3
Ovary                            9.8
Breast ca. MCF-7                 0.5
Breast ca. MDA-MB-231            0.9
Breast ca. BT 549                4.1
Breast ca. T47D                  4.6
Breast ca. MDA-N                 0.0
Breast Pool                      34.9
Trachea                          7.8
Lung                             19.1
Fetal Lung                       15.6
Lung ca. NCI-N417                0.0
Lung ca. LX-1                    0.9
Lung ca. NCI-H146                0.0
Lung ca. SHP-77                  0.2
Lung ca. A549                    0.3
Lung ca. NCI-H526                0.1
Lung ca. NCI-H23                 2.5
Lung ca. NCI-H460                0.1
Lung ca. HOP-62                  0.5
Lung ca. NCI-H522                8.7
Liver                            0.1
Fetal Liver                      0.9
Liver ca. HepG2                  0.0
Kidney Pool                      48.6
Fetal Kidney                     6.4
Renal ca. 786-0                  0.1
Renal ca. A498                   0.6
Renal ca. ACHN                   0.2
Renal ca. UO-31                  0.0
Renal ca. TK-10                  0.6
Bladder                          6.8
Gastric ca. (liver met.) NCI-N87 31.0
Gastric ca. KATO III             0.1
Colon ca. SW-948                 0.1
Colon ca. SW480                  0.8
Colon ca.* (SW480 met) SW620     0.3
Colon ca. HT29                   0.1
Colon ca. HCT-116                1.0
Colon ca. CaCo-2                 0.7
Colon cancer tissue              10.4
Colon ca. SW1116                 0.3
Colon ca. Colo-205               0.1
Colon ca. SW-48                  0.0
Colon Pool                       35.1
Small Intestine Pool             36.1
Stomach Pool                     24.7
Bone Marrow Pool                 19.1
Fetal Heart                      11.6
Heart Pool                       20.4
Lymph Node Pool                  41.8
Fetal Skeletal Muscle            5.9
Skeletal Muscle Pool             9.2
Spleen Pool                      3.1
Thymus Pool                      77.9
CNS cancer (glio/astro) U87-MG   3.8
CNS cancer (glio/astro) U-118-MG 1.0
CNS cancer (neuro;met) SK-N-AS   0.4
CNS cancer (astro) SF-539        1.3
CNS cancer (astro) SNB-75        100.0
CNS cancer (glio) SNB-19         0.4
CNS cancer (glio) SF-295         0.9
Brain (Amygdala) Pool            10.2
Brain (cerebellum)               47.0
Brain (fetal)                    22.1
Brain (Hippocampus) Pool         5.0
Cerebral Cortex Pool             17.7
Brain (Substantia nigra) Pool    8.8
Brain (Thalamus) Pool            17.7
Brain (whole)                    25.0
Spinal Cord Pool                 8.7
Adrenal Gland                    3.4
Pituitary gland Pool             1.8
Salivary Gland                   2.6
Thyroid (female)                 1.8
Pancreatic ca. CAPAN2            3.8
Pancreas Pool                    26.8

[0580]

TABLE AD
Panel 3D
                                 Rel. Exp.(%)
                                 Ag4727,
                                 Run 218912984
Daoy- Medulloblastoma            2.1
TE671- Medulloblastoma           25.5
D283 Med- Medulloblastoma        21.6
PFSK-1- Primitive Neuroectodermal 3.4
XF-498- CNS                      0.0
SNB-78- Glioma                   11.0
SF-268- Glioblastoma             0.0
T98G- Glioblastoma               0.0
SK-N-SH- Neuroblastoma (metastasis) 1.2
SF-295- Glioblastoma             0.7
Cerebellum                       84.7
Cerebellum                       100.0
NCI-H292- Mucoepidermoid lung carcinoma 0.0
DMS-114- Small cell lung cancer  15.9
DMS-79- Small cell lung cancer   87.7
NCI-H146- Small cell lung cancer 1.6
NCI-H526- Small cell lung cancer 2.0
NCI-N417- Small cell lung cancer 0.0
NCI-H82- Small cell lung cancer  2.5
NCI-H157- Squamous cell lung cancer (metastasis) 0.0
NCI-H1155- Large cell lung cancer 11.1
NCI-H1299- Large cell lung cancer 3.1
NCI-H727- Lung carcinoid         0.0
NCI-UMC-11- Lung carcinoid       0.0
LX-1- Small cell lung cancer     3.3
Colo-205- Colon cancer           0.0
KM12- Colon cancer               1.5
KM20L2- Colon cancer             0.0
NCI-H716- Colon cancer           0.0
SW-48- Colon adenocarcinoma      0.3
SW1116- Colon adenocarcinoma     0.6
LS 174T- Colon adenocarcinoma    1.1
SW-948- Colon adenocarcinoma     0.0
SW-480- Colon adenocarcinoma     1.4
NCI-SNU-5- Gastric carcinoma     0.8
KATO III- Gastric carcinoma      0.0
NCI-SNU-16- Gastric carcinoma    0.7
NCI-SNU-1- Gastric carcinoma     0.0
RF-1- Gastric adenocarcinoma     1.5
RF-48- Gastric adenocarcinoma    6.0
MKN-45- Gastric carcinoma        0.8
NCI-N87- Gastric carcinoma       5.3
OVCAR-5- Ovarian carcinoma       0.0
RL95-2- Uterine carcinoma        0.0
HelaS3- Cervical adenocarcinoma  0.0
Ca Ski- Cervical epidermoid carcinoma (metastasis) 0.9
ES-2- Ovarian clear cell carcinoma 0.0
Ramos- Stimulated with PMA/ionomycin 6 h 0.0
Ramos- Stimulated with PMA/ionomycin 14 h 0.9
MEG-01- Chronic myelogenous leukemia (megokaryoblast) 0.0
Raji- Burkitt's lymphoma         0.0
Daudi- Burkitt's lymphoma        0.7
U266- B-cell plasmacytoma        1.7
CA46- Burkitt's lymphoma         1.4
RL- non-Hodgkin's B-cell lymphoma 0.0
JM1- pre-B-cell lymphoma         0.0
Jurkat- T cell leukemia          0.0
TF-1- Erythroleukemia            1.7
HUT 78- T-cell lymphoma          0.0
U937- Histiocytic lymphoma       1.0
KU-812- Myelogenous leukemia     0.0
769-P- Clear cell renal carcinoma 0.0
Caki-2- Clear cell renal carcinoma 0.0
SW 839- Clear cell renal carcinoma 0.0
Rhabdoid kidney tumor            2.2
Hs766T- Pancreatic carcinoma (LN metastasis) 0.0
CAPAN-1- Pancreatic adenocarcinoma (liver 1.4
metastasis)
SU86.86- Pancreatic carcinoma (liver metastasis) 0.7
BxPC-3- Pancreatic adenocarcinoma 0.3
HPAC- Pancreatic adenocarcinoma  0.8
MIA PaCa-2- Pancreatic carcinoma 1.5
CFPAC-1- Pancreatic ductal adenocarcinoma 5.3
PANC-1- Pancreatic epithelioid ductal carcinoma 0.7
T24- Bladder carcinma (transitional cell) 0.0
5637- Bladder carcinoma          0.0
HT-1197- Bladder carcinoma       1.5
UM-UC-3- Bladder carcinma (transitional cell) 0.0
A204- Rhabdomyosarcoma           0.0
HT-1080- Fibrosarcoma            0.8
MG-63- Osteosarcoma              0.0
SK-LMS-1- Leiomyosarcoma (vulva) 0.8
SJRH30- Rhabdomyosarcoma (met to bone marrow) 0.0
A431- Epidermoid carcinoma       1.6
WM266-4- Melanoma                1.6
DU 145- Prostate carcinoma (brain metastasis) 0.0
MDA-MB-468- Breast adenocarcinoma 2.7
SCC-4- Squamous cell carcinoma of tongue 0.0
SCC-9- Squamous cell carcinoma of tongue 0.0
SCC-15- Squamous cell carcinoma of tongue 0.0
CAL 27- Squamous cell carcinoma of tongue 1.7

[0581]

TABLE AE
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag4727,
Tissue Name                      Run 204150153
Secondary Th1 act                0.2
Secondary Th2 act                0.3
Secondary Tr1 act                0.3
Secondary Th1 rest               2.0
Secondary Th2 rest               4.5
Secondary Tr1 rest               2.0
Primary Th1 act                  0.3
Primary Th2 act                  1.1
Primary Tr1 act                  0.4
Primary Th1 rest                 2.1
Primary Th2 rest                 4.2
Primary Tr1 rest                 11.0
CD45RA CD4 lymphocyte act        0.5
CD45RO CD4 lymphocyte act        1.2
CD8 lymphocyte act               2.0
Secondary CD8 lymphocyte rest    2.9
Secondary CD8 lymphocyte act     0.0
CD4 lymphocyte none              16.6
2ry Th1/Th2/Tr1_anti-CD95 CH11   8.3
LAK cells rest                   14.1
LAK cells IL-2                   1.0
LAK cells IL-2 + IL-12           2.3
LAK cells IL-2 + IFN gamma       4.0
LAKcells IL-2 + IL-18            4.2
LAK cells PMA/ionomycin          8.6
NK Cells IL-2 rest               7.7
Two Way MLR 3 day                2.5
Two Way MLR 5 day                3.7
Two Way MLR 7 day                0.7
PBMC rest                        4.4
PBMC PWM                         1.0
PBMC PHA-L                       0.6
Ramos (B cell) none              0.0
Ramos (B cell) ionomycin         0.0
B lymphocytes PWM                0.6
B lymphocytes CD40L and IL-4     6.6
EOL-1 dbcAMP                     0.0
EOL-1 dbcAMP PMA/ionomycin       0.3
Dendritic cells none             4.6
Dendritic cells LPS              0.1
Dendritic cells anti-CD40        2.4
Monocytes rest                   0.1
Monocytes LPS                    0.0
Macrophages rest                 0.3
Macrophages LPS                  0.0
HUVEC none                       0.0
HUVEC starved                    0.0
HUVEC IL-1beta                   0.0
HUVEC IFN gamma                  0.0
HUVEC TNF alpha + IFN gamma      0.0
HUVEC TNF alpha + IL4            0.0
HUVEC IL-11                      0.1
Lung Microvascular EC none       0.0
Lung Microvascular EC TNFalpha + IL-1beta 0.0
Microvascular Dermal EC none     0.0
Microsvasular Dermal EC TNFalpha + IL-1beta 0.0
Bronchial epithelium TNFalpha + IL1beta 0.0
Small airway epithelium none     0.0
Small airway epithelium TNFalpha + IL-1beta 0.0
Coronery artery SMC rest         0.0
Coronery artery SMC TNFalpha + IL-1beta 0.1
Astrocytes rest                  0.0
Astrocytes TNFalpha + IL-1beta   0.1
KU-812 (Basophil) rest           0.0
KU-812 (Basophil) PMA/ionomycin  0.0
CCD1106 (Keratinocytes) none     0.2
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 1.1
Liver cirrhosis                  2.4
NCI-H292 none                    0.1
NCI-H292 IL-4                    0.0
NCI-H292 IL-9                    0.0
NCI-H292 IL-13                   0.3
NCI-H292 IFN gamma               0.1
HPAEC none                       0.0
HPAEC TNF alpha + IL-1 beta      0.0
Lung fibroblast none             2.4
Lung fibroblast TNF alpha + IL-1 beta 0.6
Lung fibroblast IL-4             1.1
Lung fibroblast IL-9             1.3
Lung fibroblast IL-13            0.7
Lung fibroblast IFN gamma        1.3
Dermal fibroblast CCD1070 rest   0.1
Dermal fibroblast CCD1070 TNF alpha 1.9
Dermal fibroblast CCD1070 IL-1 beta 0.9
Dermal fibroblast IFN gamma      3.1
Dermal fibroblast IL-4           1.7
Dermal Fibroblasts rest          2.4
Neutrophils TNFa + LPS           0.3
Neutrophils rest                 0.4
Colon                            2.0
Lung                             37.6
Thymus                           100.0
Kidney                           3.6

[0582]

TABLE AF
general oncology screening panel v 2.4
                               Rel. Exp.(%)
                               Ag4727,
Tissue Name                    Run 260280475
Colon cancer 1                 9.0
Colon cancer NAT 1             1.9
Colon cancer 2                 9.3
Colon cancer NAT 2             4.3
Colon cancer 3                 17.9
Colon cancer NAT 3             12.2
Colon malignant cancer 4       8.6
Colon normal adjacent tissue 4 1.8
Lung cancer 1                  10.6
Lung NAT 1                     2.1
Lung cancer 2                  19.5
Lung NAT 2                     1.2
Squamous cell carcinoma 3      10.2
Lung NAT 3                     0.6
metastatic melanoma 1          34.9
Melanoma 2                     1.4
Melanoma 3                     4.0
metastatic melanoma 4          19.3
metastatic melanoma 5          31.9
Bladder cancer 1               3.1
Bladder cancer NAT 1           0.0
Bladder cancer 2               1.9
Bladder cancer NAT 2           0.3
Bladder cancer NAT 3           0.4
Bladder cancer NAT 4           3.8
Prostate adenocarcinoma 1      39.8
Prostate adenocarcinoma 2      3.3
Prostate adenocarcinoma 3      1.4
Prostate adenocarcinoma 4      9.3
Prostate cancer NAT 5          0.7
Prostate adenocarcinoma 6      3.3
Prostate adenocarcinoma 7      7.2
Prostate adenocarcinoma 8      1.6
Prostate adenocarcinoma 9      26.2
Prostate cancer NAT 10         0.1
Kidney cancer 1                98.6
KidneyNAT 1                    15.3
Kidney cancer 2                100.0
Kidney NAT 2                   8.6
Kidney cancer 3                35.1
Kidney NAT 3                   0.8
Kidney cancer 4                17.8
Kidney NAT 4                   2.6

[0583] CNS_neurodegeneration_v1.0 Summary: Ag4727 Two experiments with the same probe and primer produce results that are in excellent agreement. This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene appears to be slightly down-regulated in the temporal cortex of Alzheimer's disease patients. Therefore, up-regulation of this gene or its protein product, or treatment with specific agonists for this receptor may be of use in reversing the dementia, memory loss, and neuronal death associated with this disease.

[0584] General_screening_panel_v1.4 Summary: Ag4727 Highest expression of this gene is seen in a brain cancer cell line (CT=28). This gene is widely expressed in this panel, with moderate expression seen in a colon and gastric cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

[0585] Among tissues with metabolic function, this gene is expressed at moderate to low levels in pituitary, adipose, adrenal gland, pancreas, thyroid, fetal liver and adult and fetal skeletal muscle and heart. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

[0586] Interestingly, this gene is expressed at much higher levels in the fetal kidney (CT=29) when compared to the level of expression in the adult kidney (CT=32). This observation suggests that expression of this gene can be used to distinguish between the fetal and adult source of this tissue. In addition, the relative overexpression of this gene in fetal kidney suggests that the protein product may enhance kidney growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of kidney related diseases.

[0587] This gene is also expressed at moderate to low significant levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

[0588] Panel 3D Summary: Ag4727 Highest expression of this gene is seen in cerebellum (CT=30). Moderate levels of expression are seen in several cancer cell lines on this panel, in agreement with Panel 1.4. Please see that panel for discussion of this.

[0589] Panel 4.1D Summary: Ag4727 Highest expression of this gene is seen in thymus (CT=27.8), with moderate levels of expression seen in normal lung. The protein encoded for by this gene could therefore play an important role in T cell development. Therefore, therapeutic modulation of the expression or function of this gene could be utilized to modulate immune function (T cell development) and be important for organ transplant, AIDS treatment or post chemotherapy immune reconstitution.

[0590] General oncology screening panel_v—2.4 Summary: Ag4727 This gene is widely expressed in this panel, with highest expression in lung cancer (CT=32.6). In addition, this gene is more highly expressed in lung and kidney cancer than in the corresponding normal adjacent tissue. Thus, expression of this gene could be used as a marker of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene product may be useful in the treatment of lung and kidney cancer.

[0591] B. CG170490-01: Ubiquitin-Protein Ligase E3 Componen N-RECOGNIN.

[0592] Expression of gene CG170490-01 was assessed using the primer-probe set Ag6130, described in Table BA. Results of the RTQ-PCR runs are shown in Tables BB, BC and BD.

TABLE BA
Probe Name Ag6130
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-agttagttcttcctataaccacctttatct-3′	30	4349	78
Probe	TET-5′-atttgatcaccatggcacacatgctt-3′-TAMRA	26	4384	79
Reverse	5′-gtaggcctgtgtctactgtaagtagtatct-3′	30	4411	80

[0593]

TABLE BB
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag6130,
Tissue Name                    Run 253339684
AD 1 Hippo                     17.7
AD 2 Hippo                     33.7
AD 3 Hippo                     10.4
AD 4 Hippo                     7.8
AD 5 Hippo                     100.0
AD 6 Hippo                     46.7
Control 2 Hippo                22.4
Control 4 Hippo                14.5
Control (Path) 3 Hippo         6.6
AD 1 Temporal Ctx              20.0
AD 2 Temporal Ctx              30.1
AD 3 Temporal Ctx              9.1
AD 4 Temporal Ctx              20.2
AD 5 Inf Temporal Ctx          95.3
AD 5 Sup Temporal Ctx          51.4
AD 6 Inf Temporal Ctx          53.6
AD 6 Sup Temporal Ctx          51.8
Control 1 Temporal Ctx         9.9
Control 2 Temporal Ctx         33.2
Control 3 Temporal Ctx         18.2
Control 3 Temporal Ctx         7.9
Control (Path) 1 Temporal Ctx  57.0
Control (Path) 2 Temporal Ctx  37.9
Control (Path) 3 Temporal Ctx  6.7
Control (Path) 4 Temporal Ctx  35.4
AD 1 Occipital Ctx             16.3
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             7.5
AD 4 Occipital Ctx             14.4
AD 5 Occipital Ctx             36.6
AD 6 Occipital Ctx             14.4
Control 1 Occipital Ctx        5.2
Control 2 Occipital Ctx        55.5
Control 3 Occipital Ctx        22.7
Control 4 Occipital Ctx        9.0
Control (Path) 1 Occipital Ctx 85.9
Control (Path) 2 Occipital Ctx 12.7
Control (Path) 3 Occipital Ctx 4.7
Control (Path) 4 Occipital Ctx 14.7
Control 1 Parietal Ctx         6.9
Control 2 Parietal Ctx         50.0
Control 3 Parietal Ctx         16.6
Control (Path) 1 Parietal Ctx  81.8
Control (Path) 2 Parietal Ctx  24.7
Control (Path) 3 Parietal Ctx  6.1
Control (Path) 4 Parietal Ctx  34.2

[0594]

TABLE BC
General screening panel v1.5
                                 Rel. Exp.(%)
                                 Ag6130,
Tissue Name                      Run 253079166
Adipose                          28.1
Melanoma* Hs688(A).T             54.0
Melanoma* Hs688(B).T             48.3
Melanoma* M14                    32.8
Melanoma* LOXIMVI                31.2
Melanoma* SK-MEL-5               48.3
Squamous cell carcinoma SCC-4    21.5
Testis Pool                      19.8
Prostate ca.* (bone met) PC-3    76.3
Prostate Pool                    23.5
Placenta                         13.2
Uterus Pool                      25.5
Ovarian ca. OVCAR-3              47.3
Ovarian ca. SK-OV-3              84.1
Ovarian ca. OVCAR-4              17.2
Ovarian ca. OVCAR-5              86.5
Ovarian ca. IGROV-1              28.3
Ovarian ca. OVCAR-8              50.0
Ovary                            26.6
Breast ca. MCF-7                 33.7
Breast ca. MDA-MB-231            63.3
Breast ca. BT 549                25.5
Breast ca. T47D                  12.8
Breast ca. MDA-N                 17.0
Breast Pool                      41.5
Trachea                          15.9
Lung                             7.3
Fetal Lung                       40.9
Lung ca. NCI-N417                9.1
Lung ca. LX-1                    82.4
Lung ca. NCI-H146                10.5
Lung ca. SHP-77                  55.1
Lung ca. A549                    27.4
Lung ca. NCI-H526                14.5
Lung ca. NCI-H23                 41.2
Lung ca. NCI-H460                35.1
Lung ca. HOP-62                  28.9
Lung ca. NCI-H522                28.9
Liver                            2.4
Fetal Liver                      46.0
Liver ca. HepG2                  14.5
Kidney Pool                      63.7
Fetal Kidney                     34.2
Renal ca. 786-0                  25.9
Renal ca. A498                   12.3
Renal ca. ACHN                   23.0
Renal ca. UO-31                  19.1
Renal ca. TK-10                  34.4
Bladder                          39.5
Gastric ca. (liver met.) NCI-N87 79.0
Gastric ca. KATO III             76.8
Colon ca. SW-948                 10.2
Colon ca. SW480                  53.6
Colon ca.* (SW480 met) SW620     64.6
Colon ca. HT29                   62.9
Colon ca. HCT-116                76.8
Colon ca. CaCo-2                 36.1
Colon cancer tissue              36.6
Colon ca. SW1116                 15.8
Colon ca. Colo-205               12.3
Colon ca. SW-48                  10.4
Colon Pool                       51.1
Small Intestine Pool             33.7
Stomach Pool                     27.2
Bone Marrow Pool                 14.3
Fetal Heart                      45.4
Heart Pool                       18.8
Lymph Node Pool                  43.5
Fetal Skeletal Muscle            10.8
Skeletal Muscle Pool             55.1
Spleen Pool                      27.9
Thymus Pool                      36.1
CNS cancer (glio/astro) U87-MG   35.4
CNS cancer (glio/astro) U-118-MG 100.0
CNS cancer (neuro; met) SK-N-AS  58.6
CNS cancer (astro) SF-539        16.7
CNS cancer (astro) SNB-75        63.3
CNS cancer (glio) SNB-19         33.2
CNS cancer (glio) SF-295         58.6
Brain (Amygdala) Pool            15.0
Brain (cerebellum)               46.7
Brain (fetal)                    33.2
Brain (Hippocampus) Pool         17.7
Cerebral Cortex Pool             23.8
Brain (Substantia nigra) Pool    15.7
Brain (Thalamus) Pool            22.5
Brain (whole)                    8.0
Spinal Cord Pool                 22.7
Adrenal Gland                    20.2
Pituitary gland Pool             9.8
Salivary Gland                   5.4
Thyroid (female)                 12.7
Pancreatic ca. CAPAN2            21.8
Pancreas Pool                    46.3

[0595]

TABLE BD
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag6130,
Tissue Name                      Run 253307370
Secondary Th1 act                68.3
Secondary Th2 act                90.1
Secondary Tr1 act                100.0
Secondary Th1 rest               32.5
Secondary Th2 rest               10.9
Secondary Tr1 rest               17.2
Primary Th1 act                  33.9
Primary Th2 act                  73.2
Primary Tr1 act                  48.6
Primary Th1 rest                 5.0
Primary Th2 rest                 11.5
Primary Tr1 rest                 50.0
CD45RA CD4 lymphocyte act        48.6
CD45RO CD4 lymphocyte act        85.3
CD8 lymphocyte act               48.3
Secondary CD8 lymphocyte rest    74.2
Secondary CD8 lymphocyte act     15.3
CD4 lymphocyte none              8.2
2ry Th1/Th2/Tr1_anti-CD95 CH11   13.6
LAK cells rest                   21.6
LAK cells IL-2                   33.9
LAK cells IL-2 + IL-12           93.3
LAK cells IL-2 + IFN gamma       74.2
LAK cells IL-2 + IL-18           37.4
LAK cells PMA/ionomycin          80.1
NK Cells IL-2 rest               84.7
Two Way MLR 3 day                38.4
Two Way MLR 5 day                15.7
Two Way MLR 7 day                13.5
PBMC rest                        7.0
PBMC PWM                         17.4
PBMC PHA-L                       28.9
Ramos (B cell) none              30.4
Ramos (B cell) ionomycin         44.1
B lymphocytes PWM                88.9
B lymphocytes CD40L and IL-4     54.3
EOL-1 dbcAMP                     35.6
EOL-1 dbcAMP PMA/ionomycin       42.6
Dendritic cells none             31.2
Dendritic cells LPS              23.7
Dendritic cells anti-CD40        9.5
Monocytes rest                   6.7
Monocytes LPS                    58.2
Macrophages rest                 12.9
Macrophages LPS                  7.6
HUVEC none                       22.2
HUVEC starved                    38.4
HUVEC IL-1beta                   39.8
HUVEC IFN gamma                  49.0
HUVEC TNF alpha + IFN gamma      38.4
HUVEC TNF alpha + IL4            24.8
HUVEC IL-11                      19.1
Lung Microvascular EC none       82.4
Lung Microvascular EC TNFalpha + IL-1beta 22.8
Microvascular Dermal EC none     10.4
Microsvasular Dermal EC TNFalpha + IL-1beta 32.5
Bronchial epithelium TNFalpha + IL1beta 11.2
Small airway epithelium none     18.4
Small airway epithelium TNFalpha + IL-1beta 44.1
Coronery artery SMC rest         42.3
Coronery artery SMC TNFalpha + IL-1beta 48.6
Astrocytes rest                  20.0
Astrocytes TNFalpha + IL-1beta   15.7
KU-812 (Basophil) rest           51.8
KU-812 (Basophil) PMA/ionomycin  89.5
CCD1106 (Keratinocytes) none     36.9
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 27.2
Liver cirrhosis                  20.9
NCI-H292 none                    22.1
NCI-H292 IL-4                    40.9
NCI-H292 IL-9                    48.0
NCI-H292 IL-13                   41.8
NCI-H292 IFN gamma               30.8
HPAEC none                       20.3
HPAEC TNF alpha + IL-1 beta      73.7
Lung fibroblast none             43.2
Lung fibroblast TNF alpha + IL-1 beta 35.1
Lung fibroblast IL-4             21.0
Lung fibroblast IL-9             51.1
Lung fibroblast IL-13            8.7
Lung fibroblast IFN gamma        87.1
Dermal fibroblast CCD1070 rest   54.0
Dermal fibroblast CCD1070 TNF alpha 86.5
Dermal fibroblast CCD1070 IL-1 beta 50.7
Dermal fibroblast IFN gamma      49.7
Dermal fibroblast IL-4           41.5
Dermal Fibroblasts rest          38.4
Neutrophils TNFa + LPS           5.4
Neutrophils rest                 12.8
Colon                            1.4
Lung                             0.8
Thymus                           8.6
Kidney                           19.8

[0596] CNS_neurodegeneration_v1.0 Summary: Ag6130 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals.

[0597] General_screening_panel_v1.5 Summary: Ag6130 Highest expression of this gene is detected in a brain cancer U-118-MG cell line (CT=27.6). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation 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.

[0598] Among tissues with metabolic or endocrine function, this gene is expressed at moderate levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

[0599] 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.

[0600] Interestingly, this gene is expressed at much higher levels in fetal (CT=28.7) when compared to adult liver (CT=33). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult.

[0601] Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of liver related diseases.

[0602] Panel 4.1D Summary: Ag6130 Highest expression of this gene is detected in activated secondary Tr1 cells (CT=29.9). This gene is expressed at moderate to low levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members of the T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_v1.5 and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

[0603] C. CG170667-02: Putative Neuronal Cell Adhesion Molecule.

[0604] Expression of gene CG170667-02 was assessed using the primer-probe sets Ag6137 and Ag6161, described in Tables CA and CB. Results of the RTQ-PCR runs are shown in Tables CC and CD.

TABLE CA
Probe Name Ag6137
Primers	Sequences	Length	Start Position	SEQ ID No
Forward	5′-ggacagccttgagccc-3′	16	1913	81
Probe	TET-5′-catccacatcggggtcgctt-3′-TAMRA	20	1992	82
Reverse	5′-gccgaacaggaggaagag-3′	18	2029	83

[0605]

TABLE CB
Probe Name Ag6161
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-gtcaggctcaagaataacaacag-3′	23	1186	84
Probe	TET-5′-acactgaccatttctggaatcggtcc-3′-TAMRA	26	1210	85
Reverse	5′-acacactgataaatggcttcatc-3′	23	1240	86

[0606]

TABLE CC
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag6137,
Tissue Name                    Run 253574596
AD 1 Hippo                     17.2
AD 2 Hippo                     11.8
AD 3 Hippo                     5.3
AD 4 Hippo                     5.4
AD 5 Hippo                     50.0
AD 6 Hippo                     28.1
Control 2 Hippo                13.4
Control 4 Hippo                12.8
Control (Path) 3 Hippo         1.4
AD 1 Temporal Ctx              29.1
AD 2 Temporal Ctx              29.7
AD 3 Temporal Ctx              9.2
AD 4 Temporal Ctx              23.7
AD 5 Inf Temporal Ctx          53.6
AD 5 Sup Temporal Ctx          33.2
AD 6 Inf Temporal Ctx          36.1
AD 6 Sup Temporal Ctx          21.5
Control 1 Temporal Ctx         13.6
Control 2 Temporal Ctx         31.4
Control 3 Temporal Ctx         16.2
Control 3 Temporal Ctx         6.5
Control (Path) 1 Temporal Ctx  52.9
Control (Path) 2 Temporal Ctx  26.2
Control (Path) 3 Temporal Ctx  4.6
Control (Path) 4 Temporal Ctx  12.3
AD 1 Occipital Ctx             19.8
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             8.0
AD 4 Occipital Ctx             19.5
AD 5 Occipital Ctx             29.1
AD 6 Occipital Ctx             7.0
Control 1 Occipital Ctx        4.4
Control 2 Occipital Ctx        100.0
Control 3 Occipital Ctx        13.0
Control 4 Occipital Ctx        4.9
Control (Path) 1 Occipital Ctx 88.9
Control (Path) 2 Occipital Ctx 16.2
Control (Path) 3 Occipital Ctx 3.9
Control (Path) 4 Occipital Ctx 20.4
Control 1 Parietal Ctx         14.1
Control 2 Parietal Ctx         40.1
Control 3 Parietal Ctx         10.5
Control (Path) 1 Parietal Ctx  43.8
Control (Path) 2 Parietal Ctx  18.4
Control (Path) 3 Parietal Ctx  7.5
Control (Path) 4 Parietal Ctx  27.7

[0607]

TABLE CD
General screening panel v1.5
                                 Rel. Exp.(%)
                                 Ag6137,
Tissue Name                      Run 253101096
Adipose                          0.0
Melanoma* Hs688(A).T             0.0
Melanoma* Hs688(B).T             0.1
Melanoma* M14                    0.0
Melanoma* LOXIMVI                0.1
Melanoma* SK-MEL-5               0.3
Squamous cell carcinoma SCC-4    0.0
Testis Pool                      2.5
Prostate ca.* (bone met) PC-3    0.0
Prostate Pool                    0.2
Placenta                         10.4
Uterus Pool                      0.1
Ovarian ca. OVCAR-3              0.5
Ovarian ca. SK-OV-3              0.0
Ovarian ca. OVCAR-4              0.2
Ovarian ca. OVCAR-5              5.8
Ovarian ca. IGROV-1              0.9
Ovarian ca. OVCAR-8              0.3
Ovary                            0.2
Breast ca. MCF-7                 24.1
Breast ca. MDA-MB-231            0.0
Breast ca. BT 549                0.0
Breast ca. T47D                  2.1
Breast ca. MDA-N                 0.0
Breast Pool                      0.6
Trachea                          0.0
Lung                             0.1
Fetal Lung                       4.3
Lung ca. NCI-N417                1.2
Lung ca. LX-1                    3.0
Lung ca. NCI-H146                0.2
Lung ca. SHP-77                  0.0
Lung ca. A549                    0.1
Lung ca. NCI-H526                3.2
Lung ca. NCI-H23                 0.6
Lung ca. NCI-H460                0.0
Lung ca. HOP-62                  0.1
Lung ca. NCI-H522                6.1
Liver                            0.0
Fetal Liver                      3.0
Liver ca. HepG2                  100.0
Kidney Pool                      3.0
Fetal Kidney                     7.1
Renal ca. 786-0                  0.0
Renal ca. A498                   0.0
Renal ca. ACHN                   0.0
Renal ca. UO-31                  0.0
Renal ca. TK-10                  59.5
Bladder                          0.0
Gastric ca. (liver met.) NCI-N87 0.0
Gastric ca. KATO III             0.1
Colon ca. SW-948                 0.0
Colon ca. SW480                  4.2
Colon ca.* (SW480 met) SW620     11.9
Colon ca. HT29                   0.1
Colon ca. HCT-116                0.7
Colon ca. CaCo-2                 86.5
Colon cancer tissue              0.0
Colon ca. SW1116                 0.0
Colon ca. Colo-205               0.0
Colon ca. SW-48                  0.0
Colon Pool                       0.9
Small Intestine Pool             3.0
Stomach Pool                     0.5
Bone Marrow Pool                 0.2
Fetal Heart                      1.2
Heart Pool                       0.1
Lymph Node Pool                  1.1
Fetal Skeletal Muscle            0.4
Skeletal Muscle Pool             0.0
Spleen Pool                      0.0
Thymus Pool                      1.7
CNS cancer (glio/astro) U87-MG   0.0
CNS cancer (glio/astro) U-118-MG 0.3
CNS cancer (neuro; met) SK-N-AS  26.8
CNS cancer (astro) SF-539        0.0
CNS cancer (astro) SNB-75        0.7
CNS cancer (glio) SNB-19         0.5
CNS cancer (glio) SF-295         0.1
Brain (Amygdala) Pool            1.6
Brain (cerebellum)               9.0
Brain (fetal)                    27.2
Brain (Hippocampus) Pool         2.0
Cerebral Cortex Pool             3.3
Brain (Substantia nigra) Pool    2.5
Brain (Thalamus) Pool            4.0
Brain (whole)                    3.0
Spinal Cord Pool                 3.3
Adrenal Gland                    0.4
Pituitary gland Pool             1.3
Salivary Gland                   0.0
Thyroid (female)                 1.3
Pancreatic ca. CAPAN2            1.5
Pancreas Pool                    1.6

[0608] CNS_neurodegeneration_v1.0 Summary: Ag6137 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals.

[0609] General_screening_panel_v1.5 Summary: Ag6137 Highest expression of this gene is detected in liver cancer cell line (CT=28.2). Moderate to low expression of this gene is detected in number of cancer cell lines derived from pancreatic, brain, colon, renal, breast, and ovarian cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation of the expression or luinction of this gene may be effective in the treatment of pancreatic, brain, colon, renal, breast, and ovarian cancers.

[0610] Among tissues with metabolic or endocrine function, this gene is expressed at low levels in pancreas, thyroid, pituitary gland, fetal heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation 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.

[0611] D. CG170791-01: Acetyl-CoA Transporter (ACATN).

[0612] Expression of gene CG 170791-01 was assessed using the primer-probe set Ag6214, described in Table DA. Results of the RTQ-PCR runs are shown in Tables DB and DC.

TABLE DA
Probe Name Ag6214
			Start	SEQ
Primers	Sequences	Length	Position	ID No
Forward	5′-gattctaactgcaaaggttacagtgtac-3′	28	996	87
Probe	TET-5′-cctttgcattgaaagccattatagaaaca-3′-TAMRA	29	1032	88
Reverse	5′-gttcctccaataagtggatcactaa-3′	25	1061	89

[0613]

TABLE DB
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag6214,
Tissue Name                    Run 257761012
AD 1 Hippo                     0.0
AD 2 Hippo                     12.2
AD 3 Hippo                     4.4
AD 4 Hippo                     4.8
AD 5 Hippo                     31.2
AD 6 Hippo                     51.8
Control 2 Hippo                44.4
Control 4 Hippo                3.8
Control (Path) 3 Hippo         0.0
AD 1 Temporal Ctx              18.9
AD 2 Temporal Ctx              12.2
AD 3 Temporal Ctx              2.4
AD 4 Temporal Ctx              4.2
AD 5 Inf Temporal Ctx          25.2
AD 5 Sup Temporal Ctx          8.8
AD 6 Inf Temporal Ctx          48.0
AD 6 Sup Temporal Ctx          54.3
Control 1 Temporal Ctx         4.1
Control 2 Temporal Ctx         24.8
Control 3 Temporal Ctx         12.5
Control 3 Temporal Ctx         3.5
Control (Path) 1 Temporal Ctx  12.3
Control (Path) 2 Temporal Ctx  100.0
Control (Path) 3 Temporal Ctx  4.0
Control (Path) 4 Temporal Ctx  11.0
AD 1 Occipital Ctx             11.0
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             6.7
AD 4 Occipital Ctx             12.6
AD 5 Occipital Ctx             21.2
AD 6 Occipital Ctx             0.0
Control 1 Occipital Ctx        0.0
Control 2 Occipital Ctx        29.5
Control 3 Occipital Ctx        11.9
Control 4 Occipital Ctx        6.6
Control (Path) 1 Occipital Ctx 27.7
Control (Path) 2 Occipital Ctx 0.0
Control (Path) 3 Occipital Ctx 3.3
Control (Path) 4 Occipital Ctx 8.6
Control 1 Parietal Ctx         6.6
Control 2 Parietal Ctx         43.5
Control 3 Parietal Ctx         0.0
Control (Path) 1 Parietal Ctx  38.2
Control (Path) 2 Parietal Ctx  12.2
Control (Path) 3 Parietal Ctx  3.2
Control (Path) 4 Parietal Ctx  23.0

[0614]

TABLE DC
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag6214,
Tissue Name                      Run 257416087
Secondary Th1 act                27.9
Secondary Th2 act                46.7
Secondary Tr1 act                9.5
Secondary Th1 rest               0.0
Secondary Th2 rest               0.0
Secondary Tr1 rest               0.0
Primary Th1 act                  0.0
Primary Th2 act                  21.8
Primary Tr1 act                  14.7
Primary Th1 rest                 0.0
Primary Th2 rest                 5.6
Primary Tr1 rest                 0.0
CD45RA CD4 lymphocyte act        39.2
CD45RO CD4 lymphocyte act        32.3
CD8 lymphocyte act               2.2
Secondary CD8 lymphocyte rest    11.4
Secondary CD8 lymphocyte act     0.0
CD4 lymphocyte none              0.0
2ry Th1/Th2/Tr1_anti-CD95 CH11   0.0
LAK cells rest                   26.2
LAK cells IL-2                   5.9
LAK cells IL-2 + IL-12           0.0
LAK cells IL-2 + IFN gamma       6.9
LAK cells IL-2 + IL-18           3.5
LAK cells PMA/ionomycin          12.2
NK Cells IL-2 rest               18.7
Two Way MLR 3 day                5.8
Two Way MLR 5 day                3.5
Two Way MLR 7 day                0.0
PBMC rest                        0.0
PBMC PWM                         4.6
PBMC PHA-L                       3.2
Ramos (B cell) none              2.5
Ramos (B cell) ionomycin         55.1
B lymphocytes PWM                13.3
B lymphocytes CD40L and IL-4     2.5
EOL-1 dbcAMP                     17.6
EOL-1 dbcAMP PMA/ionomycin       4.0
Dendritic cells none             19.3
Dendritic cells LPS              0.0
Dendritic cells anti-CD40        0.0
Monocytes rest                   5.1
Monocytes LPS                    24.8
Macrophages rest                 4.8
Macrophages LPS                  3.9
HUVEC none                       11.0
HUVEC starved                    0.0
HUVEC IL-1beta                   42.3
HUVEC IFN gamma                  20.6
HUVEC TNF alpha + IFN gamma      2.8
HUVEC TNF alpha + IL4            4.9
HUVEC IL-11                      13.8
Lung Microvascular EC none       45.1
Lung Microvascular EC TNFalpha + IL-1beta 30.8
Microvascular Dermal EC none     1.9
Microsvasular Dermal EC TNFalpha + IL-1beta 3.4
Bronchial epithelium TNFalpha + IL1beta 68.8
Small airway epithelium none     35.4
Small airway epithelium TNFalpha + IL-1beta 37.1
Coronery artery SMC rest         33.9
Coronery artery SMC TNFalpha + IL-1beta 43.8
Astrocytes rest                  5.2
Astrocytes TNFalpha + IL-1beta   11.3
KU-812 (Basophil) rest           16.4
KU-812 (Basophil) PMA/ionomycin  42.0
CCD1106 (Keratinocytes) none     24.8
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 23.5
Liver cirrhosis                  10.5
NCI-H292 none                    2.6
NCI-H292 IL-4                    26.6
NCI-H292 IL-9                    42.3
NCI-H292 IL-13                   39.2
NCI-H292 IFN gamma               12.6
HPAEC none                       9.2
HPAEC TNF alpha + IL-1 beta      100.0
Lung fibroblast none             21.0
Lung fibroblast TNF alpha + IL-1 beta 31.9
Lung fibroblast IL-4             22.5
Lung fibroblast IL-9             39.2
Lung fibroblast IL-13            0.0
Lung fibroblast IFN gamma        28.3
Dermal fibroblast CCD1070 rest   76.3
Dermal fibroblast CCD1070 TNF alpha 74.2
Dermal fibroblast CCD1070 IL-1 beta 50.7
Dermal fibroblast IFN gamma      13.0
Dermal fibroblast IL-4           36.6
Dermal Fibroblasts rest          2.9
Neutrophils TNFa + LPS           0.0
Neutrophils rest                 8.4
Colon                            0.0
Lung                             0.0
Thymus                           6.1
Kidney                           27.2

[0615] CNS_neurodegeneration_v1.0 Summary: Ag6214 Low expression of this gene is seen in temporal cortex of a control patient (CT=34.2). Therefore, therapeutic modulation of this gene or its protein product may be useful in the treatment of neurological disorder such as seizure, Alzheimer's, schizophrenia, and forgetfulness.

[0616] Panel 4.1D Summary: Ag6214 Low expression of this gene is detected mainly in TNF alpha+IL-1 beta activated HPAEC and bronchial epithelium and TNF alpha activated dermal fibroblasts. Therefore, therapeutic modulation of this gene or its protein product may be useful in the treatment of autoimmune and inflammatory disorders that include psoriasis, chronic obstructive pulmonary disease, asthma, allergy and emphysema.

[0617] E. CG171174-01: Novel Plasma Membrane Protein.

[0618] Expression of gene CG171174-01 was assessed using the primer-probe set Ag6166, described in Table EA. Results of the RTQ-PCR runs are shown in Tables EB, EC and ED.

TABLE EA
Probe Name Ag6166
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-gctgtaattaagatgagatcagtttcttag-3′	30	6838	90
Probe	TET-5′-agctttcctgaatccctctgacgttg-3′-TAMRA	26	6872	91
Reverse	5′-gaaaagacgaatcaacagaaagatc-3′	25	6903	92

[0619]

TABLE EB
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag6166,
Tissue Name                    Run 256423585
AD 1 Hippo                     14.5
AD 2 Hippo                     26.6
AD 3 Hippo                     10.2
AD 4 Hippo                     10.9
AD 5 hippo                     62.0
AD 6 Hippo                     60.7
Control 2 Hippo                19.3
Control 4 Hippo                10.0
Control (Path) 3 Hippo         4.6
AD 1 Temporal Ctx              25.7
AD 2 Temporal Ctx              26.4
AD 3 Temporal Ctx              9.3
AD 4 Temporal Ctx              23.8
AD 5 Inf Temporal Ctx          100.0
AD 5 Sup Temporal Ctx          62.0
AD 6 Inf Temporal Ctx          44.4
AD 6 Sup Temporal Ctx          57.8
Control 1 Temporal Ctx         11.2
Control 2 Temporal Ctx         37.4
Control 3 Temporal Ctx         20.0
Control 4 Temporal Ctx         10.2
Control (Path) 1 Temporal Ctx  48.3
Control (Path) 2 Temporal Ctx  32.3
Control (Path) 3 Temporal Ctx  5.6
Control (Path) 4 Temporal Ctx  33.7
AD 1 Occipital Ctx             18.9
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             10.2
AD 4 Occipital Ctx             20.6
AD 5 Occipital Ctx             19.5
AD 6 Occipital Ctx             37.6
Control 1 Occipital Ctx        5.3
Control 2 Occipital Ctx        63.7
Control 3 Occipital Ctx        22.1
Control 4 Occipital Ctx        5.4
Control (Path) 1 Occipital Ctx 61.6
Control (Path) 2 Occipital Ctx 9.9
Control (Path) 3 Occipital Ctx 5.4
Control (Path) 4 Occipital Ctx 16.0
Control 1 Parietal Ctx         14.1
Control 2 Parietal Ctx         50.7
Control 3 Parietal Ctx         18.8
Control (Path) 1 Parietal Ctx  85.3
Control (Path) 2 Parietal Ctx  22.1
Control (Path) 3 Parietal Ctx  4.1
Control (Path) 4 Parietal Ctx  37.9

[0620]

TABLE EC
General screening panel v1.5
                                 Rel. Exp.(%)
                                 Ag6166,
Tissue Name                      Run 254396196
Adipose                          4.7
Melanoma* Hs688(A).T             17.8
Melanoma* Hs688(B).T             14.7
Melanoma* M14                    18.8
Melanoma* LOXIMVI                19.1
Melanoma* SK-MEL-5               12.7
Squamous cell carcinoma SCC-4    23.0
Testis Pool                      12.6
Prostate ca.* (bone met) PC-3    25.0
Prostate Pool                    14.1
Placenta                         6.8
Uterus Pool                      7.5
Ovarian ca. OVCAR-3              12.5
Ovarian ca. SK-OV-3              41.5
Ovarian ca. OVCAR-4              9.5
Ovarian ca. OVCAR-5              43.8
Ovarian ca. IGROV-1              12.3
Ovarian ca. OVCAR-8              7.8
Ovary                            11.4
Breast ca. MCF-7                 29.5
Breast ca. MDA-MB-231            23.7
Breast ca. BT 549                29.5
Breast ca. T47D                  7.1
Breast ca. MDA-N                 8.0
Breast Pool                      19.5
Trachea                          5.8
Lung                             9.4
Fetal Lung                       24.8
Lung ca. NCI-N417                6.0
Lung ca. LX-1                    31.4
Lung ca. NCI-H146                12.9
Lung ca. SHP-77                  23.5
Lung ca. A549                    21.9
Lung ca. NCI-H526                5.9
Lung ca. NCI-H23                 23.5
Lung ca. NCI-H460                16.7
Lung ca. HOP-62                  13.1
Lung ca. NCI-H522                24.8
Liver                            0.5
Fetal Liver                      11.3
Liver ca. HepG2                  8.0
Kidney Pool                      33.0
Fetal Kidney                     19.1
Renal ca. 786-0                  13.5
Renal ca. A498                   10.7
Renal ca. ACHN                   18.8
Renal ca. UO-31                  25.9
Renal ca. TK-10                  28.5
Bladder                          14.1
Gastric ca. (liver met.) NCI-N87 36.9
Gastric ca. KATO III             35.1
Colon ca. SW-948                 8.7
Colon ca. SW480                  31.4
Colon ca.* (SW480 met) SW620     17.4
Colon ca. HT29                   6.4
Colon ca. HCT-116                27.0
Colon ca. CaCo-2                 19.6
Colon cancer tissue              7.7
Colon ca. SW1116                 5.2
Colon ca. Colo-205               2.5
Colon ca. SW-48                  6.4
Colon Pool                       24.1
Small Intestine Pool             13.4
Stomach Pool                     10.6
Bone Marrow Pool                 8.3
Fetal Heart                      9.0
Heart Pool                       7.9
Lymph Node Pool                  19.3
Fetal Skeletal Muscle            5.8
Skeletal Muscle Pool             11.7
Spleen Pool                      11.1
Thymus Pool                      14.3
CNS cancer (glio/astro) U87-MG   10.4
CNS cancer (glio/astro) U-118-MG 38.7
CNS cancer (neuro; met) SK-N-AS  22.1
CNS cancer (astro) SF-539        9.1
CNS cancer (astro) SNB-75        39.5
CNS cancer (glio) SNB-19         14.5
CNS cancer (glio) SF-295         41.5
Brain (Amygdala) Pool            7.0
Brain (cerebellum)               100.0
Brain (fetal)                    22.5
Brain (Hippocampus) Pool         9.0
Cerebral Cortex Pool             11.3
Brain (Substantia nigra) Pool    7.5
Brain (Thalamus) Pool            11.5
Brain (whole)                    11.0
Spinal Cord Pool                 6.6
Adrenal Gland                    6.7
Pituitary gland Pool             5.2
Salivary Gland                   2.5
Thyroid (female)                 5.0
Pancreatic ca. CAPAN2            40.3
Pancreas Pool                    20.6

[0621]

TABLE ED
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag6166,
Tissue Name                      Run 255171339
Secondary Th1 act                58.6
Secondary Th2 act                84.7
Secondary Tr1 act                34.6
Secondary Th1 rest               0.0
Secondary Th2 rest               2.8
Secondary Tr1 rest               1.4
Primary Th1 act                  0.0
Primary Th2 act                  70.7
Primary Tr1 act                  66.0
Primary Th1 rest                 1.6
Primary Th2 rest                 1.5
Primary Tr1 rest                 1.5
CD45RA CD4 lymphocyte act        45.7
CD45RO CD4 lymphocyte act        45.7
CD8 lymphocyte act               6.4
Secondary CD8 lymphocyte rest    27.9
Secondary CD8 lymphocyte act     1.7
CD4 lymphocyte none              1.0
2ry Th1/Th2/Tr1_anti-CD95 CH11   7.6
LAK cells rest                   12.9
LAK cells IL-2                   13.6
LAK cells IL-2 + IL-12           0.0
LAK cells IL-2 + IFN gamma       6.4
LAK cells IL-2 + IL-18           4.4
LAK cells PMA/ionomycin          31.6
NK Cells IL-2 rest               87.7
Two Way MLR 3 day                16.5
Two Way MLR 5 day                1.8
Two Way MLR 7 day                8.7
PBMC rest                        2.1
PBMC PWM                         7.0
PBMC PHA-L                       8.1
Ramos (B cell) none              4.2
Ramos (B cell) ionomycin         74.2
B lymphocytes PWM                36.3
B lymphocytes CD40L and IL-4     62.9
EOL-1 dbcAMP                     67.8
EOL-1 dbcAMP PMA/ionomycin       10.5
Dendritic cells none             14.4
Dendritic cells LPS              4.9
Dendritic cells anti-CD40        3.3
Monocytes rest                   1.2
Monocytes LPS                    89.5
Macrophages rest                 4.8
Macrophages LPS                  10.9
HUVEC none                       23.8
HUVEC starved                    59.0
HUVEC IL-1beta                   32.5
HUVEC IFN gamma                  40.9
HUVEC TNF alpha + IFN gamma      5.9
HUVEC TNF alpha + IL4            5.9
HUVEC IL-11                      18.4
Lung Microvascular EC none       85.3
Lung Microvascular EC TNFalpha + IL-1beta 48.6
Microvascular Dermal EC none     3.3
Microsvasular Dermal EC TNFalpha + IL-1beta 13.3
Bronchial epithelium TNFalpha + IL1beta 13.3
Small airway epithelium none     17.8
Small airway epithelium TNFalpha + IL-1beta 36.9
Coronery artery SMC rest         14.8
Coronary artery SMC TNFalpha + IL-1beta 15.2
Astrocytes rest                  4.7
Astrocytes TNFalpha + IL-1beta   8.1
KU-812 (Basophil) rest           52.5
KU-812 (Basophil) PMA/ionomycin  55.5
CCD1106 (Keratinocytes) none     54.3
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 30.6
Liver cirrhosis                  9.3
NCI-H292 none                    59.5
NCI-H292 IL-4                    69.7
NCI-H292 IL-9                    58.6
NCI-H292 IL-13                   66.9
NCI-H292 IFN gamma               46.7
HPAEC none                       14.4
HPAEC TNF alpha + IL-1 beta      51.1
Lung fibroblast none             30.6
Lung fibroblast TNF alpha + IL-1 beta 26.2
Lung fibroblast IL-4             20.7
Lung fibroblast IL-9             27.4
Lung fibroblast IL-13            3.6
Lung fibroblast IFN gamma        27.9
Dermal fibroblast CCD1070 rest   42.0
Dermal fibroblast CCD1070 TNF alpha 100.0
Dermal fibroblast CCD1070 IL-1 beta 51.4
Dermal fibroblast IFN gamma      16.5
Dermal fibroblast IL-4           37.9
Dermal Fibroblasts rest          13.7
Neutrophils TNFa + LPS           2.0
Neutrophils rest                 18.7
Colon                            1.6
Lung                             1.0
Thymus                           7.7
Kidney                           20.9

[0622] CNS_neurodegeneration_v1.0 Summary: Ag6166 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene is found to be slightly upregulated in the temporal cortex of Alzheimer's disease patients. Blockade of this receptor may be of use in the treatment of this disease and decrease neuronal death.

[0623] General_screening_panel_v1.5 Summary: Ag6166 Highest expression of this gene is detected in cerebellum (CT=27). 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.

[0624] Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers.

[0625] Among tissues with metabolic or endocrine function, this gene is expressed at moderate to low levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

[0626] Interestingly, this gene is expressed at much higher levels in fetal (CT=30) when compared to adult liver (CT=34.8). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of liver related diseases.

[0627] Panel 4.1D Summary: Ag6166 Highest expression of this gene is detected in TNF alpha treated dermal fibroblasts (CT=33.3). Low expression of this gene is seen in wide range of cell types of significance in the immune response in health and disease. These cells include members of the T-cell, B-cell, endothelial cell, activated monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin. Expression of this gene is upregulated in LPS stimulated monocytes, activated Ramos B cells, activated naive and memory T cells and activated polarized T cells. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

[0628] F. CG172921-01: Interleukin-5 Receptor Alpha Chain.

[0629] Expression of gene CG172921-01 was assessed using the primer-probe sets Ag5894 and Ag6187, described in Tables FA and FB. Results of the RTQ-PCR runs are shown in Tables FC and FD. Please note that CG172921-01 represents a full length physical clone.

TABLE FA
Probe Name Ag5894
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-tcctggaacctcaattgtga-3′	20	385	93
Probe	TET-5′-tgcaccacaaacactacagaagacaa-3′-TAMRA	26	413	94
Reverse	5′-gcagtgaagggaaacttggta-3′	21	458	95

[0630]

TABLE FB
Probe Name Ag6187
				SEQ
			Start	ID
Primers	Sequences	Length	Position	No
Forward	5′-ccactaactatgaggtcctctgc-3′	23	1176	96
Probe	TET-5′-atatacatcttagattcggctgacaattttctacaa-3′-TAMRA	36	1205	97
Reverse	5′-ctcactggacccagctttctt-3′	21	1244	98

[0631]

TABLE FC
AI comprehensive panel v1.0
                                 Rel. Exp.(%)
                                 Ag5894,
Tissue Name                      Run 47756588
110967 COPD-F                    2.7
110980 COPD-F                    0.0
110968 COPD-M                    2.9
110977 COPD-M                    3.5
110989 Emphysema-F               1.5
110992 Emphysema-F               2.0
110993 Emphysema-F               0.0
110994 Emphysema-F               0.0
110995 Emphysema-F               9.1
110996 Emphysema-F               2.4
110997 Asthma-M                  1.4
111001 Asthma-F                  2.2
111002 Asthma-F                  18.0
111003 Atopic Asthma-F           4.4
111004 Atopic Asthma-F           1.8
111005 Atopic Asthma-F           12.7
111006 Atopic Asthma-F           4.8
111417 Allergy-M                 12.2
112347 Allergy-M                 0.0
112349 Normal Lung-F             0.0
112357 Normal Lung-F             0.0
112354 Normal Lung-M             5.5
112374 Crohns-F                  0.0
112389 Match Control Crohns-F    0.0
112375 Crohns-F                  0.0
112732 Match Control Crohns-F    11.7
112725 Crohns-M                  0.0
112387 Match Control Crohns-M    5.1
112378 Crohns-M                  0.0
112390 Match Control Crohns-M    19.9
112726 Crohns-M                  4.9
112731 Match Control Crohns-M    5.6
112380 Ulcer Col-F               5.7
112734 Match Control Ulcer Col-F 32.1
112384 Ulcer Col-F               39.8
112737 Match Control Ulcer Col-F 12.7
112386 Ulcer Col-F               3.4
112738 Match Control Ulcer Col-F 1.0
112381 Ulcer Col-M               0.0
112735 Match Control Ulcer Col-M 0.7
112382 Ulcer Col-M               0.9
112394 Match Control Ulcer Col-M 1.6
112383 Ulcer Col-M               21.0
112736 Match Control Ulcer Col-M 0.0
112423 Psoriasis-F               2.7
112427 Match Control Psoriasis-F 3.8
112418 Psoriasis-M               1.8
112723 Match Control Psoriasis-M 0.6
112419 Psoriasis-M               2.5
112424 Match Control Psoriasis-M 1.3
112420 Psoriasis-M               15.0
112425 Match Control Psoriasis-M 10.5
104689 (MF) OA Bone-Backus       8.9
104690 (MF) Adj “Normal” Bone-Backus 4.1
104691 (MF) OA Synovium-Backus   9.2
104692 (BA) OA Cartilage-Backus  0.0
104694 (BA) OA Bone-Backus       2.6
104695 (BA) Adj “Normal” Bone-Backus 1.5
104696 (BA) OA Synovium-Backus   11.5
104700 (SS) OA Bone-Backus       13.2
104701 (SS) Adj “Normal” Bone-Backus 3.4
104702 (SS) OA Synovium-Backus   5.2
117093 OA Cartilage Rep7         10.7
112672 OA Bone5                  25.5
112673 OA Synovium5              18.0
112674 OA Synovial Fluid cells5  10.9
117100 OA Cartilage Rep14        3.2
112756 OA Bone9                  0.0
112757 OA Synovium9              0.0
112758 OA Synovial Fluid Cells9  1.3
117125 RA Cartilage Rep2         5.3
113492 Bone2 RA                  100.0
113493 Synovium2 RA              67.8
113494 Syn Fluid Cells RA        85.9
113499 Cartilage4 RA             56.6
113500 Bone4 RA                  62.0
113501 Synovium4 RA              62.9
113502 Syn Fluid Cells4 RA       34.4
113495 Cartilage3 RA             39.5
113496 Bone3 RA                  46.3
113497 Synovium3 RA              17.7
113498 Syn Fluid Cells3 RA       82.9
117106 Normal Cartilage Rep20    3.0
113663 Bone3 Normal              0.0
113664 Synovium3 Normal          0.0
113665 Syn Fluid Cells3 Normal   0.0
117107 Normal Cartilage Rep22    0.0
113667 Bone4 Normal              3.2
113668 Synovium4 Normal          3.2
113669 Syn Fluid Cells4 Normal   4.1

[0632]

TABLE FD
Panel 4.1D
	Rel. Exp. (%)	Rel. Exp. (%)
	A5894, Run	Ag6187, Run
Tissue Name	247290701	256523052
Secondary Th1 act	0.0	0.0
Secondary Th2 act	0.0	0.0
Secondary Tr1 act	0.0	0.0
Secondary Th1 rest	0.0	0.0
Secondary Th2 rest	0.0	0.0
Secondary Tr1 rest	0.0	0.0
Primary Th1 act	0.0	0.0
Primary Th2 act	0.0	0.0
Primary Tr1 act	0.0	0.0
Primary Th1 rest	0.0	0.0
Primary Th2 rest	0.0	0.0
Primary Tr1 rest	0.0	0.0
CD45RA CD4 lymphocyte act	0.3	0.0
CD45RO CD4 lymphocyte act	0.0	0.0
CD8 lymphocyte act	0.0	0.0
Secondary CD8	1.5	0.0
lymphocyte rest
Secondary CD8	0.0	0.0
lymphocyte act
CD4 lymphocyte none	0.0	0.0
2ry Th1/Th2/Tr1_anti-CD95	0.0	0.0
CH11
LAK cells rest	0.0	0.0
LAK cells IL-2	0.7	0.0
LAK cells IL-2 + IL-12	0.0	0.0
LAK cells IL-2 + IFN gamma	0.0	0.0
LAK cells IL-2 + IL-18	0.0	0.0
LAK cells PMA/ionomycin	1.3	0.0
NK Cells IL-2 rest	0.9	0.0
Two Way MLR 3 day	0.0	0.0
Two Way MLR 5 day	0.0	0.0
Two Way MLR 7 day	0.0	0.0
PBMC rest	1.8	0.0
PBMC PWM	1.6	0.0
PBMC PHA-L	0.0	0.0
Ramos (B cell) none	1.1	0.0
Ramos (B cell) ionomycin	0.0	0.0
B lymphocytes PWM	0.7	0.0
B lymphocytes CD40L and IL-4	1.5	0.0
EOL-1 dbcAMP	0.0	0.0
EOL-1 dbcAMP PMA/ionomycin	0.0	0.0
Dendritic cells none	0.8	0.0
Dendritic cells LPS	0.0	0.0
Dendritic cells anti-CD40	0.0	0.0
Monocytes rest	0.0	0.0
Monocytes LPS	0.0	0.0
Macrophages rest	0.0	0.0
Macrophages LPS	0.0	0.0
HUVEC none	0.0	0.0
HUVEC starved	0.0	0.0
HUVEC IL-1beta	0.0	0.0
HUVEC IFN gamma	0.0	0.0
HUVEC TNF alpha +	0.0	0.0
IFN gamma
HUVEC TNF alpha + IL4	1.2	0.0
HUVEC IL-11	0.0	0.0
Lung Microvascular EC none	0.0	0.0
Lung Microvascular EC	0.0	0.0
TNFalpha + IL-1beta
Microvascular	0.0	0.0
Dermal EC none
Microsvasular Dermal EC	0.0	0.0
TNFalpha + IL-1beta
Bronchial epithelium	0.0	0.0
TNFalpha + IL1beta
Small airway epithelium none	0.0	0.0
Small airway epithelium	0.0	0.0
TNFalpha + IL-1beta
Coronery artery SMC rest	0.0	0.0
Coronery artery	0.0	0.0
SMC TNFalpha + IL-1beta
Astrocytes rest	3.7	0.0
Astrocytes	0.0	0.0
TNFalpha + IL-1beta
KU-812 (Basophil) rest	2.2	0.0
KU-812 (Basophil)	0.6	0.0
PMA/ionomycin
CCD1106 (Keratinocytes)	0.0	0.0
none
CCD1106 (Keratinocytes)	0.0	0.0
TNFalpha + IL-1beta
Liver cirrhosis	1.6	0.0
NCI-H292 none	0.0	0.0
NCI-H292 IL-4	0.0	0.0
NCI-H292 IL-9	0.0	0.0
NCI-H292 IL-13	0.0	0.0
NCI-H292 IFN gamma	0.0	0.0
HPAEC none	0.0	0.0
HPAEC TNF alpha +	0.0	0.0
IL-1 beta
Lung fibroblast none	0.0	0.0
Lung fibroblast	0.0	0.0
TNF alpha + IL-1 beta
Lung fibroblast IL-4	0.0	0.0
Lung fibroblast IL-9	0.0	0.0
Lung fibroblast IL-13	0.0	0.0
Lung fibroblast IFN gamma	0.0	0.0
Dermal fibroblast	1.7	0.0
CCD1070 rest
Dermal fibroblast	0.0	0.0
CCD1070 TNF alpha
Dermal fibroblast	0.0	0.0
CCD1070 IL-1 beta
Dermal fibroblast	0.0	0.0
IFN gamma
Dermal fibroblast	0.0	0.0
IL-4
Dermal Fibroblasts rest	0.0	0.0
Neutrophils TNFa + LPS	0.0	0.0
Neutrophils rest	100.0	100.0
Colon	0.0	0.0
Lung	1.0	0.0
Thymus	0.9	0.0
Kidney	0.0	0.0

[0633] AI_comprehensive panel_v1.0 Summary: Ag5894 Highest expression is seen in a sample from RA bone (CT=30.9). Furthermore, moderate levels of expression are seen in a cluster of RA derived samples. Thus, expression of this gene could be used to differentiate RA derived samples from other samples on this panel and as a marker of RA. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of RA.

[0634] Panel 4.1D Summary: Ag5894/Ag6187 Two experiments with two different probe and primer sets produce results that are in excellent agreement. Detectable expression is limited to resting neutrophils (CTs=31-33). This expression is reduced to background level (CT=40) in neutrophils activated by TNF-alpha+LPS. This expression profile suggest that this gene is produced by resting neutrophils but not by activated neutrophils. Therefore, the gene product may reduce activation of these inflammatory cells and be useful as a protein therapeutic to reduce or eliminate the symptoms in patients with Crohn's disease, ulcerative colitis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, emphysema, rheumatoid arthritis, lupus erythematosus, or psoriasis.

[0635] G. CG176203-01: Novel Membrane Protein.

[0636] Expression of gene CG176203-01 was assessed using the primer-probe set Ag6370, described in Table GA. Results of the RTQ-PCR runs are shown in Tables GB and GC.

TABLE GA
Probe Name Ag6370
			Start	SEQ
Primers	Sequences	Length	Position	ID No
Forward	5′-agagaaagaagtttgtaggttggaatac-3′	28	351	99
Probe	TET-5′-ccaagagagaatagtttccaaattctcca-3′-TAMRA	29	384	100
Reverse	5′-caggcaatttcactaactccattat-3′	25	425	101

[0637]

TABLE GB
General screening panel v1.6
                                 Rel. Exp.(%)
                                 Ag6370,
Tissue Name                      Run 277237205
Adipose                          16.4
Melanoma* Hs688(A).T             64.2
Melanoma* Hs688(B).T             55.9
Melanoma* M14                    17.3
Melanoma* LOXIMVI                18.3
Melanoma* SK-MEL-5               30.1
Squamous cell carcinoma SCC-4    9.8
Testis Pool                      8.8
Prostate ca.* (bone met) PC-3    21.8
Prostate Pool                    16.7
Placenta                         28.1
Uterus Pool                      10.5
Ovarian ca. OVCAR-3              27.9
Ovarian ca. SK-OV-3              100.0
Ovarian ca. OVCAR-4              18.8
Ovarian ca. OVCAR-5              66.0
Ovarian ca. IGROV-1              12.7
Ovarian ca. OVCAR-8              12.9
Ovary                            37.1
Breast ca. MCF-7                 8.7
Breast ca. MDA-MB-231            36.6
Breast ca. BT 549                63.3
Breast ca. T47D                  18.7
Breast ca. MDA-N                 7.4
Breast Pool                      30.8
Trachea                          27.9
Lung                             10.2
Fetal Lung                       47.3
Lung ca. NCI-N417                2.3
Lung ca. LX-1                    10.2
Lung ca. NCI-H146                4.7
Lung ca. SHP-77                  19.2
Lung ca. A549                    47.6
Lung ca. NCI-H526                1.3
Lung ca. NCI-H23                 50.0
Lung ca. NCI-H460                24.5
Lung ca. HOP-62                  39.0
Lung ca. NCI-H522                43.5
Liver                            0.0
Fetal Liver                      32.5
Liver ca. HepG2                  6.4
Kidney Pool                      49.3
Fetal Kidney                     19.8
Renal ca. 786-0                  25.3
Renal ca. A498                   10.6
Renal ca. ACHN                   18.3
Renal ca. UO-31                  55.1
Renal ca. TK-10                  30.6
Bladder                          37.6
Gastric ca. (liver met.) NCI-N87 34.9
Gastric ca. KATO III             16.7
Colon ca. SW-948                 6.5
Colon ca. SW480                  11.8
Colon ca.* (SW480 met) SW620     5.1
Colon ca. HT29                   1.2
Colon ca. HCT-116                8.3
Colon ca. CaCo-2                 21.0
Colon cancer tissue              17.8
Colon ca. SW1116                 4.4
Colon ca. Colo-205               1.9
Colon ca. SW-48                  4.9
Colon Pool                       41.2
Small Intestine Pool             25.2
Stomach Pool                     21.0
Bone Marrow Pool                 11.1
Fetal Heart                      18.0
Heart Pool                       12.5
Lymph Node Pool                  40.9
Fetal Skeletal Muscle            12.3
Skeletal Muscle Pool             2.2
Spleen Pool                      14.8
Thymus Pool                      25.2
CNS cancer (glio/astro) U87-MG   36.1
CNS cancer (glio/astro) U-118-MG 60.3
CNS cancer (neuro; met) SK-N-AS  17.7
CNS cancer (astro) SF-539        44.1
CNS cancer (astro) SNB-75        95.3
CNS cancer (glio) SNB-19         16.3
CNS cancer (glio) SF-295         55.1
Brain (Amygdala) Pool            10.2
Brain (cerebellum)               39.2
Brain (fetal)                    39.2
Brain (Hippocampus) Pool         11.3
Cerebral Cortex Pool             14.7
Brain (Substantia nigra) Pool    9.9
Brain (Thalamus) Pool            19.3
Brain (whole)                    19.1
Spinal Cord Pool                 10.4
Adrenal Gland                    29.5
Pituitary gland Pool             7.5
Salivary Gland                   9.9
Thyroid (female)                 14.2
Pancreatic ca. CAPAN2            47.0
Pancreas Pool                    16.8

[0638]

TABLE GC
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag6370,
Tissue Name                      Run 264778106
Secondary Th1 act                7.0
Secondary Th2 act                16.5
Secondary Tr1 act                8.8
Secondary Th1 rest               1.7
Secondary Th2 rest               1.8
Secondary Tr1 rest               1.9
Primary Th1 act                  1.7
Primary Th2 act                  11.7
Primary Tr1 act                  7.0
Primary Th1 rest                 1.2
Primary Th2 rest                 2.1
Primary Tr1 rest                 1.9
CD45RA CD4 lymphocyte act        16.2
CD45RO CD4 lymphocyte act        13.9
CD8 lymphocyte act               2.5
Secondary CD8 lymphocyte rest    4.5
Secondary CD8 lymphocyte act     0.7
CD4 lymphocyte none              4.2
2ry Th1/Th2/Tr1_anti-CD95 CH11   3.9
LAK cells rest                   7.0
LAK cells IL-2                   5.9
LAK cells IL-2 + IL-12           1.1
LAK cells IL-2 + IFN gamma       3.3
LAK cells IL-2 + IL-18           3.1
LAK cells PMA/ionomycin          26.4
NK Cells IL-2 rest               21.0
Two Way MLR 3 day                9.1
Two Way MLR 5 day                1.4
Two Way MLR 7 day                22.5
PBMC rest                        3.6
PBMC PWM                         3.4
PBMC PHA-L                       5.3
Ramos (B cell) none              6.1
Ramos (B cell) ionomycin         13.3
B lymphocytes PWM                2.6
B lymphocytes CD40L and IL-4     14.0
EOL-1 dbcAMP                     29.1
EOL-1 dbcAMP PMA/ionomycin       17.9
Dendritic cells none             24.0
Dendritic cells LPS              8.2
Dendritic cells anti-CD40        12.8
Monocytes rest                   10.1
Monocytes LPS                    27.0
Macrophages rest                 6.9
Macrophages LPS                  4.9
HUVEC none                       16.5
HUVEC starved                    39.5
HUVEC IL-1beta                   28.7
HUVEC IFN gamma                  43.2
HUVEC TNF alpha + IFN gamma      17.2
HUVEC TNF alpha + IL4            16.5
HUVEC IL-11                      21.2
Lung Microvascular EC none       100.0
Lung Microvascular EC TNFalpha + IL-1beta 39.0
Microvascular Dermal EC none     13.4
Microsvasular Dermal EC TNFalpha + IL-1beta 13.2
Bronchial epithelium TNFalpha + IL1beta 11.0
Small airway epithelium none     17.3
Small airway epithelium TNFalpha + IL-1beta 35.4
Coronery artery SMC rest         21.2
Coronery artery SMC TNFalpha + IL-1beta 36.3
Astrocytes rest                  25.0
Astrocytes TNFalpha + IL-1beta   19.1
KU-812 (Basophil) rest           20.0
KU-812 (Basophil) PMA/ionomycin  31.6
CCD1106 (Keratinocytes) none     35.8
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 10.2
Liver cirrhosis                  14.1
NCI-H292 none                    42.0
NCI-H292 IL-4                    70.2
NCI-H292 IL-9                    66.4
NCI-H292 IL-13                   85.9
NCI-H292 IFN gamma               41.5
HPAEC none                       23.0
HPAEC TNF alpha + IL-1 beta      52.5
Lung fibroblast none             56.6
Lung fibroblast TNF alpha + IL-1 beta 43.5
Lung fibroblast IL-4             20.7
Lung fibroblast IL-9             31.9
Lung fibroblast IL-13            7.2
Lung fibroblast IFN gamma        79.0
Dermal fibroblast CCD1070 rest   31.6
Dermal fibroblast CCD1070 TNF alpha 74.2
Dermal fibroblast CCD1070 IL-1 beta 29.7
Dermal fibroblast IFN gamma      37.4
Dermal fibroblast IL-4           53.6
Dermal Fibroblasts rest          33.4
Neutrophils TNFa + LPS           2.0
Neutrophils rest                 13.9
Colon                            0.9
Lung                             2.9
Thymus                           1.9
Kidney                           27.7

[0639] General_screening_panel_v1.6 Summary: Ag6370 Highest expression of this gene is detected in ovarian cancer SK-OV-3 cell line (CT=27.7). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation 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.

[0640] Among tissues with metabolic or endocrine function, this gene is expressed at moderate levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, fetal liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

[0641] 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.

[0642] Interestingly, this gene is expressed at much higher levels in fetal (CT=29.3) when compared to adult liver (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of liver related diseases.

[0643] Panel 4.1D Summary: Ag6370 Highest expression of this gene is detected in lung microvascular endothelial cells (CT=29.3). This gene is expressed at moderate to low levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members of the T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by lung and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_v1.6 and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

[0644] H. CG50691-02: CRIM1 Homolog

[0645] Expression of gene CG50691-02 was assessed using the primer-probe sets Ag155 and Ag7290, described in Tables HA and HB. Results of the RTQ-PCR runs are shown in Tables HC, HD, HE, HF and HG.

TABLE HA
Probe Name Ag155
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-tcaaacgcgatcacaatggt-3′	20	1496	102
Probe	TET-5′-tcggacctgtcagtgcataaacaccg-3′-TAMRA	26	1518	103
Reverse	5′-gccttgtttacgttctgaacatagtt-3′	26	1547	104

[0646]

TABLE HB
Probe Name Ag7290
			Start	SEQ
Primers	Sequences	Length	Position	ID No
Forward	5′-gtcttgagaaaaggccagtgtt-3′	22	2431	105
Probe	TET-5′-tccctactgcatagaaatgtatgtccca-3′-TAMRA	28	2454	106
Reverse	5′-ctcaatgggtatattggttggtt-3′	23	2483	107

[0647]

TABLE HC
Ardais Prostate 1.0
                            Rel. Exp.(%)
                            Ag7290,
Tissue Name                 Run 321632647
145904_Prostate cancer(9E2) 29.7
149776_Prostate cancer(AD5) 41.8
151135_Prostate NAT(B87)    26.6
151143_Prostate NAT(B8A)    53.2
153653_Prostate cancer(D4E) 31.2
153661_Prostate cancer(D56) 18.7
153669_Prostate NAT(D5E)    44.4
153677_Prostate NAT(D66)    13.6
153685_Prostate NAT(D6E)    22.7
145905_Prostate NAT(A0C)    33.7
151128_Prostate cancer(B8C) 40.6
151136_Prostate cancer(B8B) 44.8
151144_Prostate cancer(B8F) 78.5
153654_Prostate cancer(D4F) 11.2
153662_Prostate cancer(D57) 58.2
153670_Prostate NAT(D5F)    34.6
153678_Prostate NAT(D67)    43.8
153686_Prostate NAT(D6F)    21.8
145906_Prostate NAT(A09)    38.7
151129_Prostate NAT(B93)    72.7
151137_Prostate NAT(B86)    100.0
151145_Prostate NAT(B91)    54.7
153655_Prostate cancer(D50) 54.3
153663_Prostate cancer(D58) 87.7
153671_Prostate NAT(D60)    45.1
153679_Prostate NAT(D68)    42.0
153687_Prostate NAT(D70)    23.0
145907_Prostate cancer(A0A) 46.3
151130_Prostate cancer(B90) 43.5
151138_Prostate cancer(B8D) 18.7
153648_Prostate cancer(D49) 25.5
153656_Prostate cancer(D51) 10.7
153664_Prostate cancer(D59) 5.4
153672_Prostate NAT(D61)    27.2
153680_Prostate NAT(D69)    28.1
155799_Prostate cancer(EA8) 18.7
145909_Prostate cancer(9E7) 9.0
151131_Prostate NAT(B85)    32.3
151139_Prostate NAT(B8E)    46.7
153649_Prostate cancer(D4A) 18.8
153657_Prostate cancer(D52) 14.0
153665_Prostate cancer(D5A) 17.0
153673_Prostate NAT(D62)    23.2
153681_Prostate NAT(D6A)    15.8
145910_Prostate NAT(9C3)    21.0
151132_Prostate cancer(B88) 42.3
151140_Prostate cancer(B95) 23.0
153650_Prostate cancer(D4B) 15.1
153658_Prostate cancer(D53) 31.9
153666_Prostate cancer(D5B) 31.6
153674_Prostate NAT(D63)    55.9
153682_Prostate NAT(D6B)    32.8
149773_Prostate NAT(AD8)    0.0
151133_Prostate NAT(B94)    56.3
151141_Prostate NAT(B96)    40.9
153651_Prostate cancer(D4C) 35.4
153659_Prostate cancer(D54) 52.1
153667_Prostate cancer(D5C) 76.3
153675_Prostate NAT(D64)    21.3
153683_Prostate NAT(D6C)    42.0
149774_Prostate cancer(AD7) 22.1
151134_Prostate cancer(B92) 26.4
151142_Prostate cancer(B89) 25.7
153652_Prostate cancer(D4D) 24.8
153660_Prostate cancer(D55) 17.1
153668_Prostate NAT(D5D)    94.6
153676_Prostate NAT(D65)    37.4
153684_Prostate NAT(D6D)    40.1

[0648]

TABLE HD
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag7290
Tissue Name                    Run 298103647
AD 1 Hippo                     7.5
AD 2 Hippo                     13.6
AD 3 Hippo                     8.8
AD 4 Hippo                     3.7
AD 5 hippo                     54.3
AD 6 Hippo                     22.7
Control 2 Hippo                16.2
Control 4 Hippo                7.2
Control (Path) 3 Hippo         11.0
AD 1 Temporal Ctx              13.7
AD 2 Temporal Ctx              22.8
AD 3 Temporal Ctx              8.5
AD 4 Temporal Ctx              13.3
AD 5 Inf Temporal Ctx          100.0
AD 5 SupTemporal Ctx           39.0
AD 6 Inf Temporal Ctx          23.0
AD 6 Sup Temporal Ctx          31.6
Control 1 Temporal Ctx         9.7
Control 2 Temporal Ctx         25.0
Control 3 Temporal Ctx         9.3
Control 4 Temporal Ctx         5.7
Control (Path) 1 Temporal Ctx  37.1
Control (Path) 2 Temporal Ctx  26.1
Control (Path) 3 Temporal Ctx  4.4
Control (Path) 4 Temporal Ctx  24.0
AD 1 Occipital Ctx             14.2
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             9.3
AD 4 Occipital Ctx             10.1
AD 5 Occipital Ctx             12.9
AD 6 Occipital Ctx             26.6
Control 1 Occipital Ctx        5.8
Control 2 Occipital Ctx        21.3
Control 3 Occipital Ctx        11.3
Control 4 Occipital Ctx        6.0
Control (Path) 1 Occipital Ctx 39.2
Control (Path) 2 Occipital Ctx 11.3
Control (Path) 3 Occipital Ctx 5.3
Control (Path) 4 Occipital Ctx 14.0
Control 1 Parietal Ctx         6.1
Control 2 Parietal Ctx         49.3
Control 3 Parietal Ctx         8.5
Control (Path) 1 Parietal Ctx  45.7
Control (Path) 2 Parietal Ctx  19.3
Control (Path) 3 Parietal Ctx  3.8
Control (Path) 4 Parietal Ctx  31.4

[0649]

TABLE HE
General screening panel v1.7
                                 Rel. Exp.(%)
                                 Ag7290,
Tissue Name                      Run 318350141
Adipose                          16.5
HUVEC                            95.9
Melanoma* Hs688(A).T             0.1
Melanoma* Hs688(B).T             29.3
Melanoma (met) SK-MEL-5          0.0
Testis                           2.0
Prostate ca. (bone met) PC-3     0.8
Prostate ca. DU145               13.7
Prostate pool                    1.8
Uterus pool                      1.3
Ovarian ca. OVCAR-3              19.8
Ovarian ca. (ascites) SK-OV-3    1.0
Ovarian ca. OVCAR-4              100.0
Ovarian ca. OVCAR-5              24.8
Ovarian ca. IGROV-1              29.7
Ovarian ca. OVCAR-8              27.9
Ovary                            14.8
Breast ca. MCF-7                 3.2
Breast ca. MDA-MB-231            85.3
Breast ca. BT-549                17.7
Breast ca. T47D                  4.7
Breast pool                      1.8
Trachea                          11.0
Lung                             30.1
Fetal Lung                       16.7
Lung ca. NCI-N417                0.1
Lung ca. LX-1                    0.0
Lung ca. NCI-H146                0.5
Lung ca. SHP-77                  0.0
Lung ca. NCI-H23                 28.5
Lung ca. NCI-H460                46.0
Lung ca. HOP-62                  27.5
Lung ca. NCI-H522                15.6
Lung ca. DMS-114                 8.0
Liver                            1.1
Fetal Liver                      1.7
Kidney pool                      50.0
Fetal Kidney                     6.3
Renal ca. 786-0                  20.9
Renal ca. A498                   48.0
Renal ca. ACHN                   49.3
Renal ca. UO-31                  28.7
Renal ca. TK-10                  25.0
Bladder                          15.7
Gastric ca. (liver met.) NCI-N87 0.5
Stomach                          0.6
Colon ca. SW-948                 5.8
Colon ca. SW480                  2.7
Colon ca. (SW480 met) SW620      1.0
Colon ca. HT29                   11.0
Colon ca. HCT-116                16.6
Colon cancer tissue              0.6
Colon ca. SW1116                 1.3
Colon ca. Colo-205               2.2
Colon ca. SW-48                  3.1
Colon                            7.4
Small Intestine                  2.1
Fetal Heart                      19.8
Heart                            3.1
Lymph Node pool 1                2.4
Lymph Node pool 2                14.3
Fetal Skeletal Muscle            2.3
Skeletal Muscle pool             2.5
Skeletal Muscle                  26.4
Spleen                           9.3
Thymus                           1.2
CNS cancer (glio/astro) SF-268   37.4
CNS cancer (glio/astro) T98G     13.6
CNS cancer (neuro; met) SK-N-AS  0.7
CNS cancer (astro) SF-539        5.2
CNS cancer (astro) SNB-75        15.4
CNS cancer (glio) SNB-19         37.9
CNS cancer (glio) SF-295         10.0
Brain (Amygdala)                 6.0
Brain (Cerebellum)               9.4
Brain (Fetal)                    21.5
Brain (Hippocampus)              8.1
Cerebral Cortex pool             5.1
Brain (Substantia nigra)         2.4
Brain (Thalamus)                 7.3
Brain (Whole)                    21.5
Spinal Cord                      1.3
Adrenal Gland                    5.9
Pituitary Gland                  1.9
Salivary Gland                   4.7
Thyroid                          13.8
Pancreatic ca. PANC-1            21.3
Pancreas pool                    1.0

[0650]

TABLE HF
Panel 1
Rel. Exp.(%)
Ag155,
Run 87589113
Endothelial cells                14.6
Endothelial cells (treated)      20.0
Pancreas                         3.5
Pancreatic ca. CAPAN 2           1.4
Adrenal gland                    2.5
Thyroid                          6.0
Salivary gland                   4.5
Pituitary gland                  0.3
Brain (fetal)                    0.3
Brain (whole)                    13.9
Brain (amygdala)                 3.1
Brain (cerebellum)               20.4
Brain (hippocampus)              2.1
Brain (substantia nigra)         0.6
Brain (thalamus)                 1.2
Brain (hypothalamus)             0.0
Spinal cord                      1.0
glio/astro U87-MG                3.9
glio/astro U-118-MG              2.6
astrocytoma SW1783               15.6
neuro*; met SK-N-AS              0.6
astrocytoma SF-539               0.0
astrocytoma SNB-75               12.6
glioma SNB-19                    19.8
glioma U251                      1.9
glioma SF-295                    0.0
Heart                            3.8
Skeletal muscle                  6.4
Bone marrow                      0.0
Thymus                           6.7
Spleen                           2.0
Lymph node                       2.0
Colon (ascending)                4.2
Stomach                          6.0
Small intestine                  2.2
Colon ca. SW480                  0.0
Colon ca.* SW620 (SW480 met)     0.0
Colon ca. HT29                   0.0
Colon ca. HCT-116                0.0
Colon ca. CaCo-2                 14.6
Colon ca. HCT-15                 1.4
Colon ca. HCC-2998               0.0
Gastric ca. * (liver met) NCI-N87 3.8
Bladder                          9.7
Trachea                          1.1
Kidney                           13.3
Kidney (fetal)                   10.9
Renal ca. 786-0                  26.8
Renal ca. A498                   18.3
Renal ca. RXF 393                0.3
Renal ca. ACHN                   20.6
Renal ca. UO-31                  12.4
Renal ca. TK-10                  11.2
Liver                            0.0
Liver (fetal)                    0.0
Liver ca. (hepatoblast) HepG2    12.7
Lung                             8.7
Lung (fetal)                     3.0
Lung ca. (small cell) LX-1       0.0
Lung ca. (small cell) NCI-H69    0.0
Lung ca. (s. cell var.) SHP-77   0.0
Lung ca. (large cell)NCI-H460    0.0
Lung ca. (non-sm. cell) A549     15.9
Lung ca. (non-s. cell) NCI-H23   6.8
Lung ca. (non-s. cell) HOP-62    7.3
Lung ca. (non-s. cl) NCI-H522    16.6
Lung ca. (squam.) SW 900         6.5
Lung ca. (squam.) NCI-H596       0.0
Mammary gland                    35.1
Breast ca.* (pl. ef) MCF-7       2.0
Breast ca.* (pl. ef) MDA-MB-231  3.7
Breast ca.* (pl. ef) T47D        16.2
Breast ca. BT-549                0.0
Breast ca. MDA-N                 0.4
Ovary                            9.6
Ovarian ca. OVCAR-3              8.4
Ovarian ca. OVCAR-4              10.3
Ovarian ca. OVCAR-5              51.4
Ovarian ca. OVCAR-8              13.9
Ovarian ca. IGROV-1              0.0
Ovarian ca. (ascites) SK-OV-3    5.7
Uterus                           19.5
Placenta                         100.0
Prostate                         1.0
Prostate ca.* (bone met) PC-3    0.0
Testis                           29.1
Melanoma Hs688(A).T              19.2
Melanoma* (met) Hs688(B).T       20.2
Melanoma UACC-62                 0.0
Melanoma M14                     0.0
Melanoma LOX IMVI                38.4
Melanoma* (met) SK-MEL-5         0.0
Melanoma SK-MEL-28               0.0

[0651]

TABLE HG
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag7290,
Tissue Name                      Run 298118747
Secondary Th1 act                6.2
Secondary Th2 act                5.0
Secondary Tr1 act                2.0
Secondary Th1 rest               0.0
Secondary Th2 rest               0.8
Secondary Tr1 rest               0.6
Primary Th1 act                  1.0
Primary Th2 act                  4.5
Primary Tr1 act                  3.4
Primary Th1 rest                 0.0
Primary Th2 rest                 0.0
Primary Tr1 rest                 0.4
CD45RA CD4 lymphocyte act        31.2
CD45RO CD4 lymphocyte act        0.7
CD8 lymphocyte act               0.5
Secondary CD8 lymphocyte rest    0.0
Secondary CD8 lymphocyte act     1.6
CD4 lymphocyte none              0.0
2ry Th1/Th2/Tr1_anti-CD95 CH11   0.2
LAK cells rest                   1.0
LAK cells IL-2                   0.6
LAK cells IL-2 + IL-12           0.0
LAK cells IL-2 + IFN gamma       0.7
LAK cells IL-2 + IL-18           1.2
LAK cells PMA/ionomycin          1.7
NK Cells IL-2 rest               3.6
Two Way MLR 3 day                0.5
Two Way MLR 5 day                0.3
Two Way MLR 7 day                0.2
PBMC rest                        0.0
PBMC PWM                         0.0
PBMC PHA-L                       0.2
Ramos (B cell) none              0.2
Ramos (B cell) ionomycin         0.2
B lymphocytes PWM                0.0
B lymphocytes CD40L and IL-4     1.5
EOL-1 dbcAMP                     0.5
EOL-1 dbcAMP PMA/ionomycin       0.5
Dendritic cells none             0.5
Dendritic cells LPS              0.6
Dendritic cells anti-CD40        0.7
Monocytes rest                   0.0
Monocytes LPS                    14.6
Macrophages rest                 0.5
Macrophages LPS                  0.4
HUVEC none                       27.5
HUVEC starved                    49.3
HUVEC IL-1beta                   100.0
HUVEC IFN gamma                  74.7
HUVEC TNF alpha + IFN gamma      26.4
HUVEC TNF alpha + IL4            13.9
HUVEC IL-11                      25.0
Lung Microvascular EC none       93.3
Lung Microvascular EC TNFalpha + IL-1beta 54.7
Microvascular Dermal EC none     8.8
Microsvasular Dermal EC TNFalpha + IL-1beta 15.5
Bronchial epithelium TNFalpha + IL1beta 1.1
Small airway epithelium none     2.0
Small airway epithelium TNFalpha + IL-1beta 10.4
Coronery artery SMC rest         52.9
Coronery artery SMC TNFalpha + IL-1beta 26.6
Astrocytes rest                  6.7
Astrocytes TNFalpha + IL-1beta   10.2
KU-812 (Basophil) rest           0.0
KU-812 (Basophil) PMA/ionomycin  0.7
CCD1106 (Keratinocytes) none     7.4
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 0.0
Liver cirrhosis                  2.6
NCI-H292 none                    10.0
NCI-H292 IL-4                    22.7
NCI-H292 IL-9                    27.7
NCI-H292 IL-13                   21.3
NCI-H292 IFN gamma               8.0
HPAEC none                       14.1
HPAEC TNF alpha + IL-1 beta      74.2
Lung fibroblast none             39.2
Lung fibroblast TNF alpha + IL-1 beta 11.3
Lung fibroblast IL-4             6.0
Lung fibroblast IL-9             23.3
Lung fibroblast IL-13            14.3
Lung fibroblast IFN gamma        43.5
Dermal fibroblast CCD1070 rest   74.2
Dermal fibroblast CCD1070 TNF alpha 87.7
Dermal fibroblast CCD1070 IL-1 beta 57.8
Dermal fibroblast IFN gamma      11.1
Dermal fibroblast IL-4           10.5
Dermal Fibroblasts rest          11.0
Neutrophils TNFa + LPS           0.0
Neutrophils rest                 0.0
Colon                            0.5
Lung                             1.1
Thymus                           0.6
Kidney                           13.3

[0652] Ardais Prostate 1.0 Summary: Ag7290 Highest expression of this gene, which encodes a putative CRIM1 protein, is seen in normal tissue adjacent to a prostate carcinoma (CT=29.9). In addition, this gene is widely expressed at low levels in most of the samples on this panel, suggesting a role for this protein in cell survival and/or proliferation.

[0653] CNS_neurodegeneration_v1.0 Summary: Ag7290 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at low levels in the brain. Please see Panel 1.7 for discussion of this gene in the central nervous system.

[0654] General_screening_panel_v1.7 Summary: Ag7290 Highest expression of this gene is seen in an ovarian cancer cell line (CT=26.2). This gene is widely expressed in this panel, with moderate to high expression seen in brain, colon, gastric, lung, breast, ovarian, prostate and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. This gene encodes a novel protein with homology to Cysteine-Rich Motor Neuron Protein 1 (CRIM1)which may interact with growth factors implicated in motor neuron differentiation and survival (Kolle, G.; Georgas, K.; Holmes, G. P.; Little, M. H.; Yamada, T.: CRIM1, a novel gene encoding a cysteine-rich repeat protein, is developmentally regulated and implicated in vertebrate CNS development and organogenesis. Mech. Dev. 90: 181-193, 2000. PubMed ID: 10642437).

[0655] The Crim1 gene encodes a putative transmembrane protein with an IGF-binding protein motif and multiple VWFC domain, analogous to those of chordin and short gastrulation (sog) proteins that associate with TGFbeta superfamily members, namely Bone Morphogenic Protein (BMP). In chordin, such repeats are responsible for its dorsalising activity and for binding to bone morphogenic proteins (BMPs). Chordin is a BMP antagonist that dorsalizes early vertebrate embryonic tissues by binding to ventralizing TGF-beta-like bone morphogenetic proteins and sequestering them in latent complexes. Scott et al. (Scott, I. C.; Blitz, I. L.; Pappano, W. N.; Imamura, Y.; Clark, T. G.; Steiglitz, B. M.; Thomas, C. L.; Maas, S. A.; Takahara, K.; Cho, K. W. Y.; Greenspan, D. S.: Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 213: 283-300, 1999.) showed that BMP1 and TLL1 counteracted the dorsalizing effects of chordin upon overexpression in Xenopus embryos. They suggested that BMP1 is the major chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis. It also directly binds BMP-4 and BMP-2, and interferes with the binding to the receptors. Bone metastases are a frequent clinical problem in patients with breast, prostate, and other cancers. Formation of these lesions is a site-specific process determined by multiple cellular and molecular interactions between the cancer cells and the bone microenvironment. Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumors. The overexpression of BMP2, BMP4, and BMP6 were found in most of osteosarcoma or prostate cancer with metastases (Guo, W., Gorlick, R., Ladanyi, M., Meyers, P A., Huvos, A G., Bertino, J R., and Healey, J H., 1999, Expression of bone morphogenetic proteins and receptors in sarcomas. Clinical Orthopaedics and Related Research 365: 175-183; Hamdy, F., Autzen, P., Robinson, M C., Wilson Home, C H., Neal, D E. and Robson C N., 1997, Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue. Cancer Research 57: 4427-4431) suggesting a close association between BMPs and skeletal metastases. BMP-2, -4, -6 may be responsible, in part, for osteoblastic changes in metastatic lesions secondary to prostate cancer.

[0656] Kolle et al. (2000) proposed that CRIM1 might be involved in motor neuron survival by interacting with various growth factors. Georgas K et al. (Georgas K, Bowles J, Yamada T, Koopman P, Little M H, 2000, Characterisation of Crim1 expression in the developing mouse urogenital tract reveals a sexually dimorphic gonadal expression pattern. Dev Dyn 219(4):582-7) have demonstrated that Crim1 also displays a striking male-specific expression pattern in the fetal gonads, its expression strongest in the Sertoli cells of the developing testis.

[0657] CG50691-02 and CG50691-03 (see below) represent two novel splice variants of CRIM1 with deletion of internal 58 aa (exon 2) or 65 aa (exon 14). The encoded protein either contains all the characteristic domains as the parent CRIM1 or only lacks one of the VWFC cysteine-rich repeat domains. Therefore, it is anticipated the splice variants may have similar or altered biological functions as the parent protein that could be involved in the BMP pathway during organogenesis or cancer development. Modulation of this gene product may therefore be useful in the treatment of cancer and in particular the cancers listed above, including prostate cancer.

[0658] Among tissues with metabolic function, this gene is expressed at moderate levels in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

[0659] This gene is also expressed at moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

[0660] In addition, this gene is expressed at much higher levels in kidney (CT=27) when compared to expression in the fetal counterpart (CT=30). Thus, expression of this gene may be used to differentiate between the fetal and adult source of this tissue.

[0661] Panel 1 Summary: Ag155 Highest expression is seen in the placenta (CT=20.74). Overall, the expression profile agrees with the results for Panel 1.7. Please see that panel for discussion of this gene.

[0662] Panel 4.1D Summary: Ag7290 Highest expression is seen in an IL1-b treated sample of HUVECs (CT=30.2). Overall, this transcript is expressed at moderate levels in endothelial cells, including samples derived from HPAEC, HUVEC and lung and dermal microvascular EC. Fibroblasts also express this transcript. Therapies designed with the protein encoded by this transcript could be important in regulating endothelium function including leukocyte extravasation, a major component of inflammation during asthma, IBD, and psoriasis.

[0663] I. CG50691-03: CRIM1 Homolog.

[0664] Expression of gene CG50691-03 was assessed using the primer-probe sets Ag155 and Ag7303, described in Tables IA and IB. Results of the RTQ-PCR runs are shown in Tables IC, ID, IE and IF.

TABLE IA
Probe Name Ag155
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-tcaaacgcgatcacaatggt3′	20	1322	108
Probe	TET-5′-tcggacctgtcagtgcataaacaccg-3′-TAMRA	26	1344	109
Reverse	5′-gccttgtttacgttctgaacatagtt-3′	26	1373	110

[0665]

TABLE IB
Probe Name Ag7303
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-cgtttgcgaagaagagaagc-3′	20	360	111
Probe	TET-5′-cccgctgtgaagtccagttctctcca-3′-TAMRA	26	395	112
Reverse	5′-agcataaccctcgatcagaac3′	21	439	113

[0666]

TABLE IC
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag7303
Tissue Name                    Run 298103650
AD 1 Hippo                     5.2
AD 2 Hippo                     12.6
AD 3 Hippo                     6.7
AD 4 Hippo                     8.6
AD 5 Hippo                     83.5
AD 6 Hippo                     42.9
Control 2 Hippo                22.7
Control 4 Hippo                18.8
Control (Path) 3 Hippo         13.3
AD 1 Temporal Ctx              20.9
AD 2 Temporal Ctx              26.2
AD 3 Temporal Ctx              12.5
AD 4 Temporal Ctx              13.4
AD 5 Inf Temporal Ctx          100.0
AD 5 Sup Temporal Ctx          52.1
AD 6 Inf Temporal Ctx          76.3
AD 6 Sup Temporal Ctx          88.3
Control 1 Temporal Ctx         12.2
Control 2 Temporal Ctx         32.1
Control 3 Temporal Ctx         25.0
Control 3 Temporal Ctx         12.6
Control (Path) 1 Temporal Ctx  56.6
Control (Path) 2 Temporal Ctx  33.7
Control (Path) 3 Temporal Ctx  7.1
Control (Path) 4 Temporal Ctx  34.9
AD 1 Occipital Ctx             13.1
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             11.3
AD 4 Occipital Ctx             9.2
AD 5 Occipital Ctx             35.4
AD 6 Occipital Ctx             55.1
Control 1 Occipital Ctx        10.1
Control 2 Occipital Ctx        30.1
Control 3 Occipital Ctx        20.9
Control 4 Occipital Ctx        13.2
Control (Path) 1 Occipital Ctx 47.0
Control (Path) 2 Occipital Ctx 12.5
Control (Path) 3 Occipital Ctx 6.7
Control (Path) 4 Occipital Ctx 17.9
Control 1 Parietal Ctx         13.7
Control 2 Parietal Ctx         60.7
Control 3 Parietal Ctx         8.1
Control (Path) 1 Parietal Ctx  30.8
Control (Path) 2 Parietal Ctx  26.6
Control (Path) 3 Parietal Ctx  22.2
Control (Path) 4 Parietal Ctx  17.7

[0667]

TABLE ID
General screening panel v1.7
                                 Rel. Exp.(%)
                                 Ag7303,
Tissue Name                      Run 318350155
Adipose                          22.2
HUVEC                            39.5
Melanoma* Hs688(A).T             0.1
Melanoma* Hs688(B).T             22.2
Melanoma (met) SK-MEL-5          0.1
Testis                           2.5
Prostate ca. (bone met) PC-3     1.6
Prostate ca. DU145               17.1
Prostate pool                    1.9
Uterus pool                      1.6
Ovarian ca. OVCAR-3              10.4
Ovarian ca. (ascites) SK-OV-3    2.2
Ovarian ca. OVCAR-4              49.7
Ovarian ca. OVCAR-5              30.4
Ovarian ca. IGROV-1              26.1
Ovarian ca. OVCAR-8              40.1
Ovary                            9.0
Breast ca. MCF-7                 1.9
Breast ca. MDA-MB-231            62.4
Breast ca. BT-549                13.4
Breast ca. T47D                  5.7
Breast pool                      0.0
Trachea                          16.2
Lung                             37.6
Fetal Lung                       10.7
Lung ca. NCI-N417
Lung ca. LX-1                    0.0
Lung ca. NCI-H146                0.2
Lung ca. SHP-77                  0.0
Lung ca. NCI-H23                 20.0
Lung ca. NCI-H460                25.0
Lung ca. HOP-62                  28.3
Lung ca. NCI-H522                21.5
Lung ca. DMS-114                 10.0
Liver                            1.3
Fetal Liver                      2.0
Kidney pool                      12.2
Fetal Kidney                     6.5
Renal ca. 786-0                  13.4
Renal ca. A498                   28.5
Renal ca. ACHN                   100.0
Renal ca. UO-31                  44.1
Renal ca. TK-10                  10.1
Bladder                          9.8
Gastric ca. (liver met.) NCI-N87 0.7
Stomach                          0.2
Colon ca. SW-948                 2.4
Colon ca. SW480                  1.0
Colon ca. (SW480 met) SW620      0.4
Colon ca. HT29                   6.7
Colon ca. HCT-116                8.1
Colon cancer tissue              0.6
Colon ca. SW1116                 1.0
Colon ca. Colo-205               1.0
Colon ca. SW-48                  1.2
Colon                            2.7
Small Intestine                  1.0
Fetal Heart                      4.4
Heart                            1.4
Lymph Node pool 1                1.5
Lymph Node pool 2                10.1
Fetal Skeletal Muscle            1.9
Skeletal Muscle pool             1.2
Skeletal Muscle                  10.6
Spleen                           4.4
Thymus                           1.4
CNS cancer (glio/astro) SF-268   30.8
CNS cancer (glio/astro) T98G     10.9
CNS cancer (neuro; met) SK-N-AS  0.7
CNS cancer (astro) SF-539        4.7
CNS cancer (astro) SNB-75        12.8
CNS cancer (glio) SNB-19         36.6
CNS cancer (glio) SF-295         5.5
Brain (Amygdala)                 3.4
Brain (Cerebellum)               6.1
Brain (Fetal)                    10.2
Brain (Hippocampus)              2.4
Cerebral Cortex pool             1.7
Brain (Substantia nigra)         1.6
Brain (Thalamus)                 2.5
Brain (Whole)                    12.7
Spinal Cord                      1.2
Adrenal Gland                    4.8
Pituitary Gland                  1.2
Salivary Gland                   4.3
Thyroid                          9.6
Pancreatic ca. PANC-1            23.5
Pancreas pool                    1.0

[0668]

TABLE IE
Panel 1
Rel. Exp.(%)
Ag155,
Run 87589113
Endothelial cells                14.6
Endothelial cells (treated)      20.0
Pancreas                         3.5
Pancreatic ca. CAPAN 2           1.4
Adrenal gland                    2.5
Thyroid                          6.0
Salivary gland                   4.5
Pituitary gland                  0.3
Brain (fetal)                    0.3
Brain (whole)                    13.9
Brain (amygdala)                 3.1
Brain (cerebellum)               20.4
Brain (hippocampus)              2.1
Brain (substantia nigra)         0.6
Brain (thalamus)                 1.2
Brain (hypothalamus)             0.0
Spinal cord                      1.0
glio/astro U87-MG                3.9
glio/astro U-118-MG              2.6
astrocytoma SW1783               15.6
neuro*; met SK-N-AS              0.6
astrocytoma SF-539               0.0
astrocytoma SNB-75               12.6
glioma SNB-19                    19.8
glioma U251                      1.9
glioma SF-295                    0.0
Heart                            3.8
Skeletal muscle                  6.4
Bone marrow                      0.0
Thymus                           6.7
Spleen                           2.0
Lymph node                       2.0
Colon (ascending)                4.2
Stomach                          6.0
Small intestine                  2.2
Colon ca. SW480                  0.0
Colon ca.* SW620 (SW480 met)     0.0
Colon ca. HT29                   0.0
Colon ca. HCT-116                0.0
Colon ca. CaCo-2                 14.6
Colon ca. HCT-15                 1.4
Colon ca. HCC-2998               0.0
Gastric ca. * (liver met) NCI-N87 3.8
Bladder                          9.7
Trachea                          1.1
Kidney                           13.3
Kidney (fetal)                   10.9
Renal ca. 786-0                  26.8
Renal ca. A498                   18.3
Renal ca. RXF 393                0.3
Renal ca. ACHN                   20.6
Renal ca. UO-31                  12.4
Renal ca. TK-10                  11.2
Liver                            0.0
Liver (fetal)                    0.0
Liver ca. (hepatoblast) HepG2    12.7
Lung                             8.7
Lung (fetal)                     3.0
Lung ca. (small cell) LX-1       0.0
Lung ca. (small cell) NCI-H69    0.0
Lung ca. (s. cell var.) SHP-77   0.0
Lung ca. (large cell)NCI-H460    0.0
Lung ca. (non-sm. cell) A549     15.9
Lung ca. (non-s. cell) NCI-H23   6.8
Lung ca. (non-s. cell) HOP-62    7.3
Lung ca. (non-s. cl) NCI-H522    16.6
Lung ca. (squam.) SW 900         6.5
Lung ca. (squam.) NCI-H596       0.0
Mammary gland                    35.1
Breast ca.* (pl. ef) MCF-7       2.0
Breast ca.* (pl. ef) MDA-MB-231  3.7
Breast ca.* (pl. ef) T47D        16.2
Breast ca. BT-549                0.0
Breast ca. MDA-N                 0.4
Ovary                            9.6
Ovarian ca. OVCAR-3              8.4
Ovarian ca. OVCAR-4              10.3
Ovarian ca. OVCAR-5              51.4
Ovarian ca. OVCAR-8              13.9
Ovarian ca. IGROV-1              0.0
Ovarian ca. (ascites) SK-OV-3    5.7
Uterus                           19.5
Placenta                         100.0
Prostate                         1.0
Prostate ca.* (bone met) PC-3    0.0
Testis                           29.1
Melanoma Hs688(A).T              19.2
Melanoma* (met) Hs688(B).T       20.2
Melanoma UACC-62                 0.0
Melanoma M14                     0.0
Melanoma LOX IMVI                38.4
Melanoma* (met) SK-MEL-5         0.0
Melanoma SK-MEL-28               0.0

[0669]

TABLE IF
Panel 4.1D
                                 Rel. Exp.(%)
                                 Ag7303,
                                 Run 298129632
Secondary Th1 act                1.7
Secondary Th2 act                1.4
Secondary Tr1 act                0.9
Secondary Th1 rest               0.0
Secondary Th2 rest               0.3
Secondary Tr1 rest               0.1
Primary Th1 act                  0.2
Primary Th2 act                  0.8
Primary Tr1 act                  1.4
Primary Th1 rest                 0.0
Primary Th2 rest                 0.1
Primary Tr1 rest                 0.0
CD45RA CD4 lymphocyte act        24.5
CD45RO CD4 lymphocyte act        0.4
CD8 lymphocyte act               0.1
Secondary CD8 lymphocyte rest    0.0
Secondary CD8 lymphocyte act     0.1
CD4 lymphocyte none              0.0
2ry Th1/Th2/Tr1_anti-CD95 CH11   0.1
LAK cells rest                   0.7
LAK cells IL-2                   0.7
LAK cells IL-2 + IL-12           0.0
LAK cells IL-2 + IFN gamma       0.0
LAK cells IL-2 + IL-18           0.3
LAK cells PMA/ionomycin          1.3
NK Cells IL-2 rest               1.8
Two Way MLR 3 day                0.1
Two Way MLR 5 day                0.0
Two Way MLR 7 day                0.2
PBMC rest                        0.0
PBMC PWM                         0.2
PBMC PHA-L                       0.2
Ramos (B cell) none              0.0
Ramos (B cell) ionomycin         0.0
B lymphocytes PWM                0.1
B lymphocytes CD40L and IL-4     0.7
EOL-1 dbcAMP                     0.4
EOL-1 dbcAMP PMA/ionomycin       0.4
Dendritic cells none             0.6
Dendritic cells LPS              0.2
Dendritic cells anti-CD40        0.0
Monocytes rest                   0.0
Monocytes LPS                    12.0
Macrophages rest                 0.1
Macrophages LPS                  0.3
HUVEC none                       17.1
HUVEC starved                    55.5
HUVEC IL-1beta                   54.3
HUVEC IFN gamma                  36.1
HUVEC TNF alpha + IFN gamma      19.8
HUVEC TNF alpha + IL4            11.4
HUVEC IL-11                      11.7
Lung Microvascular EC none       100.0
Lung Microvascular EC TNFalpha + IL-1beta 54.7
Microvascular Dermal EC none     12.7
Microsvasular Dermal EC TNFalpha + IL-1beta 15.3
Bronchial epithelium TNFalpha + IL1beta 1.8
Small airway epithelium none     3.2
Small airway epithelium TNFalpha + IL-1beta 6.5
Coronery artery SMC rest         33.7
Coronery artery SMC TNFalpha + IL-1beta 28.1
Astrocytes rest                  8.6
Astrocytes TNFalpha + IL-1beta   11.3
KU-812 (Basophil) rest           0.0
KU-812 (Basophil) PMA/ionomycin  0.6
CCD1106 (Keratinocytes) none     4.5
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 1.0
Liver cirrhosis                  1.3
NCI-H292 none                    7.8
NCI-H292 IL-4                    8.1
NCI-H292 IL-9                    17.9
NCI-H292 IL-13                   10.2
NCI-H292 IFN gamma               5.4
HPAEC none                       17.6
HPAEC TNF alpha + IL-1 beta      50.0
Lung fibroblast none             16.5
Lung fibroblast TNF alpha + IL-1 beta 9.3
Lung fibroblast IL-4             8.7
Lung fibroblast IL-9             9.7
Lung fibroblast IL-13            7.1
Lung fibroblast IFN gamma        25.2
Dermal fibroblast CCD1070 rest   49.0
Dermal fibroblast CCD1070 TNF alpha 49.0
Dermal fibroblast CCD1070 IL-1 beta 28.9
Dermal fibroblast IFN gamma      5.8
Dermal fibroblast IL-4           5.4
Dermal Fibroblasts rest          9.6
Neutrophils TNFa + LPS           0.0
Neutrophils rest                 0.0
Colon                            0.1
Lung                             1.4
Thymus                           0.3
Kidney                           8.1

[0670] CNS_neurodegeneration_v1.0 Summary: Ag7303 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene is appears to be slightly upregulated in the temporal cortex of Alzheimer's disease patients. Therefore, therapeutic modulation of the expression or function of this gene may decrease neuronal death and be of use in the treatment of this disease.

[0671] General_screening_panel_v1.7 Summary: Ag7303 Highest expression of this gene is seen in a renal cancer cell line (CT=23.5). This gene is widely expressed in this panel, with high to moderate expression seen in brain, colon, gastric, lung, prostate, breast, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

[0672] Among tissues with metabolic function, this gene is expressed at high to moderate levels in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

[0673] This gene is also expressed at high to moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

[0674] This gene encodes a novel splice variant of CRIM1. Please see CG50691-02 for further discussion of this gene.

[0675] Panel 1 Summary: Ag155 Highest expression is seen in the placenta (CT=20.74). Overall, the expression profile agrees with the results for Panel 1.7. Please see that panel for discussion of this gene.

[0676] Panel 4.1D Summary: Ag7303 Highest expression of this gene is seen in untreated lung microvascular endothelial cells (CT=30). This transcript is also expressed in clusters of samples derived from HPAEC, HUVEC, lung, and dermal microvascular EC, and lung and dermal fibroblasts. Therefore, therapies designed with the protein encoded by this transcript could be important in regulating endothelium function including leukocyte extravasation, a major component of inflammation during asthma, IBD, and psoriasis.

[0677] J. CG50691-04: Cysteine-Rich Repeat-Containing Protein S52 Precursor.

[0678] Expression of gene CG50691-04 was assessed using the primer-probe sets Ag155 and Ag8164, described in Tables JA and JB. Results of the RTQ-PCR runs are shown in Tables JC, JD, JE and JF.

TABLE JA
Probe Name Ag155
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-tcaaacgcgatcacaatggt-3′	20	1496	114
Probe	TET-5′-tcggacctgtcagtgcataaacaccg-3′-TAMRA	26	1518	115
Reverse	5′-gccttgtttacgttctgaacatagtt-3′	26	1547	116

[0679]

TABLE JB
Probe Name Ag8164
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-ccacagtgtacagaagacacaatt-3′	24	2233	117
Probe	TET-5′-cactgaagtggcacaccaccttcttt-3′-TAMRA	26	2259	118
Reverse	5′-gggtgcagctgtcaaggt-3′	18	2315	119

[0680]

TABLE JC
CNS neurodegeneration v1.0
                               Rel. Exp.(%)
                               Ag8164,
Tissue Name                    Run 323199051
AD 1 Hippo                     9.7
AD 2 Hippo                     34.2
AD 3 Hippo                     12.8
AD 4 Hippo                     7.3
AD 5 hippo                     65.1
AD 6 Hippo                     86.5
Control 2 Hippo                17.6
Control 4 Hippo                3.8
Control (Path) 3 Hippo         34.2
AD 1 Temporal Ctx              8.2
AD 2 Temporal Ctx              23.2
AD 3 Temporal Ctx              8.5
AD 4 Temporal Ctx              29.1
AD 5 Inf Temporal Ctx          62.0
AD 5 SupTemporal Ctx           48.0
AD 6 Inf Temporal Ctx          95.9
AD 6 Sup Temporal Ctx          84.7
Control 1 Temporal Ctx         9.7
Control 2 Temporal Ctx         36.3
Control 3 Temporal Ctx         25.7
Control 4 Temporal Ctx         7.5
Control (Path) 1 Temporal Ctx  67.4
Control (Path) 2 Temporal Ctx  69.3
Control (Path) 3 Temporal Ctx  20.3
Control (Path) 4 Temporal Ctx  23.7
AD 1 Occipital Ctx             3.1
AD 2 Occipital Ctx (Missing)   0.0
AD 3 Occipital Ctx             21.2
AD 4 Occipital Ctx             35.4
AD 5 Occipital Ctx             61.1
AD 6 Occipital Ctx             44.8
Control 1 Occipital Ctx        15.6
Control 2 Occipital Ctx        45.1
Control 3 Occipital Ctx        19.6
Control 4 Occipital Ctx        2.8
Control (Path) 1 Occipital Ctx 97.9
Control (Path) 2 Occipital Ctx 20.7
Control (Path) 3 Occipital Ctx 6.2
Control (Path) 4 Occipital Ctx 24.7
Control 1 Parietal Ctx         56.6
Control 2 Parietal Ctx         100.0
Control 3 Parietal Ctx         27.2
Control (Path) 1 Parietal Ctx  87.7
Control (Path) 2 Parietal Ctx  21.0
Control (Path) 3 Parietal Ctx  8.9
Control (Path) 4 Parietal Ctx  30.8

[0681]

TABLE JD
Oncology cell line screening panel v3.2
                                 Rel.
                                 Exp. (%)
                                 Ag8164,
                                 Run
Tissue Name                      323342068
94905_Daoy_Medulloblastoma/Cerebellum_sscDNA 3.4
94906_TE671_Medulloblastom/Cerebellum_sscDNA 0.0
94907_D283                       1.6
Med_Medulloblastoma/Cerebellum_sscDNA
94908_PFSK-1_Primitive           2.1
Neuroectodermal/Cerebellum_sscDNA
94909_XF-498_CNS_sscDNA          28.3
94910_SNB-78_CNS/glioma_sscDNA   31.0
94911_SF-268_CNS/glioblastoma_sscDNA 52.9
94912_T98G_Glioblastoma_sscDNA   17.1
96776_SK-N-SH_Neuroblastoma      49.7
(metastasis)_sscDNA
94913_SF-295_CNS/glioblastoma_sscDNA 15.7
132565_NT2 pool_sscDNA           5.4
94914_Cerebellum_sscDNA          2.2
96777_Cerebellum_sscDNA          0.0
94916_NCI-H292_Mucoepidermoid lung 67.8
carcinoma_sscDNA
94917_DMS-114_Small cell lung cancer_sscDNA 22.1
94918_DMS-79_Small cell lung     0.0
cancer/neuroendocrine_sscDNA
94919_NCI-H146_Small cell lung   0.0
cancer/neuroendocrine_sscDNA
94920_NCI-H526_Small cell lung   1.1
cancer/neuroendocrine_sscDNA
94921_NCI-N417_Small cell lung   0.0
cancer/neuroendocrine_sscDNA
94923_NCI-H82_Small cell lung    1.2
cancer/neuroendocrine_sscDNA
94924_NCI-H157_Squamous cell lung 11.5
cancer (metastasis)_sscDNA
94925_NCI-H1155_Large cell lung  2.3
cancer/neuroendocrine_sscDNA
94926_NCI-H1299_Large cell lung  16.8
cancer/neuroendocrine_sscDNA
94927_NCI-H727_Lung carcinoid_sscDNA 23.2
94928_NCI-UMC-11_Lung carcinoid_sscDNA 2.2
94929_LX-1_Small cell lung cancer_sscDNA 0.0
94930_Colo-205_Colon cancer_sscDNA 100.0
94931_KM12_Colon cancer_sscDNA   7.9
94932_KM20L2_Colon cancer_sscDNA 0.7
94933_NCI-H716_Colon cancer_sscDNA 18.0
94935_SW-48_Colon adenocarcinoma_sscDNA 2.0
94936_SW1116_Colon adenocarcinoma_sscDNA 1.5
94937_LS 174T_Colon adenocarcinoma_sscDNA 5.6
94938_SW-948_Colon adenocarcinoma_sscDNA 0.0
94939_SW-480_Colon adenocarcinoma_sscDNA 5.4
94940_NCI-SNU-5_Gastric carcinoma_sscDNA 10.7
112197_KATO III_Stomach_sscDNA   2.0
94943_NCI-SNU-16_Gastric carcinoma_sscDNA 4.2
94944_NCI-SNU-1_Gastric carcinoma_sscDNA 3.8
94946_RF-1_Gastric adenocarcinoma_sscDNA 2.0
94947_RF-48_Gastric adenocarcinoma_sscDNA 3.1
96778_MKN-45_Gastric carcinoma_sscDNA 0.3
94949_NCI-N87_Gastric carcinoma_sscDNA 7.5
94951_OVCAR-5_Ovarian carcinoma_sscDNA 8.0
94952_RL95-2_Uterine carcinoma_sscDNA 8.8
94953_HelaS3_Cervical            24.5
adenocarcinoma_sscDNA
94954_Ca Ski_Cervical epidermoid 37.9
carcinoma (metastasis)_sscDNA
94955_ES-2_Ovarian clear cell    29.3
carcinoma_sscDNA
94957_Ramos/6 h stim_Stimulated  3.5
with PMA/ionomycin 6 h_sscDNA
94958_Ramos/14 h stim—           6.0
Stimulated with PMA/ionomycin
14 h_sscDNA
94962_MEG-01_Chronic myelogenous leukemia 0.5
(megokaryoblast)_sscDNA
94963_Raji_Burkitt's lymphoma_sscDNA 0.0
94964_Daudi_Burkitt's lymphoma_sscDNA 0.0
94965_U266_B-cell                0.0
plasmacytoma/myeloma_sscDNA
94968_CA46_Burkitt's lymphoma_sscDNA 2.3
94970_RL_non-Hodgkin's B-cell    0.0
lymphoma_sscDNA
94972_JM1_pre-B-cell             2.2
lymphoma/leukemia_sscDNA
94973_Jurkat_T cell              0.0
leukemia_sscDNA
94974_TF-1_Erythroleukemia_sscDNA 1.4
94975_HUT 78_T-cell lymphoma_sscDNA 0.0
94977_U937_Histiocytic lymphoma_sscDNA 0.0
94980_KU-812_Myelogenous         0.0
leukemia_sscDNA
94981_769-P_Clear cell renal     18.8
carcinoma_sscDNA
94983_Caki-2_Clear cell renal    15.3
carcinoma_sscDNA
94984_SW 839_Clear cell renal    27.7
carcinoma_sscDNA
94986_G401_Wilms' tumor_sscDNA   5.6
126768_293 cells_sscDNA          2.4
94987_Hs766T_Pancreatic carcinoma 24.3
(LN metastasis)_sscDNA
94988_CAPAN-1_Pancreatic adenocarcinoma 7.1
(liver metastasis)_sscDNA
94989_SU86.86_Pancreatic carcinoma 15.5
(liver metastasis)_sscDNA
94990_BxPC-3_Pancreatic          4.7
adenocarcinoma_sscDNA
94991_HPAC_Pancreatic            27.4
adenocarcinoma_sscDNA
94992_MIA PaCa-2_Pancreatic      5.5
carcinoma_sscDNA
94993_CFPAC-1_Pancreatic ductal  16.6
adenocarcinoma_sscDNA
94994_PANC-1_Pancreatic epithelioid 56.6
ductal carcinoma_sscDNA
94996_T24_Bladder carcinma       14.6
(transitional cell)_sscDNA
94997_5637_Bladder carcinoma_sscDNA 3.0
94998_HT-1197_Bladder carcinoma_sscDNA 21.3
94999_UM-UC-3_Bladder carcinma   16.0
(transitional cell)_sscDNA
95000_A204_Rhabdomyosarcoma—     0.0
sscDNA
95001_HT-1080_Fibrosarcoma_sscDNA 43.8
95002_MG-63_Osteosarcoma (bone)_sscDNA 11.2
95003_SK-LMS-1_Leiomyosarcoma    30.1
(vulva)_sscDNA
95004_SJRH30_Rhabdomyosarcoma    0.9
(met to bone marrow)_sscDNA
95005_A431_Epidermoid carcinoma_sscDNA 6.5
95007_WM266-4_Melanoma_sscDNA    0.7
112195_DU 145_Prostate_sscDNA    40.9
95012_MDA-MB-468_Breast          7.4
adenocarcinoma_sscDNA
112196_SSC-4_Tongue_sscDNA       4.4
112194_SSC-9_Tongue_sscDNA       8.5
112191_SSC-15_Tongue_sscDNA      12.2
95017_CAL 27_Squamous cell       18.0
carcinoma of tongue_sscDNA

[0682]

TABLE JE
Panel 1
Rel. Exp. (%)
Ag155, Run
87589113
Endothelial cells                14.6
Endothelial cells (treated)      20.0
Pancreas                         3.5
Pancreatic ca. CAPAN 2           1.4
Adrenal gland                    2.5
Thyroid                          6.0
Salivary gland                   4.5
Pituitary gland                  0.3
Brain (fetal)                    0.3
Brain (whole)                    13.9
Brain (amygdala)                 3.1
Brain (cerebellum)               20.4
Brain (hippocampus)              2.1
Brain (substantia nigra)         0.6
Brain (thalamus)                 1.2
Brain (hypothalamus)             0.0
Spinal cord                      1.0
glio/astro U87-MG                3.9
glio/astro U-118-MG              2.6
astrocytoma SW1783               15.6
neuro*; met SK-N-AS              0.6
astrocytoma SF-539               0.0
astrocytoma SNB-75               12.6
glioma SNB-19                    19.8
glioma U251                      1.9
glioma SF-295                    0.0
Heart                            3.8
Skeletal muscle                  6.4
Bone marrow                      0.0
Thymus                           6.7
Spleen                           2.0
Lymph node                       2.0
Colon (ascending)                4.2
Stomach                          6.0
Small intestine                  2.2
Colon ca. SW480                  0.0
Colon ca.* SW620 (SW480 met)     0.0
Colon ca. HT29                   0.0
Colon ca. HCT-116                0.0
Colon ca. CaCo-2                 14.6
Colon ca. HCT-15                 1.4
Colon ca. HCC-2998               0.0
Gastric ca. * (liver met) NCI-N87 3.8
Bladder                          9.7
Trachea                          1.1
Kidney                           13.3
Kidney (fetal)                   10.9
Renal ca. 786-0                  26.8
Renal ca. A498                   18.3
Renal ca. RXF 393                0.3
Renal ca. ACHN                   20.6
Renal ca. UO-31                  12.4
Renal ca. TK-10                  11.2
Liver                            0.0
Liver (fetal)                    0.0
Liver ca. (hepatoblast) HepG2    12.7
Lung                             8.7
Lung (fetal)                     3.0
Lung ca. (small cell) LX-1       0.0
Lung ca. (small cell) NCI-H69    0.0
Lung ca. (s. cell var.) SHP-77   0.0
Lung ca. (large cell)NCI-H460    0.0
Lung ca. (non-sm. cell) A549     15.9
Lung ca. (non-s. cell) NCI-H23   6.8
Lung ca. (non-s. cell) HOP-62    7.3
Lung ca. (non-s. cl) NCI-H522    16.6
Lung ca. (squam.) SW 900         6.5
Lung ca. (squam.) NCI-H596       0.0
Mammary gland                    35.1
Breast ca.* (pl. ef) MCF-7       2.0
Breast ca.* (pl. ef) MDA-MB-231  3.7
Breast ca.* (pl. ef) T47D        16.2
Breast ca. BT-549                0.0
Breast ca. MDA-N                 0.4
Ovary                            9.6
Ovarian ca. OVCAR-3              8.4
Ovarian ca. OVCAR-4              10.3
Ovarian ca. OVCAR-5              51.4
Ovarian ca. OVCAR-8              13.9
Ovarian ca. IGROV-1              0.0
Ovarian ca. (ascites) SK-OV-3    5.7
Uterus                           19.5
Placenta                         100.0
Prostate                         1.0
Prostate ca.* (bone met) PC-3    0.0
Testis                           29.1
Melanoma Hs688(A).T              19.2
Melanoma* (met) Hs688(B).T       20.2
Melanoma UACC-62                 0.0
Melanoma M14                     0.0
Melanoma LOX IMVI                38.4
Melanoma* (met) SK-MEL-5         0.0
Melanoma SK-MEL-28               0.0

[0683]

TABLE JF
Panel 4.1D
                                 Rel. Exp. (%)
                                 Ag8164, Run
                                 319806138
Secondary Th1 act                6.0
Secondary Th2 act                1.3
Secondary Tr1 act                2.3
Secondary Th1 rest               0.0
Secondary Th2 rest               0.0
Secondary Tr1 rest               0.0
Primary Th1 act                  0.9
Primary Th2 act                  3.3
Primary Tr1 act                  3.2
Primary Th1 rest                 0.0
Primary Th2 rest                 1.1
Primary Tr1 rest                 0.0
CD45RA CD4 lymphocyte act        32.3
CD45RO CD4 lymphocyte act        0.0
CD8 lymphocyte act               0.8
Secondary CD8 lymphocyte rest    0.0
Secondary CD8 lymphocyte act     0.5
CD4 lymphocyte none              0.0
2ry Th1/Th2/Tr1_anti-CD95 CH11   0.0
LAK cells rest                   0.7
LAK cells IL-2                   0.0
LAK cells IL-2 + IL-12           0.7
LAK cells IL-2 + IFN gamma       0.0
LAK cells IL-2 + IL-18           0.0
LAK cells PMA/ionomycin          2.1
NK Cells IL-2 rest               5.6
Two Way MLR 3 day                1.2
Two Way MLR 5 day                0.0
Two Way MLR 7 day                0.0
PBMC rest                        0.0
PBMC PWM                         0.7
PBMC PHA-L                       0.0
Ramos (B cell) none              0.0
Ramos (B cell) ionomycin         0.0
B lymphocytes PWM                0.0
B lymphocytes CD40L and IL-4     0.0
EOL-1 dbcAMP                     0.0
EOL-1 dbcAMP PMA/ionomycin       0.0
Dendritic cells none             3.4
Dendritic cells LPS              0.8
Dendritic cells anti-CD40        0.0
Monocytes rest                   0.0
Monocytes LPS                    10.0
Macrophages rest                 0.0
Macrophages LPS
HUVEC none                       33.4
HUVEC starved                    59.5
HUVEC IL-1beta                   53.2
HUVEC IFN gamma                  57.4
HUVEC TNF alpha + IFN gamma      20.2
HUVEC TNF alpha + IL4            15.6
HUVEC IL-11                      31.6
Lung Microvascular EC none       100.0
Lung Microvascular EC TNFalpha + IL-1beta 80.7
Microvascular Dermal EC none     23.2
Microsvasular Dermal EC TNFalpha + IL-1beta 34.9
Bronchial epithelium TNFalpha + IL1beta 5.3
Small airway epithelium none     2.5
Small airway epithelium TNFalpha + IL-1beta 50.0
Coronery artery SMC rest         32.8
Coronery artery SMC TNFalpha + IL-1beta 24.5
Astrocytes rest                  5.7
Astrocytes TNFalpha + IL-1beta   3.1
KU-812 (Basophil) rest           0.0
KU-812 (Basophil) PMA/ionomycin  0.0
CCD1106 (Keratinocytes) none     8.4
CCD1106 (Keratinocytes) TNFalpha + IL-1beta 6.0
Liver cirrhosis                  7.9
NCI-H292 none                    10.2
NCI-H292 IL-4                    13.3
NCI-H292 IL-9                    16.5
NCI-H292 IL-13                   21.3
NCI-H292 IFN gamma               6.6
HPAEC none                       15.2
HPAEC TNF alpha + IL-1 beta      68.3
Lung fibroblast none             30.8
Lung fibroblast TNF alpha + IL-1 beta 9.9
Lung fibroblast IL-4             24.7
Lung fibroblast IL-9             23.2
Lung fibroblast IL-13            5.2
Lung fibroblast IFN gamma        56.3
Dermal fibroblast CCD1070 rest   58.6
Dermal fibroblast CCD1070 TNF alpha 83.5
Dermal fibroblast CCD1070 IL-1 beta 45.4
Dermal fibroblast IFN gamma      4.0
Dermal fibroblast IL-4           3.6
Dermal Fibroblasts rest          5.4
Neutrophils TNFa + LPS           0.0
Neutrophils rest                 0.0
Colon                            0.0
Lung                             0.6
Thymus                           0.0
Kidney                           26.6

[0684] CNS_neurodegeneration_v1.0 Summary: Ag8164, This profile confirms the expression of this gene at low levels in the brain. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

[0685] Oncology_cell_line_screening_panel_v3.2 Summary: Ag8164 Highest expression is seen in a colon cancer cell line (CT=30). In addition, this gene is expressed at moderate to low levels in many samples on this panel, suggesting a role for this gene in cell survival and proliferation.

[0686] Panel 1 Summary: Ag155 Highest expression is seen in the placenta (CT=20.74). Overall, the expression profile agrees with the results for Panel 1.7. Please see that panel for discussion of this gene.

[0687] Panel 4.1D Summary: Ag8164 Highest expression of this gene is seen in untreated lung microvascular endothelial cells (CT=31.1). This transcript is also expressed in clusters of samples derived from HPAEC, HUVEC, lung, and dermal microvascular EC, and lung and dermal fibroblasts. Therefore, therapies designed with the protein encoded by this transcript could be important in regulating endothelium function including leukocyte extravasation, a major component of inflammation during asthma, IBD, and psoriasis.

[0688] K. CG51905-01 and CG51905-03: 28804279.0.7.

[0689] Expression of gene CG51905-01 and CG51905-03 was assessed using the primer-probe sets Ag2814 and Ag203, described in Tables KA and KB. Results of the RTQ-PCR runs are shown in Tables KC, KD and KE. Please note that CG51905-03 represents a full-length physical clone of the CG51905-01 gene, validating the prediction of the gene sequence.

TABLE KA
Probe Name Ag2814
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-gcacagctctagaagcttcaat-3′	22	200	120
Probe	TET-5′-ccacccatacatctctttgtgctctca-3′-TAMRA	27	229	121
Reverse	5′-cctgtgctgtgatggtcttatt-3′	22	276	122

[0690]

TABLE KB
Probe Name Ag203
			Start	SEQ
Primers	Sequences	Length	Position	ID No
Forward	5′-gtgctctcacacccccaca-3′	19	247	123
Probe	TET-5′-cccttctggaataagaccatcacagcacag-3′-TAMRA	30	267	124
Reverse	5′-gactggcattagacatcttgcaa-3′	23	300	125

[0691]

TABLE KC
Panel 1
Rel. Exp. (%)
Ag203, Run
90995866
Endothelial cells                0.0
Endothelial cells (treated)      0.0
Pancreas                         7.9
Pancreatic ca. CAPAN 2           0.3
Adrenal gland                    4.5
Thyroid                          0.0
Salivary gland                   0.0
Pituitary gland                  0.0
Brain (fetal)                    0.0
Brain (whole)                    0.8
Brain (amygdala)                 0.0
Brain (cerebellum)               1.1
Brain (hippocampus)              0.0
Brain (substantia nigra)         0.9
Brain (thalamus)                 0.0
Brain (hypothalamus)             0.0
Spinal cord                      0.0
glio/astro U87-MG                0.0
glio/astro U-118-MG              0.0
astrocytoma SW1783               0.0
neuro*; met SK-N-AS              1.2
astrocytoma SF-539               0.0
astrocytoma SNB-75               15.7
glioma SNB-19                    0.2
glioma U251                      0.0
glioma SF-295                    0.0
Heart                            0.0
Skeletal muscle                  0.0
Bone marrow                      0.1
Thymus                           100.0
Spleen                           0.0
Lymph node                       12.5
Colon (ascending)                6.5
Stomach                          0.6
Small intestine                  0.0
Colon ca. SW480                  0.0
Colon ca.* SW620 (SW480 met)     0.0
Colon ca. HT29                   0.0
Colon ca. HCT-116                0.0
Colon ca. CaCo-2                 0.0
Colon ca. HCT-15                 0.0
Colon ca. HCC-2998               0.3
Gastric ca. * (liver met) NCI-N87 0.3
Bladder                          0.0
Trachea                          0.7
Kidney                           0.0
Kidney (fetal)                   0.5
Renal ca. 786-0                  0.2
Renal ca. A498                   0.0
Renal ca. RXF 393                2.4
Renal ca. ACHN                   4.8
Renal ca. UO-31                  0.0
Renal ca. TK-10                  0.0
Liver                            0.0
Liver (fetal)                    0.0
Liver ca. (hepatoblast) HepG2    0.0
Lung                             0.0
Lung (fetal)                     0.0
Lung ca. (small cell) LX-1       0.0
Lung ca. (small cell) NCI-H69    0.0
Lung ca. (s. cell var.) SHP-77   7.5
Lung ca. (large cell)NCI-H460    0.0
Lung ca. (non-sm. cell) A549     0.0
Lung ca. (non-s. cell) NCI-H23   0.0
Lung ca. (non-s. cell) HOP-62    0.0
Lung ca. (non-s. cl) NCI-H522    0.0
Lung ca. (squam.) SW 900         0.0
Lung ca. (squam.) NCI-H596       0.0
Mammary gland                    1.3
Breast ca.* (pl. ef) MCF-7       0.0
Breast ca.* (pl. ef) MDA-MB-231  0.0
Breast ca.* (pl. ef) T47D        0.3
Breast ca. BT-549                0.1
Breast ca. MDA-N                 1.2
Ovary                            0.0
Ovarian ca. OVCAR-3              0.1
Ovarian ca. OVCAR-4              0.0
Ovarian ca. OVCAR-5              0.8
Ovarian ca. OVCAR-8              0.0
Ovarian ca. IGROV-1              0.0
Ovarian ca. (ascites) SK-OV-3    4.5
Uterus                           0.0
Placenta                         0.0
Prostate                         0.2
Prostate ca.* (bone met) PC-3    0.0
Testis                           1.6
Melanoma Hs688(A).T              0.0
Melanoma* (met) Hs688(B).T       0.0
Melanoma UACC-62                 0.0
Melanoma M14                     0.4
Melanoma LOX IMVI                0.0
Melanoma* (met) SK-MEL-5         1.5
Melanoma SK-MEL-28               0.3

[0692]

TABLE KD
Panel 1.3D
                                 Rel. Exp. (%)
                                 Ag2814, Run
Tissue Name                      154287717
Liver adenocarcinoma             10.7
Pancreas                         15.9
Pancreatic ca. CAPAN 2           3.7
Adrenal gland                    18.3
Thyroid                          7.7
Salivary gland                   2.2
Pituitary gland                  11.3
Brain (fetal)                    7.3
Brain (whole)                    7.9
Brain (amygdala)                 11.7
Brain (cerebellum)               14.4
Brain (hippocampus)              28.3
Brain (substantia nigra)         6.5
Brain (thalamus)                 6.7
Cerebral Cortex                  2.6
Spinal cord                      15.7
glio/astro U87-MG                12.1
glio/astro U-118-MG              26.1
astrocytoma SW1783               6.6
neuro*; met SK-N-AS              28.9
astrocytoma SF-539               14.5
astrocytoma SNB-75               22.2
glioma SNB-19                    14.5
glioma U251                      13.3
glioma SF-295                    7.4
Heart (fetal)                    0.0
Heart                            0.0
Skeletal muscle (fetal)          100.0
Skeletal muscle                  0.0
Bone marrow                      16.7
Thymus                           18.6
Spleen                           6.4
Lymph node                       17.7
Colorectal                       36.6
Stomach                          23.2
Small intestine                  11.9
Colon ca. SW480                  6.4
Colon ca.* SW620(SW480 met)      3.2
Colon ca. HT29                   0.0
Colon ca. HCT-116                8.2
Colon ca. CaCo-2                 6.7
Colon ca. tissue(ODO3866)        4.3
Colon ca. HCC-2998               15.3
Gastric ca.* (liver met) NCI-N87 23.2
Bladder                          4.5
Trachea                          14.2
Kidney                           0.0
Kidney (fetal)                   6.0
Renal ca. 786-0                  13.7
Renal ca. A498                   12.4
Renal ca. RXF 393                9.4
Renal ca. ACHN                   7.9
Renal ca. UO-31                  14.7
Renal ca. TK-10                  2.8
Liver                            0.0
Liver (fetal)                    25.2
Liver ca. (hepatoblast) HepG2    8.1
Lung                             17.6
Lung (fetal)                     17.8
Lung ca. (small cell) LX-1       8.2
Lung ca. (small cell) NCI-H69    11.9
Lung ca. (s. cell var.) SHP-77   16.4
Lung ca. (large cell)NCI-H460    1.1
Lung ca. (non-sm. cell) A549     9.2
Lung ca. (non-s. cell) NCI-H23   13.9
Lung ca. (non-s. cell) HOP-62    11.2
Lung ca. (non-s. cl) NCI-H522    1.6
Lung ca. (squam.) SW 900         3.4
Lung ca. (squam.) NCI-H596       3.4
Mammary gland                    2.1
Breast ca.* (pl. ef) MCF-7       4.6
Breast ca.* (pl. ef) MDA-MB-231  14.4
Breast ca.* (pl. ef) T47D        4.4
Breast ca. BT-549                18.9
Breast ca. MDA-N                 8.6
Ovary                            5.7
Ovarian ca. OVCAR-3              9.2
Ovarian ca. OVCAR-4              0.0
Ovarian ca. OVCAR-5              12.7
Ovarian ca. OVCAR-8              7.3
Ovarian ca. IGROV-1              14.4
Ovarian ca.* (ascites) SK-OV-3   17.9
Uterus                           12.5
Placenta                         35.4
Prostate                         3.1
Prostate ca.* (bone met)PC-3     12.9
Testis                           9.5
Melanoma Hs688(A).T              10.7
Melanoma* (met) Hs688(B).T       13.7
Melanoma UACC-62                 0.0
Melanoma M14                     8.4
Melanoma LOX IMVI                0.0
Melanoma* (met) SK-MEL-5         40.3
Adipose                          19.2

[0693]

TABLE KE
Panel 4D
	Rel. Exp. (%)	Rel. Exp. (%)
	Ag203, Run	Ag2814, Run
Tissue Name	138065785	154296871
Secondary Th1 act	7.4	27.2
Secondary Th2 act	16.4	13.1
Secondary Tr1 act	7.3	28.7
Secondary Th1 rest	0.7	7.9
Secondary Th2 rest	1.8	17.9
Secondary Tr1 rest	1.4	8.5
Primary Th1 act	14.6	35.6
Primary Th2 act	100.0	22.2
Primary Tr1 act	27.7	34.2
Primary Th1 rest	9.3	39.0
Primary Th2 rest	7.0	20.2
Primary Tr1 rest	5.8	4.9
CD45RA CD4 lymphocyte act	2.0	10.4
CD45RO CD4 lymphocyte act	13.4	37.4
CD8 lymphocyte act	3.7	10.5
Secondary CD8 lymphocyte rest	4.4	25.2
Secondary CD8 lymphocyte act	3.4	14.5
CD4 lymphocyte none	1.3	3.4
2ry Th1/Th2/Tr1_anti-CD95	1.4	15.0
CH11
LAK cells rest	3.7	6.7
LAK cells IL-2	3.6	17.7
LAK cells IL-2 + IL-12	3.9	9.7
LAK cells IL-2 + IFN gamma	7.5	33.4
LAK cells IL-2 + IL-18	1.3	23.2
LAK cells PMA/ionomycin	1.9	13.4
NK Cells IL-2 rest	1.6	13.6
Two Way MLR 3 day	4.0	10.8
Two Way MLR 5 day	1.1	9.3
Two Way MLR 7 day	2.2	28.1
PBMC rest	2.1	6.3
PBMC PWM	22.8	52.5
PBMC PHA-L	11.3	17.4
Ramos (B cell) none	29.1	68.3
Ramos (B cell) ionomycin	29.5	100.0
B lymphocytes PWM	9.3	44.4
B lymphocytes CD40L and IL-4	10.0	52.5
EOL-1 dbcAMP	0.8	5.4
EOL-1 dbcAMP	1.4	4.5
PMA/ionomycin
Dendritic cells none	1.7	1.7
Dendritic cells LPS	2.3	5.8
Dendritic cells anti-CD40	3.1	10.9
Monocytes rest	3.3	18.6
Monocytes LPS	6.0	12.9
Macrophages rest	1.5	10.5
Macrophages LPS	3.7	2.1
HUVEC none	1.5	10.2
HUVEC starved	2.6	19.6
HUVEC IL-1beta	1.2	1.1
HUVEC IFN gamma	5.5	9.3
HUVEC TNF alpha + IFN gamma	2.2	7.2
HUVEC TNF alpha + IL4	1.7	10.7
HUVEC IL-11	0.9	0.0
Lung Microvascular EC none	1.3	0.0
Lung Microvascular EC	1.0	3.9
TNFalpha + IL-1beta
Microvascular	1.0	10.5
Dermal EC none
Microsvasular Dermal EC	2.9	13.4
TNFalpha + IL-1beta
Bronchial epithelium	4.1	0.0
TNFalpha + IL1beta
Small airway	2.0	5.2
epithelium none
Small airway epithelium	12.0	51.4
TNFalpha + IL-1beta
Coronery artery SMC rest	2.2	13.0
Coronery artery	1.4	4.5
SMC TNFalpha + IL-1beta
Astrocytes rest	2.8	9.9
Astrocytes	2.4	9.0
TNFalpha + IL-1beta
KU-812 (Basophil) rest	9.3	31.2
KU-812 (Basophil)	18.7	50.7
PMA/ionomycin
CCD1106 (Keratinocytes)	3.8	9.5
none
CCD1106 (Keratinocytes)	13.0	2.6
TNFalpha + IL-1beta
Liver cirrhosis	3.8	10.2
Lupus kidney	1.4	2.2
NCI-H292 none	5.7	18.3
NCI-H292 IL-4	6.5	28.3
NCI-H292 IL-9	8.6	22.5
NCI-H292 IL-13	12.2	14.4
NCI-H292 IFN gamma	6.5	25.0
HPAEC none	3.3	5.8
HPAEC TNF alpha + IL-1 beta	3.0	5.4
Lung fibroblast none	1.5	6.9
Lung fibroblast	1.1	1.5
TNF alpha + IL-1 beta
Lung fibroblast IL-4	3.0	15.7
Lung fibroblast IL-9	5.5	9.8
Lung fibroblast IL-13	8.6	4.7
Lung fibroblast IFN gamma	4.2	15.8
Dermal fibroblast	7.0	21.0
CCD1070 rest
Dermal fibroblast	15.5	27.5
CCD1070 TNF alpha
Dermal fibroblast	9.6	9.8
CCD1070 IL-1 beta
Dermal fibroblast IFN gamma	1.0	1.7
Dermal fibroblast IL-4	2.4	5.3
IBD Colitis 2	0.6	2.8
IBD Crohn's	0.0	2.8
Colon	0.7	5.7
Lung	1.6	10.8
Thymus	4.0	13.1
Kidney	19.8	82.4

[0694] Panel 1 Summary: Ag203 Highest expression of this gene is detected in thymus (CT=29.9). Thus, this gene or its protein product could play an important role in T cell development. Small molecule therapeutics, or antibody therapeutics designed against the protein encoded for by this gene could be utilized to modulate immune function (T cell development) and be important for organ transplant, AIDS treatment or post chemotherapy immune reconstitution.

[0695] Moderate to low expression of this gene is also detected in a few cancer cell lines derived from ovarian, lung, renal and brain cancers. Therefore, therapeutic modulation of this gene or its protein product may be useful in the treatment of ovarian, lung, renal and brain cancers.

[0696] Low expression of this gene is also seen in lymph node, pancreas, and colon. Please see panel 1.3D for further discussion of this gene.

[0697] Panel 1.3D Summary: Ag2814 Highest expression of this gene in mainly seen in fetal skeletal muscle (CT=32.6). Interestingly, this gene is expressed at much higher levels in fetal when compared to adult skeletal muscle (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult skeletal muscle. In addition, the relative overexpression of this gene in fetal skeletal muscle suggests that the protein product may enhance muscular growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the GPCR encoded by this gene could be useful in treatment of muscle related diseases. More specifically, treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function.

[0698] Low expression of this gene is also seen in some of the tissues with metabolic or endocrine function including placenta, fetal liver, colorectal region, and stomach. Thus, therapeutic modulation of this gene or its protein product may be useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

[0699] In addition, this gene is expressed at much higher levels in fetal (CT=34.6) when compared to adult liver (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of liver related diseases.

[0700] Panel 4D Summary: Ag203/Ag2814 Two experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in activated primary Th2 cells and activated Ramos B cells (CTs=32-32.6). Low expression of this gene is also seen in activated primary TH1 and Tr1, activated secondary Th1, Th2 and Tr1 cells, activated memory T cells, activated PBMC cells, activated B lymphocytes, activated small airway epithelium, basophils, mucoepidermoid cells, dermal fibroblasts, thymus and kidney. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

[0701] L. CG52414-01: Rhomboid (3541612.0.88).

[0702] Expression of gene CG52414-01 was assessed using the primer-probe sets Ag2648, Ag2786, Ag2787 and Ag913, described in Tables LA, LB, LC and LD. Results of the RTQ-PCR runs are shown in Tables LE, LF, LG, LH, LI, LJ and LK.

TABLE LA
Probe Name Ag2648
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-ggtggatcaggtcaatcga-3′	19	1231	126
Probe	TET-5′-caacccagaagttctcctgctggatg-3′-TAMRA	26	1197	127
Reverse	5′-gtgtgtacgagagcgtgaagta-3′	22	1175	128

[0703]

TABLE LB
Probe Name Ag2786
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-tggctgtacatctaccccatta-3′	22	2308	129
Probe	TET-5′-ctggatcgagcacctcacctgctt-3′-TAMRA	24	2337	130
Reverse	5′-acctggtccagctcatacttct-3′	22	2384	131

[0704]

TABLE LC
Probe Name Ag2787
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-gaggtcccagatcagttctaca-3′	22	1789	132
Probe	TET-5′-tggctgtctctcttcctacatgctgg-3′-TAMRA	26	1816	133
Reverse	5′-tcaggatggtcatttgaaagac3′	22	1867	134

[0705]

TABLE LD
Probe Name Ag913
			Start	SEQ ID
Primers	Sequences	Length	Position	No
Forward	5′-cctgccacttgacaaaagtg-3′	20	1407	135
Probe	TET-5′-aaagtctccgagcagtccttccgct-3′-TAMRA	25	1379	136
Reverse	5′-gctgctgtgtccagaatgac-3′	20	1337	137

[0706]

TABLE LE
AI comprehensive panel v1.0
                                 Rel. Exp. (%)
                                 Ag2787, Run
Tissue Name                      56553035
110967 COPD-F                    6.0
110980 COPD-F                    6.7
110968 COPD-M                    4.6
110977 COPD-M                    10.7
110989 Emphysema-F               3.3
110992 Emphysema-F               5.7
110993 Emphysema-F               7.8
110994 Emphysema-F               3.8
110995 Emphysema-F               8.5
110996 Emphysema-F               1.6
110997 Asthma-M                  5.3
111001 Asthma-F                  6.0
111002 Asthma-F                  9.7
111003 Atopic Asthma-F           3.6
111004 Atopic Asthma-F           4.7
111005 Atopic Asthma-F           3.7
111006 Atopic Asthma-F           0.7
111417 Allergy-M                 6.9
112347 Allergy-M                 0.1
112349 Normal Lung-F             0.0
112357 Normal Lung-F             9.9
112354 Normal Lung-M             3.1
112374 Crohns-F                  6.3
112389 Match Control Crohns-F    27.7
112375 Crohns-F                  5.5
112732 Match Control Crohns-F    45.7
112725 Crohns-M                  0.7
112387 Match Control Crohns-M    6.0
112378 Crohns-M                  0.1
112390 Match Control Crohns-M    7.4
112726 Crohns-M                  2.0
112731 Match Control Crohns-M    2.7
112380 Ulcer Col-F               5.8
112734 Match Control Ulcer Col-F 100.0
112384 Ulcer Col-F               12.9
112737 Match Control Ulcer Col-F 1.9
112386 Ulcer Col-F               17.8
112738 Match Control Ulcer Col-F 11.7
112381 Ulcer Col-M               0.2
112735 Match Control Ulcer Col-M 3.2
112382 Ulcer Col-M               12.6
112394 Match Control Ulcer Col-M 1.5
112383 Ulcer Col-M               7.4
112736 Match Control Ulcer Col-M 10.1
112423 Psoriasis-F               4.6
112427 Match Control Psoriasis-F 12.9
112418 Psoriasis-M               4.2
112723 Match Control Psoriasis-M 4.8
112419 Psoriasis-M               7.2
112424 Match Control Psoriasis-M 4.9
112420 Psoriasis-M               6.3
112425 Match Control Psoriasis-M 5.4
104689 (MF) OA Bone-Backus       33.4
104690 (MF) Adj “Normal” Bone-Backus 21.3
104691 (MF) OA Synovium-Backus   23.8
104692 (BA) OA Cartilage-Backus  22.1
104694 (BA) OA Bone-Backus       12.9
104695 (BA) Adj “Normal” Bone-Backus 25.2
104696 (BA) OA Synovium-Backus   32.5
104700 (SS) OA Bone-Backus       13.6
104701 (SS) Adj “Normal” Bone-Backus 20.9
104702 (SS) OA Synovium-Backus   18.9
117093 OA Cartilage Rep7         2.9
112672 OA Bone5                  13.5
112673 OA Synovium5              4.2
112674 OA Synovial Fluid cells5  5.5
117100 OA Cartilage Rep14        0.7
112756 OA Bone9                  12.7
112757 OA Synovium9              4.5
112758 OA Synovial Fluid Cells9  5.8
117125 RA Cartilage Rep2         10.7
113492 Bone2 RA                  16.5
113493 Synovium2 RA              8.2
113494 Syn Fluid Cells RA        13.7
113499 Cartilage4 RA             12.6
113500 Bone4 RA                  15.7
113501 Synovium4 RA              9.9
113502 Syn Fluid Cells4 RA       6.3
113495 Cartilage3 RA             12.8
113496 Bone3 RA                  14.4
113497 Synovium3 RA              5.9
113498 Syn Fluid Cells3 RA       17.3
117106 Normal Cartilage Rep20    3.5
113663 Bone3 Normal              0.1
113664 Synovium3 Normal          0.0
113665 Syn Fluid Cells3 Normal   0.1
117107 Normal Cartilage Rep22    2.5
113667 Bone4 Normal              4.8
113668 Synovium4 Normal          3.2
113669 Syn Fluid Cells4 Normal   4.3

[0707]

TABLE LF
CNS neurodegeneration v1.0
	Rel.	Rel.	Rel.	Rel.
	Exp. (%)	Exp. (%)	Exp. (%)	Exp. (%)
	Ag2648,	Ag264,	Ag2786,	Ag2787,
	Run	Run	Run	Run
Tissue Name	206955013	219966625	209052393	206985450
AD 1 Hippo	21.0	47.6	30.6	17.7
AD 2 Hippo	27.0	44.4	23.0	34.6
AD 3 Hippo	8.4	16.8	9.7	9.3
AD 4 Hippo	12.0	15.2	6.0	6.2
AD 5 Hippo	28.3	15.5	28.9	0.0
AD 6 Hippo	88.3	100.0	100.0	100.0
Control 2 Hippo	15.5	45.1	17.0	20.7
Control 4 Hippo	22.7	47.3	34.9	21.5
Control (Path) 3 Hippo	4.2	3.7	5.3	4.4
AD 1 Temporal Ctx	18.6	35.1	14.5	23.2
AD 2 Temporal Ctx	14.8	30.6	9.8	16.4
AD 3 Temporal Ctx	5.4	10.2	5.1	5.7
AD 4 Temporal Ctx	8.8	25.3	8.0	11.0
AD 5 Inf Temporal Ctx	35.4	18.9	30.4	36.6
AD 5 Sup Temporal Ctx	39.0	24.3	40.1	50.0
AD 6 Inf Temporal Ctx	100.0	61.6	61.6	85.9
AD 6 Sup Temporal Ctx	51.4	60.3	53.6	76.8
Control 1 Temporal Ctx	2.5	11.9	4.0	3.9
Control 2 Temporal Ctx	28.5	32.1	17.0	20.7
Control 3 Temporal Ctx	12.8	21.5	12.0	13.0
Control 3 Temporal Ctx	6.5	11.3	9.2	5.1
Control (Path) 1 Temporal Ctx	8.7	9.2	7.9	11.9
Control (Path) 2 Temporal Ctx	8.3	19.8	8.5	16.0
Control (Path) 3 Temporal Ctx	3.5	8.3	7.1	4.0
Control (Path) 4 Temporal Ctx	7.1	8.3	5.1	6.3
AD 1 Occipital Ctx	11.3	23.2	9.3	11.8
AD 2 Occipital Ctx (Missing)	0.0	0.0	0.0	0.0
AD 3 Occipital Ctx	9.2	11.8	4.4	4.4
AD 4 Occipital Ctx	9.5	17.2	4.6	7.4
AD 5 Occipital Ctx	19.8	11.2	12.5	9.9
AD 6 Occipital Ctx	13.4	5.9	9.1	16.4
Control 1 Occipital Ctx	2.8	5.7	5.2	3.6
Control 2 Occipital Ctx	11.3	29.5	19.8	26.6
Control 3 Occipital Ctx	12.2	17.0	5.4	10.9
Control 4 Occipital Ctx	3.9	7.9	2.7	10.0
Control (Path) 1 Occipital Ctx	11.6	13.8	13.7	16.3
Control (Path) 2 Occipital Ctx	3.6	5.9	3.5	2.8
Control (Path) 3 Occipital Ctx	3.3	6.6	4.0	3.5
Control (Path) 4 Occipital Ctx	10.2	12.9	8.7	9.3
Control 1 Parietal Ctx	2.2	3.5	5.4	2.8
Control 2 Parietal Ctx	30.4	25.9	26.4	25.7
Control 3 Parietal Ctx	8.2	15.9	7.1	7.6
Control (Path) 1 Parietal Ctx	11.4	10.5	9.3	10.9
Control (Path) 2 Parietal Ctx	5.6	6.7	4.9	5.3
Control (Path) 3 Parietal Ctx	3.3	3.1	3.1	2.7
Control (Path) 4 Parietal Ctx	11.7	12.6	11.1	9.5

[0708]

TABLE LG
General screening panel v1.6
                                 Rel. Exp. (%)
                                 Ag913, Run
Tissue Name                      277243064
Adipose                          5.7
Melanoma* Hs688(A).T             2.1
Melanoma* Hs688(B).T             2.7
Melanoma* M14                    30.4
Melanoma* LOXIMVI                0.0
Melanoma* SK-MEL-5               37.9
Squamous cell carcinoma SCC-4    11.0
Testis Pool                      1.1
Prostate ca.* (bone met) PC-3    21.6
Prostate Pool                    1.4
Placenta                         5.0
Uterus Pool                      1.5
Ovarian ca. OVCAR-3              0.0
Ovarian ca. SK-OV-3              9.7
Ovarian ca. OVCAR-4              19.1
Ovarian ca. OVCAR-5              60.7
Ovarian ca. IGROV-1              4.9
Ovarian ca. OVCAR-8              8.9
Ovary                            1.4
Breast ca. MCF-7                 13.1
Breast ca. MDA-MB-231            31.9
Breast ca. BT 549                6.7
Breast ca. T47D                  6.8
Breast ca. MDA-N                 3.0
Breast Pool                      1.1
Trachea                          6.6
Lung                             0.3
Fetal Lung                       7.9
Lung ca. NCI-N417                2.2
Lung ca. LX-1                    81.2
Lung ca. NCI-H146                1.0
Lung ca. SHP-77                  2.5
Lung ca. A549                    29.3
Lung ca. NCI-H526                1.2
Lung ca. NCI-H23                 17.1
Lung ca. NCI-H460                5.3
Lung ca. HOP-62                  21.3
Lung ca. NCI-H522                17.0
Liver                            0.0
Fetal Liver                      2.1
Liver ca. HepG2                  20.3
Kidney Pool                      4.6
Fetal Kidney                     2.1
Renal ca. 786-0                  29.3
Renal ca. A498                   42.3
Renal ca. ACHN                   8.8
Renal ca. UO-31                  55.9
Renal ca. TK-10                  100.0
Bladder                          18.3
Gastric ca. (liver met.) NCI-N87 43.5
Gastric ca. KATO III             24.0
Colon ca. SW-948                 9.3
Colon ca. SW480                  31.6
Colon ca.* (SW480 met) SW620     29.1
Colon ca. HT29                   10.7
Colon ca. HCT-116                37.6
Colon ca. CaCo-2                 15.7
Colon cancer tissue              19.9
Colon ca. SW1116                 6.3
Colon ca. Colo-205               9.7
Colon ca. SW-48                  12.2
Colon Pool                       1.5
Small Intestine Pool             3.1
Stomach Pool                     0.8
Bone Marrow Pool                 1.5
Fetal Heart                      1.2
Heart Pool                       0.7
Lymph Node Pool                  1.5
Fetal Skeletal Muscle            1.2
Skeletal Muscle Pool             1.5
Spleen Pool                      7.6
Thymus Pool                      4.5
CNS cancer (glio/astro) U87-MG   26.4
CNS cancer (glio/astro) U-118-MG 5.7
CNS cancer (neuro;met) SK-N-AS   16.8
CNS cancer (astro) SF-539        4.9
CNS cancer (astro) SNB-75        10.3
CNS cancer (glio) SNB-19         4.6
CNS cancer (glio) SF-295         63.7
Brain (Amygdala) Pool            3.9
Brain (cerebellum)               1.9
Brain (fetal)                    1.7
Brain (Hippocampus) Pool         2.8
Cerebral Cortex Pool             1.5
Brain (Substantia nigra) Pool    2.9
Brain (Thalamus) Pool            2.1
Brain (whole)                    2.2
Spinal Cord Pool                 5.2
Adrenal Gland                    5.9
Pituitary gland Pool             0.2
Salivary Gland                   3.3
Thyroid (female)                 3.6
Pancreatic ca. CAPAN2            30.6
Pancreas Pool                    7.2

[0709]

TABLE LH
Oncology cell line screening panel v3.2
                                 Rel. Exp. (%)
                                 Ag2648, Run
Tissue Name                      268695314
94905_Daoy_Medulloblastoma/Cerebellum_sscDNA 5.8
94906_TE671_Medulloblastom/Cerebellum_sscDNA 14.5
94907_D283                       21.2
Med_Medulloblastoma/Cerebellum_sscDNA
94908_PFSK-1_Primitive           3.8
Neuroectodermal/Cerebellum_sscDNA
94909_XF-498_CNS_sscDNA          2.3
94910_SNB-78_CNS/glioma_sscDNA   0.0
94911_SF-268_CNS/glioblastoma_sscDNA 9.4
94912_T98G_Glioblastoma_sscDNA   29.7
96776_SK-N-SH_Neuroblastoma      23.7
(metastasis)_sscDNA
94913_SF-295_CNS/glioblastoma_sscDNA 45.4
132565_NT2 pool_sscDNA           12.0
94914_Cerebellum_sscDNA          5.3
96777_Cerebellum_sscDNA          2.5
94916_NCI-H292_Mucoepidermoid lung 47.3
carcinoma_sscDNA
94917_DMS-114_Small cell lung cancer_sscDNA 14.6
94918_DMS-79_Small cell lung     42.9
cancer/neuroendocrine_sscDNA
94919_NCI-H146_Small cell lung   2.9
cancer/neuroendocrine_sscDNA
94920_NCI-H526_Small cell lung   11.1
cancer/neuroendocrine_sscDNA
94921_NCI-N417_Small cell lung   5.1
cancer/neuroendocrine_sscDNA
94923_NCI-H82_Small cell lung    17.3
cancer/neuroendocrine_sscDNA
94924_NCI-H157_Squamous cell lung 34.2
cancer (metastasis)_sscDNA
94925_NCI-H1155_Large cell lung  14.6
cancer/neuroendocrine_sscDNA
94926_NCI-H1299_Large cell lung  34.6
cancer/neuroendocrine_sscDNA
94927_NCI-H727_Lung carcinoid_sscDNA 19.6
94928_NCI-UMC-11_Lung carcinoid_sscDNA 12.2
94929_LX-1_Small cell lung cancer_sscDNA 72.7
94930_Colo-205_Colon cancer_sscDNA 42.9
94931_KM12_Colon cancer_sscDNA   23.7
94932_KM20L2_Colon cancer_sscDNA 11.7
94933_NCI-H716_Colon cancer_sscDNA 23.7
94935_SW-48_Colon adenocarcinoma_sscDNA 50.3
94936_SW1116_Colon adenocarcinoma_sscDNA 14.9
94937_LS 174T_Colon adenocarcinoma_sscDNA 54.0
94938_SW-948_Colon adenocarcinoma_sscDNA 2.3
94939_SW-480_Colon adenocarcinoma_sscDNA 29.3
94940_NCI-SNU-5_Gastric carcinoma_sscDNA 30.4
112197_KATO III_Stomach_sscDNA   19.6
94943_NCI-SNU-16_Gastric carcinoma_sscDNA 9.4
94944_NCI-SNU-1_Gastric carcinoma_sscDNA 32.1
94946_RF-1_Gastric adenocarcinoma_sscDNA 16.7
94947_RF-48_Gastric adenocarcinoma_sscDNA 12.2
96778_MKN-45_Gastric carcinoma_sscDNA 34.4
94949_NCI-N87_Gastric carcinoma_sscDNA 43.5
94951_OVCAR-5_Ovarian carcinoma_sscDNA 25.9
94952_RL95-2_Uterine carcinoma_sscDNA 13.9
94953_HelaS3_Cervical            14.3
adenocarcinoma_sscDNA
94954_Ca Ski_Cervical epidermoid 20.4
carcinoma (metastasis)_sscDNA
94955_ES-2_Ovarian clear cell    11.3
carcinoma_sscDNA
94957_Ramos/6 h stim_Stimulated  13.8
with PMA/ionomycin 6 h_sscDNA
94958_Ramos/14 h stim—           24.3
Stimulated with PMA/ionomycin
14 h_sscDNA
94962_MEG-01_Chronic myelogenous 12.2
leukemia(megokaryoblast)_sscDNA
94963_Raji_Burkitt's lymphoma_sscDNA 14.9
94964_Daudi_Burkitt's lymphoma_sscDNA 36.9
94965_U266_B-cell                49.3
plasmacytoma/myeloma_sscDNA
94968_CA46_Burkitt's lymphoma_sscDNA 20.6
94970_RL_non-Hodgkin's B-cell    18.0
lymphoma_sscDNA
94972_JM1_pre-B-cell             45.7
lymphoma/leukemia_sscDNA
94973_Jurkat_T cell leukemia_sscDNA 8.0
94974_TF-1_Erythroleukemia_sscDNA 7.5
94975_HUT 78_T-cell lymphoma_sscDNA 32.3
94977_U937_Histiocytic lymphoma_sscDNA 19.5
94980_KU-812_Myelogenous leukemia_sscDNA 7.0
94981_769-P_Clear cell renal     37.9
carcinoma_sscDNA
94983_Caki-2_Clear cell renal    58.6
carcinoma_sscDNA
94984_SW 839_Clear cell renal    97.9
carcinoma_sscDNA
94986_G401_Wilms' tumor_sscDNA   12.3
126768_293 cells_sscDNA          19.5
94987_Hs766T_Pancreatic carcinoma (LN 17.1
metastasis)_sscDNA
94988_CAPAN-1_Pancreatic adenocarcinoma 60.3
(liver metastasis)_sscDNA
94989_SU86.86_Pancreatic carcinoma 100.0
(liver metastasis)_sscDNA
94990_BxPC-3_Pancreatic          21.9
adenocarcinoma_sscDNA
94991_HPAC_Pancreatic            39.5
adenocarcinoma_sscDNA
94992_MIA PaCa-2_Pancreatic      11.6
carcinoma_sscDNA
94993_CFPAC-1_Pancreatic ductal  65.1
adenocarcinoma_sscDNA
94994_PANC-1_Pancreatic epithelioid 87.7
ductal carcinoma_sscDNA
94996_T24_Bladder carcinma       18.6
(transitional cell)_sscDNA
94997_5637_Bladder carcinoma_sscDNA 74.2
94998_HT-1197_Bladder carcinoma_sscDNA 21.0
94999_UM-UC-3_Bladder carcinma   4.9
(transitional cell)_sscDNA
95000_A204_Rhabdomyosarcoma_sscDNA 26.8
95001_HT-1080_Fibrosarcoma_sscDNA 55.1
95002_MG-63_Osteosarcoma (bone)_sscDNA 17.6
95003_SK-LMS-1_Leiomyosarcoma    24.3
(vulva)_sscDNA
95004_SJRH30_Rhabdomyosarcoma    6.7
(met to bone marrow)_sscDNA
95005_A431_Epidermoid carcinoma_sscDNA 24.7
95007_WM266-4_Melanoma_sscDNA    11.7
112195_DU 145_Prostate_sscDNA    50.0
95012_MDA-MB-468_Breast          22.2
adenocarcinoma_sscDNA
112196_SSC-4_Tongue_sscDNA       20.7
112194_SSC-9_Tongue_sscDNA       84.7
112191_SSC-15_Tongue_sscDNA      83.5
95017_CAL 27_Squamous cell       50.3
carcinoma of tongue_sscDNA

[0710]

TABLE LI
Panel 1.3D
	Rel.	Rel.	Rel.
	Exp. (%)	Exp. (%)	Exp. (%)
	Ag264,	Ag2786,	Ag2787,
	Run	Run	Run
Tissue Name	156606391	165527181	165518605
Liver	20.9	16.5	19.9
adenocarcinoma
Pancreas	10.3	13.0	13.5
Pancreatic ca.	19.1	13.0	27.4
CAPAN2
Adrenal gland	14.5	18.2	15.5
Thyroid	15.2	13.8	9.3
Salivary gland	6.2	7.9	6.0
Pituitary gland	3.1	4.4	5.6
Brain (fetal)	2.9	6.6	7.7
Brain (whole)	4.7	18.6	25.2
Brain (amygdala)	17.3	32.1	27.9
Brain	2.1	9.3	8.0
(cerebellum)
Brain	50.3	29.5	24.3
(hippocampus)
Brain (substantia	5.4	34.6	32.3
nigra)
Brain (thalamus)	12.2	29.9	33.7
Cerebral Cortex	1.9	5.0	7.6
Spinal cord	11.6	50.0	49.3
glio/astro U87-MG	20.2	10.2	18.8
glio/astro	7.1	6.3	10.2
U-118-MG
astrocytoma SW1783	19.6	22.7	39.0
neuro*; met	33.2	16.7	38.2
SK-N-AS
astrocytoma	2.6	7.0	6.5
SF-539
astrocytoma	10.9	15.0	10.2
SNB-75
glioma SNB-19	0.5	6.6	6.4
glioma U251	2.5	17.1	35.6
glioma SF-295	89.5	29.3	38.7
Heart (fetal)	10.3	3.4	7.5
Heart	2.6	6.5	7.6
Skeletal muscle	78.5	8.2	5.3
(fetal)
Skeletal muscle	2.0	13.3	12.3
Bone marrow	16.6	20.9	20.6
Thymus	14.1	12.2	9.7
Spleen	55.5	52.9	62.0
Lymph node	19.8	88.3	80.7
Colorectal	10.4	4.9	9.2
Stomach	12.6	24.7	22.8
Small intestine	24.8	37.9	47.0
Colon ca. SW480	31.9	15.7	15.3
Colon ca.*	37.9	10.2	22.8
SW620(SW480 met)
Colon ca. HT29	14.0	0.9	4.0
Colon ca. HCT-116	17.1	11.2	14.3
Colon ca. CaCo-2	14.6	10.4	10.7
Colon ca.	31.0	18.3	25.9
tissue(ODO3866)
Colon ca.	21.5	11.0	11.2
HCC-2998
Gastric ca.*	44.4	46.7	52.1
(liver met)
NCI-N87
Bladder	12.7	8.1	11.0
Trachea	42.0	16.7	20.3
Kidney	3.7	7.3	5.1
Kidney (fetal)	17.1	17.7	11.0
Renal ca. 786-0	11.3	19.3	39.0
Renal ca. A498	100.0	100.0	100.0
Renal ca. RXF 393	18.7	73.2	81.8
Renal ca. ACHN	17.7	7.5	13.1
Renal ca. UO-31	63.3	34.9	42.0
Renal ca. TK-10	49.3	24.7	39.5
Liver	2.4	4.5	3.2
Liver (fetal)	7.9	10.7	5.4
Liver ca.	35.4	20.9	33.7
(hepatoblast)
HepG2
Lung	33.2	40.3	27.5
Lung (fetal)	10.2	9.1	8.9
Lung ca. (small	46.0	42.3	63.3
cell) LX-1
Lung ca. (small	6.6	0.1	2.1
cell) NCI-H69
Lung ca. (s. cell	3.0	2.0	2.6
var.) SHP-77
Lung ca. (large	2.8	11.7	17.7
cell)NCI-H460
Lung ca. (non-sm.	11.7	7.1	9.1
cell) A549
Lung ca. (non-s.	10.4	11.5	10.5
cell) NCI-H23
Lung ca. (non-s.	47.0	53.2	43.2
cell) HOP-62
Lung ca. (non-s.	20.6	2.4	7.5
cl) NCI-H522
Lung ca. (squam.)	12.0	9.0	18.6
SW 900
Lung ca. (squam.)	0.2	1.0	1.4
NCI-H596
Mammary gland	9.5	23.5	22.2
Breast ca.*	11.0	8.1	10.5
(pl. ef) MCF-7
Breast ca.*(pl.	60.7	43.5	67.4
ef) MDA-MB-231
Breast ca.*	7.8	11.2	15.1
(pl. ef) T47D
Breast ca.	9.4	6.4	7.3
BT-549
Breast ca. MDA-N	5.0	3.3	4.3
Ovary	21.9	1.7	9.4
Ovarian ca.	11.5	12.8	17.9
OVCAR-3
Ovarian ca.	6.9	15.5	17.6
OVCAR-4
Ovarian ca.	69.7	44.8	45.1
OVCAR-5
Ovarian ca.	13.3	2.5	3.3
OVCAR-8
Ovarian ca.	5.4	1.7	4.3
IGROV-1
Ovarian ca.*	2.8	6.7	9.1
(ascites)
SK-OV-3
Uterus	2.3	23.7	12.4
Placenta	24.0	19.2	13.4
Prostate	4.2	11.8	9.0
Prostate ca.*	21.8	13.2	11.2
(bone met)PC-3
Testis	13.2	14.9	2.6
Melanoma	1.2	2.2	0.6
Hs688(A).T
Melanoma* (met)	0.7	3.9	2.8
Hs688(B).T
Melanoma	3.0	10.8	13.9
UACC-62
Melanoma M14	11.1	47.6	85.9
Melanoma	7.7	2.6	4.2
LOX IMVI
Melanoma* (met)	12.3	5.8	14.9
SK-MEL-5
Adipose	11.2	10.3	10.6

[0711]

TABLE LJ
Panel 2D
	Rel.	Rel.	Rel.
	Exp. (%)	Exp. (%)	Exp. (%)
	Ag2648,	Ag2786,	Ag2787,
	Run	Run	Run
Tissue Name	156606695	162570060	163577798
Normal Colon	21.9	25.7	42.3
CC Well to Mod	26.4	30.6	18.8
Diff (ODO3866)
CC Margin (ODO3866)	7.6	6.6	3.6
CC Gr. 2	11.7	10.1	4.7
rectosigmoid
(ODO3868)
CC Margin (ODO3868)	2.3	1.6	1.4
CC Mod Diff	23.2	26.1	8.3
(ODO3920)
CC Margin	11.3	7.1	2.3
(ODO3920)
CC Gr. 2 ascend	54.7	62.4	39.8
colon (ODO3921)
CC Margin (ODO3921)	10.7	8.7	5.4
CC from Partial	58.2	44.1	37.9
Hepatectomy
(ODO4309) Mets
Liver Margin	11.3	9.9	7.7
(ODO4309)
Colon mets to	49.7	41.2	12.6
lung (OD04451-01)
Lung Margin	20.9	8.3	10.9
(OD04451-02)
Normal	8.3	37.4	35.4
Prostate 6546-1
Prostate Cancer	18.3	16.4	8.2
(OD04410)
Prostate Margin	15.3	11.3	7.5
(OD04410)
Prostate Cancer	11.0	11.6	6.3
(OD04720-01)
Prostate Margin	19.6	21.2	33.4
(OD04720-02)
Normal Lung	42.3	39.2	27.5
061010
Lung Met to	33.9	37.1	25.9
Muscle
(ODO4286)
Muscle Margin	17.0	17.4	11.3
(ODO4286)
Lung Malignant	45.4	47.0	31.0
Cancer (OD03126)
Lung Margin	44.1	29.9	33.4
(OD03126)
Lung Cancer	52.5	33.9	35.6
(OD04404)
Lung Margin	24.1	17.7	11.7
(OD04404)
Lung Cancer	42.3	31.4	20.0
(OD04565)
Lung Margin	28.3	14.0	8.2
(OD04565)
Lung Cancer	36.3	35.1	35.4
(OD04237-01)
Lung Margin	25.5	31.6	25.3
(OD04237-02)
Ocular Mel	22.1	25.0	23.7
Met to Liver
(ODO4310)
Liver Margin	10.3	7.1	7.4
(ODO4310)
Melanoma	21.3	18.0	11.3
Mets to Lung
(OD04321)
Lung Margin	39.2	34.2	33.7
(OD04321)
Normal Kidney	17.1	16.8	27.0
Kidney Ca,	85.9	77.4	78.5
Nuclear grade
2 (OD04338)
Kidney Margin	26.4	18.6	10.4
(OD04338)
Kidney Ca	56.6	46.7	19.2
Nuclear grade
1/2 (OD04339)
Kidney Margin	8.8	10.1	10.3
(OD04339)
Kidney Ca,	97.9	100.0	100.0
Clear cell type
(OD04340)
Kidney Margin	31.2	29.9	18.2
(OD04340)
Kidney Ca,	47.6	40.9	25.3
Nuclear grade
3 (OD04348)
Kidney Margin	21.6	25.2	15.5
(OD04348)
Kidney Cancer	60.3	42.6	25.5
(OD04622-01)
Kidney Margin	5.7	4.9	3.7
(OD04622-03)
Kidney Cancer	27.9	32.3	18.0
(OD04450-01)
Kidney Margin	6.8	6.1	9.5
(OD04450-03)
Kidney Cancer	37.6	29.3	44.1
8120607
Kidney Margin	10.4	7.1	8.2
8120608
Kidney Cancer	29.9	33.7	36.6
8120613
Kidney Margin	0.0	4.9	6.8
8120614
Kidney Cancer	77.9	47.6	68.8
9010320
Kidney Margin	27.0	26.1	22.1
9010321
Normal Uterus	2.8	2.0	0.8
Uterus Cancer	12.9	15.2	11.1
064011
Normal Thyroid	15.2	9.9	13.0
Thyroid Cancer	13.2	10.4	18.7
064010
Thyroid Cancer	19.2	16.0	6.1
A302152
Thyroid Margin	17.2	16.4	9.5
A302153
Normal Breast	19.2	18.8	15.7
Breast Cancer	37.6	25.2	9.7
(OD04566)
Breast Cancer	31.0	34.4	27.2
(OD04590-01)
Breast Cancer	56.3	57.0	38.2
Mets (OD04590-03)
Breast Cancer	28.3	25.7	16.2
Metastasis
(OD04655-05)
Breast Cancer	23.7	18.9	16.6
064006
Breast Cancer	16.6	17.9	14.2
1024
Breast Cancer	33.7	37.1	50.0
9100266
Breast Margin	9.9	13.0	16.8
9100265
Breast Cancer	42.9	35.4	49.0
A209073
Breast Margin	16.8	16.8	11.0
A209073
Normal Liver	4.5	7.0	3.1
Liver Cancer	6.5	4.8	6.4
064003
Liver Cancer	9.3	4.5	7.7
1025
Liver Cancer	25.0	19.6	16.5
1026
Liver Cancer	10.4	8.0	7.5
6004-T
Liver Tissue	28.1	15.5	20.0
6004-N
Liver Cancer	18.3	16.2	17.2
6005-T
Liver Tissue	6.0	7.0	3.0
6005-N
Normal Bladder	51.4	66.9	66.4
Bladder Cancer	31.0	17.4	23.3
1023
Bladder Cancer	27.4	16.2	49.0
A302173
Bladder Cancer	100.0	94.6	72.2
(OD04718-01)
Bladder Normal	24.0	22.1	14.9
Adjacent
(OD04718-03)
Normal Ovary	8.8	8.3	11.6
Ovarian Cancer	74.7	76.8	80.7
064008
Ovarian Cancer	82.4	72.2	73.2
(OD04768-07)
Ovary Margin	19.9	12.9	5.9
(OD04768-08)
Normal Stomach	11.8	9.5	14.8
Gastric Cancer	8.2	1.8	6.7
9060358
Stomach Margin	18.4	16.6	15.6
9060359
Gastric Cancer	21.6	18.6	15.9
9060395
Stomach Margin	23.5	17.7	20.6
9060394
Gastric Cancer	62.0	63.7	61.6
9060397
Stomach Margin	10.2	9.9	4.9
9060396
Gastric Cancer	24.1	26.2	33.4
064005

[0712]

TABLE LK
Panel 4D
	Rel.	Rel.	Rel.
	Exp. (%)	Exp. (%)	Exp. (%)
	Ag2648,	Ag2786,	Ag2787,
	Run	Run	Run
Tissue Name	156607036	162188411	162187585
Secondary Th1 act	18.4	11.2	10.9
Secondary Th2 act	24.8	17.6	17.8
Secondary Tr1 act	20.6	10.4	10.6
Secondary Th1 rest	9.6	8.6	6.3
Secondary Th2 rest	8.2	7.2	4.5
Secondary Tr1 rest	9.3	6.7	5.5
Primary Th1 act	12.1	9.8	11.1
Primary Th2 act	11.0	5.3	6.5
Primary Tr1 act	15.2	5.7	9.0
Primary Th1 rest	28.3	15.1	21.5
Primary Th2 rest	13.6	9.3	11.0
Primary Tr1 rest	10.4	4.5	7.0
CD45RA CD4	22.8	9.9	8.5
lymphocyte act
CD45RO CD4	16.5	7.2	10.7
lymphocyte act
CD8 lymphocyte	8.8	12.1	7.4
act
Secondary CD8	15.7	17.4	10.8
lymphocyte rest
Secondary CD8	8.5	5.1	6.7
lymphocyte act
CD4 lymphocyte none	7.1	5.6	3.4
2ry Th1/Th2/Tr1_anti-	9.0	8.2	8.4
CD95 CH11
LAK cells rest	83.5	58.6	70.2
LAK cells IL-2	19.3	11.6	19.3
LAK cells	16.0	15.8	14.1
IL-2 + IL-12
LAK cells IL-2 +	28.5	17.3	23.3
IFN gamma
LAK cells IL-2 +	27.4	21.8	13.0
IL-18
LAK cells	84.1	60.3	49.7
PMA/ionomycin
NK Cells IL-2 rest	31.9	26.6	21.9
Two Way MLR 3 day	90.1	73.2	60.7
Two Way MLR 5 day	33.9	35.6	40.1
Two Way MLR 7 day	13.5	12.4	10.6
PBMC rest	12.4	15.3	11.6
PBMC PWM	38.2	27.2	47.3
PBMC PHA-L	25.3	23.2	31.9
Ramos (B cell) none	22.7	24.1	21.2
Ramos (B cell)	49.7	11.3	49.3
ionomycin
B lymphocytes PWM	22.8	17.0	21.9
B lymphocytes	26.6	16.2	22.2
CD40L and IL-4
EOL-1 dbcAMP	2.8	1.6	2.7
EOL-1 dbcAMP	33.0	25.5	24.8
PMA/ionomycin
Dendritic cells none	48.3	42.3	42.6
Dendritic cells LPS	83.5	68.3	81.2
Dendritic cells	29.3	29.9	27.2
anti-CD40
Monocytes rest	42.9	57.0	40.9
Monocytes LPS	80.7	100.0	90.1
Macrophages rest	94.6	82.9	98.6
Macrophages LPS	100.0	94.0	100.0
HUVEC none	11.3	8.7	9.5
HUVEC starved	19.6	10.6	19.6
HUVEC IL-1beta	10.2	4.6	8.6
HUVEC IFN gamma	33.2	21.9	18.7
HUVEC TNF alpha +	76.3	59.0	66.9
IFN gamma
HUVEC TNF	34.6	29.9	26.1
alpha + IL4
HUVEC IL-11	7.4	6.3	3.8
Lung Microvascular	36.1	40.6	38.2
EC none
Lung Microvascular	57.0	57.8	49.7
EC TNFalpha +
IL-1beta
Microvascular	34.6	26.2	33.9
Dermal EC none
Microsvasular	72.7	75.8	55.5
Dermal EC
TNFalpha + IL-1beta
Bronchial epithelium	7.4	88.3	61.6
TNFalpha + IL1beta
Small airway	9.9	11.6	13.3
epithelium none
Small airway	73.2	35.6	94.6
epithelium
TNFalpha + IL-1beta
Coronery	20.7	17.4	12.2
artery SMC rest
Coronery artery SMC	20.2	9.6	10.6
TNFalpha + IL-1beta
Astrocytes rest	8.3	6.8	7.0
Astrocytes	18.2	15.8	10.3
TNFalpha + IL-1beta
KU-812	2.3	1.9	1.6
(Basophil) rest
KU-812 (Basophil)	3.4	1.9	3.3
PMA/ionomycin
CCD1106	19.5	21.8	20.7
(Keratinocytes) none
CCD1106 (Keratinocytes)	12.7	80.7	51.8
TNFalpha + IL-1beta
Liver cirrhosis	4.7	4.6	2.9
Lupus kidney	4.2	2.3	2.3
NCI-H292 none	20.0	12.1	16.4
NCI-H292 IL-4	23.5	13.5	21.5
NCI-H292 IL-9	23.3	18.7	23.5
NCI-H292 IL-13	23.7	15.9	17.4
NCI-H292 IFN gamma	58.6	38.2	39.5
HPAEC none	13.8	9.2	8.8
HPAEC TNF alpha +	89.5	71.7	72.2
IL-1 beta
Lung fibroblast none	3.1	3.2	3.0
Lung fibroblast	20.0	15.6	12.3
TNF alpha + IL-1 beta
Lung fibroblast IL-4	4.4	2.5	3.3
Lung fibroblast IL-9	4.3	3.1	3.3
Lung fibroblast IL-13	1.6	2.9	1.6
Lung fibroblast	27.9	20.3	25.3
IFN gamma
Dermal fibroblast	9.6	7.6	7.5
CCD1070 rest
Dermal fibroblast	26.6	17.8	25.2
CCD1070 TNF alpha
Dermal fibroblast	8.4	6.9	4.6
CCD1070 IL-1 beta
Dermal fibroblast	20.2	12.5	12.1
IFN gamma
Dermal	6.0	1.7	5.0
fibroblast IL-4
IBD Colitis 2	0.9	1.1	1.2
IBD Crohn's	2.4	0.3	0.3
Colon	13.7	9.8	5.4
Lung	17.9	10.8	8.8
Thymus	5.8	1.5	3.2
Kidney	11.3	11.0	8.4

[0713] AI_comprehensive panel_v1.0 Summary: Ag2787 Highest expression of this gene is detected in a matched control sample for ulcerative colitis (CT=28). Moderate to low levels of expression of this gene is also detected in samples derived from normal and orthoarthitis/rheumatoid arthritis bone and adjacent bone, cartilage, synovium and synovial fluid samples, from normal lung, COPD lung, emphysema, atopic asthma, asthma, allergy, Crohn's disease (normal matched control and diseased), ulcerative colitis (normal matched control and diseased), and psoriasis (normal matched control and diseased). Therefore, therapeutic modulation of this gene product may ameliorate symptoms/conditions associated with autoimmune and inflammatory disorders including psoriasis, allergy, asthma, inflammatory bowel disease, rheumatoid arthritis and osteoarthritis.

[0714] CNS_neurodegeneration_v1.0 Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals. However, no differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment. Please see Panel 1.3D for a discussion of the potential role of this gene in treatment of central nervous system disorders.

[0715] General_screening—panel_v1.6 Summary: Ag913 Highest expression of this gene is detected in renal cancer TK-10 cell line (CT=26.9). Moderate to high expression of this gene is seen in number of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. In addition, this gene shows widespread expression in this panel which correlates with expression seen in panel 1.3D. Please see panel 1.3D for further discussion of this gene.

[0716] Interestingly, this gene is expressed at much higher levels in fetal (CTs=30-32.5) when compared to adult lung and liver (CTs=35-40). This observation suggests that expression of this gene can be used to distinguish fetal from adult lung and liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance lung and liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of lung and liver related diseases.

[0717] Oncology_cell_line_screening_panel_v3.2 Summary: Ag2648 Highest expression of this gene is detected in pancreatic cancer SU86.86 cell line (CT=30). Moderate expression of this gene is seen in number of cancer cell lines derived from lung, bone marrow, epidermoid, vulva, bone, bladder, pancreatic, renal, B cells and T cells, leukemia, lymphoma, cervical, gastric, colon, lung and brain. This expression pattern correlates with that seen in panel 1.3D. Please see panel 1.3D for further discussion of this gene.

[0718] Panel 1.3D Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in renal cancer A498 cell line (CTs=28-28.9). Moderate levels of expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation 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.

[0719] Among tissues with metabolic or endocrine function, this gene is expressed at moderate to low levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

[0720] In addition, this gene is expressed at moderate to low levels in all regions of the central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. 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.

[0721] Panel 2D Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in bladder and kidney cancers (CTs=26.4-28). High to moderate expression of this gene is also detected in cancer and normal samples derived from colon, prostate, liver, lung, kidney, breast, thyroid, ovary and stomach. Interestingly, expression of this gene is higher in cancer samples especially gastric, bladder, breast, kidney and colon cancer compared to adjacent normal tissues. Therefore, expression of this gene may be used as diagnostic marker to detect the presence of these cancers. In addition, this gene codes for a putative protease belonging to Rhomboid family that activates growth factors ligands (Urban et al. Cell 2001 Oct. 19;107(2):173-82). Therefore this gene may play a role in tumor cell proliferation and invasion, by activating growth factors like TGFalpha and EGF that mediates cell growth and invasion. Targeting of protease encoded by this gene with a human monoclonal antibody that results in an inhibition of the activity of this protein, preferably its putative protease activity, will have therapeutic effect on tumors, preferably on colon, gastric, kidney, ovarian and bladder tumors and will result in reduced tumor cell growth, proliferation and invasion.

[0722] Panel 4D Summary: Ag2648/Ag2786/Ag2787 Three experiments with different probe-primer sets are in good agreement. Highest expression of this gene is detected in LPS activated macrophages and monocytes (CTs=27-28.5). This gene is expressed at high to moderate levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members of the T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. Expression of this gene is stimulated in activated endothelial cells, small airway epithelium and fibroblasts. The ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

Example D

Identification of Single Nucleotide Polymorphisms in NOVX Nucleic Acid Sequences

[0723] 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.

[0724] 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, HMMER, FASTA, Hybrid and other relevant programs.

[0725] 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.

[0726] 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).

[0727] 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.

TABLE SNP1
SNP Variants for CG172318-01 (NOV 7a).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13382021	916	G	A	295	Gly	Glu

[0728]

TABLE SNP2
SNP Variants for CG170791-01 (NOV 5a).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13382023	798	A	T	250	Gln	His

[0729]

TABLE SNP3
SNP Variant for CG176203-01 (NOV 11a).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13382025	2728	C	T	0
13382024	3712	C	T	0

[0730]

TABLE SNP4
SNP Variants for CG176213-01 (NOV 12a).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13382017	1105	C	G	234	Thr	Ser
13382018	1730	A	T	0

[0731]

TABLE SNP5
SNP Variant for CG50691-02 (NOV13c).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13382027	663	C	T	208	Ser	Ser

[0732]

TABLE SNP6
SNP Variants for CG52414-01 (NOV 15b).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13379509	467	T	C	60	Leu	Pro
13381817	565	G	A	93	Ala	Thr
13382069	841	C	T	185	Gln	End

[0733]

TABLE SNP7
SNP Variants for CG52552-04 (NOV 16b).
	Nucleotides	Amino Acids
Variant	Position	Initial	Modified	Position	Initial	Modified
13374454	316	C	T	104	Ala	Val
13374455	414	T	C	137	Tyr	His
13374456	468	A	G	155	Ile	Val
13374457	502	T	C	166	Phe	Ser
13374458	524	T	C	173	Ser	Ser
13382066	818	C	G	271	Pro	Pro

Example E

Inhibition of Expression of NOV15 CG52414-01 Using Antisense in an Ovarian Tumor-Derived Cell Line

[0734] Five oligonucleotides were designed and synthesized as mixed-backbone olignucleotides containing modified phosphorothioate segments at 5′ and 3′ ends and 2′-O-methyl RNA oligoribonucleotide segments located in the middle. The purity of the olignucleotides was confirmed by mass spectrophometry. The oligonucleotide sequences for CG52414-01 are:

AS1:	5′ CCCTCCCAGUCGCCGCTGAC 3′	(complementary to the	SEQ ID 138
		sequence located in 5′UTR of LIV-1):
AS2:	5′ TTCAGGCGGCCGAGCGCAT 3′	(complementary to the	SEQ ID 139
		sequence surrounding ATG start codon):
AS3:	5′ AGGTCACGCUGGCACGAGGC 3′	(complementary to the	SEQ ID 140
		sequence 3′ next to AS2):
AS4:	5′ GCCCUUGAUCUCGCAGTCCA 3′	(complementary to the	SEQ ID 141
		sequence in the middle of SLPI ORF):
AS5:	5′ AGCGGUCAGUGCAGCACCTG 3′	(complementary to the	SEQ ID 142
		sequence flanking the 3′ stop codon):

[0735] OVCAR-5 cells were seeded 10,000 cells/well in a 96 well plate in complete medium 24 hr before transfection and cultured to reach 50% confluency on the day of transfection. Oligonucleotides were diluted with Optimen to 100 and 400 nM, and mixed with Oligofectamine (Invitrogen) according to manufacturer's instructions. Cells were washed with serum-free medium. The oligo and liposome mixture were then added to cells. After 4 hr incubation period, serum were added back to cells. CellTiter 96Aqueous Non-Radioactive Cell Proliferation Assay Kit from Promega was used to determine the number of viable cells 72 hrs after transfection. Briefly, 20 μl of combined MTS solution was diluted with 100 μl complete medium, and added to each well of the 96 well plate. After 1 hr incubation at 37° C., the absorbance at 490 nm was recorded using an ELISA plate reader.

[0736] Inhibition of expression of CG52414-01 by antisense compounds AS1, AS2, AS3, AS4, AS5, alone or in combination, resulted in inhibition of OVCAR-5 cell proliferation (FIG. 1).

Example F

Relevant Pathways of NOV15 CG52414.

[0737] Materials and Methods

[0738] PathCalling™ Technology: The sequence of Ace. No CG52414-01 was derived by laboratory screening of cDNA library by the two-hybrid approach. cDNA fragments covering either the full length of the DNA sequence, or part of the sequence, or both, were 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.

[0739] The laboratory screening was performed using the methods that follow. cDNA libraries were derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then directionally cloned into the appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). Such cDNA libraries as well as commercially available cDNA libraries from Clontech (Palo Alto, Calif.) were then transferred from E. coli into a CuraGen Corporation proprietary yeast strain (disclosed in U.S. Pat. Nos. 6,057,101 and 6,083,693, incorporated herein by reference in their entireties).

[0740] 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.

[0741] 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. Pat. Nos. 6,057,101 and 6,083,693).

[0742] Interaction protein pairs are added to CuraGen's PathCalling™ Protein Interaction Database. This database allows for the discovery of novel pharmaceutical drug targets by virtue of their interactions and/or presence in pathologically related signaling pathways. Protein interactions are subsequently analyzed using bioinformatic tools within GeneScape™, which provides a means of visualization of binary protein interactions, protein complex formation, as well as complete cellular signaling pathways. The specific interactions, which constitute the specific complexes, may also be useful for therapeutic intervention through the use of recombinant protein or antibody therapies, small molecule drugs, or gene therapy approaches. Protein interactions, which are identified through the mining of the PathCalling™ database, can be screened in vitro and in vivo to provide expression, functional, biochemical, and phenotypic information. Assays may be used alone or in conjunction and include, but are not limited to the following technologies; RTQ-PCR, Transfection of recombinant proteins, Co-immunoprecipitation and mass spectrometry, FRET, Affinity Chromatography, Immunohistochemisty or Immunocytochemistry, gene CHIP hybridizations, antisense (i.e., knock-down, knock-up), GeneCalling experiments, and/or biochemical assays (phosphorylation, dephosphorylation, protease, etc.).

[0743] Proteins CG52414-01 (Rhomboid like), tousled like kinase 1 and zinc finger ZNF263 protein were found to interact and may result in the formation of a protein complex, or may constitute a series of complexes, which form in order to propagate a cellular signal, which is physiologically relevant to a disease pathology.

Other Embodiments

[0744] Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims. The claims presented are representative of the inventions disclosed herein. Other, unclaimed inventions are also contemplated. Applicants reserve the right to pursue such inventions in later claims.

Claims

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

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

3. An isolated polypeptide comprising an amino acid sequence which is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

4. An isolated polypeptide, wherein the polypeptide comprises an amino acid sequence comprising one or more conservative substitutions in the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

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

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

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

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

9. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising: (a) providing said sample; (b) introducing said sample to an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.

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

11. A method of identifying an agent that binds to the polypeptide of claim 1, the method comprising: (a) introducing said polypeptide to said agent; and (b) determining whether said agent binds to said polypeptide.

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

13. A method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of the polypeptide of claim 1, the method comprising: (a) providing a cell expressing the polypeptide of claim 1 and having a property or function ascribable to the polypeptide; (b) contacting the cell with a composition comprising a candidate substance; and (c) determining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition in the absence of the substance, the substance is identified as a potential therapeutic agent.

14. A method for screening for a modulator of activity of or of latency or predisposition to a pathology associated with the polypeptide of claim 1, said method comprising: (a) administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of claim 1, wherein said test animal recombinantly expresses the polypeptide of claim 1;(b) measuring the activity of said polypeptide in said test animal after administering the compound of step (a); and (c) comparing the activity of said polypeptide in said test animal with the activity of said polypeptide in a control animal not administered said polypeptide, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator activity of or latency or predisposition to, a pathology associated with the polypeptide of claim 1.

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

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

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

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

19. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37 or a biologically active fragment thereof.

20. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37.

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

22. A nucleic acid molecule, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2n−1, wherein n is an integer between 1 and 37.

23. An isolated nucleic acid molecule encoding the mature form of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 37.

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

25. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n−1, wherein n is an integer between 1 and 37, or a complement of said nucleotide sequence.

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

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

28. A cell comprising the vector of claim 26.

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

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

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

32. A method for determining the presence or amount of the nucleic acid molecule of claim 20 in a sample, the method comprising: (a) providing said sample; (b) introducing said sample to a probe that binds to said nucleic acid molecule; and (c) determining the presence or amount of said probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.

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

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

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

36. A method of producing the polypeptide of claim 1, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37.

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

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

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

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

41. A method of producing the polypeptide of claim 2, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n−1, wherein n is an integer between 1 and 37.

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

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

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

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